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Sample records for lysosomal alpha-n-acetylgalactosaminidase deficiency

  1. Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: no association with neuroaxonal dystrophy?

    NARCIS (Netherlands)

    Bakker, H. D.; de Sonnaville, M. L.; Vreken, P.; Abeling, N. G.; Groener, J. E.; Keulemans, J. L.; van Diggelen, O. P.

    2001-01-01

    Two new individuals with alpha-NAGA deficiency are presented. The index patient, 3 years old, has congenital cataract, slight motor retardation and secondary demyelinisation. Screening of his sibs revealed an alpha-NAGA deficiency in his 7-year-old healthy brother who had no clinical or neurological

  2. Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus.

    Science.gov (United States)

    Yamamoto, N; Naraparaju, V R; Moore, M; Brent, L H

    1997-03-01

    A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.

  3. Effect of salivary gland adenocarcinoma cell-derived alpha-N-acetylgalactosaminidase on the bioactivity of macrophage activating factor.

    Science.gov (United States)

    Matsuura, Takashi; Uematsu, Takashi; Yamaoka, Minoru; Furusawa, Kiyofumi

    2004-03-01

    The aim of this study was to clarify the effects of alpha-N-acetylgalactosaminidase (alpha-NaGalase) produced by human salivary gland adenocarcinoma (SGA) cells on the bioactivity of macrophage-activating factor (GcMAF). High exo-alpha-NaGalase activity was detected in the SGA cell line HSG. HSG alpha-NaGalase had both exo- and endo-enzyme activities, cleaving the Gal-GalNAc and GalNAc residues linked to Thr/Ser but not releasing the [NeuAc2-6]GalNac residue. Furthermore, GcMAF enzymatically prepared from the Gc protein enhanced the superoxide-generation capacity and phagocytic activity of monocytes/macrophages. However, GcMAF treated with purified alpha-NaGalase did not exhibit these effects. Thus, HSG possesses the capacity to produce larger quantities of alpha-NaGalase, which inactivates GcMAF produced from Gc protein, resulting in reduced phagocytic activity and superoxide-generation capacity of monocytes/macrophages. The present data strongly suggest that HSG alpha-NaGalase acts as an immunodeficiency factor in cancer patients.

  4. Genetics Home Reference: lysosomal acid lipase deficiency

    Science.gov (United States)

    ... lipase deficiency develop multi-organ failure and severe malnutrition and generally do not survive past 1 year. In the later-onset form of lysosomal acid lipase deficiency , signs and symptoms vary and usually begin in mid-childhood, although they can appear anytime up to late ...

  5. Clinical Features of Lysosomal Acid Lipase Deficiency

    NARCIS (Netherlands)

    Burton, Barbara K.; Deegan, Patrick B.; Enns, Gregory M.; Guardamagna, Ornella; Horslen, Simon; Hovingh, Gerard K.; Lobritto, Steve J.; Malinova, Vera; McLin, Valerie A.; Raiman, Julian; Di Rocco, Maja; Santra, Saikat; Sharma, Reena; Sykut-Cegielska, Jolanta; Whitley, Chester B.; Eckert, Stephen; Valayannopoulos, Vassili; Quinn, Anthony G.

    2015-01-01

    The aim of this study was to characterize key clinical manifestations of lysosomal acid lipase deficiency (LAL D) in children and adults. Investigators reviewed medical records of LAL D patients ages ≥5 years, extracted historical data, and obtained prospective laboratory and imaging data on living

  6. Endo-lysosomal dysfunction in human proximal tubular epithelial cells deficient for lysosomal cystine transporter cystinosin.

    Directory of Open Access Journals (Sweden)

    Ekaterina A Ivanova

    Full Text Available Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles.

  7. Lipidomic and Transcriptomic Basis of Lysosomal Dysfunction in Progranulin Deficiency

    Directory of Open Access Journals (Sweden)

    Bret M. Evers

    2017-09-01

    Full Text Available Defective lysosomal function defines many neurodegenerative diseases, such as neuronal ceroid lipofuscinoses (NCL and Niemann-Pick type C (NPC, and is implicated in Alzheimer’s disease (AD and frontotemporal lobar degeneration (FTLD-TDP with progranulin (PGRN deficiency. Here, we show that PGRN is involved in lysosomal homeostasis and lipid metabolism. PGRN deficiency alters lysosome abundance and morphology in mouse neurons. Using an unbiased lipidomic approach, we found that brain lipid composition in humans and mice with PGRN deficiency shows disease-specific differences that distinguish them from normal and other pathologic groups. PGRN loss leads to an accumulation of polyunsaturated triacylglycerides, as well as a reduction of diacylglycerides and phosphatidylserines in fibroblast and enriched lysosome lipidomes. Transcriptomic analysis of PGRN-deficient mouse brains revealed distinct expression patterns of lysosomal, immune-related, and lipid metabolic genes. These findings have implications for the pathogenesis of FTLD-TDP due to PGRN deficiency and suggest lysosomal dysfunction as an underlying mechanism.

  8. Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases.

    NARCIS (Netherlands)

    Asfaw, B.; Ledvinova, J.; Dobrovolny, R.; Bakker, H.; Desnick, R.J.; Diggelen, O.P. van; Jong, J.G.N. de; Kanzaki, T.; Chabas, A.; Maire, I.; Conzelmann, E.; Schindler, D.

    2002-01-01

    Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B

  9. Lysosome

    Directory of Open Access Journals (Sweden)

    Ursula Matte BSc, PhD

    2016-12-01

    Full Text Available Since Christian de Duve first described the lysosome in the 1950s, it has been generally presented as a membrane-bound compartment containing acid hydrolases that enables the cell to degrade molecules without being digested by autolysis. For those working on the field of lysosomal storage disorders, the lack of one such hydrolase would lead to undegraded or partially degraded substrate storage inside engorged organelles disturbing cellular function by yet poorly explored mechanisms. However, in recent years, a much more complex scenario of lysosomal function has emerged, beyond and above the cellular “digestive” system. Knowledge on how the impairment of this organelle affects cell functioning may shed light on signs and symptoms of lysosomal disorders and open new roads for therapy.

  10. Autophagic lysosome reformation dysfunction in glucocerebrosidase deficient cells: relevance to Parkinson disease.

    Science.gov (United States)

    Magalhaes, Joana; Gegg, Matthew E; Migdalska-Richards, Anna; Doherty, Mary K; Whitfield, Phillip D; Schapira, Anthony H V

    2016-08-15

    Glucocerebrosidase (GBA1) gene mutations increase the risk of Parkinson disease (PD). While the cellular mechanisms associating GBA1 mutations and PD are unknown, loss of the glucocerebrosidase enzyme (GCase) activity, inhibition of autophagy and increased α-synuclein levels have been implicated. Here we show that autophagy lysosomal reformation (ALR) is compromised in cells lacking functional GCase. ALR is a cellular process controlled by mTOR which regenerates functional lysosomes from autolysosomes formed during macroautophagy. A decrease in phopho-S6K levels, a marker of mTOR activity, was observed in models of GCase deficiency, including primary mouse neurons and the PD patient derived fibroblasts with GBA1 mutations, suggesting that ALR is compromised. Importantly Rab7, a GTPase crucial for endosome-lysosome trafficking and ALR, accumulated in GCase deficient cells, supporting the notion that lysosomal recycling is impaired. Recombinant GCase treatment reversed ALR inhibition and lysosomal dysfunction. Moreover, ALR dysfunction was accompanied by impairment of macroautophagy and chaperone-mediated autophagy, increased levels of total and phosphorylated (S129) monomeric α-synuclein, evidence of amyloid oligomers and increased α-synuclein release. Concurrently, we found increased cholesterol and altered glucosylceramide homeostasis which could compromise ALR. We propose that GCase deficiency in PD inhibits lysosomal recycling. Consequently neurons are unable to maintain the pool of mature and functional lysosomes required for the autophagic clearance of α-synuclein, leading to the accumulation and spread of pathogenic α-synuclein species in the brain. Since GCase deficiency and lysosomal dysfunction occur with ageing and sporadic PD pathology, the decrease in lysosomal reformation may be a common feature in PD. © The Author 2016. Published by Oxford University Press.

  11. Correction of lysosomal enzyme deficiency in various organs of beta-glucuronidase-deficient mice by allogeneic bone marrow transplantation

    NARCIS (Netherlands)

    Hoogerbrugge, P. M.; Poorthuis, B. J.; Mulder, A. H.; Wagemaker, G.; Dooren, L. J.; Vossen, J. M.; van Bekkum, D. W.

    1987-01-01

    The correction of lysosomal enzyme deficiency was investigated for various organs of beta-glucuronidase-deficient C3H/Rij mice after allogeneic bone marrow transplantation from an enzymatically normal donor strain (C57BL/Rij). In the hemopoietic organs, the enzyme level increased to levels found in

  12. Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn

    Science.gov (United States)

    Aittaleb, Mohamed; Chen, Po-Ju; Akaaboune, Mohammed

    2015-01-01

    ABSTRACT Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we show that rapsyn plays an AChR-independent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induces the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to colocalize with AChR clusters on the cell surface. In rapsyn-deficient (Rapsn−/−) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in Rapsn−/− cells is associated with a significant increase in lysosomal exocytosis, as evidenced by increased release of lysosomal enzymes and plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis and plasma membrane integrity. PMID:26330529

  13. A saposin deficiency model in Drosophila: Lysosomal storage, progressive neurodegeneration and sensory physiological decline.

    Science.gov (United States)

    Hindle, Samantha J; Hebbar, Sarita; Schwudke, Dominik; Elliott, Christopher J H; Sweeney, Sean T

    2017-02-01

    Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. There are four saposin proteins in humans, which are encoded by the prosaposin gene. Mutations causing an absence or impaired function of individual saposins or the whole prosaposin gene lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. We have generated and characterised a Drosophila model of saposin deficiency that shows striking similarities to the human diseases. Drosophila saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. Our data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing our understanding of the cellular pathology implicated in saposin deficiency and related LSDs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Wolman's disease and cholesteryl ester storage disorder: the phenotypic spectrum of lysosomal acid lipase deficiency.

    Science.gov (United States)

    Pericleous, Marinos; Kelly, Claire; Wang, Tim; Livingstone, Callum; Ala, Aftab

    2017-09-01

    Lysosomal acid lipase deficiency is a rare, autosomal recessive condition caused by mutations in the gene encoding lysosomal acid lipase (LIPA) that result in reduced or absent activity of this essential enzyme. The severity of the resulting disease depends on the nature of the underlying mutation and magnitude of its effect on enzymatic function. Wolman's disease is a severe disorder that presents during infancy, resulting in failure to thrive, hepatomegaly, and hepatic failure, and an average life expectancy of less than 4 months. Cholesteryl ester storage disorder arises later in life and is less severe, although the two diseases share many common features, including dyslipidaemia and transaminitis. The prevalence of these diseases has been estimated at one in 40 000 to 300 000, but many cases are undiagnosed and unreported, and awareness among clinicians is low. Lysosomal acid lipase deficiency-which can be diagnosed using dry blood spot testing-is often misdiagnosed as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hereditary dyslipidaemia, or cryptogenic cirrhosis. There are no formal guidelines for treatment of these patients, and treatment options are limited. In this Review we appraise the existing literature on Wolman's disease and cholesteryl ester storage disease, and discuss available treatments, including enzyme replacement therapy, oral lipid-lowering therapy, stem-cell transplantation, and liver transplantation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Property of lysosomal storage disease associated with midbrain pathology in the central nervous system of Lamp-2-deficient mice.

    Science.gov (United States)

    Furuta, Akiko; Kikuchi, Hisae; Fujita, Hiromi; Yamada, Daisuke; Fujiwara, Yuuki; Kabuta, Tomohiro; Nishino, Ichizo; Wada, Keiji; Uchiyama, Yasuo

    2015-06-01

    Lysosome-associated membrane protein-2 (LAMP-2) is the gene responsible for Danon disease, which is characterized by cardiomyopathy, autophagic vacuolar myopathy, and variable mental retardation. To elucidate the function of LAMP-2 in the central nervous system, we investigated the neuropathological changes in Lamp-2-deficient mice. Immunohistochemical observations revealed that Lamp-1 and cathepsin D-positive lysosomal structures increased in the large neurons of the mouse brain. Ubiquitin-immunoreactive aggregates and concanavalin A-positive materials were detected in these neurons. By means of ultrastructural studies, we found various-shaped accumulations, including lipofuscin, glycolipid-like materials, and membranous structures, in the neurons and glial cells of Lamp-2-deficient brains. In deficient mice, glycogen granules accumulated in hepatocyte lysosomes but were not observed in neurons. These pathological features indicate lysosomal storage disease; however, the findings are unlikely a consequence of deficiency of a single lysosomal enzyme. Although previous study results have shown a large amount of autophagic vacuoles in parenchymal cells of the visceral organs, these findings were rarely detected in the brain tissue except for some axons in the substantia nigra, in which abundant activated microglial cells with increased lipid peroxidation were observed. Thus, LAMP-2 in the central nervous system has a possible role in the degradation of the various macromolecules in lysosomes and an additional function concerning protection from oxidative stress, especially in the substantia nigra. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  16. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis: LAMP-2 deficient mice develop pancreatitis.

    Science.gov (United States)

    Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

    2015-11-01

    The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs) in pancreatitis. We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP de-glycosylation and degradation. LAMP cleavage by cathepsin B (CatB) was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger LAMPs' bulk de-glycosylation, but induces their degradation via CatB-mediated cleavage of LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, stimulates the basal and inhibits CCK-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis, and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction.

  17. Lysosomal Storage of Subunit c of Mitochondrial ATP Synthase in Brain-Specific Atp13a2-Deficient Mice.

    Science.gov (United States)

    Sato, Shigeto; Koike, Masato; Funayama, Manabu; Ezaki, Junji; Fukuda, Takahiro; Ueno, Takashi; Uchiyama, Yasuo; Hattori, Nobutaka

    2016-12-01

    Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of early-onset parkinsonism linked to the PARK9 locus. The causative gene for KRS is Atp13a2, which encodes a lysosomal type 5 P-type ATPase. We recently showed that KRS/PARK9-linked mutations lead to several lysosomal alterations, including reduced proteolytic processing of cathepsin D in vitro. However, it remains unknown how deficiency of Atp13a2 is connected to lysosomal impairments. To address this issue, we analyzed brain tissues of Atp13a2 conditional-knockout mice, which exhibited characteristic features of neuronal ceroid lipofuscinosis, including accumulation of lipofuscin positive for subunit c of mitochondrial ATP synthase, suggesting that a common pathogenic mechanism underlies both neuronal ceroid lipofuscinosis and Parkinson disease. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  18. Elevated TMEM106B levels exaggerate lipofuscin accumulation and lysosomal dysfunction in aged mice with progranulin deficiency.

    Science.gov (United States)

    Zhou, Xiaolai; Sun, Lirong; Brady, Owen Adam; Murphy, Kira A; Hu, Fenghua

    2017-01-26

    Mutations resulting in haploinsufficiency of progranulin (PGRN) cause frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), a devastating neurodegenerative disease. Accumulating evidence suggest a crucial role of progranulin in maintaining proper lysosomal function during aging. TMEM106B has been identified as a risk factor for frontotemporal lobar degeneration with progranulin mutations and elevated mRNA and protein levels of TMEM106B are associated with increased risk for frontotemporal lobar degeneration. Increased levels of TMEM106B alter lysosomal morphology and interfere with lysosomal degradation. However, how progranulin and TMEM106B interact to regulate lysosomal function and frontotemporal lobar degeneration (FTLD) disease progression is still unclear. Here we report that progranulin deficiency leads to increased TMEM106B protein levels in the mouse cortex with aging. To mimic elevated levels of TMEM106B in frontotemporal lobar degeneration (FTLD) cases, we generated transgenic mice expressing TMEM106B under the neuronal specific promoter, CamKII. Surprisingly, we found that the total protein levels of TMEM106B are not altered despite the expression of the TMEM106B transgene at mRNA and protein levels, suggesting a tight regulation of TMEM106B protein levels in the mouse brain. However, progranulin deficiency results in accumulation of TMEM106B protein from the transgene expression during aging, which is accompanied by exaggerated lysosomal abnormalities and increased lipofuscin accumulation. In summary, our mouse model nicely recapitulates the interaction between progranulin and TMEM106B in human patients and supports a critical role of lysosomal dysfunction in the frontotemporal lobar degeneration (FTLD) disease progression.

  19. Lysosomal acid lipase deficiency: A hidden disease among cohorts of familial hypercholesterolemia?

    Science.gov (United States)

    Chora, Joana Rita; Alves, Ana Catarina; Medeiros, Ana Margarida; Mariano, Cibelle; Lobarinhas, Goreti; Guerra, António; Mansilha, Helena; Cortez-Pinto, Helena; Bourbon, Mafalda

    Lysosomal acid lipase deficiency (LALD) is an autosomal recessive disorder and an unrecognized cause of dyslipidemia. Patients usually present with dyslipidemia and altered liver function and mutations in LIPA gene are the underlying cause of LALD. The aim of this study was to investigate LALD in individuals with severe dyslipidemia and/or liver steatosis. Coding, splice regions, and promoter region of LIPA were sequenced by Sanger sequencing in a cohort of mutation-negative familial hypercholesterolemia (FH) patients (n = 492) and in a population sample comprising individuals with several types of dyslipidemia and/or liver steatosis (n = 258). This study led to the identification of LALD in 4 children referred to the Portuguese FH Study, all with a clinical diagnosis of FH. Mild liver dysfunction was present at the age of FH diagnosis; however, a diagnosis of LALD was not considered. No adults at the time of referral have been identified with LALD. LALD is a life-threatening disorder, and early identification is crucial for the implementation of specific treatment to avoid premature mortality. FH cohorts should be investigated to identify possible LALD patients, who will need appropriate treatment. These results highlight the importance of correctly identifying the etiology of the dyslipidemia. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  20. Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques.

    Science.gov (United States)

    Gowrishankar, Swetha; Yuan, Peng; Wu, Yumei; Schrag, Matthew; Paradise, Summer; Grutzendler, Jaime; De Camilli, Pietro; Ferguson, Shawn M

    2015-07-14

    Through a comprehensive analysis of organellar markers in mouse models of Alzheimer's disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer's disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer's disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology.

  1. Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer’s disease amyloid plaques

    Science.gov (United States)

    Gowrishankar, Swetha; Yuan, Peng; Wu, Yumei; Schrag, Matthew; Paradise, Summer; Grutzendler, Jaime; De Camilli, Pietro; Ferguson, Shawn M.

    2015-01-01

    Through a comprehensive analysis of organellar markers in mouse models of Alzheimer’s disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer’s disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer’s disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology. PMID:26124111

  2. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis : LAMP-2 deficient mice develop pancreatitis

    NARCIS (Netherlands)

    Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

    2015-01-01

    BACKGROUND & AIMS: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated

  3. Disease: H00146 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H00146 Alpha-N-acetylgalactosaminidase deficiency, including: Schindler disease; K...ided into three types. Type 1 is Schindler disease, characterized by mental retardation, spasticity ,and myo...n alpha-N-acetylgalactosaminidase deficiency (Schindler/Kanzaki disease). ... JOURNAL ... J Hum Genet 49:1-8 (20... ... TITLE ... The 1.9 a structure of human alpha-N-acetylgalactosaminidase: The molecular basis of Schindler a...eland AM, de Jong JG, Wevers RA, Renier WO, Schindler D, Coll MJ, Chabas A, Sakuraba H, Suzuki Y, van Diggel

  4. Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

    Science.gov (United States)

    Maric-Biresev, Jelena; Hunn, Julia P; Krut, Oleg; Helms, J Bernd; Martens, Sascha; Howard, Jonathan C

    2016-04-20

    The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse. We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency. The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal

  5. Lysosomal acid lipase deficiency in rats: Lipid analyses and lipase activities in liver and spleen

    International Nuclear Information System (INIS)

    Kuriyama, M.; Yoshida, H.; Suzuki, M.; Fujiyama, J.; Igata, A.

    1990-01-01

    We report the biological characterization of an animal model of a genetic lipid storage disease analogous to human Wolman's disease. Affected rats accumulated cholesteryl esters (13.3-fold), free cholesterol (2.8-fold), and triglycerides (5.4-fold) in the liver, as well as cholesteryl esters (2.5-fold) and free cholesterol (1.33-fold) in the spleen. Triglycerides did not accumulate, and the levels actually decreased in the spleen. Analysis of the fatty acid composition of the cholesteryl esters and triglycerides showed high percentages of linoleic acid (18:2) and arachidonic acid (20:4) in both organs, especially in the liver. No accumulation of phospholipids, neutral glycosphingolipids, or gangliosides was found in the affected rats. Acid lipase activity for [14C]triolein, [14C]cholesteryl oleate, and 4-methyl-umbelliferyl oleate was deficient in both the liver and spleen of affected rats. Lipase activity at neutral pH was normal in both liver and spleen. Heterozygous rats showed intermediate utilization of these substrates in both organs at levels between those for affected rats and those for normal controls, although they did not accumulate any lipids. These data suggest that these rats represent an animal counterpart of Wolman's disease in humans

  6. Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

    Science.gov (United States)

    Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

    2002-05-01

    Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

  7. Lysosomal impairment in Parkinson's disease.

    Science.gov (United States)

    Dehay, Benjamin; Martinez-Vicente, Marta; Caldwell, Guy A; Caldwell, Kim A; Yue, Zhenyue; Cookson, Mark R; Klein, Christine; Vila, Miquel; Bezard, Erwan

    2013-06-01

    Impairment of autophagy-lysosomal pathways (ALPs) is increasingly regarded as a major pathogenic event in neurodegenerative diseases, including Parkinson's disease (PD). ALP alterations are observed in sporadic PD brains and in toxic and genetic rodent models of PD-related neurodegeneration. In addition, PD-linked mutations and post-translational modifications of α-synuclein impair its own lysosomal-mediated degradation, thereby contributing to its accumulation and aggregation. Furthermore, other PD-related genes, such as leucine-rich repeat kinase-2 (LRRK2), parkin, and phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), have been mechanistically linked to alterations in ALPs. Conversely, mutations in lysosomal-related genes, such as glucocerebrosidase (GBA) and lysosomal type 5 P-type ATPase (ATP13A2), have been linked to PD. New data offer mechanistic molecular evidence for such a connection, unraveling a causal link between lysosomal impairment, α-synuclein accumulation, and neurotoxicity. First, PD-related GBA deficiency/mutations initiate a positive feedback loop in which reduced lysosomal function leads to α-synuclein accumulation, which, in turn, further decreases lysosomal GBA activity by impairing the trafficking of GBA from the endoplasmic reticulum-Golgi to lysosomes, leading to neurodegeneration. Second, PD-related mutations/deficiency in the ATP13A2 gene lead to a general lysosomal impairment characterized by lysosomal membrane instability, impaired lysosomal acidification, decreased processing of lysosomal enzymes, reduced degradation of lysosomal substrates, and diminished clearance of autophagosomes, collectively contributing to α-synuclein accumulation and cell death. According to these new findings, primary lysosomal defects could potentially account for Lewy body formation and neurodegeneration in PD, laying the groundwork for the prospective development of new neuroprotective/disease-modifying therapeutic strategies

  8. Lysosome-Associated Membrane Proteins (LAMP Maintain Pancreatic Acinar Cell Homeostasis: LAMP-2–Deficient Mice Develop PancreatitisSummary

    Directory of Open Access Journals (Sweden)

    Olga A. Mareninova

    2015-11-01

    Full Text Available Background & Aims: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs in pancreatitis. Methods: We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP deglycosylation and degradation. LAMP cleavage by cathepsin B (CatB was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. Results: Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger the LAMPs’ bulk deglycosylation but induces their degradation via CatB-mediated cleavage of the LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, and stimulates the basal and inhibits cholecystokinin-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. Conclusions: The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction. Keywords: Amylase Secretion, Autophagy

  9. Mice doubly-deficient in lysosomal hexosaminidase A and neuraminidase 4 show epileptic crises and rapid neuronal loss.

    Directory of Open Access Journals (Sweden)

    Volkan Seyrantepe

    2010-09-01

    Full Text Available Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts G(M2 to G(M3 ganglioside. Hexa(-/- mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise G(M2 ganglioside via a lysosomal sialidase into glycolipid G(A2, which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4(-/-;Hexa(-/- show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa(-/- or Neu4(-/- siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating G(M2 ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa(-/- mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa(-/- mice.

  10. Lysosomal storage diseases

    Science.gov (United States)

    Ferreira, Carlos R.; Gahl, William A.

    2016-01-01

    Lysosomes are cytoplasmic organelles that contain a variety of different hydrolases. A genetic deficiency in the enzymatic activity of one of these hydrolases will lead to the accumulation of the material meant for lysosomal degradation. Examples include glycogen in the case of Pompe disease, glycosaminoglycans in the case of the mucopolysaccharidoses, glycoproteins in the cases of the oligosaccharidoses, and sphingolipids in the cases of Niemann-Pick disease types A and B, Gaucher disease, Tay-Sachs disease, Krabbe disease, and metachromatic leukodystrophy. Sometimes, the lysosomal storage can be caused not by the enzymatic deficiency of one of the hydrolases, but by the deficiency of an activator protein, as occurs in the AB variant of GM2 gangliosidosis. Still other times, the accumulated lysosomal material results from failed egress of a small molecule as a consequence of a deficient transporter, as in cystinosis or Salla disease. In the last couple of decades, enzyme replacement therapy has become available for a number of lysosomal storage diseases. Examples include imiglucerase, taliglucerase and velaglucerase for Gaucher disease, laronidase for Hurler disease, idursulfase for Hunter disease, elosulfase for Morquio disease, galsulfase for Maroteaux-Lamy disease, alglucosidase alfa for Pompe disease, and agalsidase alfa and beta for Fabry disease. In addition, substrate reduction therapy has been approved for certain disorders, such as eliglustat for Gaucher disease. The advent of treatment options for some of these disorders has led to newborn screening pilot studies, and ultimately to the addition of Pompe disease and Hurler disease to the Recommended Uniform Screening Panel (RUSP) in 2015 and 2016, respectively. PMID:29152458

  11. Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes.

    Science.gov (United States)

    Tanaka, Yoshinori; Suzuki, Genjiro; Matsuwaki, Takashi; Hosokawa, Masato; Serrano, Geidy; Beach, Thomas G; Yamanouchi, Keitaro; Hasegawa, Masato; Nishihara, Masugi

    2017-03-01

    Progranulin (PGRN) haploinsufficiency resulting from loss-of-function mutations in the PGRN gene causes frontotemporal lobar degeneration accompanied by TDP-43 accumulation, and patients with homozygous mutations in the PGRN gene present with neuronal ceroid lipofuscinosis. Although it remains unknown why PGRN deficiency causes neurodegenerative diseases, there is increasing evidence that PGRN is implicated in lysosomal functions. Here, we show PGRN is a secretory lysosomal protein that regulates lysosomal function and biogenesis by controlling the acidification of lysosomes. PGRN gene expression and protein levels increased concomitantly with the increase of lysosomal biogenesis induced by lysosome alkalizers or serum starvation. Down-regulation or insufficiency of PGRN led to the increased lysosomal gene expression and protein levels, while PGRN overexpression led to the decreased lysosomal gene expression and protein levels. In particular, the level of mature cathepsin D (CTSDmat) dramatically changed depending upon PGRN levels. The acidification of lysosomes was facilitated in cells transfected with PGRN. Then, this caused degradation of CTSDmat by cathepsin B. Secreted PGRN is incorporated into cells via sortilin or cation-independent mannose 6-phosphate receptor, and facilitated the acidification of lysosomes and degradation of CTSDmat. Moreover, the change of PGRN levels led to a cell-type-specific increase of insoluble TDP-43. In the brain tissue of FTLD-TDP patients with PGRN deficiency, CTSD and phosphorylated TDP-43 accumulated in neurons. Our study provides new insights into the physiological function of PGRN and the role of PGRN insufficiency in the pathogenesis of neurodegenerative diseases. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Mice, double deficient in lysosomal serine carboxypeptidases Scpep1 and Cathepsin A develop the hyperproliferative vesicular corneal dystrophy and hypertrophic skin thickenings.

    Directory of Open Access Journals (Sweden)

    Xuefang Pan

    Full Text Available Vasoactive and mitogenic peptide, endothelin-1 (ET-1 plays an important role in physiology of the ocular tissues by regulating the growth of corneal epithelial cells and maintaining the hemodynamics of intraocular fluids. We have previously established that ET-1 can be degraded in vivo by two lysosomal/secreted serine carboxypeptidases, Cathepsin A (CathA and Serine Carboxypeptidase 1 (Scpep1 and that gene-targeted CathAS190A /Scpep1-/- mice, deficient in CathA and Scpep1 have a prolonged half-life of circulating ET-1 associated with systemic hypertension. In the current work we report that starting from 6 months of age, ~43% of CathAS190A /Scpep1-/- mice developed corneal clouding that eventually caused vision impairment. Histological evaluation of these mice demonstrated a selective fibrotic thickening and vacuolization of the corneas, resembling human hyperproliferative vesicular corneal stromal dystrophy and coexisting with a peculiar thickening of the skin epidermis. Moreover, we found that cultured corneal epithelial cells, skin fibroblasts and vascular smooth muscle cells derived from CathA/Scpep1-deficient mice, demonstrated a significantly higher proliferative response to treatment with exogenous ET-1, as compared with cells from wild type mice. We also detected increased activation level of ERK1/2 and AKT kinases involved in cell proliferation in the ET-1-treated cultured cells from CathA/Scpep1 deficient mice. Together, results from our experimental model suggest that; in normal tissues the tandem of serine carboxypeptidases, Scpep1 and CathA likely constitutes an important part of the physiological mechanism responsible for the balanced elimination of heightened levels of ET-1 that otherwise would accumulate in tissues and consequently contribute to development of the hyper-proliferative corneal dystrophy and abnormal skin thickening.

  13. Progranulin, lysosomal regulation and neurodegenerative disease.

    Science.gov (United States)

    Kao, Aimee W; McKay, Andrew; Singh, Param Priya; Brunet, Anne; Huang, Eric J

    2017-06-01

    The discovery that heterozygous and homozygous mutations in the gene encoding progranulin are causally linked to frontotemporal dementia and lysosomal storage disease, respectively, reveals previously unrecognized roles of the progranulin protein in regulating lysosome biogenesis and function. Given the importance of lysosomes in cellular homeostasis, it is not surprising that progranulin deficiency has pleiotropic effects on neural circuit development and maintenance, stress response, innate immunity and ageing. This Progress article reviews recent advances in progranulin biology emphasizing its roles in lysosomal function and brain innate immunity, and outlines future avenues of investigation that may lead to new therapeutic approaches for neurodegeneration.

  14. Lysosomes, Lysosomal Storage Diseases, and Inflammation

    Directory of Open Access Journals (Sweden)

    Calogera M. Simonaro PhD

    2016-05-01

    Full Text Available Lysosomes were originally described in the early 1950s by de Duve who was also the first to recognize the importance of these organelles in human disease. We know now that lysosomes are involved in numerous biological processes, and abnormalities in lysosomal function may result in a broad range of diseases. This review will briefly discuss the role of lysosomes in inflammation and how disruption of normal lysosomal function in the lysosomal storage diseases (LSDs leads to abnormalities in inflammation and immunity.

  15. Sensitivity to lysosome-dependent cell death is directly regulated by lysosomal cholesterol content.

    Directory of Open Access Journals (Sweden)

    Hanna Appelqvist

    Full Text Available Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1 protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2, which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

  16. CNS-directed gene therapy for lysosomal storage diseases

    OpenAIRE

    Sands, Mark S; Haskins, Mark E

    2008-01-01

    Lysosomal storage diseases (LSDs) are a group of inherited metabolic disorders usually caused by deficient activity of a single lysosomal enzyme. As most lysosomal enzymes are ubiquitously expressed, a deficiency in a single enzyme can affect multiple organ systems, including the central nervous system (CNS). At least 75% of all LSDs have a significant CNS component. Approaches such as bone marrow transplantation (BMT) or enzyme replacement therapy (ERT) can effectively treat the systemic dis...

  17. The alpha-galactosidase type A gene aglA from Aspergillus niger encodes a fully functional alpha-N-acetylgalactosaminidase

    Czech Academy of Sciences Publication Activity Database

    Kulik, Natallia; Weignerová, L.; Filipi, Tomáš; Pompach, Petr; Novák, Petr; Mrázek, H.; Slámová, Kristýna; Bezouška, K.; Křen, Vladimír; Ettrich, Rüdiger

    2010-01-01

    Roč. 20, č. 11 (2010), s. 1410-1419 ISSN 0959-6658 R&D Projects: GA MŠk(CZ) LC06010; GA ČR GAP207/10/1934; GA ČR GAP207/10/1040; GA ČR GA303/09/0477; GA ČR GAP207/10/0321 Institutional research plan: CEZ:AV0Z60870520; CEZ:AV0Z50200510; CEZ:AV0Z10750506 Keywords : α-N-acetylgalactosaminidase * α-galactosidase * Aspergillus niger * substrate binding * molecular modeling Subject RIV: CE - Biochemistry Impact factor: 3.791, year: 2010

  18. lysosome tethering and fusion

    Indian Academy of Sciences (India)

    AMIT TULI

    LYSOSOME. MTOC. LATE ENDOSOME. Arl8b promotes the assembly of the HOPS complex on the lysosomes to mediate late endosome-lysosome fusion and cargo delivery to lysosomes. Khatter D et al., J Cell Science 2015. Khatter D et al., Cellular Logistics 2015 ...

  19. Lysosomal storage disease 2 - Pompe's disease

    NARCIS (Netherlands)

    van der Ploeg, Ans T.; Reuser, Arnold J. J.

    2008-01-01

    Pompe's disease, glycogen-storage disease type II, and acid maltase deficiency are alternative names for the same metabolic disorder. It is a pan-ethnic autosomal recessive trait characterised by acid alpha-glucosidase deficiency leading to lysosomal glycogen storage. Pompe's disease is also

  20. Pathogenic cascades in lysosomal disease-Why so complex?

    Science.gov (United States)

    Walkley, S U

    2009-04-01

    Lysosomal disease represents a large group of more than 50 clinically recognized conditions resulting from inborn errors of metabolism affecting the organelle known as the lysosome. The lysosome is an integral part of the larger endosomal/lysosomal system, and is closely allied with the ubiquitin-proteosomal and autophagosomal systems, which together comprise essential cell machinery for substrate degradation and recycling, homeostatic control, and signalling. More than two-thirds of lysosomal diseases affect the brain, with neurons appearing particularly vulnerable to lysosomal compromise and showing diverse consequences ranging from specific axonal and dendritic abnormalities to neuron death. While failure of lysosomal function characteristically leads to lysosomal storage, new studies argue that lysosomal diseases may also be appropriately viewed as 'states of deficiency' rather than simply overabundance (storage). Interference with signalling events and salvage processing normally controlled by the endosomal/lysosomal system may represent key mechanisms accounting for the inherent complexity of lysosomal disorders. Analysis of lysosomal disease pathogenesis provides a unique window through which to observe the importance of the greater lysosomal system for normal cell health.

  1. Mitochondrial respiration controls lysosomal function during inflammatory T cell responses

    Science.gov (United States)

    Baixauli, Francesc; Acín-Pérez, Rebeca; Villarroya-Beltrí, Carolina; Mazzeo, Carla; Nuñez-Andrade, Norman; Gabandé-Rodriguez, Enrique; Dolores Ledesma, Maria; Blázquez, Alberto; Martin, Miguel Angel; Falcón-Pérez, Juan Manuel; Redondo, Juan Miguel; Enríquez, Jose Antonio; Mittelbrunn, Maria

    2016-01-01

    Summary The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4+ T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration-deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward pro-inflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD+ levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify novel strategies for intervention in mitochondrial-related diseases. PMID:26299452

  2. A non-conserved miRNA regulates lysosomal function and impacts on a human lysosomal storage disorder

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Di Malta, Chiara; Wen, Jiayu

    2014-01-01

    Sulfatases are key enzymatic regulators of sulfate homeostasis with several biological functions including degradation of glycosaminoglycans (GAGs) and other macromolecules in lysosomes. In a severe lysosomal storage disorder, multiple sulfatase deficiency (MSD), global sulfatase activity...... of proteoglycan catabolism and lysosomal function. This blocks autophagy-mediated degradation, causing cytoplasmic accumulation of autophagosomes and autophagic substrates. By targeting miR-95 in cells from MSD patients, we can effectively increase residual SUMF1 expression, allowing for reactivation of sulfatase...

  3. Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration

    Science.gov (United States)

    Berg, Russell D.; Levitte, Steven; O’Sullivan, Mary P.; O’Leary, Seónadh M.; Cambier, C.J.; Cameron, James; Takaki, Kevin K.; Moens, Cecilia B.; Tobin, David M.; Keane, Joseph; Ramakrishnan, Lalita

    2016-01-01

    Summary A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers’ susceptibility to tuberculosis. PMID:27015311

  4. Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification.

    Science.gov (United States)

    Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M; Haslett, Luke J; Kumar, Asok; Sato, Yutaka; Lie, Pearl P Y; Mohan, Panaiyur; Coffey, Erin E; Kompella, Uday; Mitchell, Claire H; Lloyd-Evans, Emyr; Nixon, Ralph A

    2015-09-01

    Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Lee

    2015-09-01

    Full Text Available Presenilin 1 (PS1 deletion or Alzheimer’s disease (AD-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  6. Healthcare resource use and costs of managing children and adults with lysosomal acid lipase deficiency at a tertiary referral centre in the United Kingdom.

    Directory of Open Access Journals (Sweden)

    Julian F Guest

    Full Text Available To estimate clinical progression and resource utilisation together with the associated costs of managing children and adults with LAL Deficiency, at a tertiary referral centre in the UK.A retrospective chart review was undertaken of patients in the UK with a confirmed diagnosis of LAL Deficiency who were managed at a LAL Deficiency tertiary referral treatment centre. Patients' pathways, treatment patterns, health outcomes and resource use were quantified over differing lengths of time for each patient enabling the NHS cost of patient management in tertiary care to be estimated.The study population comprised 19 patients of whom 58% were male. Mean age at the time of initial presentation was 15.5 years and the mean age at diagnosis was 18.0 years. 63%, 53% and 42% of patients had hepatomegaly, abnormal lipid storage and splenomegaly at a mean age of presentation of 17.8, 17.1 and 20.9 years, respectively. Over a period of 50 years there were a mean of 48.5 clinician visits and 3.4 hospital admissions per patient. The mean NHS cost of patient management at a LAL Deficiency tertiary referral treatment centre, spanning a period of over 50 years was £61,454 per patient.This study provides important insights into a number of aspects of the disease that are difficult to ascertain from published case reports. Additionally, it provides the best estimate available of NHS resource use and costs with which to inform policy and budgetary decisions pertaining to managing this ultra-orphan disease.

  7. Healthcare resource use and costs of managing children and adults with lysosomal acid lipase deficiency at a tertiary referral centre in the United Kingdom.

    Science.gov (United States)

    Guest, Julian F; Ingram, Andy; Ayoub, Nadia; Hendriksz, Christian J; Murphy, Elaine; Rahman, Yusof; McKiernan, Patrick; Mundy, Helen; Deegan, Patrick

    2018-01-01

    To estimate clinical progression and resource utilisation together with the associated costs of managing children and adults with LAL Deficiency, at a tertiary referral centre in the UK. A retrospective chart review was undertaken of patients in the UK with a confirmed diagnosis of LAL Deficiency who were managed at a LAL Deficiency tertiary referral treatment centre. Patients' pathways, treatment patterns, health outcomes and resource use were quantified over differing lengths of time for each patient enabling the NHS cost of patient management in tertiary care to be estimated. The study population comprised 19 patients of whom 58% were male. Mean age at the time of initial presentation was 15.5 years and the mean age at diagnosis was 18.0 years. 63%, 53% and 42% of patients had hepatomegaly, abnormal lipid storage and splenomegaly at a mean age of presentation of 17.8, 17.1 and 20.9 years, respectively. Over a period of 50 years there were a mean of 48.5 clinician visits and 3.4 hospital admissions per patient. The mean NHS cost of patient management at a LAL Deficiency tertiary referral treatment centre, spanning a period of over 50 years was £61,454 per patient. This study provides important insights into a number of aspects of the disease that are difficult to ascertain from published case reports. Additionally, it provides the best estimate available of NHS resource use and costs with which to inform policy and budgetary decisions pertaining to managing this ultra-orphan disease.

  8. Lysosome Transport as a Function of Lysosome Diameter

    Science.gov (United States)

    Bandyopadhyay, Debjyoti; Cyphersmith, Austin; Zapata, Jairo A.; Kim, Y. Joseph; Payne, Christine K.

    2014-01-01

    Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking. We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter. PMID:24497985

  9. Lysosomal membrane permeability stimulates protein aggregate formation in neurons of a lysosomal disease.

    Science.gov (United States)

    Micsenyi, Matthew C; Sikora, Jakub; Stephney, Gloria; Dobrenis, Kostantin; Walkley, Steven U

    2013-06-26

    Protein aggregates are a common pathological feature of neurodegenerative diseases and several lysosomal diseases, but it is currently unclear what aggregates represent for pathogenesis. Here we report the accumulation of intraneuronal aggregates containing the macroautophagy adapter proteins p62 and NBR1 in the neurodegenerative lysosomal disease late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). CLN2 disease is caused by a deficiency in the lysosomal enzyme tripeptidyl peptidase I, which results in aberrant lysosomal storage of catabolites, including the subunit c of mitochondrial ATP synthase (SCMAS). In an effort to define the role of aggregates in CLN2, we evaluated p62 and NBR1 accumulation in the CNS of Cln2(-/-) mice. Although increases in p62 and NBR1 often suggest compromised degradative mechanisms, we found normal ubiquitin-proteasome system function and only modest inefficiency in macroautophagy late in disease. Importantly, we identified that SCMAS colocalizes with p62 in extra-lysosomal aggregates in Cln2(-/-) neurons in vivo. This finding is consistent with SCMAS being released from lysosomes, an event known as lysosomal membrane permeability (LMP). We predicted that LMP and storage release from lysosomes results in the sequestration of this material as cytosolic aggregates by p62 and NBR1. Notably, LMP induction in primary neuronal cultures generates p62-positive aggregates and promotes p62 localization to lysosomal membranes, supporting our in vivo findings. We conclude that LMP is a previously unrecognized pathogenic event in CLN2 disease that stimulates cytosolic aggregate formation. Furthermore, we offer a novel role for p62 in response to LMP that may be relevant for other diseases exhibiting p62 accumulation.

  10. Lysosomes and radiation injury

    International Nuclear Information System (INIS)

    Watkins, D.K.

