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Sample records for autophagy genes map1lc3b

  1. CRISPR/Cas9 Mediated GFP Knock-in at the MAP1LC3B Locus in 293FT Cells Is Better for Bona Fide Monitoring Cellular Autophagy.

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    Wu, Zhiqiang; Zhao, Jinlin; Qiu, Minghan; Mi, Zeyun; Meng, Maobin; Guo, Yu; Wang, Hui; Yuan, Zhiyong

    2018-04-19

    Accurately identifying and quantifying cellular autophagy is very important as the significance of autophagy in physiological and pathological processes becomes increasingly evident. Ectopically expressed fluorescent-tagged microtubule-associated protein light chain 3B (MAP1LC3B, LC3) is the most widely used reporter for monitoring autophagy activity thus far. However, this approach ignores the influence of constitutively overexpressed LC3 on autophagy itself and autophagy-related processes and its accuracy in indicating autophagy is questionable. Here, we generated a knock-in GFP-LC3 reporter via the CRISPR/Cas9 system in 293FT cells to add GFP to the N-terminal of and in frame with endogenous LC3. We proved that this knock-in GFP-LC3 was expressed at biological level driven by the endogenous transcriptional regulatory elements as the wild type alleles. Compared with the ectopically expressed GFP-LC3, the endogenous knock-in reporter exhibited much higher sensitivity and signal-to-noise ratio of GFP-LC3 puncta upon the induction or inhibition of autophagy at certain step for monitoring autophagy activity. Thus, according to the previous reported concerning and the results presented here, we suggest that this knock-in GFP-LC3 reporter is better for bona fide monitoring cellular autophagy and should be employed for further study of autophagy in vitro and in vivo. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

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    Rodríguez, Andrea E; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

    2016-01-01

    Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.

  3. THE DIFFERENCE OF MAP1LC3 LEVEL AS MACROPHAGE AUTOPHAGY MARKER BETWEEN RESISTANT AND SENSITIVE TUBERCULOSIS PATIENTS ON RIFAMPICIN

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    Dian novita W

    2018-04-01

    Full Text Available Mycobacterium tuberculosis (MTB is an intracelular bacteria that live in the host macrophage cells. Several organs can be affected by tuberculosis but most major illnesses are lung diseases. Immediately after infection, MTB will be phagocytosed by the alveolar macrophage cells and can survive in the phagosome. The macrophage plays a role in innate immunity towards an infection using autophagy by removing the microbe directly via phagocytosis. When bacteria phagocytosized, vacuole membrane formed double membranes called autophagosome, and followed by degradation by lysosome, which known as autolysosome. Induction of autophagy can be observed on the formation of microtubule-associated proteins 1B lightchain 3B (MAP1LC3B/LC3. MAP1LC3B is protein that have role at autophagic way for selection autophagy substrate and biogenesis. In this study we are used serum from patients TB with rifampicin resistant and rifampicin sensitive as control. Samples were divided using gene expert to differentiate between resistant and sensitive rifampicin.This research aims to compare MAP1LC3B levels in resistant and sensitive rifampicin to study macrophages respond in autophagic way in tuberculosis patients, and give information for define therapy plan to improve therapy for MDR-TB patients. Type of this research is a case control study design with cross sectional research with each groups sample is 19 from age 18-65 years old. Result, MAP1LC3B serum levels on the rifampicin resistant group are lower compared to rifampicin sensitive group. This occur because MTB is able to hide and evade innate immune defense mechanisms. MTB can maintain intracellular growth inside the phagosome by inhibiting phagolysosome formation in autophagy process especially inhibit MAP1LC3B formation by PDIM.

  4. Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

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    Periyasamy, Palsamy; Guo, Ming-Lei; Buch, Shilpa

    2016-01-01

    ABSTRACT Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation. PMID:27337297

  5. Enhanced Autophagy in Polycystic Kidneys of AQP11 Null Mice

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    Yasuko Tanaka

    2016-11-01

    Full Text Available Aquaporin-11 (AQP11 is an intracellular water channel expressed at the endoplasmic reticulum (ER of the proximal tubule. Its gene disruption in mice leads to intracellular vacuole formation at one week and the subsequent development of polycystic kidneys by three weeks. As the damaged proximal tubular cells with intracellular vacuoles form cysts later, we postulated that autophagy may play a role in the cyst formation and examined autophagy activity before and after cyst development in AQP11(−/− kidneys. PCR analysis showed the increased expression of the transcript encoding LC3 (Map1lc3b as well as other autophagy-related genes in AQP11(−/− mice. Using green fluorescent protein (GFP-LC3 transgenic mice and AQP11(−/− mice, we found that the number of GFP-LC3–positive puncta was increased in the proximal tubule of AQP11(−/− mice before the cyst formation. Interestingly, they were also observed in the cyst-lining epithelial cell. Further PCR analyses revealed the enhanced expression of apoptosis-related and ER stress–related caspase genes before and after the cyst formation, which may cause the enhanced autophagy. These results suggest the involvement of autophagy in the development and maintenance of kidney cysts in AQP11(−/− mice.

  6. Exploring autophagy with Gene Ontology

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    2018-01-01

    ABSTRACT Autophagy is a fundamental cellular process that is well conserved among eukaryotes. It is one of the strategies that cells use to catabolize substances in a controlled way. Autophagy is used for recycling cellular components, responding to cellular stresses and ridding cells of foreign material. Perturbations in autophagy have been implicated in a number of pathological conditions such as neurodegeneration, cardiac disease and cancer. The growing knowledge about autophagic mechanisms needs to be collected in a computable and shareable format to allow its use in data representation and interpretation. The Gene Ontology (GO) is a freely available resource that describes how and where gene products function in biological systems. It consists of 3 interrelated structured vocabularies that outline what gene products do at the biochemical level, where they act in a cell and the overall biological objectives to which their actions contribute. It also consists of ‘annotations’ that associate gene products with the terms. Here we describe how we represent autophagy in GO, how we create and define terms relevant to autophagy researchers and how we interrelate those terms to generate a coherent view of the process, therefore allowing an interoperable description of its biological aspects. We also describe how annotation of gene products with GO terms improves data analysis and interpretation, hence bringing a significant benefit to this field of study. PMID:29455577

  7. Autophagy-related genes in Helicobacter pylori infection.

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    Tanaka, Shingo; Nagashima, Hiroyuki; Uotani, Takahiro; Graham, David Y; Yamaoka, Yoshio

    2017-06-01

    In vitro studies have shown that Helicobacter pylori (H. pylori) infection induces autophagy in gastric epithelial cells. However, prolonged exposure to H. pylori reduces autophagy by preventing maturation of the autolysosome. The alterations of the autophagy-related genes in H. pylori infection are not yet fully understood. We analyzed autophagy-related gene expression in H. pylori-infected gastric mucosa compared with uninfected gastric mucosa obtained from 136 Bhutanese volunteers with mild dyspeptic symptoms. We also studied single nucleotide polymorphisms (SNPs) of autophagy-related gene in 283 Bhutanese participants to identify the influence on susceptibility to H. pylori infection. Microarray analysis of 226 autophagy-related genes showed that 16 genes were upregulated (7%) and nine were downregulated (4%). We used quantitative reverse transcriptase polymerase chain reaction to measure mRNA levels of the downregulated genes (ATG16L1, ATG5, ATG4D, and ATG9A) that were core molecules of autophagy. ATG16L1 and ATG5 mRNA levels in H. pylori-positive specimens (n=86) were significantly less than those in H. pylori-negative specimens (n=50). ATG16L1 mRNA levels were inversely related to H. pylori density. We also compared SNPs of ATG16L1 (rs2241880) among 206 H. pylori-positive and 77 H. pylori-negative subjects. The odds ratio for the presence of H. pylori in the GG genotype was 0.40 (95% CI: 0.18-0.91) relative to the AA/AG genotypes. Autophagy-related gene expression profiling using high-throughput microarray analysis indicated that downregulation of core autophagy machinery genes may depress autophagy functions and possibly provide a better intracellular habit for H. pylori in gastric epithelial cells. © 2017 John Wiley & Sons Ltd.

  8. Intact initiation of autophagy and mitochondrial fission by acute exercise in skeletal muscle of patientswith type 2 diabetes

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    Kruse Sørensen, Rikke; Pedersen, Andreas James Thestrup; Kristensen, Jonas Møller

    2017-01-01

    AIMS: Type 2 diabetes (T2D) is characterized by insulin resistance, mitochondrial dysregulation, and, in some studies, exercise resistance in skeletal muscle. Regulation of autophagy and mitochondrial dynamics during exercise and recovery is important for skeletal muscle homeostasis......, and these responses may be altered in T2D. MATERIALS AND METHODS: We examined the effect of acute exercise on markers of autophagy and mitochondrial fusion and fission in skeletal muscle biopsies from patients with T2D (n=13) and weight-matched controls (n=14) before, immediately after and 3h after an acute bout...... of exercise. RESULTS: While mRNA levels of most markers of autophagy ( PIK3C, MAP1LC3B, SQSTM1, BNIP3, BNIP3L ) and mitochondrial dynamics ( OPA1, FIS1 ) remained unchanged, some either increased during and after exercise (GABARAPL1 ), decreased in the recovery period ( BECN1, ATG7, DNM1L ), or both ( MFN2...

  9. A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.

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    Guo, Sujuan; Pridham, Kevin J; Virbasius, Ching-Man; He, Bin; Zhang, Liqing; Varmark, Hanne; Green, Michael R; Sheng, Zhi

    2018-02-12

    Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identifies identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.

  10. Characterization of a novel autophagy-specific gene, ATG29

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    Kawamata, Tomoko; Kamada, Yoshiaki; Suzuki, Kuninori; Kuboshima, Norihiro; Akimatsu, Hiroshi; Ota, Shinichi; Ohsumi, Mariko; Ohsumi, Yoshinori

    2005-01-01

    Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29Δ cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins

  11. A large-scale RNA interference screen identifies genes that regulate autophagy at different stages

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    Guo, Sujuan; Pridham, Kevin J; Virbasius, Ching-Man

    2018-01-01

    Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed...... with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes...... have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays...

  12. IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

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    Rovetta, Ana I; Peña, Delfina; Hernández Del Pino, Rodrigo E; Recalde, Gabriela M; Pellegrini, Joaquín; Bigi, Fabiana; Musella, Rosa M; Palmero, Domingo J; Gutierrez, Marisa; Colombo, María I; García, Verónica E

    2015-01-01

    Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen. PMID:25426782

  13. Stimulation of autophagy by the p53 target gene Sestrin2.

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    Maiuri, Maria Chiara; Malik, Shoaib Ahmad; Morselli, Eugenia; Kepp, Oliver; Criollo, Alfredo; Mouchel, Pierre-Luc; Carnuccio, Rosa; Kroemer, Guido

    2009-05-15

    The oncosuppressor protein p53 regulates autophagy in a dual fashion. The pool of cytoplasmic p53 protein represses autophagy in a transcription-independent fashion, while the pool of nuclear p53 stimulates autophagy through the transactivation of specific genes. Here we report the discovery that Sestrin2, a novel p53 target gene, is involved in the induction of autophagy. Depletion of Sestrin2 by RNA interference reduced the level of autophagy in a panel of p53-sufficient human cancer cell lines responding to distinct autophagy inducers. In quantitative terms, Sestrin2 depletion was as efficient in preventing autophagy induction as was the depletion of Dram, another p53 target gene. Knockout of either Sestrin2 or Dram reduced autophagy elicited by nutrient depletion, rapamycin, lithium or thapsigargin. Moreover, autophagy induction by nutrient depletion or pharmacological stimuli led to an increase in Sestrin2 expression levels in p53-proficient cells. In strict contrast, the depletion of Sestrin2 or Dram failed to affect autophagy in p53-deficient cells and did not modulate the inhibition of baseline autophagy by a cytoplasmic p53 mutant that was reintroduced into p53-deficient cells. We conclude that Sestrin2 acts as a positive regulator of autophagy in p53-proficient cells.

  14. SIRT5 regulation of ammonia-induced autophagy and mitophagy

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    Polletta, Lucia; Vernucci, Enza; Carnevale, Ilaria; Arcangeli, Tania; Rotili, Dante; Palmerio, Silvia; Steegborn, Clemens; Nowak, Theresa; Schutkowski, Mike; Pellegrini, Laura; Sansone, Luigi; Villanova, Lidia; Runci, Alessandra; Pucci, Bruna; Morgante, Emanuela; Fini, Massimo; Mai, Antonello; Russo, Matteo A; Tafani, Marco

    2015-01-01

    In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism. PMID:25700560

  15. 20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body

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    Tian, Ling; Ma, Li; Guo, Enen; Deng, Xiaojuan; Ma, Sanyuan; Xia, Qingyou; Cao, Yang; Li, Sheng

    2013-01-01

    Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcRDN) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body. PMID:23674061

  16. Genome-wide analysis of autophagy-related genes in banana highlights MaATG8s in cell death and autophagy in immune response to Fusarium wilt.

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    Wei, Yunxie; Liu, Wen; Hu, Wei; Liu, Guoyin; Wu, Chunjie; Liu, Wei; Zeng, Hongqiu; He, Chaozu; Shi, Haitao

    2017-08-01

    MaATG8s play important roles in hypersensitive-like cell death and immune response, and autophagy is essential for disease resistance against Foc in banana. Autophagy is responsible for the degradation of damaged cytoplasmic constituents in the lysosomes or vacuoles. Although the effects of autophagy have been extensively revealed in model plants, the possible roles of autophagy-related gene in banana remain unknown. In this study, 32 MaATGs were identified in the draft genome, and the profiles of several MaATGs in response to fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) were also revealled. We found that seven MaATG8s were commonly regulated by Foc. Through transient expression in Nicotiana benthamiana leaves, we highlight the novel roles of MaATG8s in conferring hypersensitive-like cell death, and MaATG8s-mediated hypersensitive response-like cell death is dependent on autophagy. Notablly, autophagy inhibitor 3-methyladenine (3-MA) treatment resulted in decreased disease resistance in response to Foc4, and the effect of 3-MA treatment could be rescued by exogenous salicylic acid, jasmonic acid and ethylene, indicating the involvement of autophagy-mediated plant hormones in banana resistance to Fusarium wilt. Taken together, this study may extend our understanding the putative role of MaATG8s in hypersensitive-like cell death and the essential role of autophagy in immune response against Foc in banana.

  17. Identification of autophagy genes participating in zinc-induced necrotic cell death in Saccharomyces cerevisiae.

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    Dziedzic, Slawomir A; Caplan, Allan B

    2011-05-01

    Eukaryotes use a common set of genes to perform two mechanistically similar autophagic processes. Bulk autophagy harvests proteins nonselectively and reuses their constitutents when nutrients are scarce. In contrast, different forms of selective autophagy target protein aggregates or damaged organelles that threaten to interfere with growth. Yeast uses one form of selective autophagy, called cytoplasm-to-vacuole targeting (Cvt), to engulf two vacuolar enzymes in Cvt vesicles ("CVT-somes") within which they are transported to vacuoles for maturation. While both are dispensable normally, bulk and selective autophagy help sustain life under stressful conditions. Consistent with this view, knocking out several genes participating in Cvt and specialized autophagic pathways heightened the sensitivity of Saccharomyces cerevisiae to inhibitory levels of Zn(2+). The loss of other autophagic genes, and genes responsible for apoptotic cell death, had no such effect. Unexpectedly, the loss of members of a third set of autophagy genes heightened cellular resistance to zinc as if they encoded proteins that actively contributed to zinc-induced cell death. Further studies showed that both sensitive and resistant strains accumulated similar amounts of H2O2 during zinc treatments, but that more sensitive strains showed signs of necrosis sooner. Although zinc lethality depended on autophagic proteins, studies with several reporter genes failed to reveal increased autophagic activity. In fact, microscopy analysis indicated that Zn(2+) partially inhibited fusion of Cvt vesicles with vacuoles. Further studies into how the loss of autophagic processes suppressed necrosis in yeast might reveal whether a similar process could occur in plants and animals.

  18. A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes

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    Ahnn Joohong

    2010-01-01

    Full Text Available Abstract Background Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines. Results We screened ~15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. Conclusions We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future

  19. Functional variant in the autophagy-related 5 gene promotor is associated with childhood asthma.

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    Lisa J Martin

    Full Text Available Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated.Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP.We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (p = 0.00085 and 0.0025, respectively. In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p<0.05. We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls.Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. These results provide novel evidence for a role for ATG5 in childhood asthma.

  20. Characterization of an Autophagy-related Gene MdATG8i from apple

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    Ping eWang

    2016-05-01

    Full Text Available Nutrient deficiencies restrict apple (Malus sp. tree growth and productivity in Northwest China. The process of autophagy, a conserved degradation pathway in eukaryotic cells, has important roles in nutrient-recycling and helps improve plant performance during periods of nutrient-starvation. Little is known about the functioning of autophagy-related genes (ATGs in apple. In this study, one of the ATG8 gene family members MdATG8i was isolated from M. domestica. MdATG8i has conserved putative tubulin binding sites and ATG7 interaction domains. A 1865-bp promoter region cloned from apple genome DNA was predicated to have cis-regulatory elements responsive to light, environmental stresses and hormones. MdATG8i transcriptions were induced in response to leaf senescence, nitrogen depletion, and oxidative stress. At cellular level, MdATG8i protein was expressed in the nucleus and cytoplasm of onion epidermal cells. Yeast two-hybrid tests showed that MdATG8i could interact with MdATG7a and MdATG7b. In Arabidopsis, its heterologous expression was associated with enhanced vegetative growth, leaf senescence, and tolerance to nitrogen- and carbon-starvation. MdATG8i-overexpressing ‘Orin’ apple callus lines also displayed improved tolerance to nutrient-limited conditions. Our results demonstrate that MdATG8i protein could function in autophagy in a conserved way, as a positive regulator in the response to nutrient-starvation.

  1. Altered Autophagy-Associated Genes Expression in T Cells of Oral Lichen Planus Correlated with Clinical Features

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    Ya-Qin Tan

    2016-01-01

    Full Text Available Oral lichen planus (OLP is a T cell-mediated inflammatory autoimmune disease. Autophagy has emerged as a fundamental trafficking event in mediating T cell response, which plays crucial roles in innate and adaptive immunity. The present study mainly investigated the mRNA expression of autophagy-associated genes in peripheral blood T cells of OLP patients and evaluated correlations between their expression and the clinical features of OLP. Five differentially expressed autophagy-associated genes were identified by autophagy array. Quantitative real-time RT-PCR results confirmed that IGF1 expression in the peripheral blood T cells of OLP patients was significantly higher than that in controls, especially in female and middle-aged (30–50 years old OLP patients. In addition, ATG9B mRNA levels were significantly lower in nonerosive OLP patients. However, no significant differences were found in the expression of HGS, ESR1, and SNCA between OLP patients and controls. Taken together, dysregulation of T cell autophagy may be involved in immune response of OLP and may be correlated with clinical patterns.

  2. The metastasis suppressor gene KISS-1 regulates osteosarcoma apoptosis and autophagy processes.

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    Yin, Yiran; Tang, Lian; Shi, Lei

    2017-03-01

    The expression of the metastasis suppressor gene KISS-1 in osteosarcoma cells during apoptosis and autophagy was evaluated. MG-63 osteosarcoma cells were transfected with either KISS-1 overexpression or KISS-1 knockdown expression vector in vitro, and compared with cell lines transfected with empty vector. After 12, 24, 48 and 72 h of cell culture, the cell proliferation was examined. The MTT method was used to detect apoptosis by flow cytometry, and the mRNA levels of apoptosis and autophagy markers caspase-3, Bcl-2, Bax, LC3 and Beclin1 were assessed by RT-PCR. Our results showed that cells in the control and low expression group kept proliferating during the cell culture period of 72 h, while the cells in the overexpression group progressively decreased in number. Also, the proliferation rate of the low expression group was significantly higher than that of the control group. The relative mRNA expression levels of caspase-3 and Bax mRNA in the control and low expression group showed no change (the expression was lowest in the low expression group). Moreover, the mRNA level of Bcl-2 increased in both cell groups. The mRNA expression levels of caspase-3 and Bax in the overexpression group were increased, and the level of Bcl-2 was reduced significantly. At the same time, the relative expression level of LC3 and Beclin1 mRNA in the control and low expression groups remained the same, and that of the overexpression group increased. The mRNA levels of LC3 and Beclin1 in the overexpression group were the highest, and that of the low expression group the lowest. The differences were statistically significant (Posteosarcoma in vitro, probably by accelerating the processes of apoptosis and autophagy in the cells.

  3. Autophagy in Inflammatory Diseases

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    Alexander J. S. Choi

    2011-01-01

    Full Text Available Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome-dependent degradation pathway. During starvation, autophagy exerts a homeostatic function that promotes cell survival by recycling metabolic precursors. Additionally, autophagy can interact with other vital processes such as programmed cell death, inflammation, and adaptive immune mechanisms, and thereby potentially influence disease pathogenesis. Macrophages deficient in autophagic proteins display enhanced caspase-1-dependent proinflammatory cytokine production and the activation of the inflammasome. Autophagy provides a functional role in infectious diseases and sepsis by promoting intracellular bacterial clearance. Mutations in autophagy-related genes, leading to loss of autophagic function, have been implicated in the pathogenesis of Crohn's disease. Furthermore, autophagy-dependent mechanisms have been proposed in the pathogenesis of several pulmonary diseases that involve inflammation, including cystic fibrosis and pulmonary hypertension. Strategies aimed at modulating autophagy may lead to therapeutic interventions for diseases associated with inflammation.

  4. A Variant of the Autophagy-Related 5 Gene Is Associated with Child Cerebral Palsy

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    Jianhua Xu

    2017-12-01

    Full Text Available Cerebral palsy (CP is a major cause of childhood disability in developed and developing countries, but the pathogenic mechanisms of CP development remain largely unknown. Autophagy is a highly conserved cellular self-digestion of damaged organelles and dysfunctional macromolecules. Growing evidence suggests that autophagy-related gene 5 (ATG5-dependent autophagy is involved in neural development, neuronal differentiation, and neurological degenerative diseases. The aim of this study was to analyze ATG5 protein expression and gene polymorphisms in Chinese patients with CP and to evaluate the importance of ATG5 in the development of CP. Five polymorphisms from different regions of the ATG5 gene (rs510432, rs3804338, rs573775, rs2299863, and rs6568431 were analyzed in 715 CP patients and 658 controls using MassARRAY. Of these, 58 patients and 56 controls were selected for measurement of plasma ATG5 level using ELISA. The relevance of disease-associated SNPs was evaluated using the SHEsis program. We identified a significant association between rs6568431 and CP (OR = 1.388, 95% CI = 1.173~1.643, Pallele = 0.0005, Pgenotype = 0.0015. Subgroup analysis showed a highly significant association of rs6568431 with spastic CP (n = 468, OR = 1.511, 95% CI = 1.251~1.824, Pallele = 8.50e−005, Pgenotype = 1.57e−004 and spastic quadriplegia (OR = 1.927, 95% CI = 1.533~2.421, Pallele = 7.35e−008, Pgenotype = 3.24e−009. Furthermore, mean plasma ATG5 levels were lower in CP patients than in controls, and individuals carrying the AA genotype of rs6568431 that was positively associated with CP had lower plasma ATG5 levels (P < 0.05. This study demonstrated an association of an ATG5 gene variant and low level of ATG5 protein with CP, and stronger associations with severe clinical manifestations were identified. Our results provide novel evidence for a role of ATG5 in CP and shed light on the molecular mechanisms underlying this neurodevelopmental disorder.

  5. Depletion of HPV16 early genes induces autophagy and senescence in a cervical carcinogenesis model, regardless of viral physical state.

    Science.gov (United States)

    Hanning, Jennifer E; Saini, Harpreet K; Murray, Matthew J; Caffarel, Maria M; van Dongen, Stijn; Ward, Dawn; Barker, Emily M; Scarpini, Cinzia G; Groves, Ian J; Stanley, Margaret A; Enright, Anton J; Pett, Mark R; Coleman, Nicholas

    2013-11-01

    In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral

  6. Lack of Connection Between Midgut Cell Autophagy Gene Expression and BmCPV Infection in the Midgut of Bombyx mori.

    Science.gov (United States)

    Yang, Xiaobing; Wu, Suli; Wu, Yongpeng; Liu, Yang; Qian, Yonghua; Jiao, Feng

    2015-01-01

    Autophagy is associated with multiple biological processes and has protective and defensive functions with respect to immunity, inflammation, and resistance to microbial infection. In this experiment, we wished to investigate whether autophagy is a factor in the midgut cell response of Bombyx mori to infection by the B. mori cytoplasmic polyhedrosis virus (BmCPV). Our results indicated that the expression of three autophagy-related genes (BmAtg8, BmAtg5, and BmAtg7) in the midgut did not change greatly after BmCPV infection in B. mori. Basal ATG8/ATG8PE protein expression was detected in different B. mori tissues by using western blot analysis. Immunohistochemistry showed that the ATG8/ATG8PE proteins were located mainly in the cytoplasm. ATG8/ATG8PE protein levels decreased at 12 and 16 h after BmCPV infection. Our results indicate that autophagy responded slightly to BmCPV infection, but could not prevent the invasion and replication of the virus. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

  7. GIMAP6 is required for T cell maintenance and efficient autophagy in mice.

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    Pascall, John C; Webb, Louise M C; Eskelinen, Eeva-Liisa; Innocentin, Silvia; Attaf-Bouabdallah, Noudjoud; Butcher, Geoffrey W

    2018-01-01

    The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family member GABARAPL2, and is recruited to autophagosomes upon starvation, suggesting a role for GIMAP6 in the autophagic process. To study this possibility and the function of GIMAP6 in the immune system, we have established a mouse line in which the Gimap6 gene can be inactivated by Cre-mediated recombination. In mice bred to carry the CD2Cre transgene such that the Gimap6 gene was deleted within the T and B cell lineages there was a 50-70% reduction in peripheral CD4+ and CD8+ T cells. Analysis of splenocyte-derived proteins from these mice indicated increased levels of MAP1LC3B, particularly the lipidated LC3-II form, and S405-phosphorylation of SQSTM1. Electron microscopic measurements of Gimap6-/- CD4+ T cells indicated an increased mitochondrial/cytoplasmic volume ratio and increased numbers of autophagosomes. These results are consistent with autophagic disruption in the cells. However, Gimap6-/- T cells were largely normal in character, could be effectively activated in vitro and supported T cell-dependent antibody production. Treatment in vitro of CD4+ splenocytes from GIMAP6fl/flERT2Cre mice with 4-hydroxytamoxifen resulted in the disappearance of GIMAP6 within five days. In parallel, increased phosphorylation of SQSTM1 and TBK1 was observed. These results indicate a requirement for GIMAP6 in the maintenance of a normal peripheral adaptive immune system and a significant role for the protein in normal autophagic processes. Moreover, as GIMAP6 is expressed in a cell-selective manner, this indicates the potential existence of a cell-restricted mode of autophagic regulation.

  8. Endosomal protein sorting and autophagy genes contribute to the regulation of yeast life span.

    Science.gov (United States)

    Longo, Valter D; Nislow, Corey; Fabrizio, Paola

    2010-11-01

    Accumulating evidence from various organisms points to a role for autophagy in the regulation of life span. By performing a genome-wide screen to identify novel life span determinants in Saccharomyces cerevisiae, we have obtained further insights into the autophagy-related and -unrelated degradation processes that may be important for preventing cellular senescence. The generation of multivesicular bodies and their fusion with the vacuole in the endosomal pathway emerged as novel cell functions involved in yeast chronological survival and longevity extension.

  9. In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver

    International Nuclear Information System (INIS)

    Donati, Alessio; Ventruti, Annamaria; Cavallini, Gabriella; Masini, Matilde; Vittorini, Simona; Chantret, Isabelle; Codogno, Patrice; Bergamini, Ettore

    2008-01-01

    Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5'-dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis

  10. Human vaginal epithelial cells augment autophagy marker genes in response to Candida albicans infection.

    Science.gov (United States)

    Shroff, Ankit; Sequeira, Roicy; Reddy, Kudumula Venkata Rami

    2017-04-01

    Autophagy plays an important role in clearance of intracellular pathogens. However, no information is available on its involvement in vaginal infections such as vulvo-vaginal candidiasis (VVC). VVC is intimately associated with the immune status of the human vaginal epithelial cells (VECs). The objective of our study is to decipher if autophagy process is involved during Candida albicans infection of VECs. In this study, C. albicans infection system was established using human VEC line (VK2/E6E7). Infection-induced change in the expression of autophagy markers like LC3 and LAMP-1 were analyzed by RT-PCR, q-PCR, Western blot, immunofluorescence and transmission electron microscopy (TEM) studies were carried out to ascertain the localization of autophagosomes. Multiplex ELISA was carried out to determine the cytokine profiles. Analysis of LC3 and LAMP-1 expression at mRNA and protein levels at different time points revealed up-regulation of these markers 6 hours post C. albicans infection. LC3 and LAMP-1 puncti were observed in infected VECs after 12 hours. TEM studies showed C. albicans entrapped in autophagosomes. Cytokines-TNF-α and IL-1β were up-regulated in culture supernatants of VECs at 12 hours post-infection. The results suggest that C. albicans invasion led to the activation of autophagy as a host defense mechanism of VECs. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Upregulation of autophagy-related gene-5 (ATG-5 is associated with chemoresistance in human gastric cancer.

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    Jie Ge

    Full Text Available Autophagy-related gene-5 (ATG-5 is one of the key regulators of autophagic cell death. It has been widely regarded as a protective molecular mechanism for tumor cells during the course of chemotherapy. In the present study, we investigated the expression pattern of ATG-5 and multidrug resistance-associated protein-1 (MRP-1 in 135 gastric cancers (GC patients who were treated with epirubicin, cisplatin and 5-FU adjuvant chemotherapy (ECF following surgical resection and explored their potential clinical significance. We found that both ATG-5 (77.78% and MRP-1 (79.26% were highly expressed in GC patients. ATG-5 expression was significantly associated with depth of wall invasion, TNM stages and distant metastasis of GC (P<0.05, whereas MRP-1 expression was significantly linked with tumor size, depth of wall invasion, lymph node metastasis, TNM stages and differentiation status (P<0.05. ATG-5 expression was positively correlated with MRP-1 (rp = 0.616, P<0.01. Increased expression of ATG-5 and MPR-1 was significantly correlated with poor overall survival (OS; P<0.01 and disease free survival (DFS; P<0.01 of our GC cohort. Furthermore, we demonstrated that ATG-5 was involved in drug resistant of GC cells, which was mainly through regulating autophagy. Our data suggest that upregulated expression of ATG-5, an important molecular feature of protective autophagy, is associated with chemoresistance in GC. Expression of ATG-5 and MRP-1 may be independent prognostic markers for GC treatment.

  12. Identification of Autophagy-Related Genes and Their Regulatory miRNAs Associated with Celiac Disease in Children

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    Sergio Comincini

    2017-02-01

    Full Text Available Celiac disease (CD is a severe genetic autoimmune disorder, affecting about one in 100 people, where the ingestion of gluten leads to damage in the small intestine. Diagnosing CD is quite complex and requires blood tests and intestinal biopsy examinations. Controversy exists regarding making the diagnosis without biopsy, due to the large spectrum of manifesting symptoms; furthermore, small-intestinal gastroscopy examinations have a relatively complex management in the pediatric population. To identify novel molecular markers useful to increase the sensitivity and specificity in the diagnosis of pediatric CD patients, the expression levels of two key autophagy executor genes (ATG7 and BECN1 and their regulatory validated miRNAs (miR-17 and miR-30a, respectively were analyzed by relative quantitative real-time-PCR on a cohort of confirmed CD patients compared to age-related controls. Among the investigated targets, the non-parametric Mann–Whitney U test and ROC analysis indicated the highest significant association of BECN1 with CD status in the blood, while in intestinal biopsies, all of the investigated sequences were positively associated with CD diagnosis. Nomogram-based analysis showed nearly opposite expression trends in blood compared to intestine tissue, while hierarchical clustering dendrograms enabled identifying CD and control subgroups based on specific genes and miRNA expression signatures. Next, using an established in vitro approach, through digested gliadin administration in Caco-2 cells, we also highlighted that the modulation of miR-17 endogenous levels using enriched exosomes increased the intracellular autophagosome content, thereby altering the autophagic status. Altogether, these results highlighted novel molecular markers that might be useful to increase the accuracy in CD diagnosis and in molecular-based stratification of the patients, further reinforcing the functional involvement of the regulation of the autophagy

  13. A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease.

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    Kaisa Kyöstilä

    2015-04-01

    Full Text Available Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136 in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

  14. Transcriptomic insights into the alternative splicing-mediated adaptation of the entomopathogenic fungus Beauveria bassiana to host niches: autophagy-related gene 8 as an example.

    Science.gov (United States)

    Dong, Wei-Xia; Ding, Jin-Li; Gao, Yang; Peng, Yue-Jin; Feng, Ming-Guang; Ying, Sheng-Hua

    2017-10-01

    Alternative splicing (AS) regulates various biological processes in fungi by extending the cellular proteome. However, comprehensive studies investigating AS in entomopathogenic fungi are lacking. Based on transcriptome data obtained via dual RNA-seq, the first overview of AS events was developed for Beauveria bassiana growing in an insect haemocoel. The AS was demonstrated for 556 of 8840 expressed genes, accounting for 5.4% of the total genes in B. bassiana. Intron retention was the most abundant type of AS, accounting for 87.1% of all splicing events and exon skipping events were rare, only accounting for 2.0% of all events. Functional distribution analysis indicated an association between alternatively spliced genes and several physiological processes. Notably, B. bassiana autophagy-related gene 8 (BbATG8), an indispensable gene for autophagy, was spliced at an alternative 5' splice site to generate two transcripts (BbATG8-α and BbATG8-β). The BbATG8-α transcript was necessary for fungal autophagy and oxidation tolerance, while the BbATG8-β transcript was not. These two transcripts differentially contributed to the formation of conidia or blastospores as well as fungal virulence. Thus, AS acts as a powerful post-transcriptional regulatory strategy in insect mycopathogens and significantly mediates fungal transcriptional adaption to host niches. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. Autophagy adaptor protein p62/SQSTM1 and autophagy-related gene Atg5 mediate autophagosome formation in response to Mycobacterium tuberculosis infection in dendritic cells.

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    Shintaro Seto

    Full Text Available Mycobacterium tuberculosis is an intracellular pathogen that can survive within phagocytic cells by inhibiting phagolysosome biogenesis. However, host cells can control the intracellular M. tuberculosis burden by the induction of autophagy. The mechanism of autophagosome formation to M. tuberculosis has been well studied in macrophages, but remains unclear in dendritic cells. We therefore characterized autophagosome formation in response to M. tuberculosis infection in dendritic cells. Autophagy marker protein LC3, autophagy adaptor protein p62/SQSTM1 (p62 and ubiquitin co-localized to M. tuberculosis in dendritic cells. Mycobacterial autophagosomes fused with lysosomes during infection, and major histcompatibility complex class II molecules (MHC II also localized to mycobacterial autophagosomes. The proteins p62 and Atg5 function in the initiation and progression of autophagosome formation to M. tuberculosis, respectively; p62 mediates ubiquitination of M. tuberculosis and Atg5 is involved in the trafficking of degradative vesicles and MHC II to mycobacterial autophagosomes. These results imply that the autophagosome formation to M. tuberculosis in dendritic cells promotes the antigen presentation of mycobacterial peptides to CD4(+ T lymphocytes via MHC II.

  16. Cloning, expression analysis, and RNA interference study of a HORMA domain containing autophagy-related gene 13 (ATG13) from the coleopteran beetle, Tenebrio molitor

    Science.gov (United States)

    Lee, Jung Hee; Jo, Yong Hun; Patnaik, Bharat Bhusan; Park, Ki Beom; Tindwa, Hamisi; Seo, Gi Won; Chandrasekar, Raman; Lee, Yong Seok; Han, Yeon Soo

    2015-01-01

    Autophagy is a process that is necessary during starvation, as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg) proteins that participate in the nucleation, elongation, and curving of the autophagosome membrane. In a pursuit to address the role of autophagy during development and immune resistance of the mealworm beetle, Tenebrio molitor, we screened ATG gene sequences from the whole-larva transcriptome database. We identified a homolog of ATG13 gene in T. molitor (designated as TmATG13) that comprises a cDNA of 1176 bp open reading frame (ORF) encoding a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region that was rich in regulatory phosphorylation sites. The N-terminal Atg13 domain had a HORMA (Hop1, Rev7, and Mad2) fold containing amino acid residues conserved across the Atg13 insect orthologs. A quantitative reverse-transcription-polymerase chain reaction analysis revealed that TmATG13 was expressed ubiquitously during all developmental stages of the insect. TmATG13 mRNA expression was high in the fat body and gut of the larval and adult stages of the insect. The TmATG13 transcripts were expressed at a high level until 6 days of ovarian development, followed by a significant decline. Silencing of ATG13 transcripts in T. molitor larvae showed a reduced survivability of 39 and 38% in response to Escherichia coli and Staphylococcus aureus infection. Furthermore, the role of TmAtg13 in initiating autophagy as a part of the host cell autophagic complex of the host cells against the intracellular pathogen Listeria monocytogenes is currently under study and will be critical to unfold the structure-function relationships. PMID:26136688

  17. Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

    Science.gov (United States)

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Lee, Yong Seok; Kang, Sang Sun; Han, Yeon Soo

    2015-07-01

    Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Identification, molecular cloning and expression analysis of a HORMA domain containing Autophagy-related gene 13 (ATG13 from the coleopteran beetle, Tenebrio molitor

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    Jung Hee eLee

    2015-06-01

    Full Text Available Autophagy is a process that is necessary during starvation as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg proteins that manifest in the nucleation, elongation, and curving of autophagosome membrane. We isolated a homolog of an ATG13 gene from the transcriptome database of the larva of the mealworm beetle, Tenebrio molitor (designated as TmATG13. The sequence analysis showed that TmATG13 cDNA comprises of 1,176 bp open reading frame that encodes a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region, rich in regulatory phosphorylation sites. The N-terminal Atg13 domain show a HORMA (Hop1, Rev7, and Mad2 fold containing conserved amino acid residues across the Atg13 orthologs in insects. qRT-PCR revealed that TmATG13 was expressed ubiquitously in all the developmental stages of insect. TmATG13 mRNA expression was high in fat body and gut of the larval and adult stages of the insect. During ovary development and maturation, the TmATG13 transcripts showed high expression until six days of development, followed by a significant decline. The prospective functions mediated by TmAtg13 during autophagy will be clarified by further studies in the near future.

  19. Inducing autophagy

    DEFF Research Database (Denmark)

    Harder, Lea M; Bunkenborg, Jakob; Andersen, Jens S.

    2014-01-01

    catabolism, which has recently been found to induce autophagy in an MTOR independent way and support cancer cell survival. In this study, quantitative phosphoproteomics was applied to investigate the initial signaling events linking ammonia to the induction of autophagy. The MTOR inhibitor rapamycin was used...... as a reference treatment to emphasize the differences between an MTOR-dependent and -independent autophagy-induction. By this means 5901 phosphosites were identified of which 626 were treatment-specific regulated and 175 were coregulated. Investigation of the ammonia-specific regulated sites supported that MTOR...

  20. Large-scale inference of gene function through phylogenetic annotation of Gene Ontology terms: case study of the apoptosis and autophagy cellular processes.

    Science.gov (United States)

    Feuermann, Marc; Gaudet, Pascale; Mi, Huaiyu; Lewis, Suzanna E; Thomas, Paul D

    2016-01-01

    We previously reported a paradigm for large-scale phylogenomic analysis of gene families that takes advantage of the large corpus of experimentally supported Gene Ontology (GO) annotations. This 'GO Phylogenetic Annotation' approach integrates GO annotations from evolutionarily related genes across ∼100 different organisms in the context of a gene family tree, in which curators build an explicit model of the evolution of gene functions. GO Phylogenetic Annotation models the gain and loss of functions in a gene family tree, which is used to infer the functions of uncharacterized (or incompletely characterized) gene products, even for human proteins that are relatively well studied. Here, we report our results from applying this paradigm to two well-characterized cellular processes, apoptosis and autophagy. This revealed several important observations with respect to GO annotations and how they can be used for function inference. Notably, we applied only a small fraction of the experimentally supported GO annotations to infer function in other family members. The majority of other annotations describe indirect effects, phenotypes or results from high throughput experiments. In addition, we show here how feedback from phylogenetic annotation leads to significant improvements in the PANTHER trees, the GO annotations and GO itself. Thus GO phylogenetic annotation both increases the quantity and improves the accuracy of the GO annotations provided to the research community. We expect these phylogenetically based annotations to be of broad use in gene enrichment analysis as well as other applications of GO annotations.Database URL: http://amigo.geneontology.org/amigo. © The Author(s) 2016. Published by Oxford University Press.

  1. The V471A polymorphism in autophagy-related gene ATG7 modifies age at onset specifically in Italian Huntington disease patients

    DEFF Research Database (Denmark)

    Metzger, Silke; Walter, Carolin; Riess, Olaf

    2013-01-01

    The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently......, we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second...... independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the "REGISTRY" cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing...

  2. Interleukin-1β-induced autophagy-related gene 5 regulates proliferation of embryonic stem cell-derived odontoblastic cells.

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    Nobuaki Ozeki

    Full Text Available We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL-1β induces matrix metalloproteinase (MMP-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8 and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells.

  3. Feedback regulation between autophagy and PKA.

    Science.gov (United States)

    Torres-Quiroz, Francisco; Filteau, Marie; Landry, Christian R

    2015-01-01

    Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.

  4. Canine hereditary ataxia in old english sheepdogs and gordon setters is associated with a defect in the autophagy gene encoding RAB24.

    Directory of Open Access Journals (Sweden)

    Caryline Agler

    2014-02-01

    Full Text Available Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.

  5. Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS

    Directory of Open Access Journals (Sweden)

    Yongjian Wu

    2018-02-01

    Full Text Available Beta-defensins 2 and 3 (BD2 and BD3 are inducible peptides present at the sites of infection, and they are well characterized for their antimicrobial activities and immune-regulatory functions. However, no study has thoroughly investigated their immunomodulatory effects on macrophage-mediated immune responses against Pseudomonas aeruginosa (PA. Here, we use THP-1 and RAW264.7 cell lines and demonstrate that BD2 and BD3 suppressed macrophage autophagy but enhanced the engulfment of PA and Zymosan bioparticles as well as the formation of phagolysosomes, using immunofluorescence staining and confocal microscopy. Plate count assay showed that macrophage-mediated phagocytosis and intracellular killing of PA were promoted by BD2 and BD3. Furthermore, microarray and real-time PCR showed that the expression of two genes, early growth response gene-1 (EGR1 and c-FOS, was attenuated by BD2 and BD3. Western blot revealed that BD2 and BD3 inhibited the expression and nuclear translocation of EGR1 and c-FOS. Knockdown of EGR1 and c-FOS by siRNA transfection suppressed macrophage autophagy before and after PA infection; while overexpression of these two transcription factors enhanced autophagy but reversed the role of BD2 and BD3 on macrophage-mediated PA eradication. Together, these results demonstrate a novel immune defense activity of BD2 and BD3, which promotes clearance of PA by inhibiting macrophage autophagy through downregulation of EGR1 and c-FOS.

  6. Autophagy genes Smatg8 and Smatg4 are required for fruiting-body development, vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora.

    Science.gov (United States)

    Voigt, Oliver; Pöggeler, Stefanie

    2013-01-01

    Autophagy is a tightly controlled degradation process involved in various developmental aspects of eukaryotes. However, its involvement in developmental processes of multicellular filamentous ascomycetes is largely unknown. Here, we analyzed the impact of the autophagic proteins SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. A Saccharomyces cerevisiae complementation assay demonstrated that the S. macrospora Smatg8 and Smatg4 genes can functionally replace the yeast homologs. By generating homokaryotic deletion mutants, we showed that the S. macrospora SmATG8 and SmATG4 orthologs were associated with autophagy-dependent processes. Smatg8 and Smatg4 deletions abolished fruiting-body formation and impaired vegetative growth and ascospore germination, but not hyphal fusion. We demonstrated that SmATG4 was capable of processing the SmATG8 precursor. SmATG8 was localized to autophagosomes, whereas SmATG4 was distributed throughout the cytoplasm of S. macrospora. Furthermore, we could show that Smatg8 and Smatg4 are not only required for nonselective macroautophagy, but for selective macropexophagy as well. Taken together, our results suggest that in S. macrospora, autophagy seems to be an essential and constitutively active process to sustain high energy levels for filamentous growth and multicellular development even under nonstarvation conditions.

  7. Autophagy is essential for hearing in mice.

    Science.gov (United States)

    Fujimoto, Chisato; Iwasaki, Shinichi; Urata, Shinji; Morishita, Hideaki; Sakamaki, Yuriko; Fujioka, Masato; Kondo, Kenji; Mizushima, Noboru; Yamasoba, Tatsuya

    2017-05-11

    Hearing loss is the most frequent sensory disorder in humans. Auditory hair cells (HCs) are postmitotic at late-embryonic differentiation and postnatal stages, and their damage is the major cause of hearing loss. There is no measurable HC regeneration in the mammalian cochlea, and the maintenance of cell function is crucial for preservation of hearing. Here we generated mice deficient in autophagy-related 5 (Atg5), a gene essential for autophagy, in the HCs to investigate the effect of basal autophagy on hearing acuity. Deletion of Atg5 resulted in HC degeneration and profound congenital hearing loss. In autophagy-deficient HCs, polyubiquitinated proteins and p62/SQSTM1, an autophagy substrate, accumulated as inclusion bodies during the first postnatal week, and these aggregates increased in number. These findings revealed that basal autophagy has an important role in maintenance of HC morphology and hearing acuity.

  8. Depletion of autophagy-related genes ATG3 and ATG5 in Tenebrio molitor leads to decreased survivability against an intracellular pathogen, Listeria monocytogenes.

    Science.gov (United States)

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Noh, Mi Young; Kim, Dong Hyun; Kim, Iksoo; Han, Yeon Soo; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung

    2015-01-01

    Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model. © 2014 Wiley Periodicals, Inc.

  9. Autophagy and the nutritional signaling pathway

    Directory of Open Access Journals (Sweden)

    Long HE,Shabnam ESLAMFAM,Xi MA,Defa LI

    2016-09-01

    Full Text Available During their growth and development, animals adapt to tremendous changes in order to survive. These include responses to both environmental and physiological changes and autophagy is one of most important adaptive and regulatory mechanisms. Autophagy is defined as an autolytic process to clear damaged cellular organelles and recycle the nutrients via lysosomic degradation. The process of autophagy responds to special conditions such as nutrient withdrawal. Once autophagy is induced, phagophores form and then elongate and curve to form autophagosomes. Autophagosomes then engulf cargo, fuse with endosomes, and finally fuse with lysosomes for maturation. During the initiation process, the ATG1/ULK1 (unc-51-like kinase 1 and VPS34 (which encodes a class III phosphatidylinositol (PtdIns 3-kinase complexes are critical in recruitment and assembly of other complexes required for autophagy. The process of autophagy is regulated by autophagy related genes (ATGs. Amino acid and energy starvation mediate autophagy by activating mTORC1 (mammalian target of rapamycin and AMP-activated protein kinase (AMPK. AMPK is the energy status sensor, the core nutrient signaling component and the metabolic kinase of cells. This review mainly focuses on the mechanism of autophagy regulated by nutrient signaling especially for the two important complexes, ULK1 and VPS34.

  10. bZIP transcription factor SmJLB1 regulates autophagy-related genes Smatg8 and Smatg4 and is required for fruiting-body development and vegetative growth in Sordaria macrospora.

    Science.gov (United States)

    Voigt, Oliver; Herzog, Britta; Jakobshagen, Antonia; Pöggeler, Stefanie

    2013-12-01

    Autophagy is a precisely controlled degradation process in eukaryotic cells, during which the bulk of the cytoplasm is engulfed by a double membrane vesicle, the autophagosome. Fusion of the autophagosome with the vacuole leads to breakdown of its contents, such as proteins and organelles, and the recycling of nutrients. Earlier studies of autophagic genes of the core autophagic machinery in the filamentous ascomycete Sordaria macrospora elucidated the impact of autophagy on fungal viability, vegetative growth and fruiting-body development. To gain further knowledge about the regulation of autophagy in S. macrospora, we analyzed the function of the bZIP transcription factor SmJLB1, a homolog of the Podospora anserina basic zipper-type transcription factor induced during incompatibility 4 (IDI-4) and the Aspergillus nidulans transcription factor jun-like bZIP A (JlbA). Generation of the homokaryotic deletion mutant demonstrated S. macrospora Smjlb1 is associated with autophagy-dependent processes. Deletion of Smjlb1 abolished fruiting-body formation and impaired vegetative growth. SmJLB1 is localized to the cytoplasm and to nuclei. Quantitative real-time PCR experiments revealed an upregulated expression of autophagy-related genes Smatg8 and Smatg4 in the Smjlb1 deletion mutant, suggesting a transcriptional repression function of SmJLB1. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Ketogenic diet change cPLA2/clusterin and autophagy related gene expression and correlate with cognitive deficits and hippocampal MFs sprouting following neonatal seizures.

    Science.gov (United States)

    Ni, Hong; Zhao, Dong-Jing; Tian, Tian

    2016-02-01

    Because the ketogenic diet (KD) was affecting expression of energy metabolism- related genes in hippocampus and because lipid membrane peroxidation and its associated autophagy stress were also found to be involved in energy depletion, we hypothesized that KD might exert its neuroprotective action via lipid membrane peroxidation and autophagic signaling. Here, we tested this hypothesis by examining the long-term expression of lipid membrane peroxidation-related cPLA2 and clusterin, its downstream autophagy marker Beclin-1, LC3 and p62, as well as its execution molecule Cathepsin-E following neonatal seizures and chronic KD treatment. On postnatal day 9 (P9), 48 Sprague-Dawley rats were randomly assigned to two groups: flurothyl-induced recurrent seizures group and control group. On P28, they were further randomly divided into the seizure group without ketogenic diet (RS+ND), seizure plus ketogenic diet (RS+KD), the control group without ketogenic diet (NS+ND), and the control plus ketogenic diet (NS+KD). Morris water maze test was performed during P37-P43. Then mossy fiber sprouting and the protein levels were detected by Timm staining and Western blot analysis, respectively. Flurothyl-induced RS+ND rats show a long-term lower amount of cPLA2 and LC3II/I, and higher amount of clusterin, Beclin-1, p62 and Cathepsin-E which are in parallel with hippocampal mossy fiber sprouting and cognitive deficits. Furthermore, chronic KD treatment (RS+KD) is effective in restoring these molecular, neuropathological and cognitive changes. The results imply that a lipid membrane peroxidation and autophagy-associated pathway is involved in the aberrant hippocampal mossy fiber sprouting and cognitive deficits following neonatal seizures, which might be a potential target of KD for the treatment of neonatal seizure-induced brain damage. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy.

    Science.gov (United States)

    Lee, Albert; Rayner, Stephanie L; Gwee, Serene S L; De Luca, Alana; Shahheydari, Hamideh; Sundaramoorthy, Vinod; Ragagnin, Audrey; Morsch, Marco; Radford, Rowan; Galper, Jasmin; Freckleton, Sarah; Shi, Bingyang; Walker, Adam K; Don, Emily K; Cole, Nicholas J; Yang, Shu; Williams, Kelly L; Yerbury, Justin J; Blair, Ian P; Atkin, Julie D; Molloy, Mark P; Chung, Roger S

    2018-01-01

    Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCF cyclin F ) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin F S621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin F WT . Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin F S621G -expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin F S621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin F S621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is

  13. Tumor Suppression and Promotion by Autophagy

    Directory of Open Access Journals (Sweden)

    Yenniffer Ávalos

    2014-01-01

    Full Text Available Autophagy is a highly regulated catabolic process that involves lysosomal degradation of proteins and organelles, mostly mitochondria, for the maintenance of cellular homeostasis and reduction of metabolic stress. Problems in the execution of this process are linked to different pathological conditions, such as neurodegeneration, aging, and cancer. Many of the proteins that regulate autophagy are either oncogenes or tumor suppressor proteins. Specifically, tumor suppressor genes that negatively regulate mTOR, such as PTEN, AMPK, LKB1, and TSC1/2 stimulate autophagy while, conversely, oncogenes that activate mTOR, such as class I PI3K, Ras, Rheb, and AKT, inhibit autophagy, suggesting that autophagy is a tumor suppressor mechanism. Consistent with this hypothesis, the inhibition of autophagy promotes oxidative stress, genomic instability, and tumorigenesis. Nevertheless, autophagy also functions as a cytoprotective mechanism under stress conditions, including hypoxia and nutrient starvation, that promotes tumor growth and resistance to chemotherapy in established tumors. Here, in this brief review, we will focus the discussion on this ambiguous role of autophagy in the development and progression of cancer.

  14. Tumor suppression and promotion by autophagy.

    Science.gov (United States)

    Ávalos, Yenniffer; Canales, Jimena; Bravo-Sagua, Roberto; Criollo, Alfredo; Lavandero, Sergio; Quest, Andrew F G

    2014-01-01

    Autophagy is a highly regulated catabolic process that involves lysosomal degradation of proteins and organelles, mostly mitochondria, for the maintenance of cellular homeostasis and reduction of metabolic stress. Problems in the execution of this process are linked to different pathological conditions, such as neurodegeneration, aging, and cancer. Many of the proteins that regulate autophagy are either oncogenes or tumor suppressor proteins. Specifically, tumor suppressor genes that negatively regulate mTOR, such as PTEN, AMPK, LKB1, and TSC1/2 stimulate autophagy while, conversely, oncogenes that activate mTOR, such as class I PI3K, Ras, Rheb, and AKT, inhibit autophagy, suggesting that autophagy is a tumor suppressor mechanism. Consistent with this hypothesis, the inhibition of autophagy promotes oxidative stress, genomic instability, and tumorigenesis. Nevertheless, autophagy also functions as a cytoprotective mechanism under stress conditions, including hypoxia and nutrient starvation, that promotes tumor growth and resistance to chemotherapy in established tumors. Here, in this brief review, we will focus the discussion on this ambiguous role of autophagy in the development and progression of cancer.

  15. Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

    Directory of Open Access Journals (Sweden)

    Paola Fabrizio

    2010-07-01

    Full Text Available The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

  16. Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

    Science.gov (United States)

    Fabrizio, Paola; Hoon, Shawn; Shamalnasab, Mehrnaz; Galbani, Abdulaye; Wei, Min; Giaever, Guri; Nislow, Corey; Longo, Valter D

    2010-07-15

    The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

  17. WNK1 is an unexpected autophagy inhibitor

    Science.gov (United States)

    Gallolu Kankanamalage, Sachith; Lee, A-Young; Wichaidit, Chonlarat; Lorente-Rodriguez, Andres; Shah, Akansha M.; Stippec, Steve; Whitehurst, Angelique W.; Cobb, Melanie H.

    2017-01-01

    ABSTRACT Autophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UVRAG (UV radiation resistance associated gene), which is a component of the PtdIns3K, binds to the N-terminal region of WNK1. Moreover, WNK1 partially colocalizes with UVRAG and this colocalization decreases when autophagy is stimulated in cells. The loss of WNK1 also alters the cellular distribution of UVRAG. The depletion of the downstream target of WNK1, OXSR1/OSR1 (oxidative-stress responsive 1) has no effect on autophagy, whereas the depletion of its relative STK39/SPAK (serine/threonine kinase 39) induces autophagy under nutrient-rich and starved conditions. PMID:28282258

  18. Autophagy in breast cancer and its implications for therapy

    Science.gov (United States)

    Jain, Kirti; Paranandi, Krishna S; Sridharan, Savitha; Basu, Alakananda

    2013-01-01

    Autophagy is an evolutionarily conserved process of cellular self-digestion that serves as a mechanism to clear damaged organelles and recycle nutrients. Since autophagy can promote cell survival as well as cell death, it has been linked to different human pathologies, including cancer. Although mono-allelic deletion of autophagy-related gene BECN1 in breast tumors originally indicated a tumor suppressive role for autophagy in breast cancer, the intense research during the last decade suggests a role for autophagy in tumor progression. It is now recognized that tumor cells often utilize autophagy to survive various stresses, such as oncogene-induced transformation, hypoxia, endoplasmic reticulum (ER) stress and extracellular matrix detachment. Induction of autophagy by tumor cells may also contribute to tumor dormancy and resistance to anticancer therapies, thus making autophagy inhibitors promising drug candidates for breast cancer treatment. The scientific endeavors continue to define a precise role for autophagy in breast cancer. In this article, we review the current literature on the role of autophagy during the development and progression of breast cancer, and discuss the potential of autophagy modulators for breast cancer treatment. PMID:23841025

  19. Overexpression of the autophagy-related gene SiATG8a from foxtail millet (Setaria italica L.) confers tolerance to both nitrogen starvation and drought stress in Arabidopsis.

