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Sample records for human dopaminergic sh-sy5y

  1. Neurotoxicity of "ecstasy" and its metabolites in human dopaminergic differentiated SH-SY5Y cells.

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    Ferreira, Patrícia Silva; Nogueira, Tiago Bernandes; Costa, Vera Marisa; Branco, Paula Sério; Ferreira, Luísa Maria; Fernandes, Eduarda; Bastos, Maria Lourdes; Meisel, Andreas; Carvalho, Félix; Capela, João Paulo

    2013-02-04

    "Ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) is a widely abused recreational drug, reported to produce neurotoxic effects, both in laboratory animals and in humans. MDMA metabolites can be major contributors for MDMA neurotoxicity. This work studied the neurotoxicity of MDMA and its catechol metabolites, α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) in human dopaminergic SH-SY5Y cells differentiated with retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate. Differentiation led to SH-SY5Y neurons with higher ability to accumulate dopamine and higher resistance towards dopamine neurotoxicity. MDMA catechol metabolites were neurotoxic to SH-SY5Y neurons, leading to caspase 3-independent cell death in a concentration- and time-dependent manner. MDMA did not show a concentration- and time-dependent death. Pre-treatment with the antioxidant and glutathione precursor, N-acetylcysteine (NAC), resulted in strong protection against the MDMA metabolites' neurotoxicity. Neither the superoxide radical scavenger, tiron, nor the inhibitor of the dopamine (DA) transporter, GBR 12909, prevented the metabolites' toxicity. Cells exposed to α-MeDA showed an increase in intracellular glutathione (GSH) levels, which, at the 48 h time-point, was not dependent in the activity increase of γ-glutamylcysteine synthetase (γ-GCS), revealing a possible transient effect. Importantly, pre-treatment with buthionine sulfoximine (BSO), an inhibitor of γ-GCS, prevented α-MeDA induced increase in GSH levels, but did not augment this metabolite cytotoxicity. Even so, BSO pre-treatment abolished NAC protective effects against α-MeDA neurotoxicity, which were, at least partially, due to GSH de novo synthesis. Inversely, pre-treatment of cells with BSO augmented N-Me-α-MeDA-induced neurotoxicity, but only slightly affected NAC neuroprotection. In conclusion, MDMA catechol metabolites promote differential toxic effects to differentiated dopaminergic human SH-SY

  2. SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease.

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    Xie, Hong-rong; Hu, Lin-sen; Li, Guo-yi

    2010-04-20

    To evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic (DAergic) neurons for Parkinson's disease (PD) research and to determine the effect of differentiation on this cell model. The data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals (Pubmed 1973 to 2009). After searching the literature, 60 articles were selected to address this review. The SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-beta-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium (MPP(+)) uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons. Differentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxicity and neuroprotection and are thus more relevant to experimental PD research.

  3. Neuroprotective effects of glyceryl nonivamide against microglia-like cells and 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells.

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    Lin, Yi-Chin; Uang, Hao-Wei; Lin, Rong-Jyh; Chen, Ing-Jun; Lo, Yi-Ching

    2007-12-01

    Glyceryl nonivamide (GLNVA), a vanilloid receptor (VR) agonist, has been reported to have calcitonin gene-related peptide-associated vasodilatation and to prevent subarachnoid hemorrhage-induced cerebral vasospasm. In this study, we investigated the neuroprotective effects of GLNVA on activated microglia-like cell mediated- and proparkinsonian neurotoxin 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. In coculture conditions, we used lipopolysaccharide (LPS)-stimulated BV-2 cells as a model of activated microglia. LPS-induced neuronal death was significantly inhibited by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase. However, capsazepine, the selective VR1 antagonist, did not block the neuroprotective effects of GLNVA. GLNVA reduced LPS-activated microglia-mediated neuronal death, but it lacked protection in DPI-pretreated cultures. GLNVA also decreased LPS activated microglia induced overexpression of neuronal nitric-oxide synthase (nNOS) and glycoprotein 91 phagocyte oxidase (gp91(phox)) on SH-SY5Y cells. Pretreatment of BV-2 cells with GLNVA diminished LPS-induced nitric oxide production, overexpression of inducible nitric-oxide synthase (iNOS), and gp91(phox) and intracellular reactive oxygen species (iROS). GLNVA also reduced cyclooxygenase (COX)-2 expression, inhibitor of nuclear factor (NF)-kappaB (IkappaB)alpha/IkappaBbeta degradation, NF-kappaB activation, and the overproduction of tumor necrosis factor-alpha, interleukin (IL)-1beta, and prostaglandin E2 in BV-2 cells. However, GLNVA augmented anti-inflammatory cytokine IL-10 production on LPS-stimulated BV-2 cells. Furthermore, in 6-OHDA-treated SH-SY5Y cells, GLNVA rescued the changes in condensed nuclear and apoptotic bodies, prevented the decrease in mitochondrial membrane potential, and reduced cells death. GLNVA also suppressed accumulation of iROS and up-regulated heme oxygenase-1 expression. 6-OHDA-induced overexpression of nNOS, i