    1975-01-01

    Changes in activities of lysosomal enzymes following whole-body treatment with ionizing radiation have long been recognized (e.g., Douglass and Day 1955, Okada et al., 1957). Attempts to explain nuclear damage by cytoplasmic enzyme attack, concentrated most of the earlier work on DNASE II and acid RNASE. Lysosomal enzymes have subsequently been studied in many tissues following whole-body irradiation. The observations coupled with in vitro results from isolated lysosomes, and u.v. and visible light studies on cells in culture, have led to the presentation of tentative mechanisms of action. General methods of detecting lysosomal damage have utilized the consequent activation or leakage of acid hydrolases. As this is of a temporal nature following irradiation, direct damage to the lysosomal membrane has not as yet been measured and the primary lesion either in the membrane itself or at the hypothetical site of acid hydrolase-membrane attachment has still to be discovered. Despite the accumulating evidence of lysosome disruption subsequent to treatment with radiation of various qualities, the role (if any) of these organelles in cell killing remains obscure. In the following pages a review of the many aspects of radiation damage will be presented and an attempt will be made to correlate the results and to draw general conclusions where possible. A final short section will deal with thecontribution that lysosomal damage may make in cell death and tissue injury and possible implications in radiotherapy

  11. Lysosomal storage disorders

    CERN Document Server

    Cabrera-Salazar, Mario A; Cabrera-Salazar, Mario

    2007-01-01

    This book describes the nature of the lysosomal dysfunction and diseases as well as potential future treatments and therapies. This is an invaluable resource for researchers in biochemical and molecular genetics, enzyme therapy, and gene transfer.

  12. Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes.

    Science.gov (United States)

    Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

    2016-01-01

    Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

  13. Isolating Lysosomes from Rat Liver.

    Science.gov (United States)

    Pryor, Paul R

    2016-04-01

    This protocol describes the generation of a fraction enriched in lysosomes from rat liver. The lysosomes are rapidly isolated using density-gradient centrifugation with gradient media that retain the osmolarity of the lysosomes such that they are functional and can be used in in vitro assays. © 2016 Cold Spring Harbor Laboratory Press.

  14. Lysosomal lipid storage diseases.

    Science.gov (United States)

    Schulze, Heike; Sandhoff, Konrad

    2011-06-01

    Lysosomal lipid storage diseases, or lipidoses, are inherited metabolic disorders in which typically lipids accumulate in cells and tissues. Complex lipids, such as glycosphingolipids, are constitutively degraded within the endolysosomal system by soluble hydrolytic enzymes with the help of lipid binding proteins in a sequential manner. Because of a functionally impaired hydrolase or auxiliary protein, their lipid substrates cannot be degraded, accumulate in the lysosome, and slowly spread to other intracellular membranes. In Niemann-Pick type C disease, cholesterol transport is impaired and unesterified cholesterol accumulates in the late endosome. In most lysosomal lipid storage diseases, the accumulation of one or few lipids leads to the coprecipitation of other hydrophobic substances in the endolysosomal system, such as lipids and proteins, causing a "traffic jam." This can impair lysosomal function, such as delivery of nutrients through the endolysosomal system, leading to a state of cellular starvation. Therapeutic approaches are currently restricted to mild forms of diseases with significant residual catabolic activities and without brain involvement.

  15. Molecular and clinical characterization of a series of patients with childhood-onset lysosomal acid lipase deficiency. Retrospective investigations, follow-up and detection of two novel LIPA pathogenic variants.

    Science.gov (United States)

    Pisciotta, Livia; Tozzi, Giulia; Travaglini, Lorena; Taurisano, Roberta; Lucchi, Tiziano; Indolfi, Giuseppe; Papadia, Francesco; Di Rocco, Maja; D'Antiga, Lorenzo; Crock, Patricia; Vora, Komal; Nightingale, Scott; Michelakakis, Helen; Garoufi, Anastasia; Lykopoulou, Lilia; Bertolini, Stefano; Calandra, Sebastiano

    2017-10-01

    Childhood/Adult-onset Lysosomal Acid Lipase Deficiency (LAL-D) is a recessive disorder due to loss of function variants of LAL, the enzyme which hydrolyses cholesteryl esters, derived from internalized apoB containing lipoproteins. The disease is characterized by multi-organ involvement including the liver, spleen, intestine and cardiovascular system. The aim of this study was the clinical and molecular characterization of 14 (13 unrelated) previously unreported patients with childhood-onset LAL-D. Data collected included clinical and laboratory investigations, liver imaging, liver biopsy and LIPA gene analysis. The response to lipid-lowering medications, liver transplantation and enzyme replacement therapy (ERT) was reported for some patients. LAL-D was suspected at 4.4 ± 3.3 years of age for the presence of hepatomegaly, elevated serum transaminases and hypercholesterolemia, and was confirmed by liver biopsy/imaging and LAL assay. The follow up period ranged from 3 to 40 years (mean 7.8 ± 4.0 years in 13 cases). Patients treated with statins with or without ezetimibe showed 28% reduction of plasma LDL-cholesterol without a tangible effect on liver enzymes; some patients receiving ERT showed normalized lipoprotein profile and transaminase levels. The common c.894G > A variant was observed in homozygosity or compound heterozygosity in 10 patients. We found seven previously reported variants: p.(Trp140*), p.(Arg218*), p.(Gly266*), p.(Thr288Ile), p.(Leu294Ser), p.(His295Tyr) and p.(Gly342Arg) and two novel variants: p.(Asp345Asn), affecting the LAL catalytic triad, and c.229+3A > C, affecting splicing. Homozygosity for p.(Thr288Ile) or c.229+3A > C was associated with a severe phenotype. This study provides additional data on the features of childhood-onset LAL-D and describes two novel pathogenic variants of the LIPA gene. Copyright © 2017. Published by Elsevier B.V.

  16. Lysosome and calcium dysregulation in Alzheimer's disease: partners in crime.

    Science.gov (United States)

    McBrayer, MaryKate; Nixon, Ralph A

    2013-12-01

    Early-onset FAD (familial Alzheimer's disease) is caused by mutations of PS1 (presenilin 1), PS2 (presenilin 2) and APP (amyloid precursor protein). Beyond the effects of PS1 mutations on proteolytic functions of the γ-secretase complex, mutant or deficient PS1 disrupts lysosomal function and Ca2+ homoeostasis, both of which are considered strong pathogenic factors in FAD. Loss of PS1 function compromises assembly and proton-pumping activity of the vacuolar-ATPase on lysosomes, leading to defective lysosomal acidification and marked impairment of autophagy. Additional dysregulation of cellular Ca2+ by mutant PS1 in FAD has been ascribed to altered ion channels in the endoplasmic reticulum; however, rich stores of Ca2+ in lysosomes are also abnormally released in PS1-deficient cells secondary to the lysosomal acidification defect. The resultant rise in cytosolic Ca2+ activates Ca2+-dependent enzymes, contributing substantially to calpain overactivation that is a final common pathway leading to neurofibrillary degeneration in all forms of AD (Alzheimer's disease). In the present review, we discuss the close inter-relationships among deficits of lysosomal function, autophagy and Ca2+ homoeostasis as a pathogenic process in PS1-related FAD and their relevance to sporadic AD.

  17. Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

    Science.gov (United States)

    Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

    2006-01-01

    Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

  18. Exploration of Structural and Functional Variations Owing to Point Mutations in α-NAGA.

    Science.gov (United States)

    Meshach Paul, D; Rajasekaran, R

    2018-03-01

    Schindler disease is a lysosomal storage disorder caused due to deficiency or defective activity of alpha-N-acetylgalactosaminidase (α-NAGA). Mutations in gene encoding α-NAGA cause wide range of diseases, characterized with mild to severe clinical features. Molecular effects of these mutations are yet to be explored in detail. Therefore, this study was focused on four missense mutations of α-NAGA namely, S160C, E325K, R329Q and R329W. Native and mutant structures of α-NAGA were analysed to determine geometrical deviations such as the contours of root mean square deviation, root mean square fluctuation, percentage of residues in allowed regions of Ramachandran plot and solvent accessible surface area, using conformational sampling technique. Additionally, global energy-minimized structures of native and mutants were further analysed to compute their intra-molecular interactions, hydrogen bond dilution and distribution of secondary structure. In addition, docking studies were also performed to determine variations in binding energies between native and mutants. The deleterious effects of mutants were evident due to variations in their active site residues pertaining to spatial conformation and flexibility, comparatively. Hence, variations exhibited by mutants, namely S160C, E325K, R329Q and R329W to that of native, consequently, lead to the detrimental effects causing Schindler disease. This study computationally explains the underlying reasons for the pathogenesis of the disease, thereby aiding future researchers in drug development and disease management.

  19. The Biogenesis of Lysosomes and Lysosome-Related Organelles

    Science.gov (United States)

    Luzio, J. Paul; Hackmann, Yvonne; Dieckmann, Nele M.G.; Griffiths, Gillian M.

    2014-01-01

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. PMID:25183830

  20. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

    Kallunki, T; Olsen, O D; Jaattela, Marja

    2013-01-01

    Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal function. Accordingly, transformation and cancer progression are characterized by dramatic changes in lysosomal volume, composition and cellular distribution. Depending on one's point of view, the cancer-asso......:10.1038/onc.2012.292....

  1. Ethambutol neutralizes lysosomes and causes lysosomal zinc accumulation.

    Science.gov (United States)

    Yamada, Daisuke; Saiki, Shinji; Furuya, Norihiko; Ishikawa, Kei-Ichi; Imamichi, Yoko; Kambe, Taiho; Fujimura, Tsutomu; Ueno, Takashi; Koike, Masato; Sumiyoshi, Katsuhiko; Hattori, Nobutaka

    2016-02-26

    Ethambutol is a common medicine used for the treatment of tuberculosis, which can have serious side effects, such as retinal and liver dysfunction. Although ethambutol has been reported to impair autophagic flux in rat retinal cells, the precise molecular mechanism remains unclear. Using various mammalian cell lines, we showed that ethambutol accumulated in autophagosomes and vacuolated lysosomes, with marked Zn(2+) accumulation. The enlarged lysosomes were neutralized and were infiltrated with Zn(2+) accumulations in the lysosomes, with simultaneous loss of acidification. These results suggest that EB neutralizes lysosomes leading to insufficient autophagy, implying that some of the adverse effects associated with EB in various organs may be of this mechanism. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  2. High lumenal chloride in the lysosome is critical for lysosome function.

    Science.gov (United States)

    Chakraborty, Kasturi; Leung, KaHo; Krishnan, Yamuna

    2017-07-25

    Lysosomes are organelles responsible for the breakdown and recycling of cellular machinery. Dysfunctional lysosomes give rise to lysosomal storage disorders as well as common neurodegenerative diseases. Here, we use a DNA-based, fluorescent chloride reporter to measure lysosomal chloride in Caenorhabditis elegans as well as murine and human cell culture models of lysosomal diseases. We find that the lysosome is highly enriched in chloride, and that chloride reduction correlates directly with a loss in the degradative function of the lysosome. In nematodes and mammalian cell culture models of diverse lysosomal disorders, where previously only lysosomal pH dysregulation has been described, massive reduction of lumenal chloride is observed that is ~10 3 fold greater than the accompanying pH change. Reducing chloride within the lysosome impacts Ca 2+ release from the lysosome and impedes the activity of specific lysosomal enzymes indicating a broader role for chloride in lysosomal function.

  3. Lysosomal cell death at a glance

    DEFF Research Database (Denmark)

    Aits, Sonja; Jaattela, Marja

    2013-01-01

    Lysosomes serve as the cellular recycling centre and are filled with numerous hydrolases that can degrade most cellular macromolecules. Lysosomal membrane permeabilization and the consequent leakage of the lysosomal content into the cytosol leads to so-called "lysosomal cell death". This form...... of cell death is mainly carried out by the lysosomal cathepsin proteases and can have necrotic, apoptotic or apoptosis-like features depending on the extent of the leakage and the cellular context. This article summarizes our current knowledge on lysosomal cell death with an emphasis on the upstream...... mechanisms that lead to lysosomal membrane permeabilization....

  4. Alterations in membrane trafficking and pathophysiological implications in lysosomal storage disorders.

    Science.gov (United States)

    Kuech, Eva-Maria; Brogden, Graham; Naim, Hassan Y

    2016-11-01

    Lysosomal storage disorders are a heterogeneous group of more than 50 distinct inborn metabolic diseases affecting about 1 in 5000 to 7000 live births. The diseases often result from mutations followed by functional deficiencies of enzymes or transporters within the acidic environment of the lysosome, which mediate the degradation of a wide subset of substrates, including glycosphingolipids, glycosaminoglycans, cholesterol, glycogen, oligosaccharides, peptides and glycoproteins, or the export of the respective degradation products from the lysosomes. The progressive accumulation of uncleaved substrates occurs in multiple organs and finally causes a broad spectrum of different pathologies including visceral, neurological, skeletal and hematologic manifestations. Besides deficient lysosomal enzymes and transporters other defects may lead to lysosomal storage disorders, including activator defects, membrane defects or defects in modifier proteins. In this review we concentrate on four different lysosomal storage disorders: Niemann-Pick type C, Fabry disease, Gaucher disease and Pompe disease. While the last three are caused by defective lysosomal hydrolases, Niemann-Pick type C is caused by the inability to export LDL-derived cholesterol out of the lysosome. We want to emphasise potential implications of membrane trafficking defects on the pathology of these diseases, as many mutations interfere with correct lysosomal protein trafficking and alter cellular lipid homeostasis. Current therapeutic strategies are summarised, including substrate reduction therapy as well as pharmacological chaperone therapy which directly aim to improve folding and lysosomal transport of misfolded mutant proteins. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  5. Glucosylceramide accumulation is not confined to the lysosome in fibroblasts from patients with Gaucher disease.

    Science.gov (United States)

    Fuller, Maria; Rozaklis, Tina; Lovejoy, Melanie; Zarrinkalam, Krystyna; Hopwood, John J; Meikle, Peter J

    2008-04-01

    Gaucher disease (GD) is an inborn error of glycosphingolipid metabolism resulting from a deficiency of the lysosomal enzyme beta-glucosidase leading to the accumulation of glucosylceramide (GC) in lysosomes of affected cells. In order to determine the effect of GC accumulation on intracellular lipid content in fibroblasts from patients with GD, we measured individual species of ceramide, di- and trihexosylceramide, sphingomyelin, phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol using electrospray ionisation-tandem mass spectrometry. The different subspecies of each lipid class correlated with each other and were summed to give total lipid concentrations. In addition to GC, we also noted secondary elevations in other lipids, especially in type 2 GD. Sub-cellular fractionation showed that GC was not confined to the lysosome but increased throughout the cell. The sequelae of extra-lysosomal accumulation may have implications in the pathogenic mechanisms of GD by interaction with biochemical and metabolic pathways located outside the lysosome. The elevation of ceramide in confluent type 2 GD fibroblasts redistributed from its primary site of accumulation in the lysosome to the endosomal region at four-weeks post-confluence. The accumulation of lipids in the endosome and lysosome suggests both impaired trafficking of lipids and reduced capacity of the lysosome to degrade lipids.

  6. Autophagy failure in Alzheimer's disease and the role of defective lysosomal acidification.

    Science.gov (United States)

    Wolfe, Devin M; Lee, Ju-Hyun; Kumar, Asok; Lee, Sooyeon; Orenstein, Samantha J; Nixon, Ralph A

    2013-06-01

    Autophagy is a lysosomal degradative process which recycles cellular waste and eliminates potentially toxic damaged organelles and protein aggregates. The important cytoprotective functions of autophagy are demonstrated by the diverse pathogenic consequences that may stem from autophagy dysregulation in a growing number of neurodegenerative disorders. In many of the diseases associated with autophagy anomalies, it is the final stage of autophagy-lysosomal degradation that is disrupted. In several disorders, including Alzheimer's disease (AD), defective lysosomal acidification contributes to this proteolytic failure. The complex regulation of lysosomal pH makes this process vulnerable to disruption by many factors, and reliable lysosomal pH measurements have become increasingly important in investigations of disease mechanisms. Although various reagents for pH quantification have been developed over several decades, they are not all equally well suited for measuring the pH of lysosomes. Here, we evaluate the most commonly used pH probes for sensitivity and localisation, and identify LysoSensor yellow/blue-dextran, among currently used probes, as having the optimal profile of properties for measuring lysosomal pH. In addition, we review evidence that lysosomal acidification is defective in AD and extend our original findings, of elevated lysosomal pH in presenilin 1 (PS1)-deficient blastocysts and neurons, to additional cell models of PS1 and PS1/2 deficiency, to fibroblasts from AD patients with PS1 mutations, and to neurons in the PS/APP mouse model of AD. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  7. Lysosomal exocytosis and lipid storage disorders.

    Science.gov (United States)

    Samie, Mohammad Ali; Xu, Haoxing

    2014-06-01

    Lysosomes are acidic compartments in mammalian cells that are primarily responsible for the breakdown of endocytic and autophagic substrates such as membranes, proteins, and lipids into their basic building blocks. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by genetic mutations in lysosomal hydrolases required for catabolic degradation, mutations in lysosomal membrane proteins important for catabolite export or membrane trafficking, or mutations in nonlysosomal proteins indirectly affecting these lysosomal functions. A hallmark feature of LSDs is the primary and secondary excessive accumulation of undigested lipids in the lysosome, which causes lysosomal dysfunction and cell death, and subsequently pathological symptoms in various tissues and organs. There are more than 60 types of LSDs, but an effective therapeutic strategy is still lacking for most of them. Several recent in vitro and in vivo studies suggest that induction of lysosomal exocytosis could effectively reduce the accumulation of the storage materials. Meanwhile, the molecular machinery and regulatory mechanisms for lysosomal exocytosis are beginning to be revealed. In this paper, we first discuss these recent developments with the focus on the functional interactions between lipid storage and lysosomal exocytosis. We then discuss whether lysosomal exocytosis can be manipulated to correct lysosomal and cellular dysfunction caused by excessive lipid storage, providing a potentially general therapeutic approach for LSDs. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  8. Lysosomal exocytosis and lipid storage disorders

    Science.gov (United States)

    Samie, Mohammad Ali; Xu, Haoxing

    2014-01-01

    Lysosomes are acidic compartments in mammalian cells that are primarily responsible for the breakdown of endocytic and autophagic substrates such as membranes, proteins, and lipids into their basic building blocks. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by genetic mutations in lysosomal hydrolases required for catabolic degradation, mutations in lysosomal membrane proteins important for catabolite export or membrane trafficking, or mutations in nonlysosomal proteins indirectly affecting these lysosomal functions. A hallmark feature of LSDs is the primary and secondary excessive accumulation of undigested lipids in the lysosome, which causes lysosomal dysfunction and cell death, and subsequently pathological symptoms in various tissues and organs. There are more than 60 types of LSDs, but an effective therapeutic strategy is still lacking for most of them. Several recent in vitro and in vivo studies suggest that induction of lysosomal exocytosis could effectively reduce the accumulation of the storage materials. Meanwhile, the molecular machinery and regulatory mechanisms for lysosomal exocytosis are beginning to be revealed. In this paper, we first discuss these recent developments with the focus on the functional interactions between lipid storage and lysosomal exocytosis. We then discuss whether lysosomal exocytosis can be manipulated to correct lysosomal and cellular dysfunction caused by excessive lipid storage, providing a potentially general therapeutic approach for LSDs. PMID:24668941

  9. Mechanisms and functions of lysosome positioning

    Science.gov (United States)

    Pu, Jing; Guardia, Carlos M.; Keren-Kaplan, Tal

    2016-01-01

    ABSTRACT Lysosomes have been classically considered terminal degradative organelles, but in recent years they have been found to participate in many other cellular processes, including killing of intracellular pathogens, antigen presentation, plasma membrane repair, cell adhesion and migration, tumor invasion and metastasis, apoptotic cell death, metabolic signaling and gene regulation. In addition, lysosome dysfunction has been shown to underlie not only rare lysosome storage disorders but also more common diseases, such as cancer and neurodegeneration. The involvement of lysosomes in most of these processes is now known to depend on the ability of lysosomes to move throughout the cytoplasm. Here, we review recent findings on the mechanisms that mediate the motility and positioning of lysosomes, and the importance of lysosome dynamics for cell physiology and pathology. PMID:27799357

  10. Disease: H00422 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available ria (AGU) [DS:H00145]; Fucosidosis [DS:H00141]; Schindler/ Kanzaki disease [DS:H00146] Glycoproteinoses is a...cosidase [HSA:2517] [KO:K01206] (Schindler/ Kanzaki) NAGA; alpha-N-acetylgalactosaminidase [HSA:4668] [KO:K0...PD:C00270] (alpha, beta-Mannosidosis, AGU, Fucosidosis, Schindler/ Kanzaki) Urine oligosaccharides, Vacuolat...g loss and mental retardation. (Fucosidosis) Type 1: a severe form. Type2: a mild form. (Schindler/ Kanzaki) Type1 (Schindle...lpha-N-acetylgalactosaminidase deficiency (Schindler/Kanzaki disease). ... JOURNAL ... J Hum Genet 49:1-8 (2004) DOI:10.1007/s10038-003-0098-z ...

  11. Pathogenic lysosomal depletion in Parkinson's disease.

    Science.gov (United States)

    Dehay, Benjamin; Bové, Jordi; Rodríguez-Muela, Natalia; Perier, Celine; Recasens, Ariadna; Boya, Patricia; Vila, Miquel

    2010-09-15

    Mounting evidence suggests a role for autophagy dysregulation in Parkinson's disease (PD). The bulk degradation of cytoplasmic proteins (including α-synuclein) and organelles (such as mitochondria) is mediated by macroautophagy, which involves the sequestration of cytosolic components into autophagosomes (AP) and its delivery to lysosomes. Accumulation of AP occurs in postmortem brain samples from PD patients, which has been widely attributed to an induction of autophagy. However, the cause and pathogenic significance of these changes remain unknown. Here we found in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD that AP accumulation and dopaminergic cell death are preceded by a marked decrease in the amount of lysosomes within dopaminergic neurons. Lysosomal depletion was secondary to the abnormal permeabilization of lysosomal membranes induced by increased mitochondrial-derived reactive oxygen species. Lysosomal permeabilization resulted in a defective clearance and subsequent accumulation of undegraded AP and contributed directly to neurodegeneration by the ectopic release of lysosomal proteases into the cytosol. Lysosomal breakdown and AP accumulation also occurred in PD brain samples, where Lewy bodies were strongly immunoreactive for AP markers. Induction of lysosomal biogenesis by genetic or pharmacological activation of lysosomal transcription factor EB restored lysosomal levels, increased AP clearance and attenuated 1-methyl-4-phenylpyridinium-induced cell death. Similarly, the autophagy-enhancer compound rapamycin attenuated PD-related dopaminergic neurodegeneration, both in vitro and in vivo, by restoring lysosomal levels. Our results indicate that AP accumulation in PD results from defective lysosomal-mediated AP clearance secondary to lysosomal depletion. Restoration of lysosomal levels and function may thus represent a novel neuroprotective strategy in PD.

  12. Lysosomal degradation of membrane lipids.

    Science.gov (United States)

    Kolter, Thomas; Sandhoff, Konrad

    2010-05-03

    The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Lysosomal Storage Diseases To date

    OpenAIRE

    HOFFMANN, Björn; MAYATEPEK, Ertan

    2011-01-01

    New therapeutic options and progress of approved therapies have made Lysosomal Storage Diseases (LSDs) one of the most exciting group of diseases. This review aims to summarize current achievements in these particular disorders and to give an outlook towards possible future treatment options. Enzyme replacement therapy is the gold standard for Gaucher disease, Fabry disease, Mucopolysaccharidosis type I, II, and VI, and for Pompe disease. Besides this, substrate reduction has been approved fo...

  14. Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

    Science.gov (United States)

    Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

    2017-06-16

    Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

  15. High lumenal chloride in the lysosome is critical for lysosome function

    Science.gov (United States)

    Chakraborty, Kasturi; Leung, KaHo; Krishnan, Yamuna

    2017-01-01

    Lysosomes are organelles responsible for the breakdown and recycling of cellular machinery. Dysfunctional lysosomes give rise to lysosomal storage disorders as well as common neurodegenerative diseases. Here, we use a DNA-based, fluorescent chloride reporter to measure lysosomal chloride in Caenorhabditis elegans as well as murine and human cell culture models of lysosomal diseases. We find that the lysosome is highly enriched in chloride, and that chloride reduction correlates directly with a loss in the degradative function of the lysosome. In nematodes and mammalian cell culture models of diverse lysosomal disorders, where previously only lysosomal pH dysregulation has been described, massive reduction of lumenal chloride is observed that is ~103 fold greater than the accompanying pH change. Reducing chloride within the lysosome impacts Ca2+ release from the lysosome and impedes the activity of specific lysosomal enzymes indicating a broader role for chloride in lysosomal function. DOI: http://dx.doi.org/10.7554/eLife.28862.001 PMID:28742019

  16. uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

    DEFF Research Database (Denmark)

    Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria

    2004-01-01

    appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient...

  17. Loss of Niemann-Pick C1 or C2 protein results in similar biochemical changes suggesting that these proteins function in a common lysosomal pathway.

    Directory of Open Access Journals (Sweden)

    Sayali S Dixit

    Full Text Available Niemann-Pick Type C (NPC disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely.

  18. The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

    International Nuclear Information System (INIS)

    Zeng, Jibin; Racicott, Jesse; Morales, Carlos R.

    2009-01-01

    Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM 2 AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.

  19. The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Jibin; Racicott, Jesse [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada); Morales, Carlos R., E-mail: carlos.morales@mcgill.ca [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada)

    2009-11-01

    Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM{sub 2}AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient

  20. BACE is degraded via the lysosomal pathway.

    Science.gov (United States)

    Koh, Young Ho; von Arnim, Christine A F; Hyman, Bradley T; Tanzi, Rudolph E; Tesco, Giuseppina

    2005-09-16

    Amyloid plaques are formed by aggregates of amyloid-beta-peptide, a 37-43-amino acid fragment (primarily Abeta(40) and Abeta(42)) generated by proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases. A type I transmembrane aspartyl protease, BACE (beta-site APP cleaving enzyme), has been identified to be the beta-secretase. BACE is targeted through the secretory pathway to the plasma membrane where it can be internalized to endosomes. The carboxyl terminus of BACE contains a di-leucine-based signal for sorting of transmembrane proteins to endosomes and lysosomes. In this study, we set out to determine whether BACE is degraded by the lysosomal pathway and whether the di-leucine motif is necessary for targeting BACE to the lysosomes. Here we show that lysosomal inhibitors, chloroquine and NH(4)Cl, lead to accumulation of endogenous and ectopically expressed BACE in a variety of cell types, including primary neurons. Furthermore, the inhibition of lysosomal hydrolases results in the redistribution and accumulation of BACE in the late endosomal/lysosomal compartments (lysosome-associated membrane protein 2 (LAMP2)-positive). In contrast, the BACE-LL/AA mutant, in which Leu(499) and Leu(500) in the COOH-terminal sequence (DDISLLK) were replaced by alanines, only partially co-localized with LAMP2-positive compartments following inhibition of lysosomal hydrolases. Collectively, our data indicate that BACE is transported to the late endosomal/lysosomal compartments where it is degraded via the lysosomal pathway and that the di-leucine motif plays a role in sorting BACE to lysosomes.

  1. A Molecular Mechanism to Regulate Lysosome Motility for Lysosome Positioning and Tubulation

    Science.gov (United States)

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-01-01

    To mediate the degradation of bio-macromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca2+ channel TRPML1 cause lysosome storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca2+-dependent centripetal movement of lysosomes towards the perinuclear region, where autophagosomes accumulate, upon autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca2+ sensor that associates physically with the minus-end directed dynactin-dynein motor, while PI(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PI(3,5)P2-TRPML1-ALG-2-dynein signaling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Collectively, Ca2+ release from lysosomes provides an on-demand mechanism regulating lysosome motility, positioning, and tubulation. PMID:26950892

  2. Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury.

    Science.gov (United States)

    Maejima, Ikuko; Takahashi, Atsushi; Omori, Hiroko; Kimura, Tomonori; Takabatake, Yoshitsugu; Saitoh, Tatsuya; Yamamoto, Akitsugu; Hamasaki, Maho; Noda, Takeshi; Isaka, Yoshitaka; Yoshimori, Tamotsu

    2013-08-28

    Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.

  3. Lysosomes as Oxidative Targets for Cancer Therapy.

    Science.gov (United States)

    Dielschneider, Rebecca F; Henson, Elizabeth S; Gibson, Spencer B

    2017-01-01

    Lysosomes are membrane-bound vesicles that contain hydrolases for the degradation and recycling of essential nutrients to maintain homeostasis within cells. Cancer cells have increased lysosomal function to proliferate, metabolize, and adapt to stressful environments. This has made cancer cells susceptible to lysosomal membrane permeabilization (LMP). There are many factors that mediate LMP such as Bcl-2 family member, p53; sphingosine; and oxidative stress which are often altered in cancer. Upon lysosomal disruption, reactive oxygen species (ROS) levels increase leading to lipid peroxidation, mitochondrial dysfunction, autophagy, and reactive iron. Cathepsins are also released causing degradation of macromolecules and cellular structures. This ultimately kills the cancer cell through different types of cell death (apoptosis, autosis, or ferroptosis). In this review, we will explore the contributions lysosomes play in inducing cell death, how this is regulated by ROS in cancer, and how lysosomotropic agents might be utilized to treat cancers.

  4. Mechanisms of communication between mitochondria and lysosomes.

    Science.gov (United States)

    Raimundo, Nuno; Fernández-Mosquera, Lorena; Yambire, King Faisal; Diogo, Cátia V

    2016-10-01

    Mitochondria and lysosomes have long been studied in the context of their classic functions: energy factory and recycle bin, respectively. In the last twenty years, it became evident that these organelles are much more than simple industrial units, and are indeed in charge of many of cellular processes. Both mitochondria and lysosomes are now recognized as far-reaching signaling platforms, regulating many key aspects of cell and tissue physiology. It has furthermore become clear that mitochondria and lysosomes impact each other. The mechanisms underlying the cross-talk between these organelles are only now starting to be addressed. In this review, we briefly summarize how mitochondria, lysosomes and the lysosome-related process of autophagy affect each other in physiology and pathology. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Delivery of Cargo to Lysosomes Using GNeosomes.

    Science.gov (United States)

    Hamill, Kristina M; Wexselblatt, Ezequiel; Tong, Wenyong; Esko, Jeffrey D; Tor, Yitzhak

    2017-01-01

    Liposomes have been used to improve the intracellular delivery of a variety of cargos. Encapsulation of cargos in liposomes leads to improved plasma half-lives and minimized degradation. Here, we present a method for improving the selective delivery of liposomes to the lysosomes using a guanidinylated neomycin (GNeo) transporter. The method for synthesizing GNeo-lipids, incorporating them into liposomes, and the enhanced lysosomal delivery of encapsulated cargo are presented. GNeo-liposomes, termed GNeosomes, are capable of delivering a fluorescent dye to the lysosomes of Chinese hamster ovary cells as shown using confocal microscopy. GNeosomes can also be used to deliver therapeutic quantities of lysosomal enzymes to fibroblasts isolated from patients with a lysosomal storage disorder.

  6. Loss of Mitochondrial Function Impairs Lysosomes.

    Science.gov (United States)

    Demers-Lamarche, Julie; Guillebaud, Gérald; Tlili, Mouna; Todkar, Kiran; Bélanger, Noémie; Grondin, Martine; Nguyen, Angela P; Michel, Jennifer; Germain, Marc

    2016-05-06

    Alterations in mitochondrial function, as observed in neurodegenerative diseases, lead to disrupted energy metabolism and production of damaging reactive oxygen species. Here, we demonstrate that mitochondrial dysfunction also disrupts the structure and function of lysosomes, the main degradation and recycling organelle. Specifically, inhibition of mitochondrial function, following deletion of the mitochondrial protein AIF, OPA1, or PINK1, as well as chemical inhibition of the electron transport chain, impaired lysosomal activity and caused the appearance of large lysosomal vacuoles. Importantly, our results show that lysosomal impairment is dependent on reactive oxygen species. Given that alterations in both mitochondrial function and lysosomal activity are key features of neurodegenerative diseases, this work provides important insights into the etiology of neurodegenerative diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Podocytes Degrade Endocytosed Albumin Primarily in Lysosomes

    Science.gov (United States)

    Carson, John M.; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B.; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, palbumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic

  8. Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases.

    Science.gov (United States)

    Oh, Doo-Byoung

    2015-08-01

    Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy.

  9. Podocytes degrade endocytosed albumin primarily in lysosomes.

    Science.gov (United States)

    Carson, John M; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, plysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin.

  10. Lysosomal storage and impaired autophagy lead to inflammasome activation in Gaucher macrophages.

    Science.gov (United States)

    Aflaki, Elma; Moaven, Nima; Borger, Daniel K; Lopez, Grisel; Westbroek, Wendy; Chae, Jae Jin; Marugan, Juan; Patnaik, Samarjit; Maniwang, Emerson; Gonzalez, Ashley N; Sidransky, Ellen

    2016-02-01

    Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer-amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL-1β and IL-6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase-1, led to the maturation of IL-1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small-molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL-1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65-NF-kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL-1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets. Published 2015. This article is a U.S. Government work and is in the public domain in the USA. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  11. Lysosomal regulation of cholesterol homeostasis in tuberous sclerosis complex is mediated via NPC1 and LDL-R.

    Science.gov (United States)

    Filippakis, Harilaos; Alesi, Nicola; Ogorek, Barbara; Nijmeh, Julie; Khabibullin, Damir; Gutierrez, Catherine; Valvezan, Alexander J; Cunningham, James; Priolo, Carmen; Henske, Elizabeth P

    2017-06-13

    Tuberous sclerosis complex (TSC) is a multisystem disease associated with hyperactive mTORC1. The impact of TSC1/2 deficiency on lysosome-mediated processes is not fully understood. We report here that inhibition of lysosomal function using chloroquine (CQ) upregulates cholesterol homeostasis genes in TSC2-deficient cells. This TSC2-dependent transcriptional signature is associated with increased accumulation and intracellular levels of both total cholesterol and cholesterol esters. Unexpectedly, engaging this CQ-induced cholesterol uptake pathway together with inhibition of de novo cholesterol synthesis allows survival of TSC2-deficient, but not TSC2-expressing cells. The underlying mechanism of TSC2-deficient cell survival is dependent on exogenous cholesterol uptake via LDL-R, and endosomal trafficking mediated by Vps34. Simultaneous inhibition of lysosomal and endosomal trafficking inhibits uptake of esterified cholesterol and cell growth in TSC2-deficient, but not TSC2-expressing cells, highlighting the TSC-dependent lysosome-mediated regulation of cholesterol homeostasis and pointing toward the translational potential of these pathways for the therapy of TSC.

  12. Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology

    DEFF Research Database (Denmark)

    Kirkegaard, Thomas; Roth, Anke G; Petersen, Nikolaj H T

    2010-01-01

    Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced cell death. Clues to its molecular mechanism of action may lay in the recently...... reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism......-is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds...

  13. Lysosomal enlargement and lysosomal membrane destabilisation in mussel digestive cells measured by an integrative index

    International Nuclear Information System (INIS)

    Izagirre, Urtzi; Marigomez, Ionan

    2009-01-01

    Lysosomal responses (enlargement and membrane destabilisation) in mussel digestive cells are well-known environmental stress biomarkers in pollution effects monitoring in marine ecosystems. Presently, in laboratory and field studies, both responses were measured separately (in terms of lysosomal volume density - Vv - and labilisation period -LP) and combined (lysosomal response index - LRI) in order to contribute to their understanding and to develop an index useful for decisions makers. LRI integrates Vv and LP, which are not necessarily dependent lysosomal responses. It is unbiased and more sensitive than Vv and LP alone and diminishes background due to confounding factors. LRI provides a simple numerical index (consensus reference = 0; critical threshold = 1) directly related to the pollution impact degree. Moreover, LRI can be represented in a way that allows the interpretation of lysosomal responses, which is useful for environmental scientists. - Lysosomal responses to pollutants measured by an integrative index.

  14. Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy

    Science.gov (United States)

    Cortes, Constanza J.; Qin, Kefeng; Norstrom, Eric M.; Green, William N.; Bindokas, Vytautas P.; Mastrianni, James A.

    2013-01-01

    Prion diseases are linked to the accumulation of a misfolded isoform (PrPSc) of prion protein (PrP). Evidence suggests that lysosomes are degradation endpoints and sites of the accumulation of PrPSc. We questioned whether lysosomes participate in the early quality control of newly generated misfolded PrP. We found PrP carrying the disease-associated T182A mutation (Mut-PrP) was delivered to lysosomes in a Golgi-independent manner. Time-lapse live cell imaging revealed early formation and uptake of GFP-tagged Mut-PrP aggregates into LysoTracker labeled vesicles. Compared with Wt-PrP, Mut-PrP expression was associated with an elevation in several markers of the autophagy-lysosomal pathway, and it extensively colocalized with the autophagosome-specific marker, LC3B. In autophagy deficient (ATG5−/−) mouse embryonic fibroblasts, or in normal cells treated with the autophagy-inhibitor 3-MA, Mut-PrP colocalization with lysosomes was reduced to a similar extent. Additionally, 3-MA selectively impaired the degradation of insoluble Mut-PrP, resulting in an increase in protease-resistant PrP, whereas the induction of autophagy by rapamycin reduced it. These findings suggest that autophagy might function as a quality control mechanism to limit the accumulation of misfolded PrP that normally leads to the generation of PrPSc. PMID:24454378

  15. Changes of lysosomes in the earliest stages of the development of atherosclerosis.

    Science.gov (United States)

    Bobryshev, Yuri V; Shchelkunova, Tatyana A; Morozov, Ivan A; Rubtsov, Petr M; Sobenin, Igor A; Orekhov, Alexander N; Smirnov, Alexander N

    2013-05-01

    One of hypotheses of atherosclerosis is based on a presumption that the zones prone to the development of atherosclerosis contain lysosomes which are characterized by enzyme deficiency and thus, are unable to dispose of lipoproteins. The present study was undertaken to investigate the characteristics and changes of lysosomes in the earliest stages of the development of atherosclerosis. Electron microscopic immunocytochemistry revealed that there were certain changes in the distribution of CD68 antigen in lysosomes along the 'normal intima-initial lesion-fatty streak' sequence. There were no significant changes found in the key mRNAs encoding for the components of endosome/lysosome compartment in initial atherosclerotic lesions, but in fatty streaks, the contents of EEA1 and Rab5a mRNAs were found to be diminished while the contents of CD68 and p62 mRNAs were increased, compared with the intact tissue. The study reinforces a view that changes occurring in lysosomes play a role in atherogenesis from the very earlier stages of the disease. © 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  16. Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes

    Science.gov (United States)

    Naphade, Swati; Sharma, Jay; Chevronnay, Héloïse P. Gaide; Shook, Michael A.; Yeagy, Brian A.; Rocca, Celine J.; Ur, Sarah N.; Lau, Athena J.; Courtoy, Pierre J.; Cherqui, Stephanie

    2014-01-01

    Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multi-systemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon co-culture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism. TNT formation was enhanced by contact with diseased cells. In vivo, HSCs grafted to cystinotic kidneys also generated nanotubular extensions resembling invadopodia that crossed the dense basement membranes and delivered cystinosin into diseased proximal tubular cells. This is the first report of correction of a genetic lysosomal defect by bidirectional vesicular exchange via TNTs and suggests broader potential for HSC transplantation for other disorders due to defective vesicular proteins. PMID:25186209

  17. Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

    Science.gov (United States)

    Takatori, Sho; Tatematsu, Tsuyako; Cheng, Jinglei; Matsumoto, Jun; Akano, Takuya; Fujimoto, Toyoshi

    2016-02-01

    Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. The lysosomal membrane protein SCAV-3 maintains lysosome integrity and adult longevity

    Science.gov (United States)

    Li, Yuan; Chen, Baohui; Zou, Wei; Wang, Xin; Wu, Yanwei; Zhao, Dongfeng; Sun, Yanan; Liu, Yubing

    2016-01-01

    Lysosomes degrade macromolecules and recycle metabolites as well as being involved in diverse processes that regulate cellular homeostasis. The lysosome is limited by a single phospholipid bilayer that forms a barrier to separate the potent luminal hydrolases from other cellular constituents, thus protecting the latter from unwanted degradation. The mechanisms that maintain lysosomal membrane integrity remain unknown. Here, we identified SCAV-3, the Caenorhabditis elegans homologue of human LIMP-2, as a key regulator of lysosome integrity, motility, and dynamics. Loss of scav-3 caused rupture of lysosome membranes and significantly shortened lifespan. Both of these phenotypes were suppressed by reinforced expression of LMP-1 or LMP-2, the C. elegans LAMPs, indicating that longevity requires maintenance of lysosome integrity. Remarkably, reduction in insulin/insulin-like growth factor 1 (IGF-1) signaling suppressed lysosomal damage and extended the lifespan in scav-3(lf) animals in a DAF-16–dependent manner. Our data reveal that SCAV-3 is essential for preserving lysosomal membrane stability and that modulation of lysosome integrity by the insulin/IGF-1 signaling pathway affects longevity. PMID:27810910

  19. Decreased T2 signal in the thalami may be a sign of lysosomal storage disease

    International Nuclear Information System (INIS)

    Autti, Taina; Joensuu, Raimo; Aaberg, Laura

    2007-01-01

    Lysosomal disorders are rare and are caused by genetically transmitted lysosomal enzyme deficiencies. A decreased T2 signal in the thalamus has occasionally been reported. Because the finding of bilateral abnormal signal intensity of the thalamus on T2-weighted images has not been systematically reviewed, and its value as a diagnostic tool critically evaluated, we carried out a systematic review of the literature. Articles in English with 30 trios of keywords were collected from PubMed. Exclusion criteria were lack of conventional T2-weighted images in the protocol and not being a human study. Finally, 111 articles were included. The thalamus was considered affected only if mentioned in the text or in the figure legends. Some 117 patients with various lysosomal diseases and five patients with ceruloplasmin deficiency were reported to have a bilateral decrease in T2 signal intensity. At least one article reported a bilateral decrease in signal intensity of the thalami on T2-weighted images in association with GM1 and GM2 gangliosidosis and with Krabbe's disease, aspartylglucosaminuria, mannosidosis, fucosidosis, and mucolipidosis IV. Furthermore, thalamic alteration was a consistent finding in several types of neuronal ceroid lipofuscinosis (NCL) including CLN1 (infantile NCL), CLN2 (classic late infantile NCL), CLN3 (juvenile NCL), CLN5 (Finnish variant late infantile NCL), and CLN7 (Turkish variant late infantile NCL). A decrease in T2 signal intensity in the thalami seems to be a sign of lysosomal disease. (orig.)

  20. [Application of lysosomal detection in marine pollution monitoring: research progress].

    Science.gov (United States)

    Weng, You-Zhu; Fang, Yong-Qiang; Zhang, Yu-Sheng

    2013-11-01

    Lysosome is an important organelle existing in eukaryotic cells. With the development of the study on the structure and function of lysosome in recent years, lysosome is considered as a target of toxic substances on subcellular level, and has been widely applied abroad in marine pollution monitoring. This paper summarized the biological characteristics of lysosomal marker enzyme, lysosome-autophagy system, and lysosomal membrane, and introduced the principles and methods of applying lysosomal detection in marine pollution monitoring. Bivalve shellfish digestive gland and fish liver are the most sensitive organs for lysosomal detection. By adopting the lysosomal detection techniques such as lysosomal membrane stability (LMS) test, neutral red retention time (NRRT) assay, morphological measurement (MM) of lysosome, immunohistochemical (Ih) assay of lysosomal marker enzyme, and electron microscopy (EM), the status of marine pollution can be evaluated. It was suggested that the lysosome could be used as a biomarker for monitoring marine environmental pollution. The advantages and disadvantages of lysosomal detection and some problems worthy of attention were analyzed, and the application prospects of lysosomal detection were discussed.