    Science.gov (United States)

    Li, Wei-wei; Chen, Ming; Zhong, Li; Liu, Jia-ming; Xu, Zhao-shi; Li, Lian-cheng; Zhou, Yong-Bin; Guo, Chang-Hong; Ma, You-Zhi

    2015-12-25

    Autophagy is an evolutionarily conserved biological process in all eukaryotes for the degradation of intracellular components for nutrient recycling. Autophagy is known to be involved in responses to low nitrogen stress in Arabidopsis. Foxtail millet has strong abiotic stress resistance to both low nutrient and drought stress. However, to date, there have only been a few genes reported to be related with abiotic stress resistance in foxtail millet. In this study, we identified an autophagy-related gene, SiATG8a, from foxtail millet. SiATG8a is mainly expressed in stems and its expression was dramatically induced by drought stress and nitrogen starvation treatments. SiATG8a was localized in the membrane and cytoplasm of foxtail millet. Overexpression of SiATG8a in Arabidopsis conferred tolerance to both nitrogen starvation and to drought stress. Under nitrogen starvation conditions, the SiATG8a transgenic plants had larger root and leaf areas and accumulated more total nitrogen than wild-type plants. The transgenic plants had lower total protein concentrations than did the WT plants. Under drought stress, the SiATG8a transgenic plants had higher survival rates, chlorophyll content, and proline content, but had lower MDA content than wild type plants. Taken together, our results represent the first identified case where overexpression of autophagy related gene can simultaneously improve plant resistance to low nitrogen and drought stresses. These findings implicate plant autophagy in plant stress responses to low nitrogen and drought and should be helpful in efforts to improve stresses resistance to nitrogen starvation and drought of crops by genetic transformation. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Autophagy in photodynamic therapy

    African Journals Online (AJOL)

    Autophagy is a conserved intracellular degradation process in which cellular organelles, proteins and invading microbes are degraded by lysosomes. There are three types of autophagy: macroautophagy, mitoautophagy and chaperone- mediated autophagy. This review is focused on macroautophagy which is referred to ...

  1. Autophagy: More Than a Nonselective Pathway

    Directory of Open Access Journals (Sweden)

    Fulvio Reggiori

    2012-01-01

    Full Text Available Autophagy is a catabolic pathway conserved among eukaryotes that allows cells to rapidly eliminate large unwanted structures such as aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens. The hallmark of this transport pathway is the sequestration of the cargoes that have to be degraded in the lysosomes by double-membrane vesicles called autophagosomes. The key actors mediating the biogenesis of these carriers are the autophagy-related genes (ATGs. For a long time, it was assumed that autophagy is a bulk process. Recent studies, however, have highlighted the capacity of this pathway to exclusively eliminate specific structures and thus better fulfil the catabolic necessities of the cell. We are just starting to unveil the regulation and mechanism of these selective types of autophagy, but what it is already clearly emerging is that structures targeted to destruction are accurately enwrapped by autophagosomes through the action of specific receptors and adaptors. In this paper, we will briefly discuss the impact that the selective types of autophagy have had on our understanding of autophagy.

  2. Genome-wide analysis of autophagy-associated genes in foxtail millet (Setaria italica L.) and characterization of the function of SiATG8a in conferring tolerance to nitrogen starvation in rice.

    Science.gov (United States)

    Li, Weiwei; Chen, Ming; Wang, Erhui; Hu, Liqin; Hawkesford, Malcolm J; Zhong, Li; Chen, Zhu; Xu, Zhaoshi; Li, Liancheng; Zhou, Yongbin; Guo, Changhong; Ma, Youzhi

    2016-10-12

    Autophagy is a cellular degradation process that is highly evolutionarily-conserved in yeast, plants, and animals. In plants, autophagy plays important roles in regulating intracellular degradation and recycling of amino acids in response to nutrient starvation, senescence, and other environmental stresses. Foxtail millet (Setaria italica) has strong resistance to stresses and has been proposed as an ideal material for use in the study of the physiological mechanisms of abiotic stress tolerance in plants. Although the genome sequence of foxtail millet (Setaria italica) is available, the characteristics and functions of abiotic stress-related genes remain largely unknown for this species. A total of 37 putative ATG (autophagy-associated genes) genes in the foxtail millet genome were identified. Gene duplication analysis revealed that both segmental and tandem duplication events have played significant roles in the expansion of the ATG gene family in foxtail millet. Comparative synteny mapping between the genomes of foxtail millet and rice suggested that the ATG genes in both species have common ancestors, as their ATG genes were primarily located in similar syntenic regions. Gene expression analysis revealed the induced expression of 31 SiATG genes by one or more phytohormone treatments, 26 SiATG genes by drought, salt and cold, 24 SiATG genes by darkness and 25 SiATG genes by nitrogen starvation. Results of qRT-PCR showing that among 37 SiATG genes, the expression level of SiATG8a was the highest after nitrogen starvation treatment 24 h, suggesting its potential role in tolerance to nutrient starvation. Moreover, the heterologous expression of SiATG8a in rice improved nitrogen starvation tolerance. Compared to wild type rice, the transgenic rice performed better and had higher aboveground total nitrogen content when the plants were grown under nitrogen starvation conditions. Our results deepen understanding about the characteristics and functions of ATG genes in

  3. The interplay between autophagy and ROS in tumorigenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kongara, Sameera [Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ (United States); The Cancer Institute of New Jersey, New Brunswick, NJ (United States); Karantza, Vassiliki, E-mail: karantva@umdnj.edu [Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ (United States); The Cancer Institute of New Jersey, New Brunswick, NJ (United States); Division of Medical Oncology, Department of Internal Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ (United States)

    2012-11-21

    Reactive oxygen species (ROS) at physiological levels are important cell signaling molecules. However, aberrantly high ROS are intimately associated with disease and commonly observed in cancer. Mitochondria are primary sources of intracellular ROS, and their maintenance is essential to cellular health. Autophagy, an evolutionarily conserved process whereby cytoplasmic components are delivered to lysosomes for degradation, is responsible for mitochondrial turnover and removal of damaged mitochondria. Impaired autophagy is implicated in many pathological conditions, including neurological disorders, inflammatory bowel disease, diabetes, aging, and cancer. The first reports connecting autophagy to cancer showed that allelic loss of the essential autophagy gene BECLIN1 (BECN1) is prevalent in human breast, ovarian, and prostate cancers and that Becn1{sup +/-} mice develop mammary gland hyperplasias, lymphomas, lung and liver tumors. Subsequent studies demonstrated that Atg5{sup -/-} and Atg7{sup -/-} livers give rise to adenomas, Atg4C{sup -/-} mice are susceptible to chemical carcinogenesis, and Bif1{sup -/-} mice are prone to spontaneous tumors, indicating that autophagy defects promote tumorigenesis. Due to defective mitophagy, autophagy-deficient cells accumulate damaged mitochondria and deregulated ROS levels, which likely contribute to their tumor-initiating capacity. However, the role of autophagy in tumorigenesis is complex, as more recent work also revealed tumor dependence on autophagy: autophagy-competent mutant-Ras-expressing cells form tumors more efficiently than their autophagy-deficient counterparts; similarly, FIP200 deficiency suppresses PyMT-driven mammary tumorigenesis. These latter findings are attributed to the fact that tumors driven by powerful oncogenes have high metabolic demands catered to by autophagy. In this review, we discuss the relationship between ROS and autophagy and summarize our current knowledge on their functional interactions

  4. The interplay between autophagy and ROS in tumorigenesis

    International Nuclear Information System (INIS)

    Kongara, Sameera; Karantza, Vassiliki

    2012-01-01

    Reactive oxygen species (ROS) at physiological levels are important cell signaling molecules. However, aberrantly high ROS are intimately associated with disease and commonly observed in cancer. Mitochondria are primary sources of intracellular ROS, and their maintenance is essential to cellular health. Autophagy, an evolutionarily conserved process whereby cytoplasmic components are delivered to lysosomes for degradation, is responsible for mitochondrial turnover and removal of damaged mitochondria. Impaired autophagy is implicated in many pathological conditions, including neurological disorders, inflammatory bowel disease, diabetes, aging, and cancer. The first reports connecting autophagy to cancer showed that allelic loss of the essential autophagy gene BECLIN1 (BECN1) is prevalent in human breast, ovarian, and prostate cancers and that Becn1 +/- mice develop mammary gland hyperplasias, lymphomas, lung and liver tumors. Subsequent studies demonstrated that Atg5 -/- and Atg7 -/- livers give rise to adenomas, Atg4C -/- mice are susceptible to chemical carcinogenesis, and Bif1 -/- mice are prone to spontaneous tumors, indicating that autophagy defects promote tumorigenesis. Due to defective mitophagy, autophagy-deficient cells accumulate damaged mitochondria and deregulated ROS levels, which likely contribute to their tumor-initiating capacity. However, the role of autophagy in tumorigenesis is complex, as more recent work also revealed tumor dependence on autophagy: autophagy-competent mutant-Ras-expressing cells form tumors more efficiently than their autophagy-deficient counterparts; similarly, FIP200 deficiency suppresses PyMT-driven mammary tumorigenesis. These latter findings are attributed to the fact that tumors driven by powerful oncogenes have high metabolic demands catered to by autophagy. In this review, we discuss the relationship between ROS and autophagy and summarize our current knowledge on their functional interactions in tumorigenesis.

  5. Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury

    Science.gov (United States)

    Xie, Cuicui; Ginet, Vanessa; Sun, Yanyan; Koike, Masato; Zhou, Kai; Li, Tao; Li, Hongfu; Li, Qian; Wang, Xiaoyang; Uchiyama, Yasuo; Truttmann, Anita C.; Kroemer, Guido; Puyal, Julien; Blomgren, Klas; Zhu, Changlian

    2016-01-01

    ABSTRACT Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy. PMID:26727396

  6. Autophagy as an Emerging Common Pathomechanism in Inherited Peripheral Neuropathies

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    Mansour Haidar

    2017-05-01

    Full Text Available The inherited peripheral neuropathies (IPNs comprise a growing list of genetically heterogeneous diseases. With mutations in more than 80 genes being reported to cause IPNs, a wide spectrum of functional consequences is expected to follow this genotypic diversity. Hence, the search for a common pathomechanism among the different phenotypes has become the holy grail of functional research into IPNs. During the last decade, studies on several affected genes have shown a direct and/or indirect correlation with autophagy. Autophagy, a cellular homeostatic process, is required for the removal of cell aggregates, long-lived proteins and dead organelles from the cell in double-membraned vesicles destined for the lysosomes. As an evolutionarily highly conserved process, autophagy is essential for the survival and proper functioning of the cell. Recently, neuronal cells have been shown to be particularly vulnerable to disruption of the autophagic pathway. Furthermore, autophagy has been shown to be affected in various common neurodegenerative diseases of both the central and the peripheral nervous system including Alzheimer’s, Parkinson’s, and Huntington’s diseases. In this review we provide an overview of the genes involved in hereditary neuropathies which are linked to autophagy and we propose the disruption of the autophagic flux as an emerging common pathomechanism. We also shed light on the different steps of the autophagy pathway linked to these genes. Finally, we review the concept of autophagy being a therapeutic target in IPNs, and the possibilities and challenges of this pathway-specific targeting.

  7. Immunologic manifestations of autophagy

    DEFF Research Database (Denmark)

    Deretic, Vojo; Kimura, Tomonori; Timmins, Graham

    2015-01-01

    The broad immunologic roles of autophagy span innate and adaptive immunity and are often manifested in inflammatory diseases. The immune effects of autophagy partially overlap with its roles in metabolism and cytoplasmic quality control but typically expand further afield to encompass unique...... immunologic adaptations. One of the best-appreciated manifestations of autophagy is protection against microbial invasion, but this is by no means limited to direct elimination of intracellular pathogens and includes a stratified array of nearly all principal immunologic processes. This Review summarizes...... the broad immunologic roles of autophagy. Furthermore, it uses the autophagic control of Mycobacterium tuberculosis as a paradigm to illustrate the breadth and complexity of the immune effects of autophagy....

  8. Autophagy in Drosophila: From Historical Studies to Current Knowledge

    Science.gov (United States)

    Mulakkal, Nitha C.; Nagy, Peter; Takats, Szabolcs; Tusco, Radu; Juhász, Gábor; Nezis, Ioannis P.

    2014-01-01

    The discovery of evolutionarily conserved Atg genes required for autophagy in yeast truly revolutionized this research field and made it possible to carry out functional studies on model organisms. Insects including Drosophila are classical and still popular models to study autophagy, starting from the 1960s. This review aims to summarize past achievements and our current knowledge about the role and regulation of autophagy in Drosophila, with an outlook to yeast and mammals. The basic mechanisms of autophagy in fruit fly cells appear to be quite similar to other eukaryotes, and the role that this lysosomal self-degradation process plays in Drosophila models of various diseases already made it possible to recognize certain aspects of human pathologies. Future studies in this complete animal hold great promise for the better understanding of such processes and may also help finding new research avenues for the treatment of disorders with misregulated autophagy. PMID:24949430

  9. Autophagy in Drosophila: From Historical Studies to Current Knowledge

    Directory of Open Access Journals (Sweden)

    Nitha C. Mulakkal

    2014-01-01

    Full Text Available The discovery of evolutionarily conserved Atg genes required for autophagy in yeast truly revolutionized this research field and made it possible to carry out functional studies on model organisms. Insects including Drosophila are classical and still popular models to study autophagy, starting from the 1960s. This review aims to summarize past achievements and our current knowledge about the role and regulation of autophagy in Drosophila, with an outlook to yeast and mammals. The basic mechanisms of autophagy in fruit fly cells appear to be quite similar to other eukaryotes, and the role that this lysosomal self-degradation process plays in Drosophila models of various diseases already made it possible to recognize certain aspects of human pathologies. Future studies in this complete animal hold great promise for the better understanding of such processes and may also help finding new research avenues for the treatment of disorders with misregulated autophagy.

  10. MicroRNA regulation of Autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lund, Anders H

    2012-01-01

    recently contributed to our understanding of the molecular mechanisms of the autophagy machinery, yet several gaps remain in our knowledge of this process. The discovery of microRNAs (miRNAs) established a new paradigm of post-transcriptional gene regulation and during the past decade these small non......RNAs to regulation of the autophagy pathway. This regulation occurs both through specific core pathway components as well as through less well-defined mechanisms. Although this field is still in its infancy, we are beginning to understand the potential implications of these initial findings, both from a pathological...

  11. Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase-Dependent Cell Death and Leads to Mild Growth Retardation.

    Science.gov (United States)

    Vuppalapati, Karuna K; Bouderlique, Thibault; Newton, Phillip T; Kaminskyy, Vitaliy O; Wehtje, Henrik; Ohlsson, Claes; Zhivotovsky, Boris; Chagin, Andrei S

    2015-12-01

    Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes. © 2015 American Society for Bone and Mineral Research.

  12. Autophagy in protists

    Science.gov (United States)

    Duszenko, Michael; Ginger, Michael L; Brennand, Ana; Gualdrón-López, Melisa; Colombo, Maria-Isabel; Coombs, Graham H; Coppens, Isabelle; Jayabalasingham, Bamini; Langsley, Gordon; de Castro, Solange Lisboa; Menna-Barreto, Rubem; Mottram, Jeremy C; Navarro, Miguel; Rigden, Daniel J; Romano, Patricia S; Stoka, Veronika; Turk, Boris

    2011-01-01

    Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles and defense against parasitic invaders. During the past 10–20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target. PMID:20962583

  13. Autophagy Is an Innate Mechanism Associated with Leprosy Polarization

    Science.gov (United States)

    Andrade, Priscila Ribeiro; Ferreira, Helen; Nery, José Augusto da Costa; Côrte-Real, Suzana; da Silva, Gilberto Marcelo Sperandio; Rosa, Patricia Sammarco; Fabri, Mario; Sarno, Euzenir Nunes

    2017-01-01

    Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages. PMID:28056107

  14. Dengue Virus and Autophagy

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    Nicholas S. Heaton

    2011-08-01

    Full Text Available Several independent groups have published that autophagy is required for optimal RNA replication of dengue virus (DENV. Initially, it was postulated that autophagosomes might play a structural role in replication complex formation. However, cryo-EM tomography of DENV replication complexes showed that DENV replicates on endoplasmic reticulum (ER cisternae invaginations and not on classical autophagosomes. Recently, it was reported that autophagy plays an indirect role in DENV replication by modulating cellular lipid metabolism. DENV-induced autophagosomes deplete cellular triglycerides that are stored in lipid droplets, leading to increased β-oxidation and energy production. This is the first example of a virus triggering autophagy to modulate cellular physiology. In this review, we summarize these data and discuss new questions and implications for autophagy during DENV replication.

  15. Chemical Inhibition of Autophagy

    DEFF Research Database (Denmark)

    Baek, Eric; Lin Kim, Che; Gyeom Kim, Mi

    2016-01-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical...... autophagy inhibitors on the specific productivity (qp), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine...... and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125...

  16. Prohibitin 1 modulates mitochondrial stress-related autophagy in human colonic epithelial cells.

    Directory of Open Access Journals (Sweden)

    Arwa S Kathiria

    Full Text Available Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα, both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB, which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A(1 or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells

  17. The autophagy-related genes BbATG1 and BbATG8 have different functions in differentiation, stress resistance and virulence of mycopathogen Beauveria bassiana

    Science.gov (United States)

    Ying, Sheng-Hua; Liu, Jing; Chu, Xin-Ling; Xie, Xue-Qin; Feng, Ming-Guang

    2016-01-01

    Autophagy-related proteins play significantly different roles in eukaryotes. In the entomopathogenic fungus Beauveria bassiana, autophagy is associated with fungal growth and development. BbATG1 (a serine/threonine protein kinase) and BbATG8 (a ubiquitin-like protein) have similar roles in autophagy, but different roles in other processes. Disruption mutants of BbATG1 and BbATG8 had impaired conidial germination under starvation stress. The mutant ΔBbATG8 exhibited enhanced sensitivity to oxidative stress, while a ΔBbATG1 mutant did not. BbATG1 and BbATG8 showed different roles in spore differentiation. The blastospore yield was reduced by 70% and 92% in ΔBbATG1 and ΔBbATG8 mutants, respectively, and the double mutant had a reduction of 95%. Conidial yield was reduced by approximately 90% and 50% in ΔBbATG1 and ΔBbATG8 mutants, respectively. A double mutant had a reduction similar to ΔBbATG1. Additionally, both BbATG1 and BbATG8 affected the levels of conidial protein BbCP15p required for conidiation. The virulence of each autophagy-deficient mutant was considerably weakened as indicated in topical and intrahemocoel injection assays, and showed a greater reduction in topical infection. However, BbATG1 and BbATG8 had different effects on fungal virulence. Our data indicate that these autophagy-related proteins have different functions in fungal stress response, asexual development and virulence. PMID:27197558

  18. Autophagy in Trypanosomatids

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    Paul A. M. Michels

    2012-07-01

    Full Text Available Autophagy is a ubiquitous eukaryotic process that also occurs in trypanosomatid parasites, protist organisms belonging to the supergroup Excavata, distinct from the supergroup Opistokontha that includes mammals and fungi. Half of the known yeast and mammalian AuTophaGy (ATG proteins were detected in trypanosomatids, although with low sequence conservation. Trypanosomatids such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for serious tropical diseases in humans. The parasites are transmitted by insects and, consequently, have a complicated life cycle during which they undergo dramatic morphological and metabolic transformations to adapt to the different environments. Autophagy plays a major role during these transformations. Since inhibition of autophagy affects the transformation, survival and/or virulence of the parasites, the ATGs offer promise for development of drugs against tropical diseases. Furthermore, various trypanocidal drugs have been shown to trigger autophagy-like processes in the parasites. It is inferred that autophagy is used by the parasites in an—not always successful—attempt to cope with the stress caused by the toxic compounds.

  19. Here, there be dragons: charting autophagy-related alterations in human tumors.

    Science.gov (United States)

    Lebovitz, Chandra B; Bortnik, Svetlana B; Gorski, Sharon M

    2012-03-01

    Macroautophagy (or autophagy) is a catabolic cellular process that is both homeostatic and stress adaptive. Normal cells rely on basal levels of autophagy to maintain cellular integrity (via turnover of long-lived proteins and damaged organelles) and increased levels of autophagy to buoy cell survival during various metabolic stresses (via nutrient and energy provision through lysosomal degradation of cytoplasmic components). Autophagy can function in both tumor suppression and tumor progression, and is under investigation in clinical trials as a novel target for anticancer therapy. However, its role in cancer pathogenesis has yet to be fully explored. In particular, it remains unknown whether in vitro observations will be applicable to human cancer patients. Another outstanding question is whether there exists tumor-specific selection for alterations in autophagy function. In this review, we survey reported mutations in autophagy genes and key autophagy regulators identified in human tumor samples and summarize the literature regarding expression levels of autophagy genes and proteins in various cancer tissues. Although it is too early to draw inferences from this collection of in vivo studies of autophagy-related alterations in human cancers, their results highlight the challenges that must be overcome before we can accurately assess the scope of autophagy's predicted role in tumorigenesis.

  20. A dual role of p53 in the control of autophagy.

    Science.gov (United States)

    Tasdemir, Ezgi; Chiara Maiuri, M; Morselli, Eugenia; Criollo, Alfredo; D'Amelio, Marcello; Djavaheri-Mergny, Mojgan; Cecconi, Francesco; Tavernarakis, Nektarios; Kroemer, Guido

    2008-08-01

    Genotoxic stress can induce autophagy in a p53-dependent fashion and p53 can transactivate autophagy-inducing genes. We have observed recently that inactivation of p53 by deletion, depletion or inhibition can trigger autophagy. Thus, human and mouse cells subjected to knockout, knockdown or pharmacological inhibition of p53 manifest signs of autophagy such as depletion of p62/SQSTM1, LC3 lipidation, redistribution of GFP-LC3 in cytoplasmic puncta, and accumulation of autophagosomes and autolysosomes, both in vitro and in vivo. Inhibition of p53 causes autophagy in enucleated cells, indicating that the cytoplasmic, non-nuclear pool of p53 can regulate autophagy. Accordingly, retransfection of p53(-/-) cells with wild-type p53 as well as a p53 mutant that is excluded from the nucleus (due to the deletion of the nuclear localization sequence) can inhibit autophagy, whereas retransfection with a nucleus-restricted p53 mutant (in which the nuclear localization sequence has been deleted) does not inhibit autophagy. Several distinct autophagy inducers (e.g., starvation, rapamycin, lithium, tunicamycin and thapsigargin) stimulate the rapid degradation of p53. In these conditions, inhibition of the p53-specific E3 ubiquitin ligase HDM2 can avoid p53 depletion and simultaneously prevent the activation of autophagy. Moreover, a p53 mutant that lacks the HDM2 ubiquitinylation site and hence is more stable than wild-type p53 is particularly efficient in suppressing autophagy. In conclusion, p53 plays a dual role in the control of autophagy. On the one hand, nuclear p53 can induce autophagy through transcriptional effects. On the other hand, cytoplasmic p53 may act as a master repressor of autophagy.

  1. Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

    Science.gov (United States)

    Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

    2016-07-25

    Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Polycystin-2-dependent control of cardiomyocyte autophagy.

    Science.gov (United States)

    Criollo, Alfredo; Altamirano, Francisco; Pedrozo, Zully; Schiattarella, Gabriele G; Li, Dan L; Rivera-Mejías, Pablo; Sotomayor-Flores, Cristian; Parra, Valentina; Villalobos, Elisa; Battiprolu, Pavan K; Jiang, Nan; May, Herman I; Morselli, Eugenia; Somlo, Stefan; de Smedt, Humbert; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

    2018-05-01

    Considerable evidence points to critical roles of intracellular Ca 2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca 2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca 2+ homeostasis and autophagy. Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2 F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca 2+ chelation using BAPTA-AM, whereas removal of extracellular Ca 2+ had no effect, pointing to a role of intracellular Ca 2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca 2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca 2+ -channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca 2+ . Furthermore, PC2 ablation was associated with impaired Ca 2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca 2+ stores. Finally, we provide evidence that Ca 2+ -mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. Together, this study unveils PC2 as a novel regulator of autophagy acting

  3. Kaempferol induces hepatocellular carcinoma cell death via endoplasmic reticulum stress-CHOP-autophagy signaling pathway

    OpenAIRE

    Guo, Haiqing; Lin, Wei; Zhang, Xiangying; Zhang, Xiaohui; Hu, Zhongjie; Li, Liying; Duan, Zhongping; Zhang, Jing; Ren, Feng

    2017-01-01

    Kaempferol is a flavonoid compound that has gained widespread attention due to its antitumor functions. However, the underlying mechanisms are still not clear. The present study investigated the effect of kaempferol on hepatocellular carcinoma and its underlying mechanisms. Kaempferol induced autophagy in a concentration- and time-dependent manner in HepG2 or Huh7 cells, which was evidenced by the significant increase of autophagy-related genes. Inhibition of autophagy pathway, through 3-meth...

  4. Proteomics Insights into Autophagy.

    Science.gov (United States)

    Cudjoe, Emmanuel K; Saleh, Tareq; Hawkridge, Adam M; Gewirtz, David A

    2017-10-01

    Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis.

    Directory of Open Access Journals (Sweden)

    María Cristina Vanrell

    2017-11-01

    Full Text Available Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.

  6. Autophagy in plant pathogenic fungi.

    Science.gov (United States)

    Liu, Xiao-Hong; Xu, Fei; Snyder, John Hugh; Shi, Huan-Bin; Lu, Jian-Ping; Lin, Fu-Cheng

    2016-09-01

    Autophagy is a conserved cellular process that degrades cytoplasmic constituents in vacuoles. Plant pathogenic fungi develop special infection structures and/or secrete a range of enzymes to invade their plant hosts. It has been demonstrated that monitoring autophagy processes can be extremely useful in visualizing the sequence of events leading to pathogenicity of plant pathogenic fungi. In this review, we introduce the molecular mechanisms involved in autophagy. In addition, we explore the relationship between autophagy and pathogenicity in plant pathogenic fungi. Finally, we discuss the various experimental strategies available for use in the study of autophagy in plant pathogenic fungi. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

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    Orfali, Nina [Cork Cancer Research Center, University College Cork, Cork (Ireland); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); McKenna, Sharon L. [Cork Cancer Research Center, University College Cork, Cork (Ireland); Cahill, Mary R. [Department of Hematology, Cork University Hospital, Cork (Ireland); Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu [Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); Mongan, Nigel P., E-mail: nigel.mongan@nottingham.ac.uk [Faculty of Medicine and Health Science, School of Veterinary Medicine and Science, University of Nottingham, LE12 5RD (United Kingdom); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States)

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects.

  8. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    International Nuclear Information System (INIS)

    Orfali, Nina; McKenna, Sharon L.; Cahill, Mary R.; Gudas, Lorraine J.; Mongan, Nigel P.

    2014-01-01

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects

  9. Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells

    International Nuclear Information System (INIS)

    Liu, Qinghao; Qin, Yixian; Zhou, Lei; Kou, Qiuwen; Guo, Xin; Ge, Xinna; Yang, Hanchun; Hu, Hongbo

    2012-01-01

    In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/β-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome–lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.

  10. Autophagy sustains the replication of porcine reproductive and respiratory virus in host cells

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    Liu, Qinghao; Qin, Yixian; Zhou, Lei; Kou, Qiuwen; Guo, Xin; Ge, Xinna [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Yang, Hanchun, E-mail: yanghanchun1@cau.edu.cn [Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agribiotechnology, China Agricultural University, Beijing (China); Hu, Hongbo, E-mail: hongbo@cau.edu.cn [College of Food Science and Nutritional Engineering, China Agricultural University, Beijing (China)

    2012-08-01

    In this study, we confirmed the autophagy induced by porcine reproductive and respiratory syndrome virus (PRRSV) in permissive cells and investigated the role of autophagy in the replication of PRRSV. We first demonstrated that PRRSV infection significantly results in the increased double-membrane vesicles, the accumulation of LC3 fluorescence puncta, and the raised ratio of LC3-II/{beta}-actin, in MARC-145 cells. Then we discovered that induction of autophagy by rapamycin significantly enhances the viral titers of PRRSV, while inhibition of autophagy by 3-MA and silencing of LC3 gene by siRNA reduces the yield of PRRSV. The results showed functional autolysosomes can be formed after PRRSV infection and the autophagosome-lysosome-fusion inhibitor decreases the virus titers. We also examined the induction of autophagy by PRRSV infection in pulmonary alveolar macrophages. These findings indicate that autophagy induced by PRRSV infection plays a role in sustaining the replication of PRRSV in host cells.

  11. Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro

    Directory of Open Access Journals (Sweden)

    Ying Xu

    2016-04-01

    Full Text Available It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN. Here we used shRNA transfection to knockdown the insulin receptor (IR gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN.

  12. Dopamine Oxidation and Autophagy

    Directory of Open Access Journals (Sweden)

    Patricia Muñoz

    2012-01-01

    Full Text Available The molecular mechanisms involved in the neurodegenerative process of Parkinson's disease remain unclear. Currently, there is a general agreement that mitochondrial dysfunction, α-synuclein aggregation, oxidative stress, neuroinflammation, and impaired protein degradation are involved in the neurodegeneration of dopaminergic neurons containing neuromelanin in Parkinson's disease. Aminochrome has been proposed to play an essential role in the degeneration of dopaminergic neurons containing neuromelanin by inducing mitochondrial dysfunction, oxidative stress, the formation of neurotoxic α-synuclein protofibrils, and impaired protein degradation. Here, we discuss the relationship between the oxidation of dopamine to aminochrome, the precursor of neuromelanin, autophagy dysfunction in dopaminergic neurons containing neuromelanin, and the role of dopamine oxidation to aminochrome in autophagy dysfunction in dopaminergic neurons. Aminochrome induces the following: (i the formation of α-synuclein protofibrils that inactivate chaperone-mediated autophagy; (ii the formation of adducts with α- and β-tubulin, which induce the aggregation of the microtubules required for the fusion of autophagy vacuoles and lysosomes.

  13. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  14. Autophagy in Measles Virus Infection

    Directory of Open Access Journals (Sweden)

    Aurore Rozières

    2017-11-01

    Full Text Available Autophagy is a biological process that helps cells to recycle obsolete cellular components and which greatly contributes to maintaining cellular integrity in response to environmental stress factors. Autophagy is also among the first lines of cellular defense against invading microorganisms, including viruses. The autophagic destruction of invading pathogens, a process referred to as xenophagy, involves cytosolic autophagy receptors, such as p62/SQSTM1 (Sequestosome 1 or NDP52/CALCOCO2 (Nuclear Dot 52 KDa Protein/Calcium Binding And Coiled-Coil Domain 2, which bind to microbial components and target them towards growing autophagosomes for degradation. However, most, if not all, infectious viruses have evolved molecular tricks to escape from xenophagy. Many viruses even use autophagy, part of the autophagy pathway or some autophagy-associated proteins, to improve their infectious potential. In this regard, the measles virus, responsible for epidemic measles, has a unique interface with autophagy as the virus can induce multiple rounds of autophagy in the course of infection. These successive waves of autophagy result from distinct molecular pathways and seem associated with anti- and/or pro-measles virus consequences. In this review, we describe what the autophagy–measles virus interplay has taught us about both the biology of the virus and the mechanistic orchestration of autophagy.

  15. Autophagy in C. elegans development.

    Science.gov (United States)

    Palmisano, Nicholas J; Meléndez, Alicia

    2018-04-27

    Autophagy involves the sequestration of cytoplasmic contents in a double-membrane structure referred to as the autophagosome and the degradation of its contents upon delivery to lysosomes. Autophagy activity has a role in multiple biological processes during the development of the nematode Caenorhabditis elegans. Basal levels of autophagy are required to remove aggregate prone proteins, paternal mitochondria, and spermatid-specific membranous organelles. During larval development, autophagy is required for the remodeling that occurs during dauer development, and autophagy can selectively degrade components of the miRNA-induced silencing complex, and modulate miRNA-mediated silencing. Basal levels of autophagy are important in synapse formation and in the germ line, to promote the proliferation of proliferating stem cells. Autophagy activity is also required for the efficient removal of apoptotic cell corpses by promoting phagosome maturation. Finally, autophagy is also involved in lipid homeostasis and in the aging process. In this review, we first describe the molecular complexes involved in the process of autophagy, its regulation, and mechanisms for cargo recognition. In the second section, we discuss the developmental contexts where autophagy has been shown to be important. Studies in C. elegans provide valuable insights into the physiological relevance of this process during metazoan development. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Crosstalk between apoptosis and autophagy within the Beclin 1 interactome.

    Science.gov (United States)

    Maiuri, Maria Chiara; Criollo, Alfredo; Kroemer, Guido

    2010-02-03

    Although the essential genes for autophagy (Atg) have been identified, the molecular mechanisms through which Atg proteins control 'self eating' in mammalian cells remain elusive. Beclin 1 (Bec1), the mammalian orthologue of yeast Atg6, is part of the class III phosphatidylinositol 3-kinase (PI3K) complex that induces autophagy. The first among an increasing number of Bec1-interacting proteins that has been identified is the anti-apoptotic protein Bcl-2. The dissociation of Bec1 from Bcl-2 is essential for its autophagic activity, and Bcl-2 only inhibits autophagy when it is present in the endoplasmic reticulum (ER). A paper in this issue of the EMBO Journal has identified a novel protein, NAF-1 (nutrient-deprivation autophagy factor-1), that binds Bcl-2 at the ER. NAF-1 is a component of the inositol-1,4,5 trisphosphate (IP3) receptor complex, which contributes to the interaction of Bcl-2 with Bec1 and is required for Bcl-2 to functionally antagonize Bec1-mediated autophagy. This work provides mechanistic insights into how autophagy- and apoptosis-regulatory molecules crosstalk at the ER.

  17. Regulation of the autophagy protein LC3 by phosphorylation

    Science.gov (United States)

    Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

    2010-01-01

    Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

  18. A role for autophagy in the extension of lifespan by dietary restriction in C. elegans.

    Directory of Open Access Journals (Sweden)

    Malene Hansen

    2008-02-01

    Full Text Available In many organisms, dietary restriction appears to extend lifespan, at least in part, by down-regulating the nutrient-sensor TOR (Target Of Rapamycin. TOR inhibition elicits autophagy, the large-scale recycling of cytoplasmic macromolecules and organelles. In this study, we asked whether autophagy might contribute to the lifespan extension induced by dietary restriction in C. elegans. We find that dietary restriction and TOR inhibition produce an autophagic phenotype and that inhibiting genes required for autophagy prevents dietary restriction and TOR inhibition from extending lifespan. The longevity response to dietary restriction in C. elegans requires the PHA-4 transcription factor. We find that the autophagic response to dietary restriction also requires PHA-4 activity, indicating that autophagy is a transcriptionally regulated response to food limitation. In spite of the rejuvenating effect that autophagy is predicted to have on cells, our findings suggest that autophagy is not sufficient to extend lifespan. Long-lived daf-2 insulin/IGF-1 receptor mutants require both autophagy and the transcription factor DAF-16/FOXO for their longevity, but we find that autophagy takes place in the absence of DAF-16. Perhaps autophagy is not sufficient for lifespan extension because although it provides raw material for new macromolecular synthesis, DAF-16/FOXO must program the cells to recycle this raw material into cell-protective longevity proteins.

  19. Chikungunya virus–induced autophagy delays caspase-dependent cell death

    Science.gov (United States)

    Joubert, Pierre-Emmanuel; Werneke, Scott W.; de la Calle, Claire; Guivel-Benhassine, Florence; Giodini, Alessandra; Peduto, Lucie; Levine, Beth; Schwartz, Olivier; Lenschow, Deborah J.

    2012-01-01

    Autophagy is an important survival pathway and can participate in the host response to infection. Studying Chikungunya virus (CHIKV), the causative agent of a major epidemic in India, Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limits cell death and mortality after infection. We use biochemical studies and single cell multispectral assays to demonstrate that direct infection triggers both apoptosis and autophagy. CHIKV-induced autophagy is mediated by the independent induction of endoplasmic reticulum and oxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the IRE1α–XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing of autophagy genes resulted in enhanced intrinsic and extrinsic apoptosis, favoring viral propagation in cultured cells. Providing in vivo evidence for the relevance of our findings, Atg16LHM mice, which display reduced levels of autophagy, exhibited increased lethality and showed a higher sensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that features of apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibits caspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study suggests that inducers of autophagy may limit the pathogenesis of acute Chikungunya disease. PMID:22508836

  20. Autophagy response in the liver of pigeon exposed to avermectin.

    Science.gov (United States)

    Wang, Xian-Song; Liu, Ci; Khoso, Pervez Ahmed; Zheng, Weijia; Li, Ming; Li, Shu

    2017-05-01

    Pesticide residues are an important aspect of environmental pollution. Environmental avermectin residues have produced adverse effects in organisms. Many pesticides exert their toxic effects via the mechanism of autophagy. The purpose of this study was to examine the changes in autophagy levels and in autophagy-related genes, including LC3, Beclin 1, Dynein, ATG5, TORC1, and TORC2, resulting from exposure to subchronic levels of AVM in liver tissue in the king pigeon model. We observed abundant autophagic vacuoles with extensively degraded organelles, autophagosomal vacuoles, secondary lysosomes, and double-membrane structures in the liver. The expression levels of the autophagy-related genes LC3-I, LC3-II, Beclin 1, ATG5, and Dynein were up-regulated; however, TORC1 and TORC2 expression levels were down-regulated. These changes occurred in a concentration-dependent manner after AVM exposure for 30, 60, and 90 days in pigeons. Taken together, these results suggested that AVM increased the autophagic flux and that upregulation of autophagy might be closely related to the hepatotoxicity of AVM in birds.

  1. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

  2. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  3. Regulation of Autophagy by Kinases

    Science.gov (United States)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets. PMID:24212825

  4. Regulation of Autophagy by Kinases

    Directory of Open Access Journals (Sweden)

    Savitha Sridharan

    2011-06-01

    Full Text Available Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  5. The Effects of Kaempferol-Inhibited Autophagy on Osteoclast Formation.

    Science.gov (United States)

    Kim, Chang-Ju; Shin, Sang-Hun; Kim, Bok-Joo; Kim, Chul-Hoon; Kim, Jung-Han; Kang, Hae-Mi; Park, Bong-Soo; Kim, In-Ryoung

    2018-01-02

    Kaempferol, a flavonoid compound, is derived from the rhizome of Kaempferia galanga L ., which is used in traditional medicine in Asia. Autophagy has pleiotropic functions that are involved in cell growth, survival, nutrient supply under starvation, defense against pathogens, and antigen presentation. There are many studies dealing with the inhibitory effects of natural flavonoids in bone resorption. However, no studies have explained the relationship between the autophagic and inhibitory processes of osteoclastogenesis by natural flavonoids. The present study was undertaken to investigate the inhibitory effects of osteoclastogenesis through the autophagy inhibition process stimulated by kaempferol in murin macrophage (RAW 264.7) cells. The cytotoxic effect of Kaempferol was investigated by MTT assay. The osteoclast differentiation and autophagic process were confirmed via tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, western blot, and real-time PCR. Kaempferol controlled the expression of autophagy-related factors and in particular, it strongly inhibited the expression of p62/SQSTM1. In the western blot and real time-PCR analysis, when autophagy was suppressed with the application of 3-Methyladenine (3-MA) only, osteoclast and apoptosis related factors were not significantly affected. However, we found that after cells were treated with kaempferol, these factors inhibited autophagy and activated apoptosis. Therefore, we presume that kaempferol-inhibited autophagy activated apoptosis by degradation of p62/SQSTM1. Further study of the p62/SQSTM1 gene as a target in the autophagy mechanism, may help to delineate the potential role of kaempferol in the treatment of bone metabolism disorders.

  6. Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Sukseree, Supawadee [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Rossiter, Heidemarie; Mildner, Michael [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Pammer, Johannes [Institute of Clinical Pathology, Medical University of Vienna, Vienna (Austria); Buchberger, Maria; Gruber, Florian [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Watanapokasin, Ramida [Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Tschachler, Erwin [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Eckhart, Leopold, E-mail: leopold.eckhart@meduniwien.ac.at [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. Black-Right-Pointing-Pointer Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. Black-Right-Pointing-Pointer Autophagy was required for normal terminal differentiation of preputial gland cells. Black-Right-Pointing-Pointer Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

  7. Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy

    Science.gov (United States)

    Zirin, Jonathan; Nieuwenhuis, Joppe; Perrimon, Norbert

    2013-01-01

    Several myopathies are associated with defects in autophagic and lysosomal degradation of glycogen, but it remains unclear how glycogen is targeted to the lysosome and what significance this process has for muscle cells. We have established a Drosophila melanogaster model to study glycogen autophagy in skeletal muscles, using chloroquine (CQ) to simulate a vacuolar myopathy that is completely dependent on the core autophagy genes. We show that autophagy is required for the most efficient degradation of glycogen in response to starvation. Furthermore, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of Glycogen Synthase in regulating autophagy through its interaction with Atg8. PMID:24265594

  8. Autophagy and neurodegenerative disorders

    Institute of Scientific and Technical Information of China (English)

    Evangelia Kesidou; Roza Lagoudaki; Olga Touloumi; Kyriaki-Nefeli Poulatsidou; Constantina Simeonidou

    2013-01-01

    Accumulation of aberrant proteins and inclusion bodies are hallmarks in most neurodegenerative diseases. Consequently, these aggregates within neurons lead to toxic effects, overproduction of reactive oxygen species and oxidative stress. Autophagy is a significant intracel ular mechanism that removes damaged organelles and misfolded proteins in order to maintain cel homeostasis. Excessive or insufficient autophagic activity in neurons leads to altered homeostasis and influences their survival rate, causing neurodegeneration. The review article provides an update of the role of autophagic process in representative chronic and acute neurodegenerative disorders.

  9. Stress-induced self-cannibalism: on the regulation of autophagy by endoplasmic reticulum stress.

    Science.gov (United States)

    Deegan, Shane; Saveljeva, Svetlana; Gorman, Adrienne M; Samali, Afshin

    2013-07-01

    Macroautophagy (autophagy) is a cellular catabolic process which can be described as a self-cannibalism. It serves as an essential protective response during conditions of endoplasmic reticulum (ER) stress through the bulk removal and degradation of unfolded proteins and damaged organelles; in particular, mitochondria (mitophagy) and ER (reticulophagy). Autophagy is genetically regulated and the autophagic machinery facilitates removal of damaged cell components and proteins; however, if the cell stress is acute or irreversible, cell death ensues. Despite these advances in the field, very little is known about how autophagy is initiated and how the autophagy machinery is transcriptionally regulated in response to ER stress. Some three dozen autophagy genes have been shown to be required for the correct assembly and function of the autophagic machinery; however; very little is known about how these genes are regulated by cellular stress. Here, we will review current knowledge regarding how ER stress and the unfolded protein response (UPR) induce autophagy, including description of the different autophagy-related genes which are regulated by the UPR.

  10. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Liang [East Hospital, Tongji University School of Medicine, Shanghai (China); Dong, Chuanming [East Hospital, Tongji University School of Medicine, Shanghai (China); Department of Anatomy and Neurobiology, The Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong (China); Sun, Chenxi; Ma, Rongjie; Yang, Danjing [East Hospital, Tongji University School of Medicine, Shanghai (China); Zhu, Hongwen, E-mail: hongwen_zhu@hotmail.com [Tianjin Hospital, Tianjin Academy of Integrative Medicine, Tianjin (China); Xu, Jun, E-mail: xunymc2000@yahoo.com [East Hospital, Tongji University School of Medicine, Shanghai (China)

    2015-08-21

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy.

  11. Autophagy is required for the activation of NFκB.

    Science.gov (United States)

    Criollo, Alfredo; Chereau, Fanny; Malik, Shoaib Ahmad; Niso-Santano, Mireia; Mariño, Guillermo; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Baud, Véronique; Kroemer, Guido

    2012-01-01

    It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.

  12. Pollination induces autophagy in petunia petals via ethylene.

    Science.gov (United States)

    Shibuya, Kenichi; Niki, Tomoko; Ichimura, Kazuo

    2013-02-01

    Autophagy is one of the main mechanisms of degradation and remobilization of macromolecules, and it appears to play an important role in petal senescence. However, little is known about the regulatory mechanisms of autophagy in petal senescence. Autophagic processes were observed by electron microscopy and monodansylcadaverine staining of senescing petals of petunia (Petunia hybrida); autophagy-related gene 8 (ATG8) homologues were isolated from petunia and the regulation of expression was analysed. Nutrient remobilization was also examined during pollination-induced petal senescence. Active autophagic processes were observed in the mesophyll cells of senescing petunia petals. Pollination induced the expression of PhATG8 homologues and was accompanied by an increase in ethylene production. Ethylene inhibitor treatment in pollinated flowers delayed the induction of PhATG8 homologues, and ethylene treatment rapidly upregulated PhATG8 homologues in petunia petals. Dry weight and nitrogen content were decreased in the petals and increased in the ovaries after pollination in detached flowers. These results indicated that pollination induces autophagy and that ethylene is a key regulator of autophagy in petal senescence of petunia. The data also demonstrated the translocation of nutrients from the petals to the ovaries during pollination-induced petal senescence.

  13. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Science.gov (United States)

    Wang, Jinli; Yang, Kun; Zhou, Lin; Minhaowu; Wu, Yongjian; Zhu, Min; Lai, Xiaomin; Chen, Tao; Feng, Lianqiang; Li, Meiyu; Huang, Chunyu; Zhong, Qiu; Huang, Xi

    2013-01-01

    Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  14. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Directory of Open Access Journals (Sweden)

    Jinli Wang

    Full Text Available Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7 reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb, a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  15. A close connection between the PERK and IRE arms of the UPR and the transcriptional regulation of autophagy.

    Science.gov (United States)

    Deegan, Shane; Koryga, Izabela; Glynn, Sharon A; Gupta, Sanjeev; Gorman, Adrienne M; Samali, Afshin

    2015-01-02

    Endoplasmic reticulum (ER) stress is known to lead to activation of both the unfolded protein response (UPR) and autophagy. Although regulatory connections have been identified between the UPR and autophagy, it is still unclear to what extent the UPR regulates the genes involved at the different stages of the autophagy pathway. Here, we carried out a microarray analysis of HCT116 cells subjected to ER stress and observed the transcriptional upregulation of a large cohort of autophagy-related genes. Of particular interest, we identified the transcriptional upregulation of the autophagy receptor genes SQSTM1/p62, NBR1 and BNIP3L/NIX in response to ER stress and show that the inhibition of the UPR transmembrane receptors, PERK and IRE1, abrogates this upregulation. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Cytotoxic Autophagy in Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Khushboo Sharma

    2014-06-01

    Full Text Available Autophagy is a process of cellular self-digestion, whereby the cell degrades subcellular materials in order to generate energy and metabolic precursors in order to prolong survival, classically under conditions of nutrient deprivation. Autophagy can also involve the degradation of damaged or aged organelles, and misfolded or damaged proteins to eliminate these components that might otherwise be deleterious to cellular survival. Consequently, autophagy has generally been considered a prosurvival response. Many, if not most chemotherapeutic drugs and radiation also promote autophagy, which is generally considered a cytoprotective response, in that its inhibition frequently promotes apoptotic cells death. Furthermore, it has been shown that conventional chemotherapeutic drugs and radiation alone rarely induce a form of autophagy that leads to cell death. However, there are multiple examples in the literature where newer chemotherapeutic agents, drug combinations or drugs in combination with radiation promote autophagic cell death. This review will describe autophagic cell death induced in breast tumor cells, lung cancer cells as well as glioblastoma, demonstrating that it cannot be concluded that stress induced autophagy is, of necessity, cytoprotective in function.

  17. Autophagy in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Piotr Czarny

    2015-01-01

    Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  18. Autophagy in human embryonic stem cells

    NARCIS (Netherlands)

    Tra, Thien; Gong, Lan; Kao, Lin-Pin; Li, Xue-Lei; Grandela, Catarina; Devenish, Rodney J.; Wolvetang, Ernst; Prescott, Mark

    2011-01-01

    Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of

  19. Enhanced myometrial autophagy in postpartum uterine involution

    Directory of Open Access Journals (Sweden)

    Keng-Fu Hsu

    2014-09-01

    Conclusion: Autophagy of myocytes may play an important role in uterine involution. These results have implications for our understanding of myometrial functional adaptations during pregnancy and the physiological role of autophagy in the uterine remodeling events in the postpartum period.

  20. d-limonene exhibits antitumor activity by inducing autophagy and apoptosis in lung cancer.

    Science.gov (United States)

    Yu, Xiao; Lin, Hongyan; Wang, Yu; Lv, Wenwen; Zhang, Shuo; Qian, Ying; Deng, Xiaobei; Feng, Nannan; Yu, Herbert; Qian, Biyun

    2018-01-01

    d-limonene is a plant extract with widespread application, and it has been recently reported to have antiproliferative and proapoptotic effects on cancer cells. However, the mechanisms by which d-limonene achieves these effects, especially in lung cancer, are not entirely clear. Therefore, the goal of this study was to examine the effects of d-limonene on lung cancer and explore its mechanisms of action. We examined the therapeutic effects of d-limonene on lung cancer cells and in a xenograft animal model by characterizing its effects on the pathways of apoptosis and autophagy. Cell proliferation was measured using the Cell Counting Kit-8, and apoptosis was determined by flow cytometric analysis. Levels of LC3 puncta, an autophagy marker, were analyzed by laser scanning confocal microscopy. Autophagy and apoptosis-related gene expression were assessed by real-time quantitative polymerase chain reaction and Western blot. d-limonene inhibited the growth of lung cancer cells and suppressed the growth of transplanted tumors in nude mice. Expression of apoptosis and autophagy-related genes were increased in tumors after treatment with d-limonene. Furthermore, the use of chloroquine, an autophagy inhibitor, and knockdown of the atg5 gene, suppressed the apoptosis induced by d-limonene. d-limonene may have a therapeutic effect on lung cancer as it can induce apoptosis of lung cancer cells by promoting autophagy.

  1. Autophagy attenuates the catabolic effect during inflammatory conditions in nucleus pulposus cells, as sustained by NF-κB and JNK inhibition

    Science.gov (United States)

    XU, KANG; CHEN, WEIJIAN; WANG, XIAOFEI; PENG, YAN; LIANG, ANJING; HUANG, DONGSHENG; LI, CHUNHAI; YE, WEI

    2015-01-01

    Proteoglycan degradation contributing to the pathogenesis of intervertebral disc (IVD) degeneration is induced by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Cell autophagy exists in degenerative diseases, including osteoarthritis and inter-vertebral disc degeneration. However, the autophagy induced by TNF-α and IL-1β and the corresponding molecular mechanism appear to be cell-type dependent. The effect and mechanism of autophagy regulated by TNF-α and IL-1β in IVDs remains unclear. Additionally, the impact of autophagy on the catabolic effect in inflammatory conditions also remains elusive. In the present study, autophagy activator and inhibitor were used to demonstrate the impact of autophagy on the catabolic effect induced by TNF-α. A critical role of autophagy was identified in rat nucleus pulposus (NP) cells: Inhibition of autophagy suppresses, while activation of autophagy enhances, the catabolic effect of cytokines. Subsequently, the autophagy-related gene expression in rat NP cells following TNF-α and IL-1β treatment was observed using immunofluorescence, quantitative polymerase chain reaction and western blot analysis; however, no association was present. In addition, nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases and p38 mitogen-activated protein kinase inhibitors and TNF-α were used to determine the molecular mechanism of autophagy during the inflammatory conditions, and only the NF-κB and JNK inhibitor were found to enhance the autophagy of rat NP cells. Finally, IKKβ knockdown was used to further confirm the effect of the NF-κB signal on human NP cells autophagy, and the data showed that IKKβ knockdown upregulated the autophagy of NP cells during inflammatory conditions. PMID:26165348

  2. Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast.

    Science.gov (United States)

    Matsuhara, Hirotada; Yamamoto, Ayumu

    2016-01-01

    Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen-starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy-defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down-regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  3. The role of autophagy inhibition in the enhanced cytotoxicity of temozolomide on melanoma cell lines

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    O. O. Ryabaya

    2017-01-01

    Full Text Available Background. Despite advantages in treatment of metastatic melanoma it remains resistant to current therapy. Recent evidence indicates that tumor cells could overcome death through autophagy, a process that degrades cellular proteins and organelles to maintain cellular biosynthesis during nutrient deprivation or lack of energy. Objective: to investigate the involvement of autophagy inhibitors chloroquine (CQ and LY-294.002 (LY in temozolomide (TMZ cytotoxicity in human melanoma cell lines.Materials and methods. The study was performed on patient-derived melanoma cell lines Mel Z, Mel IL and Mel MTP. The antiproliferative activity of combined TMZ and autophagy inhibitors treatment was determined by MTT assay and colony-forming assay. Cell cycle analysis, apoptosis activation and expression analysis of key autophagy markers under combined treatment was evaluated.Results. CQ and LY enhanced the cytotoxicity of TMZ and reduced colony formation in 3 melanoma cell lines, moreover both inhibitors increased cell population in G0 / G1 phase of cell cycle in Mel Z, Mel IL cell lines, but not in Mel MTP. CQ and LY synergistically activated apoptosis in all cell lines. The matrix RNA expression analysis of key autophagy genes showed autophagy involvement in enhanced cytotoxicity.Conclusions. Thus, autophagy inhibition on different stages of this process could overcome resistance to TMZ and be applicable as potent target in metastatic melanoma treatment.

  4. Kaempferol induces hepatocellular carcinoma cell death via endoplasmic reticulum stress-CHOP-autophagy signaling pathway.

    Science.gov (United States)

    Guo, Haiqing; Lin, Wei; Zhang, Xiangying; Zhang, Xiaohui; Hu, Zhongjie; Li, Liying; Duan, Zhongping; Zhang, Jing; Ren, Feng

    2017-10-10

    Kaempferol is a flavonoid compound that has gained widespread attention due to its antitumor functions. However, the underlying mechanisms are still not clear. The present study investigated the effect of kaempferol on hepatocellular carcinoma and its underlying mechanisms. Kaempferol induced autophagy in a concentration- and time-dependent manner in HepG2 or Huh7 cells, which was evidenced by the significant increase of autophagy-related genes. Inhibition of autophagy pathway, through 3-methyladenine or Atg7 siRNA, strongly diminished kaempferol-induced apoptosis. We further hypothesized that kaempferol can induce autophagy via endoplasmic reticulum (ER) stress pathway. Indeed, blocking ER stress by 4-phenyl butyric acid (4-PBA) or knockdown of CCAAT/enhancer-binding protein homologous protein (CHOP) with siRNA alleviated kaempferol-induced HepG2 or Huh7 cells autophagy; while transfection with plasmid overexpressing CHOP reversed the effect of 4-PBA on kaempferol-induced autophagy. Our results demonstrated that kaempferol induced hepatocarcinoma cell death via ER stress and CHOP-autophagy signaling pathway; kaempferol may be used as a potential chemopreventive agent for patients with hepatocellular carcinoma.

  5. Autophagy and Alzheimer’s Disease: From Molecular Mechanisms to Therapeutic Implications

    Directory of Open Access Journals (Sweden)

    Md. Sahab Uddin

    2018-01-01

    Full Text Available Alzheimer’s disease (AD is the most common cause of progressive dementia in the elderly. It is characterized by a progressive and irreversible loss of cognitive abilities and formation of senile plaques, composed mainly of amyloid β (Aβ, and neurofibrillary tangles (NFTs, composed of tau protein, in the hippocampus and cortex of afflicted humans. In brains of AD patients the metabolism of Aβ is dysregulated, which leads to the accumulation and aggregation of Aβ. Metabolism of Aβ and tau proteins is crucially influenced by autophagy. Autophagy is a lysosome-dependent, homeostatic process, in which organelles and proteins are degraded and recycled into energy. Thus, dysfunction of autophagy is suggested to lead to the accretion of noxious proteins in the AD brain. In the present review, we describe the process of autophagy and its importance in AD. Additionally, we discuss mechanisms and genes linking autophagy and AD, i.e., the mTOR pathway, neuroinflammation, endocannabinoid system, ATG7, BCL2, BECN1, CDK5, CLU, CTSD, FOXO1, GFAP, ITPR1, MAPT, PSEN1, SNCA, UBQLN1, and UCHL1. We also present pharmacological agents acting via modulation of autophagy that may show promise in AD therapy. This review updates our knowledge on autophagy mechanisms proposing novel therapeutic targets for the treatment of AD.