  4. Hydrogen Peroxide Toxicity Induces Ras Signaling in Human Neuroblastoma SH-SY5Y Cultured Cells

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    Jirapa Chetsawang

    2010-01-01

    Full Text Available It has been reported that overproduction of reactive oxygen species occurs after brain injury and mediates neuronal cells degeneration. In the present study, we examined the role of Ras signaling on hydrogen peroxide-induced neuronal cells degeneration in dopaminergic neuroblastoma SH-SY5Y cells. Hydrogen peroxide significantly reduced cell viability in SH-SY5Y cultured cells. An inhibitor of the enzyme that catalyzes the farnesylation of Ras proteins, FTI-277, and a competitive inhibitor of GTP-binding proteins, GDP-beta-S significantly decreased hydrogen peroxide-induced reduction in cell viability in SH-SY5Y cultured cells. The results of this study might indicate that a Ras-dependent signaling pathway plays a role in hydrogen peroxide-induced toxicity in neuronal cells.

  5. Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research.

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    Cheung, Yuen-Ting; Lau, Way Kwok-Wai; Yu, Man-Shan; Lai, Cora Sau-Wan; Yeung, Sze-Chun; So, Kwok-Fai; Chang, Raymond Chuen-Chung

    2009-01-01

    Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research.

  6. Ras-MAPK signaling in differentiating SH-SY5Y human neuroblastoma cells

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    Olsson, Anna-Karin

    2000-01-01

    Neuroblastoma is a malignant childhood cancer, originating from sympathetic neuroblasts of the peripheral nervous system. Neuroblastoma is a heterogenous group of tumours, while some are highly malignant others can spontaneosly mature into a more benign form or regress. Less than half of the patients survive and this statistics has improved only modestly over the past 20 years. SH-SY5Y is a human neuroblastoma cell line established from a highly malignant tumour. The cells have retained a ca...

  7. Atractylenolide-I Protects Human SH-SY5Y Cells from 1-Methyl-4-Phenylpyridinium-Induced Apoptotic Cell Death

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    Sandeep Vasant More

    2017-05-01

    Full Text Available Oxidative stress and apoptosis are the major mechanisms that induce dopaminergic cell death. Our study investigates the protective effects of atractylenolide-I (ATR-I on 1-methyl-4-phenylpyridinium (MPP+-induced cytotoxicity in human dopaminergic SH-SY5Y cells, as well as its underlying mechanism. Our experimental data indicates that ATR-I significantly inhibits the loss of cell viability induced by MPP+ in SH-SY5Y cells. To further unravel the mechanism, we examined the effect of ATR-I on MPP+-induced apoptotic cell death characterized by an increase in the Bax/Bcl-2 mRNA ratio, the release of cytochrome-c, and the activation of caspase-3 leading to elevated levels of cleaved poly(ADP-ribose polymerase (PARP resulting in SH-SY5Y cell death. Our results demonstrated that ATR-I decreases the level of pro-apoptotic proteins induced by MPP+ and also restored Bax/Bcl-2 mRNA levels, which are critical for inducing apoptosis. In addition, ATR-I demonstrated a significant increase in the protein expression of heme-oxygenase in MPP+-treated SH-SY5Y cells. These results suggest that the pharmacological effect of ATR-I may be, at least in part, caused by the reduction in pro-apoptotic signals and also by induction of anti-oxidant protein.