  1. Lysosomal Storage Disorders and Malignancy

    Directory of Open Access Journals (Sweden)

    Gregory M. Pastores

    2017-02-01

    Full Text Available Lysosomal storage disorders (LSDs are infrequent to rare conditions caused by mutations that lead to a disruption in the usual sequential degradation of macromolecules or their transit within the cell. Gaucher disease (GD, a lipidosis, is among the most common LSD, with an estimated incidence of 1 in 40,000 among the Caucasian, non-Jewish population. Studies have indicated an increased frequency of polyclonal and monoclonal gammopathy among patients with GD. It has been shown that two major sphingolipids that accumulate in GD, namely, β-glucosylceramide 22:0 (βGL1-22 and glucosylsphingosine (LGL1, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT cells. Investigations undertaken in an affected mouse model revealed βGL1-22- and LGL1-specific NKT cells were present and constitutively promoted the expression of a T-follicular helper (TFH phenotype; injection of these lipids led to downstream induction of germinal center B cells, hypergammaglobulinemia, and the production of antilipid antibodies. Subsequent studies have found clonal immunoglobulin in 33% of sporadic human monoclonal gammopathies is also specific for the lysolipids LGL1 and lysophosphatidylcholine (LPC. Furthermore, substrate reduction ameliorated GD-associated gammopathy in mice. It had been hypothesized that chronic antigenic stimulation by the abnormal lipid storage and associated immune dysregulation may be the underlying mechanism for the increased incidence of monoclonal and polyclonal gammopathies, as well as an increased incidence of multiple myeloma in patients with GD. Current observations support this proposition and illustrate the value of investigations into rare diseases, which as ‘experiments of nature’ may provide insights into conditions found in the general population that continue to remain incompletely understood.

  2. ErbB2-associated changes in the lysosomal proteome

    DEFF Research Database (Denmark)

    Nylandsted, Jesper; Becker, Andrea C; Bunkenborg, Jakob

    2011-01-01

    Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic...... purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact...... lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal...

  3. Mitochondrial Dysfunction in Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    Mario de la Mata

    2016-10-01

    Full Text Available Lysosomal storage diseases (LSDs describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS, where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm, diminished ATP production and increased generation of reactive oxygen species (ROS. Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD, the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase. Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer and glucosylsphingosine (GlcSph in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs.

  4. Lysosomal enzyme activation in irradiated mammary tumors

    International Nuclear Information System (INIS)

    Clarke, C.; Wills, E.D.

    1976-01-01

    Lysosomal enzyme activity of C3H mouse mammary tumors was measured quantitatively by a histochemical method. Following whole-body doses of 3600 rad or less no changes were observed in the lysosomal enzyme activity for 12 hr after the irradiation, but very large increases in acid phosphatase and β-naphthylamidase activity were, however, observed 24 hr after irradiation. Significant increases in enzyme activity were detected 72 hr after a dose of 300 rad and the increases of enzyme activity were dose dependent over the range 300 to 900 rad. Testosterone (80 mg/kg) injected into mice 2 hr before irradiation (850 rad) caused a significant increase of lysosomal enzyme activity over and above that of the same dose of irradiation alone. If the tumor-bearing mice were given 95 percent oxygen/5 percent carbon dioxide to breathe for 8 min before irradiation the effect of 850 rad on lysosomal acid phosphatase was increased to 160 percent/that of the irradiation given alone. Activitation of lysosomal enzymes in mammary tumors is an important primary or secondary consequence of radiation

  5. Intracellular sphingosine releases calcium from lysosomes.

    Science.gov (United States)

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-11-27

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.

  6. Lysosomes as mediators of drug resistance in cancer.

    Science.gov (United States)

    Zhitomirsky, Benny; Assaraf, Yehuda G

    2016-01-01

    Drug resistance remains a leading cause of chemotherapeutic treatment failure and cancer-related mortality. While some mechanisms of anticancer drug resistance have been well characterized, multiple mechanisms remain elusive. In this respect, passive ion trapping-based lysosomal sequestration of multiple hydrophobic weak-base chemotherapeutic agents was found to reduce the accessibility of these drugs to their target sites, resulting in a markedly reduced cytotoxic effect and drug resistance. Recently we have demonstrated that lysosomal sequestration of hydrophobic weak base drugs triggers TFEB-mediated lysosomal biogenesis resulting in an enlarged lysosomal compartment, capable of enhanced drug sequestration. This study further showed that cancer cells with an increased number of drug-accumulating lysosomes are more resistant to lysosome-sequestered drugs, suggesting a model of drug-induced lysosome-mediated chemoresistance. In addition to passive drug sequestration of hydrophobic weak base chemotherapeutics, other mechanisms of lysosome-mediated drug resistance have also been reported; these include active lysosomal drug sequestration mediated by ATP-driven transporters from the ABC superfamily, and a role for lysosomal copper transporters in cancer resistance to platinum-based chemotherapeutics. Furthermore, lysosomal exocytosis was suggested as a mechanism to facilitate the clearance of chemotherapeutics which highly accumulated in lysosomes, thus providing an additional line of resistance, supplementing the organelle entrapment of chemotherapeutics away from their target sites. Along with these mechanisms of lysosome-mediated drug resistance, several approaches were recently developed for the overcoming of drug resistance or exploiting lysosomal drug sequestration, including lysosomal photodestruction and drug-induced lysosomal membrane permeabilization. In this review we explore the current literature addressing the role of lysosomes in mediating cancer drug

  7. Pathogenic mechanisms in lysosomal disease: a reappraisal of the role of the lysosome.

    Science.gov (United States)

    Walkley, Steven U

    2007-04-01

    The view that lysosomes simply represent end organelles in the serial degradation of polymeric molecules derived from the cell surface and its interior has led to major misconceptions about the nature of lysosomal storage diseases and the pathogenic cascades that characterize them. Accordingly, lysosomal storage bodies are often considered 'inert', inducing cell dysfunction and death primarily through mechanical overcrowding of normal organelles or by other non-specific means leading to generalized cytotoxicity. However, modern studies of lysosomes and their component proteins provide evidence to support a far greater role for these organelles in cell metabolism. In intimate association with endosomal, autophagosomal and related vesicular systems, the greater lysosomal system can be conceptualized as a vital recycling centre that serves as a central metabolic coordinator, influencing literally every aspect of the cell, from signal transduction to regulation of gene expression. This broader view of the role of lysosomes in cells not only provides insight into how single gene defects impacting on lysosomal function can result in the plethora of complex cellular transformations characteristic of these diseases, but also suggests new and innovative therapies that may hold considerable promise for ameliorating disease progression.

  8. Storage Pool Deficiencies

    Science.gov (United States)

    ... Deficiency Factor V Deficiency Combined FV & FVIII Deficiencies Factor VII Deficiency Factor X Deficiency Factor XI Deficiency Factor ... Deficiency Factor V Deficiency Combined FV & FVIII Deficiencies Factor VII Deficiency Factor X Deficiency Factor XI Deficiency Factor ...

  9. Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes.

    Science.gov (United States)

    Aizawa, Shu; Contu, Viorica Raluca; Fujiwara, Yuuki; Hase, Katsunori; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

    2017-01-02

    Lysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy.

  10. Modest phenotypic improvements in ASA-deficient mice with only one UDP-galactose:ceramide-galactosyltransferase gene

    OpenAIRE

    Franken, S; Wittke, D; Mansson, JE; D'Hooge, R; De Deyn, PP; Lüllmann-Rauch, R; Matzner, U; Gieselmann, V

    2006-01-01

    Summary Background Arylsulfatase A (ASA)-deficient mice are a model for the lysosomal storage disorder metachromatic leukodystrophy. This lipidosis is characterised by the lysosomal accumulation of the sphingolipid sulfatide. Storage of this lipid is associated with progressive demyelination. We have mated ASA-deficient mice with mice heterozygous for a non-functional allele of UDP-galactose:ceramide-galactosyltransferase (CGT). This deficiency is known to lead to a decreased synthesis of gal...

  11. From bedside to cell biology: a century of history on lysosomal dysfunction.

    Science.gov (United States)

    Coutinho, Maria Francisca; Matos, Liliana; Alves, Sandra

    2015-01-15

    Lysosomal storage disorders (LSDs) are a group of rare genetic diseases, generally caused by a deficiency of specific lysosomal enzymes, which results in abnormal accumulation of undegraded substrates. The first clinical reports describing what were later shown to be LSDs were published more than a hundred years ago. In general, the history and pathophysiology of LSDs has impacted on our current knowledge of lysosomal biology. Classically, depending on the nature of the substrates, LSDs can be divided into different subgroups. The mucopolysaccharidoses (MPSs) are those caused by impaired degradation of glycosaminoglycans (GAGs). Amongst LSDs, the MPSs are a major group of pathologies with crucial historical relevance, since their study has revealed important biological pathways and highlighted interconnecting pathological cascades which are still being unveiled nowadays. Here we review the major historical discoveries in the field of LSDs and their impact on basic cellular knowledge and practical applications. Attention will be focused on the MPSs, with occasional references to other LSDs. We will show as studies on the metabolic basis of this group of diseases have increased our knowledge of the complex degradative pathways associated with the lysosome and established the basis to the development of specific therapeutic approaches aiming at correcting or, at least ameliorating their associated phenotypes. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. The GARP Complex Is Involved in Intracellular Cholesterol Transport via Targeting NPC2 to Lysosomes.

    Science.gov (United States)

    Wei, Jian; Zhang, Ying-Yu; Luo, Jie; Wang, Ju-Qiong; Zhou, Yu-Xia; Miao, Hong-Hua; Shi, Xiong-Jie; Qu, Yu-Xiu; Xu, Jie; Li, Bo-Liang; Song, Bao-Liang

    2017-06-27

    Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Analyzing Lysosome-Related Organelles by Electron Microscopy

    KAUST Repository

    Hurbain, Ilse; Romao, Maryse; Bergam, Ptissam; Heiligenstein, Xavier; Raposo, Graç a

    2017-01-01

    and their dynamics at the cellular level. Deciphering the biogenesis and functions of lysosomes and lysosome-related organelles (LROs) and their dysfunctions requires their visualization and detailed characterization at high resolution by electron microscopy. Here

  14. Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion.

    Science.gov (United States)

    Zhou, Jing; Tan, Shi-Hao; Nicolas, Valérie; Bauvy, Chantal; Yang, Nai-Di; Zhang, Jianbin; Xue, Yuan; Codogno, Patrice; Shen, Han-Ming

    2013-04-01

    Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

  15. Purification of Lysosomes Using Supraparamagnetic Iron Oxide Nanoparticles (SPIONs).

    Science.gov (United States)

    Rofe, Adam P; Pryor, Paul R

    2016-04-01

    Lysosomes can be rapidly isolated from tissue culture cells using supraparamagnetic iron oxide particles (SPIONs). In this protocol, colloidal iron dextran (FeDex) particles, a type of SPION, are taken up by cultured mouse macrophage cells via the endocytic pathway. The SPIONs accumulate in lysosomes, the end point of the endocytic pathway, permitting the lysosomes to be isolated magnetically. The purified lysosomes are suitable for in vitro fusion assays or for proteomic analysis. © 2016 Cold Spring Harbor Laboratory Press.

  16. Pathogenic Cascades in Lysosomal Disease – Why so Complex?

    OpenAIRE

    Walkley, Steven U.

    2009-01-01

    Lysosomal disease represents a large group of more than 50 clinically recognized conditions resulting from inborn errors of metabolism affecting the organelle known as the lysosome.The lysosome is an integral part of the larger endosomal/lysosomal system, and is closely allied with the ubiquitin-proteosomal and autophagosomal systems, which together comprise essential cell machinery for substrate degradation and recycling, homeostatic control, as well as signaling. More than two-thirds of lys...

  17. Neuroinflammatory paradigms in lysosomal storage diseases

    Directory of Open Access Journals (Sweden)

    Megan Elizabeth Bosch

    2015-10-01

    Full Text Available Lysosomal storage diseases (LSDs include approximately 70 distinct disorders that collectively account for 14% of all inherited metabolic diseases. LSDs are caused by mutations in various enzymes/proteins that disrupt lysosomal function, which impairs macromolecule degradation following endosome-lysosome and phagosome-lysosome fusion and autophagy, ultimately disrupting cellular homeostasis. LSDs are pathologically typified by lysosomal inclusions composed of a heterogeneous mixture of various proteins and lipids that can be found throughout the body. However, in many cases the CNS is dramatically affected, which may result from heightened neuronal vulnerability based on their post-mitotic state. Besides intrinsic neuronal defects, another emerging factor common to many LSDs is neuroinflammation, which may negatively impact neuronal survival and contribute to neurodegeneration. Microglial and astrocyte activation is a hallmark of many LSDs that affect the CNS, which often precedes and predicts regions where eventual neuron loss will occur. However, the timing, intensity, and duration of neuroinflammation may ultimately dictate the impact on CNS homeostasis. For example, a transient inflammatory response following CNS insult/injury can be neuroprotective, as glial cells attempt to remove the insult and provide trophic support to neurons. However, chronic inflammation, as seen in several LSDs, can promote neurodegeneration by creating a neurotoxic environment due to elevated levels of cytokines, chemokines, and pro-apoptotic molecules. Although neuroinflammation has been reported in several LSDs, the cellular basis and mechanisms responsible for eliciting neuroinflammatory pathways are just beginning to be defined. This review highlights the role of neuroinflammation in select LSDs and its potential contribution to neuron loss.

  18. uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

    DEFF Research Database (Denmark)

    Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria

    2004-01-01

    Collagen turnover is crucial for tissue homeostasis and remodeling and pathological processes such as cancer invasion, but the underlying molecular mechanisms are poorly understood. A major pathway appears to be internalization and degradation by fibroblasts. We now show that the endocytic...... transmembrane glycoprotein urokinase plasminogen activator receptor-associated protein (uPARAP/endo180) directs collagen IV for lysosomal delivery and degradation. In wild-type fibroblasts, fluorescently labeled collagen IV was first internalized into vesicular structures with diffuse fluorescence eventually...... appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient...

  19. Functional analysis of lysosomes during mouse preimplantation embryo development.

    Science.gov (United States)

    Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Ohta, Yuki; Wada, Ayako; Ishida, Yuka; Kito, Seiji; Nishikawa, Tetsu; Minami, Naojiro; Sato, Ken; Kokubo, Toshiaki

    2013-01-01

    Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.

  20. 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.

  1. Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway.

    Science.gov (United States)

    Liu, Haiyan; Javaheri, Ali; Godar, Rebecca J; Murphy, John; Ma, Xiucui; Rohatgi, Nidhi; Mahadevan, Jana; Hyrc, Krzysztof; Saftig, Paul; Marshall, Connie; McDaniel, Michael L; Remedi, Maria S; Razani, Babak; Urano, Fumihiko; Diwan, Abhinav

    2017-01-01

    Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

  2. Lysosomal Re-acidification Prevents Lysosphingolipid-Induced Lysosomal Impairment and Cellular Toxicity.

    Directory of Open Access Journals (Sweden)

    Christopher J Folts

    2016-12-01

    Full Text Available Neurodegenerative lysosomal storage disorders (LSDs are severe and untreatable, and mechanisms underlying cellular dysfunction are poorly understood. We found that toxic lipids relevant to three different LSDs disrupt multiple lysosomal and other cellular functions. Unbiased drug discovery revealed several structurally distinct protective compounds, approved for other uses, that prevent lysosomal and cellular toxicities of these lipids. Toxic lipids and protective agents show unexpected convergence on control of lysosomal pH and re-acidification as a critical component of toxicity and protection. In twitcher mice (a model of Krabbe disease [KD], a central nervous system (CNS-penetrant protective agent rescued myelin and oligodendrocyte (OL progenitors, improved motor behavior, and extended lifespan. Our studies reveal shared principles relevant to several LSDs, in which diverse cellular and biochemical disruptions appear to be secondary to disruption of lysosomal pH regulation by specific lipids. These studies also provide novel protective strategies that confer therapeutic benefits in a mouse model of a severe LSD.

  3. Ultraviolet induced lysosome activity in corneal epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Cullen, A.P.

    1980-01-01

    A 5.000 W Xe-Hg high pressure lamp and a double monochromator were used to produce a 3.3 nm half-bandpass ultraviolet radiation at 295 nm. Pigmented rabbit eyes were irradiated with radiant exposures from 140 Jm/sup -2/ to 10.000 Jm/sup -2/ and evaluated by slit-lamp biomicroscopy, light and electron microscopy. Corneal threshold (Hsub(c) was 200 Jm/sup -2/ and lens threshold (Hsub(L)) was 7.500 Jm/sup -2/. The most repeatable and reliable corneal response to these levels of UV was the development of corneal epithelial granules. Histological changes included a loss of superficial epithelial cells and selective UV induced autolysis of the wing cells. It is suggested that the biomicroscopically observed granules are the clinical manifestation of the secondary lysosomes revealed by light and electron microscopy. It is proposed that UV breaks down the primary lysosome membranes to release hydrolytic enzymes which in turn form the secondary lysosomes during autolysis. Extreme levels of radiant exposure at 295 nm result in indiscriminate destruction of all layers of the corneal epithelium, but the posterior cornea was spared.

  4. Ultraviolet induced lysosome activity in corneal epithelium

    International Nuclear Information System (INIS)

    Cullen, A.P.

    1980-01-01

    A 5.000 W Xe-Hg high pressure lamp and a double monochromator were used to produce a 3.3 nm half-bandpass ultraviolet radiation at 295 nm. Pigmented rabbit eyes were irradiated with radiant exposures from 140 Jm -2 to 10.000 Jm -2 and evaluated by slit-lamp biomicroscopy, light and electron microscopy. Corneal threshold (Hsub(c) was 200 Jm -2 and lens threshold (Hsub(L)) was 7.500 Jm -2 . The most repeatable and reliable corneal response to these levels of UV was the development of corneal epithelial granules. Histological changes included a loss of superficial epithelial cells and selective UV induced autolysis of the wing cells. It is suggested that the biomicroscopically observed granules are the clinical manifestation of the secondary lysosomes revealed by light and electron microscopy. It is proposed that UV breaks down the primary lysosome membranes to release hydrolytic enzymes which in turn form the secondary lysosomes during autolysis. Extreme levels of radiant exposure at 295 nm result in indiscriminate destruction of all layers of the corneal epithelium, but the posterior cornea was spared. (orig.) [de

  5. Mitochondrial–Lysosomal Axis in Acetaminophen Hepatotoxicity

    Directory of Open Access Journals (Sweden)

    Anna Moles

    2018-05-01

    Full Text Available Acetaminophen (APAP toxicity is the most common cause of acute liver failure and a major indication for liver transplantion in the United States and Europe. Although significant progress has been made in understanding the molecular mechanisms underlying APAP hepatotoxicity, there is still an urgent need to find novel and effective therapies against APAP-induced acute liver failure. Hepatic APAP metabolism results in the production of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI, which under physiological conditions is cleared by its conjugation with glutathione (GSH to prevent its targeting to mitochondria. APAP overdose or GSH limitation leads to mitochondrial NAPQI-protein adducts formation, resulting in oxidative stress, mitochondrial dysfunction, and necrotic cell death. As mitochondria are a major target of APAP hepatotoxicity, mitochondrial quality control and clearance of dysfunctional mitochondria through mitophagy, emerges as an important strategy to limit oxidative stress and the engagement of molecular events leading to cell death. Recent evidence has indicated a lysosomal–mitochondrial cross-talk that regulates APAP hepatotoxicity. Moreover, as lysosomal function is essential for mitophagy, impairment in the fusion of lysosomes with autophagosomes-containing mitochondria may compromise the clearance of dysfunctional mitochondria, resulting in exacerbated APAP hepatotoxicity. This review centers on the role of mitochondria in APAP hepatotoxicity and how the mitochondrial/lysosomal axis can influence APAP-induced liver failure.

  6. BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

    Science.gov (United States)

    Bové, Jordi; Martínez-Vicente, Marta; Dehay, Benjamin; Perier, Celine; Recasens, Ariadna; Bombrun, Agnes; Antonsson, Bruno; Vila, Miquel

    2014-05-01

    Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

  7. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes.

    Science.gov (United States)

    Abu-Remaileh, Monther; Wyant, Gregory A; Kim, Choah; Laqtom, Nouf N; Abbasi, Maria; Chan, Sze Ham; Freinkman, Elizaveta; Sabatini, David M

    2017-11-10

    The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here, we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient-replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H + -adenosine triphosphatase (V-ATPase) increased the luminal concentrations of most metabolites but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mechanistic target of rapamycin (mTOR) pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  8. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Masato, E-mail: okadam@biken.osaka-u.ac.jp [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  9. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    International Nuclear Information System (INIS)

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose; Okada, Masato

    2012-01-01

    Highlights: ► p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. ► We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. ► The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. ► Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. ► The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome–lysosome fusion, which is required for processing of various macromolecules.

  10. Cancer-associated lysosomal changes: friends or foes?

    Science.gov (United States)

    Kallunki, T; Olsen, O D; Jäättelä, M

    2013-04-18

    Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal function. Accordingly, transformation and cancer progression are characterized by dramatic changes in lysosomal volume, composition and cellular distribution. Depending on one's point of view, the cancer-associated changes in the lysosomal compartment can be regarded as friends or foes. Most of them are clearly transforming as they promote invasive growth, angiogenesis and drug resistance. The same changes can, however, strongly sensitize cells to lysosomal membrane permeabilization and thereby to lysosome-targeting anti-cancer drugs. In this review we compile our current knowledge on cancer-associated changes in lysosomal composition and discuss the consequences of these alterations to cancer progression and the possibilities they can bring to cancer therapy.

  11. Effect of cadmium on lung lysosomal enzymes in vitro

    International Nuclear Information System (INIS)

    Giri, S.N.; Hollinger, M.A.

    1995-01-01

    Labilization of lysosomal enzymes is often associated with the general process of inflammation. The present study investigated the effect of the pneumotoxin cadmium on the release and activity of two lung lysosomal enzymes. Incubation of rat lung lysosomes with cadmium resulted in the release of β-glucuronidase but not acid phosphatase. The failure to ''release'' acid phosphatase appears to be the result of a direct inhibitory effect of cadmium on this enzyme. The K I for cadmium was determined to be 26.3 μM. The differential effect of cadmium on these two lysosomal enzymes suggests that caution should be exercised in selecting the appropriate enzyme marker for assessing lysosomal fragility in the presence of this toxicant. Furthermore, the differential basal release rate of the two enzymes from lung lysosomes may reflect the cellular heterogeneity of the lung. (orig.)

  12. Magnetic resonance imaging findings of central nervous system in lysosomal storage diseases: A pictorial review.

    Science.gov (United States)

    Fagan, Nathan; Alexander, Allen; Irani, Neville; Saade, Charbel; Naffaa, Lena

    2017-06-01

    Lysosomal storage diseases (LSD) are a complex group of genetic disorders that are a result of inborn errors of metabolism. These errors result in a variety of metabolic dysfunction and build-up certain molecules within the tissues of the central nervous system (CNS). Although, they have discrete enzymatic deficiencies, symptomology and CNS imaging findings can overlap with each other, which can become challenging to radiologists. The purpose of this paper is to review the most common CNS imaging findings in LSD in order to familiarize the radiologist with their imaging findings and help narrow down the differential diagnosis. © 2016 The Royal Australian and New Zealand College of Radiologists.

  13. Impaired cholesterol esterification in primary brain cultures of the lysosomal cholesterol storage disorder (LCSD) mouse mutant

    International Nuclear Information System (INIS)

    Patel, S.C.; Suresh, S.; Weintroub, H.; Brady, R.O.; Pentchev, P.G.

    1987-01-01

    Esterification of cholesterol was investigated in primary neuroglial cultures obtained from newborn lysosomal cholesterol storage disorder (LCSD) mouse mutants. An impairment in 3 H-oleic acid incorporation into cholesteryl esters was demonstrated in cultures of homozygous LCSD brain. Primary cultures derived from other phenotypically normal pups of the carrier breeders esterified cholesterol at normal levels or at levels which were intermediary between normal and deficient indicating a phenotypic expression of the LCSD heterozygote genotype. These observations on LCSD mutant brain cells indicate that the defect in cholesterol esterification is closely related to the primary genetic defect and is expressed in neuroglial cells in culture

  14. Targeting Androgen Receptor by Lysosomal Degradation in Prostate Cancer

    Science.gov (United States)

    2015-11-01

    Preparation of the Lysosomes A673 cells were treated with 100 pM chloroquine for 12 h or left untreated. Lysosomes were prepared using the Lysosome...were treated with 100 JlM chloroquine fur 12 h or left tmtreated, and the luciferase activity was determined using the same arnotmt of protein...TFEB levels or by activating TFEB using mTORC1 kinase inhibitor, torin 1. Additionally, we determined that the same approach can be used to target

  15. GRASP55 Senses Glucose Deprivation through O-GlcNAcylation to Promote Autophagosome-Lysosome Fusion.

    Science.gov (United States)

    Zhang, Xiaoyan; Wang, Leibin; Lak, Behnam; Li, Jie; Jokitalo, Eija; Wang, Yanzhuang

    2018-04-23

    The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. SRT1720 induces lysosomal-dependent cell death of breast cancer cells.

    Science.gov (United States)

    Lahusen, Tyler J; Deng, Chu-Xia

    2015-01-01

    SRT1720 is an activator of SIRT1, a NAD(+)-dependent protein and histone deacetylase that plays an important role in numerous biologic processes. Several studies have illustrated that SRT1720 treatment could improve metabolic conditions in mouse models and in a study in cancer SRT1720 caused increased apoptosis of myeloma cells. However, the effect of SRT1720 on cancer may be complex, as some recent studies have demonstrated that SRT1720 may not directly activate SIRT1 and another study showed that SRT1720 treatment could promote lung metastasis. To further investigate the role of SRT1720 in breast cancer, we treated SIRT1 knockdown and control breast cancer cell lines with SRT1720 both in vitro and in vivo. We showed that SRT1720 more effectively decreased the viability of basal-type MDA-MB-231 and BT20 cells as compared with luminal-type MCF-7 breast cancer cells or nontumorigenic MCF-10A cells. We demonstrated that SRT1720 induced lysosomal membrane permeabilization and necrosis, which could be blocked by lysosomal inhibitors. In contrast, SRT1720-induced cell death occurred in vitro irrespective of SIRT1 status, whereas in nude mice, SRT1720 exhibited a more profound effect in inhibiting the growth of allograft tumors of SIRT1 proficient cells as compared with tumors of SIRT1-deficient cells. Thus, SRT1720 causes lysosomal-dependent necrosis and may be used as a therapeutic agent for breast cancer treatment. ©2014 American Association for Cancer Research.

  17. Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells*

    Science.gov (United States)

    Yogalingam, Gouri; Lee, Amanda R.; Mackenzie, Donald S.; Maures, Travis J.; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y.; Hague, Chuck; Christianson, Terri; Bell, Sean M.; LeBowitz, Jonathan H.

    2017-01-01

    Neutrophil myeloperoxidase (MPO) catalyzes the H2O2-dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N-retinylidene-N-retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N-retinylidene-N-retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. PMID:28115520

  18. Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells.

    Science.gov (United States)

    Yogalingam, Gouri; Lee, Amanda R; Mackenzie, Donald S; Maures, Travis J; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y; Hague, Chuck; Christianson, Terri; Bell, Sean M; LeBowitz, Jonathan H

    2017-03-10

    Neutrophil myeloperoxidase (MPO) catalyzes the H 2 O 2 -dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N -retinylidene- N -retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N -retinylidene- N -retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Clinical neurogenetics: neuropathic lysosomal storage disorders.

    Science.gov (United States)

    Pastores, Gregory M; Maegawa, Gustavo H B

    2013-11-01

    The lysosomal storage disorders are a clinically heterogeneous group of inborn errors of metabolism, associated with the accumulation of incompletely degraded macromolecules within several cellular sites. Affected individuals present with a broad range of clinical problems, including hepatosplenomegaly and skeletal dysplasia. Onset of symptoms may range from birth to adulthood. Most are associated with neurologic features. Later-onset forms are often misdiagnosed as symptoms, which might include psychiatric manifestations, are slowly progressive, and may precede other neurologic or systemic features. Symptomatic care, which remains the mainstay for most subtypes, can lead to significant improvement in quality of life. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. The lysosomal enzyme receptor protein (LERP is not essential, but is implicated in lysosomal function in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Medina Hasanagic

    2015-10-01

    Full Text Available The lysosomal enzyme receptor protein (LERP of Drosophila melanogaster is the ortholog of the mammalian cation-independent mannose 6-phosphate (Man 6-P receptor, which mediates trafficking of newly synthesized lysosomal acid hydrolases to lysosomes. However, flies lack the enzymes necessary to make the Man 6-P mark, and the amino acids implicated in Man 6-P binding by the mammalian receptor are not conserved in LERP. Thus, the function of LERP in sorting of lysosomal enzymes to lysosomes in Drosophila is unclear. Here, we analyze the consequence of LERP depletion in S2 cells and intact flies. RNAi-mediated knockdown of LERP in S2 cells had little or no effect on the cellular content or secretion of several lysosomal hydrolases. We generated a novel Lerp null mutation, LerpF6, which abolishes LERP protein expression. Lerp mutants have normal viability and fertility and display no overt phenotypes other than reduced body weight. Lerp mutant flies exhibit a 30–40% decrease in the level of several lysosomal hydrolases, and are hypersensitive to dietary chloroquine and starvation, consistent with impaired lysosome function. Loss of LERP also enhances an eye phenotype associated with defective autophagy. Our findings implicate Lerp in lysosome function and autophagy.

  1. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

    International Nuclear Information System (INIS)

    Bame, K.J.

    1986-01-01

    Acetyl-CoA:α-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal α-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from [ 3 H]CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with [ 3 H]acetyl-CoA. The acetyl group can be transferred to glucosamine, forming [ 3 H]N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism

  2. Iodine Deficiency

    Science.gov (United States)

    ... Fax/Phone Home » Iodine Deficiency Leer en Español Iodine Deficiency Iodine is an element that is needed ... world’s population remains at risk for iodine deficiency. Iodine Deficiency FAQs WHAT IS THE THYROID GLAND? The ...

  3. Spastic paraplegia proteins spastizin and spatacsin mediate autophagic lysosome reformation.

    Science.gov (United States)

    Chang, Jaerak; Lee, Seongju; Blackstone, Craig

    2014-12-01

    Autophagy allows cells to adapt to changes in their environment by coordinating the degradation and recycling of cellular components and organelles to maintain homeostasis. Lysosomes are organelles critical for terminating autophagy via their fusion with mature autophagosomes to generate autolysosomes that degrade autophagic materials; therefore, maintenance of the lysosomal population is essential for autophagy-dependent cellular clearance. Here, we have demonstrated that the two most common autosomal recessive hereditary spastic paraplegia gene products, the SPG15 protein spastizin and the SPG11 protein spatacsin, are pivotal for autophagic lysosome reformation (ALR), a pathway that generates new lysosomes. Lysosomal targeting of spastizin required an intact FYVE domain, which binds phosphatidylinositol 3-phosphate. Loss of spastizin or spatacsin resulted in depletion of free lysosomes, which are competent to fuse with autophagosomes, and an accumulation of autolysosomes, reflecting a failure in ALR. Moreover, spastizin and spatacsin were essential components for the initiation of lysosomal tubulation. Together, these results link dysfunction of the autophagy/lysosomal biogenesis machinery to neurodegeneration.

  4. The Lysosome, Elixir of Neural Stem Cell Youth.

    Science.gov (United States)

    Simic, Milos S; Dillin, Andrew

    2018-05-03

    Recently in Science, Leeman et al. find that perturbing lysosomal activity of quiescent NSCs directly impedes their ability to become activated, similar to what happens during aging. Excitingly, they could rejuvenate old quiescent NSCs by enhancing the lysosome pathway, ameliorating their ability to clear protein aggregates and become activated. Copyright © 2018. Published by Elsevier Inc.

  5. Lysosomal and Mitochondrial Liaisons in Niemann-Pick Disease

    Directory of Open Access Journals (Sweden)

    Sandra Torres

    2017-11-01

    Full Text Available Lysosomal storage disorders (LSD are characterized by the accumulation of diverse lipid species in lysosomes. Niemann-Pick type A/B (NPA/B and type C diseases Niemann-Pick type C (NPC are progressive LSD caused by loss of function of distinct lysosomal-residing proteins, acid sphingomyelinase and NPC1, respectively. While the primary cause of these diseases differs, both share common biochemical features, including the accumulation of sphingolipids and cholesterol, predominantly in endolysosomes. Besides these alterations in lysosomal homeostasis and function due to accumulation of specific lipid species, the lysosomal functional defects can have far-reaching consequences, disrupting intracellular trafficking of sterols, lipids and calcium through membrane contact sites (MCS of apposed compartments. Although MCS between endoplasmic reticulum and mitochondria have been well studied and characterized in different contexts, emerging evidence indicates that lysosomes also exhibit close proximity with mitochondria, which translates in their mutual functional regulation. Indeed, as best illustrated in NPC disease, alterations in the lysosomal-mitochondrial liaisons underlie the secondary accumulation of specific lipids, such as cholesterol in mitochondria, resulting in mitochondrial dysfunction and defective antioxidant defense, which contribute to disease progression. Thus, a better understanding of the lysosomal and mitochondrial interactions and trafficking may identify novel targets for the treatment of Niemann-Pick disease.

  6. Fluorometric Assessment Of Lysosomal Enzymes In Garlic Oil ...

    African Journals Online (AJOL)

    The effect of Garlic oil on Lysosomal enzymes in streptozotocin-induced diabetic rats were investigated fluorometrically. The serum lysosomal enzymes assayed include β-glucuronidase, N-acetylglucosaminidase (NAG) β-D-galactosidase and α-D-galactosidase. The results of the study in nMole-4Mu/hr/ml show that ...

  7. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis.

    Directory of Open Access Journals (Sweden)

    Ting-Wei Mu

    2008-02-01

    Full Text Available A lysosomal storage disease (LSD results from deficient lysosomal enzyme activity, thus the substrate of the mutant enzyme accumulates in the lysosome, leading to pathology. In many but not all LSDs, the clinically most important mutations compromise the cellular folding of the enzyme, subjecting it to endoplasmic reticulum-associated degradation instead of proper folding and lysosomal trafficking. A small molecule that restores partial mutant enzyme folding, trafficking, and activity would be highly desirable, particularly if one molecule could ameliorate multiple distinct LSDs by virtue of its mechanism of action. Inhibition of L-type Ca2+ channels, using either diltiazem or verapamil-both US Food and Drug Administration-approved hypertension drugs-partially restores N370S and L444P glucocerebrosidase homeostasis in Gaucher patient-derived fibroblasts; the latter mutation is associated with refractory neuropathic disease. Diltiazem structure-activity studies suggest that it is its Ca2+ channel blocker activity that enhances the capacity of the endoplasmic reticulum to fold misfolding-prone proteins, likely by modest up-regulation of a subset of molecular chaperones, including BiP and Hsp40. Importantly, diltiazem and verapamil also partially restore mutant enzyme homeostasis in two other distinct LSDs involving enzymes essential for glycoprotein and heparan sulfate degradation, namely alpha-mannosidosis and type IIIA mucopolysaccharidosis, respectively. Manipulation of calcium homeostasis may represent a general strategy to restore protein homeostasis in multiple LSDs. However, further efforts are required to demonstrate clinical utility and safety.

  8. Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase

    Science.gov (United States)

    Glukhova, Alisa; Hinkovska-Galcheva, Vania; Kelly, Robert; Abe, Akira; Shayman, James A.; Tesmer, John J. G.

    2015-03-01

    Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high-resolution crystal structures of human LPLA2 and a low-resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome.

  9. Effect of irradiation on lysosomal enzyme activation in cultured macrophages

    International Nuclear Information System (INIS)

    Clarke, C.; Wills, E.D.

    1980-01-01

    The effect of γrays on lysosomal enzyme activity of normal and immune macrophages of DBA/2 mice cultured in vitro has been studied. A dose of 500 rad did not significantly affect lysosomal enzyme activity 3 hours after irradiation but caused the activity to increase to 1.4 times the control value 22.5 hours after irradiation. 22.5 hours after a dose of 3000 rad the enzyme activity increased to 2.5 times the control. Lysosomal enzyme activity of the macrophages was also markedly increased by immunization of the mice with D lymphoma cells, before culture in vitro, but irradiation of these cells with a dose of 500 rad caused a further increase in lysosomal enzyme activity. The results indicate that immunization and irradiation both cause stimulation of lysosomal enzyme activity in macrophages but that the mechanisms of activation are unlikely to be identical. (author)

  10. Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

    Science.gov (United States)

    Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

    2016-01-01

    Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

  11. Prostaglandin levels and lysosomal enzyme activities in irradiated rats

    International Nuclear Information System (INIS)

    Trocha, P.J.; Catravas, G.N.

    1980-01-01

    Whole-body irradiation of rats results in the release of hydrolases from lysosomes, an increase in lysosomal enzyme activities, and changes in the prostaglandin levels in spleen and liver tissues. A transient increase in the concentration of prostaglandins E and F and leakage of lysosomal hydrolases occurred in both spleen and liver tissues 3-6 hours after the animals were irradiated. Maximal values for hydrolase activities, prostaglandin E and F content, and release of lysosomal enzymes were found 4 days postirradiation in rat spleens whereas in the liver only slight increases were observed at this time period for prostaglandin F levels. On day 7 there was a final rise in the spleen's prostaglandin E and F concentrations and leakage of hydrolases from the lysosomes before returning to near normal values on day 11. The prostaglandin F concentration in liver was also slightly elevated on the 7th day after irradiation and then decreased to control levels. (author)

  12. Lysosomal cysteine peptidases - Molecules signaling tumor cell death and survival.

    Science.gov (United States)

    Pišlar, Anja; Perišić Nanut, Milica; Kos, Janko

    2015-12-01

    Lysosomal cysteine peptidases - cysteine cathepsins - are general intracellular protein-degrading enzymes that control also a variety of specific physiological processes. They can trigger irreversible events leading to signal transduction and activation of signaling pathways, resulting in cell survival and proliferation or cell death. In cancer cells, lysosomal cysteine peptidases are involved in multiple processes during malignant progression. Their translocation from the endosomal/lysosomal pathway to nucleus, cytoplasm, plasma membrane and extracellular space enables the activation and remodeling of a variety of tumor promoting proteins. Thus, lysosomal cysteine peptidases interfere with cytokine/chemokine signaling, regulate cell adhesion and migration and endocytosis, are involved in the antitumor immune response and apoptosis, and promote cell invasion, angiogenesis and metastasis. Further, lysosomal cysteine peptidases modify growth factors and receptors involved in tyrosine kinase dependent pathways such as MAPK, Akt and JNK, thus representing key signaling tools for the activation of tumor cell growth and proliferation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function.

    Science.gov (United States)

    Bharadwaj, Rajnish; Cunningham, Kathleen M; Zhang, Ke; Lloyd, Thomas E

    2016-02-15

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content

    Science.gov (United States)

    Mundy, Dorothy I.; Li, Wei Ping; Luby-Phelps, Katherine; Anderson, Richard G. W.

    2012-01-01

    Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking. PMID:22238363

  15. Optogenetic acidification of synaptic vesicles and lysosomes.

    Science.gov (United States)

    Rost, Benjamin R; Schneider, Franziska; Grauel, M Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

    2015-12-01

    Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

  16. Health Deficiencies

    Data.gov (United States)

    U.S. Department of Health & Human Services — A list of all health deficiencies currently listed on Nursing Home Compare, including the nursing home that received the deficiency, the associated inspection date,...

  17. 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.

  18. The emerging role of lysosomes in copper homeostasis.

    Science.gov (United States)

    Polishchuk, Elena V; Polishchuk, Roman S

    2016-09-01

    The lysosomal system operates as a focal point where a number of important physiological processes such as endocytosis, autophagy and nutrient sensing converge. One of the key functions of lysosomes consists of regulating the metabolism/homeostasis of metals. Metal-containing components are carried to the lysosome through incoming membrane flows, while numerous transporters allow metal ions to move across the lysosome membrane. These properties enable lysosomes to direct metal fluxes to the sites where metal ions are either used by cellular components or sequestered. Copper belongs to a group of metals that are essential for the activity of vitally important enzymes, although it is toxic when in excess. Thus, copper uptake, supply and intracellular compartmentalization have to be tightly regulated. An increasing number of publications have indicated that these processes involve lysosomes. Here we review studies that reveal the expanding role of the lysosomal system as a hub for the control of Cu homeostasis and for the regulation of key Cu-dependent processes in health and disease.

  19. Crosstalk between Lysosomes and Mitochondria in Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Nicoletta Plotegher

    2017-12-01

    Full Text Available Parkinson's disease (PD is the most common motor neurodegenerative disorder. In most cases the cause of the disease is unknown, while in about 10% of subjects, it is associated with mutations in a number of different genes. Several different mutations in 15 genes have been identified as causing familial forms of the disease, while many others have been identified as risk factors. A striking number of these genes are either involved in the regulation of mitochondrial function or of endo-lysosomal pathways. Mutations affecting one of these two pathways are often coupled with defects in the other pathway, suggesting a crosstalk between them. Moreover, PD-linked mutations in genes encoding proteins with other functions are frequently associated with defects in mitochondrial and/or autophagy/lysosomal function as a secondary effect. Even toxins that impair mitochondrial function and cause parkinsonian phenotypes, such as rotenone, also impair lysosomal function. In this review, we explore the reciprocal relationship between mitochondrial and lysosomal pathways in PD. We will discuss the impact of mitochondrial dysfunction on the lysosomal compartment and of endo-lysosomal defects on mitochondrial function, and explore the roles of both causative genes and genes that are risk factors for PD. Understanding the pathways that govern these interactions should help to define a framework to understand the roles and mechanisms of mitochondrial and lysosomal miscommunication in the pathophysiology of PD.

  20. A quantitative assay for lysosomal acidification rates in human osteoclasts

    DEFF Research Database (Denmark)

    Jensen, Vicki Kaiser; Nosjean, Olivier; Dziegiel, Morten Hanefeld

    2011-01-01

    The osteoclast initiates resorption by creating a resorption lacuna. The ruffled border surrounding the lacunae arises from exocytosis of lysosomes. To dissolve the inorganic phase of the bone, the vacuolar adenosine triphosphatase, located in the ruffled border, pumps protons into the resorption...... assay with respect to lysosomal acidification and assess whether it is a reliable test of a compound's ability to inhibit acidification. Investigated were the expression levels of the lysosomal acidification machinery, the activation of the assay by adenosine triphosphate, H(+) and Cl(-) dependency...

  1. A lysosomal lair for a pathogenic protein pair.

    Science.gov (United States)

    Dawson, Ted M; Dawson, Valina L

    2011-07-13

    Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects movement. Although many of the causes of PD remain unclear, a consistent finding is the abnormal accumulation of the protein α-synuclein. In a recent issue of Cell, Mazzuli et al. provide a molecular explanation for the unexpected link between PD and Gaucher's disease, a glycolipid lysosomal storage disorder caused by loss of the enzyme glucocerebrosidase (GBA). They report a reciprocal connection between loss of GBA activity and the accumulation of α-synuclein in lysosomes that establishes a bidirectional positive feedback loop with pathogenic consequences. Understanding how lysosomes are implicated in PD may reveal new therapeutic targets for treating this disease.