  6. TFE3 Alleviates Hepatic Steatosis through Autophagy-Induced Lipophagy and PGC1α-Mediated Fatty Acid β-Oxidation

    OpenAIRE

    Jie Xiong; Kezhou Wang; Jiangping He; Guangya Zhang; Dandan Zhang; Fengling Chen

    2016-01-01

    Autophagy flux deficiency is closely related to the development of hepatic steatosis. Transcription factor E3 (TFE3) is reported to be a crucial gene that regulates autophagy flux and lysosome function. Therefore, we investigated the role of TFE3 in a cell model of hepatic steatosis. We constructed L02 hepatocyte lines that stably over-expressed or knocked down the expression of TFE3. Subsequently, the effects of TFE3 on hepatocellular lipid metabolism were determined by autophagy flux assay,...

  7. Cisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells.

    Science.gov (United States)

    Lin, Ji-Fan; Lin, Yi-Chia; Tsai, Te-Fu; Chen, Hung-En; Chou, Kuang-Yu; Hwang, Thomas I-Sheng

    2017-01-01

    Cisplatin-based chemotherapy is the first line treatment for several cancers including bladder cancer (BC). Autophagy induction has been implied to contribute to cisplatin resistance in ovarian cancer; and a high basal level of autophagy has been demonstrated in human bladder tumors. Therefore, it is reasonable to speculate that autophagy may account for the failure of cisplatin single treatment in BC. This study investigated whether cisplatin induces autophagy and the mechanism involved using human BC cell lines. Human BC cells (5637 and T24) were used in this study. Cell viability was detected using water soluble tetrazolium-8 reagents. Autophagy induction was detected by monitoring the levels of light chain 3 (LC3)-II and p62 by Western blot, LC3-positive puncta formation by immunofluorescence, and direct observation of the autophagolysosome (AL) formation by transmission electron microscopy. Inhibitors including bafilomycin A1 (Baf A1), chloroquine (CQ), and shRNA-based lentivirus against autophagy-related genes (ATG7 and ATG12) were utilized. Apoptosis level was detected by caspase 3/7 activity and DNA fragmentation. Cisplatin decreased cell viability and induced apoptosis of 5637 and T24 cells in a dose-and time-dependent manner. The increased LC3-II accumulation, p62 clearance, the number of LC3-positive puncta, and ALs in cisplatin-treated cells suggested that cisplatin indeed induces autophagy. Inhibition of cisplatin-induced autophagy using Baf A1, CQ, or ATG7/ATG12 shRNAs significantly enhanced cytotoxicity of cisplatin toward BC cells. These results indicated that cisplatin induced protective autophagy which may contribute to the development of cisplatin resistance and resulted in treatment failure. Mechanistically, upregulation of beclin-1 (BECN1) was detected in cisplatin-treated cells, and knockdown of BECN1 using shRNA attenuated cisplatin-induced autophagy and subsequently enhanced cisplatin-induced apoptosis. Collectively, the study results

  8. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia.

    LENUS (Irish Health Repository)

    Orfali, Nina

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies.

  9. Inhibiting ROS-TFEB-Dependent Autophagy Enhances Salidroside-Induced Apoptosis in Human Chondrosarcoma Cells.

    Science.gov (United States)

    Zeng, Wei; Xiao, Tao; Cai, Anlie; Cai, Weiliang; Liu, Huanhuan; Liu, Jingling; Li, Jie; Tan, Miduo; Xie, Li; Liu, Ying; Yang, Xiangcheng; Long, Yi

    2017-01-01

    Autophagy modulation has been considered a potential therapeutic strategy for human chondrosarcoma, and a previous study indicated that salidroside exhibits significant anti-carcinogenic activity. However, the ability of salidroside to induce autophagy and its role in human chondrosarcoma cell death remains unclear. We exposed SW1353 cells to different concentrations of salidroside (0.5, 1 and 2 mM) for 24 h. RT-PCR, Western-blotting, Immunocytofluorescence, and Luciferase Reporter Assays were used to evaluate whether salidroside activated the TFEB-dependent autophagy. We show that salidroside induced significant apoptosis in the human chondrosarcoma cell line SW1353. In addition, we demonstrate that salidroside-induced an autophagic response in SW1353 cells, as evidenced by the upregulation of LC3-II and downregulation of P62. Moreover, pharmacological or genetic blocking of autophagy enhanced salidroside -induced apoptosis, indicating the cytoprotective role of autophagy in salidroside-treated SW1353 cells. Salidroside also induced TFEB (Ser142) dephosphorylation, subsequently to activated TFEB nuclear translocation and increase of TFEB reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes. Importantly, we found that salidroside triggered the generation of ROS in SW1353 cells. Furthermore, NAC, a ROS scavenger, abrogated the effects of salidroside on TFEB-dependent autophagy. These data demonstrate that salidroside increased TFEB-dependent autophagy by activating ROS signaling pathways in human chondrosarcoma cells. These data also suggest that blocking ROS-TFEB-dependent autophagy to enhance the activity of salidroside warrants further attention in treatment of human chondrosarcoma cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

  10. Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line

    International Nuclear Information System (INIS)

    Miyamae, Yusaku; Nishito, Yukina; Nakai, Naomi; Nagumo, Yoko; Usui, Takeo; Masuda, Seiji; Kambe, Taiho; Nagao, Masaya

    2016-01-01

    Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. - Highlights: • Autophagy is closely related to lipid degradation in hepatic stellate cells. • Tetrandrine (Tet) causes lipid accumulation via blockade of autophagy in HSC-T6 cells. • Tet blocked autophagy without affecting lysosomal function unlike bafilomycin A_1. • Perilipin 1 was specifically co-localized with LC3 in Tet-treated cells. • Perilipin 1 may play potential roles in autophagy-mediated lipid degradation.

  11. DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy

    Directory of Open Access Journals (Sweden)

    Mengqiang Yu

    2014-10-01

    Full Text Available DNA damage-regulated autophagy modulator protein 1 (DRAM1, a multi-pass membrane lysosomal protein, is reportedly a tumor protein p53 (TP53 target gene involved in autophagy. During cerebral ischemia/reperfusion (I/R injury, DRAM1 protein expression is increased, and autophagy is activated. However, the functional significance of DRAM1 and the relationship between DRAM1 and autophagy in brain I/R remains uncertain. The aim of this study is to investigate whether DRAM1 mediates autophagy activation in cerebral I/R injury and to explore its possible effects and mechanisms. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R Neuro-2a cell model to mimic cerebral I/R conditions in vitro, and RNA interference is used to knock down DRAM1 expression in this model. Cell viability assay is performed using the LIVE/DEAD viability/cytotoxicity kit. Cell phenotypic changes are analyzed through Western blot assays. Autophagy flux is monitored through the tandem red fluorescent protein–Green fluorescent protein–microtubule associated protein 1 light chain 3 (RFP–GFP–LC3 construct. The expression levels of DRAM1 and microtubule associated protein 1 light chain 3II/I (LC3II/I are strongly up-regulated in Neuro-2a cells after OGD/R treatment and peaked at the 12 h reperfusion time point. The autophagy-specific inhibitor 3-Methyladenine (3-MA inhibits the expression of DRAM1 and LC3II/I and exacerbates OGD/R-induced cell injury. Furthermore, DRAM1 knockdown aggravates OGD/R-induced cell injury and significantly blocks autophagy through decreasing autophagosome-lysosome fusion. In conclusion, our data demonstrate that DRAM1 knockdown in Neuro-2a cells inhibits autophagy by blocking autophagosome-lysosome fusion and exacerbated OGD/R-induced cell injury. Thus, DRAM1 might constitute a new therapeutic target for I/R diseases.

  12. Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line

    Energy Technology Data Exchange (ETDEWEB)

    Miyamae, Yusaku, E-mail: ymiyamae@lif.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Nishito, Yukina; Nakai, Naomi [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Nagumo, Yoko; Usui, Takeo [Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki 305-8572 (Japan); Masuda, Seiji; Kambe, Taiho [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan); Nagao, Masaya, E-mail: mnagao@kais.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Oiwakecho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 (Japan)

    2016-08-12

    Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. - Highlights: • Autophagy is closely related to lipid degradation in hepatic stellate cells. • Tetrandrine (Tet) causes lipid accumulation via blockade of autophagy in HSC-T6 cells. • Tet blocked autophagy without affecting lysosomal function unlike bafilomycin A{sub 1}. • Perilipin 1 was specifically co-localized with LC3 in Tet-treated cells. • Perilipin 1 may play potential roles in autophagy-mediated lipid degradation.

  13. Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Hui [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Zhuo, Liling [College of Life Science, Zaozhuang University, Zaozhuang, Shandong, 277160 (China); Han, Tao; Hu, Di; Yang, Xiaokang; Wang, Yi; Yuan, Yan; Gu, Jianhong; Bian, Jianchun; Liu, Xuezhong [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Liu, Zongping, E-mail: liuzongping@yzu.edu.cn [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China)

    2015-04-17

    Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate. - Highlights: • GJIC and autophagy is crucial for biological processes. • Cd exposure causes GJIC inhibition and autophagy increase in BRL 3A cells. • Autophagy protects Cd induced BRL 3A cells apoptosis at an early stage. • Autophagy exacerbates Cd-induced GJIC inhibition. • GJIC plays an important role in autophagy induced cell death or survival.

  14. Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

    International Nuclear Information System (INIS)

    Wu, Chun-Yan; Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng; Guo, Xiao-Zhong; Wang, Hua

    2011-01-01

    Research highlights: → We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. → Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. → This study also supplies a possible novel therapeutic method for the treatment of pancreatic cancer using autophagy inhibitors. -- Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

  15. The intersection between growth factors, autophagy and ER stress: A new target to treat neurodegenerative diseases?

    Science.gov (United States)

    Garcia-Huerta, Paula; Troncoso-Escudero, Paulina; Jerez, Carolina; Hetz, Claudio; Vidal, Rene L

    2016-10-15

    One of the salient features of most neurodegenerative diseases is the aggregation of specific proteins in the brain. This proteostasis imbalance is proposed as a key event triggering the neurodegenerative cascade. The unfolded protein response (UPR) and autophagy pathways are emerging as critical processes implicated in handling disease-related misfolded proteins. However, in some conditions, perturbations in the buffering capacity of the proteostasis network may be part of the etiology of the disease. Thus, pharmacological or gene therapy strategies to enhance autophagy or UPR responses are becoming an attractive target for disease intervention. Here, we discuss current evidence depicting the complex involvement of autophagy and ER stress in brain diseases. Novel pathways to modulate protein misfolding are discussed including the relation between aging and growth factor signaling. This article is part of a Special Issue entitled SI:Autophagy. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Ordered bulk degradation via autophagy

    DEFF Research Database (Denmark)

    Dengjel, Jörn; Kristensen, Anders Riis; Andersen, Jens S

    2008-01-01

    During amino acid starvation, cells undergo macroautophagy which is regarded as an unspecific bulk degradation process. Lately, more and more organelle-specific autophagy subtypes such as reticulophagy, mitophagy and ribophagy have been described and it could be shown, depending on the experimental...... at proteasomal and lysosomal degradation ample cross-talk between the two degradation pathways became evident. Degradation via autophagy appeared to be ordered and regulated at the protein complex/organelle level. This raises several important questions such as: can macroautophagy itself be specific and what...

  17. Autophagy, lipophagy and lysosomal lipid storage disorders.

    Science.gov (United States)

    Ward, Carl; Martinez-Lopez, Nuria; Otten, Elsje G; Carroll, Bernadette; Maetzel, Dorothea; Singh, Rajat; Sarkar, Sovan; Korolchuk, Viktor I

    2016-04-01

    Autophagy is a catabolic process with an essential function in the maintenance of cellular and tissue homeostasis. It is primarily recognised for its role in the degradation of dysfunctional proteins and unwanted organelles, however in recent years the range of autophagy substrates has also been extended to lipids. Degradation of lipids via autophagy is termed lipophagy. The ability of autophagy to contribute to the maintenance of lipo-homeostasis becomes particularly relevant in the context of genetic lysosomal storage disorders where perturbations of autophagic flux have been suggested to contribute to the disease aetiology. Here we review recent discoveries of the molecular mechanisms mediating lipid turnover by the autophagy pathways. We further focus on the relevance of autophagy, and specifically lipophagy, to the disease mechanisms. Moreover, autophagy is also discussed as a potential therapeutic target in several key lysosomal storage disorders. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Modulation of pathogen recognition by autophagy

    Directory of Open Access Journals (Sweden)

    Ji Eun eOh

    2012-03-01

    Full Text Available Autophagy is an ancient biological process for maintaining cellular homeostasis by degradation of long-lived cytosolic proteins and organelles. Recent studies demonstrated that autophagy is availed by immune cells to regulate innate immunity. On the one hand, cells exert direct effector function by degrading intracellular pathogens; on the other hand, autophagy modulates pathogen recognition and downstream signaling for innate immune responses. Pathogen recognition via pattern recognition receptors induces autophagy. The function of phagocytic cells is enhanced by recruitment of autophagy-related proteins. Moreover, autophagy acts as a delivery system for viral replication complexes to migrate to the endosomal compartments where virus sensing occurs. In another case, key molecules of the autophagic pathway have been found to negatively regulate immune signaling, thus preventing aberrant activation of cytokine production and consequent immune responses. In this review, we focus on the recent advances in the role of autophagy in pathogen recognition and modulation of innate immune responses.

  19. Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC.

    Science.gov (United States)

    Won, Shen-Jeu; Yen, Cheng-Hsin; Lin, Ting-Yu; Jiang-Shieh, Ya-Fen; Lin, Chun-Nan; Chen, Jyun-Ti; Su, Chun-Li

    2018-01-01

    The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT-29 cells. In the present study, we observed that many autophagy-related genes in GFC-treated HT-29 cells were up- and down-regulated using a cDNA microarray containing oncogenes and kinase genes. GFC-induced autophagy of HT-29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double-membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5-Atg12 and PI3K/Beclin-1 complexes were observed using Western blot. Administration of autophagy inhibitor (3-methyladenine and shRNA Atg5) and apoptosis inhibitor Z-VAD showed that the GFC-induced autophagy was cytotoxic form and GFC-induced apoptosis enhanced GFC-induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC-induced anticancer mechanisms of human colorectal cancer. © 2017 Wiley Periodicals, Inc.

  20. Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein.

    LENUS (Irish Health Repository)

    Elzinga, Baukje M

    2013-06-01

    Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1\\/ATG7). Pharmacological inhibition of autophagy also reduced Bcr-Abl\\/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib.

  1. Antimony trichloride induces a loss of cell viability via reactive oxygen species-dependent autophagy in A549 cells.

    Science.gov (United States)

    Zhao, Xinyuan; Xing, Fengjun; Cong, Yewen; Zhuang, Yin; Han, Muxi; Wu, Zhiqiang; Yu, Shali; Wei, Haiyan; Wang, Xiaoke; Chen, Gang

    2017-12-01

    Antimony (Sb) is one of the most prevalent heavy metals and frequently leads to biological toxicity. Although autophagy is believed to be involved in metal-associated cytotoxicity, there is no evidence of its involvement following exposure. Moreover, the underlying mechanism of autophagy remains unclear. In this study, treatment with antimony trichloride caused autophagy in a dose- and time-dependent manner in A549 cells but did not affect the level of Atg5 or Atg7 mRNA expression. Furthermore, Sb enhanced autophagic flux while upregulating p62 gene and protein levels. The classic mechanistic target of rapamycin (mTOR) pathway is not involved in Sb-induced autophagy. However, Sb-induced autophagy and the upregulation of p62 were inhibited by treatment with the antioxidant N-acetylcysteine (NAC). Subsequent analyses demonstrated that the inhibition of autophagy protected A549 cells from a loss of cell viability, while the activation of autophagy by rapamycin had the opposite effect. These data suggest that reactive oxygen species-dependent autophagy mediates Sb-stimulated cell viability loss in A549 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. A Yin-Yang 1/miR-30a regulatory circuit modulates autophagy in pancreatic cancer cells.

    Science.gov (United States)

    Yang, Chuang; Zhang, Jing-Jing; Peng, Yun-Peng; Zhu, Yi; Yin, Ling-Di; Wei, Ji-Shu; Gao, Wen-Tao; Jiang, Kui-Rong; Miao, Yi

    2017-10-19

    Autophagy is a highly regulated biological process that mediates the degradation of intracellular components. It is required for tumor cell metabolism and homeostasis. Yin-Yang 1 (YY1) has been reported to be involved in autophagy in several carcinomas. However, its role in autophagy in pancreatic cancer, one of the deadliest human malignancies, is unknown. Here, we investigated the function of YY1 in pancreatic cancer cells autophagy and its mechanisms of action. The activity of cells undergoing autophagy was assessed using transmission electron microscopy, immunofluorescence, and Western blotting. A luciferase activity assay, real-time quantitative polymerase chain reaction (RT-qPCR), and chromatin immunoprecipitation (ChIP) were also used to identify putative downstream targets of YY1. YY1 was confirmed to regulate autophagy in pancreatic cancer cells. It was found to directly regulate the expression of miR-30a, a known modulator of autophagy-associated genes. Furthermore, overexpression of miR-30a attenuated the pro-autophagic effects of YY1. Cumulatively, our data suggest that miR-30a acts in a feedback loop to modulate the pro-autophagic activities of YY1. Thus, autophagy in pancreatic cancer cells may be regulated, in part, by a tightly coordinated YY1/miR-30a regulatory circuit. These findings provide a potential druggable target for the development of treatments for pancreatic cancer.

  3. Stress granules at the intersection of autophagy and ALS.

    Science.gov (United States)

    Monahan, Zachary; Shewmaker, Frank; Pandey, Udai Bhan

    2016-10-15

    Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease caused by loss of upper and lower motor neurons. The majority of ALS cases are classified as sporadic (80-90%), with the remaining considered familial based on patient history. The last decade has seen a surge in the identification of ALS-causing genes - including TARDBP (TDP-43), FUS, MATR3 (Matrin-3), C9ORF72 and several others - providing important insights into the molecular pathways involved in pathogenesis. Most of the protein products of ALS-linked genes fall into two functional categories: RNA-binding/homeostasis and protein-quality control (i.e. autophagy and proteasome). The RNA-binding proteins tend to be aggregation-prone with low-complexity domains similar to the prion-forming domains of yeast. Many also incorporate into stress granules (SGs), which are cytoplasmic ribonucleoprotein complexes that form in response to cellular stress. Mutant forms of TDP-43 and FUS perturb SG dynamics, lengthening their cytoplasmic persistence. Recent evidence suggests that SGs are regulated by the autophagy pathway, suggesting a unifying connection between many of the ALS-linked genes. Persistent SGs may give rise to intractable aggregates that disrupt neuronal homeostasis, thus failure to clear SGs by autophagic processes may promote ALS pathogenesis. This article is part of a Special Issue entitled SI:Autophagy. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Neem oil limonoids induces p53-independent apoptosis and autophagy.

    Science.gov (United States)

    Srivastava, Pragya; Yadav, Neelu; Lella, Ravi; Schneider, Andrea; Jones, Anthony; Marlowe, Timothy; Lovett, Gabrielle; O'Loughlin, Kieran; Minderman, Hans; Gogada, Raghu; Chandra, Dhyan

    2012-11-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.

  5. Neem oil limonoids induces p53-independent apoptosis and autophagy

    Science.gov (United States)

    Chandra, Dhyan

    2012-01-01

    Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells. PMID:22915764

  6. Nobel Prize Honors Autophagy Discovery.

    Science.gov (United States)

    2016-12-01

    Japanese cell biologist Yoshinori Ohsumi, PhD, was awarded this year's Nobel Prize in Physiology or Medicine for his discovery of autophagy. His groundbreaking studies in yeast cells illuminated how cells break down and recycle damaged material, a process that is critical to the survival of both normal cells and some cancer cells. ©2016 American Association for Cancer Research.

  7. Human Papilloma Virus and Autophagy

    Directory of Open Access Journals (Sweden)

    Domenico Mattoscio

    2018-06-01

    Full Text Available Human papilloma viruses (HPVs are a group of double-stranded DNA viruses known to be the primary cause of cervical cancer. In addition, evidence has now established their role in non-melanoma skin cancers, head and neck cancer (HNC, and the development of other anogenital malignancies. The prevalence of HPV-related HNC, in particular oropharyngeal cancers, is rapidly increasing, foreseeing that HPV-positive oropharyngeal cancers will outnumber uterine cervical cancers in the next 15–20 years. Therefore, despite the successful advent of vaccines originally licensed for cervical cancer prevention, HPV burden is still very high, and a better understanding of HPV biology is urgently needed. Autophagy is the physiological cellular route that accounts for removal, degradation, and recycling of damaged organelles, proteins, and lipids in lysosomal vacuoles. In addition to this scavenger function, autophagy plays a fundamental role during viral infections and cancers and is, therefore, frequently exploited by viruses to their own benefit. Recently, a link between HPV and autophagy has clearly emerged, leading to the conceivable development of novel anti-viral strategies aimed at restraining HPV infectivity. Here, recent findings on how oncogenic HPV16 usurp autophagy are described, highlighting similarities and differences with mechanisms adopted by other oncoviruses.

  8. Horning cell self-digestion: Autophagy wins the 2016 Nobel Prize in Physiology or Medicine

    Directory of Open Access Journals (Sweden)

    Po-Yuan Ke

    2017-02-01

    Full Text Available Autophagy is an evolutionarily conserved process by which eukaryotic cells eliminate intracellular components via the lysosomal degradation process. This cell self-digestion process was first discovered and morphologically characterized in the late 1950s and early 1960s. The genetic screen studies in baker's yeast in the 1990s further identified the essential genes functioning in the autophagic process. In the past two decades, the detailed molecular process involved in the completion of autophagy was delineated. Additionally, autophagy has been implied to function in many aspects of biological processes, including maintenance of organelle integrity, protein quality control, regulation of the stress response, and immunity. In addition to maintain cell homeostasis, autophagy has recently been shown to be modulated and to participate in the pathogenesis of human diseases, such as pathogen infections, neurodegenerative diseases, and tumor development. Overall, the breakthrough in autophagy research relies on the discovery of autophagy-related genes (ATGs using a genetic screening approach in Saccharomyces cerevisiae, which was established by Yoshinori Ohsumi. This year the Nobel Committee has awarded Yoshinori Ohsumi the Nobel Prize in Physiology or Medicine for his remarkable contribution to autophagy research.

  9. Induction of genomic instability and activation of autophagy in artificial human aneuploid cells

    Energy Technology Data Exchange (ETDEWEB)

    Ariyoshi, Kentaro [Hirosaki University, Institute of Radiation Emergency Medicine, 66-1 Hon-cho, Hirosaki 036-8564 (Japan); Miura, Tomisato; Kasai, Kosuke; Fujishima, Yohei [Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki 036-8564 (Japan); Oshimura, Mitsuo [Chromosome Engineering Research Center (CERC), Tottori University, Nishicho 86, Yonago, Tottori 683-8503 (Japan); Yoshida, Mitsuaki A., E-mail: ariyoshi@hirosaki-u.ac.jp [Hirosaki University, Institute of Radiation Emergency Medicine, 66-1 Hon-cho, Hirosaki 036-8564 (Japan)

    2016-08-15

    Highlights: • Clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. • Increased autophagy was observed in the artificially aneuploid clones. • Inhibition of autophagy resulted in increased genomic instability and DNA damage. • Intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones. - Abstract: Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

  10. Induction of genomic instability and activation of autophagy in artificial human aneuploid cells

    International Nuclear Information System (INIS)

    Ariyoshi, Kentaro; Miura, Tomisato; Kasai, Kosuke; Fujishima, Yohei; Oshimura, Mitsuo; Yoshida, Mitsuaki A.

    2016-01-01

    Highlights: • Clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. • Increased autophagy was observed in the artificially aneuploid clones. • Inhibition of autophagy resulted in increased genomic instability and DNA damage. • Intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones. - Abstract: Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

  11. Modulation of Autophagy by a Small Molecule Inverse Agonist of ERRα Is Neuroprotective

    Directory of Open Access Journals (Sweden)

    S. N. Suresh

    2018-04-01

    Full Text Available Mechanistic insights into aggrephagy, a selective basal autophagy process to clear misfolded protein aggregates, are lacking. Here, we report and describe the role of Estrogen Related Receptor α (ERRα, HUGO Gene Nomenclature ESRRA, new molecular player of aggrephagy, in keeping autophagy flux in check by inhibiting autophagosome formation. A screen for small molecule modulators for aggrephagy identified ERRα inverse agonist XCT 790, that cleared α-synuclein aggregates in an autophagy dependent, but mammalian target of rapamycin (MTOR independent manner. XCT 790 modulates autophagosome formation in an ERRα dependent manner as validated by siRNA mediated knockdown and over expression approaches. We show that, in a basal state, ERRα is localized on to the autophagosomes and upon autophagy induction by XCT 790, this localization is lost and is accompanied with an increase in autophagosome biogenesis. In a preclinical mouse model of Parkinson’s disease (PD, XCT 790 exerted neuroprotective effects in the dopaminergic neurons of nigra by inducing autophagy to clear toxic protein aggregates and, in addition, ameliorated motor co-ordination deficits. Using a chemical biology approach, we unrevealed the role of ERRα in regulating autophagy and can be therapeutic target for neurodegeneration.

  12. EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy

    Directory of Open Access Journals (Sweden)

    Mengtao Li

    2016-03-01

    Full Text Available Self-renewal and differentiation of neural stem cells is essential for embryonic neurogenesis, which is associated with cell autophagy. However, the mechanism by which autophagy regulates neurogenesis remains undefined. Here, we show that Eva1a/Tmem166, an autophagy-related gene, regulates neural stem cell self-renewal and differentiation. Eva1a depletion impaired the generation of newborn neurons, both in vivo and in vitro. Conversely, overexpression of EVA1A enhanced newborn neuron generation and maturation. Moreover, Eva1a depletion activated the PIK3CA-AKT axis, leading to the activation of the mammalian target of rapamycin and the subsequent inhibition of autophagy. Furthermore, addition of methylpyruvate to the culture during neural stem cell differentiation rescued the defective embryonic neurogenesis induced by Eva1a depletion, suggesting that energy availability is a significant factor in embryonic neurogenesis. Collectively, these data demonstrated that EVA1A regulates embryonic neurogenesis by modulating autophagy. Our results have potential implications for understanding the pathogenesis of neurodevelopmental disorders caused by autophagy dysregulation.

  13. Osteoporosis and autophagy: What is the relationship?

    Directory of Open Access Journals (Sweden)

    Rinaldo Florencio-Silva

    Full Text Available Summary Autophagy is a survival pathway wherein non-functional proteins and organelles are degraded in lysosomes for recycling and energy production. Therefore, autophagy is fundamental for the maintenance of cell viability, acting as a quality control process that prevents the accumulation of unnecessary structures and oxidative stress. Increasing evidence has shown that autophagy dysfunction is related to several pathologies including neurodegenerative diseases and cancer. Moreover, recent studies have shown that autophagy plays an important role for the maintenance of bone homeostasis. For instance, in vitro and animal and human studies indicate that autophagy dysfunction in bone cells is associated with the onset of bone diseases such as osteoporosis. This review had the purpose of discussing the issue to confirm whether a relationship between autophagy dysfunction and osteoporosis exits.

  14. Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.

    Directory of Open Access Journals (Sweden)

    Zhi-Hua Chen

    2008-10-01

    Full Text Available Chronic obstructive pulmonary disease (COPD is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear.Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7. Cigarette smoke extract (CSE is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1 and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1(-/- mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema.We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.

  15. GAMDB: a web resource to connect microRNAs with autophagy in gerontology.

    Science.gov (United States)

    Zhang, Lan; Xie, Tao; Tian, Mao; Li, Jingjing; Song, Sicheng; Ouyang, Liang; Liu, Bo; Cai, Haoyang

    2016-04-01

    MicroRNAs (miRNAs) are endogenous ~23 nucleotides (nt) RNAs, regulating gene expression by pairing to the mRNAs of protein-coding genes to direct their post-transcriptional repression. Both in normal and aberrant activities, miRNAs contribute to a recurring paradigm of cellular behaviors in pathological settings, especially in gerontology. Autophagy, a multi-step lysosomal degradation process with function to degrade long-lived proteins and damaged organelles, has significant impact on gerontology. Thus, elucidating how miRNAs participate in autophagy may enlarge the scope of miRNA in autophagy and facilitate researches in gerontology. Herein, based upon the published studies, predicted targets and gerontology-related diseases, we constructed a web resource named Gerontology-Autophagic-MicroRNA Database (GAMDB) (http://gamdb.liu-lab.com/index.php), which contained 836 autophagy-related miRNAs, 197 targeted genes/proteins and 56 aging-related diseases such as Parkinson' disease, Alzheimer's disease and Huntington's disease. We made use of large amounts of data to elucidate the intricate relationships between microRNA-regulated autophagic mechanisms and gerontology. This database will facilitate better understanding of autophagy regulation network in gerontology and thus promoting gerontology-related therapy in the future. © 2016 John Wiley & Sons Ltd.

  16. Autophagy in the control of food intake

    OpenAIRE

    Singh, Rajat

    2012-01-01

    The cellular nutrient sensing apparatus detects nutritional depletion and transmits this information to downstream effectors that generate energy from alternate sources. Autophagy is a crucial catabolic pathway that turns over redundant cytoplasmic components in lysosomes to provide energy to the starved cell. Recent studies have described a role for hypothalamic autophagy in the control of food intake and energy balance. Activated autophagy in hypothalamic neurons during starvation mobilized...

  17. Regulation of cardiomyocyte autophagy by calcium.

    Science.gov (United States)

    Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F G; Hill, Joseph A; Lavandero, Sergio

    2016-04-15

    Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. Copyright © 2016 the American Physiological Society.

  18. Emerging connections between RNA and autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lubas, Michal; Lund, Anders H

    2017-01-01

    in yeast, plants and animals, reviewing the molecular mechanisms and biological importance in normal physiology, stress and disease. In addition, we explore emerging evidence of core autophagy regulation mediated by RNA-binding proteins and noncoding RNAs, and point to gaps in our current knowledge......Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority...... of the connection between RNA and autophagy. Finally, we discuss the pathological implications of RNA-protein aggregation, primarily in the context of neurodegenerative disease....

  19. Involvement of Autophagy in Coronavirus Replication

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    Paul Britton

    2012-11-01

    Full Text Available Coronaviruses are single stranded, positive sense RNA viruses, which induce the rearrangement of cellular membranes upon infection of a host cell. This provides the virus with a platform for the assembly of viral replication complexes, improving efficiency of RNA synthesis. The membranes observed in coronavirus infected cells include double membrane vesicles. By nature of their double membrane, these vesicles resemble cellular autophagosomes, generated during the cellular autophagy pathway. In addition, coronavirus infection has been demonstrated to induce autophagy. Here we review current knowledge of coronavirus induced membrane rearrangements and the involvement of autophagy or autophagy protein microtubule associated protein 1B light chain 3 (LC3 in coronavirus replication.

  20. Approaches for Studying Autophagy in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Yanfang Chen

    2017-08-01

    Full Text Available Macroautophagy (hereafter referred to as autophagy is an intracellular degradative process, well conserved among eukaryotes. By engulfing cytoplasmic constituents into the autophagosome for degradation, this process is involved in the maintenance of cellular homeostasis. Autophagy induction triggers the formation of a cup-shaped double membrane structure, the phagophore, which progressively elongates and encloses materials to be removed. This double membrane vesicle, which is called an autophagosome, fuses with lysosome and forms the autolysosome. The inner membrane of the autophagosome, along with engulfed compounds, are degraded by lysosomal enzymes, which enables the recycling of carbohydrates, amino acids, nucleotides, and lipids. In response to various factors, autophagy can be induced for non-selective degradation of bulk cytoplasm. Autophagy is also able to selectively target cargoes and organelles such as mitochondria or peroxisome, functioning as a quality control system. The modification of autophagy flux is involved in developmental processes such as resistance to stress conditions, aging, cell death, and multiple pathologies. So, the use of animal models is essential for understanding these processes in the context of different cell types throughout the entire lifespan. For almost 15 years, the nematode Caenorhabditis elegans has emerged as a powerful model to analyze autophagy in physiological or pathological contexts. This review presents a rapid overview of physiological processes involving autophagy in Caenorhabditis elegans, the different assays used to monitor autophagy, their drawbacks, and specific tools for the analyses of selective autophagy.

  1. Autophagy contributes to 4-Amino-2-Trifluoromethyl-Phenyl Retinate-induced differentiation in human acute promyelocytic leukemia NB4 cells

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    Li, Yue; Li, Ge; Wang, Ke; Xie, Ya-Ya; Zhou, Ren-Peng; Meng, Yao; Ding, Ran; Ge, Jin-Fang; Chen, Fei-Hu, E-mail: cfhchina@sohu.com

    2017-03-15

    As a classic differentiation agent, all-trans retinoic acid (ATRA) has been widely used in treatment of acute promyelocytic leukemia (APL). However, clinical application of ATRA has limitations. Our previous studies suggested that 4-Amino-2-Trifluoromethyl-Phenyl Retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative designed and synthesized by our team, could induce differentiation of APL cells in vivo and in vitro. To explore the underlying mechanism of ATPR, the effect of ATPR on autophagy of APL cells was observed in the present study. The results showed that the differentiation effect of ATPR on APL cells was accompanied with autophagy induction and PML-RARα degradation via activating Notch1 signaling pathway. Moreover, inhibition of autophagy using 3-methyladenine (3-MA) or small interfering RNA (siRNA) that targets essential autophagy gene ATG5 abrogated the ATPR-induced cell differentiation. Furthermore, when pretreated with DAPT, a γ-secretase inhibitor, the Notch1 signaling pathway was blocked in APL cells, followed by the reduction of ATPR-induced autophagy and differentiation. Taken together, these results suggested that autophagy play an important role in ATPR-induced cell differentiation, which may provide a novel approach to cure APL patients. - Highlights: • ATPR induces autophagy in APL cell line NB4 cells. • Autophagy induction is essential for cell differentiation in NB4 cells. • Notch1 signaling is involved in ATPR-induced autophagy and differentiation in NB4 cells.

  2. Autophagy in muscle of glucose-infusion hyperglycemia rats and streptozotocin-induced hyperglycemia rats via selective activation of m-TOR or FoxO3.

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    Pengfei Lv

    Full Text Available Autophagy is a conserved process in eukaryotes required for metabolism and is involved in diverse diseases. To investigate autophagy in skeletal muscle under hyperglycemia status, we established two hyperglycemia-rat models that differ in their circulating insulin levels, by glucose infusion and singe high-dose streptozotocin injection. We then detected expression of autophagy related genes with real-time PCR and western blot. We found that under hyperglycemia status induced by glucose-infusion, autophagy was inhibited in rat skeletal muscle, whereas under streptozotocin-induced hyperglycemia status autophagy was enhanced. Meanwhile, hyperglycemic gastrocnemius muscle was more prone to autophagy than soleus muscle. Furthermore, inhibition of autophagy in skeletal muscle in glucose-infusion hyperglycemia rats was mediated by the m-TOR pathway while m-TOR and FoxO3 both contributed to enhancement of autophagy in gastrocnemius muscle in streptozotocin-induced hyperglycemia rats. These data shows that insulin plays a relatively more important role than hyperglycemia in regulating autophagy in hyperglycemia rat muscle through selectively activating the m-TOR or FoxO3 pathway in a fiber-selective manner.

  3. Cisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells

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    Lin JF

    2017-05-01

    Full Text Available Ji-Fan Lin,1 Yi-Chia Lin,2 Te-Fu Tsai,2,3 Hung-En Chen,2 Kuang-Yu Chou,2,3 Thomas I-Sheng Hwang2–4 1Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, 2Division of Urology, School of Medicine, Fu-Jen Catholic University, New Taipei, 3Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, 4Department of Urology, Taipei Medical University, Taipei, Taiwan Purpose: Cisplatin-based chemotherapy is the first line treatment for several cancers including bladder cancer (BC. Autophagy induction has been implied to contribute to cisplatin resistance in ovarian cancer; and a high basal level of autophagy has been demonstrated in human bladder tumors. Therefore, it is reasonable to speculate that autophagy may account for the failure of cisplatin single treatment in BC. This study investigated whether cisplatin induces autophagy and the mechanism involved using human BC cell lines.Materials and methods: Human BC cells (5637 and T24 were used in this study. Cell viability was detected using water soluble tetrazolium-8 reagents. Autophagy induction was detected by monitoring the levels of light chain 3 (LC3-II and p62 by Western blot, LC3-positive puncta formation by immunofluorescence, and direct observation of the autophagolysosome (AL formation by transmission electron microscopy. Inhibitors including bafilomycin A1 (Baf A1, chloroquine (CQ, and shRNA-based lentivirus against autophagy-related genes (ATG7 and ATG12 were utilized. Apoptosis level was detected by caspase 3/7 activity and DNA fragmentation.Results: Cisplatin decreased cell viability and induced apoptosis of 5637 and T24 cells in a dose- and time-dependent manner. The increased LC3-II accumulation, p62 clearance, the number of LC3-positive puncta, and ALs in cisplatin-treated cells suggested that cisplatin indeed induces autophagy. Inhibition of cisplatin-induced autophagy using Baf A1, CQ, or ATG7/ATG12 shRNAs significantly enhanced cytotoxicity of

  4. Increased autophagy and apoptosis contribute to muscle atrophy in a myotonic dystrophy type 1 Drosophila model

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    Ariadna Bargiela

    2015-07-01

    Full Text Available Muscle mass wasting is one of the most debilitating symptoms of myotonic dystrophy type 1 (DM1 disease, ultimately leading to immobility, respiratory defects, dysarthria, dysphagia and death in advanced stages of the disease. In order to study the molecular mechanisms leading to the degenerative loss of adult muscle tissue in DM1, we generated an inducible Drosophila model of expanded CTG trinucleotide repeat toxicity that resembles an adult-onset form of the disease. Heat-shock induced expression of 480 CUG repeats in adult flies resulted in a reduction in the area of the indirect flight muscles. In these model flies, reduction of muscle area was concomitant with increased apoptosis and autophagy. Inhibition of apoptosis or autophagy mediated by the overexpression of DIAP1, mTOR (also known as Tor or muscleblind, or by RNA interference (RNAi-mediated silencing of autophagy regulatory genes, achieved a rescue of the muscle-loss phenotype. In fact, mTOR overexpression rescued muscle size to a size comparable to that in control flies. These results were validated in skeletal muscle biopsies from DM1 patients in which we found downregulated autophagy and apoptosis repressor genes, and also in DM1 myoblasts where we found increased autophagy. These findings provide new insights into the signaling pathways involved in DM1 disease pathogenesis.

  5. Modulation of Apoptosis Pathways by Oxidative Stress and Autophagy in β Cells

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    Maorong Wang

    2012-01-01

    Full Text Available Human islets isolated for transplantation are exposed to multiple stresses including oxidative stress and hypoxia resulting in significant loss of functional β cell mass. In this study we examined the modulation of apoptosis pathway genes in islets exposed to hydrogen peroxide, peroxynitrite, hypoxia, and cytokines. We observed parallel induction of pro- and antiapoptotic pathways and identified several novel genes including BFAR, CARD8, BNIP3, and CIDE-A. As BNIP3 is an inducer of autophagy, we examined this pathway in MIN6 cells, a mouse beta cell line and in human islets. Culture of MIN6 cells under low serum conditions increased the levels of several proteins in autophagy pathway, including ATG4, Beclin 1, LAMP-2, and UVRAG. Amino acid deprivation led to induction of autophagy in human islets. Preconditioning of islets with inducers of autophagy protected them from hypoxia-induced apoptosis. However, induction of autophagy during hypoxia exacerbated apoptotic cell death. ER stress led to induction of autophagy and apoptosis in β cells. Overexpression of MnSOD, an enzyme that scavenges free radicals, resulted in protection of MIN6 cells from cytokine-induced apoptosis. Ceramide, a mediator of cytokine-induced injury, reduced the active phosphorylated form of Akt and downregulated the promoter activity of the antiapoptotic gene bcl-2. Furthermore, cytokine-stimulated JNK pathway downregulated the bcl-2 promoter activity which was reversed by preincubation with SP600125, a JNK inhibitor. Our findings suggest that β cell apoptosis by multiple stresses in islets isolated for transplantation is the result of orchestrated gene expression in apoptosis pathway.

  6. Characterization of the autophagy marker protein Atg8 reveals atypical features of autophagy in Plasmodium falciparum.

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    Rahul Navale

    Full Text Available Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8 employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64 and aspartic (pepstatin protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine, indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in

  7. Opposite Effects of Two Human ATG10 Isoforms on Replication of a HCV Sub-genomic Replicon Are Mediated via Regulating Autophagy Flux in Zebrafish

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    Yu-Chen Li

    2018-04-01

    Full Text Available Autophagy is a host mechanism for cellular homeostatic control. Intracellular stresses are symptoms of, and responses to, dysregulation of the physiological environment of the cell. Alternative gene transcription splicing is a mechanism potentially used by a host to respond to physiological or pathological challenges. Here, we aimed to confirm opposite effects of two isoforms of the human autophagy-related protein ATG10 on an HCV subgenomic replicon in zebrafish. A liver-specific HCV subreplicon model was established and exhibited several changes in gene expression typically induced by HCV infection, including overexpression of several HCV-dependent genes (argsyn, leugpcr, rasgbd, and scaf-2, as well as overexpression of several ER stress related genes (atf4, chop, atf6, and bip. Autophagy flux was blocked in the HCV model. Our results indicated that the replication of the HCV subreplicon was suppressed via a decrease in autophagosome formation caused by the autophagy inhibitor 3MA, but enhanced via dysfunction in the lysosomal degradation caused by another autophagy inhibitor CQ. Human ATG10, a canonical isoform in autophagy, facilitated the amplification of the HCV-subgenomic replicon via promoting autophagosome formation. ATG10S, a non-canonical short isoform of the ATG10 protein, promoted autophagy flux, leading to lysosomal degradation of the HCV-subgenomic replicon. Human ATG10S may therefore inhibit HCV replication, and may be an appropriate target for future antiviral drug screening.

  8. Spermidine: a novel autophagy inducer and longevity elixir.

    Science.gov (United States)

    Madeo, Frank; Eisenberg, Tobias; Büttner, Sabrina; Ruckenstuhl, Christoph; Kroemer, Guido

    2010-01-01

    Spermidine is a ubiquitous polycation that is synthesized from putrescine and serves as a precursor of spermine. Putrescine, spermidine and spermine all are polyamines that participate in multiple known and unknown biological processes. Exogenous supply of spermidine prolongs the life span of several model organisms including yeast (Saccharomyces cerevisiae), nematodes (Caenorhabditis elegans) and flies (Drosophila melanogaster) and significantly reduces age-related oxidative protein damage in mice, indicating that this agent may act as a universal anti-aging drug. Spermidine induces autophagy in cultured yeast and mammalian cells, as well as in nematodes and flies. Genetic inactivation of genes essential for autophagy abolishes the life span-prolonging effect of spermidine in yeast, nematodes and flies. These findings complement expanding evidence that autophagy mediates cytoprotection against a variety of noxious agents and can confer longevity when induced at the whole-organism level. We hypothesize that increased autophagic turnover of cytoplasmic organelles or long-lived proteins is involved in most if not all life span-prolonging therapies.

  9. Midgut morphological changes and autophagy during metamorphosis in sand flies.

    Science.gov (United States)

    Malta, Juliana; Heerman, Matthew; Weng, Ju Lin; Fernandes, Kenner M; Martins, Gustavo Ferreira; Ramalho-Ortigão, Marcelo

    2017-06-01

    During metamorphosis, holometabolous insects undergo significant remodeling of their midgut and become able to cope with changes in dietary requirements between larval and adult stages. At this stage, insects must be able to manage and recycle available food resources in order to develop fully into adults, especially when no nutrients are acquired from the environment. Autophagy has been previously suggested to play a crucial role during metamorphosis of the mosquito. Here, we investigate the overall morphological changes of the midgut of the sand fly during metamorphosis and assess the expression profiles of the autophagy-related genes ATG1, ATG6, and ATG8, which are associated with various steps of the autophagic process. Morphological changes in the midgut start during the fourth larval instar, with epithelial degeneration followed by remodeling via the differentiation of regenerative cells in pre-pupal and pupal stages. The changes in the midgut epithelium are paired with the up-regulation of ATG1, ATG6 and ATG8 during the larva-adult transition. Vein, a putative epidermal growth factor involved in regulating epithelial midgut regeneration, is also up-regulated. Autophagy has further been confirmed in sand flies via the presence of autophagosomes residing within the cytoplasmic compartment of the pupal stages. An understanding of the underlying mechanisms of this process should aid the future management of this neglected tropical vector.

  10. Regulation of autophagy by cytoplasmic p53.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, M Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

    2008-06-01

    Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

  11. Autophagy: A Sweet Process in Diabetes

    NARCIS (Netherlands)

    Meijer, Alfred J.; Codogno, Patrice

    2008-01-01

    Autophagy is inhibited by the insulin-amino acid-mTOR signaling pathway. Two papers in this issue of Cell Metabolism (Ebato et al., 2008; Jung et al., 2008) provide evidence that basal autophagy is necessary to maintain the architecture and function of pancreatic beta cells and that its induction in

  12. Autophagy, inflammation and innate immunity in inflammatory myopathies.

    Directory of Open Access Journals (Sweden)

    Cristina Cappelletti

    Full Text Available Autophagy has a large range of physiological functions and its dysregulation contributes to several human disorders, including autoinflammatory/autoimmune diseases such as inflammatory myopathies (IIMs. In order to better understand the pathogenetic mechanisms of these muscular disorders, we sought to define the role of autophagic processes and their relation with the innate immune system in the three main subtypes of IIM, specifically sporadic inclusion body myositis (sIBM, polymyositis (PM, dermatomyositis (DM and juvenile dermatomyositis (JDM. We found that although the mRNA transcript levels of the autophagy-related genes BECN1, ATG5 and FBXO32 were similar in IIM and controls, autophagy activation in all IIM subgroups was suggested by immunoblotting results and confirmed by immunofluorescence. TLR4 and TLR3, two potent inducers of autophagy, were highly increased in IIM, with TLR4 transcripts significantly more expressed in PM and DM than in JDM, sIBM and controls, and TLR3 transcripts highly up-regulated in all IIM subgroups compared to controls. Co-localization between autophagic marker, LC3, and TLR4 and TLR3 was observed not only in sIBM but also in PM, DM and JDM muscle tissues. Furthermore, a highly association with the autophagic processes was observed in all IIM subgroups also for some TLR4 ligands, endogenous and bacterial HSP60, other than the high-mobility group box 1 (HMGB1. These findings indicate that autophagic processes are active not only in sIBM but also in PM, DM and JDM, probably in response to an exogenous or endogenous 'danger signal'. However, autophagic activation and regulation, and also interaction with the innate immune system, differ in each type of IIM. Better understanding of these differences may lead to new therapies for the different IIM types.

  13. Autophagy Proteins in Phagocyte Endocytosis and Exocytosis

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    Christian Münz

    2017-09-01

    Full Text Available Autophagy was initially described as a catabolic pathway that recycles nutrients of cytoplasmic constituents after lysosomal degradation during starvation. Since the immune system monitors products of lysosomal degradation via major histocompatibility complex (MHC class II restricted antigen presentation, autophagy was found to process intracellular antigens for display on MHC class II molecules. In recent years, however, it has become apparent that the molecular machinery of autophagy serves phagocytes in many more membrane trafficking pathways, thereby regulating immunity to infectious disease agents. In this minireview, we will summarize the recent evidence that autophagy proteins regulate phagocyte endocytosis and exocytosis for myeloid cell activation, pathogen replication, and MHC class I and II restricted antigen presentation. Selective stimulation and inhibition of the respective functional modules of the autophagy machinery might constitute valid therapeutic options in the discussed disease settings.

  14. PICALM modulates autophagy activity and tau accumulation

    Science.gov (United States)

    Moreau, Kevin; Fleming, Angeleen; Imarisio, Sara; Lopez Ramirez, Ana; Mercer, Jacob L.; Jimenez-Sanchez, Maria; Bento, Carla F.; Puri, Claudia; Zavodszky, Eszter; Siddiqi, Farah; Lavau, Catherine P.; Betton, Maureen; O’Kane, Cahir J.; Wechsler, Daniel S.; Rubinsztein, David C.

    2014-01-01

    Genome-wide association studies have identified several loci associated with Alzheimer’s disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover. PMID:25241929

  15. Autophagy in endometriosis: Friend or foe?

    Science.gov (United States)

    Zhan, Lei; Li, Jun; Wei, Bing

    2018-01-01

    Endometriosis is a chronic, estrogen-dependent disease and characterized by the implantation of endometrial glands and stroma deep and haphazardly into the outside the uterine cavity. It affects an estimated 10% of the female population of reproductive age and results in obvious reduction in health-related quality of life. Unfortunately, there is no a consistent theory for the etiology of endometriosis. Furthermore, the endometriosis is hard to diagnose in early stage and the treatment methods are limited. Importantly, emerging evidence has investigated that there is a close relationship between endometriosis and autophagy. However, autophagy is a friend or foe in endometriosis is puzzling, the precise mechanism underlying autophagy in endometriosis has not been fully elucidated yet. Here, we provide an integrated view on the acquired findings of the connections between endometriosis and autophagy. We also discuss which may contribute to the abnormal level of autophagy in endometriosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

    International Nuclear Information System (INIS)

    Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo; Koya, Daisuke

    2012-01-01

    Highlights: ► SIRT1 inactivation decreases autophagy in THP-1 cell. ► Inhibition of autophagy induces inflammation. ► SIRT1 inactivation induces inflammation through NF-κB activation. ► The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-κB activation. ► SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD + -dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 through nuclear factor (NF)-κB signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-κB activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-κB activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and is implicated in decreased 5′-AMP activated kinase (AMPK) activation, leading to the impairment of autophagy. The mTOR inhibitor, rapamycin, abolishes

  17. SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

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    Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan); Koya, Daisuke, E-mail: koya0516@kanazawa-med.ac.jp [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer SIRT1 inactivation decreases autophagy in THP-1 cell. Black-Right-Pointing-Pointer Inhibition of autophagy induces inflammation. Black-Right-Pointing-Pointer SIRT1 inactivation induces inflammation through NF-{kappa}B activation. Black-Right-Pointing-Pointer The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-{kappa}B activation. Black-Right-Pointing-Pointer SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD{sup +}-dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-6 through nuclear factor (NF)-{kappa}B signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-{kappa}B activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-{kappa}B activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and

  18. Hypercholesterolemia downregulates autophagy in the rat heart.

    Science.gov (United States)

    Giricz, Zoltán; Koncsos, Gábor; Rajtík, Tomáš; Varga, Zoltán V; Baranyai, Tamás; Csonka, Csaba; Szobi, Adrián; Adameová, Adriana; Gottlieb, Roberta A; Ferdinandy, Péter

    2017-03-23

    We have previously shown that efficiency of ischemic conditioning is diminished in hypercholesterolemia and that autophagy is necessary for cardioprotection. However, it is unknown whether isolated hypercholesterolemia disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways. Therefore, we investigated whether isolated hypercholesterolemia modulates cardiac autophagy-related pathways or programmed cell death mechanisms such as apoptosis and necroptosis in rat heart. Male Wistar rats were fed either normal chow (NORM; n = 9) or with 2% cholesterol and 0.25% cholic acid-enriched diet (CHOL; n = 9) for 12 weeks. CHOL rats exhibited a 41% increase in plasma total cholesterol level over that of NORM rats (4.09 mmol/L vs. 2.89 mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out. Left ventricles were snap-frozen for determination of markers of autophagy, mTOR pathway, apoptosis, and necroptosis by Western blot. Isolated hypercholesterolemia was associated with a significant reduction in expression of cardiac autophagy markers such as LC3-II, Beclin-1, Rubicon and RAB7 as compared to controls. Phosphorylation of ribosomal S6, a surrogate marker for mTOR activity, was increased in CHOL samples. Cleaved caspase-3, a marker of apoptosis, increased in CHOL hearts, while no difference in the expression of necroptotic marker RIP1, RIP3 and MLKL was detected between treatments. This is the first comprehensive analysis of autophagy and programmed cell death pathways of apoptosis and necroptosis in hearts of hypercholesterolemic rats. Our data show that isolated hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an activated mTOR pathway. Reduced autophagy was accompanied by increased apoptosis, while cardiac necroptosis was not modulated by isolated hypercholesterolemia. Decreased basal autophagy and elevated apoptosis may be responsible for the

  19. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury.

    Science.gov (United States)

    Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury.

  20. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

    Science.gov (United States)

    Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury. PMID:26103523

  1. NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells

    International Nuclear Information System (INIS)

    Sobhakumari, Arya; Schickling, Brandon M.; Love-Homan, Laurie; Raeburn, Ayanna; Fletcher, Elise V.M.; Case, Adam J.; Domann, Frederick E.; Miller, Francis J.

    2013-01-01

    Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect. - Highlights: • Erlotinib increased LC3B-II and autophagosome formation in HNSCC cells. • Inhibition of autophagy sensitized HNSCC cells to erlotinib. • Erlotinib increased NOX4 promoter and 3′UTR luciferase activity. • Manipulating NOX4 decreases or increases autophagy

  2. NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Sobhakumari, Arya [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Schickling, Brandon M. [Department of Internal Medicine, The University of Iowa, Iowa City, IA (United States); Love-Homan, Laurie; Raeburn, Ayanna [Department of Pathology, The University of Iowa, Iowa City, IA (United States); Fletcher, Elise V.M. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Case, Adam J. [Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Domann, Frederick E. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Pathology, The University of Iowa, Iowa City, IA (United States); Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics (UIHC), Iowa City, IA (United States); Miller, Francis J. [Department of Internal Medicine, The University of Iowa, Iowa City, IA (United States); Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics (UIHC), Iowa City, IA (United States); and others

    2013-11-01

    Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect. - Highlights: • Erlotinib increased LC3B-II and autophagosome formation in HNSCC cells. • Inhibition of autophagy sensitized HNSCC cells to erlotinib. • Erlotinib increased NOX4 promoter and 3′UTR luciferase activity. • Manipulating NOX4 decreases or increases autophagy.

  3. Non-canonical autophagy: an exception or an underestimated form of autophagy?

    Science.gov (United States)

    Scarlatti, Francesca; Maffei, Roberta; Beau, Isabelle; Ghidoni, Riccardo; Codogno, Patrice

    2008-11-01

    Macroautophagy (hereafter called autophagy) is a dynamic and evolutionarily conserved process used to sequester and degrade cytoplasm and entire organelles in a sequestering vesicle with a double membrane, known as the autophagosome, which ultimately fuses with a lysosome to degrade its autophagic cargo. Recently, we have unraveled two distinct forms of autophagy in cancer cells, which we term canonical and non-canonical autophagy. In contrast to classical or canonical autophagy, non-canonical autophagy is a process that does not require the entire set of autophagy-related (Atg) proteins in particular Beclin 1, to form the autophagosome. Non-canonical autophagy is therefore not blocked by the knockdown of Beclin 1 or of its binding partner hVps34. Moreover overexpression of Bcl-2, which is known to block canonical starvation-induced autophagy by binding to Beclin 1, is unable to reverse the non-canonical autophagy triggered by the polyphenol resveratrol in the breast cancer MCF-7 cell line. In MCF-7 cells, at least, non-canonical autophagy is involved in the caspase-independent cell death induced by resveratrol.