  8. Baicalein antagonizes rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to Parkinsonism

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    Song Ju-Xian; Choi Mandy; Wong Kavin; Chung Winkie; Sze Stephen; Ng Tzi-Bun; Zhang Kalin

    2012-01-01

    Abstract Background Two active compounds, baicalein and its glycoside baicalin were found in the dried root of Scutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluate the protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to parkinsonism. Methods Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis was evaluated with a fluorescent DNA-bindi...

  9. Baicalein antagonizes rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to Parkinsonism.

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    Song, Ju-Xian; Choi, Mandy Yuen-Man; Wong, Kavin Chun-Kit; Chung, Winkie Wing-Yan; Sze, Stephen Cho-Wing; Ng, Tzi-Bun; Zhang, Kalin Yan-Bo

    2012-01-21

    Two active compounds, baicalein and its glycoside baicalin were found in the dried root of Scutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluate the protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to parkinsonism. Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis was evaluated with a fluorescent DNA-binding probe Hoechst 33258. The production of reactive oxidative species (ROS) and loss of mitochondrial membrane potential (ΔΨm) were determined by fluorescent staining with DCFH-DA and Rhodanmine 123, respectively. The expression of Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 was determined by the Western blots. Baicalein significantly increased viability and decreased rotenone-induced death of SH-SY5Y cells in a dose-dependent manner. Pre- and subsequent co-treatment with baicalein preserved the cell morphology and attenuated the nuclear apoptotic characteristics triggered by rotenone. Baicalein antagonized rotenone-induced overproduction of ROS, loss of ΔΨm, the increased expression of Bax, cleaved caspase-3 and phosphorylated ERK1/2 and the decreased expression of Bcl-2. The antioxidative effect, mitochondrial protection and modulation of anti-and pro-apoptotic proteins are related to the neuroprotective effects of baicalein against rotenone induced cell death in SH-SY5Y cells.

  10. Baicalein antagonizes rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to Parkinsonism

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    Song Ju-Xian

    2012-01-01

    Full Text Available Abstract Background Two active compounds, baicalein and its glycoside baicalin were found in the dried root of Scutellaria baicalensis Georgi, and reported to be neuroprotective in vitro and in vivo. This study aims to evaluate the protective effects of baicalein on the rotenone-induced apoptosis in dopaminergic SH-SY5Y cells related to parkinsonism. Methods Cell viability and cytotoxicity were determined by MTT assay. The degree of nuclear apoptosis was evaluated with a fluorescent DNA-binding probe Hoechst 33258. The production of reactive oxidative species (ROS and loss of mitochondrial membrane potential (ΔΨm were determined by fluorescent staining with DCFH-DA and Rhodanmine 123, respectively. The expression of Bax, Bcl-2, cleaved caspase-3 and phosphorylated ERK1/2 was determined by the Western blots. Results Baicalein significantly increased viability and decreased rotenone-induced death of SH-SY5Y cells in a dose-dependent manner. Pre- and subsequent co-treatment with baicalein preserved the cell morphology and attenuated the nuclear apoptotic characteristics triggered by rotenone. Baicalein antagonized rotenone-induced overproduction of ROS, loss of ΔΨm, the increased expression of Bax, cleaved caspase-3 and phosphorylated ERK1/2 and the decreased expression of Bcl-2. Conclusion The antioxidative effect, mitochondrial protection and modulation of anti-and pro-apoptotic proteins are related to the neuroprotective effects of baicalein against rotenone induced cell death in SH-SY5Y cells.

  11. Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line.