  2. Progranulin Gene Therapy Improves Lysosomal Dysfunction and Microglial Pathology Associated with Frontotemporal Dementia and Neuronal Ceroid Lipofuscinosis.

    Science.gov (United States)

    Arrant, Andrew E; Onyilo, Vincent C; Unger, Daniel E; Roberson, Erik D

    2018-02-28

    Loss-of-function mutations in progranulin, a lysosomal glycoprotein, cause neurodegenerative disease. Progranulin haploinsufficiency causes frontotemporal dementia (FTD) and complete progranulin deficiency causes CLN11 neuronal ceroid lipofuscinosis (NCL). Progranulin replacement is a rational therapeutic strategy for these disorders, but there are critical unresolved mechanistic questions about a progranulin gene therapy approach, including its potential to reverse existing pathology. Here, we address these issues using an AAV vector (AAV- Grn ) to deliver progranulin in Grn -/- mice (both male and female), which model aspects of NCL and FTD pathology, developing lysosomal dysfunction, lipofuscinosis, and microgliosis. We first tested whether AAV- Grn could improve preexisting pathology. Even with treatment after onset of pathology, AAV- Grn reduced lipofuscinosis in several brain regions of Grn -/- mice. AAV- Grn also reduced microgliosis in brain regions distant from the injection site. AAV-expressed progranulin was only detected in neurons, not in microglia, indicating that the microglial activation in progranulin deficiency can be improved by targeting neurons and thus may be driven at least in part by neuronal dysfunction. Even areas with sparse transduction and almost undetectable progranulin showed improvement, indicating that low-level replacement may be sufficiently effective. The beneficial effects of AAV- Grn did not require progranulin binding to sortilin. Finally, we tested whether AAV- Grn improved lysosomal function. AAV-derived progranulin was delivered to the lysosome, ameliorated the accumulation of LAMP-1 in Grn -/- mice, and corrected abnormal cathepsin D activity. These data shed light on progranulin biology and support progranulin-boosting therapies for NCL and FTD due to GRN mutations. SIGNIFICANCE STATEMENT Heterozygous loss-of-function progranulin ( GRN ) mutations cause frontotemporal dementia (FTD) and homozygous mutations cause neuronal

  3. Acidic nanoparticles are trafficked to lysosomes and restore an acidic lysosomal pH and degradative function to compromised ARPE-19 cells.

    Directory of Open Access Journals (Sweden)

    Gabriel C Baltazar

    Full Text Available Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-glycolide (PLGA 502 H, PLGA 503 H and poly (DL-lactide (PLA colocalized to lysosomes of ARPE-19 cells within 60 min. PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured 1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of lysosomal pH and improved degradative activity.

  4. Lysosomal storage diseases and the blood-brain barrier.

    Science.gov (United States)

    Begley, David J; Pontikis, Charles C; Scarpa, Maurizio

    2008-01-01

    The blood-brain barrier becomes a crucial issue in neuronopathic lysosomal storage diseases for three reasons. Firstly, the function of the blood-brain barrier may be compromised in many of the lysosomal storage diseases and this barrier dysfunction may contribute to the neuropathology seen in the diseases and accelerate cell death. Secondly, the substrate reduction therapies, which successfully reduce peripheral lysosomal storage, because of the blood-brain barrier may not have as free an access to brain cells as they do to peripheral cells. And thirdly, enzyme replacement therapy appears to have little access to the central nervous system as the mannose and mannose-6-phosphate receptors involved in their cellular uptake and transport to the lysosome do not appear to be expressed at the adult blood-brain barrier. This review will discuss in detail these issues and their context in the development of new therapeutic strategies.

  5. Lysosomal storage disorders: A review of the musculoskeletal features.

    Science.gov (United States)

    James, Rebecca A; Singh-Grewal, Davinder; Lee, Senq-J; McGill, Jim; Adib, Navid

    2016-03-01

    The lysosomal storage disorders are a collection of progressive, multisystem disorders that frequently present in childhood. Their timely diagnosis is paramount as they are becoming increasingly treatable. Musculoskeletal manifestations often occur early in the disease course, hence are useful as diagnostics clues. Non-inflammatory joint stiffness or pain, carpal tunnel syndrome, trigger fingers, unexplained pain crises and short stature should all prompt consideration of a lysosomal storage disorder. Recurrent ENT infections, hepatosplenomegaly, recurrent hernias and visual/hearing impairment - especially when clustered together - are important extra-skeletal features. As diagnostic and therapeutic options continue to evolve, children with lysosomal storage disorders and their families are facing more sophisticated options for screening and treatment. The aim of this article is to highlight the paediatric presentations of lysosomal storage disorders, with an emphasis on the musculoskeletal features. © 2016 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).

  6. What lysosomes actually tell us about Parkinson's disease?

    Science.gov (United States)

    Bourdenx, Mathieu; Dehay, Benjamin

    2016-12-01

    Parkinson's disease is a common neurodegenerative disorder of unknown origin mainly characterized by the loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta and the presence of intraneuronal proteinaceous inclusions called Lewy bodies. Lysosomes are dynamic organelles that degrade, in a controlled manner, cellular components delivered via the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Increasing amounts of evidence suggest a central role of lysosomal impairment in PD aetiology. This review provides an update on how genetic evidence support this connection and highlights how the neuropathologic and mechanistic evidence might relate to the disease process in sporadic forms of Parkinson's disease. Finally, we discuss the influence of ageing on lysosomal impairment and PD aetiology and therapeutic strategies targeting lysosomal function. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Direct uptake and degradation of DNA by lysosomes

    Science.gov (United States)

    Fujiwara, Yuuki; Kikuchi, Hisae; Aizawa, Shu; Furuta, Akiko; Hatanaka, Yusuke; Konya, Chiho; Uchida, Kenko; Wada, Keiji; Kabuta, Tomohiro

    2013-01-01

    Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered “RNautophagy,” an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term “DNautophagy,” is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa. PMID:23839276

  8. Spastic paraplegia proteins spastizin and spatacsin mediate autophagic lysosome reformation

    OpenAIRE

    Chang, Jaerak; Lee, Seongju; Blackstone, Craig

    2014-01-01

    Autophagy allows cells to adapt to changes in their environment by coordinating the degradation and recycling of cellular components and organelles to maintain homeostasis. Lysosomes are organelles critical for terminating autophagy via their fusion with mature autophagosomes to generate autolysosomes that degrade autophagic materials; therefore, maintenance of the lysosomal population is essential for autophagy-dependent cellular clearance. Here, we have demonstrated that the two most common...

  9. Production of lysosomal enzymes in plant-based expression systems

    OpenAIRE

    1996-01-01

    The invention relates to the production of enzymatically active recombinant human and animal lysosomal enzymes involving construction and expression of recombinant expression constructs comprising coding sequences of human or animal lysosomal enzymes in a plant expression system. The plant expression system provides for post-translational modification and processing to produce a recombinant gene product exhibiting enzymatic activity. The invention is demonstrated by working examples in which ...

  10. Proteasomal and Lysosomal Protein Degradation and Heart Disease

    OpenAIRE

    Wang, Xuejun; Robbins, Jeffrey

    2013-01-01

    In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out ...

  11. Activation of lysosomal cathepsins in pregnant bovine leukocytes.

    Science.gov (United States)

    Talukder, Md Abdus Shabur; Balboula, Ahmed Zaky; Shirozu, Takahiro; Kim, Sung Woo; Kunii, Hiroki; Suzuki, Toshiyuki; Ito, Tsukino; Kimura, Koji; Takahashi, Masashi

    2018-06-01

    In ruminants, interferon-tau (IFNT) - mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy. © 2018 Society for Reproduction and Fertility.

  12. Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV⃞

    OpenAIRE

    Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

    2001-01-01

    An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed ...

  13. P-selectin targeting to secretory lysosomes of Rbl-2H3 cells

    OpenAIRE

    Kaur, J.; Cutler, D. F.

    2002-01-01

    The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal...

  14. Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

    Science.gov (United States)

    Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

    2015-01-01

    Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here.

  15. The crucial impact of lysosomes in aging and longevity.

    Science.gov (United States)

    Carmona-Gutierrez, Didac; Hughes, Adam L; Madeo, Frank; Ruckenstuhl, Christoph

    2016-12-01

    Lysosomes are the main catabolic organelles of a cell and play a pivotal role in a plethora of cellular processes, including responses to nutrient availability and composition, stress resistance, programmed cell death, plasma membrane repair, development, and cell differentiation. In line with this pleiotropic importance for cellular and organismal life and death, lysosomal dysfunction is associated with many age-related pathologies like Parkinson's and Alzheimer's disease, as well as with a decline in lifespan. Conversely, targeting lysosomal functional capacity is emerging as a means to promote longevity. Here, we analyze the current knowledge on the prominent influence of lysosomes on aging-related processes, such as their executory and regulatory roles during general and selective macroautophagy, or their storage capacity for amino acids and ions. In addition, we review and discuss the roles of lysosomes as active players in the mechanisms underlying known lifespan-extending interventions like, for example, spermidine or rapamycin administration. In conclusion, this review aims at critically examining the nature and pliability of the different layers, in which lysosomes are involved as a control hub for aging and longevity. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Impact of lysosome status on extracellular vesicle content and release.

    Science.gov (United States)

    Eitan, Erez; Suire, Caitlin; Zhang, Shi; Mattson, Mark P

    2016-12-01

    Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells. Copyright © 2016. Published by Elsevier B.V.

  17. A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nanoparticles.

    Science.gov (United States)

    Walker, Mathew W; Lloyd-Evans, Emyr

    2015-01-01

    Lysosomes are an emerging and increasingly important cellular organelle. With every passing year, more novel proteins and key cellular functions are associated with lysosomes. Despite this, the methodologies for their purification have largely remained unchanged since the days of their discovery. With little advancement in this area, it is no surprise that analysis of lysosomal function has been somewhat stymied, largely in part by the change in buoyant densities that occur under conditions where lysosomes accumulate macromolecules. Such phenotypes are often associated with the lysosomal storage diseases but are increasingly being observed under conditions where lysosomal proteins or, in some cases, cellular functions associated with lysosomal proteins are being manipulated. These altered lysosomes poise a problem to the classical methods to purify lysosomes that are reliant largely on their correct sedimentation by density gradient centrifugation. Building upon a technique developed by others to purify lysosomes magnetically, we have developed a unique assay using superparamagnetic iron oxide nanoparticles (SPIONs) to purify high yields of ultrapure functional lysosomes from multiple cell types including the lysosomal storage disorders. Here we describe this method in detail, including the rationale behind using SPIONs, the potential pitfalls that can be avoided and the potential functional assays these lysosomes can be used for. Finally we also summarize the other methodologies and the exact reasons why magnetic purification of lysosomes is now the method of choice for lysosomal researchers. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes.

    Science.gov (United States)

    Young, Nathan P; Kamireddy, Anwesh; Van Nostrand, Jeanine L; Eichner, Lillian J; Shokhirev, Maxim Nikolaievich; Dayn, Yelena; Shaw, Reuben J

    2016-03-01

    Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK(-/-) EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. © 2016 Young et al.; Published by Cold Spring Harbor Laboratory Press.

  19. Iodine Deficiency

    NARCIS (Netherlands)

    Zimmermann, M.B.

    2009-01-01

    Iodine deficiency has multiple adverse effects in humans, termed iodine deficiency disorders, due to inadequate thyroid hormone production. Globally, it is estimated that 2 billion individuals have an insufficient iodine intake, and South Asia and sub-Saharan Africa are particularly affected.

  20. Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus.

    Science.gov (United States)

    Watson, G L; Sayles, J N; Chen, C; Elliger, S S; Elliger, C A; Raju, N R; Kurtzman, G J; Podsakoff, G M

    1998-12-01

    Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by a genetic deficiency of beta-glucuronidase (GUS). We used a recombinant adeno-associated virus vector (AAV-GUS) to deliver GUS cDNA to MPS VII mice. The route of vector administration had a dramatic effect on the extent and distribution of GUS activity. Intramuscular injection of AAV-GUS resulted in high, localized production of GUS, while intravenous administration produced low GUS activity in several tissues. This latter treatment of MPS VII mice reduced glycosaminoglycan levels in the liver to normal and reduced storage granules dramatically. We show that a single administration of AAV-GUS can provide sustained expression of GUS in a variety of cell types and is sufficient to reverse the disease phenotype at least in the liver.

  1. Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells.

    Science.gov (United States)

    Liu, Ji; Lu, Wennan; Guha, Sonia; Baltazar, Gabriel C; Coffey, Erin E; Laties, Alan M; Rubenstein, Ronald C; Reenstra, William W; Mitchell, Claire H

    2012-07-15

    The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTR(inh)-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4(-/-) mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.

  2. Lysosomal storage diseases: current diagnostic and therapeutic options

    International Nuclear Information System (INIS)

    Malinova, V.; Honzik, T.

    2013-01-01

    Lysosomal storage diseases are rare genetic diseases caused by insufficient activity of some of the lysosomal enzymes and/or transport proteins. Initial symptoms may appear any time from the neonatal period to late adulthood; early forms tend to have a severe course with rapid progression and unfavorable prognosis. There is multisystem involvement with continuous progression of symptoms and involvement of metabolically active organs or tissues – the bone marrow, liver, bones, skeletal muscles, myocardium, or CNS. The diagnosis is definitively confirmed by demonstration of reduced activity of the particular enzyme and by mutation analysis. Some of the storage diseases can be effectively treated by intravenous administration of recombinant enzymes or by limiting the amount of the substrate stored. In a small number of lysosomal storage diseases, bone marrow transplantation is successful. Multidisciplinary collaboration, including genetic counselling and prenatal diagnosis in patient families, is required. The first part of the paper deals with general characteristics of lysosomal storage diseases and the most common diseases that are currently treatable in the Czech Republic (Gaucher’s disease, Pompe disease, Fabry disease, Niemann–Pick disease, cholesterol ester storage disease). The second part of the paper deals with mucopolysaccharidase, another group of rare lysosomal storage diseases. (author)

  3. Proteasomal and lysosomal protein degradation and heart disease.

    Science.gov (United States)

    Wang, Xuejun; Robbins, Jeffrey

    2014-06-01

    In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out in animal models and in cell culture have begun to establish both sufficiency and, in some cases, the necessity of proteasomal functional insufficiency or lysosomal insufficiency as a major pathogenic factor in the heart. This review article highlights some recent advances in the research into proteasome and lysosome protein degradation in relation to cardiac pathology and examines the emerging evidence for enhancing degradative capacities of the proteasome and/or lysosome as a new therapeutic strategy for heart disease. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy". Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Lysosomal multienzyme complex: pros and cons of working together.

    Science.gov (United States)

    Bonten, Erik J; Annunziata, Ida; d'Azzo, Alessandra

    2014-06-01

    The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, protective protein/cathepsin A (PPCA), the sialidase, neuraminidase-1 (NEU1), and the glycosidase β-galactosidase (β-GAL). Next to this 'core' complex, the existence of sub-complexes, which may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1, and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders.

  5. Vertebrate scavenger receptor class B member 2 (SCARB2: comparative studies of a major lysosomal membrane glycoprotein

    Directory of Open Access Journals (Sweden)

    Roger Stephen Holmes

    2012-06-01

    Full Text Available Scavenger receptor class B member 2 (SCARB2 (also LIMP-2, CD36L2 or LGP85 is a major lysosomal membrane glycoprotein involved in endosomal and lysosomal biogenesis and maintenance. SCARB2 acts as a receptor for the lysosomal mannose-6-phosphate independent targeting of β-glucuronidase and enterovirus 71 and influences Parkinson’s disease and epilepsy. Genetic deficiency of this protein causes deafness and peripheral neuropathy in mice as well as myoclonic epilepsy and nephrotic syndrome in humans. Comparative SCARB2 amino acid sequences and structures and SCARB2 gene locations were examined using data from several vertebrate genome projects. Vertebrate SCARB2 sequences shared 43-100% identity as compared with 30-36% sequence identities with other CD36-like superfamily members, SCARB1 and CD36. At least 10 N-glycosylation sites were conserved among most vertebrate SCARB2 proteins examined. Sequence alignments, key amino acid residues and conserved predicted secondary structures were examined, including cytoplasmic, transmembrane and external lysosomal membrane sequences: cysteine disulfide residues, thrombospondin (THP1 binding sites and 16 proline and 20 glycine conserved residues, which may contribute to short loop formation within the exomembrane SCARB2 sequences. Vertebrate SCARB2 genes contained 12 coding exons. The human SCARB2 gene contained a CpG island (CpG100, ten microRNA-binding sites and several transcription factor binding sites (including PPARA which may contribute to a higher level (2.4 times average of gene expression. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate SCARB2 gene with vertebrate SCARB1 and CD36 genes. These suggested that SCARB2 originated from duplications of the CD36 gene in an ancestral genome forming three vertebrate CD36 gene family members: SCARB1, SCARB2 and CD36.

  6. Fiber type conversion by PGC-1α activates lysosomal and autophagosomal biogenesis in both unaffected and Pompe skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Shoichi Takikita

    2010-12-01

    Full Text Available PGC-1α is a transcriptional co-activator that plays a central role in the regulation of energy metabolism. Our interest in this protein was driven by its ability to promote muscle remodeling. Conversion from fast glycolytic to slow oxidative fibers seemed a promising therapeutic approach in Pompe disease, a severe myopathy caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA which is responsible for the degradation of glycogen. The recently approved enzyme replacement therapy (ERT has only a partial effect in skeletal muscle. In our Pompe mouse model (KO, the poor muscle response is seen in fast but not in slow muscle and is associated with massive accumulation of autophagic debris and ineffective autophagy. In an attempt to turn the therapy-resistant fibers into fibers amenable to therapy, we made transgenic KO mice expressing PGC-1α in muscle (tgKO. The successful switch from fast to slow fibers prevented the formation of autophagic buildup in the converted fibers, but PGC-1α failed to improve the clearance of glycogen by ERT. This outcome is likely explained by an unexpected dramatic increase in muscle glycogen load to levels much closer to those observed in patients, in particular infants, with the disease. We have also found a remarkable rise in the number of lysosomes and autophagosomes in the tgKO compared to the KO. These data point to the role of PGC-1α in muscle glucose metabolism and its possible role as a master regulator for organelle biogenesis - not only for mitochondria but also for lysosomes and autophagosomes. These findings may have implications for therapy of lysosomal diseases and other disorders with altered autophagy.

  7. Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress.

    Science.gov (United States)

    Pascua-Maestro, Raquel; Diez-Hermano, Sergio; Lillo, Concepción; Ganfornina, Maria D; Sanchez, Diego

    2017-02-01

    Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular

  8. Unconventional Trafficking of Mammalian Phospholipase D3 to Lysosomes

    Directory of Open Access Journals (Sweden)

    Adriana Carolina Gonzalez

    2018-01-01

    Full Text Available Variants in the phospholipase D3 (PLD3 gene have genetically been linked to late-onset Alzheimer's disease. We present a detailed biochemical analysis of PLD3 and reveal its endogenous localization in endosomes and lysosomes. PLD3 reaches lysosomes as a type II transmembrane protein via a (for mammalian cells uncommon intracellular biosynthetic route that depends on the ESCRT (endosomal sorting complex required for transport machinery. PLD3 is sorted into intraluminal vesicles of multivesicular endosomes, and ESCRT-dependent sorting correlates with ubiquitination. In multivesicular endosomes, PLD3 is subjected to proteolytic cleavage, yielding a stable glycosylated luminal polypeptide and a rapidly degraded N-terminal membrane-bound fragment. This pathway closely resembles the delivery route of carboxypeptidase S to the yeast vacuole. Our experiments reveal a biosynthetic route of PLD3 involving proteolytic processing and ESCRT-dependent sorting for its delivery to lysosomes in mammalian cells.

  9. Cellular proteostasis: degradation of misfolded proteins by lysosomes

    Science.gov (United States)

    Jackson, Matthew P.

    2016-01-01

    Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

  10. Salinomycin kills cancer stem cells by sequestering iron in lysosomes

    Science.gov (United States)

    Mai, Trang Thi; Hamaï, Ahmed; Hienzsch, Antje; Cañeque, Tatiana; Müller, Sebastian; Wicinski, Julien; Cabaud, Olivier; Leroy, Christine; David, Amandine; Acevedo, Verónica; Ryo, Akihide; Ginestier, Christophe; Birnbaum, Daniel; Charafe-Jauffret, Emmanuelle; Codogno, Patrice; Mehrpour, Maryam; Rodriguez, Raphaël

    2017-10-01

    Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.

  11. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

    Science.gov (United States)

    Garrity, Abigail G; Wang, Wuyang; Collier, Crystal MD; Levey, Sara A; Gao, Qiong; Xu, Haoxing

    2016-01-01

    Impaired homeostasis of lysosomal Ca2+ causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca2+ are not known. We developed a physiological assay to monitor lysosomal Ca2+ store refilling using specific activators of lysosomal Ca2+ channels to repeatedly induce lysosomal Ca2+ release. In contrast to the prevailing view that lysosomal acidification drives Ca2+ into the lysosome, inhibiting the V-ATPase H+ pump did not prevent Ca2+ refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca2+ prevented lysosomal Ca2+ stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca2+ refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca2+ or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca2+for the lysosome. DOI: http://dx.doi.org/10.7554/eLife.15887.001 PMID:27213518

  12. Specific lysosomal transport of small neutral amino acids

    International Nuclear Information System (INIS)

    Pisoni, R.L.; Flickinger, K.S.; Thoene, J.G.; Christensen, H.N.

    1986-01-01

    Studies of amino acid exodus from lysosomes have allowed us previously to describe transport systems specific for cystine and another for cationic amino acids in fibroblast lysosomes. They are now able to study amino acid uptake into highly purified fibroblast lysosomes obtained by separating crude granular fraction on gradients formed by centrifugation in 35% isoosmotic Percoll solutions. Analog inhibition and saturation studies indicate that L-[ 14 C]proline (50 μM) uptake by fibroblast lysosomes at 37 0 C in 50 mM citrate/tris pH 7.0 buffer containing 0.25 M sucrose is mediated by two transport systems, one largely specific for L-proline and the other for which transport is shared with small neutral amino acids such as alanine, serine and threonine. At 7 mM, L-proline inhibits L-[ 14 C]proline uptake almost completely, whereas ala, ser, val, thr, gly, N-methylalanine and sarcosine inhibit proline uptake by 50-65%. The system shared by alanine, serine and threonine is further characterized by these amino acids strongly inhibiting the uptakes of each other. Lysosomal proline transport is selective for the L-isomer of the amino acid, and is scarcely inhibited by 7 mM arg, glu, asp, leu, phe, his, met, (methylamino) isobutyrate, betaine or N,N-dimethylglycine. Cis or trans-4-hydroxy-L-proline inhibit proline uptake only slightly. In sharp contrast to the fibroblast plasma membrane in which Na + is required for most proline and alanine transport, lysosomal uptake of these amino acids occurs independently of Na +

  13. Purification and primary structure determination of human lysosomal dipeptidase.

    Science.gov (United States)

    Dolenc, Iztok; Mihelic, Marko

    2003-02-01

    The lysosomal metallopeptidase is an enzyme that acts preferentially on dipeptides with unsubstituted N- and C-termini. Its activity is highest in slightly acidic pH. Here we describe the isolation and characterization of lysosomal dipeptidase from human kidney. The isolated enzyme has the amino-terminal sequence DVAKAIINLAVY and is a homodimer with a molecular mass of 100 kDa. So far no amino acid sequence has been determined for this metallopeptidase. The complete primary structure as deduced from the nucleotide sequence revealed that the isolated dipeptidase is similar to blood plasma glutamate carboxypeptidase.

  14. Analyzing Lysosome-Related Organelles by Electron Microscopy

    KAUST Repository

    Hurbain, Ilse

    2017-04-29

    Intracellular organelles have a particular morphological signature that can only be appreciated by ultrastructural analysis at the electron microscopy level. Optical imaging and associated methodologies allow to explore organelle localization and their dynamics at the cellular level. Deciphering the biogenesis and functions of lysosomes and lysosome-related organelles (LROs) and their dysfunctions requires their visualization and detailed characterization at high resolution by electron microscopy. Here, we provide detailed protocols for studying LROs by transmission electron microscopy. While conventional electron microscopy and its recent improvements is the method of choice to investigate organelle morphology, immunoelectron microscopy allows to localize organelle components and description of their molecular make up qualitatively and quantitatively.

  15. Quantitative modeling of selective lysosomal targeting for drug design

    DEFF Research Database (Denmark)

    Trapp, Stefan; Rosania, G.; Horobin, R.W.

    2008-01-01

    log K ow. These findings were validated with experimental results and by a comparison to the properties of antimalarial drugs in clinical use. For ten active compounds, nine were predicted to accumulate to a greater extent in lysosomes than in other organelles, six of these were in the optimum range...... predicted by the model and three were close. Five of the antimalarial drugs were lipophilic weak dibasic compounds. The predicted optimum properties for a selective accumulation of weak bivalent bases in lysosomes are consistent with experimental values and are more accurate than any prior calculation...

  16. Common and uncommon pathogenic cascades in lysosomal storage diseases.

    Science.gov (United States)

    Vitner, Einat B; Platt, Frances M; Futerman, Anthony H

    2010-07-02

    Lysosomal storage diseases (LSDs), of which about 50 are known, are caused by the defective activity of lysosomal proteins, resulting in accumulation of unmetabolized substrates. As a result, a variety of pathogenic cascades are activated such as altered calcium homeostasis, oxidative stress, inflammation, altered lipid trafficking, autophagy, endoplasmic reticulum stress, and autoimmune responses. Some of these pathways are common to many LSDs, whereas others are only altered in a subset of LSDs. We now review how these cascades impact upon LSD pathology and suggest how intervention in the pathways may lead to novel therapeutic approaches.

  17. Lysosomes are associated with microtubules and not with intermediate filaments in cultured fibroblasts.

    OpenAIRE

    Collot, M; Louvard, D; Singer, S J

    1984-01-01

    Double immunofluorescent labeling experiments for lysosomes and either microtubules or vimentin intermediate filaments in cultured well-spread fibroblasts show a remarkable degree of superposition of the lysosomes and the microtubules. Under two different sets of conditions where the microtubules and intermediate filaments are well segregated from one another, the lysosomes remain codistributed with the microtubules. It is suggested that this specific association of lysosomes with microtubule...

  18. Determination of the lysosomal role in tumor accumulation of 67Ga by dual-tracer studies

    International Nuclear Information System (INIS)

    Ando, Atsushi; Ando, Itsuko; Hiraki, Tatsunosuke; Yamada, Norihisa; Hisada, Kinichi

    1989-01-01

    The lysosomal role in tumor accumulation of 67 Ga was determined by dual-tracer( 67 Ga and 46 Sc) studies. It became clear that 67 Ga essentially did not accumulate in the tumor lysosome, and that the lysosome did not play a major role in tumor accumulation of 67 Ga. In addition, it was revealed that tumor lysosome was hardly disrupted at all in some phases of fractionation procedures. (author)

  19. Inhibition of substrate synthesis as a strategy for glycolipid lysosomal storage disease therapy

    NARCIS (Netherlands)

    Platt, F. M.; Jeyakumar, M.; Andersson, U.; Priestman, D. A.; Dwek, R. A.; Butters, T. D.; Cox, T. M.; Lachmann, R. H.; Hollak, C.; Aerts, J. M.; van Weely, S.; Hrebícek, M.; Moyses, C.; Gow, I.; Elstein, D.; Zimran, A.

    2001-01-01

    The glycosphingolipid (GSL) lysosomal storage diseases are caused by mutations in the genes encoding the glycohydrolases that catabolize GSLs within lysosomes. In these diseases the substrate for the defective enzyme accumulates in the lysosome and the stored GSL leads to cellular dysfunction and

  20. Structure of human saposin A at lysosomal pH

    International Nuclear Information System (INIS)

    Hill, Chris H.; Read, Randy J.; Deane, Janet E.

    2015-01-01

    A 1.8 Å resolution structure of the sphingolipid activator protein saposin A has been determined at pH 4.8, the physiologically relevant lysosomal pH for hydrolase enzyme activation and lipid-transfer activity. The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme β-galactocerebrosidase (GALC), which catalyzes the breakdown of β-d-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a ‘closed’ to an ‘open’ conformation. However, previous structures were determined at non-lysosomal pH. This work describes a 1.8 Å resolution X-ray crystal structure determined at the physiologically relevant lysosomal pH 4.8. In the absence of lipid or detergent at pH 4.8, SapA is observeed to adopt a conformation closely resembling the previously determined ‘closed’ conformation, showing that pH alone is not sufficient for the transition to the ‘open’ conformation. Structural alignments reveal small conformational changes, highlighting regions of flexibility

  1. Conversion of diphosphatidylglycerol to bis(monoacylglyceryl)phosphate by lysosomes

    NARCIS (Netherlands)

    Poorthuis, B. J.; Hostetler, K. Y.

    1978-01-01

    Diphosphatidyl[1',2',3'-14C]glycerol (cardiolipin) is converted to bis(monoacylglyceryl)phosphate when incubated in vitro with rat lysosomes at pH 4.4. The stereochemical configuration of the product is unknown. This reaction probably takes place via lysophosphatidylglycerol, one of the major

  2. Assessments of lysosomal membrane responses to stresses with ...

    African Journals Online (AJOL)

    In marine bivalves, it has been demonstrated that their lysosomal membrane stability are very susceptible to many internal and external environmental changes and this physiological response can be quantified by the neutral red retention (NRR) assay. This assay has been applied in many recent studies in the areas of ...

  3. Structure of human saposin A at lysosomal pH

    Energy Technology Data Exchange (ETDEWEB)

    Hill, Chris H.; Read, Randy J.; Deane, Janet E., E-mail: jed55@cam.ac.uk [University of Cambridge, Wellcome Trust/MRC Building, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY (United Kingdom)

    2015-06-27

    A 1.8 Å resolution structure of the sphingolipid activator protein saposin A has been determined at pH 4.8, the physiologically relevant lysosomal pH for hydrolase enzyme activation and lipid-transfer activity. The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme β-galactocerebrosidase (GALC), which catalyzes the breakdown of β-d-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a ‘closed’ to an ‘open’ conformation. However, previous structures were determined at non-lysosomal pH. This work describes a 1.8 Å resolution X-ray crystal structure determined at the physiologically relevant lysosomal pH 4.8. In the absence of lipid or detergent at pH 4.8, SapA is observeed to adopt a conformation closely resembling the previously determined ‘closed’ conformation, showing that pH alone is not sufficient for the transition to the ‘open’ conformation. Structural alignments reveal small conformational changes, highlighting regions of flexibility.

  4. The second report of a new hypomyelinating disease due to a defect in the VPS11 gene discloses a massive lysosomal involvement.

    Science.gov (United States)

    Hörtnagel, Konstanze; Krägeloh-Mann, Inge; Bornemann, Antje; Döcker, Miriam; Biskup, Saskia; Mayrhofer, Heidi; Battke, Florian; du Bois, Gabriele; Harzer, Klaus

    2016-11-01

    Vesicular protein sorting-associated proteins (VPS, including VPS11) are indispensable in the endocytic network, in particular the endosome-lysosome biogenesis. Exome sequencing revealed the homozygous variant p.Leu387_ Gly395del in the VPS11 gene in two siblings. On immunoblotting, the mutant VPS11 protein showed a distinctly reduced immunostaining intensity. The children presented with primary and severe developmental delay associated with myoclonic seizures, spastic tetraplegia, trunk and neck hypotonia, blindness, hearing loss, and microcephaly. Neuro-imaging showed severe hypomyelination affecting cerebral and cerebellar white matter and corpus callosum, in the absence of a peripheral neuropathy. Electron microscopy of a skin biopsy revealed clusters of membranous cytoplasmic bodies in dermal unmyelinated nerve axons, and numbers of vacuoles in eccrine sweat glands, similar to what is seen in a classic lysosomal storage disease (LSD). Bone marrow cytology showed a high number of storage macrophages with a micro-vacuolated cytoplasm. Biochemically, changes in urinary glycosphingolipids were reminiscent of those in prosaposin deficiency (another LSD). The clinical and neuro-imaged features in our patients were almost identical to those in some recently reported patients with another variant in the VPS11 gene, p.Cys846Gly; underlining the presumed pathogenic potential of VPS11 defects. A new feature was the morphological evidence for lysosomal storage in VPS11 deficiency: This newly characterised disease can be viewed as belonging to the complex field of LSD.

  5. Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

    Science.gov (United States)

    Park, Soonhong; Ahuja, Malini; Kim, Min Seuk; Brailoiu, G Cristina; Jha, Archana; Zeng, Mei; Baydyuk, Maryna; Wu, Ling-Gang; Wassif, Christopher A; Porter, Forbes D; Zerfas, Patricia M; Eckhaus, Michael A; Brailoiu, Eugen; Shin, Dong Min; Muallem, Shmuel

    2016-02-01

    Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV. © 2015 The Authors.

  6. What Are Rare Clotting Factor Deficiencies?

    Science.gov (United States)

    ... Deficiency Factor V Deficiency Combined FV & FVIII Deficiencies Factor VII Deficiency Factor X Deficiency Factor XI Deficiency Factor ... Deficiency Factor V Deficiency Combined FV & FVIII Deficiencies Factor VII Deficiency Factor X Deficiency Factor XI Deficiency Factor ...

  7. Imaging Lysosomal pH Alteration in Stressed Cells with a Sensitive Ratiometric Fluorescence Sensor.

    Science.gov (United States)

    Xue, Zhongwei; Zhao, Hu; Liu, Jian; Han, Jiahuai; Han, Shoufa

    2017-03-24

    The organelle-specific pH is crucial for cell homeostasis. Aberrant pH of lysosomes has been manifested in myriad diseases. To probe lysosome responses to cell stress, we herein report the detection of lysosomal pH changes with a dual colored probe (CM-ROX), featuring a coumarin domain with "always-on" blue fluorescence and a rhodamine-lactam domain activatable to lysosomal acidity to give red fluorescence. With sensitive ratiometric signals upon subtle pH changes, CM-ROX enables discernment of lysosomal pH changes in cells undergoing autophagy, cell death, and viral infection.

  8. Autophagic dysfunction in a lysosomal storage disorder due to impaired proteolysis.

    Science.gov (United States)

    Elrick, Matthew J; Lieberman, Andrew P

    2013-02-01

    Alterations in macroautophagy (hereafter referred to as "autophagy") are a common feature of lysosomal storage disorders, and have been hypothesized to play a major role in the pathogenesis of these diseases. We have recently reported multiple defects in autophagy contributing to the lysosomal storage disorder Niemann-Pick type C (NPC). These include increased formation of autophagosomes, slowed turnover of autophagosomes secondary to impaired lysosomal proteolysis, and delivery of stored lipids to the lysosome via autophagy. The study summarized here describes novel methods for the interrogation of individual stages of the autophagic pathway, and suggests mechanisms by which lipid storage may result in broader lysosomal dysfunction.

  9. Lysosomes in cancer-living on the edge (of the cell).

    Science.gov (United States)

    Hämälistö, Saara; Jäättelä, Marja

    2016-04-01

    The lysosomes have definitely polished their status inside the cell. Being discovered as the last resort of discarded cellular biomass, the steady rising of this versatile signaling organelle is currently ongoing. This review discusses the recent data on the unconventional functions of lysosomes, focusing mainly on the less studied lysosomes residing in the cellular periphery. We emphasize our discussion on the emerging paths the lysosomes have taken in promoting cancer progression to metastatic disease. Finally, we address how the altered cancerous lysosomes in metastatic cancers may be specifically targeted and what are the pending questions awaiting for elucidation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. A lysosome-locating and acidic pH-activatable fluorescent probe for visualizing endogenous H2O2 in lysosomes.

    Science.gov (United States)

    Liu, Jun; Zhou, Shunqing; Ren, Jing; Wu, Chuanliu; Zhao, Yibing

    2017-11-20

    There is increasing evidence indicating that lysosomal H 2 O 2 is closely related to autophagy and apoptotic pathways under both physiological and pathological conditions. Therefore, fluorescent probes that can be exploited to visualize H 2 O 2 in lysosomes are potential tools for exploring diverse roles of H 2 O 2 in cells. However, functional exploration of lysosomal H 2 O 2 is limited by the lack of fluorescent probes capable of compatibly sensing H 2 O 2 under weak acidic conditions (pH = 4.5) of lysosomes. Lower spatial resolution of the fluorescent visualization of lysosomal H 2 O 2 might be caused by the interference of signals from cytosolic and mitochondrial H 2 O 2 , as well as the non-specific distribution of the probes in cells. In this work, we developed a lysosome-locating and acidic-pH-activatable fluorescent probe for the detection and visualization of H 2 O 2 in lysosomes, which consists of a H 2 O 2 -responsive boronate unit, a lysosome-locating morpholine group, and a pH-activatable benzorhodol fluorophore. The response of the fluorescent probe to H 2 O 2 is significantly more pronounced under acidic pH conditions than that under neutral pH conditions. Notably, the present probe enables the fluorescence sensing of endogenous lysosomal H 2 O 2 in living cells without external stimulations, with signal interference from the cytoplasm and other intracellular organelles being negligible.

  11. Chitotriosidase activity as additional biomarker in the diagnosis of lysosomal storage diseases

    Directory of Open Access Journals (Sweden)

    N. V. Olkhovych

    2016-02-01

    Full Text Available To date, several genetic variants that lead to a deficiency of chitotriosidase activity have been described. The duplication of 24 bp (dup24bp in exon 10 of the CHIT1 gene, which causes a complete loss of enzymatic activity of the gene product, is the most common among the European population. The aim of the study was to evaluate the possibility of using chitotriosidase activity as an additional biomarker in diagnosis of lysosomal storage diseases (LSDs in Ukraine, to determine this parameter in blood plasma of the patients with various lysosomal diseases and to assess the effect of the presence of dup24bp in the CHIT1 gene on this parameter. It has been shown that chitotriosidase activity in blood plasma is a convenient additional biochemical marker in the diagnosis of some LSDs, namely Gaucher disease, Niemann-Pick disease A, B, C and GM1-gangliosidosis. Reference ranges of the normal chitotriosidase activity were determined in blood plasma of Ukrainian population and found to be 8.0-53.1 nmol 4-methylumbelliferone/h·ml of plasma. The total allele frequency of the dup24bp in the CHIT1 gene in Ukrainian population was determined, which amounted to 0.26 (323/1244 that is higher than in European population. It was indicated that molecular-genetic screening of dup24bp in the CHIT1 gene is a necessary stage in a protocol for the laboratory diagnosis of Gaucher disease, Niemann-Pick disease A, B, C as well as GM1-gangliosidosis to avoid incorrect diagnosis.

  12. Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Vuong, Tram Thu [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway); Bertelsen, Vibeke; Rødland, Marianne Skeie [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Stang, Espen [Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene, E-mail: i.h.madshus@medisin.uio.no [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway)

    2013-02-01

    The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub{sub 4}) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub{sub 4} chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub{sub 4} was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub{sub 4} was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub{sub 4} was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis. -- Highlights: ► A chimera containing ErbB2 and a tetra-Ubiquitin chain internalizes constitutively. ► Receptor fragmentation is not required for endocytosis of ErbB2. ► Ubiquitination is sufficient to induce endocytosis and degradation of ErbB2. ► ErbB2-Ub4 is internalized clathrin-dependently.

  13. Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes

    International Nuclear Information System (INIS)

    Vuong, Tram Thu; Berger, Christian; Bertelsen, Vibeke; Rødland, Marianne Skeie; Stang, Espen; Madshus, Inger Helene

    2013-01-01

    The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub 4 ) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub 4 chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub 4 was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub 4 was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub 4 was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis. -- Highlights: ► A chimera containing ErbB2 and a tetra-Ubiquitin chain internalizes constitutively. ► Receptor fragmentation is not required for endocytosis of ErbB2. ► Ubiquitination is sufficient to induce endocytosis and degradation of ErbB2. ► ErbB2-Ub4 is internalized clathrin-dependently.

  14. Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules

    International Nuclear Information System (INIS)

    Mithieux, G.; Rousset, B.

    1989-01-01

    We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes, a process which is inhibited by ATP. Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells

  15. TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

    Science.gov (United States)

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms

  16. 19q13.12 microdeletion syndrome fibroblasts display abnormal storage of cholesterol and sphingolipids in the endo-lysosomal system.

    Science.gov (United States)

    Zhao, Kexin; van der Spoel, Aarnoud; Castiglioni, Claudia; Gale, Sarah; Fujiwara, Hideji; Ory, Daniel S; Ridgway, Neale D

    2018-06-01

    Microdeletions in 19q12q13.12 cause a rare and complex haploinsufficiency syndrome characterized by intellectual deficiency, developmental delays, and neurological movement disorders. Variability in the size and interval of the deletions makes it difficult to attribute the complex clinical phenotype of this syndrome to an underlying gene(s). As an alternate approach, we examined the biochemical and metabolic features of fibroblasts from an affected individual to derive clues as to the molecular basis for the syndrome. Immunofluorescence and electron microscopy of affected fibroblasts revealed an abnormal endo-lysosomal compartment that was characterized by rapid accumulation of lysosomotropic dyes, elevated LAMP1 and LAMP2 expression and vacuoles containing membrane whorls, common features of lysosomal lipid storage disorders. The late endosomes-lysosomes (LE/LY) of affected fibroblasts accumulated low-density lipoprotein cholesterol, and displayed reduced cholesterol esterification and increased de novo cholesterol synthesis, indicative of defective cholesterol transport to the endoplasmic reticulum. Affected fibroblasts also had increased ceramide and sphingolipid mass, altered glycosphingolipid species and accumulation of a fluorescent lactosylceramide probe in LE/LY. Autophagosomes also accumulated in affected fibroblasts because of decreased fusion with autolysosomes, a defect associated with other lysosomal storage diseases. Attempts to correct the cholesterol/sphingolipid storage defect in fibroblasts with cyclodextrin, sphingolipid synthesis inhibitors or by altering ion transport were unsuccessful. Our data show that 19q13.12 deletion fibroblasts have abnormal accumulation of cholesterol and sphingolipids in the endo-lysosomal system that compromises organelle function and could be an underlying cause of the clinical features of the syndrome. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. BCM-95 and (2-hydroxypropyl)-β-cyclodextrin reverse autophagy dysfunction and deplete stored lipids in Sap C-deficient fibroblasts.