  4. Interplay between cell cycle and autophagy induced by boswellic acid analog

    Science.gov (United States)

    Pathania, Anup S.; Guru, Santosh K.; Kumar, Suresh; Kumar, Ashok; Ahmad, Masroor; Bhushan, Shashi; Sharma, Parduman R.; Mahajan, Priya; Shah, Bhahwal A.; Sharma, Simmi; Nargotra, Amit; Vishwakarma, Ram; Korkaya, Hasan; Malik, Fayaz

    2016-01-01

    In this study, we investigated the role of autophagy induced by boswellic acid analog BA145 on cell cycle progression in pancreatic cancer cells. BA145 induced robust autophagy in pancreatic cancer cell line PANC-1 and exhibited cell proliferation inhibition by inducing cells to undergo G2/M arrest. Inhibition of G2/M progression was associated with decreased expression of cyclin A, cyclin B, cyclin E, cdc2, cdc25c and CDK-1. Pre-treatment of cells with autophagy inhibitors or silencing the expression of key autophagy genes abrogated BA145 induced G2/M arrest and downregulation of cell cycle regulatory proteins. It was further observed that BA145 induced autophagy by targeting mTOR kinase (IC50 1 μM), leading to reduced expression of p-mTOR, p-p70S6K (T389), p-4EBP (T37/46) and p-S6 (S240/244). Notably, inhibition of mTOR signalling by BA145 was followed by attendant activation of AKT and its membrane translocation. Inhibition of Akt through pharmacological inhibitors or siRNAs enhanced BA145 mediated autophagy, G2/M arrest and reduced expression of G2/M regulators. Further studies revealed that BA145 arbitrated inhibition of mTOR led to the activation of Akt through IGFR/PI3k/Akt feedback loop. Intervention in IGFR/PI3k/Akt loop further depreciated Akt phosphorylation and its membrane translocation that culminates in augmented autophagy with concomitant G2/M arrest and cell death. PMID:27680387

  5. EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy

    NARCIS (Netherlands)

    Byrne, Susan; Jansen, Lara; U-King-Im, Jean-Marie; Siddiqui, Ata; Lidov, Hart G. W.; Bodi, Istvan; Smith, Luke; Mein, Rachael; Cullup, Thomas; Dionisi-Vici, Carlo; Al-Gazali, Lihadh; Al-Owain, Mohammed; Bruwer, Zandre; Al Thihli, Khalid; El-Garhy, Rana; Flanigan, Kevin M.; Manickam, Kandamurugu; Zmuda, Erik; Banks, Wesley; Gershoni-Baruch, Ruth; Mandel, Hanna; Dagan, Efrat; Raas-Rothschild, Annick; Barash, Hila; Filloux, Francis; Creel, Donnell; Harris, Michael; Hamosh, Ada; Kölker, Stefan; Ebrahimi-Fakhari, Darius; Hoffmann, Georg F.; Manchester, David; Boyer, Philip J.; Manzur, Adnan Y.; Lourenco, Charles Marques; Pilz, Daniela T.; Kamath, Arveen; Prabhakar, Prab; Rao, Vamshi K.; Rogers, R. Curtis; Ryan, Monique M.; Brown, Natasha J.; McLean, Catriona A.; Said, Edith; Schara, Ulrike; Stein, Anja; Sewry, Caroline; Travan, Laura; Wijburg, Frits A.; Zenker, Martin; Mohammed, Shehla; Fanto, Manolis; Gautel, Mathias; Jungbluth, Heinz

    2016-01-01

    Vici syndrome is a progressive neurodevelopmental multisystem disorder due to recessive mutations in the key autophagy gene EPG5. We report genetic, clinical, neuroradiological, and neuropathological features of 50 children from 30 families, as well as the neuronal phenotype of EPG5 knock-down in

  6. Autophagy and bacterial clearance: a not so clear picture

    OpenAIRE

    Mostowy, Serge

    2012-01-01

    Autophagy, an intracellular degradation process highly conserved from yeast to humans, is viewed as an important defence mechanism to clear intracellular bacteria. However, recent work has shown that autophagy may have different roles during different bacterial infections that restrict bacterial replication (antibacterial autophagy), act in cell autonomous signalling (non-bacterial autophagy) or support bacterial replication (pro-bacterial autophagy). This review will focus on newfound intera...

  7. Enhanced Production of Bovine Chymosin by Autophagy Deficiency in the Filamentous Fungus Aspergillus oryzae

    Science.gov (United States)

    Maruyama, Jun-ichi; Kitamoto, Katsuhiko

    2013-01-01

    Aspergillus oryzae has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, A. oryzae often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production. In the present study, we examined the production of heterologous protein in several autophagy (Aoatg) gene disruptants of A. oryzae. We transformed A. oryzae gene disruptants of Aoatg1, Aoatg13, Aoatg4, Aoatg8, or Aoatg15, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the Aoatg gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed Aoatg gene-conditional expression strains in which the promoter region of the Aoatg gene was replaced with the thiamine-controllable thiA promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the Aoatg gene disruptants. Consequently, we succeeded in the construction of A. oryzae strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing heterologous protein production in A. oryzae. PMID:23658635

  8. Autophagy and Neurodegeneration: Insights from a Cultured Cell Model of ALS

    Directory of Open Access Journals (Sweden)

    Francesca Navone

    2015-08-01

    Full Text Available Autophagy plays a major role in the elimination of cellular waste components, the renewal of intracellular proteins and the prevention of the build-up of redundant or defective material. It is fundamental for the maintenance of homeostasis and especially important in post-mitotic neuronal cells, which, without competent autophagy, accumulate protein aggregates and degenerate. Many neurodegenerative diseases are associated with defective autophagy; however, whether altered protein turnover or accumulation of misfolded, aggregate-prone proteins is the primary insult in neurodegeneration has long been a matter of debate. Amyotrophic lateral sclerosis (ALS is a fatal disease characterized by selective degeneration of motor neurons. Most of the ALS cases occur in sporadic forms (SALS, while 10%–15% of the cases have a positive familial history (FALS. The accumulation in the cell of misfolded/abnormal proteins is a hallmark of both SALS and FALS, and altered protein degradation due to autophagy dysregulation has been proposed to contribute to ALS pathogenesis. In this review, we focus on the main molecular features of autophagy to provide a framework for discussion of our recent findings about the role in disease pathogenesis of the ALS-linked form of the VAPB gene product, a mutant protein that drives the generation of unusual cytoplasmic inclusions.

  9. Beclin1-induced autophagy abrogates radioresistance of lung cancer cells by suppressing osteopontin

    International Nuclear Information System (INIS)

    Chang, Seung-Hee; Minai-Tehrani, Arash; Shin, Ji-Young

    2012-01-01

    Osteopontin (OPN) serves as an indicator of resistance to radiotherapy. However, the role of OPN in the development of acquired radioresistance in human lung cancer cells has not yet been fully elucidated. Therefore, the potential importance of OPN as a marker of lung cancer with a potential significant role in the development of radioresistance against repeated radiotherapy has prompted us to define the pathways by which OPN regulates lung cancer cell growth. In addition, autophagy has been reported to play a key role in the radiosensitization of cancer cells. Here, we report that increased OPN expression through induction of nuclear p53 following irradiation was inhibited by exogenous beclin-1 (BECN1). Our results clearly show that BECN1 gene expression led to induction of autophagy and inhibition of cancer cell growth and angiogenesis. Our results suggest that the induction of autophagy abrogated the radioresistance of the cancer cells. Interestingly, we showed that knockdown of OPN by lentivirus-mediated shRNA induced the autophagy of human lung cancer cell. Taken together, these results suggest that OPN and BECN1 can be molecular targets for overcoming radioresistance by controlling autophagy. (author)

  10. Autophagy response: manipulating the mTOR-controlled machinery by amino acids and pathogens.

    Science.gov (United States)

    Fader, Claudio Marcelo; Aguilera, Milton Osmar; Colombo, María Isabel

    2015-10-01

    Macroautophagy is a self-degradative process that normally maintains cellular homeostasis via a lysosomal pathway. It is induced by different stress signals, including nutrients and growth factors' restriction as well as pathogen invasions. These stimuli are modulated by the serine/threonine protein kinase mammalian target of rapamycin (mTOR) which control not only autophagy but also protein translation and gene expression. This review focuses on the important role of mTOR as a master regulator of cell growth and the autophagy pathway. Here, we have discussed the role of intracellular amino acid availability and intracellular pH in the redistribution of autophagic structures, which may contribute to mammalian target of rapamycin complex 1 (mTORC1) activity regulation. We have also discussed that mTORC1 complex and components of the autophagy machinery are localized at the lysosomal surface, representing a fascinating mechanism to control the metabolism, cellular clearance and also to restrain invading intracellular pathogens.

  11. Parkinson disease: a role for autophagy?

    Science.gov (United States)

    Yang, Qian; Mao, Zixu

    2010-08-01

    Autophagy is a term used to describe the process by which lysosomes degrade intracellular components. Known originally as an adaptive response to nutrient deprivation, autophagy has now been recognized to play important roles in several human disorders including neurodegenerative diseases. Experimental results from genetic, cellular, and toxicological studies indicate that many of the etiological factors associated with Parkinson disease (PD) can perturb the autophagic process in various model systems. Thus, the emerging data support the view that dysregulation of autophagy may play a critical role in the pathogenic process of PD.

  12. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  13. The dual role of autophagy under hypoxia-involvement of interaction between autophagy and apoptosis.

    Science.gov (United States)

    Li, Mengmeng; Tan, Jin; Miao, Yuyang; Lei, Ping; Zhang, Qiang

    2015-06-01

    Hypoxia is one of severe cellular stress and it is well known to be associated with a worse outcome since a lack of oxygen accelerates the induction of apoptosis. Autophagy, an important and evolutionarily conserved mechanism for maintaining cellular homeostasis, is closely related to the apoptosis caused by hypoxia. Generally autophagy blocks the induction of apoptosis and inhibits the activation of apoptosis-associated caspase which could reduce cellular injury. However, in special cases, autophagy or autophagy-relevant proteins may help to induce apoptosis, which could aggravate cell damage under hypoxia condition. In addition, the activation of apoptosis-related proteins-caspase can also degrade autophagy-related proteins, such as Atg3, Atg4, Beclin1 protein, inhibiting autophagy. Although the relationship between autophagy and apoptosis has been known for rather complex for more than a decade, the underlying regulatory mechanisms have not been clearly understood. This short review discusses and summarizes the dual role of autophagy and the interaction and molecular regulatory mechanisms between autophagy and apoptosis under hypoxia.

  14. Oxidative stress-induced autophagy: Role in pulmonary toxicity

    International Nuclear Information System (INIS)

    Malaviya, Rama; Laskin, Jeffrey D.; Laskin, Debra L.

    2014-01-01

    Autophagy is an evolutionarily conserved catabolic process important in regulating the turnover of essential proteins and in elimination of damaged organelles and protein aggregates. Autophagy is observed in the lung in response to oxidative stress generated as a consequence of exposure to environmental toxicants. Whether autophagy plays role in promoting cell survival or cytotoxicity is unclear. In this article recent findings on oxidative stress-induced autophagy in the lung are reviewed; potential mechanisms initiating autophagy are also discussed. A better understanding of autophagy and its role in pulmonary toxicity may lead to the development of new strategies to treat lung injury associated with oxidative stress. - Highlights: • Exposure to pulmonary toxicants is associated with oxidative stress. • Oxidative stress is known to induce autophagy. • Autophagy is upregulated in the lung following exposure to pulmonary toxicants. • Autophagy may be protective or pathogenic

  15. Oxidative stress-induced autophagy: Role in pulmonary toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Malaviya, Rama [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States)

    2014-03-01

    Autophagy is an evolutionarily conserved catabolic process important in regulating the turnover of essential proteins and in elimination of damaged organelles and protein aggregates. Autophagy is observed in the lung in response to oxidative stress generated as a consequence of exposure to environmental toxicants. Whether autophagy plays role in promoting cell survival or cytotoxicity is unclear. In this article recent findings on oxidative stress-induced autophagy in the lung are reviewed; potential mechanisms initiating autophagy are also discussed. A better understanding of autophagy and its role in pulmonary toxicity may lead to the development of new strategies to treat lung injury associated with oxidative stress. - Highlights: • Exposure to pulmonary toxicants is associated with oxidative stress. • Oxidative stress is known to induce autophagy. • Autophagy is upregulated in the lung following exposure to pulmonary toxicants. • Autophagy may be protective or pathogenic.

  16. GLP-1 Treatment Improves Diabetic Retinopathy by Alleviating Autophagy through GLP-1R-ERK1/2-HDAC6 Signaling Pathway.

    Science.gov (United States)

    Cai, Xiangsheng; Li, Jingjing; Wang, Mingzhu; She, Miaoqin; Tang, Yongming; Li, Jinlong; Li, Hongwei; Hui, Hongxiang

    2017-01-01

    Objective: Apoptosis and autophagy of retinal cells, which may be induced by oxidative stress, are tightly associated with the pathogenesis of diabetic retinopathy (DR). The autophagy induced by oxidative stress is considered as excessively stimulated autophagy, which accelerates the progression of DR. This study aims to investigate the protective effect of GLP-1 treatment on alleviating apoptosis and autophagy of retinal cells in type 2 diabetic rats and reveals its possible mechanism. Methods: Type 2 diabetic rats were induced by fed with high sugar, high fat diet and followed with streptozotocin injection. GLP-1 was applied to treat the diabetic rats for one week after the onset of diabetes. The expressions of oxidative stress-related enzymes, retinal GLP-1R, mitochondria-dependent apoptosis- related genes, autophagy markers, and autophagy-associated pathway genes were studied by Western blotting or immunohistochemistry analysis. Results: GLP-1treatment reduced the levels of NOX3 and SOD2 in DR. The expression of BCL2 was increased, while the levels of caspase3 and LC3B were reduced through GLP-1 treatment in DR . GLP-1 treatment restored the GLP-1R expression and decreased the levels of phosphorylated AKT and phosphorylated ERK1/2, which was accompanied with the reduction of the HDAC6 levels in DR. Conclusions: GLP-1 treatment can alleviate autophagy which may be induced by oxidative stress; this protective effect is likely through GLP-1R-ERK1/2-HDAC6 signaling pathway.

  17. How Parkinsonian Toxins Dysregulate the Autophagy Machinery

    Directory of Open Access Journals (Sweden)

    Ruben K. Dagda

    2013-11-01

    Full Text Available Since their discovery, Parkinsonian toxins (6-hydroxydopamine, MPP+, paraquat, and rotenone have been widely employed as in vivo and in vitro chemical models of Parkinson’s disease (PD. Alterations in mitochondrial homeostasis, protein quality control pathways, and more recently, autophagy/mitophagy have been implicated in neurotoxin models of PD. Here, we highlight the molecular mechanisms by which different PD toxins dysregulate autophagy/mitophagy and how alterations of these pathways play beneficial or detrimental roles in dopamine neurons. The convergent and divergent effects of PD toxins on mitochondrial function and autophagy/mitophagy are also discussed in this review. Furthermore, we propose new diagnostic tools and discuss how pharmacological modulators of autophagy/mitophagy can be developed as disease-modifying treatments for PD. Finally, we discuss the critical need to identify endogenous and synthetic forms of PD toxins and develop efficient health preventive programs to mitigate the risk of developing PD.

  18. Intersection of autophagy with pathways of antigen presentation.

    Science.gov (United States)

    Patterson, Natalie L; Mintern, Justine D

    2012-12-01

    Traditionally, macroautophagy (autophagy) is viewed as a pathway of cell survival. Autophagy ensures the elimination of damaged or unwanted cytosolic components and provides a source of cellular nutrients during periods of stress. Interestingly, autophagy can also directly intersect with, and impact, other major pathways of cellular function. Here, we will review the contribution of autophagy to pathways of antigen presentation. The autophagy machinery acts to modulate both MHCI and MHCII antigen presentation. As such autophagy is an important participant in pathways that elicit host cell immunity and the elimination of infectious pathogens.

  19. SIRT6 reduces macrophage foam cell formation by inducing autophagy and cholesterol efflux under ox-LDL condition.

    Science.gov (United States)

    He, Jiangping; Zhang, Guangya; Pang, Qi; Yu, Cong; Xiong, Jie; Zhu, Jing; Chen, Fengling

    2017-05-01

    SIRT6 is a pivotal regulator of lipid metabolism. It is also closely connected to cardiovascular diseases, which are the main cause of death in diabetic patients. We observed a decrease in the expression of SIRT6 and key autophagy effectors (ATG5, LC3B, and LAMP1) in ox-LDL-induced foam cells, a special form of lipid-laden macrophages. In these cells, SIRT6 WT but not SIRT6 H133Y overexpression markedly reduced foam cell formation, as shown by Oil Red O staining, while inducing autophagy flux, as determined by both mRFP-GFP-LC3 labeling and transmission electron microscopy. Silencing the key autophagy initiation gene ATG5, reversed the autophagy-promoting effect of SIRT6 in ox-LDL-treated THP1 cells, as evidenced by an increase in foam cells. Cholesterol efflux assays indicated that SIRT6 overexpression in foam cells promoted cholesterol efflux, increased the levels of ABCA1 and ABCG1, and reduced miR-33 levels. By transfecting miR-33 into cells overexpressing SIRT6, we observed that reduced foam cell formation and autophagy flux induction were largely reversed. These data imply that SIRT6 plays an essential role in protecting against atherosclerosis by reducing foam cell formation through an autophagy-dependent pathway. © 2017 Federation of European Biochemical Societies.

  20. Administration of Ketamine Causes Autophagy and Apoptosis in the Rat Fetal Hippocampus and in PC12 Cells

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    Xinran Li

    2018-02-01

    Full Text Available Drug abuse during pregnancy is a serious problem. Like alcohol, anticonvulsants, sedatives, and anesthetics, such as ketamine, can pass through the placental barrier and affect the growing fetus. However, the mechanism by which ketamine causes damage to fetal rats is not well understood. Therefore, in this study, we anesthetized pregnant rats with ketamine and evaluated the Total Antioxidant Capacity (T-AOC, Reactive Oxygen Species (ROS, and Malondialdehyde (MDA. Moreover, we determined changes in the levels of Cleaved-Caspase-3 (C-Caspase-3, Beclin-1, B-cell lymphoma-2 (Bcl-2, Bcl-2 Associated X Protein (Bax, Autophagy-related gene 4 (Atg4, Atg5, p62 (SQSTM1, and marker of autophagy Light Chain 3 (LC3. In addition, we cultured PC12 cells in vitro to determine the relationship between ROS, autophagy, and apoptosis following ketamine treatment. The results showed that ketamine induced changes in autophagy- and apoptosis-related proteins, reduced T-AOC, and generated excessive levels of ROS and MDA. In vitro experiments showed similar results, indicating that apoptosis levels can be inhibited by 3-MA. We also found that autophagy and apoptosis can be inhibited by N-acetyl-L-cysteine (Nac. Thus, anesthesia with ketamine in pregnant rats may increase the rate of autophagy and apoptosis in the fetal hippocampus and the mechanism may be through inhibition of antioxidant activity and ROS accumulation.

  1. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells.

    Science.gov (United States)

    Kang, Minyong; Lee, Kyoung-Hwa; Lee, Hye Sun; Jeong, Chang Wook; Kwak, Cheol; Kim, Hyeon Hoe; Ku, Ja Hyeon

    2017-02-04

    Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquine (CQ) and 3-methyladenine (3-MA) were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8) assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI) was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA) remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating a novel

  2. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Minyong Kang

    2017-02-01

    Full Text Available Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1, chloroquine (CQ and 3-methyladenine (3-MA were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8 assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating

  3. Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    Science.gov (United States)

    Chen, Song; Wang, Chenran; Yeo, Syn; Liang, Chun-Chi; Okamoto, Takako; Sun, Shaogang; Wen, Jian; Guan, Jun-Lin

    2016-01-01

    Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582–585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo. PMID:27013233

  4. Kinases Involved in Both Autophagy and Mitosis.

    Science.gov (United States)

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  5. Kinases Involved in Both Autophagy and Mitosis

    Directory of Open Access Journals (Sweden)

    Zhiyuan Li

    2017-08-01

    Full Text Available Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases, Aurora kinases, PLK-1 (polo-like kinase 1, BUB1 (budding uninhibited by benzimidazoles 1, MAPKs (mitogen-activated protein kinases, mTORC1 (mechanistic target of rapamycin complex 1, AMPK (AMP-activated protein kinase, PI3K (phosphoinositide-3 kinase and protein kinase B (AKT. By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  6. The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Kim, In-Ryoung; Kim, Seong-Eon; Baek, Hyun-Su; Kim, Bok-Joo; Kim, Chul-Hoon; Chung, In-Kyo; Park, Bong-Soo; Shin, Sang-Hun

    2016-08-31

    Kaempferol, a kind of flavonol, has been reported to possess various osteogenic biological activities, such as inhibiting bone resorption of osteoclasts and promoting the differentiation and mineralization of preosteoblasts. However, the precise cellular mechanism of action of kaempferol in osteogenesis is elusive. Autophagy is a major intracellular degradation system, which plays an important role in cell growth, survival, differentiation and homeostasis in mammals. Recent studies showed that autophagy appeared to be involved in the degradation of osteoclasts, osteoblasts and osteocytes, potentially pointing to a new pathogenic mechanism of bone homeostasis and bone marrow disease. The potential correlation between autophagy, osteogenesis and flavonoids is unclear. The present study verified that kaempferol promoted osteogenic differentiation and mineralization and that it elevated osteogenic gene expression based on alkaline phosphatase (ALP) activity, alizarin red staining and quantitative PCR. And then we found that kaempferol induced autophagy by acridine orange (AO) and monodansylcadaverine (MDC) staining and autophagy-related protein expression. The correlation between kaempferol-induced autophagy and the osteogenic process was confirmed by the autophagy inhibitor 3-methyladenine (3-MA). Kaempferol promoted the proliferation, differentiation and mineralization of osteoblasts at a concentration of 10 μM. Kaempferol showed cytotoxic properties at concentrations above 50 μM. Concentrations above 10 μM decreased ALP activity, whereas those up to 10 μM increased ALP activity. Kaempferol at concentrations up to 10 μM also increased the expression of the osteoblast- activated factors RUNX-2, osterix, BMP-2 and collagen I according to RT-PCR analyses. 10 μM or less, the higher of the concentration and over time, kaempferol promoted the activity of osteoblasts. Kaempferol induced autophagy. It also increased the expression of the autophagy-related factors

  7. Ribosomal protein mutations induce autophagy through S6 kinase inhibition of the insulin pathway.

    Directory of Open Access Journals (Sweden)

    Harry F Heijnen

    Full Text Available Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA, for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS. The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS. We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies.

  8. Agent-based modeling of autophagy reveals emergent regulatory behavior of spatio-temporal autophagy dynamics.

    Science.gov (United States)

    Börlin, Christoph S; Lang, Verena; Hamacher-Brady, Anne; Brady, Nathan R

    2014-09-10

    Autophagy is a vesicle-mediated pathway for lysosomal degradation, essential under basal and stressed conditions. Various cellular components, including specific proteins, protein aggregates, organelles and intracellular pathogens, are targets for autophagic degradation. Thereby, autophagy controls numerous vital physiological and pathophysiological functions, including cell signaling, differentiation, turnover of cellular components and pathogen defense. Moreover, autophagy enables the cell to recycle cellular components to metabolic substrates, thereby permitting prolonged survival under low nutrient conditions. Due to the multi-faceted roles for autophagy in maintaining cellular and organismal homeostasis and responding to diverse stresses, malfunction of autophagy contributes to both chronic and acute pathologies. We applied a systems biology approach to improve the understanding of this complex cellular process of autophagy. All autophagy pathway vesicle activities, i.e. creation, movement, fusion and degradation, are highly dynamic, temporally and spatially, and under various forms of regulation. We therefore developed an agent-based model (ABM) to represent individual components of the autophagy pathway, subcellular vesicle dynamics and metabolic feedback with the cellular environment, thereby providing a framework to investigate spatio-temporal aspects of autophagy regulation and dynamic behavior. The rules defining our ABM were derived from literature and from high-resolution images of autophagy markers under basal and activated conditions. Key model parameters were fit with an iterative method using a genetic algorithm and a predefined fitness function. From this approach, we found that accurate prediction of spatio-temporal behavior required increasing model complexity by implementing functional integration of autophagy with the cellular nutrient state. The resulting model is able to reproduce short-term autophagic flux measurements (up to 3

  9. Autophagy Deficiency Compromises Alternative Pathways of Respiration following Energy Deprivation in Arabidopsis thaliana.

    Science.gov (United States)

    Barros, Jessica A S; Cavalcanti, João Henrique F; Medeiros, David B; Nunes-Nesi, Adriano; Avin-Wittenberg, Tamar; Fernie, Alisdair R; Araújo, Wagner L

    2017-09-01

    Under heterotrophic conditions, carbohydrate oxidation inside the mitochondrion is the primary energy source for cellular metabolism. However, during energy-limited conditions, alternative substrates are required to support respiration. Amino acid oxidation in plant cells plays a key role in this by generating electrons that can be transferred to the mitochondrial electron transport chain via the electron transfer flavoprotein/ubiquinone oxidoreductase system. Autophagy, a catabolic mechanism for macromolecule and protein recycling, allows the maintenance of amino acid pools and nutrient remobilization. Although the association between autophagy and alternative respiratory substrates has been suggested, the extent to which autophagy and primary metabolism interact to support plant respiration remains unclear. To investigate the metabolic importance of autophagy during development and under extended darkness, Arabidopsis ( Arabidopsis thaliana ) mutants with disruption of autophagy ( atg mutants) were used. Under normal growth conditions, atg mutants showed lower growth and seed production with no impact on photosynthesis. Following extended darkness, atg mutants were characterized by signatures of early senescence, including decreased chlorophyll content and maximum photochemical efficiency of photosystem II coupled with increases in dark respiration. Transcript levels of genes involved in alternative pathways of respiration and amino acid catabolism were up-regulated in atg mutants. The metabolite profiles of dark-treated leaves revealed an extensive metabolic reprogramming in which increases in amino acid levels were partially compromised in atg mutants. Although an enhanced respiration in atg mutants was observed during extended darkness, autophagy deficiency compromises protein degradation and the generation of amino acids used as alternative substrates to the respiration. © 2017 American Society of Plant Biologists. All Rights Reserved.

  10. Sirtuin1 and autophagy protect cells from fluoride-induced cell stress

    Science.gov (United States)

    Suzuki, Maiko; Bartlett, John D.

    2014-01-01

    Sirtuin1 (SIRT1) is an (NAD+)-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum stress and oxidative stress. Previously, we reported that fluoride induces endoplasmic reticulum (ER) stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augmented SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50 and 100 ppm) in drinking water for 6 weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis. PMID:24296261

  11. Sirtuin1 and autophagy protect cells from fluoride-induced cell stress.

    Science.gov (United States)

    Suzuki, Maiko; Bartlett, John D

    2014-02-01

    Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum (ER) stress and oxidative stress. Previously, we reported that fluoride induces ER-stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augment SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50, 100 and 125ppm) in drinking water for 6weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. MicroRNA-125a Inhibits Autophagy Activation and Antimicrobial Responses during Mycobacterial Infection.

    Science.gov (United States)

    Kim, Jin Kyung; Yuk, Jae-Min; Kim, Soo Yeon; Kim, Tae Sung; Jin, Hyo Sun; Yang, Chul-Su; Jo, Eun-Kyeong

    2015-06-01

    MicroRNAs (miRNAs) are small noncoding nucleotides that play critical roles in the regulation of diverse biological functions, including the response of host immune cells. Autophagy plays a key role in activating the antimicrobial host defense against Mycobacterium tuberculosis. Although the pathways associated with autophagy must be tightly regulated at a posttranscriptional level, the contribution of miRNAs and whether they specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that M. tuberculosis infection of macrophages leads to increased expression of miRNA-125a-3p (miR-125a), which targets UV radiation resistance-associated gene (UVRAG), to inhibit autophagy activation and antimicrobial responses to M. tuberculosis. Forced expression of miR-125a significantly blocked M. tuberculosis-induced activation of autophagy and phagosomal maturation in macrophages, and inhibitors of miR-125a counteracted these effects. Both TLR2 and MyD88 were required for biogenesis of miR-125a during M. tuberculosis infection. Notably, activation of the AMP-activated protein kinase significantly inhibited the expression of miR-125a in M. tuberculosis-infected macrophages. Moreover, either overexpression of miR-125a or silencing of UVRAG significantly attenuated the antimicrobial effects of macrophages against M. tuberculosis. Taken together, these data indicate that miR-125a regulates the innate host defense by inhibiting the activation of autophagy and antimicrobial effects against M. tuberculosis through targeting UVRAG. Copyright © 2015 by The American Association of Immunologists, Inc.

  13. A Dual Role of P53 in Regulating Colistin-Induced Autophagy in PC-12 Cells

    Directory of Open Access Journals (Sweden)

    Ziyin Lu

    2017-10-01

    Full Text Available This study aimed to investigate the mechanism of p53 in regulating colistin-induced autophagy in PC-12 cells. Importantly, cells were treated with 125 μg/ml colistin for 12 and 24 h after transfection with p53 siRNA or recombinant plasmid. The hallmarks of autophagy and apoptosis were examined by real-time PCR and western blot, fluorescence/immunofluorescence microscopy, and electron microscopy. The results showed that silencing of p53 leads to down-regulation of Atg5 and beclin1 for 12 h while up-regulation at 24 h and up-regulation of p62 noted. The ratio of LC3-II/I and autophagic vacuoles were significantly increased at 24 h, but autophagy flux was blocked. The cleavage of caspase3 and PARP (poly ADP-ribose polymerase were enhanced, while PC-12-sip53 cells exposed to 3-MA showed down-regulation of apoptosis. By contrast, the expression of autophagy-related genes and protein reduced in p53 overexpressing cells following a time dependent manner. Meanwhile, there was an increase in the expression of activated caspase3 and PARP, condensed and fragmented nuclei were evident. Conclusively, the data supported that silencing of p53 promotes impaired autophagy, which acts as a pro-apoptotic induction factor in PC-12 cells treated with colistin for 24 h, and overexpression of p53 inhibits autophagy and accelerates apoptosis. Hence, it has been suggested that p53 could not act as a neuro-protective target in colistin-induced neurotoxicity.

  14. Neither eosinophils nor neutrophils require ATG5-dependent autophagy for extracellular DNA trap formation.

    Science.gov (United States)

    Germic, Nina; Stojkov, Darko; Oberson, Kevin; Yousefi, Shida; Simon, Hans-Uwe

    2017-11-01

    The importance of extracellular traps (ETs) in innate immunity is well established, but the molecular mechanisms responsible for their formation remain unclear and in scientific dispute. ETs have been defined as extracellular DNA scaffolds associated with the granule proteins of eosinophils or neutrophils. They are capable of killing bacteria extracellularly. Based mainly on results with phosphoinositide 3-kinase (PI3K) inhibitors such as 3-methyladenine (3-MA) and wortmannin, which are commonly used to inhibit autophagy, several groups have reported that autophagy is required for neutrophil extracellular trap (NET) formation. We decided to investigate this apparent dependence on autophagy for ET release and generated genetically modified mice that lack, specifically in eosinophils or neutrophils, autophagy-related 5 (Atg5), a gene encoding a protein essential for autophagosome formation. Interestingly, neither eosinophils nor neutrophils from Atg5-deficient mice exhibited abnormalities in ET formation upon physiological activation or exposure to low concentrations of PMA, although we could confirm that human and mouse eosinophils and neutrophils, after pre-treatment with inhibitors of class III PI3K, show a block both in reactive oxygen species (ROS) production and in ET formation. The so-called late autophagy inhibitors bafilomycin A1 and chloroquine, on the other hand, were without effect. These data indicate that ET formation occurs independently of autophagy and that the inhibition of ROS production and ET formation in the presence of 3-MA and wortmannin is probably owing to their additional ability to block the class I PI3Ks, which are involved in signalling cascades initiated by triggers of ET formation. © 2017 John Wiley & Sons Ltd.

  15. Re-expression of ARHI (DIRAS3) induces autophagy in breast cancer cells and enhances the inhibitory effect of paclitaxel

    International Nuclear Information System (INIS)

    Zou, Chun-Fang; Yu, Yinhua; Jia, Luoqi; Jin, Hongyan; Yao, Ming; Zhao, Naiqing; Huan, Jin; Lu, Zhen; Bast, Robert C Jr; Feng, Youji

    2011-01-01

    ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma in situ (DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel. Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts. ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest. ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest

  16. Regulation of Autophagy by Glucose in Mammalian Cells

    OpenAIRE

    Moruno, Félix; Pérez-Jiménez, Eva; Knecht, Erwin

    2012-01-01

    Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focu...

  17. Sorafenib-induced defective autophagy promotes cell death by necroptosis

    OpenAIRE

    Kharaziha, Pedram; Chioureas, Dimitris; Baltatzis, George; Fonseca, Pedro; Rodriguez, Patricia; Gogvadze, Vladimir; Lennartsson, Lena; Bj?rklund, Ann-Charlotte; Zhivotovsky, Boris; Grand?r, Dan; Egevad, Lars; Nilsson, Sten; Panaretakis, Theocharis

    2015-01-01

    Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencin...

  18. Cisplatin-induced downregulation of miR-199a-5p increases drug resistance by activating autophagy in HCC cell

    International Nuclear Information System (INIS)

    Xu, Ning; Zhang, Jianjun; Shen, Conghuan; Luo, Yi; Xia, Lei; Xue, Feng; Xia, Qiang

    2012-01-01

    Highlights: ► miR-199a-5p levels were significantly decreased after cisplatin treatment. ► Cisplatin treatment induced autophagy activation. ► Cisplatin-induced downregulation of miR-199a-5p increases drug resistance by activating autophagy in HCC cell. -- Abstract: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Systemic chemotherapy plays an important role in the treatment of patients with advanced liver cancer. However, chemoresistance to cisplatin is a major limitation of cisplatin-based chemotherapy in the clinic, and the underlying mechanism of such resistance is not fully understood. In the study, we found that miR-199a-5p levels were significantly reduced in HCC patients treated with cisplatin-based chemotherapy. Cisplatin treatment also resulted in decreased miR-199a-5p levels in human HCC cell lines. Forced expression of miR-199a-5p promoted cisplatin-induced inhibition of cell proliferation. Cisplatin treatment activated autophagy in Huh7 and HepG2 cells, which increased cell proliferation. We further demonstrated that downregulated miR-199a-5p enhanced autophagy activation by targeting autophagy-associated gene 7 (ATG7). More important, autophagy inhibition abrogated miR-199a-5p downregulation-induced cell proliferation. These data demonstrated that miR-199a-5p/autophagy signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of HCC.

  19. Autophagy and Retromer Components in Plant Innate Immunity

    DEFF Research Database (Denmark)

    Munch, David

    -hormone salicylic acid. Here, I present data that make it clear that NPR1 does not directly regulate autophagy, but instead control stress responses that indirectly activate autophagy. The observations presented will also clarify why autophagy has been described as being both a pro-death and pro-life pathway under...

  20. Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

    Directory of Open Access Journals (Sweden)

    Chan Shih-Hung

    2012-07-01

    Full Text Available Abstract Background Insulin receptor substrate (IRS-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels. Methods and results In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3, aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress

  1. Antioxidant Supplement Inhibits Skeletal Muscle Constitutive Autophagy rather than Fasting-Induced Autophagy in Mice

    Directory of Open Access Journals (Sweden)

    Zhengtang Qi

    2014-01-01

    Full Text Available In this study, we tested the hypothesis that NAC administration leads to reduced oxidative stress and thus to decreased expression of autophagy markers in young mice. Our results reveal that NAC administration results in reduced muscle mRNA levels of several autophagy markers, including Beclin-1, Atg7, LC3, Atg9, and LAMP2. However, NAC supplement fails to block the activation of skeletal muscle autophagy in response to fasting, because fasting significantly increases the mRNA level of several autophagy markers and LC3 lipidation. We further examined the effects of NAC administration on mitochondrial antioxidant capacity in fed and 24-hour fasted mice. Our results clearly show that NAC administration depresses the expression of manganese superoxide dismutase (MnSOD and TP53-induced glycolysis and apoptosis regulator (TIGAR, both of which play a predominant antioxidant role in mitochondria by reducing ROS level. In addition, we found no beneficial effect of NAC supplement on muscle mass but it can protect from muscle loss in response to fasting. Collectively, our findings indicate that ROS is required for skeletal muscle constitutive autophagy, rather than starvation-induced autophagy, and that antioxidant NAC inhibits constitutive autophagy by the regulation of mitochondrial ROS production and antioxidant capacity.

  2. Hyperosmotic stress stimulates autophagy via polycystin-2.

    Science.gov (United States)

    Peña-Oyarzun, Daniel; Troncoso, Rodrigo; Kretschmar, Catalina; Hernando, Cecilia; Budini, Mauricio; Morselli, Eugenia; Lavandero, Sergio; Criollo, Alfredo

    2017-08-22

    Various intracellular mechanisms are activated in response to stress, leading to adaptation or death. Autophagy, an intracellular process that promotes lysosomal degradation of proteins, is an adaptive response to several types of stress. Osmotic stress occurs under both physiological and pathological conditions, provoking mechanical stress and activating various osmoadaptive mechanisms. Polycystin-2 (PC2), a membrane protein of the polycystin family, is a mechanical sensor capable of activating the cell signaling pathways required for cell adaptation and survival. Here we show that hyperosmotic stress provoked by treatment with hyperosmolar concentrations of sorbitol or mannitol induces autophagy in HeLa and HCT116 cell lines. In addition, we show that mTOR and AMPK, two stress sensor proteins involved modulating autophagy, are downregulated and upregulated, respectively, when cells are subjected to hyperosmotic stress. Finally, our findings show that PC2 is required to promote hyperosmotic stress-induced autophagy. Downregulation of PC2 prevents inhibition of hyperosmotic stress-induced mTOR pathway activation. In conclusion, our data provide new insight into the role of PC2 as a mechanosensor that modulates autophagy under hyperosmotic stress conditions.

  3. A Molecular View of Autophagy in Lepidoptera

    Directory of Open Access Journals (Sweden)

    Davide Romanelli

    2014-01-01

    Full Text Available Metamorphosis represents a critical phase in the development of holometabolous insects, during which the larval body is completely reorganized: in fact, most of the larval organs undergo remodeling or completely degenerate before the final structure of the adult insect is rebuilt. In the past, increasing evidence emerged concerning the intervention of autophagy and apoptosis in the cell death processes that occur in larval organs of Lepidoptera during metamorphosis, but a molecular characterization of these pathways was undertaken only in recent years. In addition to developmentally programmed autophagy, there is growing interest in starvation-induced autophagy. Therefore we are now entering a new era of research on autophagy that foreshadows clarification of the role and regulatory mechanisms underlying this self-digesting process in Lepidoptera. Given that some of the most important lepidopteran species of high economic importance, such as the silkworm, Bombyx mori, belong to this insect order, we expect that this information on autophagy will be fully exploited not only in basic research but also for practical applications.

  4. Autophagy induction for the treatment of cancer.

    Science.gov (United States)

    Pietrocola, Federico; Pol, Jonathan; Vacchelli, Erika; Baracco, Elisa E; Levesque, Sarah; Castoldi, Francesca; Maiuri, Maria Chiara; Madeo, Frank; Kroemer, Guido

    2016-10-02

    Cancer can be viewed in 2 rather distinct ways, namely (i) as a cell-autonomous disease in which malignant cells have escaped control from cell-intrinsic barriers against proliferation and dissemination or (ii) as a systemic disease that involves failing immune control of aberrant cells. Since macroautophagy/autophagy generally increases the fitness of cells as well as their resistance against endogenous or iatrogenic (i.e., relating to illness due to medical intervention) stress, it has been widely proposed that inhibition of autophagy would constitute a valid strategy for sensitizing cancer cells to chemotherapy or radiotherapy. Colliding with this cell-autonomous vision, however, we found that immunosurveillance against transplantable, carcinogen-induced or genetically engineered cancers can be improved by pharmacologically inducing autophagy with caloric restriction mimetics. This positive effect depends on autophagy induction in cancer cells and is mediated by alterations in extracellular ATP metabolism, namely increased release of immunostimulatory ATP and reduced adenosine-dependent recruitment of immunosuppressive regulatory T cells into the tumor bed. The combination of autophagy inducers and chemotherapeutic agents is particularly efficient in reducing cancer growth through the stimulation of CD8 + T lymphocyte-dependent anticancer immune responses.

  5. Autophagy and Liver Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Raffaele Cursio

    2015-01-01

    Full Text Available Liver ischemia-reperfusion (I-R injury occurs during liver resection, liver transplantation, and hemorrhagic shock. The main mode of liver cell death after warm and/or cold liver I-R is necrosis, but other modes of cell death, as apoptosis and autophagy, are also involved. Autophagy is an intracellular self-digesting pathway responsible for removal of long-lived proteins, damaged organelles, and malformed proteins during biosynthesis by lysosomes. Autophagy is found in normal and diseased liver. Although depending on the type of ischemia, warm and/or cold, the dynamic process of liver I-R results mainly in adenosine triphosphate depletion and in production of reactive oxygen species (ROS, leads to both, a local ischemic insult and an acute inflammatory-mediated reperfusion injury, and results finally in cell death. This process can induce liver dysfunction and can increase patient morbidity and mortality after liver surgery and hemorrhagic shock. Whether autophagy protects from or promotes liver injury following warm and/or cold I-R remains to be elucidated. The present review aims to summarize the current knowledge in liver I-R injury focusing on both the beneficial and the detrimental effects of liver autophagy following warm and/or cold liver I-R.

  6. Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway.

    Science.gov (United States)

    Liu, Kuang-Kai; Qiu, Wei-Ru; Naveen Raj, Emmanuel; Liu, Huei-Fang; Huang, Hou-Syun; Lin, Yu-Wei; Chang, Chien-Jen; Chen, Ting-Hua; Chen, Chinpiao; Chang, Huan-Cheng; Hwang, Jenn-Kang; Chao, Jui-I

    2017-01-02

    Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.

  7. Degradation of AF1Q by chaperone-mediated autophagy

    International Nuclear Information System (INIS)

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru; Li, Huanjie; Cui, Taixing; Li Wang, Xing; Tang, Dongqi; Ji, Chunyan

    2014-01-01

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components

  8. Constitutive upregulation of chaperone-mediated autophagy in Huntington's disease.

    Science.gov (United States)

    Koga, Hiroshi; Martinez-Vicente, Marta; Arias, Esperanza; Kaushik, Susmita; Sulzer, David; Cuervo, Ana Maria

    2011-12-14

    Autophagy contributes to the removal of prone-to-aggregate proteins, but in several instances these pathogenic proteins have been shown to interfere with autophagic activity. In the case of Huntington's disease (HD), a congenital neurodegenerative disorder resulting from mutation in the huntingtin protein, we have previously described that the mutant protein interferes with the ability of autophagic vacuoles to recognize cytosolic cargo. Growing evidence supports the existence of cross talk among autophagic pathways, suggesting the possibility of functional compensation when one of them is compromised. In this study, we have identified a compensatory upregulation of chaperone-mediated autophagy (CMA) in different cellular and mouse models of HD. Components of CMA, namely the lysosome-associated membrane protein type 2A (LAMP-2A) and lysosomal-hsc70, are markedly increased in HD models. The increase in LAMP-2A is achieved through both an increase in the stability of this protein at the lysosomal membrane and transcriptional upregulation of this splice variant of the lamp-2 gene. We propose that CMA activity increases in response to macroautophagic dysfunction in the early stages of HD, but that the efficiency of this compensatory mechanism may decrease with age and so contribute to cellular failure and the onset of pathological manifestations.

  9. Degradation of AF1Q by chaperone-mediated autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Li, Peng; Ji, Min; Lu, Fei; Zhang, Jingru [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Li, Huanjie; Cui, Taixing; Li Wang, Xing [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Tang, Dongqi, E-mail: tangdq@sdu.edu.cn [Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China); Center for Stem Cell and Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250033 (China); Ji, Chunyan, E-mail: jichunyan@sdu.edu.cn [Department of Hematology, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012 (China)

    2014-09-10

    AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA. - Highlights: • Chaperone-mediated autophagy (CMA) is involved in the degradation of AF1Q. • Macroautophagy does not contribute to the AF1Q degradation. • AF1Q has a KFERQ-like motif that is recognized by CMA core components.

  10. The Cytoskeleton-Autophagy Connection.

    Science.gov (United States)

    Kast, David J; Dominguez, Roberto

    2017-04-24

    Actin cytoskeleton dynamics play vital roles in most forms of intracellular trafficking by promoting the biogenesis and transport of vesicular cargoes. Mounting evidence indicates that actin dynamics and membrane-cytoskeleton scaffolds also have essential roles in macroautophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. Branched actin polymerization is necessary for the biogenesis of autophagosomes from the endoplasmic reticulum (ER) membrane. Actomyosin-based transport is then used to feed the growing phagophore with pre-selected cargoes and debris derived from different membranous organelles inside the cell. Finally, mature autophagosomes detach from the ER membrane by an as yet unknown mechanism, undergo intracellular transport and then fuse with lysosomes, endosomes and multivesicular bodies through mechanisms that involve actin- and microtubule-mediated motility, cytoskeleton-membrane scaffolds and signaling proteins. In this review, we highlight the considerable progress made recently towards understanding the diverse roles of the cytoskeleton in autophagy. Published by Elsevier Ltd.

  11. Saturated fatty acid palmitate negatively regulates autophagy by promoting ATG5 protein degradation in meniscus cells.

    Science.gov (United States)

    Mallik, Aritra; Yammani, Raghunatha R

    2018-07-20

    Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Human stefin B role in cell's response to misfolded proteins and autophagy.

    Directory of Open Access Journals (Sweden)

    Mira Polajnar

    Full Text Available Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

  13. Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy.

    Science.gov (United States)

    Zabirnyk, Olga; Liu, Wei; Khalil, Shadi; Sharma, Anit; Phang, James M

    2010-03-01

    Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated proline oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible for the latter. To elucidate the role of POX in oxLDL-mediated cytotoxicity, we knocked down POX via small interfering RNA and found that this (i) further reduced viability of cancer cells treated with oxLDL; (ii) decreased oxLDL-associated reactive oxygen species generation; (iii) decreased autophagy measured via beclin-1 protein level and light-chain 3 protein (LC3)-I into LC3-II conversion. Using POX-expressing cell model, we established that single POX overexpression was sufficient to activate autophagy. Thus, it led to autophagosomes accumulation and increased conversion of LC3-I into LC3-II. Moreover, beclin-1 gene expression was directly dependent on POX catalytic activity, namely the generation of POX-dependent superoxide. We conclude that POX is critical in the cellular response to the noxious effects of oxLDL by activating protective autophagy.

  14. Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication.

    Science.gov (United States)

    Meng, Chunchun; Zhou, Zhizhi; Jiang, Ke; Yu, Shengqing; Jia, Lijun; Wu, Yantao; Liu, Yanqing; Meng, Songshu; Ding, Chan

    2012-06-01

    Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages of infection. However, the interaction between NDV and cells in early stages of infection is not well understood. Here, we report that, shortly after infection, NDV triggers the formation of autophagosomes in U251 glioma cells, as demonstrated by an increased number of double-membrane vesicles, GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) a dot formations, and elevated production of LC3II. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine or small interfering RNAs targeting the genes critical for autophagosome formation (Atg5 and Beclin-1) affects virus production, indicating that autophagy may be utilized by NDV to facilitate its own production. Furthermore, the class III phosphatidylinositol 3-kinase (PI3K)/Beclin-1 pathway plays a role in NDV-induced autophagy and virus production. Collectively, our data provide a unique example of a paramyxovirus that uses autophagy to enhance its production.

  15. Nano rare-earth oxides induced size-dependent vacuolization: an independent pathway from autophagy.

    Science.gov (United States)

    Zhang, Ying; Yu, Chenguang; Huang, Guanyi; Wang, Changli; Wen, Longping

    2010-09-07

    Four rare earth oxides have been shown to induce autophagy. Interestingly, we often noticed plentiful vacuolization, which was not always involved in this autophagic process. In this study, we investigated three other rare-earth elements, including Yttrium (Y), Ytterbium (Yb), and Lanthanum (La). Autophagic effect could be induced by all of them but only Y(2)O(3) and Yb(2)O(3) could cause massive vacuolization. Y(2)O(3) and Yb(2)O(3) treated by sonication or centrifugation to reduce particle size were used to test vacuolization level in HeLa cell lines. The results showed that rare earth oxides-induced vacuolization is size-dependent and differs from autophagic pathway. To further clarify the characteristics of this autophagic process, we used MEF Atg-5 (autophagy associated gene 5) knockout cell line, and the result showed that the autophagic process induced by rare earth oxides is Atg-5-dependent and the observed vacuolization was independent from autophagy. Similar results could also be observed in our tests on 3-methyladenine(3-MA), a well-known autophagy inhibitor. In conclusion, for the first time, we clarified the relationship between massive vacuolization and autophagic process induced by rare earth oxides and pointed out the size effect of rare earth oxides on the formation of vacuoles, which give clues to further investigation on the mechanisms underlying their biological effects.

  16. Dihydroptychantol A, a macrocyclic bisbibenzyl derivative, induces autophagy and following apoptosis associated with p53 pathway in human osteosarcoma U2OS cells

    International Nuclear Information System (INIS)

    Li Xia; Wu, William K.K.; Sun Bin; Cui Min; Liu Shanshan; Gao Jian; Lou Hongxiang

    2011-01-01

    Dihydroptychantol A (DHA), a novel macrocyclic bisbibenzyl compound extracted from liverwort Asterella angusta, has antifungal and multi-drug resistance reversal properties. Here, the chemically synthesized DHA was employed to test its anti-cancer activities in human osteosarcoma U2OS cells. Our results demonstrated that DHA induced autophagy followed by apoptotic cell death accompanied with G 2 /M-phase cell cycle arrest in U2OS cells. DHA-induced autophagy was morphologically characterized by the formation of double membrane-bound autophagic vacuoles recognizable at the ultrastructural level. DHA also increased the levels of LC3-II, a marker of autophagy. Surprisingly, DHA-mediated apoptotic cell death was potentiated by the autophagy inhibitor 3-methyladenine, suggesting that autophagy may play a protective role that impedes the eventual cell death. Furthermore, p53 was shown to be involved in DHA-meditated autophagy and apoptosis. In this connection, DHA increased nuclear expression of p53, induced p53 phosphorylation, and upregulated p53 target gene p21 Waf1/Cip1 . In contrast, cytoplasmic p53 was reduced by DHA, which contributed to the stimulation of autophagy. In relation to the cell cycle, DHA decreased the expression of cyclin B 1 , a cyclin required for progression through the G 2 /M phase. Taken together, DHA induces G 2 /M-phase cell cycle arrest and apoptosis in U2OS cells. DHA-induced apoptosis was preceded by the induction of protective autophagy. DHA-mediated autophagy and apoptosis are associated with the cytoplasmic and nuclear functions of p53.

  17. Downregulation of B-cell lymphoma/leukemia-2 by overexpressed microRNA 34a enhanced titanium dioxide nanoparticle-induced autophagy in BEAS-2B cells

    Science.gov (United States)

    Bai, Wenlin; Chen, Yujiao; Sun, Pengling; Gao, Ai

    2016-01-01

    Titanium dioxide (TiO2) nanoparticles (TNPs) are manufactured worldwide for a wide range of applications and the toxic effect of TNPs on biological systems is gaining attention. Autophagy is recognized as an emerging toxicity mechanism triggered by nanomaterials. MicroRNA 34a (miR34a) acts as a tumor suppressor gene by targeting many oncogenes, but how it affects autophagy induced by TNPs is not completely understood. Here, we observed the activation of TNP-induced autophagy through monodansylcadaverine staining and LC3-I/LC3-II conversion. Meanwhile, the transmission electron microscope ultrastructural analysis showed typical morphological characteristics in autophagy process. We detected the expression of miR34a and B-cell lymphoma/leukemia-2 (Bcl-2). In addition, the underlying mechanism of TNP-induced autophagy was performed using overexpression of miR34a by lentivirus vector transfection. Results showed that TNPs induced autophagy generation evidently. Typical morphological changes in the process of autophagy were observed by the transmission electron microscope ultrastructural analysis and LC3-I/LC3-II conversion increased significantly in TNP-treated cells. Meanwhile, TNPs induced the downregulation of miR34a and increased the expression of Bcl-2. Furthermore, overexpressed miR34a decreased the expression of Bcl-2 both in messenger RNA and protein level, following which the level of autophagy and cell death rate increased after the transfected cells were incubated with TNPs for 24 hours. These findings provide the first evidence that overexpressed miR34a enhanced TNP-induced autophagy and cell death through targeted downregulation of Bcl-2 in BEAS-2B cells. PMID:27226226

  18. Autophagy in health and disease: focus on the cardiovascular system.

    Science.gov (United States)

    Mialet-Perez, Jeanne; Vindis, Cécile

    2017-12-12

    Autophagy is a highly conserved mechanism of lysosome-mediated protein and organelle degradation that plays a crucial role in maintaining cellular homeostasis. In the last few years, specific functions for autophagy have been identified in many tissues and organs. In the cardiovascular system, autophagy appears to be essential to heart and vessel homeostasis and function; however defective or excessive autophagy activity seems to contribute to major cardiovascular disorders including heart failure (HF) or atherosclerosis. Here, we review the current knowledge on the role of cardiovascular autophagy in physiological and pathophysiological conditions. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  19. Emerging role of autophagy in kidney function, diseases and aging

    Science.gov (United States)

    Huber, Tobias B.; Edelstein, Charles L.; Hartleben, Björn; Inoki, Ken; Jiang, Man; Koya, Daisuke; Kume, Shinji; Lieberthal, Wilfred; Pallet, Nicolas; Quiroga, Alejandro; Ravichandran, Kameswaran; Susztak, Katalin; Yoshida, Sei; Dong, Zheng

    2012-01-01

    Autophagy is a highly conserved process that degrades cellular long-lived proteins and organelles. Accumulating evidence indicates that autophagy plays a critical role in kidney maintenance, diseases and aging. Ischemic, toxic, immunological, and oxidative insults can cause an induction of autophagy in renal epithelial cells modifying the course of various kidney diseases. This review summarizes recent insights on the role of autophagy in kidney physiology and diseases alluding to possible novel intervention strategies for treating specific kidney disorders by modifying autophagy. PMID:22692002

  20. Autophagy in the light of sphingolipid metabolism

    DEFF Research Database (Denmark)

    Harvald, Eva Bang; Olsen, Anne Sofie Braun; Færgeman, Nils J.

    2015-01-01

    Maintenance of cellular homeostasis requires tight and coordinated control of numerous metabolic pathways, which are governed by interconnected networks of signaling pathways and energy-sensing regulators. Autophagy, a lysosomal degradation pathway by which the cell self-digests its own components......, has over the past decade been recognized as an essential part of metabolism. Autophagy not only rids the cell of excessive or damaged organelles, misfolded proteins, and invading microorganisms, it also provides nutrients to maintain crucial cellular functions. Besides serving as essential structural...... moieties of biomembranes, lipids including sphingolipids are increasingly being recognized as central regulators of a number of important cellular processes, including autophagy. In the present review we describe how sphingolipids, with special emphasis on ceramides and sphingosine-1-phosphate, can act...

  1. Induction of autophagy by spermidine promotes longevity.

    Science.gov (United States)

    Eisenberg, Tobias; Knauer, Heide; Schauer, Alexandra; Büttner, Sabrina; Ruckenstuhl, Christoph; Carmona-Gutierrez, Didac; Ring, Julia; Schroeder, Sabrina; Magnes, Christoph; Antonacci, Lucia; Fussi, Heike; Deszcz, Luiza; Hartl, Regina; Schraml, Elisabeth; Criollo, Alfredo; Megalou, Evgenia; Weiskopf, Daniela; Laun, Peter; Heeren, Gino; Breitenbach, Michael; Grubeck-Loebenstein, Beatrix; Herker, Eva; Fahrenkrog, Birthe; Fröhlich, Kai-Uwe; Sinner, Frank; Tavernarakis, Nektarios; Minois, Nadege; Kroemer, Guido; Madeo, Frank

    2009-11-01

    Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

  2. IKK connects autophagy to major stress pathways.

    Science.gov (United States)

    Criollo, Alfredo; Senovilla, Laura; Authier, Hélène; Maiuri, Maria Chiara; Morselli, Eugenia; Vitale, Ilio; Kepp, Oliver; Tasdemir, Ezgi; Galluzzi, Lorenzo; Shen, Shensi; Tailler, Maximilien; Delahaye, Nicolas; Tesniere, Antoine; De Stefano, Daniela; Younes, Aména Ben; Harper, Francis; Pierron, Gérard; Lavandero, Sergio; Zitvogel, Laurence; Israel, Alain; Baud, Véronique; Kroemer, Guido

    2010-01-01

    Cells respond to stress by activating cytoplasmic mechanisms as well as transcriptional programs that can lead to adaptation or death. Autophagy represents an important cytoprotective response that is regulated by both transcriptional and transcription-independent pathways. NFkappaB is perhaps the transcription factor most frequently activated by stress and has been ascribed with either pro- or anti-autophagic functions, depending on the cellular context. Our results demonstrate that activation of the IKK (IkappaB kinase) complex, which is critical for the stress-elicited activation of NFkappaB, is sufficient to promote autophagy independent of NFkappaB, and that IKK is required for the optimal induction of autophagy by both physiological and pharmacological autophagic triggers.