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    Shipley, Mackenzie M; Mangold, Colleen A; Szpara, Moriah L

    2016-02-17

    Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

  12. Rosiglitazone protects human neuroblastoma SH-SY5Y cells against acetaldehyde-induced cytotoxicity

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    Jung, Tae Woo; Lee, Ji Young; Shim, Wan Sub; Kang, Eun Seok; Kim, Soo Kyung; Ahn, Chul Woo; Lee, Hyun Chul; Cha, Bong Soo

    2006-01-01

    Acetaldehyde, an inhibitor of mitochondrial function, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with elevation of the intracellular reactive oxygen species level and apoptosis. Rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist, has been known to show various non-hypoglycemic effects, including anti-inflammatory, anti-atherogenic, and anti-apoptotic. In this study, we investigated the protective effects of rosiglitazone on acetaldehyde-induced apoptosis in human neuroblastoma SH-SY5Y cells and attempted to examine its mechanism. Acetaldehyde-induced apoptosis was moderately reversed by rosiglitazone treatment. Our results suggest that the protective effects of rosiglitazone on acetaldehyde-induced apoptosis may be ascribed to ability to induce the expression of anti-oxidant enzymes and to regulate Bcl-2 and Bax expression. These data indicate that rosiglitazone may provide a useful therapeutic strategy for the prevention of progressive neurodegenerative disease such as Parkinson's disease

  13. Neuronal differentiation and long-term culture of the human neuroblastoma line SH-SY5Y.

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    Constantinescu, R; Constantinescu, A T; Reichmann, H; Janetzky, B

    2007-01-01

    Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in industrialized countries. Present cell culture models for PD rely on either primary cells or immortal cell lines, neither of which allow for long-term experiments on a constant population, a crucial requisite for a realistic model of slowly progressing neurodegenerative diseases. We differentiated SH-SY5Y human dopaminergic neuroblastoma cells to a neuronal-like state in a perfusion culture system using a combination of retinoic acid and mitotic inhibitors. The cells could be cultivated for two months without the need for passage. We show, by various means, that the differentiated cells exhibit, at the molecular level, many neuronal properties not characteristic to the starting line. This approach opens the possibility to develop chronic models, in which the effect of perturbations and putative counteracting strategies can be monitored over long periods of time in a quasi-stable cell population.

  14. Presenilin-1 mutations alter K+ currents in the human neuroblastoma cell line, SH-SY5Y

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    Plant, Leigh D; Boyle, John P; Thomas, Natasha M

    2002-01-01

    Mutations in presenilin 1 (PS1) are the major cause of autosomal dominant Alzheimer's disease. We have measured the voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y using whole-cell patch-clamp. When cells were stably transfected to over-express PS1, no change in K+ current...

  15. Mitochondrial Effects of PGC-1alpha Silencing in MPP+ Treated Human SH-SY5Y Neuroblastoma Cells

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    Qinyong Ye

    2017-05-01

    Full Text Available The dopaminergic neuron degeneration and loss that occurs in Parkinson’s disease (PD has been tightly linked to mitochondrial dysfunction. Although the aged-related cause of the mitochondrial defect observed in PD patients remains unclear, nuclear genes are of potential importance to mitochondrial function. Human peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC-1α is a multi-functional transcription factor that tightly regulates mitochondrial biogenesis and oxidative capacity. The goal of the present study was to explore the potential pathogenic effects of interference by the PGC-1α gene on N-methyl-4-phenylpyridinium ion (MPP+-induced SH-SY5Y cells. We utilized RNA interference (RNAi technology to probe the pathogenic consequences of inhibiting PGC-1α in the SH-SY5Y cell line. Remarkably, a reduction in PGC-1α resulted in the reduction of mitochondrial membrane potential, intracellular ATP content and intracellular H2O2 generation, leading to the translocation of cytochrome c (cyt c to the cytoplasm in the MPP+-induced PD cell model. The expression of related proteins in the signaling pathway (e.g., estrogen-related receptor α (ERRα, nuclear respiratory factor 1 (NRF-1, NRF-2 and Peroxisome proliferator-activated receptor γ (PPARγ also decreased. Our finding indicates that small interfering RNA (siRNA interference targeting the PGC-1α gene could inhibit the function of mitochondria in several capacities and that the PGC-1α gene may modulate mitochondrial function by regulating the expression of ERRα, NRF-1, NRF-2 and PPARγ. Thus, PGC-1α can be considered a potential therapeutic target for PD.