    Science.gov (United States)

    Tatti, Massimo; Motta, Marialetizia; Scarpa, Susanna; Di Bartolomeo, Sabrina; Cianfanelli, Valentina; Tartaglia, Marco; Salvioli, Rosa

    2015-08-01

    Saposin (Sap) C deficiency is a rare variant form of Gaucher disease caused by impaired Sap C expression or accelerated degradation, and associated with accumulation of glucosylceramide and other lipids in the endo/lysosomal compartment. No effective therapies are currently available for the treatment of Sap C deficiency. We previously reported that a reduced amount and enzymatic activity of cathepsin (Cath) B and Cath D, and defective autophagy occur in Sap C-deficient fibroblasts. Here, we explored the use of two compounds, BCM-95, a curcumin derivative, and (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), to improve lysosomal function of Sap C-deficient fibroblasts. Immunofluorescence and biochemical studies documented that each compound promotes an increase of the expression levels and activities of Cath B and Cath D, and efficient clearance of cholesterol (Chol) and ceramide (Cer) in lysosomes. We provide evidence that BCM-95 and HP-β-CD enhance lysosomal function promoting autophagic clearance capacity and lysosome reformation. Our findings suggest a novel pharmacological approach to Sap C deficiency directed to treat major secondary pathological aspects in this disorder. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. A Novel Method of Imaging Lysosomes in Living Human Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Kristine Glunde

    2003-01-01

    Full Text Available Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2. The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.

  19. A novel transgenic mouse model of lysosomal storage disorder

    OpenAIRE

    Ortiz-Miranda, Sonia; Ji, Rui; Jurczyk, Agata; Aryee, Ken-Edwin; Mo, Shunyan; Fletcher, Terry; Shaffer, Scott A.; Greiner, Dale L.; Bortell, Rita; Gregg, Ronald G.; Cheng, Alan; Hennings, Leah J.; Rittenhouse, Ann R.

    2016-01-01

    We provide an explanation for striking pathology found in a subset of genetically engineered mice homozygous for a rat CaVβ2a transgene (Tg+/+). Multiple transgene (Tg) copies inserted into chromosome 19; at this same site a large deletion occurred, ablating cholesterol 25-hydroxylase and partially deleting lysosomal acid lipase and CD95. Their loss of function can account for lipid build up and immune system hypertrophy, which defines this phenotype and serendipitously provides a novel model...

  20. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    Science.gov (United States)

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. © 2015 Tewari et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  1. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function

    OpenAIRE

    Bharadwaj, Rajnish; Cunningham, Kathleen M.; Zhang, Ke; Lloyd, Thomas E.

    2015-01-01

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 mi...

  2. Doxorubicin Blocks Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification.

    Science.gov (United States)

    Li, Dan L; Wang, Zhao V; Ding, Guanqiao; Tan, Wei; Luo, Xiang; Criollo, Alfredo; Xie, Min; Jiang, Nan; May, Herman; Kyrychenko, Viktoriia; Schneider, Jay W; Gillette, Thomas G; Hill, Joseph A

    2016-04-26

    The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1(+/-) mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. © 2016 American Heart Association, Inc.

  3. Discriminating lysosomal membrane protein types using dynamic neural network.

    Science.gov (United States)

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

    This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

  4. The position of lysosomes within the cell determines their luminal pH.

    Science.gov (United States)

    Johnson, Danielle E; Ostrowski, Philip; Jaumouillé, Valentin; Grinstein, Sergio

    2016-03-14

    We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H(+)-adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility. © 2016 Johnson et al.

  5. Effects of misonidazole, irradiation and hyperthermia on lysosomal enzyme activity in mouse tumours

    International Nuclear Information System (INIS)

    Barratt, G.M.; Wills, E.D.

    1981-01-01

    Male C3H mice bearing transplanted tumours were treated with hyperthermia, gamma radiation and the radiosensitising drug misonidazole. The activity of tumour lysosomal acid phosphatase and β-glucuronidase was determined using quantitative cytochemical techniques which measure both lysosomal membrane permeability and enzyme activity. Misonidazole had no effect on the membrane permeability or enzyme activity of tumour lysosomes 1 hr after injection; but 25 hr after the drug treatment the permeability of the lysosomal membrane to the substrate was increased to 1.7 times control. Increases in the lysosomal enzyme activity and membrane permeability were observed 1 hr after combined treatment with misonidazole and irradiation, although neither the drug nor irradiation given alone affected the lysosomes 1 hr after treatment. Twenty-five hours after treatment of tumours with misonidazole given 25 minutes before irradiation of tumours, permeability of the lysosomal membrane had increased to 2.3 times the control. The effects of the irradiation and the radio-sensitisers were thus synergistic. Hyperthermic treatment of tumours increased and misonidazole decreased the lysosomal membrane permeability and enzyme activity measured immediately after exposure. Thus misonidazole and irradiation act synergistically to cause increased lysosomal activity but misonidazole depresses the effect of hyperthermia on lysosomes. (author)

  6. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    International Nuclear Information System (INIS)

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-01-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts

  7. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... Research Home / < Back To Health Topics / Iron-Deficiency Anemia Iron-Deficiency Anemia Also known as Leer en español Iron-deficiency ... iron-deficiency anemia. Blood tests to screen for iron-deficiency anemia To screen for iron-deficiency anemia, your doctor ...

  8. Boronated protoporphyrin (BOPP): localization in lysosomes of the human glioma cell line SF-767 with uptake modulated by lipoprotein levels

    International Nuclear Information System (INIS)

    Callahan, Daniel E.; Forte, Trudy M.; Javed Afzal, S.M.; Deen, Dennis F.; Kahl, Stephen B.; Bjornstad, Kathleen A.; Bauer, William F.; Blakely, Eleanor A.

    1999-01-01

    Purpose: Boronated protoporphyrin (BOPP) is a candidate for use in both boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) of glioblastoma multiforme (GBM). Our objectives are to identify factors that influence the uptake and retention of BOPP in vitro and to determine BOPP distribution in a human glioma cell line in vitro. This information will aid the development of compounds and treatment strategies that increase the effectiveness of BNCT therapy for GBM. Methods and Materials: The amount, distribution pattern, and site of internalization of BOPP were assessed using fluorescence microscopy. Living human glioma (SF-767) cells were imaged after a 24-h exposure to BOPP (20-135.6 μg/ml, normal serum). Dose-dependent uptake of BOPP was determined using both fluorescence microscopy of individual living cells and inductively-coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of cell pellets. Lysosome- or mitochondria-specific fluorescent probes were used to identify the cellular compartment containing BOPP. Two human fibroblast cell lines, AG-1522 (LDL receptor-positive) and GM019-15C (LDL receptor-deficient), were used to investigate LDL receptor-dependent BOPP uptake. The dependence of BOPP uptake on lipoproteins in the media was determined by exposing each of the three cell types to BOPP in medium containing either normal (NS) or lipoprotein deficient serum (LPDS). Results: BOPP accumulated in the lysosomes of human glioma cells in vitro, and not in the mitochondria, as reported for C6 rat glioma cells in vitro. BOPP uptake was concentration-dependent and was also dependent on the amount of lipoproteins in the medium. Over the range of incubation concentrations studied and at the single exposure duration time point investigated (24 h), all cells retained a similar amount of BOPP. At the lowest incubation concentration (20 μg/ml, NS), the amount of boron retained was near 10 9 atoms per cell (15 μg B/g cells). Lysosomes containing high

  9. Iron deficiency

    DEFF Research Database (Denmark)

    Schou, Morten; Bosselmann, Helle; Gaborit, Freja

    2015-01-01

    BACKGROUND: Both iron deficiency (ID) and cardiovascular biomarkers are associated with a poor outcome in heart failure (HF). The relationship between different cardiovascular biomarkers and ID is unknown, and the true prevalence of ID in an outpatient HF clinic is probably overlooked. OBJECTIVES.......043). CONCLUSION: ID is frequent in an outpatient HF clinic. ID is not associated with cardiovascular biomarkers after adjustment for traditional confounders. Inflammation, but not neurohormonal activation is associated with ID in systolic HF. Further studies are needed to understand iron metabolism in elderly HF...

  10. Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes.

    Science.gov (United States)

    Squillaro, Tiziana; Antonucci, Ivana; Alessio, Nicola; Esposito, Anna; Cipollaro, Marilena; Melone, Mariarosa Anna Beatrice; Peluso, Gianfranco; Stuppia, Liborio; Galderisi, Umberto

    2017-12-01

    Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity. © 2017 Wiley Periodicals, Inc.

  11. Lamp-2 deficiency prevents high-fat diet-induced obese diabetes via enhancing energy expenditure

    International Nuclear Information System (INIS)

    Yasuda-Yamahara, Mako; Kume, Shinji; Yamahara, Kosuke; Nakazawa, Jun; Chin-Kanasaki, Masami; Araki, Hisazumi; Araki, Shin-ichi; Koya, Daisuke; Haneda, Masakzu; Ugi, Satoshi; Maegawa, Hiroshi; Uzu, Takashi

    2015-01-01

    Autophagy process is essential for maintaining intracellular homeostasis and consists of autophagosome formation and subsequent fusion with lysosome for degradation. Although the role of autophagosome formation in the pathogenesis of diabetes has been recently documented, the role of the latter process remains unclear. This study analyzed high-fat diet (HFD)-fed mice lacking lysosome-associated membrane protein-2 (lamp-2), which is essential for the fusion with lysosome and subsequent degradation of autophagosomes. Although lamp-2 deficient mice showed little alteration in glucose metabolism under normal diet feeding, they showed a resistance against high-fat diet (HFD)-induced obesity, hyperinsulinemic hyperglycemia and tissues lipid accumulation, accompanied with higher energy expenditure. The expression levels of thermogenic genes in brown adipose tissue were significantly increased in HFD-fed lamp-2-deficient mice. Of some serum factors related to energy expenditure, the serum level of fibroblast growth factor (FGF) 21 and its mRNA expression level in the liver were significantly higher in HFD-fed lamp-2-deficient mice in an ER stress-, but not PPARα-, dependent manner. In conclusion, a lamp-2-depenedent fusion and degradation process of autophagosomes is involved in the pathogenesis of obese diabetes, providing a novel insight into autophagy and diabetes. - Highlights: • Lamp-2 is essential for autophagosome fusion with lysosome and its degradation. • Lamp-2 deficiency lead to a resistance to diet-induced obese diabetes in mice. • Lamp-2 deficiency increased whole body energy expenditure under HFD-feeding. • Lamp-2 deficiency elevated the serum level of FGF21 under HFD-feeding

  12. Lamp-2 deficiency prevents high-fat diet-induced obese diabetes via enhancing energy expenditure

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda-Yamahara, Mako [Department of Medicine, Shiga University of Medical Science, Otsu, Shiga (Japan); Kume, Shinji, E-mail: skume@belle.shiga-med.ac.jp [Department of Medicine, Shiga University of Medical Science, Otsu, Shiga (Japan); Yamahara, Kosuke; Nakazawa, Jun; Chin-Kanasaki, Masami; Araki, Hisazumi; Araki, Shin-ichi [Department of Medicine, Shiga University of Medical Science, Otsu, Shiga (Japan); Koya, Daisuke [Department of Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan); Haneda, Masakzu [Division of Metabolism and Biosystemic Science, Asahikawa Medical University, Asahikawa, Hokkaido (Japan); Ugi, Satoshi; Maegawa, Hiroshi; Uzu, Takashi [Department of Medicine, Shiga University of Medical Science, Otsu, Shiga (Japan)

    2015-09-18

    Autophagy process is essential for maintaining intracellular homeostasis and consists of autophagosome formation and subsequent fusion with lysosome for degradation. Although the role of autophagosome formation in the pathogenesis of diabetes has been recently documented, the role of the latter process remains unclear. This study analyzed high-fat diet (HFD)-fed mice lacking lysosome-associated membrane protein-2 (lamp-2), which is essential for the fusion with lysosome and subsequent degradation of autophagosomes. Although lamp-2 deficient mice showed little alteration in glucose metabolism under normal diet feeding, they showed a resistance against high-fat diet (HFD)-induced obesity, hyperinsulinemic hyperglycemia and tissues lipid accumulation, accompanied with higher energy expenditure. The expression levels of thermogenic genes in brown adipose tissue were significantly increased in HFD-fed lamp-2-deficient mice. Of some serum factors related to energy expenditure, the serum level of fibroblast growth factor (FGF) 21 and its mRNA expression level in the liver were significantly higher in HFD-fed lamp-2-deficient mice in an ER stress-, but not PPARα-, dependent manner. In conclusion, a lamp-2-depenedent fusion and degradation process of autophagosomes is involved in the pathogenesis of obese diabetes, providing a novel insight into autophagy and diabetes. - Highlights: • Lamp-2 is essential for autophagosome fusion with lysosome and its degradation. • Lamp-2 deficiency lead to a resistance to diet-induced obese diabetes in mice. • Lamp-2 deficiency increased whole body energy expenditure under HFD-feeding. • Lamp-2 deficiency elevated the serum level of FGF21 under HFD-feeding.

  13. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

    DEFF Research Database (Denmark)

    Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G

    2013-01-01

    Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present...... study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded...... in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem...

  14. TFEB and TFE3: Linking Lysosomes to Cellular Adaptation to Stress.

    Science.gov (United States)

    Raben, Nina; Puertollano, Rosa

    2016-10-06

    In recent years, our vision of lysosomes has drastically changed. Formerly considered to be mere degradative compartments, they are now recognized as key players in many cellular processes. The ability of lysosomes to respond to different stimuli revealed a complex and coordinated regulation of lysosomal gene expression. This review discusses the participation of the transcription factors TFEB and TFE3 in the regulation of lysosomal function and biogenesis, as well as the role of the lysosomal pathway in cellular adaptation to a variety of stress conditions, including nutrient deprivation, mitochondrial dysfunction, protein misfolding, and pathogen infection. We also describe how cancer cells make use of TFEB and TFE3 to promote their own survival and highlight the potential of these transcription factors as therapeutic targets for the treatment of neurological and lysosomal diseases.

  15. Modest phenotypic improvements in ASA-deficient mice with only one UDP-galactose:ceramide-galactosyltransferase gene

    Directory of Open Access Journals (Sweden)

    De Deyn PP

    2006-08-01

    Full Text Available Summary Background Arylsulfatase A (ASA-deficient mice are a model for the lysosomal storage disorder metachromatic leukodystrophy. This lipidosis is characterised by the lysosomal accumulation of the sphingolipid sulfatide. Storage of this lipid is associated with progressive demyelination. We have mated ASA-deficient mice with mice heterozygous for a non-functional allele of UDP-galactose:ceramide-galactosyltransferase (CGT. This deficiency is known to lead to a decreased synthesis of galactosylceramide and sulfatide, which should reduce sulfatide storage and improve pathology in ASA-deficient mice. Results ASA-/- CGT+/- mice, however, showed no detectable decrease in sulfatide storage. Neuronal degeneration of cells in the spiral ganglion of the inner ear, however, was decreased. Behavioural tests showed small but clear improvements of the phenotype in ASA-/- CGT+/- mice. Conclusion Thus the reduction of galactosylceramide and sulfatide biosynthesis by genetic means overall causes modest improvements of pathology.

  16. Autophagic dysfunction in a lysosomal storage disorder due to impaired proteolysis

    OpenAIRE

    Elrick, Matthew J.; Lieberman, Andrew P.

    2013-01-01

    Alterations in macroautophagy (hereafter referred to as “autophagy”) are a common feature of lysosomal storage disorders, and have been hypothesized to play a major role in the pathogenesis of these diseases. We have recently reported multiple defects in autophagy contributing to the lysosomal storage disorder Niemann-Pick type C (NPC). These include increased formation of autophagosomes, slowed turnover of autophagosomes secondary to impaired lysosomal proteolysis, and delivery of stored lip...

  17. Disruption of Lysosome Function Promotes Tumor Growth and Metastasis in Drosophila *

    OpenAIRE

    Chi, Congwu; Zhu, Huanhu; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2010-01-01

    Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the RasV12 cells....

  18. Involvement of the endosomal-lysosomal system correlates with regional pathology in Creutzfeldt-Jakob disease

    DEFF Research Database (Denmark)

    Kovács, Gábor G; Gelpi, Ellen; Ströbel, Thomas

    2007-01-01

    The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes...... correlate with regional pathology. Overloading of this system might impair the function of lysosomal enzymes and thus may mimic some features of lysosomal storage disorders. Udgivelsesdato: 2007-Jul...

  19. Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload

    International Nuclear Information System (INIS)

    Gross, J.B. Jr.; Myers, B.M.; Kost, L.J.; Kuntz, S.M.; LaRusso, N.F.

    1989-01-01

    We investigated the hypothesis that lysosomes are the main source of biliary copper in conditions of hepatic copper overload. We used a rat model of oral copper loading and studied the relationship between the biliary output of copper and lysosomal hydrolases. Male Sprague-Dawley rats were given tap water with or without 0.125% copper acetate for up to 36 wk. Copper loading produced a 23-fold increase in the hepatic copper concentration and a 30-65% increase in hepatic lysosomal enzyme activity. Acid phosphatase histochemistry showed that copper-loaded livers contained an increased number of hepatocyte lysosomes; increased copper concentration of these organelles was confirmed directly by both x ray microanalysis and tissue fractionation. The copper-loaded rats showed a 16-fold increase in biliary copper output and a 50-300% increase in biliary lysosomal enzyme output. In the basal state, excretory profiles over time were similar for biliary outputs of lysosomal enzymes and copper in the copper-loaded animals but not in controls. After pharmacologic stimulation of lysosomal exocytosis, biliary outputs of copper and lysosomal hydrolases in the copper-loaded animals remained coupled: injection of colchicine or vinblastine produced an acute rise in the biliary output of both lysosomal enzymes and copper to 150-250% of baseline rates. After these same drugs, control animals showed only the expected increase in lysosomal enzyme output without a corresponding increase in copper output. We conclude that the hepatocyte responds to an increased copper load by sequestering excess copper in an increased number of lysosomes that then empty their contents directly into bile. The results provide direct evidence that exocytosis of lysosomal contents into biliary canaliculi is the major mechanism for biliary copper excretion in hepatic copper overload

  20. Blood group does not correlate with disease severity in patients with Fabry disease (alpha-galactosidase A deficiency)

    NARCIS (Netherlands)

    Linthorst, Gabor E.; Folman, Claudia C.; Aerts, Johannes M. F. G.; Hollak, Carla E. M.

    2003-01-01

    Blood groups B and P1 are substrates for the lysosomal enzyme alpha-galactosidase A. Therefore, patients with alpha-Gal A deficiency and blood groups B or P1 may exhibit more severe disease. In 48 Fabry patients distribution of blood group was not different from that in the Dutch population. No

  1. SILAC-Based Comparative Proteomic Analysis of Lysosomes from Mammalian Cells Using LC-MS/MS.

    Science.gov (United States)

    Thelen, Melanie; Winter, Dominic; Braulke, Thomas; Gieselmann, Volkmar

    2017-01-01

    Mass spectrometry-based proteomics of lysosomal proteins has led to significant advances in understanding lysosomal function and pathology. The ever-increasing sensitivity and resolution of mass spectrometry in combination with labeling procedures which allow comparative quantitative proteomics can be applied to shed more light on the steadily increasing range of lysosomal functions. In addition, investigation of alterations in lysosomal protein composition in the many lysosomal storage diseases may yield further insights into the molecular pathology of these disorders. Here, we describe a protocol which allows to determine quantitative differences in the lysosomal proteome of cells which are genetically and/or biochemically different or have been exposed to certain stimuli. The method is based on stable isotope labeling of amino acids in cell culture (SILAC). Cells are exposed to superparamagnetic iron oxide particles which are endocytosed and delivered to lysosomes. After homogenization of cells, intact lysosomes are rapidly enriched by passing the cell homogenates over a magnetic column. Lysosomes are eluted after withdrawal of the magnetic field and subjected to mass spectrometry.

  2. Endo-lysosomal and autophagic dysfunction: a driving factor in Alzheimer's disease?

    Science.gov (United States)

    Whyte, Lauren S; Lau, Adeline A; Hemsley, Kim M; Hopwood, John J; Sargeant, Timothy J

    2017-03-01

    Alzheimer's disease (AD) is the most common cause of dementia, and its prevalence will increase significantly in the coming decades. Although important progress has been made, fundamental pathogenic mechanisms as well as most hereditary contributions to the sporadic form of the disease remain unknown. In this review, we examine the now substantial links between AD pathogenesis and lysosomal biology. The lysosome hydrolyses and processes cargo delivered by multiple pathways, including endocytosis and autophagy. The endo-lysosomal and autophagic networks are central to clearance of cellular macromolecules, which is important given there is a deficit in clearance of amyloid-β in AD. Numerous studies show prominent lysosomal dysfunction in AD, including perturbed trafficking of lysosomal enzymes and accumulation of the same substrates that accumulate in lysosomal storage disorders. Examination of the brain in lysosomal storage disorders shows the accumulation of amyloid precursor protein metabolites, which further links lysosomal dysfunction with AD. This and other evidence leads us to hypothesise that genetic variation in lysosomal genes modifies the disease course of sporadic AD. © 2016 International Society for Neurochemistry.

  3. Lysosomal responses in the digestive gland of the freshwater mussel, Dreissena polymorpha, experimentally exposed to cadmium

    International Nuclear Information System (INIS)

    Giamberini, Laure; Cajaraville, Miren P.

    2005-01-01

    In order to examine the possible use of lysosomal response as a biomarker of freshwater quality, structural changes of lysosomes were measured by image analysis in the digestive gland of the zebra mussel, Dreissena polymorpha, exposed in laboratory conditions to cadmium. Mussels were exposed to the metal (10 and 200 μg/L) for 3 weeks and randomly collected after 7 and 21 days. At each treatment day, digestive tissues were excised and β-glucuronidase activity was revealed in cryotome sections. Four stereological parameters were calculated: lysosomal volume density, lysosomal surface density, lysosomal surface to volume ratio, and lysosomal numerical density. The changes observed in this study reflected a general activation of the lysosomal system, including an increase in both the number and the size of lysosomes in the digestive gland cells of mussels exposed to cadmium. The digestive lysosomal response in zebra mussels was related to exposure time and to metal concentration, demonstrating the potential of this biomarker in freshwater biomonitoring

  4. Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

    Science.gov (United States)

    Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

    1996-08-01

    To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

  5. A novel transgenic mouse model of lysosomal storage disorder.

    Science.gov (United States)

    Ortiz-Miranda, Sonia; Ji, Rui; Jurczyk, Agata; Aryee, Ken-Edwin; Mo, Shunyan; Fletcher, Terry; Shaffer, Scott A; Greiner, Dale L; Bortell, Rita; Gregg, Ronald G; Cheng, Alan; Hennings, Leah J; Rittenhouse, Ann R

    2016-11-01

    Knockout technology has proven useful for delineating functional roles of specific genes. Here we describe and provide an explanation for striking pathology that occurs in a subset of genetically engineered mice expressing a rat Ca V β2a transgene under control of the cardiac α-myosin heavy chain promoter. Lesions were limited to mice homozygous for transgene and independent of native Cacnb2 genomic copy number. Gross findings included an atrophied pancreas; decreased adipose tissue; thickened, orange intestines; and enlarged liver, spleen, and abdominal lymph nodes. Immune cell infiltration and cell engulfment by macrophages were associated with loss of pancreatic acinar cells. Foamy macrophages diffusely infiltrated the small intestine's lamina propria, while similar macrophage aggregates packed liver and splenic red pulp sinusoids. Periodic acid-Schiff-positive, diastase-resistant, iron-negative, Oil Red O-positive, and autofluorescent cytoplasm was indicative of a lipid storage disorder. Electron microscopic analysis revealed liver sinusoids distended by clusters of macrophages containing intracellular myelin "swirls" and hepatocytes with enlarged lysosomes. Additionally, build up of cholesterol, cholesterol esters, and triglycerides, along with changes in liver metabolic enzyme levels, were consistent with a lipid processing defect. Because of this complex pathology, we examined the transgene insertion site. Multiple transgene copies inserted into chromosome 19; at this same site, an approximate 180,000 base pair deletion occurred, ablating cholesterol 25-hydroxylase and partially deleting lysosomal acid lipase and CD95 Loss of gene function can account for the altered lipid processing, along with hypertrophy of the immune system, which define this phenotype, and serendipitously provides a novel mouse model of lysosomal storage disorder. Copyright © 2016 the American Physiological Society.

  6. Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; Olsen, Ole D; Groth-Pedersen, Line

    2013-01-01

    Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome......-destabilizing experimental anticancer agent siramesine inhibits ASM by interfering with the binding of ASM to its essential lysosomal cofactor, bis(monoacylglycero)phosphate. Like siramesine, several clinically relevant ASM inhibitors trigger cancer-specific lysosomal cell death, reduce tumor growth in vivo, and revert...

  7. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... To Health Topics / Iron-Deficiency Anemia Iron-Deficiency Anemia Also known as Leer en español Iron-deficiency ... anemia. Blood tests to screen for iron-deficiency anemia To screen for iron-deficiency anemia, your doctor ...

  8. Iron-Deficiency Anemia

    Science.gov (United States)

    ... To Health Topics / Iron-Deficiency Anemia Iron-Deficiency Anemia Also known as Leer en español Iron-deficiency ... anemia. Blood tests to screen for iron-deficiency anemia To screen for iron-deficiency anemia, your doctor ...

  9. From Lysosomal Storage Diseases to NKT Cell Activation and Back

    Directory of Open Access Journals (Sweden)

    Cátia S. Pereira

    2017-02-01

    Full Text Available Lysosomal storage diseases (LSDs are inherited metabolic disorders characterized by the accumulation of different types of substrates in the lysosome. With a multisystemic involvement, LSDs often present a very broad clinical spectrum. In many LSDs, alterations of the immune system were described. Special emphasis was given to Natural Killer T (NKT cells, a population of lipid-specific T cells that is activated by lipid antigens bound to CD1d (cluster of differentiation 1 d molecules at the surface of antigen-presenting cells. These cells have important functions in cancer, infection, and autoimmunity and were altered in a variety of LSDs’ mouse models. In some cases, the observed decrease was attributed to defects in either lipid antigen availability, trafficking, processing, or loading in CD1d. Here, we review the current knowledge about NKT cells in the context of LSDs, including the alterations detected, the proposed mechanisms to explain these defects, and the relevance of these findings for disease pathology. Furthermore, the effect of enzyme replacement therapy on NKT cells is also discussed.

  10. Biogenesis and proteolytic processing of lysosomal DNase II.

    Directory of Open Access Journals (Sweden)

    Susumu Ohkouchi

    Full Text Available Deoxyribonuclease II (DNase II is a key enzyme in the phagocytic digestion of DNA from apoptotic nuclei. To understand the molecular properties of DNase II, particularly the processing, we prepared a polyclonal antibody against carboxyl-terminal sequences of mouse DNase II. In the present study, partial purification of DNase II using Con A Sepharose enabled the detection of endogenous DNase II by Western blotting. It was interesting that two forms of endogenous DNase II were detected--a 30 kDa form and a 23 kDa form. Neither of those forms carried the expected molecular weight of 45 kDa. Subcellular fractionation showed that the 23 kDa and 30 kDa proteins were localized in lysosomes. The processing of DNase II in vivo was also greatly altered in the liver of mice lacking cathepsin L. DNase II that was extracellularly secreted from cells overexpressing DNase II was detected as a pro-form, which was activated under acidic conditions. These results indicate that DNase II is processed and activated in lysosomes, while cathepsin L is involved in the processing of the enzyme.

  11. Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection.

    Science.gov (United States)

    Di Cristina, Manlio; Dou, Zhicheng; Lunghi, Matteo; Kannan, Geetha; Huynh, My-Hang; McGovern, Olivia L; Schultz, Tracey L; Schultz, Aric J; Miller, Alyssa J; Hayes, Beth M; van der Linden, Wouter; Emiliani, Carla; Bogyo, Matthew; Besteiro, Sébastien; Coppens, Isabelle; Carruthers, Vern B

    2017-06-19

    Globally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii 1 . This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses 2 and cognitive impairment 3 . There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of the essential pathways that maintain long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here, we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection, and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. This work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacological development.

  12. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... you are diagnosed with iron-deficiency anemia. Risk Factors You may have an increased risk for iron- ... iron-deficiency anemia if you have certain risk factors , including pregnancy. To prevent iron-deficiency anemia, your ...

  13. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... to moderate iron-deficiency anemia, or red blood cell transfusion for severe iron-deficiency anemia. You may ... body needs iron to make healthy red blood cells. Iron-deficiency anemia usually develops over time because ...

  14. Vitamin Deficiency Anemia

    Science.gov (United States)

    ... are unique to specific vitamin deficiencies. Folate-deficiency anemia risk factors include: Undergoing hemodialysis for kidney failure. ... the metabolism of folate. Vitamin B-12 deficiency anemia risk factors include: Lack of intrinsic factor. Most ...

  15. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

    DEFF Research Database (Denmark)

    Jensen, Stine Skov; Christensen, Karina; Aaberg-Jessen, Charlotte

    Targeting lysosomes is a novel approach in cancer therapy providing a possible way of killing the otherwise apoptosis-resistant cancer cells. Recent research has thus shown that lysosome targeting compounds induce cell death in a cervix cancer cell line. Tumor stem cells in glioblastomas have...

  16. Lysosome stabilization in slices of rat liver when incubated with vitamin A excess

    International Nuclear Information System (INIS)

    Morre, D.M.; Morre, D.J.; Bowen, S.; Reutter, W.

    1986-01-01

    An organ culture of slices of livers from adult rats was used to study effect of vitamin A (all-trans retinol) on lysosome stability. Lysosomes were purified by centrifugation in Percoll gradients. Preparations were monitored by electron microscopy and evaluated by morphometry and assays of marker enzymes. Enrichments relative to homogenates and crude pellets were estimated from latent (triton X-100) acid p-nitrophenylphosphatase specific activities. Lysosomes prepared from unincubated slices were enriched 50-fold in latent acid phosphatase relative to homogenates. In contrast, lysosomes prepared from slices incubated for 30 min in PBS alone were enriched only 20-fold. When 25 μg/ml retinol was included in the incubation medium, enrichments of 40-fold were obtained. The integrity of the slices was monitored by electron microscopy and their viability was confirmed by a sustained uptake and incorporation of [ 3 H]leucine into protein (up to 2 h in culture). The loss of lysosomes from homogenates of slices incubated in the absence of retinol was accompanied by a loss of acid phosphatase from the lysosomal pellet to the supernatant during purification. Addition of retinol to slices just prior to homogenization was without effect. The results demonstrate a stabilizing influence of vitamin A on lysosomes during incubation of licer slices. The findings contrast earlier reports of retinol-induced lysosome fragility in other in vitro systems

  17. Expression Pattern of Lysosomal Protective Protein/Cathepsin A: Implications for the analysis of hnman galactosialidosis

    NARCIS (Netherlands)

    R.J. Rottier (Robbert)

    1998-01-01

    textabstractThe lysosome represents a well characterized, membrane-contained intracellular digestive system. Iu this important organelle a battery of lysosomal hydro lases and accessory proteins work in concert on the step-wise conversion of macromolecular substrates into small biological building

  18. Drug-drug interactions involving lysosomes: mechanisms and potential clinical implications.

    Science.gov (United States)

    Logan, Randall; Funk, Ryan S; Axcell, Erick; Krise, Jeffrey P

    2012-08-01

    Many commercially available, weakly basic drugs have been shown to be lysosomotropic, meaning they are subject to extensive sequestration in lysosomes through an ion trapping-type mechanism. The extent of lysosomal trapping of a drug is an important therapeutic consideration because it can influence both activity and pharmacokinetic disposition. The administration of certain drugs can alter lysosomes such that their accumulation capacity for co-administered and/or secondarily administered drugs is altered. In this review the authors explore what is known regarding the mechanistic basis for drug-drug interactions involving lysosomes. Specifically, the authors address the influence of drugs on lysosomal pH, volume and lipid processing. Many drugs are known to extensively accumulate in lysosomes and significantly alter their structure and function; however, the therapeutic and toxicological implications of this remain controversial. The authors propose that drug-drug interactions involving lysosomes represent an important potential source of variability in drug activity and pharmacokinetics. Most evaluations of drug-drug interactions involving lysosomes have been performed in cultured cells and isolated tissues. More comprehensive in vivo evaluations are needed to fully explore the impact of this drug-drug interaction pathway on therapeutic outcomes.

  19. Quantitative proteomic profiling for clarification of the crucial roles of lysosomes in microbial infections.

    Science.gov (United States)

    Xu, Benhong; Gao, Yanpan; Zhan, Shaohua; Ge, Wei

    2017-07-01

    Lysosomes play vital roles in both innate and adaptive immunity. It is widely accepted that lysosomes do not function exclusively as a digestive organelle. It is also involved in the process of immune cells against pathogens. However, the changes in the lysosomal proteome caused by infection with various microbes are still largely unknown, and our understanding of the proteome of the purified lysosome is another obstacle that needs to be resolved. Here, we performed a proteomic study on lysosomes enriched from THP1 cells after infection with Listeria monocytogenes (L.m), Herpes Simplex Virus 1 (HSV-1) and Vesicular Stomatitis Virus (VSV). In combination with the gene ontology (GO) analysis, we identified 284 lysosomal-related proteins from a total of 4560 proteins. We also constructed the protein-protein interaction networks for the differentially expressed proteins and revealed the core lysosomal proteins, including SRC in the L. m treated group, SRC, GLB1, HEXA and HEXB in the HSV-1 treated group and GLB1, CTSA, CTSB, HEXA and HEXB in the VSV treated group, which are involved in responding to diverse microbial infections. This study not only reveals variable lysosome responses depending on the bacterial or virus infection, but also provides the evidence based on which we propose a novel approach to proteome research for investigation of the function of the enriched organelles. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Degradation of Alzheimer's amyloid fibrils by microglia requires delivery of ClC-7 to lysosomes

    Science.gov (United States)

    Majumdar, Amitabha; Capetillo-Zarate, Estibaliz; Cruz, Dana; Gouras, Gunnar K.; Maxfield, Frederick R.

    2011-01-01

    Incomplete lysosomal acidification in microglia inhibits the degradation of fibrillar forms of Alzheimer's amyloid β peptide (fAβ). Here we show that in primary microglia a chloride transporter, ClC-7, is not delivered efficiently to lysosomes, causing incomplete lysosomal acidification. ClC-7 protein is synthesized by microglia but it is mistargeted and appears to be degraded by an endoplasmic reticulum–associated degradation pathway. Activation of microglia with macrophage colony-stimulating factor induces trafficking of ClC-7 to lysosomes, leading to lysosomal acidification and increased fAβ degradation. ClC-7 associates with another protein, Ostm1, which plays an important role in its correct lysosomal targeting. Expression of both ClC-7 and Ostm1 is increased in activated microglia, which can account for the increased delivery of ClC-7 to lysosomes. Our findings suggest a novel mechanism of lysosomal pH regulation in activated microglia that is required for fAβ degradation. PMID:21441306

  1. The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes

    Science.gov (United States)

    Schwenk, Benjamin M; Lang, Christina M; Hogl, Sebastian; Tahirovic, Sabina; Orozco, Denise; Rentzsch, Kristin; Lichtenthaler, Stefan F; Hoogenraad, Casper C; Capell, Anja; Haass, Christian; Edbauer, Dieter

    2014-01-01

    TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP-43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule-associated protein 6 (MAP6) as novel interacting protein for TMEM106B. MAP6 over-expression inhibits dendritic branching similar to TMEM106B knockdown. MAP6 knockdown fully rescues the dendritic phenotype of TMEM106B knockdown, supporting a functional interaction between TMEM106B and MAP6. Live imaging reveals that TMEM106B knockdown and MAP6 overexpression strongly increase retrograde transport of lysosomes in dendrites. Downregulation of MAP6 in TMEM106B knockdown neurons restores the balance of anterograde and retrograde lysosomal transport and thereby prevents loss of dendrites. To strengthen the link, we enhanced anterograde lysosomal transport by expressing dominant-negative Rab7-interacting lysosomal protein (RILP), which also rescues the dendrite loss in TMEM106B knockdown neurons. Thus, TMEM106B/MAP6 interaction is crucial for controlling dendritic trafficking of lysosomes, presumably by acting as a molecular brake for retrograde transport. Lysosomal misrouting may promote neurodegeneration in patients with TMEM106B risk variants. PMID:24357581

  2. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation

    Directory of Open Access Journals (Sweden)

    Fang Chen

    2016-04-01

    Full Text Available Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O, has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu and Cat D inhibitor (pepA inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p < 0.05 in lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome.

  3. Vps33B is required for delivery of endocytosed cargo to lysosomes

    NARCIS (Netherlands)

    Galmes, Romain; ten Brink, Corlinda; Oorschot, Viola; Veenendaal, Tineke; Jonker, Caspar; van der Sluijs, Peter; Klumperman, Judith

    2015-01-01

    In mammalian cells Vps33B forms a complex with VIPAS-39 that is recruited to recycling endosomes. Here we show that when Vps33B is expressed together with Rab7-interacting lysosomal protein (RILP) it is recruited to late endosomes-lysosomes and that depletion of Vps33B impairs late

  4. [Influence of delta-sleep inducing peptide on the state of lysosomal membranes and intensity of lysosomal proteolysis in different rat tissues during physiological aging of the organism].

    Science.gov (United States)

    Kutilin, D S; Bondarenko, T I; Mikhaleva, I I

    2014-01-01

    It is shown that subcutaneous injection of exogenous delta-sleep inducing peptide (DSIP) to rats aged 2-24 months in a dose of 100 μg/kg animal body weight by courses of 5 consecutive days per month has a stabilizing effect on the state of lysosomal membranes in rat tissues (brain, heart muscle and liver) at different ontogenetic stages, and this effect is accompanied by increasing intensity of lysosomal proteolysis in these tissues.

  5. Activation of lysosomal P2X4 by ATP transported into lysosomes via VNUT/SLC17A9 using V‐ATPase generated voltage gradient as the driving force

    Science.gov (United States)

    Zhong, Xi Zoë; Cao, Qi; Sun, Xue

    2016-01-01

    Key points SLC17A9 proteins function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation.P2X4 receptors act as lysosomal ion channels activated by luminal ATP.SLC17A9‐mediated ATP transport across the lysosomal membrane is suppressed by Bafilomycin A1, the V‐ATPase inhibitor.SLC17A9 mainly uses voltage gradient but not pH gradient generated by the V‐ATPase as the driving force to transport ATP into the lysosome to activate P2X4. Abstract The lysosome contains abundant ATP which plays important roles in lysosome functions and in cell signalling. Recently, solute carrier family 17 member 9 (SLC17A9, also known as VNUT for vesicular nucleotide transporter) proteins were suggested to function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation, and P2X4 receptors were suggested to be lysosomal ion channels that are activated by luminal ATP. However, the molecular mechanism of SLC17A9 transporting ATP and the regulatory mechanism of lysosomal P2X4 are largely unknown. In this study, we report that SLC17A9‐mediated ATP transport across lysosomal membranes is suppressed by Bafilomycin A1, the V‐ATPase inhibitor. By measuring P2X4 activity, which is indicative of ATP transport across lysosomal membranes, we further demonstrated that SLC17A9 mainly uses voltage gradient but not pH gradient as the driving force to transport ATP into lysosomes. This study provides a molecular mechanism for lysosomal ATP transport mediated by SLC17A9. It also suggests a regulatory mechanism of lysosomal P2X4 by SLC17A9. PMID:27477609

  6. Characterizing Adversity of Lysosomal Accumulation in Nonclinical Toxicity Studies: Results from the 5th ESTP International Expert Workshop

    Science.gov (United States)

    Lysosomes have a central role in cellular catabolism, trafficking, and processing of foreign particles. Accumulation of endogenous and exogenous materials in lysosomes represents a common finding in nonclinical toxicity studies. Histologically, these accumulations often lack dist...

  7. CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

    Science.gov (United States)

    Zhang, Hanrui; Shi, Jianting; Hachet, Melanie A; Xue, Chenyi; Bauer, Robert C; Jiang, Hongfeng; Li, Wenjun; Tohyama, Junichiro; Millar, John; Billheimer, Jeffrey; Phillips, Michael C; Razani, Babak; Rader, Daniel J; Reilly, Muredach P

    2017-11-01

    To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA ( LIPA -/- ) had barely detectable LAL enzymatic activity. Control and LIPA -/- IPSDM were loaded with [ 3 H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [ 3 H]-cholesterol to apolipoprotein A-I was abolished in LIPA -/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [ 3 H]-cholesterol-labeled AcLDL, [ 3 H]-cholesterol efflux was, however, not different between control and LIPA -/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA -/- IPSDM. In nonlipid loaded state, LIPA -/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA -/- IPSDM. LIPA -/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B , IL6 , and CCL5. CONCLUSIONS: LIPA -/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human

  8. Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex

    International Nuclear Information System (INIS)

    Hostetler, K.Y.; Gardner, M.F.; Giordano, J.R.

    1986-01-01

    Phospholipase A has been isolated from a crude lysosomal fraction from rat kidney cortex and purified 7600-fold with a recovery of 9.8% of the starting activity. The purified enzyme is a glycoprotein having an isoelectric point of pH 5.4 and an apparent molecular weight of 30,000 by high-pressure liquid chromatography gel permeation. Naturally occurring inhibitors of lysosomal phospholipase A are present in two of the lysosomal-soluble protein fractions obtained in the purification. They inhibit hydrolysis of 1,2-di[1- 14 C]oleoylphosphatidylcholine by purified phospholipase A 1 with IC 50 values of 7-11 μg. The inhibition is abolished by preincubation with trypsin at 37 0 C, but preincubation with trypsin at 4 0 C has no effect, providing evidence that the inhibitors are proteins. The results suggest that the activity of lysosomal phospholipase A may be regulated in part by inhibitory proteins. Lysosomal phospholipase A from rat kidney hydrolyzes the sn-1 acyl group of phosphatidylcholine, does not require divalent cations for full activity, and is not inhibited by ethylenediaminetetraacetic acid. It has an acid pH optimum of 3.6-3.8. Neither rho-bromophenacyl bromide, diisopropyl fluorophosphate, nor mercuric ion inhibits phospholipase A 1 . In contrast to rat liver, which has two major isoenzymes of acid phospholipase A 1 , kidney cortex has only one isoenzyme of lysosomal phospholipase A 1

  9. Effect of whole-body X-irradiation on lysosomal enzymes

    Energy Technology Data Exchange (ETDEWEB)

    D' souza, D W; Vakil, U K; Srinivasan, A [Bhabha Atomic Research Centre, Bombay (India). Biochemistry and Food Technology Div.

    1974-06-01

    Effects of whole-body x irradiation with sublethal dose (400 rad) on three intestinal lysosomal enzymes, namely, arylsulphatase, cathepsin and acid phosphatases, have been studied. They are almost equally distributed throughout the entire small intestine region. X irradiation adversely affects the integrity of lysosomal membranes. ''Free'' and ''total'' lysosomal enzyme activities exhibit maxima on 6th day. These activities return to normal level on 14th day when there is rapid generation of villi, indicating that lysosomal activities correlate with the progression of injury and of repair mechanism after sublethal dose of x irradiation. The increase in total lysosomal activity may be due to its decreased breakdown, since the rate of protein synthesis in intestinal mucosa is reduced. This is evidenced by reduced incorporation of orally fed /sup 14/C leucine into acid insoluble proteins. (auth)

  10. BORC/kinesin-1 ensemble drives polarized transport of lysosomes into the axon.

    Science.gov (United States)

    Farías, Ginny G; Guardia, Carlos M; De Pace, Raffaella; Britt, Dylan J; Bonifacino, Juan S

    2017-04-04

    The ability of lysosomes to move within the cytoplasm is important for many cellular functions. This ability is particularly critical in neurons, which comprise vast, highly differentiated domains such as the axon and dendrites. The mechanisms that control lysosome movement in these domains, however, remain poorly understood. Here we show that an ensemble of BORC, Arl8, SKIP, and kinesin-1, previously shown to mediate centrifugal transport of lysosomes in nonneuronal cells, specifically drives lysosome transport into the axon, and not the dendrites, in cultured rat hippocampal neurons. This transport is essential for maintenance of axonal growth-cone dynamics and autophagosome turnover. Our findings illustrate how a general mechanism for lysosome dispersal in nonneuronal cells is adapted to drive polarized transport in neurons, and emphasize the importance of this mechanism for critical axonal processes.