  3. Myocardial Autophagy after Severe Burn in Rats

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    Zhang, Qiong; Shi, Xiao-hua; Huang, Yue-sheng

    2012-01-01

    Background Autophagy plays a major role in myocardial ischemia and hypoxia injury. The present study investigated the effects of autophagy on cardiac dysfunction in rats after severe burn. Methods Protein expression of the autophagy markers LC3 and Beclin 1 were determined at 0, 1, 3, 6, and 12 h post-burn in Sprague Dawley rats subjected to 30% total body surface area 3rd degree burns. Autophagic, apoptotic, and oncotic cell death were evaluated in the myocardium at each time point by immunofluorescence. Changes of cardiac function were measured in a Langendorff model of isolated heart at 6 h post-burn, and the autophagic response was measured following activation by Rapamycin and inhibition by 3-methyladenine (3-MA). The angiotensin converting enzyme inhibitor enalaprilat, the angiotensin receptor I blocker losartan, and the reactive oxygen species inhibitor diphenylene iodonium (DPI) were also applied to the ex vivo heart model to examine the roles of these factors in post-burn cardiac function. Results Autophagic cell death was first observed in the myocardium at 3 h post-burn, occurring in 0.008 ± 0.001% of total cardiomyocytes, and continued to increase to a level of 0.022 ± 0.005% by 12 h post-burn. No autophagic cell death was observed in control hearts. Compared with apoptosis, autophagic cell death occurred earlier and in larger quantities. Rapamycin enhanced autophagy and decreased cardiac function in isolated hearts 6 h post-burn, while 3-MA exerted the opposite response. Enalaprilat, losartan, and DPI all inhibited autophagy and enhanced heart function. Conclusion Myocardial autophagy is enhanced in severe burns and autophagic cell death occurred early at 3 h post-burn, which may contribute to post-burn cardiac dysfunction. Angiotensin II and reactive oxygen species may play important roles in this process by regulating cell signaling transduction. PMID:22768082

  4. Plac8 Links Oncogenic Mutations to Regulation of Autophagy and Is Critical to Pancreatic Cancer Progression

    Directory of Open Access Journals (Sweden)

    Conan Kinsey

    2014-05-01

    Full Text Available Mutations in p53 and RAS potently cooperate in oncogenic transformation, and correspondingly, these genetic alterations frequently coexist in pancreatic ductal adenocarcinoma (PDA and other human cancers. Previously, we identified a set of genes synergistically activated by combined RAS and p53 mutations as frequent downstream mediators of tumorigenesis. Here, we show that the synergistically activated gene Plac8 is critical for pancreatic cancer growth. Silencing of Plac8 in cell lines suppresses tumor formation by blocking autophagy, a process essential for maintaining metabolic homeostasis in PDA, and genetic inactivation in an engineered mouse model inhibits PDA progression. We show that Plac8 is a critical regulator of the autophagic machinery, localizing to the lysosomal compartment and facilitating lysosome-autophagosome fusion. Plac8 thus provides a mechanistic link between primary oncogenic mutations and the induction of autophagy, a central mechanism of metabolic reprogramming, during PDA progression.

  5. Inhibition of autophagy initiation potentiates chemosensitivity in mesothelioma.

    Science.gov (United States)

    Follo, Carlo; Cheng, Yao; Richards, William G; Bueno, Raphael; Broaddus, Virginia Courtney

    2018-03-01

    The benefits of inhibiting autophagy in cancer are still controversial, with differences in outcome based on the type of tumor, the context and the particular stage of inhibition. Here, we investigated the impact of inhibiting autophagy at different stages on chemosensitivity using 3-dimensional (3D) models of mesothelioma, including ex vivo human tumor fragment spheroids. As shown by LC3B accumulation, we successfully inhibited autophagy using either an early stage ULK1/2 inhibitor (MRT 68921) or a late stage inhibitor (hydroxychloroquine). We found that inhibition of autophagy at the early stage, but not at late stage, potentiated chemosensitivity. This effect was seen only in those spheroids with high autophagy and active initiation at steady state. Inhibition of autophagy alone, at either early or late stage, did not cause cell death, showing that the inhibitors were non-toxic and that mesothelioma did not depend on autophagy at baseline, at least over 24 h. Using ATG13 puncta analysis, we found that autophagy initiation identified tumors that are more chemosensitive at baseline and after autophagy inhibition. Our results highlight a potential role of autophagy initiation in supporting mesothelioma cells during chemotherapy. Our work also highlights the importance of testing the inhibition of different stages in order to uncover the role of autophagy and the potential of its modulation in the treatment of cancer. © 2017 Wiley Periodicals, Inc.

  6. Wheat homologs of yeast ATG6 function in autophagy and are implicated in powdery mildew immunity.

    Science.gov (United States)

    Yue, Jieyu; Sun, Hong; Zhang, Wei; Pei, Dan; He, Yang; Wang, Huazhong

    2015-04-01

    Autophagy-related ATG6 proteins are pleiotropic proteins functioning in autophagy and the phosphatidylinositol 3-phosphate-signaling pathways. Arabidopsis ATG6 regulates normal plant growth, pollen development and germination, and plant responses to biotic/abiotic stresses. However, the ATG6 functions in wheat (Triticum aestivum L.), an important food crop, are lacking. We identified three members, TaATG6a-6c, of the ATG6 family from common wheat. TaATG6a, 6b and 6c were localized on homeologous chromosomes 3DL, 3BL and 3AL, respectively, of the allo-hexaploid wheat genome, and evidence was provided for their essential role in autophagy. The TaATG6a-GFP fusion protein was found in punctate pre-autophagosomal structures. The expression of each TaATG6 gene restored the accumulation of autophagic bodies in atg6-mutant yeast. Additionally, TaATG6 knockdown plants showed impaired constitutive and pathogen-induced autophagy and growth abnormalities under normal conditions. We also examined the expression patterns of wheat ATG6s for clues to their physiological roles, and found that their expression was induced by the fungus Blumeria graminis f. sp. tritici (Bgt), the causal agent of powdery mildew, and by abiotic stress factors. A role for TaATG6s in wheat immunity to powdery mildew was further implied when knockdowns of TaATG6s weakly compromised the broad-spectrum powdery mildew resistance gene Pm21-triggered resistance response and, conversely and significantly, enhanced the basal resistance of susceptible plants. In addition, leaf cell death was sometimes induced by growth-retarded small Bgt mycelia on susceptible TaATG6 knockdown plants after a long period of interaction. Thus, we provide an important extension of the previous characterization of plant ATG6 genes in wheat, and observed a role for autophagy genes in wheat immune responses to fungal pathogens. Three wheat ATG6s were identified and shown to be essential for autophagy biogenesis. Wheat ATG6s are

  7. Autophagy: one more Nobel Prize for yeast

    Directory of Open Access Journals (Sweden)

    Andreas Zimmermann

    2016-12-01

    Full Text Available The recent announcement of the 2016 Nobel Prize in Physiology or Medicine, awarded to Yoshinori Ohsumi for the discoveries of mechanisms governing autophagy, underscores the importance of intracellular degradation and recycling. At the same time, it further cements yeast, in which this field decisively developed, as a prolific model organism. Here we provide a quick historical overview that mirrors both the importance of autophagy as a conserved and essential process for cellular life and death as well as the crucial role of yeast in its mechanistic characterization.

  8. Molecular Interactions of Autophagy with the Immune System and Cancer

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    Yunho Jin

    2017-08-01

    Full Text Available Autophagy is a highly conserved catabolic mechanism that mediates the degradation of damaged cellular components by inducing their fusion with lysosomes. This process provides cells with an alternative source of energy for the synthesis of new proteins and the maintenance of metabolic homeostasis in stressful environments. Autophagy protects against cancer by mediating both innate and adaptive immune responses. Innate immune receptors and lymphocytes (T and B are modulated by autophagy, which represent innate and adaptive immune responses, respectively. Numerous studies have demonstrated beneficial roles for autophagy induction as well as its suppression of cancer cells. Autophagy may induce either survival or death depending on the cell/tissue type. Radiation therapy is commonly used to treat cancer by inducing autophagy in human cancer cell lines. Additionally, melatonin appears to affect cancer cell death by regulating programmed cell death. In this review, we summarize the current understanding of autophagy and its regulation in cancer.

  9. Role of autophagy in development and progression of acute pancreatitis

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    YANG Shuli

    2014-08-01

    Full Text Available Acute pancreatitis is considered an autodigestive disorder in which inappropriate activation of trypsinogen to trypsin within pancreatic acinar cells leads to the development of pancreatitis. Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents, and it is one of the early pathological processes in acute pancreatitis. Autophagic flux is impaired in acute pancreatitis, which mediates the key pathologic responses of this disease. Impaired autophagy, dysfunction of lysosomes, and dysregulation of autophagy suggest a disorder of the endolysosomal pathway in acute pancreatitis. The role of autophagy in acute pancreatitis is discussed from the aspects of autophagic process, autophagy and activation of trypsinogen, impaired autophagy and acute pancreatitis, and defective autophagy promoting inflammation.

  10. A comparative in-silico analysis of autophagy proteins in ciliates

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    Erhan Aslan

    2017-01-01

    Full Text Available Autophagy serves as a turnover mechanism for the recycling of redundant and/or damaged macromolecules present in eukaryotic cells to re-use them under starvation conditions via a double-membrane structure known as autophagosome. A set of eukaryotic genes called autophagy-related genes (ATGs orchestrate this highly elaborative process. The existence of these genes and the role they play in different eukaryotes are well-characterized. However, little is known of their role in some eukaryotes such as ciliates. Here, we report the computational analyses of ATG genes in five ciliate genomes to understand their diversity. Our results show that Oxytricha trifallax is the sole ciliate which has a conserved Atg12 conjugation system (Atg5-Atg12-Atg16. Interestingly, Oxytricha Atg16 protein includes WD repeats in addition to its N-terminal Atg16 domain as is the case in multicellular organisms. Additionally, phylogenetic analyses revealed that E2-like conjugating protein Atg10 is only present in Tetrahymena thermophila. We fail to find critical autophagy components Atg5, Atg7 and Atg8 in the parasitic ciliate Ichthyophthirius multifiliis. Contrary to previous reports, we also find that ciliate genomes do not encode typical Atg1 since all the candidate sequences lack an Atg1-specific C-terminal domain which is essential for Atg1 complex formation. Consistent with the absence of Atg1, ciliates also lack other members of the Atg1 complex. However, the presence of Atg6 in all ciliates examined here may rise the possibility that autophagosome formation could be operated through Atg6 in ciliates, since Atg6 has been shown as an alternative autophagy inducer. In conclusion, our results highlight that Atg proteins are partially conserved in ciliates. This may provide a better understanding for the autophagic destruction of the parental macronucleus, a developmental process also known as programmed nuclear death in ciliates.

  11. LC3B is indispensable for selective autophagy of p62 but not basal autophagy

    International Nuclear Information System (INIS)

    Maruyama, Yoko; Sou, Yu-Shin; Kageyama, Shun; Takahashi, Takao; Ueno, Takashi; Tanaka, Keiji; Komatsu, Masaaki; Ichimura, Yoshinobu

    2014-01-01

    Highlights: • Knockdown of LC3 or GABARAP families did not affect the basal autophagy. • LC3B has a higher affinity for the autophagy-specific substrate, p62, than GABARAPs. • siRNA-mediated knockdown of LC3B, but not that of GABARAPs, resulted in significant accumulation of p62. - Abstract: Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs

  12. LC3B is indispensable for selective autophagy of p62 but not basal autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yoko [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Department of Pediatrics, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Sou, Yu-Shin; Kageyama, Shun [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Takahashi, Takao [Department of Pediatrics, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Ueno, Takashi [Division of Proteomics and Biomolecular Science, Center for Biomedical Research Resources, Juntendo University Graduate School of Medicine, Tokyo 113-8421 (Japan); Tanaka, Keiji [Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Komatsu, Masaaki, E-mail: komatsu-ms@igakuken.or.jp [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Department of Biochemistry, School of Medicine, Niigata University, Niigata 951-8510 (Japan); Ichimura, Yoshinobu, E-mail: ichimura-ys@igakuken.or.jp [Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan)

    2014-03-28

    Highlights: • Knockdown of LC3 or GABARAP families did not affect the basal autophagy. • LC3B has a higher affinity for the autophagy-specific substrate, p62, than GABARAPs. • siRNA-mediated knockdown of LC3B, but not that of GABARAPs, resulted in significant accumulation of p62. - Abstract: Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.

  13. Spliced XBP1 promotes macrophage survival and autophagy by interacting with Beclin-1

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    Tian, Ping-Ge [Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Zhi-Xin [Centre Laboratory, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China); Li, Jian-Hua [Department of Geriatric Cardiology, Chinese PLA General Hosptial, Beijing 100853 (China); Zhou, Zhe, E-mail: zhouzhe76@126.com [Laboratory of Biotechnology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Zhang, Qing-Hua, E-mail: 1056055170@qq.com [Department of Cardiology, The 305th Hospital of the People' s Liberation Army, Beijing 100017 (China)

    2015-08-07

    Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that the sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE−/− mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis. - Highlights: • XBP1 was up-regulated in atherosclerotic plaques of ApoE−/− mice. • Transient spliced XBP1 overexpression induced macrophages autophagy via Beclin-1. • Sustained spliced XBP1 overexpression triggered macrophages apoptosis. • Spliced XBP1 plays a key role in maintaining the macrophages survival.

  14. Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum.

    Science.gov (United States)

    Corral-Ramos, Cristina; Roca, M Gabriela; Di Pietro, Antonio; Roncero, M Isabel G; Ruiz-Roldán, Carmen

    2015-01-01

    In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum.

  15. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

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    Tashiro, Kanae [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Shishido, Mayumi [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Fujimoto, Keiko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan); Hirota, Yuko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Yo, Kazuyuki; Gomi, Takamasa [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Tanaka, Yoshitaka, E-mail: tanakay@bioc.phar.kyushu-u.ac.jp [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan)

    2014-01-03

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

  16. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    International Nuclear Information System (INIS)

    Tashiro, Kanae; Shishido, Mayumi; Fujimoto, Keiko; Hirota, Yuko; Yo, Kazuyuki; Gomi, Takamasa; Tanaka, Yoshitaka

    2014-01-01

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility

  17. The roles of autophagy and hypoxia in human inflammatory periapical lesions.

    Science.gov (United States)

    Huang, H Y; Wang, W C; Lin, P Y; Huang, C P; Chen, C Y; Chen, Y K

    2018-02-01

    To determine the expressions of hypoxia-related [hypoxia-inducible transcription factors (HIF)-1α, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and phospho-adenosine monophosphate activated protein kinase (pAMPK)] and autophagy-related [microtubule-associated protein 1 light chain 3 (LC3), beclin-1 (BECN-1), autophagy-related gene (Atg)5-12, and p62] proteins in human inflammatory periapical lesions. Fifteen samples of radicular cysts (RCs) and 21 periapical granulomas (PGs), combined with 17 healthy dental pulp tissues, were examined. Enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin (IL)-1β cytokine; immunohistochemical (IHC) and Western blot (WB) analyses were employed to examine autophagy-related and hypoxia-related proteins. Transmission electron microscopy (TEM) was used to explore the ultrastructural morphology of autophagy in periapical lesions. Nonparametric Kruskal-Wallis tests and Mann-Whitney U-tests were used for statistical analyses. ELISA revealed a significantly higher (P periapical lesions than in normal pulp tissue. Immunoscores of IHC expressions of pAMPK, HIF-1α, BNIP3, BECN-1 and Atg5-12 proteins in periapical lesions were significantly higher (P periapical lesions were noted as compared to normal pulp tissue. Upon TEM, ultrastructural double-membrane autophagosomes and autolysosomes were observed in PGs and RCs. Autophagy associated with hypoxia may play a potential causative role in the development and maintenance of inflamed periapical lesions. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  18. Arsenite-induced autophagy is associated with proteotoxicity in human lymphoblastoid cells

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    Bolt, Alicia M.; Zhao, Fei; Pacheco, Samantha; Klimecki, Walter T., E-mail: klimecki@pharmacy.arizona.edu

    2012-10-15

    Epidemiological studies of arsenic-exposed populations have provided evidence that arsenic exposure in humans is associated with immunosuppression. Previously, we have reported that arsenite-induced toxicity is associated with the induction of autophagy in human lymphoblastoid cell lines (LCL). Autophagy is a cellular process that functions in the degradation of damaged cellular components, including protein aggregates formed by misfolded or damaged proteins. Accumulation of misfolded or damaged proteins in the endoplasmic reticulum (ER) lumen causes ER stress and activates the unfolded protein response (UPR). In an effort to investigate the mechanism of autophagy induction by arsenite in the LCL model, we examined the potential contribution of ER stress and activation of the UPR. LCL exposed to sodium arsenite for 8-days induced expression of UPR-activated genes, including CHOP and GRP78, at the RNA and the protein level. Evidence for activation of the three arms of the UPR was observed. The arsenite-induced activation of the UPR was associated with an accumulation of protein aggregates containing p62 and LC3, proteins with established roles in the sequestration and autophagic clearance of protein aggregates. Taken together, these data provide evidence that arsenite-induced autophagy is associated with the generation of ER stress, activation of the UPR, and formation of protein aggregates that may be targeted to the lysosome for degradation. -- Highlights: ► Arsenite induces endoplasmic reticulum stress and the unfolded protein response. ► Arsenite induces the formation of protein aggregates that contain p62 and LC3-II. ► Time-course data suggests that arsenite-induced autophagy precedes ER stress.

  19. The activation of autophagy protects neurons and astrocytes against bilirubin-induced cytotoxicity.

    Science.gov (United States)

    Qaisiya, Mohammed; Mardešić, Paula; Pastore, Beatrice; Tiribelli, Claudio; Bellarosa, Cristina

    2017-11-20

    Unconjugated bilirubin (UCB) neurotoxicity involves oxidative stress, calcium signaling and ER-stress. The same insults can also induce autophagy, a process of "self-eating", with both a pro-survival or a pro-apoptotic role. Our aim was to study the outcome of autophagy activation by UCB in the highly sensitive neuronal SH-SY5Y cells and in the resistant astrocytoma U87 cells. Upon treatment with a toxic dose of UCB, the conversion of LC3-I to LC3-II was detected in both cell lines. Inhibition of autophagy by E64d before UCB treatment increased SH-SY5Y cell mortality and made U87 cells sensitive to UCB. In SH-SY5Y autophagy related genes ATG8 (5 folds), ATG18 (5 folds), p62 (3 folds) and FAM 129A (4.5 folds) were induced 8h after UCB treatment while DDIT4 upregulation (13 folds) started at 4h. mTORC1 inactivation by UCB was confirmed by phosphorylation of 4EBP1. UCB induced LC3-II conversion was completely prevented by pretreating cells with the calcium chelator BAPTA and reduced by 65% using the ER-stress inhibitor 4-PBA. Pretreatment with the PKC inhibitor reduced LC3 mRNA by 70% as compared to cells exposed to UCB alone. Finally, autophagy induction by Trifluoroperazine (TFP) increased the cell viability of rat hippocampal primary neurons upon UCB treatment from 60% to 80%. In SH-SY5Y cells, TFP pretreatment blocked the UCB-induced cleaved caspase-3 protein expression, decreased LDH release from 50% to 23%, reduced the UCB-induction of HO1, CHOP and IL-8 mRNAs by 85%, 70% and 97%. Collectively these data indicate that the activation of autophagy protects neuronal cells from UCB cytotoxicity. The mechanisms of autophagy activation by UCB involves mTOR/ER-stress/PKC/calcium signaling. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Shear Stress Induces Phenotypic Modulation of Vascular Smooth Muscle Cells via AMPK/mTOR/ULK1-Mediated Autophagy.

    Science.gov (United States)

    Sun, Liqian; Zhao, Manman; Liu, Aihua; Lv, Ming; Zhang, Jingbo; Li, Youxiang; Yang, Xinjian; Wu, Zhongxue

    2018-03-01

    Phenotypic modulation of vascular smooth muscle cells (VSMCs) is involved in the pathophysiological processes of the intracranial aneurysms (IAs). Although shear stress has been implicated in the proliferation, migration, and phenotypic conversion of VSMCs, the molecular mechanisms underlying these events are currently unknown. In this study, we investigated whether shear stress(SS)-induced VSMC phenotypic modulation was mediated by autophagy involved in adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) pathway. The results show that shear stress could inhibit the expression of key VSMC contractile genes and induce pro-inflammatory/matrix-remodeling genes levels, contributing to VSMCs phenotypic switching from a contractile to a synthetic phenotype. More importantly, Shear stress also markedly increased the levels of the autophagy marker microtubule-associated protein light chain 3-II (LC3II), Beclin-1, and p62 degradation. The autophagy inhibitor 3-methyladenine (3-MA) significantly blocked shear-induced phenotypic modulation of VSMCs. To further explore the molecular mechanism involved in shear-induced autophagy, we found that shear stress could activate AMPK/mTOR/ULK1 signaling pathway in VSMCs. Compound C, a pharmacological inhibitor of AMPK, significantly reduced the levels of p-AMPK and p-ULK, enhanced p-mTOR level, and finally decreased LC3II and Beclin-1 level, which suggested that activated AMPK/mTOR/ULK1 signaling was related to shear-mediated autophagy. These results indicate that shear stress promotes VSMC phenotypic modulation through the induction of autophagy involved in activating the AMPK/mTOR/ULK1 pathway.

  1. Enhancement of Autophagy by Simvastatin through Inhibition of Rac1-mTOR Signaling Pathway in Coronary Arterial Myocytes

    Directory of Open Access Journals (Sweden)

    Yu-Miao Wei

    2013-06-01

    Full Text Available Background/Aims: In addition to their action of lowering blood cholesterol levels, statins modulate biological characteristics and functions of arterial myocytes such as viability, proliferation, apoptosis, survival and contraction. The present study tested whether simvastatin, as a prototype statin, enhances autophagy in coronary arterial myocytes (CAMs to thereby exert their beneficial effects in atherosclerosis. Methods and Results: Using flow cytometry, we demonstrated that simvastatin significantly increased the autophagsome formation in CAMs. Western blot analysis confirmed that simvastatin significantly increased protein expression of typical autophagy markers LC3B and Beclin1 in these CAMs. Confocal microscopy further demonstrated that simvastatin increased fusion of autophagosomes with lysosomes, which was blocked by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Simvastatin reduced mammalian target of rapamycin (mTOR activity, which was reversed by Rac1-GTPase overexpression and the mTOR agonist phosphatidic acid. Moreover, both Rac1-GTPase overexpression and activation of mTOR by phosphatidic acid drastically blocked simvastatin-induced autophagosome formation in CAMs. Interestingly, simvastatin increased protein expression of a contractile phenotype marker calponin in CAMs, which was blocked by autophagy inhibitor 3-methyladenine. Simvastatin markedly reduced proliferation of CAMs under both control and proatherogenic stimulation. However, this inhibitory effect of simvastatin on CAM proliferation was blocked by by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Lastly, animal experiments demonstrated that simvastatin increased protein expression of LC3B and calponin in mouse coronary arteries. Conclusion: Our results indicate that simvastatin inhibits the Rac1-mTOR pathway and thereby increases autophagy in CAMs which may stabilize CAMs in the contractile phenotype to prevent proliferation and growth

  2. Autophagy resolves early retinal inflammation in Igf1-deficient mice

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    Ana I. Arroba

    2016-09-01

    Full Text Available Insulin-like growth factor-1 (IGF-1 is a growth factor with differentiating, anti-apoptotic and metabolic functions in the periphery, and anti-inflammatory properties in the nervous system. Mice that have mutations in the Igf1 gene, rendering the gene product inactive (Igf1−/−, present with age-related visual loss accompanied by structural alterations in the first synapses of the retinal pathway. Recent advances have revealed a crucial role of autophagy in immunity and inflammation. Keeping in mind this close relationship, we aimed to decipher these processes in the context of the defects that occur during ageing in the retina of Igf1−/− mice. Tnfa and Il1b mRNAs, and phosphorylation of JNK and p38 MAPK were elevated in the retinas of 6- and 12-month old Igf1−/− mice compared to those in age-matched Igf1+/+ controls. In 6-month-old Igf1−/− retinas, increased mRNA levels of the autophagy mediators Becn1, Atg9, Atg5 and Atg4, decreased p62 (also known as SQSTM1 protein expression together with an increased LC3-II:LC3-I ratio reflected active autophagic flux. However, in retinas from 12-month-old Igf1−/− mice, Nlrp3 mRNA, processing of the IL1β pro-form and immunostaining of active caspase-1 were elevated compared to those in age-matched Igf1+/+ controls, suggesting activation of the inflammasome. This effect concurred with accumulation of autophagosomes and decreased autophagic flux in the retina. Microglia localization and status of activation in the retinas of 12-month-old Igf1+/+ and Igf1−/− mice, analyzed by immunostaining of Cd11b and Iba-1, showed a specific distribution pattern in the outer plexiform layer (OPL, inner plexiform layer (IPL and inner nuclear layer (INL, and revealed an increased number of activated microglia cells in the retina of 12-month-old blind Igf1−/− mice. Moreover, reactive gliosis was exclusively detected in the retinas from 12-month-old blind Igf1−/− mice. In conclusion, this study

  3. CD4 T cell autophagy is integral to memory maintenance.

    Science.gov (United States)

    Murera, Diane; Arbogast, Florent; Arnold, Johan; Bouis, Delphine; Muller, Sylviane; Gros, Frédéric

    2018-04-13

    Studies of mice deficient for autophagy in T cells since thymic development, concluded that autophagy is integral to mature T cell homeostasis. Basal survival and functional impairments in vivo, limited the use of these models to delineate the role of autophagy during the immune response. We generated Atg5 f/f distal Lck (dLck)-cre mice, with deletion of autophagy only at a mature stage. In this model, autophagy deficiency impacts CD8 + T cell survival but has no influence on CD4 + T cell number and short-term activation. Moreover, autophagy in T cells is dispensable during early humoral response but critical for long-term antibody production. Autophagy in CD4 + T cells is required to transfer humoral memory as shown by injection of antigen-experienced cells in naive mice. We also observed a selection of autophagy-competent cells in the CD4 + T cell memory compartment. We performed in vitro differentiation of memory CD4 + T cells, to better characterize autophagy-deficient memory cells. We identified mitochondrial and lipid load defects in differentiated memory CD4 + T cells, together with a compromised survival, without any collapse of energy production. We then propose that memory CD4 + T cells rely on autophagy for their survival to regulate toxic effects of mitochondrial activity and lipid overload.

  4. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

  5. Autophagy and Microglia: Novel Partners in Neurodegeneration and Aging.

    Science.gov (United States)

    Plaza-Zabala, Ainhoa; Sierra-Torre, Virginia; Sierra, Amanda

    2017-03-09

    Autophagy is emerging as a core regulator of Central Nervous System (CNS) aging and neurodegeneration. In the brain, it has mostly been studied in neurons, where the delivery of toxic molecules and organelles to the lysosome by autophagy is crucial for neuronal health and survival. However, we propose that the (dys)regulation of autophagy in microglia also affects innate immune functions such as phagocytosis and inflammation, which in turn contribute to the pathophysiology of aging and neurodegenerative diseases. Herein, we first describe the basic concepts of autophagy and its regulation, discuss key aspects for its accurate monitoring at the experimental level, and summarize the evidence linking autophagy impairment to CNS senescence and disease. We focus on acute, chronic, and autoimmunity-mediated neurodegeneration, including ischemia/stroke, Alzheimer's, Parkinson's, and Huntington's diseases, and multiple sclerosis. Next, we describe the actual and potential impact of autophagy on microglial phagocytic and inflammatory function. Thus, we provide evidence of how autophagy may affect microglial phagocytosis of apoptotic cells, amyloid-β, synaptic material, and myelin debris, and regulate the progression of age-associated neurodegenerative diseases. We also discuss data linking autophagy to the regulation of the microglial inflammatory phenotype, which is known to contribute to age-related brain dysfunction. Overall, we update the current knowledge of autophagy and microglia, and highlight as yet unexplored mechanisms whereby autophagy in microglia may contribute to CNS disease and senescence.

  6. Impairment of autophagy: From hereditary disorder to drug intoxication

    International Nuclear Information System (INIS)

    Aki, Toshihiko; Funakoshi, Takeshi; Unuma, Kana; Uemura, Koichi

    2013-01-01

    At first, the molecular mechanism of autophagy was unveiled in a unicellular organism Saccharomyces cerevisiae (budding yeast), followed by the discovery that the basic mechanism of autophagy is conserved in multicellular organisms including mammals. Although autophagy was considered to be a non-selective bulk protein degradation system to recycle amino acids during periods of nutrient starvation, it is also believed to be an essential mechanism for the selective elimination of proteins/organelles that are damaged under pathological conditions. Research advances made using autophagy-deficient animals have revealed that impairments of autophagy often underlie the pathogenesis of hereditary disorders such as Danon, Parkinson's, Alzheimer's, and Huntington's diseases, and amyotrophic lateral sclerosis. On the other hand, there are many reports that drugs and toxicants, including arsenic, cadmium, paraquat, methamphetamine, and ethanol, induce autophagy during the development of their toxicity on many organs including heart, brain, lung, kidney, and liver. Although the question as to whether autophagic machinery is involved in the execution of cell death or not remains controversial, the current view of the role of autophagy during cell/tissue injury is that it is an important, often essential, cytoprotective reaction; disturbances in cytoprotective autophagy aggravate cell/tissue injuries. The purpose of this review is to provide (1) a gross summarization of autophagy processes, which are becoming more important in the field of toxicology, and (2) examples of important studies reporting the involvement of perturbations in autophagy in cell/tissue injuries caused by acute as well as chronic intoxication

  7. Induction of cytoprotective autophagy in PC-12 cells by cadmium

    International Nuclear Information System (INIS)

    Wang, Qiwen; Zhu, Jiaqiao; Zhang, Kangbao; Jiang, Chenyang; Wang, Yi; Yuan, Yan; Bian, Jianchun; Liu, Xuezhong; Gu, Jianhong; Liu, Zongping

    2013-01-01

    Highlights: •Cadmium can promote early upregulation of autophagy in PC-12 cells. •Autophagy precedes apoptosis in cadmium-treated PC-12 cells. •Cadmium-induced autophagy is cytoprotective in PC-12 cells. •Class III PI3K/beclin-1/Bcl-2 signaling pathway plays a positive role in cadmium-triggered autophagy. -- Abstract: Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity

  8. Brucella Melitensis 16M Regulates the Effect of AIR Domain on Inflammatory Factors, Autophagy, and Apoptosis in Mouse Macrophage through the ROS Signaling Pathway.

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    Tiansen Li

    Full Text Available Brucellosis is a highly contagious zoonosis caused by Brucella. Brucella can invade and persist inside host cells, which results in chronic infection. We constructed AIR interference and overexpression lentiviruses to acquire AIR interference, overexpression, and rescue stable expression cell lines. We also established a Brucella melitensis 16M-infected macrophage model, which was treated with either the vehicle control or NAC (ROS scavenger N-acetylcysteine (NAC for 0, 3, 6, 12, and 24 h. Confocal laser microscopy, transmission electron microscopy, fluorescence quantitative PCR, flow cytometry, ELISA, and Western blot were used to detect inflammation, cell autophagy and apoptosis-related protein expression levels, ROS levels, and the distribution of mitochondria. It was found that after interference and overexpression of AIR, ROS release was significantly changed, and mitochondria became abnormally aggregated. B. melitensis 16M activated the NLRP3/AIM2 inflammatory complex, and induced RAW264.7 cells to secrete IL-1β and IL-18 through the ROS pathway. B. melitensis 16M also altered autophagy-related gene expression, increased autophagy activity, and induced cell apoptosis through the ROS pathway. The results showed that after B. melitensis 16M infection, ROS induced apoptosis, inflammation, and autophagy while AIR inhibited autophagosome maturation and autophagy initiation. Autophagy negatively regulated the activation of inflammasomes and prevented inflammation from occurring. In addition, mitophagy could promote cell apoptosis.

  9. Chloroquine enhances the efficacy of cisplatin by suppressing autophagy in human adrenocortical carcinoma treatment

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    Qin L

    2016-03-01

    Full Text Available Liang Qin,1,* Tianyuan Xu,1,* Leilei Xia,1 Xianjin Wang,1 Xiang Zhang,1 Xiaohua Zhang,1 Zhaowei Zhu,1 Shan Zhong,1 Chuandong Wang,2 Zhoujun Shen1 1Department of Urology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 2Key Laboratory of Stem Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: It has been demonstrated that chloroquine (CQ enhances the efficacy of chemotherapy. However, little is known about whether CQ could enhance the efficacy of cisplatin (DDP in the treatment of adrenocortical carcinoma (ACC. In this study, we explore the efficacy and mechanism by which CQ affects DDP sensitivity in human ACC in vitro and in vivo.Methods: The autophagic gene Beclin-1 expression was detected by immunohistochemistry, and the protein levels were analyzed using immunoblotting assays of ACC tissues and normal adrenal cortex tissues. The ACC SW13 cells were treated with DDP and/or CQ. The cell viability assay was performed using the MTT method. Qualitative autophagy detection was performed by monodansylcadaverine staining of autophagic vacuoles. Annexin V-fluorescein isothiocyanate/propidium iodide double staining was used to count cell apoptosis by flow cytometry. The autophagy-related protein (Beclin-1, LC3, and p62 and apoptosis relative protein (Bax and Bcl-2 levels were evaluated with Western blot analysis. Furthermore, a murine model of nude BALB/c mice bearing SW13 cell xenografts was established to evaluate the efficacy of concomitant therapy.Results: The expression of the autophagic gene Beclin-1 was significantly downregulated in ACC tissues compared to normal adrenal cortex tissues. The Beclin-1 protein level in ACC tissues was lower than that in normal adrenal cortex tissues (P<0.05. In vitro concomitant therapy (DDP and CQ was more

  10. Methods for assessing autophagy and autophagic cell death.

    Science.gov (United States)

    Tasdemir, Ezgi; Galluzzi, Lorenzo; Maiuri, M Chiara; Criollo, Alfredo; Vitale, Ilio; Hangen, Emilie; Modjtahedi, Nazanine; Kroemer, Guido

    2008-01-01

    Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. We also review the techniques currently available to detect autophagy, including transmission electron microscopy, half-life assessments of long-lived proteins, detection of LC3 maturation/aggregation, fluorescence microscopy, and colocalization of mitochondrion- or endoplasmic reticulum-specific markers with lysosomal proteins. Massive autophagic vacuolization may cause cellular stress and represent a frustrated attempt of adaptation. In this case, cell death occurs with (or in spite of) autophagy. When cell death occurs through autophagy, on the contrary, the inhibition of the autophagic process should prevent cellular demise. Accordingly, we describe a strategy for discriminating cell death with autophagy from cell death through autophagy.

  11. Autophagy as a potential target for sarcoma treatment.

    Science.gov (United States)

    Min, Li; Choy, Edwin; Pollock, Raphael E; Tu, Chongqi; Hornicek, Francis; Duan, Zhenfeng

    2017-08-01

    Autophagy is a constitutively active, evolutionary conserved, catabolic process for maintaining homeostasis in cellular stress responses and cell survival. Although its mechanism has not been fully illustrated, recent work on autophagy in various types of sarcomas has demonstrated that autophagy exerts an important role in sarcoma cell growth and proliferation, in pro-survival response to therapies and stresses, and in therapeutic resistance of sarcoma. Thus, the autophagic process is being seen as a possibly novel therapeutic target of sarcoma. Additionally, some co-regulators of autophagy have also been investigated as promising biomarkers for the diagnosis and prognosis of sarcoma. In this review, we summarize contemporary advances in the role of autophagy in sarcoma and discuss the potential of autophagy as a new target for sarcoma treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Autophagy Facilitates Salmonella Replication in HeLa Cells

    Science.gov (United States)

    Yu, Hong B.; Croxen, Matthew A.; Marchiando, Amanda M.; Ferreira, Rosana B. R.; Cadwell, Ken; Foster, Leonard J.; Finlay, B. Brett

    2014-01-01

    ABSTRACT Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. PMID:24618251

  13. Forms, Crosstalks, and the Role of Phospholipid Biosynthesis in Autophagy

    Directory of Open Access Journals (Sweden)

    Leanne Pereira

    2012-01-01

    Full Text Available Autophagy is a highly conserved cellular process occurring during periods of stress to ensure a cell's survival by recycling cytosolic constituents and making products that can be used in energy generation and other essential processes. Three major forms of autophagy exist according to the specific mechanism through which cytoplasmic material is transported to a lysosome. Chaperone-mediated autophagy is a highly selective form of autophagy that delivers specific proteins for lysosomal degradation. Microautophagy is a less selective form of autophagy that occurs through lysosomal membrane invaginations, forming tubes and directly engulfing cytoplasm. Finally, macroautophagy involves formation of new membrane bilayers (autophagosomes that engulf cytosolic material and deliver it to lysosomes. This review provides new insights on the crosstalks between different forms of autophagy and the significance of bilayer-forming phospholipid synthesis in autophagosomal membrane formation.

  14. Uvrag targeting by Mir125a and Mir351 modulates autophagy associated with Ewsr1 deficiency.

    Science.gov (United States)

    Kim, Yunha; Kang, Young-Sook; Lee, Na-Young; Kim, Ki Yoon; Hwang, Yu Jin; Kim, Hyun-Wook; Rhyu, Im Joo; Her, Song; Jung, Min-Kyung; Kim, Sun; Lee, Chai-Jin; Ko, Seyoon; Kowall, Neil W; Lee, Sean Bong; Lee, Junghee; Ryu, Hoon

    2015-01-01

    The EWSR1 (EWS RNA-binding protein 1/Ewing Sarcoma Break Point Region 1) gene encodes a RNA/DNA binding protein that is ubiquitously expressed and involved in various cellular processes. EWSR1 deficiency leads to impairment of development and accelerated senescence but the mechanism is not known. Herein, we found that EWSR1 modulates the Uvrag (UV radiation resistance associated) gene at the post-transcription level. Interestingly, EWSR1 deficiency led to the activation of the DROSHA-mediated microprocessor complex and increased the level of Mir125a and Mir351, which directly target Uvrag. Moreover, the Mir125a- and Mir351-mediated reduction of Uvrag was associated with the inhibition of autophagy that was confirmed in ewsr1 knockout (KO) MEFs and ewsr1 KO mice. Taken together, our data indicate that EWSR1 is involved in the post-transcriptional regulation of Uvrag via a miRNA-dependent pathway, resulting in the deregulation of autophagy inhibition. The mechanism of Uvrag and autophagy regulation by EWSR1 provides new insights into the role of EWSR1 deficiency-related cellular dysfunction.

  15. Autophagy provides nutrients for nonassimilating fungal structures and is necessary for plant colonization but not for infection in the necrotrophic plant pathogen Fusarium graminearum

    DEFF Research Database (Denmark)

    Josefsen, Lone; Droce, Aida; Sondergaard, Teis Esben

    2012-01-01

    starvation is severely inhibited in the Delta Fgatg8 strain demonstrating autophagy-dependent lipid utilization, lipophagy, in fungi. Radial growth rate of the Delta Fgatg8 strain is reduced compared with the wild type and the mutant does not grow over inert plastic surfaces in contrast to the wild type....... The ability to infect barley and wheat is normal but the mutant is unable to spread from spikelet to spikelet in wheat. Complementation by inserting the F. graminearum atg8 gene into a region adjacent to the actin gene in Delta Fgatg8 fully restores the WT phenotype. The results showed that autophagy plays...

  16. Parkinson's Disease: Leucine-Rich Repeat Kinase 2 and Autophagy, Intimate Enemies

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    José M. Bravo-San Pedro

    2012-01-01

    Full Text Available Parkinson's disease is the second common neurodegenerative disorder, after Alzheimer's disease. It is a clinical syndrome characterized by loss of dopamine-generating cells in the substancia nigra, a region of the midbrain. The etiology of Parkinson's disease has long been through to involve both genetic and environmental factors. Mutations in the leucine-rich repeat kinase 2 gene cause late-onset Parkinson's disease with a clinical appearance indistinguishable from Parkinson's disease idiopathic. Autophagy is an intracellular catabolic mechanism whereby a cell recycles or degrades damage proteins and cytoplasmic organelles. This degradative process has been associated with cellular dysfunction in neurodegenerative processes including Parkinson's disease. We discuss the role of leucine-rich repeat kinase 2 in autophagy, and how the deregulations of this degradative mechanism in cells can be implicated in the Parkinson's disease etiology.

  17. Induction of autophagy is essential for monocyte-macrophage differentiation

    OpenAIRE

    Zhang, Yan; Morgan, Michael J.; Chen, Kun; Choksi, Swati; Liu, Zheng-gang

    2012-01-01

    Monocytes are programmed to undergo apoptosis in the absence of stimulation. Stimuli that promote monocyte-macrophage differentiation not only cause cellular changes, but also prevent the default apoptosis of monocytes. In the present study, we demonstrate that autophagy is induced when monocytes are triggered to differentiate and that the induction of autophagy is pivotal for the survival and differentiation of monocytes. We also show that inhibition of autophagy results in apoptosis of cell...

  18. Targeting Pediatric Glioma with Apoptosis and Autophagy Manipulation

    Science.gov (United States)

    2014-10-01

    that chloroquine treatments give the most reliable inhibition of autophagy without being directly cytotoxic. Bafilomycin can continue to be used for...in pediatric glioma and its interaction with RTK inhibition and apoptotic pathway activation will enable us to develop efficacious clinical trials...of autophagy, Rab7 and Lamp 2. We are now introducing siRNA against Rab7 and Lamp2 to reiterate the effects of Chloroquine inhibition of autophagy

  19. The Mucosal Immune System and Its Regulation by Autophagy.

    Science.gov (United States)

    Kabat, Agnieszka M; Pott, Johanna; Maloy, Kevin J

    2016-01-01

    The gastrointestinal tract presents a unique challenge to the mucosal immune system, which has to constantly monitor the vast surface for the presence of pathogens, while at the same time maintaining tolerance to beneficial or innocuous antigens. In the intestinal mucosa, specialized innate and adaptive immune components participate in directing appropriate immune responses toward these diverse challenges. Recent studies provide compelling evidence that the process of autophagy influences several aspects of mucosal immune responses. Initially described as a "self-eating" survival pathway that enables nutrient recycling during starvation, autophagy has now been connected to multiple cellular responses, including several aspects of immunity. Initial links between autophagy and host immunity came from the observations that autophagy can target intracellular bacteria for degradation. However, subsequent studies indicated that autophagy plays a much broader role in immune responses, as it can impact antigen processing, thymic selection, lymphocyte homeostasis, and the regulation of immunoglobulin and cytokine secretion. In this review, we provide a comprehensive overview of mucosal immune cells and discuss how autophagy influences many aspects of their physiology and function. We focus on cell type-specific roles of autophagy in the gut, with a particular emphasis on the effects of autophagy on the intestinal T cell compartment. We also provide a perspective on how manipulation of autophagy may potentially be used to treat mucosal inflammatory disorders.

  20. Glucocorticoids induce autophagy in rat bone marrow mesenchymal stem cells

    DEFF Research Database (Denmark)

    Wang, L.; Fan, J.; Lin, Y. S.

    2015-01-01

    Glucocorticoidinduced osteoporosis (GIOP) is a widespread clinical complication following glucocorticoid therapy. This irreversible damage to boneforming and resorbing cells is essential in the pathogenesis of osteoporosis. Autophagy is a physiological process involved in the regulation of cells...... and their responses to diverse stimuli, however, the role of autophagy in glucocorticoidinduced damage to bone marrow mesenchymal stem cells (BMSCs) remains unclear. The current study confirmed that glucocorticoid administration impaired the proliferation of BMSCs. Transmission electron microscopy...... that in response to glucocorticoid administration, induced autophagy aids to maintain proliferation and prevent apoptosis of BMSCs. Thus, it is hypothesized that autophagy may be a novel target in the treatment or prevention of osteoporosis....

  1. Heme oxygenase-1 prevents cardiac dysfunction in streptozotocin-diabetic mice by reducing inflammation, oxidative stress, apoptosis and enhancing autophagy.

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    Yanli Zhao

    Full Text Available Heme oxygenase-1 (HO-1 has been implicated in cardiac dysfunction, oxidative stress, inflammation, apoptosis and autophagy associated with heart failure, and atherosclerosis, in addition to its recognized role in metabolic syndrome and diabetes. Numerous studies have presented contradictory findings about the role of HO-1 in diabetic cardiomyopathy (DCM. In this study, we explored the role of HO-1 in myocardial dysfunction, myofibril structure, oxidative stress, inflammation, apoptosis and autophagy using a streptozotocin (STZ-induced diabetes model in mice systemically overexpressing HO-1 (Tg-HO-1 or mutant HO-1 (Tg-mutHO-1. The diabetic mouse model was induced by multiple peritoneal injections of STZ. Two months after injection, left ventricular (LV function was measured by echocardiography. In addition, molecular biomarkers related to oxidative stress, inflammation, apoptosis and autophagy were evaluated using classical molecular biological/biochemical techniques. Mice with DCM exhibited severe LV dysfunction, myofibril structure disarray, aberrant cardiac oxidative stress, inflammation, apoptosis, autophagy and increased levels of HO-1. In addition, we determined that systemic overexpression of HO-1 ameliorated left ventricular dysfunction, myofibril structure disarray, oxidative stress, inflammation, apoptosis and autophagy in DCM mice. Furthermore, serine/threonine-specific protein kinase (Akt and AMP-activated protein kinase (AMPK phosphorylation is normally inhibited in DCM, but overexpression of the HO-1 gene restored the phosphorylation of these kinases to normal levels. In contrast, the functions of HO-1 in DCM were significantly reversed by overexpression of mutant HO-1. This study underlines the unique roles of HO-1, including the inhibition of oxidative stress, inflammation and apoptosis and the enhancement of autophagy, in the pathogenesis of DCM.

  2. Dihydrocapsaicin (DHC), a saturated structural analog of capsaicin, induces autophagy in human cancer cells in a catalase-regulated manner.

    Science.gov (United States)

    Oh, Seon Hee; Kim, Young Soon; Lim, Sung Chul; Hou, Yi Feng; Chang, In Youb; You, Ho Jin

    2008-11-01

    Although capsaicin, a pungent component of red pepper, is known to induce apoptosis in several types of cancer cells, the mechanisms underlying capsaicin-induced cytotoxicity are unclear. Here, we showed that dihydrocapsaicin (DHC), an analog of capsaicin, is a potential inducer of autophagy. DHC was more cytotoxic than capsaicin in HCT116, MCF-7 and WI38 cell lines. Capsaicin and DHC did not affect the sub-G(1) apoptotic peak, but induced G(0)/G(1) arrest in HCT116 and MCF-7 cells. DHC caused the artificial autophagosome marker GFP-LC3 to redistribute and upregulated expression of autophagy-related proteins. Blocking of autophagy by 3-methyladenine (3MA) as well as siRNA Atg5 induced a high level of caspase-3 activation. Although pretreatment with zVAD completely inhibited caspase-3 activation by 3MA, it did not prevent cell death. DHC-induced autophagy was enhanced by zVAD pretreatment, as shown by increased accumulation of LC3-II protein. DHC attenuated basal ROS levels through catalase induction; this effect was enhanced by antioxidants, which increased both LC3-II expression and caspase-3 activation. The catalase inhibitor 3-amino-1,2,4-triazole (3AT) abrogated DHC-induced expression of LC3-II, overexpression of the catalase gene increased expression of LC3-II protein, and knockdown decreased it. Additionally, DHC-induced autophagy was independent of p53 status. Collectively, DHC activates autophagy in a p53-independent manner and that may contribute to cytotoxicity of DHC.

  3. Grouping annotations on the subcellular layered interactome demonstrates enhanced autophagy activity in a recurrent experimental autoimmune uveitis T cell line.

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    Xiuzhi Jia

    Full Text Available Human uveitis is a type of T cell-mediated autoimmune disease that often shows relapse-remitting courses affecting multiple biological processes. As a cytoplasmic process, autophagy has been seen as an adaptive response to cell death and survival, yet the link between autophagy and T cell-mediated autoimmunity is not certain. In this study, based on the differentially expressed genes (GSE19652 between the recurrent versus monophasic T cell lines, whose adoptive transfer to susceptible animals may result in respective recurrent or monophasic uveitis, we proposed grouping annotations on a subcellular layered interactome framework to analyze the specific bioprocesses that are linked to the recurrence of T cell autoimmunity. That is, the subcellular layered interactome was established by the Cytoscape and Cerebral plugin based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The analysis showed that significant bioprocesses with autophagy were orchestrated in the cytoplasmic layered interactome and that mTOR may have a regulatory role in it. Furthermore, by setting up recurrent and monophasic uveitis in Lewis rats, we confirmed by transmission electron microscopy that, in comparison to the monophasic disease, recurrent uveitis in vivo showed significantly increased autophagy activity and extended lymphocyte infiltration to the affected retina. In summary, our framework methodology is a useful tool to disclose specific bioprocesses and molecular targets that can be attributed to a certain disease. Our results indicated that targeted inhibition of autophagy pathways may perturb the recurrence of uveitis.

  4. Dysregulation of Autophagy Contributes to Anal Carcinogenesis.

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    Evie H Carchman

    Full Text Available Autophagy is an intracellular catabolic process that removes and recycles unnecessary/dysfunctional cellular components, contributing to cellular health and survival. Autophagy is a highly regulated cellular process that responds to several intracellular signals, many of which are deregulated by human papillomavirus (HPV infection through the expression of HPV-encoded oncoproteins. This adaptive inhibitory response helps prevent viral clearance. A strong correlation remains between HPV infection and the development of squamous cell carcinoma (SCC of the anus, particularly in HIV positive and other immunosuppressed patients. We hypothesize that autophagy is inhibited by HPV-encoded oncoproteins thereby promoting anal carcinogenesis (Fig 1.HPV16 transgenic mice (K14E6/E7 and non-transgenic mice (FVB/N, both of which do not spontaneously develop anal tumors, were treated topically with the chemical carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA, to induce anal cancer. The anuses at different time points of treatment (5, 10, 15 and 20 weeks were analyzed using immunofluorescence (IF for two key autophagy marker proteins (LC3β and p62 in addition to histological grading. The anuses from the K14E6/E7 mice were also analyzed for visual evidence of autophagic activity by electron microscopy (EM. To see if there was a correlation to humans, archival anal specimens were assessed histologically for grade of dysplasia and then analyzed for LC3β and p62 protein content. To more directly examine the effect of autophagic inhibition on anal carcinogenesis, nontransgenic mice that do not develop anal cancer with DMBA treatment were treated with a known pharmacologic inhibitor of autophagy, chloroquine, and examined for tumor development and analyzed by IF for autophagic proteins.Histologically, we observed the progression of normal anoderm to invasive SCC with DMBA treatment in K14E6/E7 mice but not in nontransgenic, syngeneic FVB/N background control mice

  5. Autophagy and phagocytosis converge for better vision.

    Science.gov (United States)

    Ferguson, Thomas A; Green, Douglas R

    2014-01-01

    The retinal pigment epithelium (RPE) is a single layer of nonregenerating cells essential to homeostasis in the retina and the preservation of vision. While the RPE perform a number of important functions, 2 essential processes are phagocytosis, which removes the most distal tips of the photoreceptors to support disk renewal, and the visual cycle, which maintains the supply of chromophore for regeneration of photo-bleached visual pigments. We recently reported that these processes are linked by a noncanonical form of autophagy termed LC3-associated phagocytosis (LAP) in which components of the autophagy pathway are co-opted by phagocytosis to recover vitamin A in support of optimal vision. Here we summarize these findings.

  6. Quiltophagy--autophagy as folk art.

    Science.gov (United States)

    Crumrine, Barbara M; Klionsky, Daniel J

    2015-01-01

    Over the years macroautophagy (hereafter autophagy) has been depicted artistically through painting, music, dance, videos, and poetry. A unifying idea behind these different aesthetic approaches is that people learn in different ways. Thus, some learners may be engaged by a detailed, but static, painting, whereas others may find insight through the dynamic visualization provided by a dance. While each of these formats has advantages, they also have a common weakness--whether delivered through watercolor on a canvas, words on a paper, or movement captured in a video, they are all 2-dimensional. Yet, some people are tactile learners. In this paper, a quilter describes a project she created with the goal of demonstrating autophagy using a 3-dimensional approach, in which different fiber textures could be used to elaborate certain parts of the process.

  7. A Molecular View of Autophagy in Lepidoptera

    OpenAIRE

    Romanelli, Davide; Casati, Barbara; Franzetti, Eleonora; Tettamanti, Gianluca

    2014-01-01

    Metamorphosis represents a critical phase in the development of holometabolous insects, during which the larval body is completely reorganized: in fact, most of the larval organs undergo remodeling or completely degenerate before the final structure of the adult insect is rebuilt. In the past, increasing evidence emerged concerning the intervention of autophagy and apoptosis in the cell death processes that occur in larval organs of Lepidoptera during metamorphosis, but a molecular characteri...

  8. β-Cell Autophagy in Diabetes Pathogenesis.

    Science.gov (United States)

    Marasco, Michelle R; Linnemann, Amelia K

    2018-05-01

    Nearly 100 years have passed since Frederick Banting and Charles Best first discovered and purified insulin. Their discovery and subsequent improvements revolutionized the treatment of diabetes, and the field continues to move at an ever-faster pace with respect to unique treatments for both type 1 and type 2 diabetes. Despite these advances, we still do not fully understand how apoptosis of the insulin-producing β-cells is triggered, presenting a challenge in the development of preventative measures. In recent years, the process of autophagy has generated substantial interest in this realm due to discoveries highlighting its clear role in the maintenance of cellular homeostasis. As a result, the number of studies focused on islet and β-cell autophagy has increased substantially in recent years. In this review, we will discuss what is currently known regarding the role of β-cell autophagy in type 1 and type 2 diabetes pathogenesis, with an emphasis on new and exciting developments over the past 5 years. Further, we will discuss how these discoveries might be translated into unique treatments in the coming years.

  9. Autophagy: a new player in skeletal maintenance?

    Science.gov (United States)

    Hocking, Lynne J; Whitehouse, Caroline; Helfrich, Miep H

    2012-07-01

    Imbalances between bone resorption and formation lie at the root of disorders such as osteoporosis, Paget's disease of bone (PDB), and osteopetrosis. Recently, genetic and functional studies have implicated proteins involved in autophagic protein degradation as important mediators of bone cell function in normal physiology and in pathology. Autophagy is the conserved process whereby aggregated proteins, intracellular pathogens, and damaged organelles are degraded and recycled. This process is important both for normal cellular quality control and in response to environmental or internal stressors, particularly in terminally-differentiated cells. Autophagic structures can also act as hubs for the spatial organization of recycling and synthetic process in secretory cells. Alterations to autophagy (reduction, hyperactivation, or impairment) are associated with a number of disorders, including neurodegenerative diseases and cancers, and are now being implicated in maintenance of skeletal homoeostasis. Here, we introduce the topic of autophagy, describe the new findings that are starting to emerge from the bone field, and consider the therapeutic potential of modifying this pathway for the treatment of age-related bone disorders. Copyright © 2012 American Society for Bone and Mineral Research.

  10. Autophagy in the eye: implications for ocular cell health.

    Science.gov (United States)

    Frost, Laura S; Mitchell, Claire H; Boesze-Battaglia, Kathleen

    2014-07-01

    Autophagy, a catabolic process by which a cell "eats" itself, turning over its own cellular constituents, plays a key role in cellular homeostasis. In an effort to maintain normal cellular function, autophagy is often up-regulated in response to environmental stresses and excessive organelle damage to facilitate aggregated protein removal. In the eye, virtually all cell types from those comprising the cornea in the front of the eye to the retinal pigment epithelium (RPE) providing a protective barrier for the retina at the back of the eye, rely on one or more aspects of autophagy to maintain structure and/or normal physiological function. In the lens autophagy plays a critical role in lens fiber cell maturation and the formation of the organelle free zone. Numerous studies delineating the role of Atg5, Vsp34 as well as FYCO1 in maintenance of lens transparency are discussed. Corneal endothelial dystrophies are also characterized as having elevated levels of autophagic proteins. Therefore, novel modulators of autophagy such as lithium and melatonin are proposed as new therapeutic strategies for this group of dystrophies. In addition, we summarize how corneal Herpes Simplex Virus (HSV-1) infection subverts the cornea's response to infection by inhibiting the normal autophagic response. Using glaucoma models we analyze the relative contribution of autophagy to cell death and cell survival. The cytoprotective role of autophagy is further discussed in an analysis of photoreceptor cell heath and function. We focus our analysis on the current understanding of autophagy in photoreceptor and RPE health, specifically on the diverse role of autophagy in rods and cones as well as its protective role in light induced degeneration. Lastly, in the RPE we highlight hybrid phagocytosis-autophagy pathways. This comprehensive review allows us to speculate on how alterations in various stages of autophagy contribute to glaucoma and retinal degenerations. Copyright © 2014 Elsevier Ltd

  11. Combined effects of starvation and butyrate on autophagy-dependent gingival epithelial cell death.

    Science.gov (United States)

    Evans, M; Murofushi, T; Tsuda, H; Mikami, Y; Zhao, N; Ochiai, K; Kurita-Ochiai, T; Yamamoto, M; Otsuka, K; Suzuki, N

    2017-06-01

    Bacteria in the dental biofilm surrounding marginal gingival grooves cause periodontal diseases. Numerous bacteria within the biofilm consume nutrients from the gingival crevicular fluid. Furthermore, some gram-negative bacteria in mature dental biofilms produce butyrate. Thus, gingival epithelial cells in close proximity to mature dental biofilms are at risk of both starvation and exposure to butyrate. In the present study, we determined the combined effects of starvation and butyrate exposure on gingival epithelial cell death and the underlying mechanisms. The Ca9-22 cell line was used as an in vitro counterpart of gingival epithelial cells. Cell death was measured as the amount of total DNA in the dead cells using SYTOX Green dye, which penetrates through membranes of dead cells and emits fluorescence when it intercalates into double-stranded DNA. AMP-activated protein kinase (AMPK) activity, the amount of autophagy, and acetylation of histone H3 were determined using western blot. Gene expression levels of microtubule-associated protein 1 light chain 3b (lc3b) were determined using quantitative reverse transcription-polymerase chain reaction. Butyrate-induced cell death occurred in a dose-dependent manner whether cells were starved or fed. However, the induction of cell death was two to four times higher when cells were placed under starvation conditions compared to when they were fed. Moreover, both starvation and butyrate exposure induced AMPK activity and autophagy. While AMPK inactivation resulted in decreased autophagy and butyrate-induced cell death under conditions of starvation, AMPK activation resulted in butyrate-induced cell death when cells were fed. Combined with the results of our previous report, which demonstrated butyrate-induced autophagy-dependent cell death, the results of this study suggest that the combination of starvation and butyrate exposure activates AMPK inducing autophagy and subsequent cell death. Notably, this combination markedly

  12. Sonic Hedgehog in cancer stem cells: a novel link with autophagy

    Directory of Open Access Journals (Sweden)

    Luis A Milla

    2012-01-01

    Full Text Available The Sonic Hegdehog/GLI (SHH/GLI pathway has been extensively studied for its role in developmental and cancer biology. During early embryonic development the SHH pathway is involved mainly in pattern formation, while in latter stages its function in stem cell and progenitor proliferation becomes increasingly relevant. During postnatal development and in adult tissues, SHH/GLI promotes cell homeostasis by actively regulating gene transcription, recapitulating the function observed during normal tissue growth. In this review, we will briefly discuss the fundamental importance of SHH/GLI in tumor growth and cancer evolution and we will then provide insights into a possible novel mechanism of SHH action in cancer through autophagy modulation in cancer stem cells. Autophagy is a homeostatic mechanism that when disrupted can promote and accelerate tumor progression in both cancer cells and the stroma that harbors tumorigenesis. Understanding possible new targets for SHH signaling and its contribution to cancer through modulation of autophagy might provide better strategies in order to design combined treatments and perform clinical trials.