  16. Neuroprotective Effects of Germinated Brown Rice against Hydrogen Peroxide Induced Cell Death in Human SH-SY5Y Cells

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    Shahid Iqbal

    2012-08-01

    Full Text Available The neuroprotective and antioxidative effects of germinated brown rice (GBR, brown rice (BR and commercially available γ-aminobutyric acid (GABA against cell death induced by hydrogen peroxide (H2O2 in human neuroblastoma SH-SY5Y cells have been investigated. Results show that GBR suppressed H2O2-mediated cytotoxicity and induced G0/G1 phase cell cycle arrest in SH-SY5Y cells. Moreover, GBR reduced mitochondrial membrane potential (MMP and prevented phosphatidylserine (PS translocation in SH-SY5Y cells, key features of apoptosis, and subsequent cell death. GBR exhibited better neuroprotective and antioxidative activities as compared to BR and GABA. These results indicate that GBR possesses high antioxidative activities and suppressed cell death in SH-SY5Y cells by blocking the cell cycle re-entry and apoptotic mechanisms. Therefore, GBR could be developed as a value added functional food to prevent neurodegenerative diseases caused by oxidative stress and apoptosis.

  17. Protective Effect of Neuropeptide Apelin-13 on 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Dopaminergic Cells: Involvement of Its Antioxidant and Antiapoptotic Properties.

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    Pouresmaeili-Babaki, Elham; Esmaeili-Mahani, Saeed; Abbasnejad, Mehdi; Ravan, Hadi

    2018-04-01

    Parkinson's disease (PD) is a severe neurodegenerative disorder characterized by the loss of brain dopaminergic neurons. Beside pharmacologic and symptomatic treatment of PD the neuroprotective therapy has recently attracted more attention. Apelin, a novel neuropeptide, and its receptors have numerous reported roles in regulating brain functions. In addition, this peptide has potent neuroprotective effects in some neurodegenerative situations. In this study, the effects of apelin-13 were investigated in a cell model of PD. Human neuroblastoma SH-SY5Y cell damage was induced by 150 μM 6-hydroxydopamine (6-OHDA) and the cells viability was examined by MTT assay. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by fluorescence spectrophotometry method. Immunoblotting analysis was also employed to evaluate cytochrome c release and caspase-3 activity. Data showed that 6-OHDA could decrease cell viability and mitochondrial membrane potential and increase intracellular ROS, cytochrome c, and cleaved caspase-3 levels. Pretreatment of SH-SY5Y cells with apelin-13 (5 and 10 nM) significantly prevented the mentioned biochemical and molecular markers of 6-OHDA-induced neurotoxicity. Furthermore, the results showed that apelin receptor and PI3K signaling contributed to the observed protective effects of apelin. The results suggest that apelin-13 has protective effects against dopaminergic neural toxicity and its antioxidant and antiapoptotic properties are involved, at least in part, in such protection.

  18. Effect of ellagic acid on proliferation, cell adhesion and apoptosis in SH-SY5Y human neuroblastoma cells.

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    Fjaeraa, Christina; Nånberg, Eewa

    2009-05-01

    Ellagic acid, a polyphenolic compound found in berries, fruits and nuts, has been shown to possess growth-inhibiting and apoptosis promoting activities in cancer cell lines in vitro. The objective of this study was to investigate the effect of ellagic acid in human neuroblastoma SH-SY5Y cells. In cultures of SH-SY5Y cells incubated with ellagic acid, time- and concentration-dependent inhibitory effects on cell number were demonstrated. Ellagic acid induced cell detachment, decreased cell viability and induced apoptosis as measured by DNA strand breaks. Ellagic acid-induced alterations in cell cycle were also observed. Simultaneous treatment with all-trans retinoic acid did not rescue the cells from ellagic acid effects. Furthermore, the results suggested that pre-treatment with all-trans retinoic acid to induce differentiation and cell cycle arrest did not rescue the cells from ellagic acid-induced cell death.