  11. Enzymatic and ultrastructural study of lysosomes in rats bearing radiation-induced thyroid follicular carcinoma

    International Nuclear Information System (INIS)

    Starling, J.R.; Clifton, K.H.; Norback, D.H.

    1981-01-01

    Radiation-induced well-differentiated and poorly differentiated follicular thyroid cancers were transplanted into the intrascapular fat pads of male Fisher 144 rats. The tumors grew in the recipient rats and after a time interval were removed and studied along with normal rat thyroids for lysosomal activity and ultrastructural characteristics. Plasma from experimental and control rats was also studied for lysosomal activity. Rats with radiation-induced thyroid carcinoma had a decrease in growth rate compared with normal rats. There was no significant increase in plasma lysosomal enzymes in the experimental rats. Well-differentiated thyroid carcinomatous tissue showed increased total activities of lysosomal enzymes as well as a difference in subcellular distribution compared with normal and poorly differentiated carcinomatous tissue. Electron microscopy of normal and carcinomatous tissue demonstrated the greatest number of lysosomes in the well-differentiated carcinoma and the fewest in the poorly differentiated carcinoma

  12. BORC/kinesin-1 ensemble drives polarized transport of lysosomes into the axon

    Science.gov (United States)

    Farías, Ginny G.; Guardia, Carlos M.; De Pace, Raffaella; Britt, Dylan J.; Bonifacino, Juan S.

    2017-01-01

    The ability of lysosomes to move within the cytoplasm is important for many cellular functions. This ability is particularly critical in neurons, which comprise vast, highly differentiated domains such as the axon and dendrites. The mechanisms that control lysosome movement in these domains, however, remain poorly understood. Here we show that an ensemble of BORC, Arl8, SKIP, and kinesin-1, previously shown to mediate centrifugal transport of lysosomes in nonneuronal cells, specifically drives lysosome transport into the axon, and not the dendrites, in cultured rat hippocampal neurons. This transport is essential for maintenance of axonal growth-cone dynamics and autophagosome turnover. Our findings illustrate how a general mechanism for lysosome dispersal in nonneuronal cells is adapted to drive polarized transport in neurons, and emphasize the importance of this mechanism for critical axonal processes. PMID:28320970

  13. CNS penetration of intrathecal-lumbar idursulfase in the monkey, dog and mouse: implications for neurological outcomes of lysosomal storage disorder.

    Directory of Open Access Journals (Sweden)

    Pericles Calias

    Full Text Available A major challenge for the treatment of many central nervous system (CNS disorders is the lack of convenient and effective methods for delivering biological agents to the brain. Mucopolysaccharidosis II (Hunter syndrome is a rare inherited lysosomal storage disorder resulting from a deficiency of iduronate-2-sulfatase (I2S. I2S is a large, highly glycosylated enzyme. Intravenous administration is not likely to be an effective therapy for disease-related neurological outcomes that require enzyme access to the brain cells, in particular neurons and oligodendrocytes. We demonstrate that intracerebroventricular and lumbar intrathecal administration of recombinant I2S in dogs and nonhuman primates resulted in widespread enzyme distribution in the brain parenchyma, including remarkable deposition in the lysosomes of both neurons and oligodendrocytes. Lumbar intrathecal administration also resulted in enzyme delivery to the spinal cord, whereas little enzyme was detected there after intraventricular administration. Mucopolysaccharidosis II model is available in mice. Lumbar administration of recombinant I2S to enzyme deficient animals reduced the storage of glycosaminoglycans in both superficial and deep brain tissues, with concurrent morphological improvements. The observed patterns of enzyme transport from cerebrospinal fluid to the CNS tissues and the resultant biological activity (a warrant further investigation of intrathecal delivery of I2S via lumbar catheter as an experimental treatment for the neurological symptoms of Hunter syndrome and (b may have broader implications for CNS treatment with biopharmaceuticals.

  14. Mild MPP+ exposure impairs autophagic degradation through a novel lysosomal acidity-independent mechanism.

    Science.gov (United States)

    Miyara, Masatsugu; Kotake, Yaichiro; Tokunaga, Wataru; Sanoh, Seigo; Ohta, Shigeru

    2016-10-01

    Parkinson's disease (PD) is the second most common neurodegenerative disorder, but its underlying cause remains unknown. Although recent studies using PD-related neurotoxin MPP + suggest autophagy involvement in the pathogenesis of PD, the effect of MPP + on autophagic processes under mild exposure, which mimics the slow progressive nature of PD, remains largely unclear. We examined the effect of mild MPP + exposure (10 and 200 μM for 48 h), which induces a more slowly developing cell death, on autophagic processes and the mechanistic differences with acute MPP + toxicity (2.5 and 5 mM for 24 h). In SH-SY5Y cells, mild MPP + exposure predominantly inhibited autophagosome degradation, whereas acute MPP + exposure inhibited both autophagosome degradation and basal autophagy. Mild MPP + exposure reduced lysosomal hydrolase cathepsin D activity without changing lysosomal acidity, whereas acute exposure decreased lysosomal density. Lysosome biogenesis enhancers trehalose and rapamycin partially alleviated mild MPP + exposure induced impaired autophagosome degradation and cell death, but did not prevent the pathogenic response to acute MPP + exposure, suggesting irreversible lysosomal damage. We demonstrated impaired autophagic degradation by MPP + exposure and mechanistic differences between mild and acute MPP + toxicities. Mild MPP + toxicity impaired autophagosome degradation through novel lysosomal acidity-independent mechanisms. Sustained mild lysosomal damage may contribute to PD. We examined the effects of MPP + on autophagic processes under mild exposure, which mimics the slow progressive nature of Parkinson's disease, in SH-SY5Y cells. This study demonstrated impaired autophagic degradation through a reduction in lysosomal cathepsin D activity without altering lysosomal acidity by mild MPP + exposure. Mechanistic differences between acute and mild MPP + toxicity were also observed. Sustained mild damage of lysosome may be an underlying cause of Parkinson

  15. Incorporation of lysosomal sequestration in the mechanistic model for prediction of tissue distribution of basic drugs.

    Science.gov (United States)

    Assmus, Frauke; Houston, J Brian; Galetin, Aleksandra

    2017-11-15

    The prediction of tissue-to-plasma water partition coefficients (Kpu) from in vitro and in silico data using the tissue-composition based model (Rodgers & Rowland, J Pharm Sci. 2005, 94(6):1237-48.) is well established. However, distribution of basic drugs, in particular into lysosome-rich lung tissue, tends to be under-predicted by this approach. The aim of this study was to develop an extended mechanistic model for the prediction of Kpu which accounts for lysosomal sequestration and the contribution of different cell types in the tissue of interest. The extended model is based on compound-specific physicochemical properties and tissue composition data to describe drug ionization, distribution into tissue water and drug binding to neutral lipids, neutral phospholipids and acidic phospholipids in tissues, including lysosomes. Physiological data on the types of cells contributing to lung, kidney and liver, their lysosomal content and lysosomal pH were collated from the literature. The predictive power of the extended mechanistic model was evaluated using a dataset of 28 basic drugs (pK a ≥7.8, 17 β-blockers, 11 structurally diverse drugs) for which experimentally determined Kpu data in rat tissue have been reported. Accounting for the lysosomal sequestration in the extended mechanistic model improved the accuracy of Kpu predictions in lung compared to the original Rodgers model (56% drugs within 2-fold or 88% within 3-fold of observed values). Reduction in the extent of Kpu under-prediction was also evident in liver and kidney. However, consideration of lysosomal sequestration increased the occurrence of over-predictions, yielding overall comparable model performances for kidney and liver, with 68% and 54% of Kpu values within 2-fold error, respectively. High lysosomal concentration ratios relative to cytosol (>1000-fold) were predicted for the drugs investigated; the extent differed depending on the lysosomal pH and concentration of acidic phospholipids among

  16. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... Iron-Deficiency Anemia Iron-Deficiency Anemia Also known as Leer en español Iron-deficiency anemia is a ... address the cause of your iron deficiency, such as any underlying bleeding. If undiagnosed or untreated, iron- ...

  17. Liver and Skin Histopathology in Adults with Acid Sphingomyelinase Deficiency (Niemann-Pick Disease Type B)

    OpenAIRE

    Thurberg, Beth L.; Wasserstein, Melissa P.; Schiano, Thomas; O’Brien, Fanny; Richards, Susan; Cox, Gerald F.; McGovern, Margaret M.

    2012-01-01

    Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder characterized by the pathologic accumulation of sphingomyelin in multiple cells types, and occurs most prominently within the liver, spleen and lungs, leading to significant clinical disease. Seventeen ASMD patients underwent a liver biopsy during baseline screening for a Phase 1 trial of recombinant human acid sphingomyelinase (rhASM) in adults with Niemann-Pick disease type B. Eleven of the 17 were enrolled in the trial...

  18. Progranulin acts as a shared chaperone and regulates multiple lysosomal enzymes

    Directory of Open Access Journals (Sweden)

    Jinlong Jian

    2017-09-01

    Full Text Available Multifunctional factor progranulin (PGRN plays an important role in lysosomes, and its mutations and insufficiency are associated with lysosomal storage diseases, including neuronal ceroid lipofuscinosis and Gaucher disease (GD. The first breakthrough in understanding the molecular mechanisms of PGRN as regulator of lysosomal storage diseases came unexpectedly while investigating the role of PGRN in inflammation. Challenged PGRN null mice displayed typical features of GD. In addition, GRN gene variants were identified in GD patients and the serum levels of PGRN were significantly lower in GD patients. PGRN directly binds to and functions as a chaperone of the lysosomal enzyme β-glucocerebrosidase (GCaase, whose mutations cause GD. In addition, its C-terminus containing granulin E domain, termed Pcgin (PGRN C-terminus for GCase Interaction, is required for the association between PGRN and GCase. The concept that PGRN acts as a chaperone of lysosomal enzymes was further supported and extended by a recent article showing that PGRN acts as a chaperone molecule of lysosomal enzyme cathepsin D (CSTD, and the association between PGRN and CSTD is also mediated by PGRN's C-terminal granulin E domain. Collectively, these reports suggest that PGRN may act as a shared chaperone and regulates multiple lysosomal enzymes.

  19. Chlamydia species-dependent differences in the growth requirement for lysosomes.

    Directory of Open Access Journals (Sweden)

    Scot P Ouellette

    2011-03-01

    Full Text Available Genome reduction is a hallmark of obligate intracellular pathogens such as Chlamydia, where adaptation to intracellular growth has resulted in the elimination of genes encoding biosynthetic enzymes. Accordingly, chlamydiae rely heavily on the host cell for nutrients yet their specific source is unclear. Interestingly, chlamydiae grow within a pathogen-defined vacuole that is in close apposition to lysosomes. Metabolically-labeled uninfected host cell proteins were provided as an exogenous nutrient source to chlamydiae-infected cells, and uptake and subsequent labeling of chlamydiae suggested lysosomal degradation as a source of amino acids for the pathogen. Indeed, Bafilomycin A1 (BafA1, an inhibitor of the vacuolar H(+/ATPase that blocks lysosomal acidification and functions, impairs the growth of C. trachomatis and C. pneumoniae, and these effects are especially profound in C. pneumoniae. BafA1 induced the marked accumulation of material within the lysosomal lumen, which was due to the inhibition of proteolytic activities, and this response inhibits chlamydiae rather than changes in lysosomal acidification per se, as cathepsin inhibitors also inhibit the growth of chlamydiae. Finally, the addition of cycloheximide, an inhibitor of eukaryotic protein synthesis, compromises the ability of lysosomal inhibitors to block chlamydial growth, suggesting chlamydiae directly access free amino acids in the host cytosol as a preferred source of these nutrients. Thus, chlamydiae co-opt the functions of lysosomes to acquire essential amino acids.

  20. Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons.

    Science.gov (United States)

    Cai, Qian; Lu, Li; Tian, Jin-Hua; Zhu, Yi-Bing; Qiao, Haifa; Sheng, Zu-Hang

    2010-10-06

    Neuron maintenance and survival require late endocytic transport from distal processes to the soma where lysosomes are predominantly localized. Here, we report a role for Snapin in attaching dynein to late endosomes through its intermediate chain (DIC). snapin(-/-) neurons exhibit aberrant accumulation of immature lysosomes, clustering and impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis due to impaired delivery of internalized proteins and hydrolase precursors from late endosomes to lysosomes, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene, but not the DIC-binding-defective Snapin-L99K mutant. Snapin overexpression in wild-type neurons enhances late endocytic transport and lysosomal function, whereas expressing the mutant defective in Snapin-DIC coupling shows a dominant-negative effect. Altogether, our study highlights new mechanistic insights into how Snapin-DIC coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function, and thus neuronal homeostasis. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Iowa Mutant Apolipoprotein A-I (ApoA-IIowa) Fibrils Target Lysosomes.

    Science.gov (United States)

    Kameyama, Hirokazu; Nakajima, Hiroyuki; Nishitsuji, Kazuchika; Mikawa, Shiho; Uchimura, Kenji; Kobayashi, Norihiro; Okuhira, Keiichiro; Saito, Hiroyuki; Sakashita, Naomi

    2016-07-28

    The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIowa) is the first mutation that was associated with familial AApoA1 amyloidosis. The N-terminal fragments (amino acid residues 1-83) of apoA-I containing this mutation deposit as amyloid fibrils in patients' tissues and organs, but the mechanisms of cellular degradation and cytotoxicity have not yet been clarified. In this study, we demonstrated degradation of apoA-IIowa fibrils via the autophagy-lysosomal pathway in human embryonic kidney 293 cells. ApoA-IIowa fibrils induced an increase in lysosomal pH and the cytosolic release of the toxic lysosomal protease cathepsin B. The mitochondrial dysfunction caused by apoA-IIowa fibrils depended on cathepsin B and was ameliorated by increasing the degradation of apoA-IIowa fibrils. Thus, although apoA-IIowa fibril transport to lysosomes and fibril degradation in lysosomes may have occurred, the presence of an excess number of apoA-IIowa fibrils, more than the lysosomes could degrade, may be detrimental to cells. Our results thus provide evidence that the target of apoA-IIowa fibrils is lysosomes, and we thereby gained a novel insight into the mechanism of AApoA1 amyloidosis.

  2. Vps33B is required for delivery of endocytosed cargo to lysosomes.

    Science.gov (United States)

    Galmes, Romain; ten Brink, Corlinda; Oorschot, Viola; Veenendaal, Tineke; Jonker, Caspar; van der Sluijs, Peter; Klumperman, Judith

    2015-12-01

    Lysosomes are the main degradative compartments of eukaryotic cells. The CORVET and HOPS tethering complexes are well known for their role in membrane fusion in the yeast endocytic pathway. Yeast Vps33p is part of both complexes, and has two mammalian homologues: Vps33A and Vps33B. Vps33B is required for recycling of apical proteins in polarized cells and a causative gene for ARC syndrome. Here, we investigate whether Vps33B is also required in the degradative pathway. By fluorescence and electron microscopy we show that Vps33B depletion in HeLa cells leads to significantly increased numbers of late endosomes that together with lysosomes accumulate in the perinuclear region. Degradation of endocytosed cargo is impaired in these cells. By electron microscopy we show that endocytosed BSA-gold reaches late endosomes, but is decreased in lysosomes. The increase in late endosome numbers and the lack of internalized cargo in lysosomes are indicative for a defect in late endosomal-lysosomal fusion events, which explains the observed decrease in cargo degradation. A corresponding phenotype was found after Vps33A knock down, which in addition also resulted in decreased lysosome numbers. We conclude that Vps33B, in addition to its role in endosomal recycling, is required for late endosomal-lysosomal fusion events. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Activity-dependent trafficking of lysosomes in dendrites and dendritic spines.

    Science.gov (United States)

    Goo, Marisa S; Sancho, Laura; Slepak, Natalia; Boassa, Daniela; Deerinck, Thomas J; Ellisman, Mark H; Bloodgood, Brenda L; Patrick, Gentry N

    2017-08-07

    In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins. © 2017 Goo et al.

  4. The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes

    Science.gov (United States)

    Khatter, Divya; Raina, Vivek B.; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

    2015-01-01

    The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit–subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. PMID:25908847

  5. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    International Nuclear Information System (INIS)

    Swanson, J.; Bushnell, A.; Silverstein, S.C.

    1987-01-01

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of ≅ 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4 0 C or in medium containing 5 μM colchicine or nocodazole at 37 0 C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37 0 C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures

  6. Disturbances in lysosomal enzymes activity in rats, following experimental postradiation disease

    International Nuclear Information System (INIS)

    Drozdz, M.; Piwowarczyk, B.; Olczyk, K.; Pikula-Zachara, M.

    1981-01-01

    The studies were aimed at detecting the biological effects of radiation on rat's organism, through studying the activity of lysosomal enzymes in blood plasma and some organs. The contemporary studies suggest that lysosomes play an important role in the occurrence and course of postradiation disease. The obtained results suggest the multidirectional gamma-rays effects on lysosomal enzymes response in serum, leucocytes, liver lysosomes and in liver, kidneys, lungs, heart. Increased activity of acid phosphatase, beta-glucoronidase and beta-acetyl-glucosaminase in the tissues of irradiated animals indicates that gamma rays labilizate the lysosomal membrane. The range of changes indicates a selective nature of this phenomenon. Kidneys, lungs and liver appeared the most ray-sensitive organs. The activity of acid phosphatase was found to be most increased in blood serum and leucocytes. The activity of all examined enzymes in liver lysosomes was decreased. Acid phosphatase exhibited the greatest activity increases. Lysosomal responses are indicative of the degree of destructive or regenerative changes in the organism. (author)

  7. The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes.

    Science.gov (United States)

    Khatter, Divya; Raina, Vivek B; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

    2015-05-01

    The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit-subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. © 2015. Published by The Company of Biologists Ltd.

  8. Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

    International Nuclear Information System (INIS)

    Haylett, T.; Thilo, L.

    1986-01-01

    Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

  9. Antagonistic control of lysosomal fusion by Rab14 and the Lyst-related protein LvsB

    OpenAIRE

    Kypri, Elena; Falkenstein, Kristin; De Lozanne, Arturo

    2013-01-01

    While loss of the protein Lyst causes abnormal lysosomes in patients with Chediak-Higashi Syndrome, the contribution of Lyst to lysosome biology is not known. Previously we found that the Dictyostelium ortholog of Lyst, LvsB, is a cytosolic protein that associates with lysosomes and post-lysosomes to prevent their inappropriate fusion. Here we provide three lines of evidence that indicate that LvsB contributes to lysosome function by antagonizing the function of DdRab14, a protein that promot...

  10. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    Directory of Open Access Journals (Sweden)

    Christine Burkard

    2014-11-01

    Full Text Available Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs. Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV. Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

  11. Triptolide induces lysosomal-mediated programmed cell death in MCF-7 breast cancer cells

    Directory of Open Access Journals (Sweden)

    Owa C

    2013-09-01

    Full Text Available Chie Owa, Michael E Messina Jr, Reginald HalabyDepartment of Biology, Montclair State University, Montclair, NJ, USABackground: Breast cancer is a major cause of death; in fact, it is the most common type, in order of the number of global deaths, of cancer in women worldwide. This research seeks to investigate how triptolide, an extract from the Chinese herb Tripterygium wilfordii Hook F, induces apoptosis in MCF-7 human breast cancer cells. Accumulating evidence suggests a role for lysosomal proteases in the activation of apoptosis. However, there is also some controversy regarding the direct participation of lysosomal proteases in activation of key apoptosis-related caspases and release of mitochondrial cytochrome c. In the present study, we demonstrate that triptolide induces an atypical, lysosomal-mediated apoptotic cell death in MCF-7 cells because they lack caspase-3.Methods: MCF-7 cell death was characterized via cellular morphology, chromatin condensation, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide colorimetric cell growth inhibition assay and the expression levels of proapoptotic proteins. Acridine orange and LysoTracker® staining were performed to visualize lysosomes. Lysosomal enzymatic activity was monitored using an acid phosphatase assay and western blotting of cathepsin B protein levels in the cytosolic fraction, which showed increased enzymatic activity in drug-treated cells.Results: These experiments suggest that triptolide-treated MCF-7 cells undergo atypical apoptosis and that, during the early stages, lysosomal enzymes leak into the cytosol, indicating lysosomal membrane permeability.Conclusion: Our results suggest that further studies are warranted to investigate triptolide's potential as an anticancer therapeutic agent.Keywords: triptolide, MCF-7 breast cancer cells, apoptosis, lysosomes, lysosomal membrane permeabilization (LMP

  12. Effect of Phosphodiesterase in Regulating the Activity of Lysosomes in the HeLa Cell Line.

    Science.gov (United States)

    Hong, Eun-Seon; Kim, Bit-Na; Kim, Yang-Hoon; Min, Jiho

    2017-02-28

    The transport of lysosomal enzymes into the lysosomes depends on the phosphorylation of their chains and the binding of the phosphorylated residues to mannose-6-phosphate receptors. The efficiency of separation depends more on the phosphodiesterases (PDEs) than on the activity of the phosphorylation of mannose residues and can be determined in vitro. PDEs play important roles in regulation of the activation of lysosomes. The expression of proteins was confirmed by western blotting. All PDE4 series protein expression was reduced in high concentrations of rolipram. As a result of observing the fluorescence intensity after rolipram treatment, the lysosomal enzyme was activated at low concentrations and suppressed at high concentrations. High concentrations of rolipram recovered the original function. Antimicrobial activity was not shown in either 10 or 100 µ concentrations of rolipram in treated HeLa cells in vitro. However, the higher anticancer activity at lower rolipram concentration was shown in lysosomal enzyme treated with 10 µ of rolipram. The anticancer activity was confirmed through cathepsin B and D assay. Tranfection allowed examination of the relationship between PDE4 and lysosomal activity in more detail. Protein expression was confirmed to be reduced. Fluorescence intensity showed decreased activity of lysosomes and ROS in cells transfected with the antisense sequences of PDE4 A, B, C, and D. PDE4A showed anticancer activity, whereas lysosome from cells transfected with the antisense sequences of PDE4 B, C, and D had decreased anticancer activity. These results showed the PDE4 A, B, C, and D are conjunctly related with lysosomal activity.

  13. Positive lysosomal modulation as a unique strategy to treat age-related protein accumulation diseases.

    Science.gov (United States)

    Bahr, Ben A; Wisniewski, Meagan L; Butler, David

    2012-04-01

    Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ(1-38) peptide corresponded with decreased levels of Aβ(1-42), supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders to enhance

  14. Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GSDII KO mice

    NARCIS (Netherlands)

    A.G.A. Bijvoet (Agnes); M.A. Kroos (Marian); F.R. Pieper (Frank); M. Van der Vliet (Martin); H.A. de Boer (Herman); A.T. van der Ploeg (Ans); M.Ph. Verbeet (Martin); A.J.J. Reuser (Arnold)

    1998-01-01

    textabstractGlycogen storage disease type II (GSDII) is caused by lysosomal acid alpha-glucosidase deficiency. Patients have a rapidly fatal or slowly progressive impairment of muscle function. Enzyme replacement therapy is under investigation. For large-scale, cost-effective

  15. Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis

    Energy Technology Data Exchange (ETDEWEB)

    Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan, E-mail: ionan.marigomez@ehu.es

    2014-04-01

    Highlights: • Thermal stress and Cd caused lysosomal enlargement and membrane destabilisation. • hex, gusb and ctsl but not hsp70 were up-regulated at elevated temperature but down-regulated by Cd. • Thermal stress influenced lysosomal responses to Cd exposure. • The presence of Cd jeopardised responsiveness against thermal stress. - Abstract: In estuaries and coastal areas, intertidal organisms may be subject to thermal stress resulting from global warming, together with pollution. In the present study, the combined effects of thermal stress and exposure to Cd were investigated in the endo-lysosomal system of digestive cells in mussels, Mytilus galloprovincialis. Mussels were maintained for 24 h at 18 °C and 26 °C seawater temperature in absence and presence of 50 μg Cd/L seawater. Cadmium accumulation in digestive gland tissue, lysosomal structural changes and membrane stability were determined. Semi-quantitative PCR was applied to reveal the changes elicited by the different experimental conditions in hexosaminidase (hex), β-glucuronidase (gusb), cathepsin L (ctsl) and heat shock protein 70 (hsp70) gene transcription levels. Thermal stress provoked lysosomal enlargement whilst Cd-exposure led to fusion of lysosomes. Both thermal stress and Cd-exposure caused lysosomal membrane destabilisation. hex, gusb and ctsl genes but not hsp70 gene were transcriptionally up-regulated as a result of thermal stress. In contrast, all the studied genes were transcriptionally down-regulated in response to Cd-exposure. Cd bioaccumulation was comparable at 18 °C and 26 °C seawater temperatures but interactions between thermal stress and Cd-exposure were remarkable both in lysosomal biomarkers and in gene transcription. hex, gusb and ctsl genes, reacted to elevated temperature in absence of Cd but not in Cd-exposed mussels. Therefore, thermal stress resulting from global warming might influence the use and interpretation of lysosomal biomarkers in marine pollution

  16. Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space.

    Science.gov (United States)

    Coppens, Isabelle; Dunn, Joe Dan; Romano, Julia D; Pypaert, Marc; Zhang, Hui; Boothroyd, John C; Joiner, Keith A

    2006-04-21

    The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.

  17. Lipid Involvement in Neurodegenerative Diseases of the Motor System: Insights from Lysosomal Storage Diseases.

    Science.gov (United States)

    Dodge, James C

    2017-01-01

    Lysosomal storage diseases (LSDs) are a heterogeneous group of rare inherited metabolic diseases that are frequently triggered by the accumulation of lipids inside organelles of the endosomal-autophagic-lysosomal system (EALS). There is now a growing realization that disrupted lysosomal homeostasis (i.e., lysosomal cacostasis) also contributes to more common neurodegenerative disorders such as Parkinson disease (PD). Lipid deposition within the EALS may also participate in the pathogenesis of some additional neurodegenerative diseases of the motor system. Here, I will highlight the lipid abnormalities and clinical manifestations that are common to LSDs and several diseases of the motor system, including amyotrophic lateral sclerosis (ALS), atypical forms of spinal muscular atrophy, Charcot-Marie-Tooth disease (CMT), hereditary spastic paraplegia (HSP), multiple system atrophy (MSA), PD and spinocerebellar ataxia (SCA). Elucidating the underlying basis of intracellular lipid mislocalization as well as its consequences in each of these disorders will likely provide innovative targets for therapeutic research.

  18. The clinical spectrum and pathophysiology of skeletal complications in lysosomal storage disorders

    NARCIS (Netherlands)

    Clarke, Lorne A.; Hollak, Carla E. M.

    2015-01-01

    Lysosomal storage disorders affect multiple organs including the skeleton. Disorders with prominent skeletal symptoms are type 1 and 3 Gaucher disease, the mucopolysaccharidoses, the glycoproteinoses and pycnodysostosis. Clinical manifestations range from asymptomatic radiographical evidence of bone

  19. SEASONAL VARIATION IN LYSOSOMAL DESTABILIZATION IN OYSTERS, CRASSOSTREA VIRGINICA. (R826201)

    Science.gov (United States)

    Lysosomal destabilization assays have been used as valuable biomarkers of pollutant exposures in a variety of bivalve and fish species. The responses of oysters, Crassostrea virginica, deployed at and native to various reference and degraded sites were evaluated for lys...

  20. Efficient routing of glucocerebrosidase to lysosomes requires complex oligosaccharide chain formation

    NARCIS (Netherlands)

    Aerts, J. M.; Brul, S.; Donker-Koopman, W. E.; van Weely, S.; Murray, G. J.; Barranger, J. A.; Tager, J. M.; Schram, A. W.

    1986-01-01

    The biosynthesis and intracellular transport of the membrane-associated lysosomal enzyme glucocerebrosidase was studied in the monoblast cell line U937. Addition to the cultures of the oligosaccharide trimming inhibitors swainsonine or deoxymannojirimycin led to an increased intracellular activity

  1. A new fluorescent pH probe for imaging lysosomes in living cells.

    Science.gov (United States)

    Lv, Hong-Shui; Huang, Shu-Ya; Xu, Yu; Dai, Xi; Miao, Jun-Ying; Zhao, Bao-Xiang

    2014-01-15

    A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5-4.1 with the pKa value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no 'alkalizing effect' on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. The endo-lysosomal system of brain endothelial cells is influenced by astrocytes in vitro

    DEFF Research Database (Denmark)

    Toth, Andrea E; Siupka, Piotr; P Augustine, Thomas J

    2018-01-01

    Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes......, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture....... Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain....

  3. Lysosomal membrane permeabilization in cell death: new evidence and implications for health and disease.

    Science.gov (United States)

    Serrano-Puebla, Ana; Boya, Patricia

    2016-05-01

    Recent studies have demonstrated that, in addition to their central role in cellular catabolic reactions, lysosomes are implicated in many cellular processes, including metabolism, membrane repair, and cell death. Lysosomal membrane permeabilization (LMP) has emerged as a pathway by which cell demise is regulated under physiological conditions and contributes to cell death in many pathological situations. Here, we review the latest evidence on LMP-mediated cell death, the upstream and downstream signals involved, and the role of LMP in the normal physiology of organisms. We also discuss the contributions of lysosomal damage and LMP to the pathogenic features of several disease states, such as lysosomal storage disorders and other neurodegenerative conditions. © 2015 New York Academy of Sciences.

  4. Frontotemporal dementia caused by CHMP2B mutation is characterised by neuronal lysosomal storage pathology

    DEFF Research Database (Denmark)

    Clayton, Emma L.; Mizielinska, Sarah; Edgar, James R.

    2015-01-01

    Mutations in the charged multivesicular body protein 2B (CHMP2B) cause frontotemporal dementia (FTD). We report that mice which express FTD-causative mutant CHMP2B at physiological levels develop a novel lysosomal storage pathology characterised by large neuronal autofluorescent aggregates...... in human CHMP2B mutation brain than in neurodegenerative disease or age-matched control brains. These data suggest that lysosomal storage pathology is the major neuronal pathology in FTD caused by CHMP2B mutation. Recent evidence suggests that two other genes associated with FTD, GRN and TMEM106B...... are important for lysosomal function. Our identification of lysosomal storage pathology in FTD caused by CHMP2B mutation now provides evidence that endolysosomal dysfunction is a major degenerative pathway in FTD....

  5. Dysregulation of Nutrient Sensing and CLEARance in Presenilin Deficiency

    Directory of Open Access Journals (Sweden)

    Kavya Reddy

    2016-03-01

    Full Text Available Attenuated auto-lysosomal system has been associated with Alzheimer disease (AD, yet all underlying molecular mechanisms leading to this impairment are unknown. We show that the amino acid sensing of mechanistic target of rapamycin complex 1 (mTORC1 is dysregulated in cells deficient in presenilin, a protein associated with AD. In these cells, mTORC1 is constitutively tethered to lysosomal membranes, unresponsive to starvation, and inhibitory to TFEB-mediated clearance due to a reduction in Sestrin2 expression. Normalization of Sestrin2 levels through overexpression or elevation of nuclear calcium rescued mTORC1 tethering and initiated clearance. While CLEAR network attenuation in vivo results in buildup of amyloid, phospho-Tau, and neurodegeneration, presenilin-knockout fibroblasts and iPSC-derived AD human neurons fail to effectively initiate autophagy. These results propose an altered mechanism for nutrient sensing in presenilin deficiency and underline an importance of clearance pathways in the onset of AD.

  6. Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex

    Energy Technology Data Exchange (ETDEWEB)

    Hostetler, K.Y.; Gardner, M.F.; Giordano, J.R.

    1986-10-21

    Phospholipase A has been isolated from a crude lysosomal fraction from rat kidney cortex and purified 7600-fold with a recovery of 9.8% of the starting activity. The purified enzyme is a glycoprotein having an isoelectric point of pH 5.4 and an apparent molecular weight of 30,000 by high-pressure liquid chromatography gel permeation. Naturally occurring inhibitors of lysosomal phospholipase A are present in two of the lysosomal-soluble protein fractions obtained in the purification. They inhibit hydrolysis of 1,2-di(1-/sup 14/C)oleoylphosphatidylcholine by purified phospholipase A/sub 1/ with IC/sub 50/ values of 7-11 ..mu..g. The inhibition is abolished by preincubation with trypsin at 37/sup 0/C, but preincubation with trypsin at 4/sup 0/C has no effect, providing evidence that the inhibitors are proteins. The results suggest that the activity of lysosomal phospholipase A may be regulated in part by inhibitory proteins. Lysosomal phospholipase A from rat kidney hydrolyzes the sn-1 acyl group of phosphatidylcholine, does not require divalent cations for full activity, and is not inhibited by ethylenediaminetetraacetic acid. It has an acid pH optimum of 3.6-3.8. Neither rho-bromophenacyl bromide, diisopropyl fluorophosphate, nor mercuric ion inhibits phospholipase A/sub 1/. In contrast to rat liver, which has two major isoenzymes of acid phospholipase A/sub 1/, kidney cortex has only one isoenzyme of lysosomal phospholipase A/sub 1/.

  7. Comparative study on lysosomal accumulation of 67Ga and 111In in Morris hepatoma 7316A

    International Nuclear Information System (INIS)

    Takeda, S.; Uchida, T.; Matsuzawa, T.

    1977-01-01

    Intracellular localization of 67 Ga and 111 In was investigated in Morris hepatoma 7316A and in normal Buffalo rat liver cells by a cell fractionation method at 48 hr after an intraperitoneal injection of the nuclides. Lysosomal fractions of the tumor and normal liver cells had the highest relative specific radioactivities of the nuclides (p 67 Ga (p 67 Ga seemed to indicate that 67 Ga determines lysosomal functions of tumor cells more precisely than 111 In

  8. Trapping of oxidized LDL in lysosomes of Kupffer cells is a trigger for hepatic inflammation.

    Science.gov (United States)

    Bieghs, Veerle; Walenbergh, Sofie M A; Hendrikx, Tim; van Gorp, Patrick J; Verheyen, Fons; Olde Damink, Steven W; Masclee, Ad A; Koek, Ger H; Hofker, Marten H; Binder, Christoph J; Shiri-Sverdlov, Ronit

    2013-08-01

    Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation. The transition from steatosis towards NASH represents a key step in pathogenesis, as it will set the stage for further severe liver damage. Under normal conditions, lipoproteins that are endocytosed by Kupffer cells (KCs) are easily transferred from the lysosomes into the cytoplasm. Oxidized LDL (oxLDL) that is taken up by the macrophages in vitro is trapped within the lysosomes, while acetylated LDL (acLDL) is leading to normal lysosomal hydrolysis, resulting in cytoplasmic storage. We have recently demonstrated that hepatic inflammation is correlated with lysosomal trapping of lipids. So far, a link between lysosomal trapping of oxLDL and inflammation was not established. We hypothesized that lysosomal trapping of oxLDL in KCs will lead to hepatic inflammation. Ldlr(-/-) mice were injected with LDL, acLDL and oxLDL and sacrificed after 2, 6 and 24 h. Electron microscopy of KCs demonstrated that after oxLDL injection, small lipid inclusions were present inside the lysosomes after all time points and were mostly pronounced after 6 and 24 h. In contrast, no lipid inclusions were present inside KCs after LDL or acLDL injection. Hepatic expression of several inflammatory genes and scavenger receptors was higher after oxLDL injections compared with LDL or acLDL. These data suggest that trapping of oxLDL inside lysosomes of KCs in vivo is causally linked to increased hepatic inflammatory gene expression. Our novel observations provide new bases for prevention and treatment of NASH. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. EGFRvIII escapes down-regulation due to impaired internalization and sorting to lysosomes

    DEFF Research Database (Denmark)

    Grandal, Michael V; Zandi, Roza; Pedersen, Mikkel W

    2007-01-01

    . Moreover, internalized EGFRvIII is recycled rather than delivered to lysosomes. EGFRvIII binds the ubiquitin ligase c-Cbl via Grb2, whereas binding via phosphorylated tyrosine residue 1045 seems to be limited. Despite c-Cbl binding, the receptor fails to become effectively ubiquitinylated. Thus, our...... results suggest that the long lifetime of EGFRvIII is caused by inefficient internalization and impaired sorting to lysosomes due to lack of effective ubiquitinylation....

  10. The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization.

    Science.gov (United States)

    Stamelos, Vasileios A; Fisher, Natalie; Bamrah, Harnoor; Voisey, Carolyn; Price, Joshua C; Farrell, William E; Redman, Charles W; Richardson, Alan

    2016-01-01

    Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal

  11. Two-Photon Probes for Lysosomes and Mitochondria: Simultaneous Detection of Lysosomes and Mitochondria in Live Tissues by Dual-Color Two-Photon Microscopy Imaging.

    Science.gov (United States)

    Lim, Chang Su; Hong, Seung Taek; Ryu, Seong Shick; Kang, Dong Eun; Cho, Bong Rae

    2015-10-01

    Novel two-photon (TP) probes were developed for lysosomes (PLT-yellow) and mitochondria (BMT-blue and PMT-yellow). These probes emitted strong TP-excited fluorescence in cells at widely separated wavelength regions and displayed high organelle selectivity, good cell permeability, low cytotoxicity, and pH insensitivity. The BMT-blue and PLT-yellow probes could be utilized to detect lysosomes and mitochondria simultaneously in live tissues by using dual-color two-photon microscopy, with minimum interference from each other. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... fatigue or tiredness, shortness of breath, or chest pain. If your doctor diagnoses you with iron-deficiency ... Common symptoms of iron-deficiency anemia include: Chest pain Coldness in the hands and feet Difficulty concentrating ...

  13. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... heart failure . Increased risk of infections Motor or cognitive development delays in children Pregnancy complications, such as ... for iron-deficiency anemia. Learn about exciting research areas that NHLBI is exploring about iron-deficiency anemia. ...

  14. Factor VII deficiency

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000548.htm Factor VII deficiency To use the sharing features on this page, please enable JavaScript. Factor VII (seven) deficiency is a disorder caused by a ...

  15. Iron-Deficiency Anemia

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    Full Text Available ... deficiency anemia can cause serious complications, including heart failure and development delays in children. Explore this Health ... lead to iron-deficiency anemia include: End-stage kidney failure, where there is blood loss during dialysis. ...

  16. Iron-Deficiency Anemia

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    Full Text Available ... for iron-deficiency anemia. Lifestyle habits Certain lifestyle habits may increase your risk for iron-deficiency anemia, including: Vegetarian or vegan eating patterns. Not eating enough iron-rich foods, such ...

  17. Iron-Deficiency Anemia

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    Full Text Available ... deficiency anemia can cause serious complications, including heart failure and development delays in children. Explore this Health ... to iron-deficiency anemia include: End-stage kidney failure, where there is blood loss during dialysis. People ...

  18. Iron-Deficiency Anemia

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    Full Text Available ... view the colon directly. What if my doctor thinks something else is causing my iron-deficiency anemia? ... deficiency anemia early in life affects later behavior, thinking, and mood during adolescence. Treating anemia in premature ...

  19. Iron-Deficiency Anemia

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    Full Text Available ... mg and women need 18 mg. After age 51, both men and women need 8 mg. Pregnant ... for iron-deficiency anemia. Learn about exciting research areas that NHLBI is exploring about iron-deficiency anemia. ...

  20. Iron-Deficiency Anemia

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    Full Text Available ... iron-deficiency anemia. These conditions include: Intestinal and digestive conditions, such as celiac disease; inflammatory bowel diseases, ... iron-deficiency anemia , such as bleeding in the digestive or urinary tract or heavy menstrual bleeding, your ...

  1. Iron-Deficiency Anemia

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    Full Text Available ... conditions that can cause iron-deficiency anemia. Blood tests to screen for iron-deficiency anemia To screen ... the size of your liver and spleen. Blood tests Based on results from blood tests to screen ...

  2. Iron-Deficiency Anemia

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    Full Text Available ... from developing iron-deficiency anemia. Foods that are good sources of iron include dried beans, dried fruits, eggs, lean red meat, ... signs of iron-deficiency anemia include: Brittle nails ...

  3. Iron-Deficiency Anemia

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    Full Text Available ... your doctor may recommend you eat heart-healthy foods or control other conditions that can cause iron-deficiency anemia. Blood tests to screen for iron-deficiency anemia To screen ...

  4. Fire Safety Deficiencies

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    U.S. Department of Health & Human Services — A list of all fire safety deficiencies currently listed on Nursing Home Compare, including the nursing home that received the deficiency, the associated inspection...

  5. Iron-Deficiency Anemia

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    Full Text Available ... learning how having iron-deficiency anemia early in life affects later behavior, thinking, and mood during adolescence. ... iron-deficiency anemia in blood donors affects the quality of donated red blood cells, such as how ...

  6. Iron-Deficiency Anemia

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    Full Text Available ... iron-deficiency anemia in blood donors affects the quality of donated red blood cells, such as how ... Cells From Iron-deficient Donors: Recovery and Storage Quality. Learn more about participating in a clinical trial . ...

  7. Iron-Deficiency Anemia

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    Full Text Available ... leaving cells where it is stored or from being absorbed in the duodenum, the first part of ... treatments for iron-deficiency anemia. Living With After being diagnosed with iron-deficiency anemia, it is important ...

  8. Lysosome-associated hypertrophic cardiomyopathy (Danon's disease in two siblings

    Directory of Open Access Journals (Sweden)

    I. V. Leontyeva

    2015-01-01

    Full Text Available The paper presents a clinical observation of two siblings with Danon's disease (lysosome-associated cardiomyopathy verified by genetic examination. Heart lesion in Danon's disease bears a phenotypic similarity to the primary forms of hypertrophic cardiomyopathy; in this connection the correct etiology of the disease has remained long unestablished. The presence of laboratory markers as the significantly raised levels of transaminases, creatine phosphokinase, and lactate dehydrogenase was as a guide for suspecting the metabolic origin of the disease. Two siblings with a similar LAMP gene mutation were observed to have a different clinical course: a severer clinical course of cardiomyopathy with extreme myocardial hypertrophy, myocardial electric instability, and mental development retardation in one case and a more favorable course in the other; although a 2-year follow-up also revealed negative changes. For the prevention of sudden cardiac death, a cardioverter defibrulator was implanted and continuous therapy with p-adrenoblockers was performed. The specific feature of the cases was no symptoms of skeletal myopathy, moderate mental retardation only in the elder brother, no evidence of an accessory atrioventricular junction despite the fact that there were ECG manifestations of Wolff-Parkinson-White syndrome

  9. Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    Maria Francisca Coutinho

    2016-07-01

    Full Text Available Lysosomal storage diseases (LSDs are a group of rare, life-threatening genetic disorders, usually caused by a dysfunction in one of the many enzymes responsible for intralysosomal digestion. Even though no cure is available for any LSD, a few treatment strategies do exist. Traditionally, efforts have been mainly targeting the functional loss of the enzyme, by injection of a recombinant formulation, in a process called enzyme replacement therapy (ERT, with no impact on neuropathology. This ineffectiveness, together with its high cost and lifelong dependence is amongst the main reasons why additional therapeutic approaches are being (and have to be investigated: chaperone therapy; gene enhancement; gene therapy; and, alternatively, substrate reduction therapy (SRT, whose aim is to prevent storage not by correcting the original enzymatic defect but, instead, by decreasing the levels of biosynthesis of the accumulating substrate(s. Here we review the concept of substrate reduction, highlighting the major breakthroughs in the field and discussing the future of SRT, not only as a monotherapy but also, especially, as complementary approach for LSDs.