  13. Autophagy in RAW264.7 Cells Treated with Surface-Functionalized Graphene Oxides

    Directory of Open Access Journals (Sweden)

    Chang Seok Park

    2015-01-01

    Full Text Available This study investigated cytotoxicity, particularly autophagy, in RAW264.7 cells exposed to graphene oxide (GO and its derivatives (dodecylamine-GO (DA-GO, reduced GO (rGO, and sodium dodecyl sulfate-rGO (SDS-rGO. Appearance of amine stretching bands, out-of-plane C-H stretching vibrations, and S=O stretching bands in infrared spectra indicated the formation of DA-GO, rGO, and SDS-rGO, respectively. Light microscopy and microculture tetrazolium assay showed that all the GO types exerted cytotoxic effects on RAW264.7 cells in a concentration-dependent manner. Higher concentrations of the GO types downregulated the expression of PU.1, a unique transcription factor in monocytes and macrophages, and decreased the conversion of LC3A/B-I to LC3A/B-II, suggesting that PU.1 was associated with autophagy in RAW264.7 cells. These results suggested that surface-functionalized GOs exerted cytotoxic effects in a concentration-dependent manner by changing the expression of critical genes and inducing autophagy in macrophages.

  14. Molecular characterization and expression of Rab7 from Clonorchis sinensis and its potential role in autophagy.

    Science.gov (United States)

    Jia, Feifei; Li, Ye; Huang, Yan; Chen, Tingjin; Li, Shan; Xu, Yanquan; Wu, Zhongdao; Li, Xuerong; Yu, Xinbing

    2013-07-01

    Accumulating evidences suggest that Rab7 GTPase is important for the normal progression of autophagy. However, the role of Rab7 GTPase in regulation of autophagy in Clonorchis sinensis is not known. In this study, a gene encoding Rab7 was isolated from C. sinensis adult cDNA. Recombinant CsRab7 was expressed and purified from Escherichia coli. CsRab7 transcripts were detected in the cDNA of adult worm, metacercaria, cercaria, and egg of C. sinensis, and were highly expressed in the metacercaria. Immunohistochemical localization results revealed that CsRab7 was specifically deposited on the vitellarium and eggs of adult worm. Furthermore, EGFP signal of CsRab7WT and the active mutant CsRab7Q67L were associated with autophagic vesicles in transiently transfected 293T cells. It is concluded from the present study that CsRab7 GTPase possibly contributes to the development of C. sinensis and that the autophagy pathway could be an important site of action with respect to the developmental role of CsRab7 in C. sinensis.

  15. Dysregulation of the Autophagy-Endolysosomal System in Amyotrophic Lateral Sclerosis and Related Motor Neuron Diseases

    Directory of Open Access Journals (Sweden)

    Asako Otomo

    2012-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a heterogeneous group of incurable motor neuron diseases (MNDs characterized by a selective loss of upper and lower motor neurons in the brain and spinal cord. Most cases of ALS are sporadic, while approximately 5–10% cases are familial. More than 16 causative genes for ALS/MNDs have been identified and their underlying pathogenesis, including oxidative stress, endoplasmic reticulum stress, excitotoxicity, mitochondrial dysfunction, neural inflammation, protein misfolding and accumulation, dysfunctional intracellular trafficking, abnormal RNA processing, and noncell-autonomous damage, has begun to emerge. It is currently believed that a complex interplay of multiple toxicity pathways is implicated in disease onset and progression. Among such mechanisms, ones that are associated with disturbances of protein homeostasis, the ubiquitin-proteasome system and autophagy, have recently been highlighted. Although it remains to be determined whether disease-associated protein aggregates have a toxic or protective role in the pathogenesis, the formation of them results from the imbalance between generation and degradation of misfolded proteins within neuronal cells. In this paper, we focus on the autophagy-lysosomal and endocytic degradation systems and implication of their dysfunction to the pathogenesis of ALS/MNDs. The autophagy-endolysosomal pathway could be a major target for the development of therapeutic agents for ALS/MNDs.

  16. Timing of autophagy and apoptosis during posterior silk gland degeneration in Bombyx mori.

    Science.gov (United States)

    Montali, Aurora; Romanelli, Davide; Cappellozza, Silvia; Grimaldi, Annalisa; de Eguileor, Magda; Tettamanti, Gianluca

    2017-07-01

    Over the years, the silkworm, Bombyx mori, has been manipulated by means of chemical and genetic approaches to improve silk production both quantitatively and qualitatively. The silk is produced by the silk gland, which degenerates quickly once the larva has finished spinning the cocoon. Thus, interfering with this degeneration process could help develop new technologies aimed at ameliorating silk yield. To this end, in this work we studied the cell death processes that lead to the demise of the posterior silk gland of B. mori, directing in particular our attention to autophagy and apoptosis. We focused on this portion of the gland because it produces fibroin, the main component of the silk thread. By using multiple markers, we provide a morphological, biochemical and molecular characterization of the apoptotic and autophagic processes and define their timing in this biological setting. Our data demonstrate that the activation of both autophagy and apoptosis is preceded by a transcriptional rise in key regulatory genes. Moreover, while autophagy is maintained active for several days and progressively digests silk gland cells, apoptosis is only switched on at a very late stage of silk gland demise. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Loss of Autophagy in Proopiomelanocortin Neurons Perturbs Axon Growth and Causes Metabolic Dysregulation

    Science.gov (United States)

    Coupé, Bérengère; Ishii, Yuko; Dietrich, Marcelo O; Komatsu, Masaaki; Horvath, Tamas L.; Bouret, Sebastien G.

    2012-01-01

    Summary The hypothalamic melanocortin system, which includes neurons that produce proopiomelanocortin (POMC)-derived peptides, is a major negative regulator of energy balance. POMC neurons begin to acquire their unique properties during neonatal life. The formation of functional neural systems requires massive cytoplasmic remodeling that may involve autophagy, an important intracellular mechanism for the degradation of damaged proteins and organelles. Here we investigated the functional and structural effects of the deletion of an essential autophagy gene, Atg7, in POMC neurons. Lack of Atg7 in POMC neurons caused higher post-weaning body weight, increased adiposity, and glucose intolerance. These metabolic impairments were associated with an age-dependant accumulation of ubiquitin/p62-positive aggregates in the hypothalamus and a disruption in the maturation of POMC-containing axonal projections. Together, these data provide direct genetic evidence that Atg7 in POMC neurons is required for normal metabolic regulation and neural development, and they implicate hypothalamic autophagy deficiency in the pathogenesis of obesity. PMID:22285542

  18. Loss of autophagy in pro-opiomelanocortin neurons perturbs axon growth and causes metabolic dysregulation.

    Science.gov (United States)

    Coupé, Bérengère; Ishii, Yuko; Dietrich, Marcelo O; Komatsu, Masaaki; Horvath, Tamas L; Bouret, Sebastien G

    2012-02-08

    The hypothalamic melanocortin system, which includes neurons that produce pro-opiomelanocortin (POMC)-derived peptides, is a major negative regulator of energy balance. POMC neurons begin to acquire their unique properties during neonatal life. The formation of functional neural systems requires massive cytoplasmic remodeling that may involve autophagy, an important intracellular mechanism for the degradation of damaged proteins and organelles. Here we investigated the functional and structural effects of the deletion of an essential autophagy gene, Atg7, in POMC neurons. Lack of Atg7 in POMC neurons caused higher postweaning body weight, increased adiposity, and glucose intolerance. These metabolic impairments were associated with an age-dependent accumulation of ubiquitin/p62-positive aggregates in the hypothalamus and a disruption in the maturation of POMC-containing axonal projections. Together, these data provide direct genetic evidence that Atg7 in POMC neurons is required for normal metabolic regulation and neural development, and they implicate hypothalamic autophagy deficiency in the pathogenesis of obesity. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Preservation of autophagy should not direct nutritional therapy

    NARCIS (Netherlands)

    McClave, S.A.; Weijs, P.J.M.

    2015-01-01

    PURPOSE OF REVIEW: Recent reports in the literature have proposed that forced mandatory feeding should be avoided in the first week of critical illness to preserve autophagy, in order to maximize responses to oxidative stress, preserve organ function, and improve outcomes. RECENT FINDINGS: Autophagy

  20. Regulation of Autophagy by Glucose in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Erwin Knecht

    2012-07-01

    Full Text Available Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focus on the signalling pathways by which environmental glucose directly, i.e., independently of insulin and glucagon, regulates autophagy in mammalian cells, but we will also briefly mention some data in yeast. Although glucose deprivation mainly induces autophagy via AMPK activation and the subsequent inhibition of mTORC1, we will also comment other signalling pathways, as well as evidences indicating that, under certain conditions, autophagy can be activated by glucose. A better understanding on how glucose regulates autophagy not only will expand our basic knowledge of this important cell process, but it will be also relevant to understand common human disorders, such as cancer and diabetes, in which glucose levels play an important role.

  1. Anti- and pro-tumor functions of autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Galluzzi, Lorenzo; Kepp, Oliver; Vicencio, José-Miguel; Criollo, Alfredo; Maiuri, Maria Chiara; Kroemer, Guido

    2009-09-01

    Autophagy constitutes one of the major responses to stress in eukaryotic cells, and is regulated by a complex network of signaling cascades. Not surprisingly, autophagy is implicated in multiple pathological processes, including infection by pathogens, inflammatory bowel disease, neurodegeneration and cancer. Both oncogenesis and tumor survival are influenced by perturbations of the molecular machinery that controls autophagy. Numerous oncoproteins, including phosphatidylinositol 3-kinase, Akt1 and anti-apoptotic members of the Bcl-2 family suppress autophagy. Conversely, several tumor suppressor proteins (e.g., Atg4c; beclin 1; Bif-1; BH3-only proteins; death-associated protein kinase 1; LKB1/STK11; PTEN; UVRAG) promote the autophagic pathway. This does not entirely apply to p53, one of the most important tumor suppressor proteins, which regulates autophagy in an ambiguous fashion, depending on its subcellular localization. Irrespective of the controversial role of p53, basal levels of autophagy appear to inhibit tumor development. On the contrary, chemotherapy- and metabolic stress-induced activation of the autophagic pathway reportedly contribute to the survival of formed tumors, thereby favoring resistance. In this context, autophagy inhibition would represent a major therapeutic target for chemosensitization. Here, we will review the current knowledge on the dual role of autophagy as an anti- and pro-tumor mechanism.

  2. Autophagy mediates pharmacological lifespan extension by spermidine and resveratrol.

    Science.gov (United States)

    Morselli, Eugenia; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Maiuri, Maria Chiara; Tavernarakis, Nektarios; Madeo, Frank; Kroemer, Guido

    2009-12-23

    Although autophagy has widely been conceived as a self-destructive mechanism that causes cell death, accumulating evidence suggests that autophagy usually mediates cytoprotection, thereby avoiding the apoptotic or necrotic demise of stressed cells. Recent evidence produced by our groups demonstrates that autophagy is also involved in pharmacological manipulations that increase longevity. Exogenous supply of the polyamine spermidine can prolong the lifespan of (while inducing autophagy in) yeast, nematodes and flies. Similarly, resveratrol can trigger autophagy in cells from different organisms, extend lifespan in nematodes, and ameliorate the fitness of human cells undergoing metabolic stress. These beneficial effects are lost when essential autophagy modulators are genetically or pharmacologically inactivated, indicating that autophagy is required for the cytoprotective and/or anti-aging effects of spermidine and resveratrol. Genetic and functional studies indicate that spermidine inhibits histone acetylases, while resveratrol activates the histone deacetylase Sirtuin 1 to confer cytoprotection/longevity. Although it remains elusive whether the same histones (or perhaps other nuclear or cytoplasmic proteins) act as the downstream targets of spermidine and resveratrol, these results point to an essential role of protein hypoacetylation in autophagy control and in the regulation of longevity.

  3. Autophagy contributes to resistance of tumor cells to ionizing radiation.

    Science.gov (United States)

    Chaachouay, Hassan; Ohneseit, Petra; Toulany, Mahmoud; Kehlbach, Rainer; Multhoff, Gabriele; Rodemann, H Peter

    2011-06-01

    Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used. Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS-PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3. LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells. Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  4. VALSARTAN REGULATES MYOCARDIAL AUTOPHAGY AND MITOCHONDRIAL TURNOVER IN EXPERIMENTAL HYPERTENSION

    Science.gov (United States)

    Zhang, Xin; Li, Zi-Lun; Crane, John A.; Jordan, Kyra L.; Pawar, Aditya S.; Textor, Stephen C.; Lerman, Amir; Lerman, Lilach O.

    2014-01-01

    Renovascular hypertension alters cardiac structure and function. Autophagy is activated during left ventricular hypertrophy and linked to adverse cardiac function. The Angiotensin II receptor blocker Valsartan lowers blood pressure and is cardioprotective, but whether it modulates autophagy in the myocardium is unclear. We hypothesized that Valsartan would alleviate autophagy and improve left ventricular myocardial mitochondrial turnover in swine renovascular hypertension. Domestic pigs were randomized to control, unilateral renovascular hypertension, and renovascular hypertension treated with Valsartan (320 mg/day) or conventional triple therapy (Reserpine+hydralazine+hydrochlorothiazide) for 4 weeks post 6-weeks of renovascular hypertension (n=7 each group). Left ventricular remodeling, function and myocardial oxygenation and microcirculation were assessed by multi-detector computer tomography, blood-oxygen-level-dependent magnetic resonance imaging and microcomputer tomography. Myocardial autophagy, markers for mitochondrial degradation and biogenesis, and mitochondrial respiratory-chain proteins were examined ex vivo. Renovascular hypertension induced left ventricular hypertrophy and myocardial hypoxia, enhanced cellular autophagy and mitochondrial degradation, and suppressed mitochondrial biogenesis. Valsartan and triple therapy similarly decreased blood pressure, but Valsartan solely alleviated left ventricular hypertrophy, ameliorated myocardial autophagy and mitophagy, and increased mitochondrial biogenesis. In contrast, triple therapy only slightly attenuated autophagy and preserved mitochondrial proteins, but elicited no improvement in mitophagy. These data suggest a novel potential role of Valsartan in modulating myocardial autophagy and mitochondrial turnover in renovascular hypertension-induced hypertensive heart disease, which may possibly bolster cardiac repair via a blood pressure-independent manner. PMID:24752430

  5. Autophagy in Negative-Strand RNA Virus Infection

    Directory of Open Access Journals (Sweden)

    Yupeng Wang

    2018-02-01

    Full Text Available Autophagy is a homoeostatic process by which cytoplasmic material is targeted for degradation by the cell. Viruses have learned to manipulate the autophagic pathway to ensure their own replication and survival. Although much progress has been achieved in dissecting the interplay between viruses and cellular autophagic machinery, it is not well understood how the cellular autophagic pathway is utilized by viruses and manipulated to their own advantage. In this review, we briefly introduce autophagy, viral xenophagy and the interaction among autophagy, virus and immune response, then focus on the interplay between NS-RNA viruses and autophagy during virus infection. We have selected some exemplary NS-RNA viruses and will describe how these NS-RNA viruses regulate autophagy and the role of autophagy in NS-RNA viral replication and in immune responses to virus infection. We also review recent advances in understanding how NS-RNA viral proteins perturb autophagy and how autophagy-related proteins contribute to NS-RNA virus replication, pathogenesis and antiviral immunity.

  6. Autophagy-dependent secretion: contribution to tumor progression

    Directory of Open Access Journals (Sweden)

    Tom Keulers

    2016-11-01

    Full Text Available Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular homeostasis. During autophagy, cytoplasmic content is recognized and packed in autophagic vacuoles, or autophagosomes, and targeted for degradation. However, during the last years, it has become evident that the role of autophagy is not restricted to degradation alone but also mediates unconventional forms of secretion. Furthermore, cells with defects in autophagy apparently are able to reroute their cargo, like mitochondria, to the extracellular environment; effects that contribute to an array of pathologies. In this review we discuss the current knowledge of the physiological roles of autophagy-dependent secretion, i.e. the effect on inflammation and insulin/ hormone secretion. Finally, we focus on the effects of autophagy-dependent secretion on the tumour microenvironment and tumour progression. The autophagy mediated secreted factors may stimulate cellular proliferation via auto- and paracrine signaling. The autophagy mediated release of immune modulating proteins change the immunosuppresive tumor microenvironment and may promote an invasive phenotype. These effects may be either direct or indirect through facilitating formation of the mobilized vesicle, aid in anterograde trafficking or alterations in homeostasis and/or autonomous cell signaling.

  7. Role of Autophagy in the Control of Body Metabolism

    Directory of Open Access Journals (Sweden)

    Wenying Quan

    2013-03-01

    Full Text Available Autophagy plays a crucial role in the maintenance of cellular nutrient balance and the function of organelles such as mitochondria or the endoplasmic reticulum, which are important in intracellular metabolism, insulin release, and insulin sensitivity. In the insulin-producing pancreatic β-cells, autophagy is important in the maintenance of β-cell mass, structure, and function. Mice with deficiencies in β-cell-specific autophagy show reduced β-cell mass and defects in insulin secretion that lead to hypoinsulinemia and hyperglycemia but not diabetes. However, these mice developed diabetes when bred with ob/ob mice, suggesting that autophagy-deficient β-cells have defects in dealing with the increased metabolic stress imposed by obesity. These results also imply that autophagy deficiency in β-cells could be a factor in the progression from obesity to diabetes. Another important function of autophagy is in hypothalamic neurons for the central control of energy expenditure, appetite, and body weight. In addition, mice with autophagy deficiencies in the target tissues of insulin have yielded diverse phenotypes. Taken together, these results suggest that autophagy is important in the control of whole body energy and nutrient homeostasis, and its dysregulation could play a role in the development of metabolic disorders and diabetes.

  8. The Nobel Prize for understanding autophagy, a cellular mechanism ...

    Indian Academy of Sciences (India)

    The lysosome was identified by Christian de Duve in the 1950s as a membrane bound organelle in thecell that contains degradative enzymes such as proteases, lipases, acid phosphatases, etc. (de Duve, 2005).The term autophagy was coined by Christian de Duve in 1963. Autophagy generally occurs at low level, butit ...

  9. Autophagy Primes Neutrophils for Neutrophil Extracellular Trap Formation during Sepsis.

    Science.gov (United States)

    Park, So Young; Shrestha, Sanjeeb; Youn, Young-Jin; Kim, Jun-Kyu; Kim, Shin-Yeong; Kim, Hyun Jung; Park, So-Hee; Ahn, Won-Gyun; Kim, Shin; Lee, Myung Goo; Jung, Ki-Suck; Park, Yong Bum; Mo, Eun-Kyung; Ko, Yousang; Lee, Suh-Young; Koh, Younsuck; Park, Myung Jae; Song, Dong-Keun; Hong, Chang-Won

    2017-09-01

    Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined. We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia. Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis. Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism. These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.

  10. Inhibition of autophagy as a treatment strategy for p53 wild-type acute myeloid leukemia

    NARCIS (Netherlands)

    Folkerts, Hendrik; Hilgendorf, Susan; Wierenga, Albertus T J; Jaques, Jennifer; Mulder, André B; Coffer, Paul J; Schuringa, Jan Jacob; Vellenga, Edo

    2017-01-01

    Here we have explored whether inhibition of autophagy can be used as a treatment strategy for acute myeloid leukemia (AML). Steady-state autophagy was measured in leukemic cell lines and primary human CD34(+) AML cells with a large variability in basal autophagy between AMLs observed. The autophagy

  11. miR-26a suppresses autophagy in swine Sertoli cells by targeting ULK2.

    Science.gov (United States)

    Ran, M; Li, Z; Cao, R; Weng, B; Peng, F; He, C; Chen, B

    2018-05-14

    A large number of microRNAs (miRNAs) have been detected from porcine testicular tissues thanks to the development of high-throughput sequencing technology. However, the regulatory roles of most identified miRNAs in swine testicular development or spermatogenesis are poorly understood. In our previous study, ULK2 (uncoordinated-51-like kinase 2) was predicted as a target gene of miR-26a. In this study, we aimed to investigate the role of miR-26a in swine Sertoli cell autophagy. The relative expression of miR-26a and ULK2 levels has a significant negative correlation (R 2  = .5964, p ≤ .01) in nine developmental stages of swine testicular tissue. Dual-luciferase reporter assay results show that miR-26a directly targets the 3'UTR of the ULK2 gene (position 618-624). In addition, both the mRNA and protein expression of ULK2 were downregulated by miR-26a in swine Sertoli cells. These results indicate that miR-26a targets the ULK2 gene and downregulates its expression in swine Sertoli cells. Based on the expression of marker genes (LC3, p62 and Beclin-1), overexpression of miR-26a or knock-down of ULK2 inhibits swine Sertoli cell autophagy. Taken together, these findings demonstrate that miR-26a suppresses autophagy in swine Sertoli cells by targeting ULK2. © 2018 Blackwell Verlag GmbH.

  12. Does autophagy work in synaptic plasticity and memory?

    Science.gov (United States)

    Shehata, Mohammad; Inokuchi, Kaoru

    2014-01-01

    Many studies have reported the roles played by regulated proteolysis in neural plasticity and memory. Within this context, most of the research focused on the ubiquitin-proteasome system and the endosome-lysosome system while giving lesser consideration to another major protein degradation system, namely, autophagy. Although autophagy intersects with many of the pathways known to underlie synaptic plasticity and memory, only few reports related autophagy to synaptic remodeling. These pathways include PI3K-mTOR pathway and endosome-dependent proteolysis. In this review, we will discuss several lines of evidence supporting a physiological role of autophagy in memory processes, and the possible mechanistic scenarios for how autophagy could fulfill this function.

  13. Modulation of Autophagy-Like Processes by Tumor Viruses

    Directory of Open Access Journals (Sweden)

    Karl Munger

    2012-06-01

    Full Text Available Autophagy is an intracellular degradation pathway for long-lived proteins and organelles. This process is activated above basal levels upon cell intrinsic or environmental stress and dysregulation of autophagy has been linked to various human diseases, including those caused by viral infection. Many viruses have evolved strategies to directly interfere with autophagy, presumably to facilitate their replication or to escape immune detection. However, in some cases, modulation of autophagy appears to be a consequence of the virus disturbing the cell’s metabolic signaling networks. Here, we summarize recent advances in research at the interface of autophagy and viral infection, paying special attention to strategies that human tumor viruses have evolved.

  14. Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver.

    Science.gov (United States)

    Blaber, Elizabeth A; Pecaut, Michael J; Jonscher, Karen R

    2017-09-27

    Increased oxidative stress is an unavoidable consequence of exposure to the space environment. Our previous studies showed that mice exposed to space for 13.5 days had decreased glutathione levels, suggesting impairments in oxidative defense. Here we performed unbiased, unsupervised and integrated multi-'omic analyses of metabolomic and transcriptomic datasets from mice flown aboard the Space Shuttle Atlantis. Enrichment analyses of metabolite and gene sets showed significant changes in osmolyte concentrations and pathways related to glycerophospholipid and sphingolipid metabolism, likely consequences of relative dehydration of the spaceflight mice. However, we also found increased enrichment of aminoacyl-tRNA biosynthesis and purine metabolic pathways, concomitant with enrichment of genes associated with autophagy and the ubiquitin-proteasome. When taken together with a downregulation in nuclear factor (erythroid-derived 2)-like 2-mediated signaling, our analyses suggest that decreased hepatic oxidative defense may lead to aberrant tRNA post-translational processing, induction of degradation programs and senescence-associated mitochondrial dysfunction in response to the spaceflight environment.

  15. Spaceflight Activates Autophagy Programs and the Proteasome in Mouse Liver

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Blaber

    2017-09-01

    Full Text Available Increased oxidative stress is an unavoidable consequence of exposure to the space environment. Our previous studies showed that mice exposed to space for 13.5 days had decreased glutathione levels, suggesting impairments in oxidative defense. Here we performed unbiased, unsupervised and integrated multi-‘omic analyses of metabolomic and transcriptomic datasets from mice flown aboard the Space Shuttle Atlantis. Enrichment analyses of metabolite and gene sets showed significant changes in osmolyte concentrations and pathways related to glycerophospholipid and sphingolipid metabolism, likely consequences of relative dehydration of the spaceflight mice. However, we also found increased enrichment of aminoacyl-tRNA biosynthesis and purine metabolic pathways, concomitant with enrichment of genes associated with autophagy and the ubiquitin-proteasome. When taken together with a downregulation in nuclear factor (erythroid-derived 2-like 2-mediated signaling, our analyses suggest that decreased hepatic oxidative defense may lead to aberrant tRNA post-translational processing, induction of degradation programs and senescence-associated mitochondrial dysfunction in response to the spaceflight environment.

  16. GAIP interacting protein C-terminus regulates autophagy and exosome biogenesis of pancreatic cancer through metabolic pathways.

    Directory of Open Access Journals (Sweden)

    Santanu Bhattacharya

    Full Text Available GAIP interacting protein C terminus (GIPC is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.

  17. Catalase and NO CATALASE ACTIVITY1 Promote Autophagy-Dependent Cell Death in Arabidopsis[C][W][OPEN

    Science.gov (United States)

    Hackenberg, Thomas; Juul, Trine; Auzina, Aija; Gwiżdż, Sonia; Małolepszy, Anna; Van Der Kelen, Katrien; Dam, Svend; Bressendorff, Simon; Lorentzen, Andrea; Roepstorff, Peter; Lehmann Nielsen, Kåre; Jørgensen, Jan-Elo; Hofius, Daniel; Breusegem, Frank Van; Petersen, Morten; Andersen, Stig Uggerhøj

    2013-01-01

    Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea-induced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death. PMID:24285797

  18. Subcellular localization of acyl-CoA binding protein in Aspergillus oryzae is regulated by autophagy machinery.

    Science.gov (United States)

    Kawaguchi, Kouhei; Kikuma, Takashi; Higuchi, Yujiro; Takegawa, Kaoru; Kitamoto, Katsuhiko

    2016-11-04

    In eukaryotic cells, acyl-CoA binding protein (ACBP) is important for cellular activities, such as in lipid metabolism. In the industrially important fungus Aspergillus oryzae, the ACBP, known as AoACBP, has been biochemically characterized, but its physiological function is not known. In the present study, although we could not find any phenotype of AoACBP disruptants in the normal growth conditions, we examined the subcellular localization of AoACBP to understand its physiological function. Using an enhanced green fluorescent protein (EGFP)-tagged AoACBP construct we showed that AoACBP localized to punctate structures in the cytoplasm, some of which moved inside the cells in a microtubule-dependent manner. Further microscopic analyses showed that AoACBP-EGFP co-localized with the autophagy marker protein AoAtg8 tagged with red fluorescent protein (mDsRed). Expression of AoACBP-EGFP in disruptants of autophagy-related genes revealed aggregation of AoACBP-EGFP fluorescence in the cytoplasm of Aoatg1, Aoatg4 and Aoatg8 disruptant cells. However, in cells harboring disruption of Aoatg15, which encodes a lipase for autophagic body, puncta of AoACBP-EGFP fluorescence accumulated in vacuoles, indicating that AoACBP is transported to vacuoles via the autophagy machinery. Collectively, these results suggest the existence of a regulatory mechanism between AoACBP localization and autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Pravastatin Protects Against Avascular Necrosis of Femoral Head via Autophagy.

    Science.gov (United States)

    Liao, Yun; Zhang, Ping; Yuan, Bo; Li, Ling; Bao, Shisan

    2018-01-01

    Autophagy serves as a stress response and may contribute to the pathogenesis of avascular necrosis of the femoral head induced by steroids. Statins promote angiogenesis and ameliorate endothelial functions through apoptosis inhibition and necrosis of endothelial progenitor cells, however the process used by statins to modulate autophagy in avascular necrosis of the femoral head remains unclear. This manuscript determines whether pravastatin protects against dexamethasone-induced avascular necrosis of the femoral head by activating endothelial progenitor cell autophagy. Pravastatin was observed to enhance the autophagy activity in endothelial progenitor cells, specifically by upregulating LC3-II/Beclin-1 (autophagy related proteins), and autophagosome formation in vivo and in vitro . An autophagy inhibitor, 3-MA, reduced pravastatin protection in endothelial progenitor cells exposed to dexamethasone by attenuating pravastatin-induced autophagy. Adenosine monophosphate-activated protein kinase (AMPK) is a key autophagy regulator by sensing cellular energy changes, and indirectly suppressing activation of the mammalian target of rapamycin (mTOR). We found that phosphorylation of AMPK was upregulated however phosphorylation of mTOR was downregulated in pravastatin-treated endothelial progenitor cells, which was attenuated by AMPK inhibitor compound C. Furthermore, liver kinase B1 (a phosphorylase of AMPK) knockdown eliminated pravastatin regulated autophagy protein LC3-II in endothelial progenitor cells in vitro . We therefore demonstrated pravastatin rescued endothelial progenitor cells from dexamethasone-induced autophagy dysfunction through the AMPK-mTOR signaling pathway in a liver kinase B1-dependent manner. Our results provide useful information for the development of novel therapeutics for management of glucocorticoids-induced avascular necrosis of the femoral head.

  20. Cisplatin-induced downregulation of miR-199a-5p increases drug resistance by activating autophagy in HCC cell

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ning; Zhang, Jianjun; Shen, Conghuan; Luo, Yi; Xia, Lei; Xue, Feng [Department of Transplantation and Hepatic Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, 1630 Dongfang Road, Shanghai 200127, People' s Republic of China (China); Xia, Qiang, E-mail: xiaqiang1@yahoo.com.cn [Department of Transplantation and Hepatic Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, 1630 Dongfang Road, Shanghai 200127, People' s Republic of China (China)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer miR-199a-5p levels were significantly decreased after cisplatin treatment. Black-Right-Pointing-Pointer Cisplatin treatment induced autophagy activation. Black-Right-Pointing-Pointer Cisplatin-induced downregulation of miR-199a-5p increases drug resistance by activating autophagy in HCC cell. -- Abstract: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Systemic chemotherapy plays an important role in the treatment of patients with advanced liver cancer. However, chemoresistance to cisplatin is a major limitation of cisplatin-based chemotherapy in the clinic, and the underlying mechanism of such resistance is not fully understood. In the study, we found that miR-199a-5p levels were significantly reduced in HCC patients treated with cisplatin-based chemotherapy. Cisplatin treatment also resulted in decreased miR-199a-5p levels in human HCC cell lines. Forced expression of miR-199a-5p promoted cisplatin-induced inhibition of cell proliferation. Cisplatin treatment activated autophagy in Huh7 and HepG2 cells, which increased cell proliferation. We further demonstrated that downregulated miR-199a-5p enhanced autophagy activation by targeting autophagy-associated gene 7 (ATG7). More important, autophagy inhibition abrogated miR-199a-5p downregulation-induced cell proliferation. These data demonstrated that miR-199a-5p/autophagy signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of HCC.

  1. Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains▿ †

    OpenAIRE

    Bellot, Grégory; Garcia-Medina, Raquel; Gounon, Pierre; Chiche, Johanna; Roux, Danièle; Pouysségur, Jacques; Mazure, Nathalie M.

    2009-01-01

    While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy,...

  2. Elucidating the composition and conservation of the autophagy pathway in photosynthetic eukaryotes

    Science.gov (United States)

    Shemi, Adva; Ben-Dor, Shifra; Vardi, Assaf

    2015-01-01

    Aquatic photosynthetic eukaryotes represent highly diverse groups (green, red, and chromalveolate algae) derived from multiple endosymbiosis events, covering a wide spectrum of the tree of life. They are responsible for about 50% of the global photosynthesis and serve as the foundation for oceanic and fresh water food webs. Although the ecophysiology and molecular ecology of some algal species are extensively studied, some basic aspects of algal cell biology are still underexplored. The recent wealth of genomic resources from algae has opened new frontiers to decipher the role of cell signaling pathways and their function in an ecological and biotechnological context. Here, we took a bioinformatic approach to explore the distribution and conservation of TOR and autophagy-related (ATG) proteins (Atg in yeast) in diverse algal groups. Our genomic analysis demonstrates conservation of TOR and ATG proteins in green algae. In contrast, in all 5 available red algal genomes, we could not detect the sequences that encode for any of the 17 core ATG proteins examined, albeit TOR and its interacting proteins are conserved. This intriguing data suggests that the autophagy pathway is not conserved in red algae as it is in the entire eukaryote domain. In contrast, chromalveolates, despite being derived from the red-plastid lineage, retain and express ATG genes, which raises a fundamental question regarding the acquisition of ATG genes during algal evolution. Among chromalveolates, Emiliania huxleyi (Haptophyta), a bloom-forming coccolithophore, possesses the most complete set of ATG genes, and may serve as a model organism to study autophagy in marine protists with great ecological significance. PMID:25915714

  3. Canonical autophagy does not contribute to cellular radioresistance

    International Nuclear Information System (INIS)

    Schaaf, Marco B.E.; Jutten, Barry; Keulers, Tom G.; Savelkouls, Kim G.M.; Peeters, Hanneke J.M.; Beucken, Twan van den; Schooten, Frederik-Jan van; Godschalk, Roger W.; Vooijs, Marc; Rouschop, Kasper M.A.

    2015-01-01

    Background: (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines. Materials and methods: Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. Results: IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair. Conclusion: Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation

  4. Distinct Contributions of Autophagy Receptors in Measles Virus Replication.

    Science.gov (United States)

    Petkova, Denitsa S; Verlhac, Pauline; Rozières, Aurore; Baguet, Joël; Claviere, Mathieu; Kretz-Remy, Carole; Mahieux, Renaud; Viret, Christophe; Faure, Mathias

    2017-05-22

    Autophagy is a potent cell autonomous defense mechanism that engages the lysosomal pathway to fight intracellular pathogens. Several autophagy receptors can recognize invading pathogens in order to target them towards autophagy for their degradation after the fusion of pathogen-containing autophagosomes with lysosomes. However, numerous intracellular pathogens can avoid or exploit autophagy, among which is measles virus (MeV). This virus induces a complete autophagy flux, which is required to improve viral replication. We therefore asked how measles virus interferes with autophagy receptors during the course of infection. We report that in addition to NDP52/CALCOCO₂ and OPTINEURIN/OPTN, another autophagy receptor, namely T6BP/TAXIBP1, also regulates the maturation of autophagosomes by promoting their fusion with lysosomes, independently of any infection. Surprisingly, only two of these receptors, NDP52 and T6BP, impacted measles virus replication, although independently, and possibly through physical interaction with MeV proteins. Thus, our results suggest that a restricted set of autophagosomes is selectively exploited by measles virus to replicate in the course of infection.

  5. The inositol trisphosphate receptor in the control of autophagy.

    Science.gov (United States)

    Criollo, Alfredo; Vicencio, José Miguel; Tasdemir, Ezgi; Maiuri, M Chiara; Lavandero, Sergio; Kroemer, Guido

    2007-01-01

    The second messenger myo-inositol-1,4,5-trisphosphate (IP(3)) acts on the IP(3) receptor (IP(3)R), an IP(3)-activated Ca(2+) channel of the endoplasmic reticulum (ER). The IP(3)R agonist IP(3) inhibits starvation-induced autophagy. The IP(3)R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-XL, two proteins that physically interact with IP(3)R. Autophagy can also be induced by depletion of the IP(3)R by small interfering RNAs. Autophagy induction by IP(3)R blockade cannot be explained by changes in steady state levels of Ca(2+) in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP(3)R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

  6. The IKK complex contributes to the induction of autophagy.

    Science.gov (United States)

    Criollo, Alfredo; Senovilla, Laura; Authier, Hélène; Maiuri, Maria Chiara; Morselli, Eugenia; Vitale, Ilio; Kepp, Oliver; Tasdemir, Ezgi; Galluzzi, Lorenzo; Shen, Shensi; Tailler, Maximilien; Delahaye, Nicolas; Tesniere, Antoine; De Stefano, Daniela; Younes, Aména Ben; Harper, Francis; Pierron, Gérard; Lavandero, Sergio; Zitvogel, Laurence; Israel, Alain; Baud, Véronique; Kroemer, Guido

    2010-02-03

    In response to stress, cells start transcriptional and transcription-independent programs that can lead to adaptation or death. Here, we show that multiple inducers of autophagy, including nutrient depletion, trigger the activation of the IKK (IkappaB kinase) complex that is best known for its essential role in the activation of the transcription factor NF-kappaB by stress. Constitutively active IKK subunits stimulated autophagy and transduced multiple signals that operate in starvation-induced autophagy, including the phosphorylation of AMPK and JNK1. Genetic inhibition of the nuclear translocation of NF-kappaB or ablation of the p65/RelA NF-kappaB subunit failed to suppress IKK-induced autophagy, indicating that IKK can promote the autophagic pathway in an NF-kappaB-independent manner. In murine and human cells, knockout and/or knockdown of IKK subunits (but not that of p65) prevented the induction of autophagy in response to multiple stimuli. Moreover, the knockout of IKK-beta suppressed the activation of autophagy by food deprivation or rapamycin injections in vivo, in mice. Altogether, these results indicate that IKK has a cardinal role in the stimulation of autophagy by physiological and pharmacological stimuli.

  7. Laser stimulation can activate autophagy in HeLa cells

    International Nuclear Information System (INIS)

    Wang, Yisen; Hu, Minglie; Wang, Chingyue; Lan, Bei; Cao, Youjia; He, Hao

    2014-01-01

    For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca 2+ dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

  8. Nanomaterials and Autophagy: New Insights in Cancer Treatment

    International Nuclear Information System (INIS)

    Panzarini, Elisa; Inguscio, Valentina; Tenuzzo, Bernardetta Anna; Carata, Elisabetta; Dini, Luciana

    2013-01-01

    Autophagy represents a cell’s response to stress. It is an evolutionarily conserved process with diversified roles. Indeed, it controls intracellular homeostasis by degradation and/or recycling intracellular metabolic material, supplies energy, provides nutrients, eliminates cytotoxic materials and damaged proteins and organelles. Moreover, autophagy is involved in several diseases. Recent evidences support a relationship between several classes of nanomaterials and autophagy perturbation, both induction and blockade, in many biological models. In fact, the autophagic mechanism represents a common cellular response to nanomaterials. On the other hand, the dynamic nature of autophagy in cancer biology is an intriguing approach for cancer therapeutics, since during tumour development and therapy, autophagy has been reported to trigger both an early cell survival and a late cell death. The use of nanomaterials in cancer treatment to deliver chemotherapeutic drugs and target tumours is well known. Recently, autophagy modulation mediated by nanomaterials has become an appealing notion in nanomedicine therapeutics, since it can be exploited as adjuvant in chemotherapy or in the development of cancer vaccines or as a potential anti-cancer agent. Herein, we summarize the effects of nanomaterials on autophagic processes in cancer, also considering the therapeutic outcome of synergism between nanomaterials and autophagy to improve existing cancer therapies

  9. Autophagy in the immune response to tuberculosis: clinical perspectives.

    LENUS (Irish Health Repository)

    Ní Cheallaigh, C

    2011-06-01

    A growing body of evidence points to autophagy as an essential component in the immune response to tuberculosis. Autophagy is a direct mechanism of killing intracellular Mycobacterium tuberculosis and also acts as a modulator of proinflammatory cytokine secretion. In addition, autophagy plays a key role in antigen processing and presentation. Autophagy is modulated by cytokines; it is stimulated by T helper type 1 (Th1) cytokines such as tumour necrosis factor (TNF)-α and interferon (IFN)-γ, and is inhibited by the Th2 cytokines interleukin (IL)-4 and IL-13 and the anti-inflammatory cytokine IL-10. Vitamin D, via cathelicidin, can also induce autophagy, as can Toll-like receptor (TLR)-mediated signals. Autophagy-promoting agents, administered either locally to the lungs or systemically, could have a clinical application as adjunctive treatment of drug-resistant and drug-sensitive tuberculosis. Moreover, vaccines which effectively induce autophagy could be more successful in preventing acquisition or reactivation of latent tuberculosis.

  10. Laser stimulation can activate autophagy in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yisen; Hu, Minglie; Wang, Chingyue [Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin (China); Lan, Bei; Cao, Youjia [Key Laboratory of Microbial Functional Genomics of Ministry of Education, College of Life Sciences, Nankai University, Tianjin (China); He, Hao, E-mail: haohe@tju.edu.cn [Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin (China); Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China)

    2014-10-27

    For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca{sup 2+} dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

  11. ATM kinase sustains breast cancer stem-like cells by promoting ATG4C expression and autophagy.

    Science.gov (United States)

    Antonelli, Martina; Strappazzon, Flavie; Arisi, Ivan; Brandi, Rossella; D'Onofrio, Mara; Sambucci, Manolo; Manic, Gwenola; Vitale, Ilio; Barilà, Daniela; Stagni, Venturina

    2017-03-28

    The efficacy of Ataxia-Telangiectasia Mutated (ATM) kinase signalling inhibition in cancer therapy is tempered by the identification of new emerging functions of ATM, which suggests that the role of this protein in cancer progression is complex. We recently demonstrated that this tumor suppressor gene could act as tumor promoting factor in HER2 (Human Epidermal Growth Factor Receptor 2) positive breast cancer. Herein we put in evidence that ATM expression sustains the proportion of cells with a stem-like phenotype, measured as the capability to form mammospheres, independently of HER2 expression levels. Transcriptomic analyses revealed that, in mammospheres, ATM modulates the expression of cell cycle-, DNA repair- and autophagy-related genes. Among these, the silencing of the autophagic gene, autophagy related 4C cysteine peptidase (ATG4C), impairs mammosphere formation similarly to ATM depletion. Conversely, ATG4C ectopic expression in cells silenced for ATM expression, rescues mammospheres growth. Finally, tumor array analyses, performed using public data, identify a significant correlation between ATM and ATG4C expression levels in all human breast cancer subtypes, except for the basal-like one.Overall, we uncover a new connection between ATM kinase and autophagy regulation in breast cancer. We demonstrate that, in breast cancer cells, ATM and ATG4C are essential drivers of mammosphere formation, suggesting that their targeting may improve current approaches to eradicate breast cancer cells with a stem-like phenotype.

  12. Autophagy contributes to gefitinib-induced glioma cell growth inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Cheng-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Graduate Institute of Pharmaceutical Science and Technology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan (China); Kuan, Yu-Hsiang [Department of Pharmacology, School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Pharmacy, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Ou, Yen-Chuan; Li, Jian-Ri [Division of Urology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Wu, Chih-Cheng [Department of Anesthesiology, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Department of Financial and Computational Mathematics, Providence University, Taichung 433, Taiwan (China); Pan, Pin-Ho [Department of Pediatrics, Tungs’ Taichung MetroHarbor Hospital, Taichung 435, Taiwan (China); Chen, Wen-Ying [Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Huang, Hsuan-Yi [Department of Surgery, Fong-Yuan Hospital, Taichung 420, Taiwan (China); Chen, Chun-Jung, E-mail: cjchen@vghtc.gov.tw [Department of Medical Research, Taichung Veterans General Hospital, Taichung 407, Taiwan (China); Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan (China); Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan (China); Center for General Education, Tunghai University, Taichung 407, Taiwan (China); Department of Nursing, HungKuang University, Taichung 433, Taiwan (China)

    2014-09-10

    Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK.

  13. Autophagy contributes to gefitinib-induced glioma cell growth inhibition

    International Nuclear Information System (INIS)

    Chang, Cheng-Yi; Kuan, Yu-Hsiang; Ou, Yen-Chuan; Li, Jian-Ri; Wu, Chih-Cheng; Pan, Pin-Ho; Chen, Wen-Ying; Huang, Hsuan-Yi; Chen, Chun-Jung

    2014-01-01

    Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation. - Highlights: • Gefitinib causes cytotoxic and cytostatic effect on glioma. • Gefitinib induces autophagy. • Gefitinib causes cytostatic effect through autophagy. • Gefitinib induces autophagy involving AMPK

  14. Oxidative Stress, Redox Signaling, and Autophagy: Cell Death Versus Survival

    Science.gov (United States)

    Navarro-Yepes, Juliana; Burns, Michaela; Anandhan, Annadurai; Khalimonchuk, Oleh; del Razo, Luz Maria; Quintanilla-Vega, Betzabet; Pappa, Aglaia; Panayiotidis, Mihalis I.

    2014-01-01

    Abstract Significance: The molecular machinery regulating autophagy has started becoming elucidated, and a number of studies have undertaken the task to determine the role of autophagy in cell fate determination within the context of human disease progression. Oxidative stress and redox signaling are also largely involved in the etiology of human diseases, where both survival and cell death signaling cascades have been reported to be modulated by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Recent Advances: To date, there is a good understanding of the signaling events regulating autophagy, as well as the signaling processes by which alterations in redox homeostasis are transduced to the activation/regulation of signaling cascades. However, very little is known about the molecular events linking them to the regulation of autophagy. This lack of information has hampered the understanding of the role of oxidative stress and autophagy in human disease progression. Critical Issues: In this review, we will focus on (i) the molecular mechanism by which ROS/RNS generation, redox signaling, and/or oxidative stress/damage alter autophagic flux rates; (ii) the role of autophagy as a cell death process or survival mechanism in response to oxidative stress; and (iii) alternative mechanisms by which autophagy-related signaling regulate mitochondrial function and antioxidant response. Future Directions: Our research efforts should now focus on understanding the molecular basis of events by which autophagy is fine tuned by oxidation/reduction events. This knowledge will enable us to understand the mechanisms by which oxidative stress and autophagy regulate human diseases such as cancer and neurodegenerative disorders. Antioxid. Redox Signal. 21, 66–85. PMID:24483238

  15. Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy.

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    Sung Won Lee

    Full Text Available Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence

  16. Autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

    Science.gov (United States)

    Xie, Xiaolei; Le, Li; Fan, Yanxin; Lv, Lin; Zhang, Junjie

    2012-07-01

    Mitoribosome in mammalian cells is responsible for synthesis of 13 mtDNA-encoded proteins, which are integral parts of four mitochondrial respiratory chain complexes (I, III, IV and V). ERAL1 is a nuclear-encoded GTPase important for the formation of the 28S small mitoribosomal subunit. Here, we demonstrate that knockdown of ERAL1 by RNA interference inhibits mitochondrial protein synthesis and promotes reactive oxygen species (ROS) generation, leading to autophagic vacuolization in HeLa cells. Cells that lack ERAL1 expression showed a significant conversion of LC3-I to LC3-II and an enhanced accumulation of autophagic vacuoles carrying the LC3 marker, all of which were blocked by the autophagy inhibitor 3-MA as well as by the ROS scavenger NAC. Inhibition of mitochondrial protein synthesis either by ERAL1 siRNA or chloramphenicol (CAP), a specific inhibitor of mitoribosomes, induced autophagy in HTC-116 TP53 (+/+) cells, but not in HTC-116 TP53 (-/-) cells, indicating that tumor protein 53 (TP53) is essential for the autophagy induction. The ROS elevation resulting from mitochondrial protein synthesis inhibition induced TP53 expression at transcriptional levels by enhancing TP53 promoter activity, and increased TP53 protein stability by suppressing TP53 ubiquitination through MAPK14/p38 MAPK-mediated TP53 phosphorylation. Upregulation of TP53 and its downstream target gene DRAM1, but not CDKN1A/p21, was required for the autophagy induction in ERAL1 siRNA or CAP-treated cells. Altogether, these data indicate that autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

  17. Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus

    Science.gov (United States)

    Hagiwara, D; Arima, H; Morishita, Y; Wenjun, L; Azuma, Y; Ito, Y; Suga, H; Goto, M; Banno, R; Sugimura, Y; Shiota, A; Asai, N; Takahashi, M; Oiso, Y

    2014-01-01

    Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30–40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy

  18. Methamphetamine exposure triggers apoptosis and autophagy in neuronal cells by activating the C/EBPβ-related signaling pathway.

    Science.gov (United States)

    Xu, Xiang; Huang, Enping; Luo, Baoying; Cai, Dunpeng; Zhao, Xu; Luo, Qin; Jin, Yili; Chen, Ling; Wang, Qi; Liu, Chao; Lin, Zhoumeng; Xie, Wei-Bing; Wang, Huijun

    2018-06-25

    Methamphetamine (Meth) is a widely abused psychoactive drug that primarily damages the nervous system, notably causing dopaminergic neuronal apoptosis. CCAAT-enhancer binding protein (C/EBPβ) is a transcription factor and an important regulator of cell apoptosis and autophagy. Insulin-like growth factor binding protein (IGFBP5) is a proapoptotic factor that mediates Meth-induced neuronal apoptosis, and Trib3 (tribbles pseudokinase 3) is an endoplasmic reticulum (ER) stress-inducible gene involved in autophagic cell death through the mammalian target of rapamycin (mTOR) signaling pathway. To test the hypothesis that C/EBPβ is involved in Meth-induced IGFBP5-mediated neuronal apoptosis and Trib3-mediated neuronal autophagy, we measured the protein expression of C/EBPβ after Meth exposure and evaluated the effects of silencing C/EBPβ, IGFBP5, or Trib3 on Meth-induced apoptosis and autophagy in neuronal cells and in the rat striatum after intrastriatal Meth injection. We found that, at relatively high doses, Meth exposure increased C/EBPβ protein expression, which was accompanied by increased neuronal apoptosis and autophagy; triggered the IGFBP5-mediated, p53-up-regulated modulator of apoptosis (PUMA)-related mitochondrial apoptotic signaling pathway; and stimulated the Trib3-mediated ER stress signaling pathway through the Akt-mTOR signaling axis. We also found that autophagy is an early response to Meth-induced stress upstream of apoptosis and plays a detrimental role in Meth-induced neuronal cell death. These results suggest that Meth exposure induces C/EBPβ expression, which plays an essential role in the neuronal apoptosis and autophagy induced by relatively high doses of Meth; however, relatively low concentrations of Meth did not change the expression of C/EBPβ in vitro. Further studies are needed to elucidate the role of C/EBPβ in low-dose Meth-induced neurotoxicity.-Xu, X., Huang, E., Luo, B., Cai, D., Zhao, X., Luo, Q., Jin, Y., Chen, L., Wang, Q

  19. ER stress, autophagy, and RNA viruses

    Directory of Open Access Journals (Sweden)

    Jia-Rong eJheng

    2014-08-01

    Full Text Available Endoplasmic reticulum (ER stress is a general term for representing the pathway by which various stimuli affect ER functions. ER stress induces the evolutionarily conserved signaling pathways, called the unfolded protein response (UPR, which compromises the stimulus and then determines whether the cell survives or dies. In recent years, ongoing research has suggested that these pathways may be linked to the autophagic response, which plays a key role in the cell’s response to various stressors. Autophagy performs a self-digestion function, and its activation protects cells against certain pathogens. However, the link between the UPR and autophagy may be more complicated. These two systems may act dependently, or the induction of one system may interfere with the other. Experimental studies have found that different viruses modulate these mechanisms to allow them to escape the host immune response or, worse, to exploit the host’s defense to their advantage; thus, this topic is a critical area in antiviral research. In this review, we summarize the current knowledge about how RNA viruses, including influenza virus, poliovirus, coxsackievirus, enterovirus 71, Japanese encephalitis virus, hepatitis C virus, and dengue virus, regulate these processes. We also discuss recent discoveries and how these will produce novel strategies for antiviral treatment.

  20. Autophagy in HCV Infection: Keeping Fat and Inflammation at Bay

    Directory of Open Access Journals (Sweden)

    Tiziana Vescovo

    2014-01-01

    Full Text Available Hepatitis C virus (HCV infection is one of the main causes of chronic liver disease. Viral persistence and pathogenesis rely mainly on the ability of HCV to deregulate specific host processes, including lipid metabolism and innate immunity. Recently, autophagy has emerged as a cellular pathway, playing a role in several aspects of HCV infection. This review summarizes current knowledge on the molecular mechanisms that link the HCV life cycle with autophagy machinery. In particular, we discuss the role of HCV/autophagy interaction in dysregulating inflammation and lipid homeostasis and its potential for translational applications in the treatment of HCV-infected patients.

  1. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome.

    Science.gov (United States)

    Morselli, Eugenia; Mariño, Guillermo; Bennetzen, Martin V; Eisenberg, Tobias; Megalou, Evgenia; Schroeder, Sabrina; Cabrera, Sandra; Bénit, Paule; Rustin, Pierre; Criollo, Alfredo; Kepp, Oliver; Galluzzi, Lorenzo; Shen, Shensi; Malik, Shoaib Ahmad; Maiuri, Maria Chiara; Horio, Yoshiyuki; López-Otín, Carlos; Andersen, Jens S; Tavernarakis, Nektarios; Madeo, Frank; Kroemer, Guido

    2011-02-21

    Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.

  2. SPBP is a sulforaphane induced transcriptional coactivator of NRF2 regulating expression of the autophagy receptor p62/SQSTM1.

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    Sagar Ramesh Darvekar

    Full Text Available Organisms exposed to oxidative stress respond by orchestrating a stress response to prevent further damage. Intracellular levels of antioxidant agents increase, and damaged components are removed by autophagy induction. The KEAP1-NRF2 signaling pathway is the main pathway responsible for cell defense against oxidative stress and for maintaining the cellular redox balance at physiological levels. Sulforaphane, an isothiocyanate derived from cruciferous vegetables, is a potent inducer of KEAP1-NRF2 signaling and antioxidant response element driven gene expression. In this study, we show that sulforaphane enhances the expression of the transcriptional coregulator SPBP. The expression curve peaks 6-8 hours post stimulation, and parallels the sulforaphane-induced expression of NRF2 and the autophagy receptor protein p62/SQSTM1. Reporter gene assays show that SPBP stimulates the expression of p62/SQSTM1 via ARE elements in the promoter region, and siRNA mediated knock down of SPBP significantly decreases the expression of p62/SQSTM1 and the formation of p62/SQSTM1 bodies in HeLa cells. Furthermore, SPBP siRNA reduces the sulforaphane induced expression of NRF2, and the expression of the autophagy marker protein LC3B. Both these proteins contain ARE-like elements in their promoter regions. Over-expressed SPBP and NRF2 acts synergistically on the p62/SQSTM1 promoter and colocalize in nuclear speckles in HeLa cells. Collectively, these results suggest that SPBP is a coactivator of NRF2, and hence may be important for securing enhanced and sustained expression of NRF2 induced genes such as proteins involved in selective autophagy.