  19. Cyclophilin B protects SH-SY5Y human neuroblastoma cells against MPP(+)-induced neurotoxicity via JNK pathway.

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    Oh, Yoojung; Jeong, Kwon; Kim, Kiyoon; Lee, Young-Seok; Jeong, Suyun; Kim, Sung Soo; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug; Choe, Wonchae

    2016-09-23

    Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. PD involves a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyidine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) inhibit the complex I of the mitochondrial electron transport chain, and have been widely used to construct PD models. Cyclophilin B (CypB) is an endoplasmic reticulum protein that binds to cyclosporine A as a cyclophilin family member. CypB has peptidyl-prolyl cis-trans isomerase (PPIase) activity. We investigated the protective effects of overexpressed CypB on MPP+-induced neurocytotoxicity in SH-SY5Y human neuroblastoma cells. Overexpressed CypB decreased MPP(+)-induced oxidative stress through the modulation of antioxidant enzymes including manganese superoxide dismutase and catalase, and prevented neurocytotoxicity via mitogen-activated protein kinase, especially the c-Jun N-terminal kinase pathway. In addition, CypB inhibited the activation of MPP(+)-induced the pro-apoptotic molecules poly (ADP-ribose) polymerase, Bax, and Bcl-2, and attenuated MPP(+)-induced mitochondrial dysfunction. The data suggest that overexpressed CypB protects neuronal cells from MPP+-induced dopaminergic neuronal cell death. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Presenilin expression during induced differentiation of the human neuroblastoma SH-SY5Y cell line.

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    Flood, Fiona; Sundström, Erik; Samuelsson, Eva-Britt; Wiehager, Birgitta; Seiger, Ake; Johnston, Janet A; Cowburn, Richard F

    2004-06-01

    Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.

  1. Dibutyryl cyclic AMP induces differentiation of human neuroblastoma SH-SY5Y cells into a noradrenergic phenotype.

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    Kume, Toshiaki; Kawato, Yuka; Osakada, Fumitaka; Izumi, Yasuhiko; Katsuki, Hiroshi; Nakagawa, Takayuki; Kaneko, Shuji; Niidome, Tetsuhiro; Takada-Takatori, Yuki; Akaike, Akinori

    2008-10-10

    Dibutyryl cyclic AMP (dbcAMP) and retinoic acid (RA) have been demonstrated to be the inducers of morphological differentiation in SH-SY5Y cells, a human catecholaminergic neuroblastoma cell line. However, it remains unclear whether morphologically differentiated SH-SY5Y cells by these compounds acquire catecholaminergic properties. We focused on the alteration of tyrosine hydroxylase (TH) expression and intracellular content of noradrenaline (NA) as the indicators of functional differentiation. Three days treatment with dbcAMP (1mM) and RA (10microM) induced morphological changes and an increase of TH-positive cells using immunocytochemical analysis in SH-SY5Y cells. The percentage of TH-expressing cells in dbcAMP (1mM) treatment was larger than that in RA (10microM) treatment. In addition, dbcAMP increased intracellular NA content, whereas RA did not. The dbcAMP-induced increase in TH-expressing cells is partially inhibited by KT5720, a protein kinase A (PKA) inhibitor. We also investigated the effect of butyrate on SH-SY5Y cells, because dbcAMP is enzymatically degraded by intracellular esterase, thereby resulting in the formation of butyrate. Butyrate induced the increase of NA content at lower concentrations than dbcAMP, although the increase in TH-expressing cells by butyrate was smaller than that by dbcAMP. The dbcAMP (1mM)- and butyrate (0.3mM)-induced increase in NA content was completely suppressed by alpha-methyl-p-tyrosine (1mM), an inhibitor of TH. These results suggest that dbcAMP induces differentiation into the noradrenergic phenotype through both PKA activation and butyrate.

  2. Salicin from Willow Bark can Modulate Neurite Outgrowth in Human Neuroblastoma SH-SY5Y Cells.

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    Wölfle, Ute; Haarhaus, Birgit; Kersten, Astrid; Fiebich, Bernd; Hug, Martin J; Schempp, Christoph M