  10. Impulse control disorder, lysosomal malfunction and ATP13A2 insufficiency in Parkinsonism.

    Science.gov (United States)

    Liu, Jun-Ping; Li, Jianfeng; Lu, Yanhua; Wang, Lihui; Chen, Gang

    2017-02-01

    Lysosomal transport of cargos in neurons is essential for neuronal proteostasis, transmission and functional motors and behaviours. Lysosomal malfunction including storage disorders is involved in the pathogenesis of Parkinson's disease (PD). Given the unclear molecular mechanisms of diverse defects in PD phenotypes, especially behavioural deficits, this mini review explores the cellular contexts of PD impulse control disorders and the molecular aspects of lysosomal cross-membrane transports. Focuses are paid to trace metal involvements in α-synuclein assembly in Lewy bodies, the functions and molecular interactions of ATP13A2 as ATPase transporters in lysosomal membranes for cross-membrane trafficking and lysosomal homeostasis, and our current understandings of the neural circuits in ICD. Erroneously polarized distributions of cargos such as metals and lipids on each side of lysosomal membranes triggered by gene mutations and deregulated expression of ATP13A2 may thus instigate sensing protein structural changes such as aggregations, organelle degeneration, and specific neuronal ageing and death in Parkinsonism. © 2016 John Wiley & Sons Australia, Ltd.

  11. Comprehensive proteome analysis of lysosomes reveals the diverse function of macrophages in immune responses.

    Science.gov (United States)

    Gao, Yanpan; Chen, Yanyu; Zhan, Shaohua; Zhang, Wenhao; Xiong, Feng; Ge, Wei

    2017-01-31

    Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses.

  12. TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes.

    Science.gov (United States)

    Hennigar, Stephen R; Kelleher, Shannon L

    2015-10-01

    Mammary epithelial cells undergo widespread lysosomal-mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor-α (TNFα), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study is to determine how TNFα retargets ZnT2 to lysosomes. We tested the hypothesis that TNFα signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C-terminus of ZnT2, allowing adaptor protein complex-3 (AP-3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNFα redistributed wild-type (WT) ZnT2 from late endosomes (Pearson's coefficient = 0.202 ± 0.05 and 0.097 ± 0.03; Plysosomes (0.292 ± 0.03 and 0.649 ± 0.03; Plysosomal Zn (Plysosomes, increase lysosomal Zn, or activate LCD. Moreover, TNFα increased (Plysosomes and activate LCD. Our findings suggest that women with variation in the C-terminus of ZnT2 may be at risk for inadequate involution and breast disease due the inability to traffic ZnT2 to lysosomes. © 2015 Wiley Periodicals, Inc.

  13. [Changes in active cysteine cathepsins in lysosomes from tissues thyroid papillary carcinomas with various biological characteristics].

    Science.gov (United States)

    Kalinichenko, O V; Myshunina, T M; Tron'ko, M D

    2013-01-01

    To clarify possible role of cysteine cathepsin H, B and L in the proteolytic processes that contribute to the progression of tumor growth in the thyroid, we studied their activity in lysosomes isolated from the tissue of papillary carcinomas. It was shown that for these enzymes there is a dependence of the changes in their activity on a number of biological characteristics of the tumors. Thus, the sharp increase in the activity ofcathepsin H observed in lysosomes of tissue carcinomas category T2 and T3, with intra-and ekstrathyroid and lymphatic invasion of tumor cells. An increase in the activity of cathepsin B is set in the lysosomes of tissue heterogeneous follicular structure, especially in the presence of solid areas, in comparison with typical papillary tumors and in the lysosomes of tissue carcinomas in intrathyroid and cathepsin L-at extrathyroid invasion. A common feature of the enzymes is to increase the activity of cathepsins in lysosomes of tissue nonencapsulated papillary carcinomas. These enzymes probably do not take part in the invasion of tumor cells into blood vessels and in the mechanisms of tumor metastasis to regional lymph nodes. The latter shows no changes in the activity of cathepsins in lysosomes of tissue carcinomas category N1. The results indicate the different role of cathepsin H, B and L in thyroid carcinogenesis, where each enzyme has its specific function.

  14. The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes.

    Science.gov (United States)

    Marwaha, Rituraj; Arya, Subhash B; Jagga, Divya; Kaur, Harmeet; Tuli, Amit; Sharma, Mahak

    2017-04-03

    Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion. © 2017 Marwaha et al.

  15. ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes.

    Science.gov (United States)

    Liu, Jun; Liu, Wenjing; Yang, Jun

    2016-02-11

    We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1-3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca(2+)-dependent lysosomal exocytosis.

  16. PIKfyve mediates the motility of late endosomes and lysosomes in neuronal dendrites.

    Science.gov (United States)

    Tsuruta, Fuminori; Dolmetsch, Ricardo E

    2015-09-25

    The endosome/lysosome system in the nervous system is critically important for a variety of neuronal functions such as neurite outgrowth, retrograde transport, and synaptic plasticity. In neurons, the endosome/lysosome system is crucial for the activity-dependent internalization of membrane proteins and contributes to the regulation of lipid level on the plasma membrane. Although homeostasis of membrane dynamics plays important roles in the properties of central nervous systems, it has not been elucidated how endosome/lysosome system is regulated. Here, we report that phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) mediates the motility of late endosomes and lysosomes in neuronal dendrites. Endosomes and lysosomes are highly motile in resting neurons, however knockdown of PIKfyve led to a significant reduction in late endosomes and lysosomes motility. We also found that vesicle acidification is crucial for their motility and PIKfyve is associated with this process indirectly. These data suggest that PIKfyve mediates vesicle motility through the regulation of vesicle integrity in neurons. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Effects of ambroxol on the autophagy-lysosome pathway and mitochondria in primary cortical neurons.

    Science.gov (United States)

    Magalhaes, J; Gegg, M E; Migdalska-Richards, A; Schapira, A H

    2018-01-23

    Glucocerebrosidase (GBA1) mutations are the major genetic risk factor for Parkinson's Disease (PD). The pathogenic mechanism is still unclear, but alterations in lysosomal-autophagy processes are implicated due to reduction of mutated glucocerebrosidase (GCase) in lysosomes. Wild-type GCase activity is also decreased in sporadic PD brains. Small molecule chaperones that increase lysosomal GCase activity have potential to be disease-modifying therapies for GBA1-associated and sporadic PD. Therefore we have used mouse cortical neurons to explore the effects of the chaperone ambroxol. This chaperone increased wild-type GCase mRNA, protein levels and activity, as well as increasing other lysosomal enzymes and LIMP2, the GCase transporter. Transcription factor EB (TFEB), the master regulator of the CLEAR pathway involved in lysosomal biogenesis was also increased upon ambroxol treatment. Moreover, we found macroautophagy flux blocked and exocytosis increased in neurons treated with ambroxol. We suggest that ambroxol is blocking autophagy and driving cargo towards the secretory pathway. Mitochondria content was also found to be increased by ambroxol via peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α). Our data suggest that ambroxol, besides being a GCase chaperone, also acts on other pathways, such as mitochondria, lysosomal biogenesis, and the secretory pathway.

  18. Uptake and degradation of cytoplasmic RNA by lysosomes in the perfused rat liver

    International Nuclear Information System (INIS)

    Heydrick, S.J.; Lardeux, B.; Mortimore, G.E.

    1987-01-01

    The release of [ 14 C]cytidine has been shown previously to be a valid marker for RNA degradation in rat hepatocytes. The breakdown of RNA measured with this marker in perfused livers prelabeled in vivo with [6- 14 C]orotic acid was found to be regulated acutely by perfusate amino acids over a wide range, from 0.29 to 3.48%/h. This regulation paralleled that of lysosomal proteolysis. Chloroquine inhibited RNA degradation 60-70%. In subsequent cell fractionation studies labelled cytidine was released; the distribution of this release paralleled that of a lysosomal marker enzyme. The release plateaued after two hours, defining a distinct lysosomal pool of RNA. The lysosomal location of the RNA pool was confirmed in experiments where a 22% increase in the apparent pool size was obtained by lowering the homogenate pH from 7.0 to 5.5. The pool size correlated linearly with the rate of RNA degradation measured during perfusion, giving a turnover constant in reasonable agreement with values reported for autophagy. These results indicate that cytoplasmic RNA degradation occurs primarily in the lysosome and is regulated under these conditions by the amino acid control of lysosomal sequestration of cytoplasm

  19. Activation of lysosomal enzymes and tumour regression caused by irradiation and steroid hormones

    International Nuclear Information System (INIS)

    Ball, A.; Barratt, G.M.; Wills, E.D.

    1982-01-01

    The lysosomal enzyme activity and membrane permeability of mouse C3H mammary tumours has been studied using quantitative cytochemical methods following irradiation of the tumours with doses of 1500, 3500 or 6000 rad ν rays. No change in the lysosomal enzyme activity was observed immediately after irradiation, but increased enzyme activity and increased membrane permeability were observed 24 hr after irradiation with doses of 3500 or 6000 rad. Twenty-four hours after injection of prednisolone there was a marked increase of lysosomal membrane permeability and enzyme activity, and injection of prednisolone soon after irradiation enhanced the effect of irradiation. After a dose of 6000 rad and prednisolone, the lysosomal membrane permeability increased to 191% of the control and the enzyme activity to 326% of the value of the control tumours. Measurement of tumour size after irradiation or after a combined treatment with irradiation and prednisolone showed that a close correlation exists between tumour regression and lysosomal enzyme activity. The experiments support the view that lysosomal enzymes play an important role in tumour regression following irradiation. (author)

  20. Detection of Misdistribution of Tyrosinase from Melanosomes to Lysosomes and Its Upregulation under Psoralen/Ultraviolet A with a Melanosome-Targeting Tyrosinase Fluorescent Probe.

    Science.gov (United States)

    Zhou, Jin; Shi, Wen; Li, Lihong; Gong, Qiuyu; Wu, Xiaofeng; Li, Xiaohua; Ma, Huimin

    2016-04-19

    Tyrosinase is regarded as an important biomarker of melanoma cancer, and its metabolism is closely related to some severe skin diseases such as vitiligo. Since tyrosinase is mainly located in the melanosomes of melanocytes, a probe that can specifically detect and image tysosinase in melanosomes would be in urgent demand to study the behavior of the enzyme in cells, but unfortunately, no melanosome-targeting tyrosinase fluorescent probe has been reported so far to the best of our knowledge. In this work, we have developed such a new probe, Mela-TYR, which bears morpholine as a melanosome-targeting group and 4-aminophenol as a tyrosinase reaction group. The probe exhibits not only a highly sensitive and selective off-on response to tyrosinase via oxidization cleavage, but also an accurate targeting ability toward the acidic organelles of melanosomes and lyososomes, which is validated by colocalization experiments with mCherry-tagged melanosomes as well as DND-99 (a commercial dye). The probe has been used to image the relative contents of tyrosinase in different cells. Notably, because of the tyrosinase deficiency in normal lysosomes, the probe only fluoresces in melanosomes in principle although it can accumulate in other acidic organelles like lysosomes. By virtue of this property, the misdistribution of tyrosinase from melanosomes to lysosomes in murine melanoma B16 cells under the stimulation of inulavosin is imaged in real time for the first time. Moreover, the upregulation of melanosomal tyrosinase in live B16 cells under the stimulation of psoralen/ultraviolet A is detected with our probe, and this upregulation is further verified by standard colorimetric assay. The probe provides a simple, visual method to study the metabolism of tyrosinase in cells and shows great potential in clinical diagnosis and treatments of tyrosinase-associated diseases.

  1. Autophagy fails to prevent glucose deprivation/glucose reintroduction-induced neuronal death due to calpain-mediated lysosomal dysfunction in cortical neurons.

    Science.gov (United States)

    Gerónimo-Olvera, Cristian; Montiel, Teresa; Rincon-Heredia, Ruth; Castro-Obregón, Susana; Massieu, Lourdes

    2017-06-29

    Autophagy is triggered during nutrient and energy deprivation in a variety of cells as a homeostatic response to metabolic stress. In the CNS, deficient autophagy has been implicated in neurodegenerative diseases and ischemic brain injury. However, its role in hypoglycemic damage is poorly understood and the dynamics of autophagy during the hypoglycemic and the glucose reperfusion periods, has not been fully described. In the present study, we analyzed the changes in the content of the autophagy proteins BECN1, LC3-II and p62/SQSTM1 by western blot, and autophagosome formation was followed through time-lapse experiments, during glucose deprivation (GD) and glucose reintroduction (GR) in cortical cultures. According to the results, autophagosome formation rapidly increased during GD, and was followed by an active autophagic flux early after glucose replenishment. However, cells progressively died during GR and autophagy inhibition reduced neuronal death. Neurons undergoing apoptosis during GR did not form autophagosomes, while those surviving up to late GR showed autophagosomes. Calpain activity strongly increased during GR and remained elevated during progressive neuronal death. Its activation led to the cleavage of LAMP2 resulting in lysosome membrane permeabilization (LMP) and release of cathepsin B to the cytosol. Calpain inhibition prevented LMP and increased the number of neurons containing lysosomes and autophagosomes increasing cell viability. Taken together, the present results suggest that calpain-mediated lysosome dysfunction during GR turns an adaptive autophagy response to energy stress into a defective autophagy pathway, which contributes to neuronal death. In these conditions, autophagy inhibition results in the improvement of cell survival.

  2. TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Magini, Alessandro [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Polchi, Alice; Urbanelli, Lorena [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Cesselli, Daniela; Beltrami, Antonio [Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Tancini, Brunella [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Emiliani, Carla, E-mail: carla.emiliani@unipg.it [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy)

    2013-10-18

    Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.

  3. TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

    International Nuclear Information System (INIS)

    Magini, Alessandro; Polchi, Alice; Urbanelli, Lorena; Cesselli, Daniela; Beltrami, Antonio; Tancini, Brunella; Emiliani, Carla

    2013-01-01

    Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane

  4. A fluorescence resonance energy transfer-based approach for investigating late endosome–lysosome retrograde fusion events

    Science.gov (United States)

    Kaufmann, A.M.; Goldman, S.D.B.; Krise, J.P.

    2009-01-01

    Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome–lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders—Niemann–Pick type C, mucolipidosis type IV, and Sandhoff’s disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies. PMID:19109922

  5. A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events.

    Science.gov (United States)

    Kaufmann, A M; Goldman, S D B; Krise, J P

    2009-03-01

    Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders-Niemann-Pick type C, mucolipidosis type IV, and Sandhoff's disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.

  6. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... if you are diagnosed with iron-deficiency anemia. Risk Factors You may have an increased risk for iron-deficiency anemia because of your age, ... or sex. Age You may be at increased risk for iron deficiency at certain ages: Infants between ...

  7. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... Home / < Back To Health Topics / Iron-Deficiency Anemia Iron-Deficiency Anemia Also known as Leer en español ... bleeding Consuming less than recommended daily amounts of iron Iron-deficiency anemia can be caused by getting ...

  8. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... Topics News & Resources Intramural Research Home / < Back To Health Topics / Iron-Deficiency Anemia Iron-Deficiency Anemia Also known as Leer ... and symptoms as well as complications from iron-deficiency anemia. Research for Your Health The NHLBI is part of the U.S. Department ...

  9. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... anemia, your doctor may order the following blood tests to diagnose iron-deficiency anemia: Complete blood count (CBC) to ... than normal when viewed under a microscope. Different tests help your doctor diagnose iron-deficiency anemia. In iron-deficiency anemia, blood ...

  10. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... for iron-deficiency anemia if you have certain risk factors , including pregnancy. To prevent iron-deficiency anemia, your doctor may recommend you eat heart-healthy foods or control other conditions that can cause iron-deficiency anemia. ...

  11. Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

    Directory of Open Access Journals (Sweden)

    Bárbara Hissa

    Full Text Available BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages and non-professional (epithelial phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of

  12. Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II.

    Science.gov (United States)

    Markmann, Sandra; Krambeck, Svenja; Hughes, Christopher J; Mirzaian, Mina; Aerts, Johannes M F G; Saftig, Paul; Schweizer, Michaela; Vissers, Johannes P C; Braulke, Thomas; Damme, Markus

    2017-03-01

    The efficient receptor-mediated targeting of soluble lysosomal proteins to lysosomes requires the modification with mannose 6-phosphate (M6P) residues. Although the absence of M6P results in misrouting and hypersecretion of lysosomal enzymes in many cells, normal levels of lysosomal enzymes have been reported in liver of patients lacking the M6P-generating phosphotransferase (PT). The identity of lysosomal proteins depending on M6P has not yet been comprehensively analyzed. In this study we purified lysosomes from liver of PT-defective mice and 67 known soluble lysosomal proteins were identified that illustrated quantitative changes using an ion mobility-assisted data-independent label-free LC-MS approach. After validation of various differentially expressed lysosomal components by Western blotting and enzyme activity assays, the data revealed a small number of lysosomal proteins depending on M6P, including neuraminidase 1, cathepsin F, Npc2, and cathepsin L, whereas the majority reach lysosomes by alternative pathways. These data were compared with findings on cultured hepatocytes and liver sinusoid endothelial cells isolated from the liver of wild-type and PT-defective mice. Our findings show that the relative expression, targeting efficiency and lysosomal localization of lysosomal proteins tested in cultured hepatic cells resemble their proportion in isolated liver lysosomes. Hypersecretion of newly synthesized nonphosphorylated lysosomal proteins suggest that secretion-recapture mechanisms contribute to maintain major lysosomal functions in liver. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Carnitine Deficiency and Pregnancy

    Directory of Open Access Journals (Sweden)

    Anouk de Bruyn

    2015-01-01

    Full Text Available We present two cases of carnitine deficiency in pregnancy. In our first case, systematic screening revealed L-carnitine deficiency in the first born of an asymptomatic mother. In the course of her second pregnancy, maternal carnitine levels showed a deficiency as well. In a second case, a mother known with carnitine deficiency under supplementation was followed throughout her pregnancy. Both pregnancies had an uneventful outcome. Because carnitine deficiency can have serious complications, supplementation with carnitine is advised. This supplementation should be continued throughout pregnancy according to plasma concentrations.

  14. Determination of glucose deficiency-induced cell death by mitochondrial ATP generation-driven proton homeostasis

    Institute of Scientific and Technical Information of China (English)

    Yanfen Cui; Yuanyuan Wang; Miao Liu; Li Qiu; Pan Xing; Xin Wang; Guoguang Ying; Binghui Li

    2017-01-01

    Glucose is one of major nutrients and its catabolism provides energy and/or building bricks for cell proliferation.Glucose deficiency results in cell death.However,the underlying mechanism still remains elusive.By using our recently developed method to monitor real-time cellular apoptosis and necrosis,we show that glucose deprivation can directly elicit necrosis,which is promoted by mitochondrial impairment,depending on mitochondrial adenosine triphosphate (ATP) generation instead of ATP depletion.We demonstrate that glucose metabolism is the major source to produce protons.Glucose deficiency leads to lack of proton provision while mitochondrial electron transfer chain continues consuming protons to generate energy,which provokes a compensatory iysosomal proton effiux and resultant increased lysosomal pH.This lysosomal alkalinization can trigger apoptosis or necrosis depending on the extent of alkalinization.Taken together,our results build up a metabolic connection between glycolysis,mitochondrion,and lysosome,and reveal an essential role of glucose metabolism in maintaining proton homeostasis to support cell survival.

  15. Lysosomal abnormalities in hereditary spastic paraplegia types SPG15 and SPG11

    Science.gov (United States)

    Renvoisé, Benoît; Chang, Jaerak; Singh, Rajat; Yonekawa, Sayuri; FitzGibbon, Edmond J; Mankodi, Ami; Vanderver, Adeline; Schindler, Alice B; Toro, Camilo; Gahl, William A; Mahuran, Don J; Blackstone, Craig; Pierson, Tyler Mark

    2014-01-01

    Objective Hereditary spastic paraplegias (HSPs) are among the most genetically diverse inherited neurological disorders, with over 70 disease loci identified (SPG1-71) to date. SPG15 and SPG11 are clinically similar, autosomal recessive disorders characterized by progressive spastic paraplegia along with thin corpus callosum, white matter abnormalities, cognitive impairment, and ophthalmologic abnormalities. Furthermore, both have been linked to early-onset parkinsonism. Methods We describe two new cases of SPG15 and investigate cellular changes in SPG15 and SPG11 patient-derived fibroblasts, seeking to identify shared pathogenic themes. Cells were evaluated for any abnormalities in cell division, DNA repair, endoplasmic reticulum, endosomes, and lysosomes. Results Fibroblasts prepared from patients with SPG15 have selective enlargement of LAMP1-positive structures, and they consistently exhibited abnormal lysosomal storage by electron microscopy. A similar enlargement of LAMP1-positive structures was also observed in cells from multiple SPG11 patients, though prominent abnormal lysosomal storage was not evident. The stabilities of the SPG15 protein spastizin/ZFYVE26 and the SPG11 protein spatacsin were interdependent. Interpretation Emerging studies implicating these two proteins in interactions with the late endosomal/lysosomal adaptor protein complex AP-5 are consistent with shared abnormalities in lysosomes, supporting a converging mechanism for these two disorders. Recent work with Zfyve26−/− mice revealed a similar phenotype to human SPG15, and cells in these mice had endolysosomal abnormalities. SPG15 and SPG11 are particularly notable among HSPs because they can also present with juvenile parkinsonism, and this lysosomal trafficking or storage defect may be relevant for other forms of parkinsonism associated with lysosomal dysfunction. PMID:24999486

  16. Ethambutol-induced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells

    International Nuclear Information System (INIS)

    Chung, Hyewon; Yoon, Young Hee; Hwang, Jung Jin; Cho, Kyung Sook; Koh, Jae Young; Kim, June-Gone

    2009-01-01

    Ethambutol, an efficacious antituberculosis agent, can cause irreversible visual loss in a small but significant fraction of patients. However, the mechanism of ocular toxicity remains to be established. We previously reported that ethambutol caused severe vacuole formation in cultured retinal cells, and that the addition of zinc along with ethambutol aggravated vacuole formation whereas addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), reduced vacuole formation. To investigate the origin of vacuoles and to obtain an understanding of drug toxicity, we used cultured primary retinal cells from newborn Sprague-Dawley rats and imaged ethambutol-treated cells stained with FluoZin-3, zinc-specific fluorescent dye, under a confocal microscope. Almost all ethambutol-induced vacuoles contained high levels of labile zinc. Double staining with LysoTracker or MitoTracker revealed that almost all zinc-containing vacuoles were lysosomes and not mitochondria. Intracellular zinc chelation with TPEN markedly blocked both vacuole formation and zinc accumulation in the vacuole. Immunocytochemistry with antibodies to lysosomal-associated membrane protein-2 (LAMP-2) and cathepsin D, an acid lysosomal hydrolase, disclosed lysosomal activation after exposure to ethambutol. Immunoblotting after 12 h exposure to ethambutol showed that cathepsin D was released into the cytosol. In addition, cathepsin inhibitors attenuated retinal cell toxicity induced by ethambutol. This is consistent with characteristics of lysosomal membrane permeabilization (LMP). TPEN also inhibited both lysosomal activation and LMP. Thus, accumulation of zinc in lysosomes, and eventual LMP, may be a key mechanism of ethambutol-induced retinal cell death

  17. Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

    DEFF Research Database (Denmark)

    Zheng, Lin; Terman, Alexei; Hallbeck, Martin

    2011-01-01

    and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production...... and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration....

  18. VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy.

    Science.gov (United States)

    Papadopoulos, Chrisovalantis; Kirchner, Philipp; Bug, Monika; Grum, Daniel; Koerver, Lisa; Schulze, Nina; Poehler, Robert; Dressler, Alina; Fengler, Sven; Arhzaouy, Khalid; Lux, Vanda; Ehrmann, Michael; Weihl, Conrad C; Meyer, Hemmo

    2017-01-17

    Rupture of endosomes and lysosomes is a major cellular stress condition leading to cell death and degeneration. Here, we identified an essential role for the ubiquitin-directed AAA-ATPase, p97, in the clearance of damaged lysosomes by autophagy. Upon damage, p97 translocates to lysosomes and there cooperates with a distinct set of cofactors including UBXD1, PLAA, and the deubiquitinating enzyme YOD1, which we term ELDR components for Endo-Lysosomal Damage Response. Together, they act downstream of K63-linked ubiquitination and p62 recruitment, and selectively remove K48-linked ubiquitin conjugates from a subpopulation of damaged lysosomes to promote autophagosome formation. Lysosomal clearance is also compromised in MEFs harboring a p97 mutation that causes inclusion body myopathy and neurodegeneration, and damaged lysosomes accumulate in affected patient tissue carrying the mutation. Moreover, we show that p97 helps clear late endosomes/lysosomes ruptured by endocytosed tau fibrils. Thus, our data reveal an important mechanism of how p97 maintains lysosomal homeostasis, and implicate the pathway as a modulator of degenerative diseases. © 2016 The Authors.

  19. Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes.

    Science.gov (United States)

    Starling, Georgina P; Yip, Yan Y; Sanger, Anneri; Morton, Penny E; Eden, Emily R; Dodding, Mark P

    2016-06-01

    The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt-Hoge-Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri-nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi-associated small GTPase Rab34. Rab34-positive peri-nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34-induced peri-nuclear lysosome clustering. FLCN interacts directly via its C-terminal DENN domain with the Rab34 effector RILP Using purified recombinant proteins, we show that the FLCN-DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP We propose a model whereby starvation-induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34-positive peri-nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  20. Permeation of lysosomal membranes in the course of photo-sensitization with methylene blue and hematoporphyrin: study by cellular microspectrofluorometry

    International Nuclear Information System (INIS)

    Santus, R.; Kohen, C.; Kohen, E.; Morliere, P.; Dubertret, L.; Tocci, P.M.

    1983-01-01

    The photodynamically-induced liberation of lysosomal enzymes using β-galactosidase as marker for the lysosomal enzymes has been studied by microspectrofluorometry on mouse L cells. Similar studies have been carried out using N-acetyl-β-glucosaminidase as marker for the lysosomal enzymes of human fibroblasts. The high sensitivity of the fluorescence detection makes it possible to use 4-methylumbelliferyl substrates for the enzymes contained in a single cell. Methylene blue and hematoporphyrin readily incorporate into both cells and upon excitation, sensitize lysosomal membrane damages, leading to enzyme release accompanying strong morphological changes. (author)

  1. Cathepsin E deficiency impairs autophagic proteolysis in macrophages.

    Directory of Open Access Journals (Sweden)

    Takayuki Tsukuba

    Full Text Available Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE(-/- mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE(-/- macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE(-/- macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR, Akt, and extracellular signal-related kinase (ERK. Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE(-/- macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE(-/- cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE(-/- macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE(-/- macrophages showed increased reactive oxygen species (ROS production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.

  2. Lysosome destruction and lipoperoxide formation due to active oxygen generated from haematoporphyrin and UV irradiation

    International Nuclear Information System (INIS)

    Torinuki, W.; Miura, T.; Seiji, M.

    1980-01-01

    The lysosomal enzymes, acid-phosphates and β-glucuronidase, were released from rat liver lysosome when exposed to 400 nm irradiation in the presence of haematoporphyrin and the release was prevented by adding vitamin E, diazabicyclo-octane, bovine serum albumin, superoxide dismutase or D-mannitol to the reaction mixture. Monochromatic irradiation with wavelengths from 380 to 410 nm caused no significant differences in the release of lysosomal enzymes, but 420 nm irradiation caused three-fifths of that of 400 nm irradiation. The malondialdeyhde level in rat liver homogenate increased after 400 nm irradiation in the presence of haematoporphyrin Reduction of nitroblue-tetrazolium was not observed when haematoporphyrin was excited by 400 nm; it was considered that superoxide anion radical (0 2 - ) was not primarily generated. The following mechanism was assumed; that porphyrin which had been excited by 400 nm, converted ground-state molecular oxygen ( 3 0 2 ) to excited singlet oxygen ( 1 0 2 ), which formed lipid peroxides in lysosomal membrane resulting in destruction of the membrane; skin changes would occur from these released lysosomal enzymes. (author)

  3. Disruption of lysosome function promotes tumor growth and metastasis in Drosophila.

    Science.gov (United States)

    Chi, Congwu; Zhu, Huanhu; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2010-07-09

    Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the Ras(V12) cells. Knocking down either of the two other components of the Class C VPS complex, carnation (car) and vps16A, also renders Ras(V12) cells capable for uncontrolled growth and metastatic behavior. Finally, chemical disruption of the lysosomal function by feeding animals with antimalarial drugs, chloroquine or monensin, leads to malignant tumor growth of the Ras(V12) cells. Taken together, our data provide evidence for a causative role of lysosome dysfunction in tumor growth and invasion and indicate that members of the Class C VPS complex behave as tumor suppressors.

  4. Color reduction of melanin by lysosomal and peroxisomal enzymes isolated from mammalian cells.

    Science.gov (United States)

    Park, Dong Jun; Sekhon, Simranjeet Singh; Yoon, Jihee; Kim, Yang-Hoon; Min, Jiho

    2016-02-01

    Lysosomes and peroxisomes are organelles with many functions in all eukaryotic cells. Lysosomes contain hydrolytic enzymes (lysozyme) that degrade molecules, whereas peroxisomes contain enzymes such as catalase that convert hydrogen peroxide (H2O2) to water and oxygen and neutralize toxicity. In contrast, melanin is known as a helpful element to protect the skin against harmful ultraviolet rays. However, a high quantity of melanin leads to hyperpigmentation or skin cancer in human. New materials have already been discovered to inhibit tyrosinase in melanogenesis; however, melanin reduction does not suggest their preparation. In this study, we report that the color intensity because of melanin decreased by the cellular activation of lysosomes and peroxisomes. An increase in the superficial intensity of lysosome and peroxisome activities of HeLa cells was observed. In addition, a decrease in the amount of melanin has also been observed in mammalian cells without using any other chemical, showing that the process can work in vivo for treating melanin. Therefore, the results of this study indicate that the amount of melanin decreases by the lysosome and peroxisome activity after entering the cells, and functional organelles are effective in color reduction. This mechanism can be used in vivo for treating melanin.

  5. Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes

    Science.gov (United States)

    Caviston, Juliane P.; Zajac, Allison L.; Tokito, Mariko; Holzbaur, Erika L.F.

    2011-01-01

    Huntingtin (Htt) is a membrane-associated scaffolding protein that interacts with microtubule motors as well as actin-associated adaptor molecules. We examined a role for Htt in the dynein-mediated intracellular trafficking of endosomes and lysosomes. In HeLa cells depleted of either Htt or dynein, early, recycling, and late endosomes (LE)/lysosomes all become dispersed. Despite altered organelle localization, kinetic assays indicate only minor defects in intracellular trafficking. Expression of full-length Htt is required to restore organelle localization in Htt-depleted cells, supporting a role for Htt as a scaffold that promotes functional interactions along its length. In dynein-depleted cells, LE/lysosomes accumulate in tight patches near the cortex, apparently enmeshed by cortactin-positive actin filaments; Latrunculin B-treatment disperses these patches. Peripheral LE/lysosomes in dynein-depleted cells no longer colocalize with microtubules. Htt may be required for this off-loading, as the loss of microtubule association is not seen in Htt-depleted cells or in cells depleted of both dynein and Htt. Inhibition of kinesin-1 relocalizes peripheral LE/lysosomes induced by Htt depletion but not by dynein depletion, consistent with their detachment from microtubules upon dynein knockdown. Together, these data support a model of Htt as a facilitator of dynein-mediated trafficking that may regulate the cytoskeletal association of dynamic organelles. PMID:21169558

  6. Activity-Dependent Exocytosis of Lysosomes Regulates the Structural Plasticity of Dendritic Spines.

    Science.gov (United States)

    Padamsey, Zahid; McGuinness, Lindsay; Bardo, Scott J; Reinhart, Marcia; Tong, Rudi; Hedegaard, Anne; Hart, Michael L; Emptage, Nigel J

    2017-01-04

    Lysosomes have traditionally been viewed as degradative organelles, although a growing body of evidence suggests that they can function as Ca 2+ stores. Here we examined the function of these stores in hippocampal pyramidal neurons. We found that back-propagating action potentials (bpAPs) could elicit Ca 2+ release from lysosomes in the dendrites. This Ca 2+ release triggered the fusion of lysosomes with the plasma membrane, resulting in the release of Cathepsin B. Cathepsin B increased the activity of matrix metalloproteinase 9 (MMP-9), an enzyme involved in extracellular matrix (ECM) remodelling and synaptic plasticity. Inhibition of either lysosomal Ca 2+ signaling or Cathepsin B release prevented the maintenance of dendritic spine growth induced by Hebbian activity. This impairment could be rescued by exogenous application of active MMP-9. Our findings suggest that activity-dependent exocytosis of Cathepsin B from lysosomes regulates the long-term structural plasticity of dendritic spines by triggering MMP-9 activation and ECM remodelling. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  7. Distribution of Cathepsin D Activity between Lysosomes and a Soluble Fraction of Marinating Brine.

    Science.gov (United States)

    Szymczak, Mariusz

    2016-08-01

    This paper is the first ever to describe the phenomenon of bimodal distribution of cathepsin D in the lysosomal and soluble fractions of brine left after herring marinating. Up to 2 times higher cathepsin D activity was observed in the lysosome fraction. Activity of cathepsin D in brine increased according to the logarithmic function during low frequency-high power ultrasounds treatment or according to the linear function after multiple freezing-thawing of brine. Activity enhancement was achieved only in the brine devoid of lipids and suspension. Study results show also that measurement of lysosomal cathepsin D activity in the marinating brine requires also determining cathepsin E activity. Decreasing pore size of microfilter from 2.7 to 0.3 μm significantly reduced the lysosome content in the brine. The presence of lysosomes and the possibility of their separation as well as the likely release of cathepsins shall be considered during industrial application of the marinating brine, as new cathepsins preparations in fish and meat technology. © 2016 Institute of Food Technologists®

  8. Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning.

    Science.gov (United States)

    Dodson, Mark W; Zhang, Ting; Jiang, Changan; Chen, Shengdi; Guo, Ming

    2012-03-15

    LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. Here, we show that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2(G2019S) allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD.

  9. The lysosome among targets of metformin: new anti-inflammatory uses for an old drug?

    Science.gov (United States)

    Lockwood, Thomas D

    2010-05-01

    Rheumatoid arthritis and type-2 diabetes exhibit progressive co-morbidity. Chloroquine (CQ) reportedly improves both. CQ inhibits lysosomal function in cultured cells at supra-therapeutic concentration; however, this is doubted as target mechanism. Some anti-diabetic biguanides are metal-interactive lysosomal inhibitors; and all bind Zn(2+). i) To bioassay the potency of CQ using (3)H-leucine release from perfused myocardial tissue. ii) To determine whether metformin (MET) is CQ-mimetic, and interactive with Zn(2+). Therapeutic CQ concentration (0.1 - 0.5 microM) clearly does cause lysosomal inhibition although delayed and submaximal. MET alone (10 microM) caused sub-maximal inhibition. Supra-physiological extracellular Zn(2+) (5 - 50 microM) alone increased tissue Zn(2+) content, and inhibited lysosomal proteolysis. Physiological equivalent Zn(2+) (approximately 1 microM) had no effect. MET (use as an anti-inflammatory agent are suggested. Guanidylguanidine is a practical pharmacophore for synthesis of future anti-lysosomal agents.

  10. A lysosomal switch triggers proteostasis renewal in the immortal C. elegans germ lineage.

    Science.gov (United States)

    Bohnert, K Adam; Kenyon, Cynthia

    2017-11-30

    Although individuals age and die with time, an animal species can continue indefinitely, because of its immortal germ-cell lineage. How the germline avoids transmitting damage from one generation to the next remains a fundamental question in biology. Here we identify a lysosomal switch that enhances germline proteostasis before fertilization. We find that Caenorhabditis elegans oocytes whose maturation is arrested by the absence of sperm exhibit hallmarks of proteostasis collapse, including protein aggregation. Remarkably, sperm-secreted hormones re-establish oocyte proteostasis once fertilization becomes imminent. Key to this restoration is activation of the vacuolar H + -ATPase (V-ATPase), a proton pump that acidifies lysosomes. Sperm stimulate V-ATPase activity in oocytes by signalling the degradation of GLD-1, a translational repressor that blocks V-ATPase synthesis. Activated lysosomes, in turn, promote a metabolic shift that mobilizes protein aggregates for degradation, and reset proteostasis by enveloping and clearing the aggregates. Lysosome acidification also occurs during Xenopus oocyte maturation; thus, a lysosomal switch that enhances oocyte proteostasis in anticipation of fertilization may be conserved in other species.

  11. Not nanocarbon but dispersant induced abnormality in lysosome in macrophages in vivo

    Science.gov (United States)

    Yudasaka, Masako; Zhang, Minfang; Matsumura, Sachiko; Yuge, Ryota; Ichihashi, Toshinari; Irie, Hiroshi; Shiba, Kiyotaka; Iijima, Sumio

    2015-05-01

    The properties of nanocarbons change from hydrophobic to hydrophilic as a result of coating them with dispersants, typically phospholipid polyethylene glycols, for biological studies. It has been shown that the dispersants remain attached to the nanocarbons when they are injected in mice and influence the nanocarbons’ biodistribution in vivo. We show in this report that the effects of dispersants also appear at the subcellular level in vivo. Carbon nanohorns (CNHs), a type of nanocarbon, were dispersed with ceramide polyethylene glycol (CPEG) and intravenously injected in mice. Histological observations and electron microscopy with energy dispersive x-ray analysis revealed that, in liver and spleen, the lysosome membranes were damaged, and the nanohorns formed a complex with hemosiderin in the lysosomes of the macrophages. It is inferred that the lysosomal membrane was damaged by sphigosine generated as a result of CPEG decomposition, which changed the intra lysosomal conditions, inducing the formation of the CPEG-CNH and hemosiderin complex. For comparison, when glucose was used instead of CPEG, neither the nanohorn-hemosiderin complex nor lysosomal membrane damage was found. Our results suggest that surface functionalization can control the behavior of nancarbons in cells in vivo and thereby improve their suitability for medical applications.

  12. Not nanocarbon but dispersant induced abnormality in lysosome in macrophages in vivo

    International Nuclear Information System (INIS)

    Yudasaka, Masako; Zhang, Minfang; Iijima, Sumio; Matsumura, Sachiko; Shiba, Kiyotaka; Yuge, Ryota; Ichihashi, Toshinari; Irie, Hiroshi

    2015-01-01

    The properties of nanocarbons change from hydrophobic to hydrophilic as a result of coating them with dispersants, typically phospholipid polyethylene glycols, for biological studies. It has been shown that the dispersants remain attached to the nanocarbons when they are injected in mice and influence the nanocarbons’ biodistribution in vivo. We show in this report that the effects of dispersants also appear at the subcellular level in vivo. Carbon nanohorns (CNHs), a type of nanocarbon, were dispersed with ceramide polyethylene glycol (CPEG) and intravenously injected in mice. Histological observations and electron microscopy with energy dispersive x-ray analysis revealed that, in liver and spleen, the lysosome membranes were damaged, and the nanohorns formed a complex with hemosiderin in the lysosomes of the macrophages. It is inferred that the lysosomal membrane was damaged by sphigosine generated as a result of CPEG decomposition, which changed the intra lysosomal conditions, inducing the formation of the CPEG-CNH and hemosiderin complex. For comparison, when glucose was used instead of CPEG, neither the nanohorn–hemosiderin complex nor lysosomal membrane damage was found. Our results suggest that surface functionalization can control the behavior of nancarbons in cells in vivo and thereby improve their suitability for medical applications. (paper)

  13. Burn-induced stimulation of lysosomal enzyme synthesis in skeletal muscle

    International Nuclear Information System (INIS)

    Odessey, R.

    1986-01-01

    A localized burn injury to a rat hindlimb results in atrophy of soleus muscle (in the absence of cellular damage) which is attributable to an increase in muscle protein breakdown. Previous work has shown that lysosomal enzyme activities (cathepsins B, H, L, and D) are elevated in muscle from the burned leg by 50% to 100%. There is no change in endogenous neutral protease activity (+/- Ca ++ ). The increase in protease activity can not be attributed to changes in endogenous protease inhibitors. The latency [(Triton X100 treated - control)/triton treated] of lysosomal enzymes is approximately 50% and is not altered by burn injury. The rate of sucrose uptake is also not altered by burn. These experiments suggest that the rate of substrate supply to the lysosomal apparatus via endocytosis or autophagocytosis is not altered by burn. When muscles are preincubated with 3 H-phenylalanine or 3 H-mannose burn increased incorporation into protein of the fraction containing lysosomes by 100%. Preincubation in the presence of tunicamycin (an inhibitor of glycoprotein synthesis) inhibited incorporation of both labels into a microsomal fraction of the muscle from the burned leg, but has little effect on incorporation in the control muscle. These findings are consistent with the hypothesis that the burn-induced increase in protein breakdown is caused by an increase in lysosomal protease synthesis

  14. The influence of gamma radiation on catheptic activity and on ultrastructure of lysosomes and postmortem skeletal muscle of poultry Gallus domesticus

    International Nuclear Information System (INIS)

    Ali, Mumtaz.

    1975-01-01

    A three-part study is presented dealing with radiation-induced release of cathepsins from isolated lysosomes, irradiation inactivation of cathepsins, and ultrastructural changes in irradiated lysosomes and skeletal muscle. After chicken liver lysosomes were irradiated with 0.1 to 1.0 Mrad of gamma radiation a decrease in absorbance at 540 nm of lysosomal suspensions and an increase of free enzyme activity due to a release of cathepsins were noted. Examination of irradiated isolated lysosomes by electron microscopy showed leakage of material from weak points in the lysosomal membrane. Examination of irradiated chicken pectoralis muscle revealed an increase in interfibrillar spaces and some breaks in the myofibres. (LL)

  15. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... increased need for iron during growth spurts. Older adults, especially those over age ... athletes. Athletes, especially young females, are at risk for iron deficiency. Endurance ...

  16. Iodine deficiency disorders

    Energy Technology Data Exchange (ETDEWEB)

    Ali, S M [Pakistan Council for Science and Technology, Islamabad (Pakistan)

    1994-12-31

    Iodine deficiency (IDD) is one of the common problem in the diet. Iodine deficiency as prevalence of goiter in population occurs in the mountainous areas. There is consensus that 800 million people are at risk of IDD from living in iodine deficient area and 190 million from goiter. Very high prevalence of IDD in different parts of the world are striking. It has generally observed that in iodine-deficient areas about 50% are affected with goiter, 1-5% from cretinsim and 20% from impaired mental and/or mortor function. (A.B.).