  3. Functional loss of two ceramide synthases elicits autophagy-dependent lifespan extension in C. elegans.

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    Mai-Britt Mosbech

    Full Text Available Ceramide and its metabolites constitute a diverse group of lipids, which play important roles as structural entities of biological membranes as well as regulators of cellular growth, differentiation, and development. The C. elegans genome comprises three ceramide synthase genes; hyl-1, hyl-2, and lagr-1. HYL-1 function is required for synthesis of ceramides and sphingolipids containing very long acyl-chains (≥C24, while HYL-2 is required for synthesis of ceramides and sphingolipids containing shorter acyl-chains (≤C22. Here we show that functional loss of HYL-2 decreases lifespan, while loss of HYL-1 or LAGR-1 does not affect lifespan. We show that loss of HYL-1 and LAGR-1 functions extend lifespan in an autophagy-dependent manner, as knock down of the autophagy-associated gene ATG-12 abolishes hyl-1;lagr-1 longevity. The transcription factors PHA-4/FOXA, DAF-16/FOXO, and SKN-1 are also required for the observed lifespan extension, as well as the increased number of autophagosomes in hyl-1;lagr-1 animals. Both autophagic events and the transcription factors PHA-4/FOXA, DAF-16, and SKN-1 have previously been associated with dietary restriction-induced longevity. Accordingly, we find that hyl-1;lagr-1 animals display reduced feeding, increased resistance to heat, and reduced reproduction. Collectively, our data suggest that specific sphingolipids produced by different ceramide synthases have opposing roles in determination of C. elegans lifespan. We propose that loss of HYL-1 and LAGR-1 result in dietary restriction-induced autophagy and consequently prolonged longevity.

  4. Interplay between apoptosis and autophagy in colorectal cancer.

    Science.gov (United States)

    Qian, Hao-Ran; Shi, Zhao-Qi; Zhu, He-Pan; Gu, Li-Hu; Wang, Xian-Fa; Yang, Yi

    2017-09-22

    Autophagy and apoptosis are two pivotal mechanisms in mediating cell survival and death. Cross-talk of autophagy and apoptosis has been documented in the tumorigenesis and progression of cancer, while the interplay between the two pathways in colorectal cancer (CRC) has not yet been comprehensively summarized. In this study, we outlined the basis of apoptosis and autophagy machinery firstly, and then reviewed the recent evidence in cellular settings or animal studies regarding the interplay between them in CRC. In addition, several key factors that modulate the cross-talk between autophagy and apoptosis as well as its significance in clinical practice were discussed. Understanding of the interplay between the cell death mechanisms may benefit the translation of CRC treatment from basic research to clinical use.

  5. Autophagy-Related Deubiquitinating Enzymes Involved in Health and Disease

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    Fouzi El Magraoui

    2015-10-01

    Full Text Available Autophagy is an evolutionarily-conserved process that delivers diverse cytoplasmic components to the lysosomal compartment for either recycling or degradation. This involves the removal of protein aggregates, the turnover of organelles, as well as the elimination of intracellular pathogens. In this situation, when only specific cargoes should be targeted to the lysosome, the potential targets can be selectively marked by the attachment of ubiquitin in order to be recognized by autophagy-receptors. Ubiquitination plays a central role in this process, because it regulates early signaling events during the induction of autophagy and is also used as a degradation-tag on the potential autophagic cargo protein. Here, we review how the ubiquitin-dependent steps of autophagy are balanced or counteracted by deubiquitination events. Moreover, we highlight the functional role of the corresponding deubiquitinating enzymes and discuss how they might be involved in the occurrence of cancer, neurodegenerative diseases or infection with pathogenic bacteria.

  6. Autophagy and Neurodegeneration: Pathogenic Mechanisms and Therapeutic Opportunities.

    Science.gov (United States)

    Menzies, Fiona M; Fleming, Angeleen; Caricasole, Andrea; Bento, Carla F; Andrews, Stephen P; Ashkenazi, Avraham; Füllgrabe, Jens; Jackson, Anne; Jimenez Sanchez, Maria; Karabiyik, Cansu; Licitra, Floriana; Lopez Ramirez, Ana; Pavel, Mariana; Puri, Claudia; Renna, Maurizio; Ricketts, Thomas; Schlotawa, Lars; Vicinanza, Mariella; Won, Hyeran; Zhu, Ye; Skidmore, John; Rubinsztein, David C

    2017-03-08

    Autophagy is a conserved pathway that delivers cytoplasmic contents to the lysosome for degradation. Here we consider its roles in neuronal health and disease. We review evidence from mouse knockout studies demonstrating the normal functions of autophagy as a protective factor against neurodegeneration associated with intracytoplasmic aggregate-prone protein accumulation as well as other roles, including in neuronal stem cell differentiation. We then describe how autophagy may be affected in a range of neurodegenerative diseases. Finally, we describe how autophagy upregulation may be a therapeutic strategy in a wide range of neurodegenerative conditions and consider possible pathways and druggable targets that may be suitable for this objective. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Autophagy as a Therapeutic Target in Cardiovascular Disease

    Science.gov (United States)

    Nemchenko, Andriy; Chiong, Mario; Turer, Aslan; Lavandero, Sergio; Hill, Joseph A.

    2011-01-01

    The epidemic of heart failure continues apace, and development of novel therapies with clinical efficacy has lagged. Now, important insights into the molecular circuitry of cardiovascular autophagy have raised the prospect that this cellular pathway of protein quality control may be a target of clinical relevance. Whereas basal levels of autophagy are required for cell survival, excessive levels – or perhaps distinct forms of autophagic flux – contribute to disease pathogenesis. Our challenge will be to distinguish mechanisms that drive adaptive versus maladaptive autophagy and to manipulate those pathways for therapeutic gain. Recent evidence suggests this may be possible. Here, we review the fundamental biology of autophagy and its role in a variety of forms of cardiovascular disease. We discuss ways in which this evolutionarily conserved catabolic mechanism can be manipulated, discuss studies presently underway in heart disease, and provide our perspective on where this exciting field may lead in the future. PMID:21723289

  8. In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

    KAUST Repository

    Castagnetti, Francesco; Fiacco, Elisabetta; Imbriano, Carol; Latella, Lucia

    2017-01-01

    with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor

  9. Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

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    Seung Eun Lee

    2014-05-01

    Full Text Available Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68 or control oocytes (44 h IVM; 42.14±4.40 significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68 (p<0.05. Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK, and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1 compared with untreated, 24 h-aged IVM oocytes (p<0.05. Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS activity and DNA fragmentation (p<0.05, and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05 and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1, anti-apoptosis (BCL2L1 and BIRC5; p<0.05, and development (NANOG and SOX2; p<0.05 genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3 compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

  10. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  11. Are mitochondrial reactive oxygen species required for autophagy?

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Jianfei, E-mail: jjf73@pitt.edu [Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh (United States); Maeda, Akihiro; Ji, Jing [Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh (United States); Baty, Catherine J.; Watkins, Simon C. [Center for Biologic Imaging, Department of Cell Biology and Physiology, University of Pittsburgh (United States); Greenberger, Joel S. [Department of Radiation Oncology, University of Pittsburgh (United States); Kagan, Valerian E., E-mail: kagan@pitt.edu [Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh (United States)

    2011-08-19

    Highlights: {yields} Autophageal and apoptotic pathways were dissected in cytochrome c deficient cells. {yields} Staurosporine (STS)-induced autophagy was not accompanied by ROS generation. {yields} Autophagy was detectable in mitochondrial DNA deficient {rho}{sup 0} cells. {yields} Mitochondrial ROS are not required for the STS-induced autophagy in HeLa cells. -- Abstract: Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H{sub 2}O{sub 2} was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient {rho}{sup o} HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.

  12. Are mitochondrial reactive oxygen species required for autophagy?

    International Nuclear Information System (INIS)

    Jiang, Jianfei; Maeda, Akihiro; Ji, Jing; Baty, Catherine J.; Watkins, Simon C.; Greenberger, Joel S.; Kagan, Valerian E.

    2011-01-01

    Highlights: → Autophageal and apoptotic pathways were dissected in cytochrome c deficient cells. → Staurosporine (STS)-induced autophagy was not accompanied by ROS generation. → Autophagy was detectable in mitochondrial DNA deficient ρ 0 cells. → Mitochondrial ROS are not required for the STS-induced autophagy in HeLa cells. -- Abstract: Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H 2 O 2 was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ o HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.

  13. Autophagy-Related Deubiquitinating Enzymes Involved in Health and Disease

    OpenAIRE

    El Magraoui, Fouzi; Reidick, Christina; Meyer, Hemut E.; Platta, Harald W.

    2015-01-01

    Autophagy is an evolutionarily-conserved process that delivers diverse cytoplasmic components to the lysosomal compartment for either recycling or degradation. This involves the removal of protein aggregates, the turnover of organelles, as well as the elimination of intracellular pathogens. In this situation, when only specific cargoes should be targeted to the lysosome, the potential targets can be selectively marked by the attachment of ubiquitin in order to be recognized by autophagy-recep...

  14. p53 and ARF: Unexpected players in autophagy

    OpenAIRE

    Balaburski, Gregor M.; Hontz, Robert D.; Murphy, Maureen E.

    2010-01-01

    p53 and ARF are well-established tumor suppressor proteins that function together in the negative regulation of cancer. Recently, both of these proteins were found to play surprising roles in autophagy. Autophagy (“self-eating”) is a critical response of eukaryotic cells to metabolic and other stress. During this process, portions of the cytosol are sequestered into characteristic double membrane vesicles that are delivered to the lysosome for degradation, leading to the release of free amino...

  15. p53-Mediated Molecular Control of Autophagy in Tumor Cells

    Directory of Open Access Journals (Sweden)

    Maria Mrakovcic

    2018-03-01

    Full Text Available Autophagy is an indispensable mechanism of the eukaryotic cell, facilitating the removal and renewal of cellular components and thereby balancing the cell’s energy consumption and homeostasis. Deregulation of autophagy is now regarded as one of the characteristic key features contributing to the development of tumors. In recent years, the suppression of autophagy in combination with chemotherapeutic treatment has been approached as a novel therapy in cancer treatment. However, depending on the type of cancer and context, interference with the autophagic machinery can either promote or disrupt tumorigenesis. Therefore, disclosure of the major signaling pathways that regulate autophagy and control tumorigenesis is crucial. To date, several tumor suppressor proteins and oncogenes have emerged as eminent regulators of autophagy whose depletion or mutation favor tumor formation. The mammalian cell “janitor” p53 belongs to one of these tumor suppressors that are most commonly mutated in human tumors. Experimental evidence over the last decade convincingly reports that p53 can act as either an activator or an inhibitor of autophagy depending on its subcellular localization and its mode of action. This finding gains particular significance as p53 deficiency or mutant variants of p53 that accumulate in the cytoplasm of tumor cells enable activation of autophagy. Accordingly, we recently identified p53 as a molecular hub that regulates autophagy and apoptosis in histone deacetylase inhibitor-treated uterine sarcoma cells. In light of this novel experimental evidence, in this review, we focus on p53 signaling as a mediator of the autophagic pathway in tumor cells.

  16. Blue-Print Autophagy: Potential for Cancer Treatment

    OpenAIRE

    Nadia Ruocco; Susan Costantini; Maria Costantini

    2016-01-01

    The marine environment represents a very rich source of biologically active compounds with pharmacological applications. This is due to its chemical richness, which is claiming considerable attention from the health science communities. In this review we give a general overview on the marine natural products involved in stimulation and inhibition of autophagy (a type of programmed cell death) linked to pharmacological and pathological conditions. Autophagy represents a complex multistep cellu...

  17. Myostatin Activates the Ubiquitin-Proteasome and Autophagy-Lysosome Systems Contributing to Muscle Wasting in Chronic Kidney Disease

    Science.gov (United States)

    Wang, Dong-Tao; Yang, Ya-Jun; Huang, Ren-Hua; Zhang, Zhi-Hua; Lin, Xin

    2015-01-01

    Our evidence demonstrated that CKD upregulated the expression of myostatin, TNF-α, and p-IkBa and downregulated the phosphorylation of PI3K, Akt, and FoxO3a, which were also associated with protein degradation and muscle atrophy. The autophagosome formation and protein expression of autophagy-related genes were increased in muscle of CKD rats. The mRNA level and protein expression of MAFbx and MuRF-1 were also upregulated in CKD rats, as well as proteasome activity of 26S. Moreover, activation of myostatin elicited by TNF-α induces C2C12 myotube atrophy via upregulating the expression of autophagy-related genes, including MAFbx and MuRF1 and proteasome subunits. Inactivation of FoxO3a triggered by PI3K inhibitor LY294002 prevented the myostatin-induced increase of expression of MuRF1, MAFbx, and LC3-II protein in C2C12 myotubes. The findings were further consolidated by using siRNA interference and overexpression of myostatin. Additionally, expression of myostatin was activated by TNF-α via a NF-κB dependent pathway in C2C12 myotubes, while inhibition of NF-κB activity suppressed myostatin and improved myotube atrophy. Collectively, myostatin mediated CKD-induced muscle catabolism via coordinate activation of the autophagy and the ubiquitin-proteasome systems. PMID:26448817

  18. Curcumin attenuates paraquat-induced cell death in human neuroblastoma cells through modulating oxidative stress and autophagy.

    Science.gov (United States)

    Jaroonwitchawan, Thiranut; Chaicharoenaudomrung, Nipha; Namkaew, Jirapat; Noisa, Parinya

    2017-01-01

    Paraquat is a neurotoxic agent, and oxidative stress plays an important role in neuronal cell death after paraquat exposure. In this study, we assessed the neuroprotective effect of curcumin against paraquat and explored the underlying mechanisms of curcumin in vitro. Curcumin treatment prevented paraquat-induced reactive oxygen species (ROS) and apoptotic cell death. Curcumin also exerted a neuroprotective effect by increasing the expression of anti-apoptotic and antioxidant genes. The pretreatment of curcumin significantly decreased gene expression and protein production of amyloid precursor protein. The activation of autophagy process was found defective in paraquat-induced cells, indicated by the accumulation and reduction of LC3I/II. Noteworthy, curcumin restored LC3I/II expression after the pretreatment. Collectively, curcumin demonstrated as a prominent suppressor of ROS, and could reverse autophagy induction in SH-SY5Y cells. The consequences of this were the reduction of APP production and prevention of SH-SY5Y cells from apoptosis. Altogether, curcumin potentially serves as a therapeutic agent of neurodegenerative diseases, associated with ROS overproduction and autophagy dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Targeting autophagy in cancer management – strategies and developments

    International Nuclear Information System (INIS)

    Ozpolat, Bulent; Benbrook, Doris M

    2015-01-01

    Autophagy is a highly regulated catabolic process involving lysosomal degradation of intracellular components, damaged organelles, misfolded proteins, and toxic aggregates, reducing oxidative stress and protecting cells from damage. The process is also induced in response to various conditions, including nutrient deprivation, metabolic stress, hypoxia, anticancer therapeutics, and radiation therapy to adapt cellular conditions for survival. Autophagy can function as a tumor suppressor mechanism in normal cells and dysregulation of this process (ie, monoallelic Beclin-1 deletion) may lead to malignant transformation and carcinogenesis. In tumors, autophagy is thought to promote tumor growth and progression by helping cells to adapt and survive in metabolically-challenged and harsh tumor microenvironments (ie, hypoxia and acidity). Recent in vitro and in vivo studies in preclinical models suggested that modulation of autophagy can be used as a therapeutic modality to enhance the efficacy of conventional therapies, including chemo and radiation therapy. Currently, more than 30 clinical trials are investigating the effects of autophagy inhibition in combination with cytotoxic chemotherapies and targeted agents in various cancers. In this review, we will discuss the role, molecular mechanism, and regulation of autophagy, while targeting this process as a novel therapeutic modality, in various cancers

  20. Overweight in elderly people induces impaired autophagy in skeletal muscle.

    Science.gov (United States)

    Potes, Yaiza; de Luxán-Delgado, Beatriz; Rodriguez-González, Susana; Guimarães, Marcela Rodrigues Moreira; Solano, Juan J; Fernández-Fernández, María; Bermúdez, Manuel; Boga, Jose A; Vega-Naredo, Ignacio; Coto-Montes, Ana

    2017-09-01

    Sarcopenia is the gradual loss of skeletal muscle mass, strength and quality associated with aging. Changes in body composition, especially in skeletal muscle and fat mass are crucial steps in the development of chronic diseases. We studied the effect of overweight on skeletal muscle tissue in elderly people without reaching obesity to prevent this extreme situation. Overweight induces a progressive protein breakdown reflected as a progressive withdrawal of anabolism against the promoted catabolic state leading to muscle wasting. Protein turnover is regulated by a network of signaling pathways. Muscle damage derived from overweight displayed by oxidative and endoplasmic reticulum (ER) stress induces inflammation and insulin resistance and forces the muscle to increase requirements from autophagy mechanisms. Our findings showed that failure of autophagy in the elderly deprives it to deal with the cell damage caused by overweight. This insufficiently efficient autophagy leads to an accumulation of p62 and NBR1, which are robust markers of protein aggregations. This impaired autophagy affects myogenesis activity. Depletion of myogenic regulatory factors (MRFs) without links to variations in myostatin levels in overweight patients suggest a possible reduction of satellite cells in muscle tissue, which contributes to declined muscle quality. This discovery has important implications that improve the understanding of aged-related atrophy caused by overweight and demonstrates how impaired autophagy is one of the main responsible mechanisms that aggravate muscle wasting. Therefore, autophagy could be an interesting target for therapeutic interventions in humans against muscle impairment diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity

    Directory of Open Access Journals (Sweden)

    Stern Stephan T

    2012-06-01

    Full Text Available Abstract The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences.

  2. Autophagy as a Possible Underlying Mechanism of Nanomaterial Toxicity

    Directory of Open Access Journals (Sweden)

    Vanessa Cohignac

    2014-07-01

    Full Text Available The rapid development of nanotechnologies is raising safety concerns because of the potential effects of engineered nanomaterials on human health, particularly at the respiratory level. Since the last decades, many in vivo studies have been interested in the pulmonary effects of different classes of nanomaterials. It has been shown that some of them can induce toxic effects, essentially depending on their physico-chemical characteristics, but other studies did not identify such effects. Inflammation and oxidative stress are currently the two main mechanisms described to explain the observed toxicity. However, the exact underlying mechanism(s still remain(s unknown and autophagy could represent an interesting candidate. Autophagy is a physiological process in which cytoplasmic components are digested via a lysosomal pathway. It has been shown that autophagy is involved in the pathogenesis and the progression of human diseases, and is able to modulate the oxidative stress and pro-inflammatory responses. A growing amount of literature suggests that a link between nanomaterial toxicity and autophagy impairment could exist. In this review, we will first summarize what is known about the respiratory effects of nanomaterials and we will then discuss the possible involvement of autophagy in this toxicity. This review should help understand why autophagy impairment could be taken as a promising candidate to fully understand nanomaterials toxicity.

  3. The role of autophagy in microbial infection and immunity

    Directory of Open Access Journals (Sweden)

    Desai M

    2015-01-01

    Full Text Available Mayura Desai,1 Rong Fang,2 Jiaren Sun11Department of Microbiology and Immunology, 2Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, TX, USAAbstract: The autophagy pathway represents an evolutionarily conserved cell recycling process that is activated in response to nutrient deprivation and other stress signals. Over the years, it has been linked to an array of cellular functions. Equally, a wide range of cell-intrinsic, as well as extracellular, factors have been implicated in the induction of the autophagy pathway. Microbial infections represent one such factor that can not only activate autophagy through specific mechanisms but also manipulate the response to the invading microbe's advantage. Moreover, in many cases, particularly among viruses, the pathway has been shown to be intricately involved in the replication cycle of the pathogen. Conversely, autophagy also plays a role in combating the infection process, both through direct destruction of the pathogen and as one of the key mediating factors in the host defense mechanisms of innate and adaptive immunity. Further, the pathway also plays a role in controlling the pathogenesis of infectious diseases by regulating inflammation. In this review, we discuss various interactions between pathogens and the cellular autophagic response and summarize the immunological functions of the autophagy pathway.Keywords: autophagy, xenophagy, antiviral, antibacterial

  4. Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases

    Science.gov (United States)

    Woo, Jongchan; Park, Eunsook; Dinesh-Kumar, S. P.

    2014-01-01

    Autophagy is a highly conserved biological process during which double membrane bound autophagosomes carry intracellular cargo material to the vacuole or lysosome for degradation and/or recycling. Autophagosome biogenesis requires Autophagy 4 (Atg4) cysteine protease-mediated processing of ubiquitin-like Atg8 proteins. Unlike single Atg4 and Atg8 genes in yeast, the Arabidopsis genome contains two Atg4 (AtAtg4a and AtAtg4b) and nine Atg8 (AtAtg8a–AtAtg8i) genes. However, we know very little about specificity of different AtAtg4s for processing of different AtAtg8s. Here, we describe a unique bioluminescence resonance energy transfer-based AtAtg8 synthetic substrate to assess AtAtg4 activity in vitro and in vivo. In addition, we developed a unique native gel assay of superhRLUC catalytic activity assay to monitor cleavage of AtAtg8s in vitro. Our results indicate that AtAtg4a is the predominant protease and that it processes AtAtg8a, AtAtg8c, AtAtg8d, and AtAtg8i better than AtAtg4b in vitro. In addition, kinetic analyses indicate that although both AtAtg4s have similar substrate affinity, AtAtg4a is more active than AtAtg4b in vitro. Activity of AtAtg4s is reversibly inhibited in vitro by reactive oxygen species such as H2O2. Our in vivo bioluminescence resonance energy transfer analyses in Arabidopsis transgenic plants indicate that the AtAtg8 synthetic substrate is efficiently processed and this is AtAtg4 dependent. These results indicate that the synthetic AtAtg8 substrate is used efficiently in the biogenesis of autophagosomes in vivo. Transgenic Arabidopsis plants expressing the AtAtg8 synthetic substrate will be a valuable tool to dissect autophagy processes and the role of autophagy during different biological processes in plants. PMID:24379391

  5. Rapamycin and chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show promising results in valosin containing protein multisystem proteinopathy.

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    Angèle Nalbandian

    Full Text Available Mutations in the valosin containing protein (VCP gene cause hereditary Inclusion body myopathy (hIBM associated with Paget disease of bone (PDB, frontotemporal dementia (FTD, more recently termed multisystem proteinopathy (MSP. Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem

  6. Resveratrol and caloric restriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating endoplasmic reticulum stress in high-fat diet-fed rats.

    Science.gov (United States)

    Ding, Shibin; Jiang, Jinjin; Zhang, Guofu; Bu, Yongjun; Zhang, Guanghui; Zhao, Xiangmei

    2017-01-01

    Studies have demonstrated that resveratrol (a natural polyphenol) and caloric restriction activate Sirtuin-1 (SIRT1) and induce autophagy. Furthermore, autophagy is induced by the SIRT1-FoxO signaling pathway and was recently shown to be a critical protective mechanism against non-alcoholic fatty liver disease (NAFLD) development. We aimed to compare the effects of resveratrol and caloric restriction on hepatic lipid metabolism and elucidate the mechanism by which resveratrol supplementation and caloric restriction alleviate hepatosteatosis by examining the molecular interplay between SIRT1 and autophagy. Eight-week-old male Wistar rats (40) were divided into four groups: the STD group, which was fed a standard chow diet; the HFD group, which was fed a high-fat diet; HFD-RES group, which was fed a high-fat diet plus resveratrol (200 mg/kg.bw); and the HFD-CR group, which was fed a high-fat diet in portions containing 70% of the mean intake of the HFD group rats. The groups were maintained for 18 weeks. Metabolic parameters, Oil Red O and hematoxylin-eosin staining of the liver, and the mRNA and protein expression of SIRT1, autophagy markers and endoplasmic reticulum(ER) stress-associated genes in the liver were assessed after the 18-week treatment. We found that resveratrol (200 mg/kg bw) and caloric restriction (30%) partially prevented hepatic steatosis and hepatocyte ballooning, increased the expression of SIRT1 and autophagy markers while decreasing ER stress markers in the liver and alleviated lipid metabolism disorder. Moreover, caloric restriction provided superior protection against HFD-induced hepatic fatty accumulation compared with resveratrol and the effects were associated with decreased total energy intake and body weight. We conclude that the SIRT1-autophagy pathway and decreased ER stress are universally required for the protective effects of moderate caloric restriction (30%) and resveratrol (a pharmacological SIRT1 activator) supplementation

  7. Resveratrol and caloric restriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating endoplasmic reticulum stress in high-fat diet-fed rats.

    Directory of Open Access Journals (Sweden)

    Shibin Ding

    Full Text Available Studies have demonstrated that resveratrol (a natural polyphenol and caloric restriction activate Sirtuin-1 (SIRT1 and induce autophagy. Furthermore, autophagy is induced by the SIRT1-FoxO signaling pathway and was recently shown to be a critical protective mechanism against non-alcoholic fatty liver disease (NAFLD development. We aimed to compare the effects of resveratrol and caloric restriction on hepatic lipid metabolism and elucidate the mechanism by which resveratrol supplementation and caloric restriction alleviate hepatosteatosis by examining the molecular interplay between SIRT1 and autophagy.Eight-week-old male Wistar rats (40 were divided into four groups: the STD group, which was fed a standard chow diet; the HFD group, which was fed a high-fat diet; HFD-RES group, which was fed a high-fat diet plus resveratrol (200 mg/kg.bw; and the HFD-CR group, which was fed a high-fat diet in portions containing 70% of the mean intake of the HFD group rats. The groups were maintained for 18 weeks. Metabolic parameters, Oil Red O and hematoxylin-eosin staining of the liver, and the mRNA and protein expression of SIRT1, autophagy markers and endoplasmic reticulum(ER stress-associated genes in the liver were assessed after the 18-week treatment. We found that resveratrol (200 mg/kg bw and caloric restriction (30% partially prevented hepatic steatosis and hepatocyte ballooning, increased the expression of SIRT1 and autophagy markers while decreasing ER stress markers in the liver and alleviated lipid metabolism disorder. Moreover, caloric restriction provided superior protection against HFD-induced hepatic fatty accumulation compared with resveratrol and the effects were associated with decreased total energy intake and body weight.We conclude that the SIRT1-autophagy pathway and decreased ER stress are universally required for the protective effects of moderate caloric restriction (30% and resveratrol (a pharmacological SIRT1 activator

  8. Inhibition of the Autophagy Pathway Synergistically Potentiates the Cytotoxic Activity of Givinostat(ITF2357on Human Glioblastoma Cancer Stem Cells

    Directory of Open Access Journals (Sweden)

    Francesca Angeletti

    2016-10-01

    Full Text Available Increasing evidence highlighted the role of cancer stem cells (CSCs in the development of tumor resistance to therapy, particularly in glioblastoma (GBM. Therefore, the development of new therapies, specifically directed against GBM CSCs, constitutes an important research avenue. Considering the extended range of cancer-related pathways modulated by histone acetylation/deacetylation processes, we studied the anti-proliferative and pro-apoptotic efficacy of givinostat (GVS, a pan-histone deacetylase inhibitor, on cell cultures enriched in CSCs, isolated from nine human GBMs. We report that GVS induced a significant reduction of viability and self-renewal ability in all GBM CSC cultures; conversely, GVS exposure did not cause a significant cytotoxic activity toward differentiated GBM cells and normal mesenchymal human stem cells.Analysing the cellular and molecular mechanisms involved, we demonstrated that GVS affected CSC viability through the activation of programmed cell death pathways. In particular, a marked stimulation of macroautophagy was observed after GVS treatment. To understand the functional link between GVS treatment and autophagy activation, different genetic and pharmacological interfering strategies were used. We show that the up-regulation of the autophagy process, obtained by deprivation of growth factors, induced a reduction of CSC sensitivity to GVS, while the pharmacological inhibition of the autophagy pathway and the silencing of the key autophagy gene ATG7, increased the cell death rate induced by GVS. Altogether these findings suggest that autophagy represents a pro-survival mechanism activated by GBM CSCs to counteract the efficacy of the anti-proliferative activity of GVS. In conclusion, we demonstrate that GVS is a novel pharmacological tool able to target GBM CSC viability and its efficacy can be enhanced by autophagy inhibitory strategies.

  9. Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines

    Science.gov (United States)

    Hirasawa, Kazuhiro; Moriya, Shota; Miyahara, Kana; Kazama, Hiromi; Hirota, Ayako; Takemura, Jun; Abe, Akihisa; Inazu, Masato; Hiramoto, Masaki; Tsukahara, Kiyoaki

    2016-01-01

    Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”. PMID

  10. A standardized extract of Butea monosperma (Lam.) flowers suppresses the IL-1β-induced expression of IL-6 and matrix-metalloproteases by activating autophagy in human osteoarthritis chondrocytes.

    Science.gov (United States)

    Ansari, Mohammad Y; Khan, Nazir M; Haqqi, Tariq M

    2017-12-01

    Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1β. The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1β induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that

  11. Role of autophagy in disease resistance and hypersensitive response-associated cell death

    DEFF Research Database (Denmark)

    Hofius, Daniel; Munch, David; Bressendorff, Simon

    2011-01-01

    Ancient autophagy pathways are emerging as key defense modules in host eukaryotic cells against microbial pathogens. Apart from actively eliminating intracellular intruders, autophagy is also responsible for cell survival, for example by reducing the deleterious effects of endoplasmic reticulum...

  12. Autophagy is essential for the differentiation of porcine PSCs into insulin-producing cells.

    Science.gov (United States)

    Ren, Lipeng; Yang, Hong; Cui, Yanhua; Xu, Shuanshuan; Sun, Fen; Tian, Na; Hua, Jinlian; Peng, Sha

    2017-07-01

    Porcine pancreatic stem cells (PSCs) are seed cells with potential use for diabetes treatment. Stem cell differentiation requires strict control of protein turnover and lysosomal digestion of organelles. Autophagy is a highly conserved process that controls the turnover of organelles and proteins within cells and contributes to the balance of cellular components. However, whether autophagy plays roles in PSC differentiation remains unknown. In this study, we successfully induced porcine PSCs into insulin-producing cells and found that autophagy was activated during the second induction stage. Inhibition of autophagy in the second stage resulted in reduced differentiational efficiency and impaired glucose-stimulated insulin secretion. Moreover, the expression of active β-catenin increased while autophagy was activated but was suppressed when autophagy was inhibited. Therefore, autophagy is essential to the formation of insulin-producing cells, and the effects of autophagy on differentiation may be regulated by canonical Wnt signalling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

    Energy Technology Data Exchange (ETDEWEB)

    Han, Jun; Wang, Qiao-Chu; Zhu, Cheng-Cheng; Liu, Jun; Zhang, Yu [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Cui, Xiang-Shun; Kim, Nam-Hyung [Department of Animal Science, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Sun, Shao-Chen, E-mail: sunsc@njau.edu.cn [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China)

    2016-06-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. - Highlights: • DON exposure disrupted meiotic spindle by reducing p-MAPK expression. • DON exposure caused retardation of cell cycle progression in porcine oocytes. • DON triggered autophagy and early-apoptosis in porcine oocytes. • DON exposure led to aberrant epigenetic modifications in porcine oocytes.

  14. Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

    International Nuclear Information System (INIS)

    Han, Jun; Wang, Qiao-Chu; Zhu, Cheng-Cheng; Liu, Jun; Zhang, Yu; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2016-01-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. - Highlights: • DON exposure disrupted meiotic spindle by reducing p-MAPK expression. • DON exposure caused retardation of cell cycle progression in porcine oocytes. • DON triggered autophagy and early-apoptosis in porcine oocytes. • DON exposure led to aberrant epigenetic modifications in porcine oocytes.

  15. Bortezomib initiates endoplasmic reticulum stress, elicits autophagy and death in Echinococcus granulosus larval stage.

    Directory of Open Access Journals (Sweden)

    María Celeste Nicolao

    Full Text Available Cystic echinococcosis (CE is a worldwide distributed helminthic zoonosis caused by Echinococcus granulosus. Benzimidazole derivatives are currently the only drugs for chemotherapeutic treatment of CE. However, their low efficacy and the adverse effects encourage the search for new therapeutic targets. We evaluated the in vitro efficacy of Bortezomib (Bz, a proteasome inhibitor, in the larval stage of the parasite. After 96 h, Bz showed potent deleterious effects at a concentration of 5 μM and 0.5 μM in protoscoleces and metacestodes, respectively (P < 0.05. After 48 h of exposure to this drug, it was triggered a mRNA overexpression of chaperones (Eg-grp78 and Eg-calnexin and of Eg-ire2/Eg-xbp1 (the conserved UPR pathway branch in protoscoleces. No changes were detected in the transcriptional expression of chaperones in Bz-treated metacestodes, thus allowing ER stress to be evident and viability to highly decrease in comparison with protoscoleces. We also found that Bz treatment activated the autophagic process in both larval forms. These facts were evidenced by the increase in the amount of transcripts of the autophagy related genes (Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16 together with the increase in Eg-Atg8-II detected by western blot and by in toto immunofluorescence labeling. It was further confirmed by direct observation of autophagic structures by electronic microscopy. Finally, in order to determine the impact of autophagy induction on Echinococcus cell viability, we evaluated the efficacy of Bz in combination with rapamycin and a synergistic cytotoxic effect on protoscolex viability was observed when both drugs were used together. In conclusion, our findings demonstrated that Bz induced endoplasmic reticulum stress, autophagy and subsequent death allowing to identify unstudied parasite-host pathways that could provide a new insight for control of parasitic diseases.

  16. The 'selfish brain' is regulated by aquaporins and autophagy under nutrient deprivation.

    Science.gov (United States)

    Ye, Qiao; Wu, Yonghong; Gao, Yan; Li, Zhihui; Li, Weiguang; Zhang, Chenggang

    2016-05-01

    The brain maintains its mass and physiological functional capacity compared with other organs under harsh conditions such as starvation, a mechanism termed the 'selfish brain' theory. To further investigate this phenomenon, mice were examined following water and/or food deprivation. Although the body weights of the mice, the weight of the organs except the brain and blood glucose levels were significantly reduced in the absence of water and/or food, the brain weight maintained its original state. Furthermore, no significant differences in the water content of the brain or its energy balance were observed when the mice were subjected to water and/or food deprivation. To further investigate the mechanism underlying the brain maintenance of water and substance homeostasis, the expression levels of aquaporins (AQPs) and autophagy‑specific protein long‑chain protein 3 (LC3) were examined. During the process of water and food deprivation, no significant differences in the transcriptional levels of AQPs were observed. However, autophagy activity levels were initially stimulated, then suppressed in a time‑dependent manner. LC3 and AQPs have important roles for the survival of the brain under conditions of food and water deprivation, which provided further understanding of the mechanism underlying the 'selfish brain' phenomenon. Although not involved in the energy regulation of the 'selfish brain', AQPs were observed to have important roles in water and food deprivation, specifically with regards to the control of water content. Additionally, the brain exhibits an 'unselfish strategy' using autophagy during water and/or food deprivation. The present study furthered current understanding of the 'selfish brain' theory, and identified additional regulating target genes of AQPs and autophagy, with the aim of providing a basis for the prevention of nutrient shortage in humans and animals.

  17. Sinomenine Hydrochloride Protects against Polymicrobial Sepsis via Autophagy

    Directory of Open Access Journals (Sweden)

    Yu Jiang

    2015-01-01

    Full Text Available Sepsis, a systemic inflammatory response to infection, is the major cause of death in intensive care units (ICUs. The mortality rate of sepsis remains high even though the treatment and understanding of sepsis both continue to improve. Sinomenine (SIN is a natural alkaloid extracted from Chinese medicinal plant Sinomenium acutum, and its hydrochloride salt (Sinomenine hydrochloride, SIN-HCl is widely used to treat rheumatoid arthritis (RA. However, its role in sepsis remains unclear. In the present study, we investigated the role of SIN-HCl in sepsis induced by cecal ligation and puncture (CLP in BALB/c mice and the corresponding mechanism. SIN-HCl treatment improved the survival of BALB/c mice that were subjected to CLP and reduced multiple organ dysfunction and the release of systemic inflammatory mediators. Autophagy activities were examined using Western blotting. The results showed that CLP-induced autophagy was elevated, and SIN-HCl treatment further strengthened the autophagy activity. Autophagy blocker 3-methyladenine (3-MA was used to investigate the mechanism of SIN-HCl in vitro. Autophagy activities were determined by examining the autophagosome formation, which was shown as microtubule-associated protein light chain 3 (LC3 puncta with green immunofluorescence. SIN-HCl reduced lipopolysaccharide (LPS-induced inflammatory cytokine release and increased autophagy in peritoneal macrophages (PM. 3-MA significantly decreased autophagosome formation induced by LPS and SIN-HCl. The decrease of inflammatory cytokines caused by SIN-HCl was partially aggravated by 3-MA treatment. Taken together, our results indicated that SIN-HCl could improve survival, reduce organ damage, and attenuate the release of inflammatory cytokines induced by CLP, at least in part through regulating autophagy activities.

  18. Increased autophagy in placentas of intrauterine growth-restricted pregnancies.

    Directory of Open Access Journals (Sweden)

    Tai-Ho Hung

    Full Text Available Unexplained intrauterine growth restriction (IUGR may be a consequence of placental insufficiency; however, its etiology is not fully understood. We surmised that defective placentation in IUGR dysregulates cellular bioenergic homeostasis, leading to increased autophagy in the villous trophoblast. The aims of this work were (1 to compare the differences in autophagy, p53 expression, and apoptosis between placentas of women with normal or IUGR pregnancies; (2 to study the effects of hypoxia and the role of p53 in regulating trophoblast autophagy; and (3 to investigate the relationship between autophagy and apoptosis in hypoxic trophoblasts.Compared with normal pregnant women, women with IUGR had higher placental levels of autophagy-related proteins LC3B-II, beclin-1, and damage-regulated autophagy modulator (DRAM, with increased p53 and caspase-cleaved cytokeratin 18 (M30. Furthermore, cytotrophoblasts cultured under hypoxia (2% oxygen in the presence or absence of nutlin-3 (a p53 activity stimulator had higher levels of LC3B-II, DRAM, and M30 proteins and increased Bax mRNA expression compared with controls cultured under standard conditions. In contrast, administration of pifithrin-α (a p53 activity inhibitor during hypoxia resulted in protein levels that were similar to those of the control groups. Moreover, cytotrophoblasts transfected with LC3B, beclin-1, or DRAM siRNA had higher levels of M30 compared with the controls under hypoxia. However, transfection with Bcl-2 or Bax siRNA did not cause any significant change in the levels of LC3B-II in hypoxic cytotrophoblasts.Together, these results suggest that there is a crosstalk between autophagy and apoptosis in IUGR and that p53 plays a pivotal and complex role in regulating trophoblast cell turnover in response to hypoxic stress.

  19. RITA plus 3-MA overcomes chemoresistance of head and neck cancer cells via dual inhibition of autophagy and antioxidant systems.

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    Shin, Daiha; Kim, Eun Hye; Lee, Jaewang; Roh, Jong-Lyel

    2017-10-01

    Reactivation of p53 and induction of tumor cell apoptosis (RITA) is a small molecule that blocks p53-MDM2 interaction, thereby reactivating p53 in tumors. RITA can induce exclusive apoptosis in cancer cells independently of the p53 pathway; however, the resistance of cancer cells remains a major drawback. Here, we found a novel resistance mechanism of RITA treatment and an effective combined treatment to overcome RITA resistance in head and neck cancer (HNC) cells. The effects of RITA and 3-methyladenine (3-MA) were tested in different HNC cell lines, including cisplatin-resistant and acquired RITA-resistant HNC cells. The effects of each drug alone and in combination were assessed by measuring cell viability, apoptosis, cell cycle, glutathione, reactive oxygen species, protein expression, genetic inhibition of p62 and Nrf2, and a mouse xenograft model of cisplatin-resistant HNC. RITA induced apoptosis of HNC cells at different levels without significantly inhibiting normal cell viability. Following RITA treatment, RITA-resistant HNC cells exhibited a sustained expression of other autophagy-related proteins, overexpressed p62, and displayed activation of the Keap1-Nrf2 antioxidant pathway. The autophagy inhibitor 3-MA sensitized resistant HNC cells to RITA treatment via the dual inhibition of molecules related to the autophagy and antioxidant systems. Silencing of the p62 gene augmented the combined effects. The effective antitumor activity of RITA plus 3-MA was also confirmed in vivo in mouse xenograft models transplanted with resistant HNC cells, showing increased oxidative stress and DNA damage. The results indicate that RITA plus 3-MA can help overcome RITA resistance in HNC cells. This study revealed a novel RITA resistant mechanism associated with the sustained induction of autophagy, p62 overexpression, and Keap1-Nrf2 antioxidant system activation. The combined treatment of RITA with the autophagy inhibitor 3-methyladenine overcomes RITA resistance via dual

  20. Inhibition of autophagy exerts anti-colon cancer effects via apoptosis induced by p53 activation and ER stress

    International Nuclear Information System (INIS)

    Sakitani, Kosuke; Hirata, Yoshihiro; Hikiba, Yohko; Hayakawa, Yoku; Ihara, Sozaburo; Suzuki, Hirobumi; Suzuki, Nobumi; Serizawa, Takako; Kinoshita, Hiroto; Sakamoto, Kei; Nakagawa, Hayato; Tateishi, Keisuke; Maeda, Shin; Ikenoue, Tsuneo; Kawazu, Shoji; Koike, Kazuhiko

    2015-01-01

    Although some molecularly targeted drugs for colorectal cancer are used clinically and contribute to a better prognosis, the current median survival of advanced colorectal cancer patients is not sufficient. Autophagy, a basic cell survival mechanism mediated by recycling of cellular amino acids, plays an important role in cancer. Recently, autophagy has been highlighted as a promising new molecular target. The unfolded protein response (UPR) reportedly act in complementary fashion with autophagy in intestinal homeostasis. However, the roles of UPR in colon cancer under autophagic inhibition remain to be elucidated. We aim to clarify the inhibitory effect of autophagy on colon cancer. We crossed K19 CreERT and Atg5 flox/flox mice to generate Atg5 flox/flox /K19 CreERT mice. Atg5 flox/flox /K19 CreERT mice were first treated with azoxymethane/dextran sodium sulfate and then injected with tamoxifen to inhibit autophagy in CK19-positive epithelial cells. To examine the anti-cancer mechanisms of autophagic inhibition, we used colon cancer cell lines harboring different p53 gene statuses, as well as small interfering RNAs (siRNAs) targeting Atg5 and immunoglobulin heavy-chain binding protein (BiP), a chaperone to aid folding of unfolded proteins. Colon tumors in Atg5 flox/flox /K19 CreERT mice showed loss of autophagic activity and decreased tumor size (the total tumor diameter was 28.1 mm in the control and 20.7 mm in Atg5 flox/flox /K19 CreERT mice, p = 0.036). We found that p53 and UPR/endoplasmic reticulum (ER) stress-related proteins, such as cleaved caspase 3, and CAAT/enhancer-binding protein homologous protein, are up-regulated in colon tumors of Atg5 flox/flox /K19 CreERT mice. Although Atg5 and BiP silencing, respectively, increased apoptosis in p53 wild type cells, Atg5 silencing alone did not show the same effect on apoptosis in p53 mutant cells. However, co-transfection of Atg5 and BiP siRNAs led to increased apoptosis in p53 mutant cells. Blocking autophagy

  1. Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation.

    Science.gov (United States)

    Lavieu, Grégory; Scarlatti, Francesca; Sala, Giusy; Carpentier, Stéphane; Levade, Thierry; Ghidoni, Riccardo; Botti, Joëlle; Codogno, Patrice

    2006-03-31

    The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1(wt)-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation.

  2. Distinct patterns of autophagy evoked by two benzoxazine derivatives in vascular endothelial cells.

    Science.gov (United States)

    Wang, Li; Dong, ZhiWu; Huang, Bin; Zhao, BaoXiang; Wang, Hua; Zhao, Jing; Kung, HsiangFu; Zhang, ShangLi; Miao, JunYing

    2010-11-01

    Macroautophagy (referred to as autophagy) is an evolutionarily conserved, bulk-destruction process in eukaryotes. During this process, the cytoplasm containing long-lived proteins and organelles is engulfed into double-membrane autophagosomes, and ultimately undergoes enzymatic degradation within lysosomes. Autophagy serves as a prosurvival machinery, or it may contribute to cell death. Accumulating evidence indicates that autophagy is involved in the pathogenesis and intervention of various human diseases. Pharmacological autophagy modulators are arousing interest from biologists and clinical physicians in light of their potential for disease therapy and increasing our understanding of the mechanism of autophagy. In this study, we identified two autophagy enhancers, 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) and 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO), in human umbilical vein endothelial cells (HUVEC s) by autophagy assays, and demonstrate that ABO and DBO could stimulate autophagy in an mtor-independent and mtor-dependent manner, respectively; ABO-stimulated autophagy was attributed to the elevation of the Ca2+ channel annexin A7 (ANXA7), whereas DBO's effect was due to the level of intracellular reactive oxygen species (ROS). Importantly, we found that ANXA7 was essential for autophagy induction via modulating the intracellular calcium concentration ([Ca2+]i) in HUVEC s. In summary, our work introduced two distinct autophagy enhancers and highlighted the critical role of ANXA7 in endothelial autophagy.

  3. Targeting autophagy in obesity: from pathophysiology to management.

    Science.gov (United States)

    Zhang, Yingmei; Sowers, James R; Ren, Jun

    2018-04-23

    Obesity poses a severe threat to human health, including the increased prevalence of hypertension, insulin resistance, diabetes mellitus, cancer, inflammation, sleep apnoea and other chronic diseases. Current therapies focus mainly on suppressing caloric intake, but the efficacy of this approach remains poor. A better understanding of the pathophysiology of obesity will be essential for the management of obesity and its complications. Knowledge gained over the past three decades regarding the aetiological mechanisms underpinning obesity has provided a framework that emphasizes energy imbalance and neurohormonal dysregulation, which are tightly regulated by autophagy. Accordingly, there is an emerging interest in the role of autophagy, a conserved homeostatic process for cellular quality control through the disposal and recycling of cellular components, in the maintenance of cellular homeostasis and organ function by selectively ridding cells of potentially toxic proteins, lipids and organelles. Indeed, defects in autophagy homeostasis are implicated in metabolic disorders, including obesity, insulin resistance, diabetes mellitus and atherosclerosis. In this Review, the alterations in autophagy that occur in response to nutrient stress, and how these changes alter the course of obesogenesis and obesity-related complications, are discussed. The potential of pharmacological modulation of autophagy for the management of obesity is also addressed.

  4. The Role of Autophagy in the Pathogenesis of Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Kosuke Yamahara

    2013-01-01

    Full Text Available Diabetic nephropathy is a leading cause of end-stage renal disease worldwide. The multipronged drug approach targeting blood pressure and serum levels of glucose, insulin, and lipids fails to fully prevent the onset and progression of diabetic nephropathy. Therefore, a new therapeutic target to combat diabetic nephropathy is required. Autophagy is a catabolic process that degrades damaged proteins and organelles in mammalian cells and plays a critical role in maintaining cellular homeostasis. The accumulation of proteins and organelles damaged by hyperglycemia and other diabetes-related metabolic changes is highly associated with the development of diabetic nephropathy. Recent studies have suggested that autophagy activity is altered in both podocytes and proximal tubular cells under diabetic conditions. Autophagy activity is regulated by both nutrient state and intracellular stresses. Under diabetic conditions, an altered nutritional state due to nutrient excess may interfere with the autophagic response stimulated by intracellular stresses, leading to exacerbation of organelle dysfunction and diabetic nephropathy. In this review, we discuss new findings showing the relationships between autophagy and diabetic nephropathy and suggest the therapeutic potential of autophagy in diabetic nephropathy.

  5. Autophagy: not good OR bad, but good AND bad.

    Science.gov (United States)

    Altman, Brian J; Rathmell, Jeffrey C

    2009-05-01

    Autophagy is a well-established mechanism to degrade intracellular components and provide a nutrient source to promote survival of cells in metabolic distress. Such stress can be caused by a lack of available nutrients or by insufficient rates of nutrient uptake. Indeed, growth factor deprivation leads to internalization and degradation of nutrient transporters, leaving cells with limited means to access extracellular nutrients even when plentiful.This loss of growth factor signaling and extracellular nutrients ultimately leads to apoptosis, but also activates autophagy, which may degrade intracellular components and provide fuel for mitochondrial bioenergetics. The precise metabolic role of autophagy and how it intersects with the apoptotic pathways in growth factor withdrawal, however, has been uncertain. Our recent findings ingrowth factor-deprived hematopoietic cells show that autophagy can simultaneously contribute to cell metabolism and initiate a pathway to sensitize cells to apoptotic death. This pathway may promote tissue homeostasis by ensuring that only cells with high resistance to apoptosis may utilize autophagy as a survival mechanism when growth factors are limiting and nutrient uptake decreases.

  6. Inhibition of autophagy by TAB2 and TAB3.

    Science.gov (United States)

    Criollo, Alfredo; Niso-Santano, Mireia; Malik, Shoaib Ahmad; Michaud, Mickael; Morselli, Eugenia; Mariño, Guillermo; Lachkar, Sylvie; Arkhipenko, Alexander V; Harper, Francis; Pierron, Gérard; Rain, Jean-Christophe; Ninomiya-Tsuji, Jun; Fuentes, José M; Lavandero, Sergio; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Kroemer, Guido

    2011-11-11

    Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory 'switch' whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.

  7. In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

    KAUST Repository

    Castagnetti, Francesco

    2017-06-13

    Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and agerelated muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

  8. Regorafenib delays the proliferation of hepatocellular carcinoma by inducing autophagy.

    Science.gov (United States)

    Han, Rui; Li, Shixin

    2018-04-02

    The aim of the present study was to investigate the effects of regorafenib on hepatocellular carcinoma autophagy, thereby supressing the malignancy of HCC. First, HepG2 and Hep3B cell autophagy was investigated using GFP-LC3 transfection after the treatment of regorafenib. Then, the activation of Akt/mTOR signaling was analyzed using western blot. Our data showed that liver cancer cell autophagy was significantly induced by 20 μM regorafenib using GFP-LC3 transfection. Meanwhile, regorafenib-induced cell death could largely be abolished by 3-MA or CQ treatment, suggesting that regorafenib-induced HepG2 cell death was partially dependent on autophagy. Moreover, the activation of Akt/mTOR signaling was inhibited by regorafenib pre-incubation. MTT assay showed the combination use of regorafenib and CDDP led to a stronger growth inhibitory effect on HepG2 and Hep3B cells. In summary, regorafenib may acts an adjunctive therapy for liver cancer patients via modulating autophagy-dependent cell death even when apoptosis resistance is induced in cancer cells.

  9. Staying young at heart: autophagy and adaptation to cardiac aging.

    Science.gov (United States)

    Leon, Leonardo J; Gustafsson, Åsa B

    2016-06-01

    Aging is a predominant risk factor for developing cardiovascular disease. Therefore, the cellular processes that contribute to aging are attractive targets for therapeutic interventions that can delay or prevent the development of age-related diseases. Our understanding of the underlying mechanisms that contribute to the decline in cell and tissue functions with age has greatly advanced over the past decade. Classical hallmarks of aging cells include increased levels of reactive oxygen species, DNA damage, accumulation of dysfunctional organelles, oxidized proteins and lipids. These all contribute to a progressive decline in the normal physiological function of the cell and to the onset of age-related conditions. A major cause of the aging process is progressive loss of cellular quality control. Autophagy is an important quality control pathway and is necessary to maintain cardiac homeostasis and to adapt to stress. A reduction in autophagy has been observed in a number of aging models and there is compelling evidence that enhanced autophagy delays aging and extends life span. Enhancing autophagy counteracts age-associated accumulation of protein aggregates and damaged organelles in cells. In this review, we discuss the functional role of autophagy in maintaining homeostasis in the heart, and how a decline is associated with accelerated cardiac aging. We also evaluate therapeutic approaches being researched in an effort to maintain a healthy young heart. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Ammonia Induces Autophagy through Dopamine Receptor D3 and MTOR

    Science.gov (United States)

    Li, Zhiyuan; Ji, Xinmiao; Wang, Wenchao; Liu, Juanjuan; Liang, Xiaofei; Wu, Hong; Liu, Jing; Eggert, Ulrike S.; Liu, Qingsong

    2016-01-01

    Hyperammonemia is frequently seen in tumor microenvironments as well as in liver diseases where it can lead to severe brain damage or death. Ammonia induces autophagy, a mechanism that tumor cells may use to protect themselves from external stresses. However, how cells sense ammonia has been unclear. Here we show that culture medium alone containing Glutamine can generate milimolar of ammonia at 37 degrees in the absence of cells. In addition, we reveal that ammonia acts through the G protein-coupled receptor DRD3 (Dopamine receptor D3) to induce autophagy. At the same time, ammonia induces DRD3 degradation, which involves PIK3C3/VPS34-dependent pathways. Ammonia inhibits MTOR (mechanistic target of Rapamycin) activity and localization in cells, which is mediated by DRD3. Therefore, ammonia has dual roles in autophagy: one to induce autophagy through DRD3 and MTOR, the other to increase autophagosomal pH to inhibit autophagic flux. Our study not only adds a new sensing and output pathway for DRD3 that bridges ammonia sensing and autophagy induction, but also provides potential mechanisms for the clinical consequences of hyperammonemia in brain damage, neurodegenerative diseases and tumors. PMID:27077655

  11. Ghrelin improves vascular autophagy in rats with vascular calcification.

    Science.gov (United States)

    Xu, Mingming; Liu, Lin; Song, Chenfang; Chen, Wei; Gui, Shuyan

    2017-06-15

    This study aimed to investigate whether ghrelin ameliorated vascular calcification (VC) through improving autophagy. VC model was induced by nicotine plus vitamin D 3 in rats and β-glycerophosphate in vascular smooth muscle cell (VSMC). Calcium deposition was detected by von Kossa staining or alizarin red S staining. ALP activity was also detected. Western blot was used to assess the protein expression. Ghrelin treatment attenuated the elevation of calcium deposition and ALP activity in VC model both in vivo and in vitro. Interesting, the protein levels of autophagy markers, LC3 and beclin1 were significantly upregulated by ghrelin in VC model. An autophagy inhibitor, 3-methyladenine blocks the ameliorative effect of ghrelin on VC. Furthermore, protein expressions of phosphate-AMPK were increased by ghrelin treatment both in calcified aorta and VSMC. The effect of ghrelin on autophagy induction and VC attenuation was prevented by AMPK inhibitor, compound C. Our results suggested that ghrelin improved autophagy through AMPK activation, which was resulted in VC amelioration. These data maybe throw light on prevention and therapy of VC. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Methods to Monitor and Manipulate TFEB Activity During Autophagy.

    Science.gov (United States)

    Medina, D L; Settembre, C; Ballabio, A

    2017-01-01

    Macroautophagy is a catabolic process deputed to the turnover of intracellular components. Recent studies have revealed that transcriptional regulation is a major mechanism controlling autophagy. Currently, more than 20 transcription factors have been shown to modulate cellular autophagy levels. Among them, the transcription factor EB (TFEB) appears to have the broadest proautophagy role, given its capacity to control the biogenesis of lysosomes and autophagosomes, the two main organelles required for the autophagy pathway. TFEB has attracted major attention owing to its ability to enhance cellular clearance of pathogenic substrates in a variety of animal models of disease, such as lysosomal storage disorders, Parkinson's, Alzheimer's, α1-antitrypsin, obesity as well as others, suggesting that the TFEB pathway represents an extraordinary possibility for future development of innovative therapies. Importantly, the subcellular localization and activity of TFEB are regulated by its phosphorylation status, suggesting that TFEB activity can be pharmacologically targeted. Given the growing list of common and rare diseases in which manipulation of autophagy may be beneficial, in this chapter we describe a set of validated protocols developed to modulate and analyze TFEB-mediated enhancement of autophagy both in vitro and in vivo conditions. © 2017 Elsevier Inc. All rights reserved.

  13. Blue-Print Autophagy: Potential for Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Nadia Ruocco

    2016-07-01

    Full Text Available The marine environment represents a very rich source of biologically active compounds with pharmacological applications. This is due to its chemical richness, which is claiming considerable attention from the health science communities. In this review we give a general overview on the marine natural products involved in stimulation and inhibition of autophagy (a type of programmed cell death linked to pharmacological and pathological conditions. Autophagy represents a complex multistep cellular process, wherein a double membrane vesicle (the autophagosome captures organelles and proteins and delivers them to the lysosome. This natural and destructive mechanism allows the cells to degrade and recycle its cellular components, such as amino acids, monosaccharides, and lipids. Autophagy is an important mechanism used by cells to clear pathogenic organism and deal with stresses. Therefore, it has also been implicated in several diseases, predominantly in cancer. In fact, pharmacological stimulation or inhibition of autophagy have been proposed as approaches to develop new therapeutic treatments of cancers. In conclusion, this blue-print autophagy (so defined because it is induced and/or inhibited by marine natural products represents a new strategy for the future of biomedicine and of biotechnology in cancer treatment.