  17. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) programs. Our ... more information about Donor Iron Deficiency Study - Red Blood Cells ...

  18. A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis.

    Directory of Open Access Journals (Sweden)

    Xuejun Tian

    2015-03-01

    Full Text Available Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj, causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.

  19. Presence of the propeptide on recombinant lysosomal dipeptidase controls both activation and dimerization.

    Science.gov (United States)

    Dolenc, Iztok; Pain, Roger; Turk, Vito

    2007-01-01

    Lysosomal dipeptidase catalyzes the hydrolysis of dipeptides with unsubstituted terminals. It is a homodimer and binds zinc. Dimerization is an important issue in understanding the enzyme's function. In this study, we investigated the influence of the propeptide on the folding and dimerization of recombinant lysosomal dipeptidase. For this purpose, we separately cloned and overexpressed the mature protein and the proenzyme. The overexpressed proteins were localized exclusively to insoluble inclusion bodies. Refolding of the urea-solubilized inclusion bodies showed that only dipeptidase lacking the propeptide was dimeric. The soluble renatured proenzyme was a monomer, although circular dichroism and fluorescence spectra of the proenzyme indicated the formation of secondary and tertiary structure. The propeptide thus controls dimerization, as well as activation, of lysosomal dipeptidase.

  20. Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes.

    Science.gov (United States)

    Abounit, Saïda; Bousset, Luc; Loria, Frida; Zhu, Seng; de Chaumont, Fabrice; Pieri, Laura; Olivo-Marin, Jean-Christophe; Melki, Ronald; Zurzolo, Chiara

    2016-10-04

    Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that α-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α-synuclein fibrils are able to seed soluble α-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies. © 2016 The Authors.

  1. Fluorescence methods for analysis of interactions between Ca(2+) signaling, lysosomes, and endoplasmic reticulum.

    Science.gov (United States)

    Prole, David L; López-Sanjurjo, Cristina I; Tovey, Stephen C; Taylor, Colin W

    2015-01-01

    The endoplasmic reticulum (ER) is both the major source of intracellular Ca(2+) for cell signaling and the organelle that forms the most extensive contacts with the plasma membrane and other organelles. Lysosomes fulfill important roles in degrading cellular materials and in cholesterol handling, but they also contribute to Ca(2+) signaling by both releasing and sequestering Ca(2+). Interactions between ER and other Ca(2+)-transporting membranes, notably mitochondria and the plasma membrane, often occur at sites where the two membranes are closely apposed, allowing local Ca(2+) signaling between them. These interactions are often facilitated by scaffold proteins. Recent evidence suggests similar local interactions between ER and lysosomes. We describe simple fluorescence-based methods that allow the interplay between Ca(2+) signals, the ER, and lysosomes to be examined. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Effects of heavy water on ultrastructural and functional status of Hep 2 and CHO cells lysosomes

    International Nuclear Information System (INIS)

    Buzgariu, Wanda; Caloianu, Maria; Zarnescu, Otilia; Cimpean, Anisoara; Titescu, Gh.; Stefanescu, I.

    2002-01-01

    The heavy water effects on the ultrastructure and function of Hep 2 and CHO lysosomal cell compartment were investigated using electron microscopy and enzymatic studies. The cell viability, measured by neutral red uptake assay, and the total protein content determination, have shown a dose dependent decrease in cell growth for both studied cell types. The electron microscopy study has revealed a progressive increase in number and size of lysosomes and autophagosomes after 96 h exposure to different deuterium concentration media in a dose dependent manner. The enzymatic determination in the lysosomal pellet revealed an increased acid phosphatase activity in both cell types (15% and 33% for Hep 2 and 24% and 52% for CHO, respectively) exposed to media with high (65%, 90%) D 2 O content. (authors)

  3. Rapid recycling of Ca2+ between IP3-sensitive stores and lysosomes.

    Directory of Open Access Journals (Sweden)

    Cristina I López Sanjurjo

    Full Text Available Inositol 1,4,5-trisphosphate (IP3 evokes release of Ca2+ from the endoplasmic reticulum (ER, but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

  4. Rapid recycling of Ca2+ between IP3-sensitive stores and lysosomes.

    Science.gov (United States)

    López Sanjurjo, Cristina I; Tovey, Stephen C; Taylor, Colin W

    2014-01-01

    Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

  5. Altered lysosome distribution is an early neuropathological event in neurological forms of Gaucher disease.

    Science.gov (United States)

    Zigdon, Hila; Meshcheriakova, Anna; Farfel-Becker, Tamar; Volpert, Giora; Sabanay, Helena; Futerman, Anthony H

    2017-03-01

    In the lysosomal storage disorder Gaucher disease (GD), glucosylceramide (GlcCer) accumulates due to the defective activity of glucocerebrosidase. A subset of GD patients develops neuropathology. We now show mislocalization of Limp2-positive puncta and a large reduction in the number of Lamp1-positive puncta, which are associated with impaired tubulin. These changes occur at an early stage in animal models of GD, prior to development of overt symptoms and considerably earlier than neuronal loss. Altered lysosomal localization and cytoskeleton disruption precede the neuroinflammatory pathways, axonal dystrophy and neuronal loss previously characterized in neuronal forms of GD. © 2017 Federation of European Biochemical Societies.

  6. SUMO-1 is associated with a subset of lysosomes in glial protein aggregate diseases.

    Science.gov (United States)

    Wong, Mathew B; Goodwin, Jacob; Norazit, Anwar; Meedeniya, Adrian C B; Richter-Landsberg, Christiane; Gai, Wei Ping; Pountney, Dean L

    2013-01-01

    Oligodendroglial inclusion bodies characterize a subset of neurodegenerative diseases. Multiple system atrophy (MSA) is characterized by α-synuclein glial cytoplasmic inclusions and progressive supranuclear palsy (PSP) is associated with glial tau inclusions. The ubiquitin homologue, SUMO-1, has been identified in inclusion bodies in MSA, located in discrete sub-domains in α-synuclein-positive inclusions. We investigated SUMO-1 associated with oligodendroglial inclusion bodies in brain tissue from MSA and PSP and in glial cell models. We examined MSA and PSP cases and compared to age-matched normal controls. Fluorescence immunohistochemistry revealed frequent SUMO-1 sub-domains within and surrounding inclusions bodies in both diseases and showed punctate co-localization of SUMO-1 and the lysosomal marker, cathepsin D, in affected brain regions. Cell counting data revealed that 70-75 % of lysosomes in inclusion body-positive oligodendrocytes were SUMO-1-positive consistently across MSA and PSP cases, compared to 20 % in neighbouring inclusion body negative oligodendrocytes and 10 % in normal brain tissue. Hsp90 co-localized with some SUMO-1 puncta. We examined the SUMO-1 status of lysosomes in 1321N1 human glioma cells over-expressing α-synuclein and in immortalized rat oligodendrocyte cells over-expressing the four repeat form of tau following treatment with the proteasome inhibitor, MG132. We also transfected 1321N1 cells with the inherently aggregation-prone huntingtin exon 1 mutant, HttQ74-GFP. Each cell model showed the association of SUMO-1-positive lysosomes around focal cytoplasmic accumulations of α-synuclein, tau or HttQ74-GFP, respectively. Association of SUMO-1 with lysosomes was also detected in glial cells bearing α-synuclein aggregates in a rotenone-lesioned rat model. SUMO-1 labelling of lysosomes showed a major increase between 24 and 48 h post-incubation of 1321N1 cells with MG132 resulting in an increase in a 90 kDa SUMO-1-positive band

  7. Effects of the lysosomal destabilizing drug siramesine on glioblastoma in vitro and in vivo

    DEFF Research Database (Denmark)

    Jensen, Stine S.; Asferg Petterson, Stine; Halle, Bo

    2017-01-01

    confirmed by immunohistochemical staining of histological sections of spheroids, spheroids in brain slice cultures and tumors in mice brains. Results: The results showed that siramesine killed standard glioma cell lines in vitro, and loss of acridine orange staining suggested a compromised lysosomal...... cell death and inhibited tumor cell migration. This could not be reproduced in the organotypic three dimensional spheroid-brain slice culture model or in the mice xenograft model. Conclusions: In conclusion the in vitro results obtained with tumor cells and spheroids suggest a potential of lysosomal...

  8. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... exploring about iron-deficiency anemia. Read more New treatments for disorders that lead to iron-deficiency anemia. We are ... and other pathways. This could help develop new therapies for conditions that ... behavior, thinking, and mood during adolescence. Treating anemia in ...

  9. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  10. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... loss and lead to iron-deficiency anemia. Common causes of blood loss that lead to iron-deficiency anemia include: Bleeding in your GI tract, from an ulcer, colon cancer, or regular use of medicines such as aspirin ...

  11. Iron-Deficiency Anemia

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    Full Text Available ... iron-deficiency anemia. Search the NIH Research Portfolio Online Reporting Tools (RePORT) to learn about research that ... iron-deficiency anemia in blood donors affects the quality of donated red blood cells, such as how ...

  12. Iron-Deficiency Anemia

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    Full Text Available ... blocks the intestine from taking up iron. Other medical conditions Other medical conditions that may lead to iron-deficiency anemia ... daily amount of iron. If you have other medical conditions that cause iron-deficiency anemia , such as ...

  13. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... anemia if you have certain risk factors , including pregnancy. To prevent iron-deficiency anemia, your doctor may recommend you eat heart-healthy foods or control other conditions that can cause iron-deficiency anemia. Blood tests to screen for ...

  14. Iron-Deficiency Anemia

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    Full Text Available ... Are you curious about how inflammation from chronic diseases can cause iron-deficiency anemia? Read more When there is ... DBDR) is a leader in research on the causes, prevention, and treatment of blood diseases, including iron-deficiency anemia. Search the NIH Research ...

  15. Nutritional iron deficiency

    NARCIS (Netherlands)

    Zimmermann, M.B.; Hurrell, R.F.

    2007-01-01

    Iron deficiency is one of the leading risk factors for disability and death worldwide, affecting an estimated 2 billion people. Nutritional iron deficiency arises when physiological requirements cannot be met by iron absorption from diet. Dietary iron bioavailability is low in populations consuming

  16. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... Topics section only, or the News and Resources section. NHLBI Entire Site NHLBI Entire Site Health ... español Iron-deficiency anemia is a common type of anemia that occurs if you do not have enough iron in your body. People with mild or moderate iron-deficiency anemia ...

  17. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... for iron-deficiency anemia. Lifestyle habits Certain lifestyle habits may increase your risk for iron-deficiency anemia, including: Vegetarian or vegan eating patterns. Not eating enough iron-rich foods, such as meat and fish, may result in ...

  18. Iron-Deficiency Anemia

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    Full Text Available ... blood cells. Iron-deficiency anemia usually develops over time because your body’s intake of iron is too ... clamping of your newborn’s umbilical cord at the time of delivery. This may help prevent iron-deficiency ...

  19. Iron-Deficiency Anemia

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    Full Text Available ... other conditions that can cause iron-deficiency anemia. Blood tests to screen for iron-deficiency anemia To screen ... check the size of your liver and spleen. Blood tests Based on results from blood tests to screen ...

  20. Iron-Deficiency Anemia

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    Full Text Available ... also are hoping to determine which iron supplements work best to treat iron-deficiency anemia in children who do not consume the daily recommended amount of iron. Read less Participate in NHLBI Clinical Trials We lead or sponsor many studies related to iron-deficiency anemia. See if you ...

  1. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... en español Iron-deficiency anemia is a common type of anemia that occurs if you do not ... iron-deficiency anemia and help rule out other types of anemia. Treatment will explain treatment-related complications ...

  2. Iron deficiency in childhood

    NARCIS (Netherlands)

    Uijterschout, L.

    2015-01-01

    Iron deficiency (ID) is the most common micronutrient deficiency in the world. Iron is involved in oxygen transport, energy metabolism, immune response, and plays an important role in brain development. In infancy, ID is associated with adverse effects on cognitive, motor, and behavioral development

  3. Iron-Deficiency Anemia

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    Full Text Available ... anemia. Return to Signs, Symptoms, and Complications to review signs and symptoms as well as complications from iron-deficiency ... NIH]) Heavy Menstrual Bleeding (Centers for Disease Control and ... Dietary Supplement Fact Sheet (NIH) Iron-Deficiency Anemia (National Library ...

  4. Iron deficiency anemia

    Science.gov (United States)

    Anemia - iron deficiency ... iron from old red blood cells. Iron deficiency anemia develops when your body's iron stores run low. ... You may have no symptoms if the anemia is mild. Most of the time, ... slowly. Symptoms may include: Feeling weak or tired more often ...

  5. Iron-Deficiency Anemia

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    Full Text Available ... be at risk for iron-deficiency anemia. Lifestyle habits Certain lifestyle habits may increase your risk for iron-deficiency anemia, ... you are experiencing side effects such as a bad metallic taste, vomiting, diarrhea, constipation, or upset stomach. ...

  6. Iron-Deficiency Anemia

    Medline Plus

    Full Text Available ... how we are using current research and advancing research to prevent iron-deficiency anemia. Participate in NHLBI Clinical Trials will explain our ongoing clinical studies that are investigating prevention strategies for iron-deficiency anemia. Signs, Symptoms, and Complications ...

  7. Cocaine induces a mixed lysosomal lipidosis in cultured fibroblasts, by inactivation of acid sphingomyelinase and inhibition of phospholipase A1

    International Nuclear Information System (INIS)

    Nassogne, Marie-Cecile; Lizarraga, Chantal; N'Kuli, Francisca; Van Bambeke, Francoise; Van Binst, Roger; Wallemacq, Pierre; Tulkens, Paul M.; Mingeot-Leclercq, Marie-Paule; Levade, Thierry; Courtoy, Pierre J.

    2004-01-01

    This paper reports that cocaine may induce a lysosomal storage disorder. Indeed, culture of Rat-1 fibroblasts with 250-500 μM cocaine induced after 2-3 days a major accumulation in lysosomes of electron-dense lamellar structures. By subcellular fractionation, this was reflected by a selective decrease of the buoyant density of several lysosomal enzymes, indicating lysosomal lipid overload. Biochemical analysis confirmed an increased cellular content of major phospholipids and sphingomyelin, but not of cholesterol. Cocaine, a membrane-permeant weak base, is concentrated by acidotropic sequestration, because its accumulation was abrogated by the proton ionophore, monensin and the vacuolar ATPase inhibitor, bafilomycin A 1 . At its estimated lysosomal concentration, cocaine almost completely inhibited phospholipase A 1 activity on liposomes. Cell incubation with cocaine, but not with its inactive metabolite, benzoylecgonine, rapidly inactivated acid sphingomyelinase, as reflected by a 10-fold decrease in V max with identical K m . Acid sphingomyelinase inactivation was fully prevented by the thiol proteinases inhibitors, leupeptin and E64, indicating that cocaine induces selective sphingomyelinase proteolysis. Upon cocaine removal, acid sphingomyelinase activity was rapidly restored, pointing to its fast turnover. In contrast, the cellular content of several other lysosomal hydrolases was increased up to 2-fold. Together, these data show that acidotropic accumulation of cocaine in lysosomes rapidly inhibits acid phospholipase A 1 and inactivates acid sphingomyelinase, which can explain induction of a mixed lysosomal lipidosis

  8. Protective effect of squalene on certain lysosomal hydrolases and free amino acids in isoprenaline-induced myocardial infarction in rats

    DEFF Research Database (Denmark)

    Farvin, Sabeena; Surendraraj, A.; Anandan, R.

    2010-01-01

    This study was aimed to evaluate the preventive role of squalene on free amino acids and lysosomal alterations in experimentally induced myocardial infarction in rats. The levels of lysosomal enzymes (beta-glucuronidase, beta-galactosidase, beta-glucosidase, acid phosphatase and cathepsin D) in p...

  9. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro.

    Science.gov (United States)

    Canfrán-Duque, Alberto; Barrio, Luis C; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A; Busto, Rebeca

    2016-03-18

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes' internal milieu induced by haloperidol affects lysosomal functionality.

  10. Transcription factor EB: from master coordinator of lysosomal pathways to candidate therapeutic target in degenerative storage diseases.

    Science.gov (United States)

    Sardiello, Marco

    2016-05-01

    The lysosome is the main catabolic hub of the cell. Owing to its role in fundamental processes such as autophagy, plasma membrane repair, mTOR signaling, and maintenance of cellular homeostasis, the lysosome has a profound influence on cellular metabolism and human health. Indeed, inefficient or impaired lysosomal function has been implicated in the pathogenesis of a number of degenerative diseases affecting various organs and tissues, most notably the brain, liver, and muscle. The discovery of the coordinated lysosomal expression and regulation (CLEAR) genetic program and its master controller, transcription factor EB (TFEB), has provided an unprecedented tool to study and manipulate lysosomal function. Most lysosome-based processes-including macromolecule degradation, autophagy, lysosomal exocytosis, and proteostasis-are under the transcriptional control of TFEB. Interestingly, impaired TFEB signaling has been suggested to be a contributing factor in the pathogenesis of several degenerative storage diseases. Preclinical studies based on TFEB exogenous expression to reinstate TFEB activity or promote CLEAR network-based lysosomal enhancement have highlighted TFEB as a candidate therapeutic target for the treatment of various degenerative storage diseases. © 2016 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals, Inc. on behalf of New York Academy of Sciences.

  11. Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis.

    Science.gov (United States)

    Yang, Dun-Sheng; Stavrides, Philip; Mohan, Panaiyur S; Kaushik, Susmita; Kumar, Asok; Ohno, Masuo; Schmidt, Stephen D; Wesson, Daniel W; Bandyopadhyay, Urmi; Jiang, Ying; Pawlik, Monika; Peterhoff, Corrinne M; Yang, Austin J; Wilson, Donald A; St George-Hyslop, Peter; Westaway, David; Mathews, Paul M; Levy, Efrat; Cuervo, Ana M; Nixon, Ralph A

    2011-07-01

    The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.

  12. Iron content and acid phosphatase activity in hepatic parenchymal lysosomes of patients with hemochromatosis before and after phlebotomy treatment

    International Nuclear Information System (INIS)

    Cleton, M.I.; de Bruijn, W.C.; van Blokland, W.T.; Marx, J.J.; Roelofs, J.M.; Rademakers, L.H.

    1988-01-01

    Lysosomal structures in liver parenchymal cells of 3 patients with iron overload and of 3 subjects without iron-storage disorders were investigated. A combination of enzyme cytochemistry--with cerium as a captive ion to demonstrate lysosomal acid phosphatase activity--and electron probe X-ray microanalysis (EPMA) was used. We were able (1) to define and quantify lysosomal structures as lysosomes, siderosomes, or residual bodies, (2) to quantify the amount of iron and cerium simultaneously in these structures, and (3) to evaluate a possible relation between iron storage and enzyme activity. With histopathologically increased iron storage, the number of siderosomes had increased at the cost of lysosomes, with a corresponding increase in acid phosphatase activity in both organelles. In histopahtologically severe iron overload, however, acid phosphatase activity was low or not detectable and most of the iron was stored in residual bodies. After phlebotomy treatment, the number of siderosomes had decreased in favor of the lysosomes, approaching values obtained in control subjects, and acid phosphatase activity was present in all iron-containing structures. In this way a relationship between iron storage and enzyme activity was established. The iron content of the individual lysosomal structures per unit area had increased with histopathologically increased iron storage and had decreased after phlebotomy treatment. From this observation, it is concluded that the iron status of the patient is not only reflected by the amount of iron-containing hepatocytes but, as well, by the iron content lysosomal unit area

  13. The Possible "Proton Sponge " Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH

    DEFF Research Database (Denmark)

    Søndergaard, Rikke Vicki; Mattebjerg, Maria Ahlm; Henriksen, Jonas Rosager

    2013-01-01

    is still elusive. The "proton sponge " hypothesis remains the most generally accepted mechanism, although it is heavily debated. This hypothesis is associated with the large buffering capacity of PEI and other polycations, which has been interpreted to cause an increase in lysosomal pH even though...... no conclusive proof has been provided. In the present study, we have used a nanoparticle pH sensor that was developed for pH measurements in the endosomal/lysosomal pathway. We have carried out quantitative measurements of lysosomal pH as a function of PEI content and correlate the results to the "proton sponge...... " hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the "proton sponge " effect is the dominant mechanism of polyplex escape.Molecular Therapy (2012); doi:10.1038/mt.2012.185....

  14. Role of lysosomal enzymes released by alveolar macrophages in the pathogenesis of the acute phase of hypersensitivity pneumonitis

    Directory of Open Access Journals (Sweden)

    J. L. Pérez-Arellano

    1995-01-01

    Full Text Available Hydrolytic enzymes are the major constituents of alveolar macrophages (AM and have been shown to be involved in many aspects of the inflammatory pulmonary response. The aim of this study was to evaluate the role of lysosomal enzymes in the acute phase of hypersensitivity pneumonitis (HPs. An experimental study on AM lysosomal enzymes of an HP-guinea-pig model was performed. The results obtained both in vivo and in vitro suggest that intracellular enzymatic activity decrease is, at least partly, due to release of lysosomal enzymes into the medium. A positive but slight correlation was found between extracellular lysosomal activity and four parameters of lung lesion (lung index, bronchoalveolar fluid total (BALF protein concentration, BALF LDH and BALF alkaline phosphatase activities. All the above findings suggest that the AM release of lysosomal enzymes during HP is a factor involved, although possibly not the only one, in the pulmonary lesions appearing in this disease.

  15. In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

    Directory of Open Access Journals (Sweden)

    Rita-Eva Varga

    2015-08-01

    Full Text Available Hereditary spastic paraplegia (HSP is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs. Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

  16. HEPES activates a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells: A call for caution.

    Science.gov (United States)

    Tol, Marc J; van der Lienden, Martijn J C; Gabriel, Tanit L; Hagen, Jacob J; Scheij, Saskia; Veenendaal, Tineke; Klumperman, Judith; Donker-Koopman, Wilma E; Verhoeven, Arthur J; Overkleeft, Hermen; Aerts, Johannes M; Argmann, Carmen A; van Eijk, Marco

    2018-01-01

    In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which MTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of "master lysosomal regulators". Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated. Here, we identify HEPES, a chemical buffering agent that is broadly applied in cell culture, as a potent inducer of lysosome biogenesis. Supplementation of HEPES to cell growth media is sufficient to decouple the MiT/TFE family members-TFEB, TFE3 and MITF-from regulatory mechanisms that control their cytosolic retention. Increased MiT/TFE nuclear import in turn drives the expression of a global network of lysosomal-autophagic and innate host-immune response genes, altering lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling. In addition, siRNA-mediated MiT/TFE knockdown effectively blunted HEPES-induced lysosome biogenesis and gene expression profiles. Mechanistically, we show that MiT/TFE activation in response to HEPES requires its macropinocytic ingestion and aberrant lysosomal storage/pH, but is independent of MTORC1 signaling. Altogether, our data underscore the cautionary use of chemical buffering agents in cell culture media due to their potentially confounding effects on experimental results.

  17. Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis--an approach for slowing Alzheimer disease?

    Science.gov (United States)

    Bendiske, Jennifer; Bahr, Ben A

    2003-05-01

    Previous reports suggest that age-related lysosomal disturbances contribute to Alzheimer-type accumulations of protein species, blockage of axonal/dendritic transport, and synaptic decline. Here, we tested the hypothesis that lysosomal enzymes are upregulated as a compensatory response to pathogenic protein accumulation. In the hippocampal slice model, tau deposits and amyloidogenic fragments induced by the lysosomal inhibitor chloroquine were accompanied by disrupted microtubule integrity and by corresponding declines in postsynaptic glutamate receptors and the presynaptic marker synaptophysin. In the same slices, cathepsins B, D, and L, beta-glucuronidase, and elastase were upregulated by 70% to 135%. To address whether this selective activation of the lysosomal system represents compensatory signaling, N-Cbz-L-phenylalanyl-L-alanyl-diazomethylketone (PADK) was used to enhance the lysosome response, generating 4- to 8-fold increases in lysosomal enzymes. PADK-mediated lysosomal modulation was stable for weeks while synaptic components remained normal. When PADK and chloroquine were co-infused, chloroquine no longer increased cellular tau levels. To assess pre-existing pathology, chloroquine was applied for 6 days after which its removal resulted in continued degeneration. In contrast, enhancing lysosomal activation by replacing chloroquine after 6 days with PADK led to clearance of accumulated protein species and restored microtubule integrity. Transport processes lost during chloroquine exposure were consequently re-established, resulting in marked recovery of synaptic components. These data indicate that compensatory activation of lysosomes follows protein accumulation events, and that lysosomal modulation represents a novel approach for treating Alzheimer disease and other protein deposition diseases.

  18. Vitamin B12 deficiency

    DEFF Research Database (Denmark)

    Green, Ralph; Allen, Lindsay H; Bjørke-Monsen, Anne-Lise

    2017-01-01

    , subclinical deficiency affects between 2.5% and 26% of the general population depending on the definition used, although the clinical relevance is unclear. B12 deficiency can affect individuals at all ages, but most particularly elderly individuals. Infants, children, adolescents and women of reproductive age...... remain debated. Management depends on B12 supplementation, either via high-dose oral routes or via parenteral administration. This Primer describes the current knowledge surrounding B12 deficiency, and highlights improvements in diagnostic methods as well as shifting concepts about the prevalence, causes...

  19. Excessive burden of lysosomal storage disorder gene variants in Parkinson's disease

    NARCIS (Netherlands)

    Robak, L.A.; Jansen, I.E.; Rooij, J van; Uitterlinden, A.G.; Kraaij, R.; Jankovic, J.; Heutink, P.; Shulman, J.M.; Bloem, B.; Post, B.; Scheffer, H.; Warrenburg, B.P.C. van de; et al.,

    2017-01-01

    Mutations in the glucocerebrosidase gene (GBA), which cause Gaucher disease, are also potent risk factors for Parkinson's disease. We examined whether a genetic burden of variants in other lysosomal storage disorder genes is more broadly associated with Parkinson's disease susceptibility. The

  20. High-throughput assay of 9 lysosomal enzymes for newborn screening.

    Science.gov (United States)

    Spacil, Zdenek; Tatipaka, Haribabu; Barcenas, Mariana; Scott, C Ronald; Turecek, Frantisek; Gelb, Michael H

    2013-03-01

    There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS). The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre-HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard-specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode. Analysis of blood spots from 58 random newborns and lysosomal storage disease-affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories. HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.

  1. Structural and functional analysis of lysosomal ss-galactosidase and its relation to the protective protein.

    NARCIS (Netherlands)

    H. Morreau (Hans)

    1992-01-01

    textabstractLysosomal B-galactosidase is the glycosidase, that cleaves B-linked galactosyl mmenes from a variety of natural and synthetic substrates. In normal tissues of various species this enzyme appears to associate with two other hydrolases, N-acetyl-o:-neuraminidase and the protective protein.

  2. Effects of mercury on lysosomal protein digestion in the kidney proximal tubule

    International Nuclear Information System (INIS)

    Madsen, K.M.; Christensen, E.I.

    1978-01-01

    The effect of mercury on renal lysosomal protein digestion was studied after administration of mercury in vitro and in vivo. Mercuric chloride or methylmercury chloride was added in vitro to lysosomal enzymes isolated from normal rats, and subsequently, digestion experiments were carried out using 125 I-labeled lysozyme or cytochrome c as substrate proteins. Both mercury compounds produced a concentration-dependent inhibition of the degradation of the proteins, mercuric chloride being the strongest inhibitor. Mercuric chloride was also administered to rats in vivo for 5 to 8 months. Renal lysosomal enzymes from these animals also had a decreased ability to digest the two substrate proteins. Furthermore, the digestion of lysozyme intravenously injected into mercury-intoxicated rats was decreased in renal cortical slices incubated in vitro. Electron microscope autoradiography showed that intravenously injected labeled lysozyme was located primarily over lysosomes in proximal tubule cells 1 hour after injection in both control animals and mercury-intoxicated rats. These results suggest a decreased catabolism of low molecular weight proteins in the kidney during chronic mercury intoxication

  3. Use of complementary and alternative medicine by patients with lysosomal storage diseases.

    Science.gov (United States)

    Balwani, Manisha; Fuerstman, Laura; Desnick, Robert J; Buckley, Brian; McGovern, Margaret M

    2009-10-01

    To evaluate the extent of complementary and alternative medicine use and perceived effectiveness in patients with lysosomal storage diseases. A 26-item survey was distributed to 495 patients with type 1 Gaucher, Fabry, and type B Niemann-Pick diseases who were seen at the Lysosomal Storage Disease Program at the Mount Sinai School of Medicine. Survey responses were entered into an access database and analyzed using descriptive statistics. Surveys were completed by 167 respondents with an overall response rate of 34%. Complementary and alternative medicines were used by 45% of patients with type 1 Gaucher disease, 41% of patients with Fabry disease, and 47% of patients with type B Niemann-Pick for symptoms related to their disease. Complementary and alternative medicines were used most frequently by adult females (55%), in patients who reported having one or more invasive procedures due to their disease, patients who use one or more conventional medical therapies, or those with depression and/or anxiety. Overall perceived effectiveness of complementary and alternative medicine supplements was low; however, complementary and alternative medicine therapies were perceived as effective. Complementary and alternative medicines are commonly used among patients with lysosomal storage diseases. Assessment of the effectiveness of these approaches in the lysosomal storage diseases is needed, and physicians should be aware of complementary and alternative medicine therapies used by patients to evaluate safety and possible drug interactions.

  4. The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro.

    Science.gov (United States)

    Toth, Andrea E; Siupka, Piotr; P Augustine, Thomas J; Venø, Susanne T; Thomsen, Louiza B; Moos, Torben; Lohi, Hannes T; Madsen, Peder; Lykke-Hartmann, Karin; Nielsen, Morten S

    2018-03-20

    Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture and in co-culture with primary rat astrocytes. The presence of astrocytes changed the intraendothelial vesicular network and significantly impacted vesicular number, morphology, and distribution. Additionally, gene set enrichment analysis revealed that 60 genes associated with vesicular trafficking showed altered expression in co-cultured BEC. Cytosolic proteins involved in subcellular trafficking were investigated to mark transport routes, such as RAB25 for transcytosis. Strikingly, the adaptor protein called AP1-μ1B, important for basolateral sorting in epithelial cells, was not expressed in BEC. Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain.

  5. Osteoclasts derive from hematopoietic stem cells according to marker, giant lysosomes of beige mice

    International Nuclear Information System (INIS)

    Ash, P.; Loutit, J.F.; Townsend, K.M.

    1981-01-01

    To ascertain the origin of multinucleated osteoclasts from hematopoietic stem cells, giant lysosomes peculiar to cells of beige mice (bg bg) were used as marker cells of that provenance. Radiation chimeras were established reciprocally between bg bg mice and osteopetrotic mi mi mice with defective osteoclasts. As a result, all the derivative cells of the hematopoietic stem cell would depend on the donor's cell line, whereas osteogenesis would remain the province of the host. It was affirmed in the chimeras mi mi/bg bg that the osteopetrosis was cured within six weeks. Thereafter the definitive osteoclasts of the chimeras contained giant lysosomes attributable to the beige cell line. However, the cure was well advanced before donor osteoclasts were prominent, for which several reasons are offered. In the mouse chimeras, bg bg/mi mi, there was a delay of some six weeks before osteopetrosis became evident, histologically before radiologically, at the major metaphyseal growth centers. During the period one to two months after establishment, osteoclasts appeared to be a mixture of two cell lines according to quantitative assessments for giant lysosomes. Assessments consisted of measurements of the percentage area of osteoclasts occupied by lysosomes over 1 micrometer diameter. The means were 0.018% +/- 0.008% for nonbeige stock and 2.09% +/- 0.58% for beige stock

  6. Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates.

    Science.gov (United States)

    DeVay, Rachel M; Delaria, Kathy; Zhu, Guoyun; Holz, Charles; Foletti, Davide; Sutton, Janette; Bolton, Gary; Dushin, Russell; Bee, Christine; Pons, Jaume; Rajpal, Arvind; Liang, Hong; Shelton, David; Liu, Shu-Hui; Strop, Pavel

    2017-04-19

    Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.

  7. Lysosome-Targeting Amplifiers of Reactive Oxygen Species as Anticancer Prodrugs

    Czech Academy of Sciences Publication Activity Database

    Daum, S.; Reshetnikov, M.S.V.; Šíša, Miroslav; Dumych, T.; Lootsik, M. D.; Bilyy, R.; Bila, E.; Janko, C.; Alexiou, C.; Herrmann, M.; Sellner, L.; Mokhir, A.

    2017-01-01

    Roč. 56, č. 49 (2017), s. 15545-15549 ISSN 1433-7851 Institutional support: RVO:61389030 Keywords : aminoferrocene * cancer * lysosomes * prodrugs * reactive oxygen species Subject RIV: ED - Physiology OBOR OECD: Organic chemistry Impact factor: 11.994, year: 2016

  8. Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules.

    Science.gov (United States)

    Pulipparacharuvil, Suprabha; Akbar, Mohammed Ali; Ray, Sanchali; Sevrioukov, Evgueny A; Haberman, Adam S; Rohrer, Jack; Krämer, Helmut

    2005-08-15

    Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.

  9. Generation of specific deoxynojirimycin-type inhibitors of the non-lysosomal glucosylceramidase

    NARCIS (Netherlands)

    Overkleeft, H. S.; Renkema, G. H.; Neele, J.; Vianello, P.; Hung, I. O.; Strijland, A.; van der Burg, A. M.; Koomen, G. J.; Pandit, U. K.; Aerts, J. M.

    1998-01-01

    The existence of a non-lysosomal glucosylceramidase in human cells has been documented (van Weely, S., Brandsma, M., Strijland, A., Tager, J. M., and Aerts, J. M. F. G. (1993) Biochim. Biophys. Acta 1181, 55-62). Hypothetically, the activity of this enzyme, which is localized near the cell surface,

  10. Giant Lysosomes as a Chemotherapy Resistance Mechanism in Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Colombo, Federico; Trombetta, Elena; Cetrangolo, Paola; Maggioni, Marco; Razini, Paola; De Santis, Francesca; Torrente, Yvan; Prati, Daniele; Torresani, Erminio; Porretti, Laura

    2014-01-01

    Despite continuous improvements in therapeutic protocols, cancer-related mortality is still one of the main problems facing public health. The main cause of treatment failure is multi-drug resistance (MDR: simultaneous insensitivity to different anti-cancer agents), the underlying molecular and biological mechanisms of which include the activity of ATP binding cassette (ABC) proteins and drug compartmentalisation in cell organelles. We investigated the expression of the main ABC proteins and the role of cytoplasmic vacuoles in the MDR of six hepatocellular carcinoma (HCC) cell lines, and confirmed the accumulation of the yellow anti-cancer drug sunitinib in giant (four lines) and small cytoplasmic vacuoles of lysosomal origin (two lines). ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes. MTT assays showed that the cell lines with giant lysosomes were more resistant to sorafenib treatment than those with small lysosomes (plysosomes in drug sequestration and MDR in HCC cell lines. The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment.

  11. Lysosomal-associated transmembrane protein 5 (LAPTM5 is a molecular partner of CD1e.

    Directory of Open Access Journals (Sweden)

    Catherine Angénieux

    Full Text Available The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.

  12. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    Science.gov (United States)

    Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

    2014-07-01

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  13. 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.

  14. TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion.

    Science.gov (United States)

    Xia, Qin; Wang, Hongfeng; Hao, Zongbing; Fu, Cheng; Hu, Qingsong; Gao, Feng; Ren, Haigang; Chen, Dong; Han, Junhai; Ying, Zheng; Wang, Guanghui

    2016-01-18

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration. © 2015 The Authors.

  15. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells

    DEFF Research Database (Denmark)

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth

    2012-01-01

    Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library...... in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 si......), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide...

  16. Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

    International Nuclear Information System (INIS)

    Isenman, L.D.; Dice, J.F.

    1989-01-01

    We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [ 3 H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides

  17. The effect of hyperthermia and radiation on lysosomal enzyme activity of mouse mammary tumours

    International Nuclear Information System (INIS)

    Barratt, G.M.; Wills, E.D.

    1979-01-01

    The effects of hyperthermia and radiation have been studied on the acid phosphatase and β-glucuronidase activities in lysosomes of C3H mice mammary tumours and of the spleen. Quantitative histochemical methods have been used. Hyperthermic treatment of both spontaneous and transplanted tumours caused an increase in the activity of both acid phosphatase and β-glucuronase when measured immediately after treatment, but the activities returned to normal after 24 hours. In contrast a radiation dose of 3500 rad did not cause an increase in activity of either enzyme immediately, but a large activation was observed after 24 hr. Combination of hyperthermic and radiation treatment caused increases in enzyme activities which were dependent on the time after treatment. Hyperthermic treatment of the lower body of mice bearing tumours also caused activation of lysosomal enzymes in the spleen. This may be hormone mediated. It is considered that the increased lysosomal enzyme activity observed after hyperthermia may be a consequence of increased permeability of the lysosomal membrane caused by hyperthermia. (author)

  18. Anticancer actions of lysosomally targeted inhibitor, LCL521, of acid ceramidase.

    Science.gov (United States)

    Bai, Aiping; Mao, Cungui; Jenkins, Russell W; Szulc, Zdzislaw M; Bielawska, Alicja; Hannun, Yusuf A

    2017-01-01

    Acid ceramidase, which catalyzes ceramide hydrolysis to sphingosine and free fatty acid mainly in the lysosome, is being recognized as a potential therapeutic target for cancer. B13 is an effective and selective acid ceramidase inhibitor in vitro, but not as effective in cells due to poor access to the lysosomal compartment. In order to achieve targeting of B13 to the lysosome, we designed lysosomotropic N, N-dimethyl glycine (DMG)-conjugated B13 prodrug LCL521 (1,3-di-DMG-B13). Our previous results indicated the efficient delivery of B13 to the lysosome resulted in augmented effects of LCL521 on cellular acid ceramidase as evaluated by effects on substrate/product levels. Our current studies indicate that functionally, this translated into enhanced inhibition of cell proliferation. Moreover, there were greater synergistic effects of LCL521 with either ionizing radiation or Tamoxifen. Taken together, these results clearly indicate that compartmental targeting for the inhibition of acid ceramidase is an efficient and valuable therapeutic strategy.

  19. Anticancer actions of lysosomally targeted inhibitor, LCL521, of acid ceramidase.

    Directory of Open Access Journals (Sweden)

    Aiping Bai

    Full Text Available Acid ceramidase, which catalyzes ceramide hydrolysis to sphingosine and free fatty acid mainly in the lysosome, is being recognized as a potential therapeutic target for cancer. B13 is an effective and selective acid ceramidase inhibitor in vitro, but not as effective in cells due to poor access to the lysosomal compartment. In order to achieve targeting of B13 to the lysosome, we designed lysosomotropic N, N-dimethyl glycine (DMG-conjugated B13 prodrug LCL521 (1,3-di-DMG-B13. Our previous results indicated the efficient delivery of B13 to the lysosome resulted in augmented effects of LCL521 on cellular acid ceramidase as evaluated by effects on substrate/product levels. Our current studies indicate that functionally, this translated into enhanced inhibition of cell proliferation. Moreover, there were greater synergistic effects of LCL521 with either ionizing radiation or Tamoxifen. Taken together, these results clearly indicate that compartmental targeting for the inhibition of acid ceramidase is an efficient and valuable therapeutic strategy.

  20. Apoptosis and signalling in acid sphingomyelinase deficient cells

    Directory of Open Access Journals (Sweden)

    Sillence Dan J

    2001-11-01

    Full Text Available Abstract Background Recent evidence suggests that the activation of a non-specific lipid scramblase during apoptosis induces the flipping of sphingomyelin from the cell surface to the cytoplasmic leaftet of the plasma membrane. Inner leaflet sphingomyelin is then cleaved to ceramide by a neutral sphingomyelinase. The production of this non-membrane forming lipid induces blebbing of the plasma membrane to aid rapid engulfment by professional phagocytes. However contrary evidence suggests that cells which are deficient in acid sphingomyelinase are defective in apoptosis signalling. This data has been interpreted as support for the activation of acid sphingomyelinase as an early signal in apoptosis. Hypothesis An alternative explanation is put forward whereby the accumulation of intracellular sphingomyelin in sphingomyelinase deficient cells leads to the formation of intracellular rafts which lead to the sequestration of important signalling molecules that are normally present on the cell surface where they perform their function. Testing the hypothesis It is expected that the subcellular distribution of important signalling molecules is altered in acid sphingomyelinase deficient cells, leading to their sequestration in late endosomes / lysosomes. Other sphingolipid storage diseases such as Niemann-Pick type C which have normal acid sphingomyelinase activity would also be expected to show the same phenotype. Implications of the hypothesis If true the hypothesis would provide a mechanism for the pathology of the sphingolipid storage diseases at the cellular level and also have implications for the role of ceramide in apoptosis.

  1. Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease.

    Science.gov (United States)

    Yi, Haiqing; Sun, Tao; Armstrong, Dustin; Borneman, Scott; Yang, Chunyu; Austin, Stephanie; Kishnani, Priya S; Sun, Baodong

    2017-05-01

    Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease. FabGAA can be delivered to both the cytoplasm and lysosomes in cultured cells. FabGAA equally reduced lysosomal glycogen accumulation as rhGAA in GAA-KO mice. FabGAA has the beneficial potential over rhGAA to clear cytoplasmic glycogen. This study suggests a novel antibody-enzyme fusion protein therapy

  2. Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter.

    Science.gov (United States)

    Seebacher, Nicole A; Lane, Darius J R; Jansson, Patric J; Richardson, Des R

    2016-02-19

    Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in

  3. Iron-Deficiency Anemia

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  8. Vitamin D Deficiency

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  9. Iron-Deficiency Anemia

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  6. Iron-Deficiency Anemia

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  7. Factor V deficiency

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    ... this page: //medlineplus.gov/ency/article/000550.htm Factor V deficiency To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  8. Factor II deficiency

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  9. Factor X deficiency

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    ... this page: //medlineplus.gov/ency/article/000553.htm Factor X deficiency To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  10. Iron-Deficiency Anemia

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  12. Iron-Deficiency Anemia

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  13. Iron-Deficiency Anemia

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  14. Iron-Deficiency Anemia

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  18. Iron-Deficiency Anemia

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  19. Iron-Deficiency Anemia

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  20. Iron-Deficiency Anemia

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  1. Iron-Deficiency Anemia

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  2. Iron-Deficiency Anemia

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  3. Iron-Deficiency Anemia

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  4. Iron-Deficiency Anemia

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  6. Iron-Deficiency Anemia

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  7. Iron-Deficiency Anemia

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  8. Iron-Deficiency Anemia

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  9. Iron-Deficiency Anemia

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  12. Iron-Deficiency Anemia

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  13. Iron-Deficiency Anemia

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    Full Text Available ... improved health for people with iron-deficiency anemia. Recipient Epidemiology Donor Studies program findings help to protect blood donors . NHLBI’s Recipient Epidemiology Donor Studies (REDS) program , which began in ...

  14. Iron-Deficiency Anemia

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  15. Iron-Deficiency Anemia

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  16. Iron-Deficiency Anemia

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  17. Iron-Deficiency Anemia

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  18. Iron-Deficiency Anemia

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  20. Iron-Deficiency Anemia

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