  14. System-wide Benefits of Intermeal Fasting by Autophagy.

    Science.gov (United States)

    Martinez-Lopez, Nuria; Tarabra, Elena; Toledo, Miriam; Garcia-Macia, Marina; Sahu, Srabani; Coletto, Luisa; Batista-Gonzalez, Ana; Barzilai, Nir; Pessin, Jeffrey E; Schwartz, Gary J; Kersten, Sander; Singh, Rajat

    2017-12-05

    Autophagy failure is associated with metabolic insufficiency. Although caloric restriction (CR) extends healthspan, its adherence in humans is poor. We established an isocaloric twice-a-day (ITAD) feeding model wherein ITAD-fed mice consume the same food amount as ad libitum controls but at two short windows early and late in the diurnal cycle. We hypothesized that ITAD feeding will provide two intervals of intermeal fasting per circadian period and induce autophagy. We show that ITAD feeding modifies circadian autophagy and glucose/lipid metabolism that correlate with feeding-driven changes in circulating insulin. ITAD feeding decreases adiposity and, unlike CR, enhances muscle mass. ITAD feeding drives energy expenditure, lowers lipid levels, suppresses gluconeogenesis, and prevents age/obesity-associated metabolic defects. Using liver-, adipose-, myogenic-, and proopiomelanocortin neuron-specific autophagy-null mice, we mapped the contribution of tissue-specific autophagy to system-wide benefits of ITAD feeding. Our studies suggest that consuming two meals a day without CR could prevent the metabolic syndrome. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Fluoride-Induced Autophagy via the Regulation of Phosphorylation of Mammalian Targets of Rapamycin in Mice Leydig Cells.

    Science.gov (United States)

    Zhang, Jianhai; Zhu, Yuchen; Shi, Yan; Han, Yongli; Liang, Chen; Feng, Zhiyuan; Zheng, Heping; Eng, Michelle; Wang, Jundong

    2017-10-11

    Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.

  16. Melipona quadrifasciata (Hymenoptera: Apidae) fat body persists through metamorphosis with a few apoptotic cells and an increased autophagy.

    Science.gov (United States)

    Santos, Douglas Elias; Azevedo, Dihego Oliveira; Campos, Lúcio Antônio Oliveira; Zanuncio, José Cola; Serrão, José Eduardo

    2015-03-01

    Fat body, typically comprising trophocytes, provides energy during metamorphosis. The fat body can be renewed once the larval phase is complete or recycled and relocated to form the fat body of the adult insect. This study aims to identify the class of programmed cell death that occurs within the fat body cells during the metamorphosis of the stingless bee Melipona quadrifasciata. Using immunodetection techniques, the fat body of the post-defecating larvae and the white-, pink-, brown-, and black-eyed pupae were tested for cleaved caspase-3 and DNA integrity, followed by ultrastructural analysis and identification of autophagy using RT-PCR for the Atg1 gene. The fat body of M. quadrifasciata showed some apoptotic cells positive for cleaved caspase-3, although without DNA fragmentation. During development, the fat body cells revealed an increased number of mitochondria and free ribosomes, in addition to higher amounts of autophagy Atg1 mRNA, than that of the pupae. The fat body of M. quadrifasciata showed few cells which underwent apoptosis, but there was evidence of increased autophagy at the completion of the larval stage. All together, these data show that some fat body cells persist during metamorphosis in the stingless bee M. quadrifasciata.

  17. Taurine protects against As2O3-induced autophagy in pancreas of rat offsprings through Nrf2/Trx pathway.

    Science.gov (United States)

    Bai, Jie; Yao, Xiaofeng; Jiang, Liping; Qiu, Tianming; Liu, Shuang; Qi, Baoxu; Zheng, Yue; Kong, Yuan; Yang, Guang; Chen, Min; Liu, Xiaofang; Sun, Xiance

    2016-04-01

    Arsenic was increasingly to blame as a risk factor for type 2 diabetes mellitus. In our previous study, we had found iAs stimulated autophagic flux and caused autophagic cell death through ROS pathway in INS-1 cells. Since NF-E2-related factor 2 (Nrf2) and the thioredoxin (Trx) system was a crucial line of defense against ROS, we investigated whether Nrf2/Trx pathway contributed to As2O3-stimulated autophagy and the role of taurine in this study. After treatment with 2 mg/kg BW-8 mg/kg BW As2O3 for 57 d, the expression of Nrf2 protein was decreased significantly in offsprings' pancreas. The expression of Trx gene was decreased significantly in pancreas subsequently. Finally, the generation of reactive oxygen species stimulated autophagy in arsenic-treated pancreas. Taurine could reverse arsenic-inhibited Nrf2 and Trx and inhibit autophagy. In short, inhibition of Nrf2/Trx pathway might play an important role in the pathogenesis of arsenic-related diabetes. Taurine could serve as nutrition supplementation against arsenic-related diabetes in high arsenic exposure area. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  18. Enterovirus 71 induces autophagy by regulating has-miR-30a expression to promote viral replication.

    Science.gov (United States)

    Fu, Yuxuan; Xu, Wentao; Chen, Deyan; Feng, Chunhong; Zhang, Li; Wang, Xiaohui; Lv, Xiaowen; Zheng, Nan; Jin, Yu; Wu, Zhiwei

    2015-12-01

    Enterovirus 71 (EV71), the etiological agent of hand-foot-and-mouth disease, has increasingly become a public health challenge around the world. Previous studies reported that EV71 infection can induce autophagic machinery to enhance viral replication in vitro and in vivo, but did not address the underlying mechanisms. Increasing evidence suggests that autophagy, in a virus-specific manner, may function to degrade viruses or facilitate viral replication. In this study, we reported that EV71 infection of human epidermoid carcinoma (Hep2) and African green monkey kidney cells (Vero) induced autophagy, which is beneficial for viral replication. Our investigation of the mechanisms revealed that EV71 infection resulted in the reduction of cellular miR-30a, which led to the inhibition of Beclin-1, a key autophagy-promoting gene that plays important roles at the early phase of autophagosome formation. We provided further evidence that by modulating cellular miR-30a level through either overexpression or inhibition, one can inhibit or promote EV71 replication, respectively, through regulating autophagic activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Sexual dimorphism in activation of placental autophagy in obese women with evidence for fetal programming from a placenta-specific mouse model.

    Science.gov (United States)

    Muralimanoharan, Sribalasubashini; Gao, Xiaoli; Weintraub, Susan; Myatt, Leslie; Maloyan, Alina

    2016-05-03

    The incidence of maternal obesity and its co-morbidities (diabetes, cardiovascular disease) continues to increase at an alarming rate, with major public health implications. In utero exposure to maternal obesity has been associated with development of cardiovascular and metabolic diseases in the offspring as a result of developmental programming. The placenta regulates maternal-fetal metabolism and shows significant changes in its function with maternal obesity. Autophagy is a cell-survival process, which is responsible for the degradation of damaged organelles and misfolded proteins. Here we show an activation of autophagosomal formation and autophagosome-lysosome fusion in placentas of males but not females from overweight (OW) and obese (OB) women vs. normal weight (NW) women. However, total autophagic activity in these placentas appeared to be decreased as it showed an increase in SQSTM1/p62 and a decrease in lysosomal biogenesis. A mouse model with a targeted deletion of the essential autophagy gene Atg7 in placental tissue showed significant placental abnormalities comparable to those seen in human placenta with maternal obesity. These included a decrease in expression of mitochondrial genes and antioxidants, and decreased lysosomal biogenesis. Strikingly, the knockout mice were developmentally programmed as they showed an increased sensitivity to high-fat diet-induced obesity, hyperglycemia, hyperinsulinemia, increased adiposity, and cardiac remodeling. In summary, our results indicate a sexual dimorphism in placental autophagy in response to maternal obesity. We also show that autophagy plays an important role in placental function and that inhibition of placental autophagy programs the offspring to obesity, and to metabolic and cardiovascular diseases.

  20. Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

    Science.gov (United States)

    Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

    2009-07-01

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

  1. SnRK1 activates autophagy via the TOR signaling pathway in Arabidopsis thaliana.

    Science.gov (United States)

    Soto-Burgos, Junmarie; Bassham, Diane C

    2017-01-01

    Autophagy is a degradation process in which cells break down and recycle their cytoplasmic contents when subjected to environmental stress or during cellular remodeling. The Arabidopsis thaliana SnRK1 complex is a protein kinase that senses changes in energy levels and triggers downstream responses to enable survival. Its mammalian ortholog, AMPK, and yeast ortholog, Snf-1, activate autophagy in response to low energy conditions. We therefore hypothesized that SnRK1 may play a role in the regulation of autophagy in response to nutrient or energy deficiency in Arabidopsis. To test this hypothesis, we determined the effect of overexpression or knockout of the SnRK1 catalytic subunit KIN10 on autophagy activation by abiotic stresses, including nutrient deficiency, salt, osmotic, oxidative, and ER stress. While wild-type plants had low basal autophagy activity in control conditions, KIN10 overexpression lines had increased autophagy under these conditions, indicating activation of autophagy by SnRK1. A kin10 mutant had a basal level of autophagy under control conditions similar to wild-type plants, but activation of autophagy by most abiotic stresses was blocked, indicating that SnRK1 is required for autophagy induction by a wide variety of stress conditions. In mammals, TOR is a negative regulator of autophagy, and AMPK acts to activate autophagy both upstream of TOR, by inhibiting its activity, and in a parallel pathway. Inhibition of Arabidopsis TOR leads to activation of autophagy; inhibition of SnRK1 did not block this activation. Furthermore, an increase in SnRK1 activity was unable to induce autophagy when TOR was also activated. These results demonstrate that SnRK1 acts upstream of TOR in the activation of autophagy in Arabidopsis.

  2. TOR-Dependent and -Independent Pathways Regulate Autophagy in Arabidopsis thaliana.

    Science.gov (United States)

    Pu, Yunting; Luo, Xinjuan; Bassham, Diane C

    2017-01-01

    Autophagy is a critical process for recycling of cytoplasmic materials during environmental stress, senescence and cellular remodeling. It is upregulated under a wide range of abiotic stress conditions and is important for stress tolerance. Autophagy is repressed by the protein kinase target of rapamycin (TOR), which is activated in response to nutrients and in turn upregulates cell growth and translation and inhibits autophagy. Down-regulation of TOR in Arabidopsis thaliana leads to constitutive autophagy and to decreased growth, but the relationship to stress conditions is unclear. Here, we assess the extent to which TOR controls autophagy activation by abiotic stress. Overexpression of TOR inhibited autophagy activation by nutrient starvation, salt and osmotic stress, indicating that activation of autophagy under these conditions requires down-regulation of TOR activity. In contrast, TOR overexpression had no effect on autophagy induced by oxidative stress or ER stress, suggesting that activation of autophagy by these conditions is independent of TOR function. The plant hormone auxin has been shown previously to up-regulate TOR activity. To confirm the existence of two pathways for activation of autophagy, dependent on the stress conditions, auxin was added exogenously to activate TOR, and the effect on autophagy under different conditions was assessed. Consistent with the effect of TOR overexpression, the addition of the auxin NAA inhibited autophagy during nutrient deficiency, salt and osmotic stress, but not during oxidative or ER stress. NAA treatment was unable to block autophagy induced by a TOR inhibitor or by a mutation in the TOR complex component RAPTOR1B , indicating that auxin is upstream of TOR in the regulation of autophagy. We conclude that repression of auxin-regulated TOR activity is required for autophagy activation in response to a subset of abiotic stress conditions.

  3. Hibiscus sabdariffa leaf polyphenolic extract induces human melanoma cell death, apoptosis, and autophagy.

    Science.gov (United States)

    Chiu, Chun-Tang; Hsuan, Shu-Wen; Lin, Hui-Hsuan; Hsu, Cheng-Chin; Chou, Fen-Pi; Chen, Jing-Hsien

    2015-03-01

    Melanoma is the least common but most fatal form of skin cancer. Previous studies have indicated that an aqueous extract of Hibiscus sabdariffa leaves possess hypoglycemic, hypolipidemic, and antioxidant effects. In this study, we want to investigate the anticancer activity of Hibiscus leaf polyphenolic (HLP) extract in melanoma cells. First, HLP was exhibited to be rich in epicatechin gallate (ECG) and other polyphenols. Apoptotic and autophagic activities of HLP and ECG were further evaluated by DAPI stain, cell-cycle analysis, and acidic vascular organelle (AVO) stain. Our results revealed that both HLP and ECG induced the caspases cleavages, Bcl-2 family proteins regulation, and Fas/FasL activation in A375 cells. In addition, we also revealed that the cells presented AVO-positive after HLP treatments. HLP could increase the expressions of autophagy-related proteins autophagy-related gene 5 (ATG5), Beclin1, and light chain 3-II (LC3-II), and induce autophagic cell death in A375 cells. These data indicated that the anticancer effect of HLP, partly contributed by ECG, in A375 cells. HLP potentially could be developed as an antimelanoma agent. © 2015 Institute of Food Technologists®

  4. Genetic perspective on the role of the autophagy-lysosome pathway in Parkinson disease.

    Science.gov (United States)

    Gan-Or, Ziv; Dion, Patrick A; Rouleau, Guy A

    2015-01-01

    Parkinson disease (PD), once considered as a prototype of a sporadic disease, is now known to be considerably affected by various genetic factors, which interact with environmental factors and the normal process of aging, leading to PD. Large studies determined that the hereditary component of PD is at least 27%, and in some populations, single genetic factors are responsible for more than 33% of PD patients. Interestingly, many of these genetic factors, such as LRRK2, GBA, SMPD1, SNCA, PARK2, PINK1, PARK7, SCARB2, and others, are involved in the autophagy-lysosome pathway (ALP). Some of these genes encode lysosomal enzymes, whereas others correspond to proteins that are involved in transport to the lysosome, mitophagy, or other autophagic-related functions. Is it possible that all these factors converge into a single pathway that causes PD? In this review, we will discuss these genetic findings and the role of the ALP in the pathogenesis of PD and will try to answer this question. We will suggest a novel hypothesis for the pathogenic mechanism of PD that involves the lysosome and the different autophagy pathways.

  5. Inhibition of autophagy enhances the cytotoxic effect of PA-MSHA in breast cancer

    International Nuclear Information System (INIS)

    Xu, Wen-Huan; Liu, Zhe-Bin; Hou, Yi-Feng; Hong, Qi; Hu, Da-Li; Shao, Zhi-Ming

    2014-01-01

    PA-MSHA, a genetically engineered Pseudomonas aeruginosa (PA) strain, is currently under investigation as a new anti-cancer drug. It can induce cell cycle arrest and apoptosis in different human cancer cells, including hormone receptor negative breast cancer cells. However, the underlying mechanism of tumor lethality mediated by PA-MSHA remains to be fully investigated. The effect of PA-MSHA on human hormone receptor negative breast cancer cells was analyzed by morphological measurement, western blot, cell proliferation assay and mouse xenograft model. PA-MSHA was found to induce endoplasmic reticulum (ER) stress in breast cancer cell lines through the IRE1 signaling pathway. Inhibiting autophagy potentiated the cytotoxic effect of PA-MSHA while treating breast cancer cell lines. In mouse xenograft model, PA-MSHA produced more pronounced tumor suppression in mice inoculated with IRE1 gene knockdown. MDA-MB-231HM cells. These findings demonstrated inhibiting autophagy together with PA-MSHA might be a promising therapeutic strategy in treating hormone receptor negative breast cancer cells

  6. The composition of the vaginal microbiome in first trimester pregnant women influences the level of autophagy and stress in vaginal epithelial cells.

    Science.gov (United States)

    Nasioudis, Dimitrios; Forney, Larry J; Schneider, G Maria; Gliniewicz, Karol; France, Michael T; Boester, Allison; Sawai, Mio; Scholl, Jessica; Witkin, Steven S

    2017-09-01

    Epithelial cells lining the vagina are major components of genital tract immunity. The influence of the vaginal microbiome on properties of host epithelial cells is largely unexplored. We evaluated whether differences in the most abundant lactobacilli species or bacterial genera in the vagina of first trimester pregnant women were associated with variations in the extent of stress and autophagy in vaginal epithelial cells. Vaginal swabs from 154 first trimester pregnant women were analyzed for bacterial composition by amplification and sequencing of the V1-V3 region of bacterial 16S rRNA genes. Vaginal epithelial cells were lysed and autophagy quantitated by measurement of p62. Intracellular levels of the inducible 70kDa heat shock protein (hsp70), an indicator of cell stress and an autophagy inhibitor, were determined. When Lactobacillus crispatus was the most abundant member of the vaginal microbiota, epithelial p62 and hsp70 levels were lowest as compared to when other bacterial taxa were most abundant. The highest concentrations of p62 and hsp70 were associated with Streptococcus and Bifidobacterium abundance. The p62 level associated with Gardnerella abundance was lower than that observed when lactobacilli other than L. crispatus were most abundant. In conclusion, in the first trimester of pregnancy the abundance of different bacterial taxa is associated with variations in autophagy and magnitude of the stress response in vaginal epithelial cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy

    DEFF Research Database (Denmark)

    Farkas, Thomas; Daugaard, Mads; Jaattela, Marja

    2011-01-01

    Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered...... for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new...

  8. An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor

    Science.gov (United States)

    Dagdas, Yasin F; Belhaj, Khaoula; Maqbool, Abbas; Chaparro-Garcia, Angela; Pandey, Pooja; Petre, Benjamin; Tabassum, Nadra; Cruz-Mireles, Neftaly; Hughes, Richard K; Sklenar, Jan; Win, Joe; Menke, Frank; Findlay, Kim; Banfield, Mark J; Kamoun, Sophien; Bozkurt, Tolga O

    2016-01-01

    Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses. DOI: http://dx.doi.org/10.7554/eLife.10856.001 PMID:26765567

  9. Roles of autophagy in male reproductive development in plants

    Directory of Open Access Journals (Sweden)

    Shigeru eHanamata

    2014-09-01

    Full Text Available Autophagy, a major catabolic pathway in eukaryotic cells, is essential in development, maintenance of cellular homeostasis, immunity and programmed cell death (PCD in multicellular organisms. In plant cells, autophagy plays roles in recycling of proteins and metabolites including lipids, and is involved in many physiological processes such as abiotic and biotic stress responses. However, its roles during reproductive development had remained poorly understood. Quantitative live cell imaging techniques for the autophagic flux and genetic studies in several plant species have recently revealed significant roles of autophagy in developmental processes, regulation of PCD and lipid metabolism. We here review the novel roles of autophagic fluxes in plant cells, and discuss their possible significance in PCD and metabolic regulation, with particular focus on male reproductive development during the pollen maturation.

  10. Characterization of early autophagy signaling by quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer Tg; Zarei, Mostafa; Sprenger, Adrian

    2014-01-01

    . To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon...... revealing regulated phosphorylation sites on proteins involved in a wide range of cellular processes and an impact of the treatments on the kinome. To approach the potential function of the identified phosphorylation sites we performed a screen for MAP1LC3-interacting proteins and identified a group...... induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis...

  11. Autophagy regulates the stemness of cervical cancer stem cells

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    Yang Y

    2017-06-01

    Full Text Available Yi Yang,1,2 Li Yu,1 Jin Li,1 Ya Hong Yuan,1 Xiao Li Wang,1 Shi Rong Yan,1 Dong Sheng Li,1 Yan Ding1 1Hubei Key Laboratory of Embryonic Stem Cell Research, 2Reproductive Center, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China Abstract: Cancer stem cells (CSCs are a rare population of multipotent cells with the capacity to self-renew. It has been reported that there are CSCs in cervical cancer cells. Pluripotency-associated (PA transcription factors such as Oct4, Sox2, Nanog and CD44 have been used to isolate CSCs subpopulations. In this study, we showed that autophagy plays an important role in the biological behavior of cervical cancer cells. The expression of the autophagy protein Beclin 1 and LC3B was higher in tumorspheres established from human cervical cancers cell lines (and CaSki than in the parental adherent cells. It was also observed that the basal and starvation-induced autophagy flux was higher in tumorspheres than in the bulk population. Autophagy could regulate the expression level of PA proteins in cervical CSCs. In addition, CRISPR/Cas 9-mediated Beclin 1 knockout enhanced the malignancy of HeLa cells, leading to accumulation of PA proteins and promoted tumorsphere formation. Our findings suggest that autophagy modulates homeostasis of PA proteins, and Beclin 1 is critical for CSC maintenance and tumor development in nude mice. This demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance. Keywords: cervical cancer, autophagy, cancer stem cell, LC3, Oct4

  12. Shock Wave Therapy Promotes Cardiomyocyte Autophagy and Survival during Hypoxia

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    Ling Du

    2017-06-01

    Full Text Available Background: Autophagy plays an important role in cardiovascular disease. Controversy still exists regarding the effect of autophagy on ischemic/hypoxic myocardium. Cardiac shock wave therapy (CSWT is an effective alternative treatment for refractory ischemic heart disease. Whether CSWT can regulate cardiomyocyte autophagy under hypoxic conditions is not clear. We established a myocardial hypoxia model using the H9c2 cell line and performed shock waves (SWs treatment to evaluate the effect of SW on autophagy. Methods: The H9c2 cells were incubated under hypoxic conditions, and SW treatment was then performed at energies of 0.02, 0.05, or 0.10 mJ/mm2. The cell viability and intracellular ATP level were examined. Western blot analysis was used to assess the expression of LC3B, AMPK, mTOR, Beclin-1, Sirt1, and HIF-1α. Autophagic vacuoles were visualized by monodansylcadaverine staining. Results: After the 24-hour hypoxic period, cardiomyocyte viability and ATP levels were decreased and autophagy was significantly increased in H9c2 cells. SW treatment with an energy of 0.05 mJ/mm2 significantly increased the cellular viability, ATP level, LC3B-II/I, and number of autophagic vacuoles. In addition, phosphorylated AMPK and Sirt1 were increased and phosphorylated mTOR and HIF-1α were decreased after SW treatment. Conclusion: SW treatment can potentially promote cardiomyocyte autophagy during hypoxia and protect cardiomyocyte function by regulating the AMPK/mTOR pathway.

  13. Tuning flux: autophagy as a target of heart disease therapy

    Science.gov (United States)

    Xie, Min; Morales, Cyndi R.; Lavandero, Sergio; Hill, Joseph A.

    2013-01-01

    Purpose of review Despite maximum medical and mechanical support therapy, heart failure remains a relentlessly progressive disorder with substantial morbidity and mortality. Autophagy, an evolutionarily conserved process of cellular cannibalization, has been implicated in virtually all forms of cardiovascular disease. Indeed, its role is context dependent, antagonizing or promoting disease depending on the circumstance. Here, we review current understanding of the role of autophagy in the pathogenesis of heart failure and explore this pathway as a target of therapeutic intervention. Recent findings In preclinical models of heart disease, cardiomyocyte autophagic flux is activated; indeed, its role in disease pathogenesis is the subject of intense investigation to define mechanism. Similarly, in failing human heart of a variety of etiologies, cardiomyocyte autophagic activity is upregulated, and therapy, such as with mechanical support systems, elicits declines in autophagy activity. However, when suppression of autophagy is complete, rapid and catastrophic cell death occurs, consistent with a model in which basal autophagic flux is required for proteostasis. Thus, a narrow zone of ‘optimal’ autophagy seems to exist. The challenge moving forward is to tune the stress-triggered autophagic response within that ‘sweet spot’ range for therapeutic benefit. Summary Whereas we have known for some years of the participation of lysosomal mechanisms in heart disease, it is only recently that upstream mechanisms (autophagy) are being explored. The challenge for the future is to dissect the underlying circuitry and titrate the response into an optimal, proteostasis-promoting range in hopes of mitigating the ever-expanding epidemic of heart failure. PMID:21415729

  14. The cell on the edge of life and death: Crosstalk between autophagy and apoptosis.

    Science.gov (United States)

    Kasprowska-Liśkiewicz, Daniela

    2017-09-21

    Recently, the crosstalk between autophagy and apoptosis has attracted broader attention. Basal autophagy serves to maintain cell homeostasis, while the upregulation of this process is an element of stress response that enables the cell to survive under adverse conditions. Autophagy may also determine the fate of the cell through its interactions with cell death pathways. The protein networks that control the initiation and the execution phase of these two processes are highly interconnected. Several scenarios for the crosstalk between autophagy and apoptosis exist. In most cases, the activation of autophagy represents an attempt of the cell to cope with stress, and protects the cell from apoptosis or delays its initiation. Generally, the simultaneous activation of pro-survival and pro-death pathways is prevented by the mutual inhibitory crosstalk between autophagy and apoptosis. But in some circumstances, autophagy or the proteins of the core autophagic machinery may promote cellular demise through excessive self-digestion (so-called "autophagic cell death") or by stimulating the activation of other cell death pathways. It is controversial whether cells actually die via autophagy, which is why the term "autophagic cell death" has been under intense debate lately. This review summarizes the recent findings on the multilevel crosstalk between autophagy and apoptosis in aspects of common regulators, mutual inhibition of these processes, the stimulation of apoptosis by autophagy or autophagic proteins and finally the role of autophagy as a death-execution mechanism.

  15. Autophagy deficiency in macrophages enhances NLRP3 inflammasome activity and chronic lung disease following silica exposure

    International Nuclear Information System (INIS)

    Jessop, Forrest; Hamilton, Raymond F.; Rhoderick, Joseph F.; Shaw, Pamela K.; Holian, Andrij

    2016-01-01

    Autophagy is an important metabolic mechanism that can promote cellular survival following injury. The specific contribution of autophagy to silica-induced inflammation and disease is not known. The objective of these studies was to determine the effects of silica exposure on the autophagic pathway in macrophages, as well as the general contribution of autophagy in macrophages to inflammation and disease. Silica exposure enhanced autophagic activity in vitro in Bone Marrow derived Macrophages and in vivo in Alveolar Macrophages isolated from silica-exposed mice. Impairment of autophagy in myeloid cells in vivo using Atg5 fl/fl LysM-Cre + mice resulted in enhanced cytotoxicity and inflammation after silica exposure compared to littermate controls, including elevated IL-18 and the alarmin HMGB1 in the whole lavage fluid. Autophagy deficiency caused some spontaneous inflammation and disease. Greater silica-induced acute inflammation in Atg5 fl/fl LysM-Cre + mice correlated with increased fibrosis and chronic lung disease. These studies demonstrate a critical role for autophagy in suppressing silica-induced cytotoxicity and inflammation in disease development. Furthermore, this data highlights the importance of basal autophagy in macrophages and other myeloid cells in maintaining lung homeostasis. - Highlights: • Silica exposure increases autophagy in macrophages. • Autophagy deficient mice have enhanced inflammation and silicosis. • Autophagy deficiency in macrophages results in greater silica-induced cytotoxicity. • Autophagy deficiency in macrophages increases extracellular IL-18 and HMGB1.

  16. The potential regulatory roles of NAD(+) and its metabolism in autophagy.

    Science.gov (United States)

    Zhang, Dong-Xia; Zhang, Jia-Ping; Hu, Jiong-Yu; Huang, Yue-Sheng

    2016-04-01

    (Macro)autophagy mediates the bulk degradation of defective organelles, long-lived proteins and protein aggregates in lysosomes and plays a critical role in cellular and tissue homeostasis. Defective autophagy processes have been found to contribute to a variety of metabolic diseases. However, the regulatory mechanisms of autophagy are not fully understood. Increasing data indicate that nicotinamide adenine nucleotide (NAD(+)) homeostasis correlates intimately with autophagy. NAD(+) is a ubiquitous coenzyme that functions primarily as an electron carrier of oxidoreductase in multiple redox reactions. Both NAD(+) homeostasis and its metabolism are thought to play critical roles in regulating autophagy. In this review, we discuss how the regulation of NAD(+) and its metabolism can influence autophagy. We focus on the regulation of NAD(+)/NADH homeostasis and the effects of NAD(+) consumption by poly(ADP-ribose) (PAR) polymerase-1 (PARP-1), NAD(+)-dependent deacetylation by sirtuins and NAD(+) metabolites on autophagy processes and the underlying mechanisms. Future studies should provide more direct evidence for the regulation of autophagy processes by NAD(+). A better understanding of the critical roles of NAD(+) and its metabolites on autophagy will shed light on the complexity of autophagy regulation, which is essential for the discovery of new therapeutic tools for autophagy-related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation*

    Science.gov (United States)

    Chang, Yu-Ping; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Chen, Chia-Ling; Lin, Yee-Shin; Kai, Jui-In; Hsieh, Chia-Yuan; Cheng, Yi-Lin; Choi, Pui-Ching; Chen, Shun-Hua; Chang, Shih-Ping; Liu, Hsiao-Sheng; Lin, Chiou-Feng

    2010-01-01

    Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. PMID:20592027

  18. Autophagy induction under carbon starvation conditions is negatively regulated by carbon catabolite repression.

    Science.gov (United States)

    Adachi, Atsuhiro; Koizumi, Michiko; Ohsumi, Yoshinori

    2017-12-01

    Autophagy is a conserved process in which cytoplasmic components are sequestered for degradation in the vacuole/lysosomes in eukaryotic cells. Autophagy is induced under a variety of starvation conditions, such as the depletion of nitrogen, carbon, phosphorus, zinc, and others. However, apart from nitrogen starvation, it remains unclear how these stimuli induce autophagy. In yeast, for example, it remains contentious whether autophagy is induced under carbon starvation conditions, with reports variously suggesting both induction and lack of induction upon depletion of carbon. We therefore undertook an analysis to account for these inconsistencies, concluding that autophagy is induced in response to abrupt carbon starvation when cells are grown with glycerol but not glucose as the carbon source. We found that autophagy under these conditions is mediated by nonselective degradation that is highly dependent on the autophagosome-associated scaffold proteins Atg11 and Atg17. We also found that the extent of carbon starvation-induced autophagy is positively correlated with cells' oxygen consumption rate, drawing a link between autophagy induction and respiratory metabolism. Further biochemical analyses indicated that maintenance of intracellular ATP levels is also required for carbon starvation-induced autophagy and that autophagy plays an important role in cell viability during prolonged carbon starvation. Our findings suggest that carbon starvation-induced autophagy is negatively regulated by carbon catabolite repression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. From Oxidative Stress Damage to Pathways, Networks, and Autophagy via MicroRNAs

    Directory of Open Access Journals (Sweden)

    Nikolai Engedal

    2018-01-01

    Full Text Available Oxidative stress can alter the expression level of many microRNAs (miRNAs, but how these changes are integrated and related to oxidative stress responses is poorly understood. In this article, we addressed this question by using in silico tools. We reviewed the literature for miRNAs whose expression is altered upon oxidative stress damage and used them in combination with various databases and software to predict common gene targets of oxidative stress-modulated miRNAs and affected pathways. Furthermore, we identified miRNAs that simultaneously target the predicted oxidative stress-modulated miRNA gene targets. This generated a list of novel candidate miRNAs potentially involved in oxidative stress responses. By literature search and grouping of pathways and cellular responses, we could classify these candidate miRNAs and their targets into a larger scheme related to oxidative stress responses. To further exemplify the potential of our approach in free radical research, we used our explorative tools in combination with ingenuity pathway analysis to successfully identify new candidate miRNAs involved in the ubiquitination process, a master regulator of cellular responses to oxidative stress and proteostasis. Lastly, we demonstrate that our approach may also be useful to identify novel candidate connections between oxidative stress-related miRNAs and autophagy. In summary, our results indicate novel and important aspects with regard to the integrated biological roles of oxidative stress-modulated miRNAs and demonstrate how this type of in silico approach can be useful as a starting point to generate hypotheses and guide further research on the interrelation between miRNA-based gene regulation, oxidative stress signaling pathways, and autophagy.

  20. Crosstalk between autophagy and inflammatory signalling pathways: balancing defence and homeostasis.

    Science.gov (United States)

    Cadwell, Ken

    2016-11-01

    Autophagy has broad functions in immunity, ranging from cell-autonomous defence to coordination of complex multicellular immune responses. The successful resolution of infection and avoidance of autoimmunity necessitates efficient and timely communication between autophagy and pathways that sense the immune environment. The recent literature indicates that a variety of immune mediators induce or repress autophagy. It is also becoming increasingly clear that immune signalling cascades are subject to regulation by autophagy, and that a return to homeostasis following a robust immune response is critically dependent on this pathway. Importantly, examples of non-canonical forms of autophagy in mediating immunity are pervasive. In this article, the progress in elucidating mechanisms of crosstalk between autophagy and inflammatory signalling cascades is reviewed. Improved mechanistic understanding of the autophagy machinery offers hope for treating infectious and inflammatory diseases.

  1. Autophagy: Friend or Foe in Breast Cancer Development, Progression, and Treatment

    International Nuclear Information System (INIS)

    Berardi, D.E.; Campodonico, P.B.; Bessone, M.I.D.; Urtreger, A.J.; Todaro, L.B.

    2011-01-01

    Autophagy is a catabolic process responsible for the degradation and recycling of long-lived proteins and organelles by lysosomes. This degradative pathway sustains cell survival during nutrient deprivation, but in some circumstances, autophagy leads to cell death. Thereby, autophagy can serve as tumor suppressor, as the reduction in autophagic capacity causes malignant transformation and spontaneous tumors. On the other hand, this process also functions as a protective cell-survival mechanism against environmental stress causing resistance to antineoplastic therapies. Although autophagy inhibition, combined with anticancer agents, could be therapeutically beneficial in some cases, autophagy induction by itself could lead to cell death in some apoptosis-resistant cancers, indicating that autophagy induction may also be used as a therapy. This paper summarizes the most important findings described in the literature about autophagy and also discusses the importance of this process in clinical settings

  2. LAMP-2 deficiency leads to hippocampal dysfunction but normal clearance of neuronal substrates of chaperone-mediated autophagy in a mouse model for Danon disease

    OpenAIRE

    Rothaug, Michelle; Stroobants, Stijn; Schweizer, Michaela; Peters, Judith; Zunke, Friederike; Allerding, Mirka; D?Hooge, Rudi; Saftig, Paul; Blanz, Judith

    2015-01-01

    The Lysosomal Associated Membrane Protein type-2 (LAMP-2) is an abundant lysosomal membrane protein with an important role in immunity, macroautophagy (MA) and chaperone-mediated autophagy (CMA). Mutations within the Lamp2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by (cardio)myopathy and intellectual dysfunction. The pathological hallmark of this disease is an accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscle that, along with ...

  3. The lack of autophagy triggers precocious activation of Notch signaling during Drosophila oogenesis

    Directory of Open Access Journals (Sweden)

    Barth Julia MI

    2012-12-01

    Full Text Available Abstract Background The proper balance of autophagy, a lysosome-mediated degradation process, is indispensable for oogenesis in Drosophila. We recently demonstrated that egg development depends on autophagy in the somatic follicle cells (FC, but not in the germline cells (GCs. However, the lack of autophagy only affects oogenesis when FCs are autophagy-deficient but GCs are wild type, indicating that a dysfunctional signaling between soma and germline may be responsible for the oogenesis defects. Thus, autophagy could play an essential role in modulating signal transduction pathways during egg development. Results Here, we provide further evidence for the necessity of autophagy during oogenesis and demonstrate that autophagy is especially required in subsets of FCs. Generation of autophagy-deficient FCs leads to a wide range of phenotypes that are similar to mutants with defects in the classical cell-cell signaling pathways in the ovary. Interestingly, we observe that loss of autophagy leads to a precocious activation of the Notch pathway in the FCs as monitored by the expression of Cut and Hindsight, two downstream effectors of Notch signaling. Conclusion Our findings point to an unexpected function for autophagy in the modulation of the Notch signaling pathway during Drosophila oogenesis and suggest a function for autophagy in proper receptor activation. Egg development is affected by an imbalance of autophagy between signal sending (germline and signal receiving cell (FC, thus the lack of autophagy in the germline is likely to decrease the amount of active ligand and accordingly compensates for increased signaling in autophagy-defective follicle cells.

  4. MicroRNA-144-3p inhibits autophagy activation and enhances Bacillus Calmette-Guérin infection by targeting ATG4a in RAW264.7 macrophage cells.

    Science.gov (United States)

    Guo, Le; Zhou, Linlin; Gao, Qian; Zhang, Aijun; Wei, Jun; Hong, Dantong; Chu, Yuankui; Duan, Xiangguo; Zhang, Ying; Xu, Guangxian

    2017-01-01

    MicroRNAs (miRNAs) are small noncoding nucleotides that play major roles in the response of host immune cells. Autophagy plays a key role in activating the antimicrobial host defense against Mycobacterium tuberculosis (M. tuberculosis). Whether miRNAs specifically influence the activation of macrophage autophagy during M. tuberculosis infection is largely unknown. In the present study, we demonstrate that Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection of macrophages leads to increased expression of miR-144-3p, which targets autophagy-related gene 4a (ATG4a), to inhibit autophagy activation and antimicrobial responses to BCG. Overexpression of miR-144-3p significantly decreased both mRNA and protein levels of ATG4a, inhibited the formation of autophagosomes in RAW264.7 cells and increased intracellular survival of BCG. However, transfection with miR-144-3p inhibitor led to an increase in ATG4a levels, accelerated the autophagic response in macrophages, and decreased BCG survival in macrophages. The experimental results of this study reveal a novel role of miR-144-3p in inhibiting autophagy activation by targeting ATG4a and enhancing BCG infection, and provide potential targets for developing improved treatment.

  5. No evidence for activated autophagy in left ventricular myocardium at early reperfusion with protection by remote ischemic preconditioning in patients undergoing coronary artery bypass grafting.

    Directory of Open Access Journals (Sweden)

    Nilgün Gedik

    Full Text Available Remote ischemic preconditioning (RIPC by repeated brief limb ischemia/reperfusion reduces myocardial injury in patients undergoing coronary artery bypass grafting (CABG. Activation of signal transducer and activator of transcription 5 (STAT5 in left ventricular (LV myocardium at early reperfusion is associated with such protection. Autophagy, i.e., removal of dysfunctional cellular components through lysosomes, has been proposed as one mechanism of cardioprotection. Therefore, we analyzed whether or not the protection by RIPC is associated with activated autophagy.CABG patients were randomized to undergo RIPC (3×5 min blood pressure cuff inflation/5 min deflation or placebo (cuff deflated before skin incision (n = 10/10. Transmural myocardial biopsies were taken from the LV before cardioplegia (baseline and at early (5-10 min reperfusion. RIPC-induced protection was reflected by decreased serum troponin I concentration area under the curve (194±17 versus 709±129 ng/ml × 72 h, p = 0.002. Western blotting for beclin-1-phosphorylation and protein expression of autophagy-related gene 5-12 (ATG5-12 complex, light chain 3 (LC3, parkin, and p62 was performed. STAT3-, STAT5- and extracellular signal-regulated protein kinase 1/2 (ERK1/2-phosphorylation was used as positive control to confirm signal activation by ischemia/reperfusion.Signals of all analyzed autophagy proteins did not differ between baseline and early reperfusion and not between RIPC and placebo. STAT5-phosphorylation was greater at early reperfusion only with RIPC (2.2-fold, p = 0.02. STAT3- and ERK1/2-phosphorylation were greater at early reperfusion with placebo and RIPC (≥2.7-fold versus baseline, p≤0.05.Protection through RIPC in patients undergoing CABG surgery does not appear to be associated with enhanced autophagy in LV myocardium at early reperfusion.

  6. Proteasome inhibitors activate autophagy involving inhibition of PI3K-Akt-mTOR pathway as an anti-oxidation defense in human RPE cells.

    Directory of Open Access Journals (Sweden)

    Bingrong Tang

    Full Text Available The two major intracellular protein degradation systems, the ubiquitin-proteasome system (UPS and autophagy, work collaboratively in many biological processes including development, apoptosis, aging, and countering oxidative injuries. We report here that, in human retinal pigment epithelial cells (RPE, ARPE-19 cells, proteasome inhibitors, clasto-lactacystinβ-lactone (LA or epoxomicin (Epo, at non-lethal doses, increased the protein levels of autophagy-specific genes Atg5 and Atg7 and enhanced the conversion of microtubule-associated protein light chain (LC3 from LC3-I to its lipidative form, LC3-II, which was enhanced by co-addition of the saturated concentration of Bafilomycin A1 (Baf. Detection of co-localization for LC3 staining and labeled-lysosome further confirmed autophagic flux induced by LA or Epo. LA or Epo reduced the phosphorylation of the protein kinase B (Akt, a downstream target of phosphatidylinositol-3-kinases (PI3K, and mammalian target of rapamycin (mTOR in ARPE-19 cells; by contrast, the induced changes of autophagy substrate, p62, showed biphasic pattern. The autophagy inhibitor, Baf, attenuated the reduction in oxidative injury conferred by treatment with low doses of LA and Epo in ARPE-19 cells exposed to menadione (VK3 or 4-hydroxynonenal (4-HNE. Knockdown of Atg7 with siRNA in ARPE-19 cells reduced the protective effects of LA or Epo against VK3. Overall, our results suggest that treatment with low levels of proteasome inhibitors confers resistance to oxidative injury by a pathway involving inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy.

  7. Isogenic FUS-eGFP iPSC Reporter Lines Enable Quantification of FUS Stress Granule Pathology that Is Rescued by Drugs Inducing Autophagy

    Directory of Open Access Journals (Sweden)

    Lara Marrone

    2018-02-01

    Full Text Available Summary: Perturbations in stress granule (SG dynamics may be at the core of amyotrophic lateral sclerosis (ALS. Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments. : Sterneckert and colleagues generate isogenic FUS-eGFP reporter iPSCs that enable the identification of stress granule (SG phenotypes specifically induced by the ALS mutation FUS P525L. Compound screening shows that modulation of the PI3K/AKT/mTOR pathway regulating autophagy ameliorates SG phenotypes. A second screen identifies similarly acting brain-penetrant US FDA-approved drugs that could be repurposed to treat ALS. Keywords: amyotrophic lateral sclerosis, induced pluripotent stem cells, FUS, stress granules, autophagy, gene editing, CRISPR/Cas9n

  8. The role of autophagy in chloroplast degradation and chlorophagy in immune defenses during Pst DC3000 (AvrRps4 infection.

    Directory of Open Access Journals (Sweden)

    Junjian Dong

    Full Text Available BACKGROUND: Chlorosis of leaf tissue normally observed during pathogen infection may result from the degradation of chloroplasts. There is a growing evidence to suggest that the chloroplast plays a significant role during pathogen infection. Although most degradation of the organelles and cellular structures in plants is mediated by autophagy, its role in chloroplast catabolism during pathogen infection is largely unknown. RESULTS: In this study, we investigated the function of autophagy in chloroplast degradation during avirulent Pst DC3000 (AvrRps4 infection. We examined the expression of defensive marker genes and suppression of bacterial growth using the electrolyte leakage assay in normal light (N and low light (L growing environments of wild-type and atg5-1 plants during pathogen treatment. Stroma-targeted GFP proteins (CT-GFP were observed with LysoTracker Red (LTR staining of autophagosome-like structures in the vacuole. The results showed that Arabidopsis expressed a significant number of small GFP-labeled bodies when infected with avirulent Pst DC3000 (AvrRps4. While barely detectable, there were small GFP-labeled bodies in plants with the CT-GFP expressing atg5-1 mutation. The results showed that chloroplast degradation depends on autophagy and this may play an important role in inhibiting pathogen growth. CONCLUSION: Autophagy plays a role in chloroplast degradation in Arabidopsis during avirulent Pst DC3000 (AvrRps4 infection. Autophagy dependent chloroplast degradation may be the primary source of reactive oxygen species (ROS as well as the pathogen-response signaling molecules that induce the defense response.

  9. Modulation of inflammation and autophagy pathways by trehalose containing eye drop formulation in corneal epithelial cells: implications for dry eye disease

    Directory of Open Access Journals (Sweden)

    Trailokyanath Panigrahi

    2017-10-01

    Full Text Available Ocular surface inflammation is an immunological perturbation activated in response to various adverse conditions and is a key biomarker to understand the disease pathology and its underlying immunological landscape [1]. The molecular link between Inflammation and autophagy, often implicated in disease conditions, is poorly understood. The aim of this study is to understand the regulation of inflammation signaling pathways by using a well-established modulator of autophagy, trehalose (TRE, on desiccation stress-induced inflammation in SV40 immortalized human corneal epithelial cells. To mimic the dry eye condition, HCE cells were exposed to desiccation stress at 80% confluency in a six well tissue culture plate. The medium was completely aspirated and cells were kept for drying at room temperature for 10 min. Fresh medium with TRE was added and incubated for 6 hrs. The regulation of induced inflammatory and autophagic gene expression and protein activation by TRE formulation (1.2% was studied. Optimal drug treatment concentrations were determined by dose escalation cytotoxicity studies. Gene expression was evaluated by quantitative PCR, while protein expression and functions were tested by immunoblotting and fluorescence imaging (Cyto-ID, Lysotracker Red. TRE formulation was able to rescue the morphological changes due to desiccation stress. Live to dead cell ratio increased upon TRE treatment. TRE treatment reduced inflammation induced gene expression of IL-6 (2%, MCP-1 (33.31%, IL-8 (9.56%, MMP-9 (18.96%, and TNFα (58.16% in HCE. Active form of p38, p44/42, and p65 protein levels were altered significantly by TRE treatment. LAMP1 and LC3 autophagy protein markers were also altered with desiccation stress and TRE treatment. The data demonstrate that TRE formulation is effective in reducing desiccation stress induced inflammation in HCE. Further increased phosphorylation of p38, p44/42 and elevated levels of LC3 and LAMP1 suggest that induction

  10. Yeast Methylotrophy and Autophagy in a Methanol-Oscillating Environment on Growing Arabidopsis thaliana Leaves

    Science.gov (United States)

    Kawaguchi, Kosuke; Yurimoto, Hiroya; Oku, Masahide; Sakai, Yasuyoshi

    2011-01-01

    The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes in the methanol assimilation pathway, but not formaldehyde dissimilation or anti-oxidizing enzymes, were necessary for yeast proliferation at the phyllosphere. Furthermore, both peroxisome assembly and pexophagy, a selective autophagy pathway that degrades peroxisomes, were necessary for phyllospheric proliferation. Thus, the present study sheds light on the life cycle and physiology of yeast in the natural environment at both the molecular and cellular levels. PMID:21966472

  11. Catalase and NO CATALASE ACTIVITY1 Promote Autophagy-Dependent Cell Death in Arabidopsis

    DEFF Research Database (Denmark)

    Hackenberg, Thomas; Juul, Trine Maxel; Auzina, Aija

    2013-01-01

    Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea-induced cell death. Here, we identify...... an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase...... activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation...

  12. Inhibition of mammalian S6 kinase by resveratrol suppresses autophagy.

    Science.gov (United States)

    Armour, Sean M; Baur, Joseph A; Hsieh, Sherry N; Land-Bracha, Abigail; Thomas, Sheila M; Sinclair, David A

    2009-06-03

    Resveratrol is a plant-derived polyphenol that promotes health and disease resistance in rodent models, and extends lifespan in lower organisms. A major challenge is to understand the biological processes and molecular pathways by which resveratrol induces these beneficial effects. Autophagy is a critical process by which cells turn over damaged components and maintain bioenergetic requirements. Disruption of the normal balance between pro- and anti-autophagic signals is linked to cancer, liver disease, and neurodegenerative disorders. Here we show that resveratrol attenuates autophagy in response to nutrient limitation or rapamycin in multiple cell lines through a pathway independent of a known target, SIRT1. In a large-scalein vitro kinase screen we identified p70 S6 kinase (S6K1) as a target of resveratrol. Blocking S6K1 activity by expression of a dominant-negative mutant or RNA interference is sufficient to disrupt autophagy to a similar extent as resveratrol. Furthermore, co-administration of resveratrol with S6K1 knockdown does not produce an additive effect. These data indicate that S6K1 is important for the full induction of autophagy in mammals and raise the possibility that some of the beneficial effects of resveratrol are due to modulation of S6K1 activity.

  13. Exocyst and autophagy-related membrane trafficking in plants.

    Science.gov (United States)

    Pecenková, Tamara; Markovic, Vedrana; Sabol, Peter; Kulich, Ivan; Žárský, Viktor

    2017-12-18

    Endomembrane traffic in eukaryotic cells functions partially as a means of communication; delivery of membrane in one direction has to be balanced with a reduction at the other end. This effect is typically the case during the defence against pathogens. To combat pathogens, cellular growth and differentiation are suppressed, while endomembrane traffic is poised towards limiting the pathogen attack. The octameric exocyst vesicle-tethering complex was originally discovered as a factor facilitating vesicle-targeting and vesicle-plasma membrane (PM) fusion during exocytosis prior to and possibly during SNARE complex formation. Interestingly, it was recently implicated both in animals and plants in autophagy membrane traffic. In animal cells, the exocyst is integrated into the mTOR-regulated energy metabolism stress/starvation pathway, participating in the formation and especially initiation of an autophagosome. In plants, the first functional link was to autophagy-related anthocyanin import to the vacuole and to starvation. In this concise review, we summarize the current knowledge of exocyst functions in autophagy and defence in plants that might involve unconventional secretion and compare it with animal conditions. Formation of different exocyst complexes during undisturbed cell growth, as opposed to periods of cellular stress reactions involving autophagy, might contribute to the coordination of endomembrane trafficking pathways. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  14. Autophagy Therapeutic Potential of Garlic in Human Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Yung-Lin Chu

    2013-07-01

    Full Text Available Cancer is one of the deadliest diseases against humans. To tackle this menace, humans have developed several high-technology therapies, such as chemotherapy, tomotherapy, targeted therapy, and antibody therapy. However, all these therapies have their own adverse side effects. Therefore, recent years have seen increased attention being given to the natural food for complementary therapy, which have less side effects. Garlic 大 蒜 Dà Suàn; Allium sativum, is one of most powerful food used in many of the civilizations for both culinary and medicinal purpose. In general, these foods induce cancer cell death by apoptosis, autophagy, or necrosis. Studies have discussed how natural food factors regulate cell survival or death by autophagy in cancer cells. From many literature reviews, garlic could not only induce apoptosis but also autophagy in cancer cells. Autophagy, which is called type-II programmed cell death, provides new strategy in cancer therapy. In conclusion, we wish that garlic could be the pioneer food of complementary therapy in clinical cancer treatment and increase the life quality of cancer patients.

  15. Autophagy: A Novel Therapeutic Target for Diabetic Nephropathy.

    Science.gov (United States)

    Kume, Shinji; Koya, Daisuke

    2015-12-01

    Diabetic nephropathy is a leading cause of end stage renal disease and its occurance is increasing worldwide. The most effective treatment strategy for the condition is intensive treatment to strictly control glycemia and blood pressure using renin-angiotensin system inhibitors. However, a fraction of patients still go on to reach end stage renal disease even under such intensive care. New therapeutic targets for diabetic nephropathy are, therefore, urgently needed. Autophagy is a major catabolic pathway by which mammalian cells degrade macromolecules and organelles to maintain intracellular homeostasis. The accumulation of damaged proteins and organelles is associated with the pathogenesis of diabetic nephropathy. Autophagy in the kidney is activated under some stress conditions, such as oxidative stress and hypoxia in proximal tubular cells, and occurs even under normal conditions in podocytes. These and other accumulating findings have led to a hypothesis that autophagy is involved in the pathogenesis of diabetic nephropathy. Here, we review recent findings underpinning this hypothesis and discuss the advantages of targeting autophagy for the treatment of diabetic nephropathy.

  16. Autophagy: A Novel Therapeutic Target for Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Shinji Kume

    2015-12-01

    Full Text Available Diabetic nephropathy is a leading cause of end stage renal disease and its occurance is increasing worldwide. The most effective treatment strategy for the condition is intensive treatment to strictly control glycemia and blood pressure using renin-angiotensin system inhibitors. However, a fraction of patients still go on to reach end stage renal disease even under such intensive care. New therapeutic targets for diabetic nephropathy are, therefore, urgently needed. Autophagy is a major catabolic pathway by which mammalian cells degrade macromolecules and organelles to maintain intracellular homeostasis. The accumulation of damaged proteins and organelles is associated with the pathogenesis of diabetic nephropathy. Autophagy in the kidney is activated under some stress conditions, such as oxidative stress and hypoxia in proximal tubular cells, and occurs even under normal conditions in podocytes. These and other accumulating findings have led to a hypothesis that autophagy is involved in the pathogenesis of diabetic nephropathy. Here, we review recent findings underpinning this hypothesis and discuss the advantages of targeting autophagy for the treatment of diabetic nephropathy.

  17. System-wide Benefits of Intermeal Fasting by Autophagy

    NARCIS (Netherlands)

    Martinez-Lopez, Nuria; Tarabra, Elena; Toledo, Miriam; Garcia-Macia, Marina; Sahu, Srabani; Coletto, Luisa; Batista-Gonzalez, Ana; Barzilai, Nir; Pessin, Jeffrey E.; Schwartz, Gary J.; Kersten, Sander; Singh, Rajat

    2017-01-01

    Autophagy failure is associated with metabolic insufficiency. Although caloric restriction (CR) extends healthspan, its adherence in humans is poor. We established an isocaloric twice-a-day (ITAD) feeding model wherein ITAD-fed mice consume the same food amount as ad libitum controls but at two

  18. Chlorogenic acid alleviates autophagy and insulin resistance by ...

    Indian Academy of Sciences (India)

    Hua Yan

    2018-04-07

    Apr 7, 2018 ... In this study, a high-fat diet-induced NAFLD rat model was used to investigate the ... CG can improve lipid and glucose meta- ... Healthy female Sprague–Dawley rats (age, 8–9 weeks; .... NAFLD, the protein levels of autophagy-related markers .... adipose tissue dysfunction, vitamin D deficiency and the.

  19. Research progress of hydroxychloroquine and autophagy inhibitors on cancer.

    Science.gov (United States)

    Shi, Ting-Ting; Yu, Xiao-Xu; Yan, Li-Jun; Xiao, Hong-Tao

    2017-02-01

    Hydroxychloroquine (HCQ), the analog of chloroquine, augments the effect of chemotherapies and radiotherapy on various tumors identified in the current clinical trials. Meanwhile, the toxicity of HCQ retinopathy raises concern worldwide. Thus, the potent autophagy inhibitors are urgently needed. A systematic review was related to 'hydroxychloroquine' or 'chloroquine' with 'clinical trials,' 'retinopathy' and 'new autophagy inhibitors.' This led to many cross-references involving HCQ, and these data have been incorporated into the following study. Many preclinical studies indicate that the combination of HCQ with chemotherapies or radiotherapies may enhance the effect of anticancer, providing base for launching cancer clinical trials involving HCQ. The new and more sensitive diagnostic techniques report a prevalence of HCQ retinopathy up to 7.5%. Lys05, SAR405, verteporfin, VATG-027, mefloquine and spautin-1 may be potent autophagy inhibitors. Additional mechanistic studies of HCQ in preclinical models are still required in order to answer these questions whether HCQ actually inhibits autophagy in non-selective tumors and whether the extent of inhibition would be sufficient to alter chemotherapy or radiotherapy sensitivity.

  20. microRNA-101 is a potent inhibitor of autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Wen, Jiayu; Lees, Michael

    2011-01-01

    performed a functional screen in search of microRNAs (miRNAs), which regulate the autophagic flux in breast cancer cells. In this study, we identified the tumour suppressive miRNA, miR-101, as a potent inhibitor of basal, etoposide- and rapamycin-induced autophagy. Through transcriptome profiling, we...

  1. Exocyst and autophagy-related membrane trafficking in plants

    Czech Academy of Sciences Publication Activity Database

    Pečenková, Tamara; Marković, Vedrana; Sabol, P.; Kulich, I.; Žárský, Viktor

    2018-01-01

    Roč. 69, č. 1 (2018), s. 47-57 ISSN 0022-0957 R&D Projects: GA ČR(CZ) GA15-14886S Institutional support: RVO:61389030 Keywords : Autophagy * endomembranes * exocyst * plant defence * secretory transport * ups Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 5.830, year: 2016

  2. Autophagy: A double-edged sword in Alzheimer's disease

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... This article briefly summarizes seminal discoveries that are shedding new light on the critical and unique roles of autophagy in AD and potential therapeutic ... those of the staff, the journals, various programmes, and Current Science, has changed from 'ias.ernet.in' (or 'academy.ias.ernet.in') to 'ias.ac.in'.

  3. N-n-butyl haloperidol iodide protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy.

    Science.gov (United States)

    Wang, Bin; Zhong, Shuping; Zheng, Fuchun; Zhang, Yanmei; Gao, Fenfei; Chen, Yicun; Lu, Binger; Xu, Han; Shi, Ganggang

    2015-09-22

    N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Our results indicated that treatment with F2 inhibited autophagy in H9c2 cells exposed to H/R. 3-methyladenine, an inhibitor of autophagy, suppressed H/R-induced autophagy, and decreased apoptosis, whereas rapamycin, a classical autophagy sensitizer, increased autophagy and apoptosis. Mechanistically, macrophage migration inhibitory factor (MIF) was inhibited by F2 treatment after H/R. Accordingly, small interfering RNA (siRNA)-mediated MIF knockdown decreased H/R-induced autophagy. In summary, F2 protects cardiomyocytes during H/R injury through suppressing autophagy activation. Our results provide a new mechanistic insight into a functional role of F2 against H/R-induced cardiomyocyte