    2015-10-01

    Salicin from willow bark has been used throughout centuries in China and Europe for the treatment of pain, headache, and inflammatory conditions. Recently, it could be demonstrated that salicin binds and activates the bitter taste receptor TAS2R16. Studies on rodent tissues showed the general expression of bitter taste receptors (TAS2Rs) in rodent brain. Here, we demonstrate the expression of hTAS2R16 in human neuronal tissues and the neuroblastoma cell line SH-SY5Y. The functionality was analyzed in the neuroblastoma cell line SH-SY5Y after stimulation with salicin, a known TAS2R16 agonist. In this setting salicin induced in SH-SY5Y cells phosphorylation of ERK and CREB, the key transcription factor of neuronal differentiation. PD98059, an inhibitor of the ERK pathway, as well as probenecid, a TAS2R16 antagonist, inhibited receptor phosphorylation as well as neurite outgrowth. These data show that salicin might modulate neurite outgrowth by bitter taste receptor activation. Copyright © 2015 John Wiley & Sons, Ltd.

  3. The Neuroprotective Effects of Brazilian Green Propolis on Neurodegenerative Damage in Human Neuronal SH-SY5Y Cells

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    Junjun Ni

    2017-01-01

    Full Text Available Oxidative stress and synapse dysfunction are the major neurodegenerative damage correlated to cognitive impairment in Alzheimer’s disease (AD. We have found that Brazilian green propolis (propolis improves the cognitive functions of mild cognitive impairment patients living at high altitude; however, mechanism underlying the effects of propolis is unknown. In the present study, we investigated the effects of propolis on oxidative stress, expression of brain-derived neurotrophic factor (BDNF, and activity-regulated cytoskeleton-associated protein (Arc, the critical factors of synapse efficacy, using human neuroblastoma SH-SY5Y cells. Pretreatment with propolis significantly ameliorated the hydrogen peroxide- (H2O2- induced cytotoxicity in SH-SY5Y cells. Furthermore, propolis significantly reduced the H2O2-generated reactive oxygen species (ROS derived from mitochondria and 8-oxo-2′-deoxyguanosine (8-oxo-dG, the DNA oxidative damage marker but significantly reversed the fibrillar β-amyloid and IL-1β-impaired BDNF-induced Arc expression in SH-SY5Y cells. Furthermore, propolis significantly upregulated BDNF mRNA expression in time- and dose-dependent manners. In addition, propolis induced Arc mRNA and protein expression via phosphoinositide-3 kinase (PI3K. These observations strongly suggest that propolis protects from the neurodegenerative damage in neurons through the properties of various antioxidants. The present study provides a potential molecular mechanism of Brazilian green propolis in prevention of cognitive impairment in AD as well as aging.

  4. Enhanced oxidative stress and aberrant mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells during methamphetamine induced apoptosis

    International Nuclear Information System (INIS)

    Wu, C.-W.; Ping, Y.-H.; Yen, J.-C.; Chang, C.-Y.; Wang, S.-F.; Yeh, C.-L.; Chi, C.-W.; Lee, H.-C.

    2007-01-01

    Methamphetamine (METH) is an abused drug that may cause psychiatric and neurotoxic damage, including degeneration of monoaminergic terminals and apoptosis of non-monoaminergic cells in Brain. The cellular and molecular mechanisms underlying these METH-induced neurotoxic effects remain to be clarified. In this study, we performed a time course assessment to investigate the effects of METH on intracellular oxidative stress and mitochondrial alterations in a human dopaminergic neuroblastoma SH-SY5Y cell line. We characterized that METH induces a temporal sequence of several cellular events including, firstly, a decrease in mitochondrial membrane potential within 1 h of the METH treatment, secondly, an extensive decline in mitochondrial membrane potential and increase in the level of reactive oxygen species (ROS) after 8 h of the treatment, thirdly, an increase in mitochondrial mass after the drug treatment for 24 h, and finally, a decrease in mtDNA copy number and mitochondrial proteins per mitochondrion as well as the occurrence of apoptosis after 48 h of the treatment. Importantly, vitamin E attenuated the METH-induced increases in intracellular ROS level and mitochondrial mass, and prevented METH-induced cell death. Our observations suggest that enhanced oxidative stress and aberrant mitochondrial biogenesis may play critical roles in METH-induced neurotoxic effects

  5. Differentiated Human SH-SY5Y Cells Provide a Reductionist Model of Herpes Simplex Virus 1 Neurotropism.

    Science.gov (United States)

    Shipley, Mackenzie M; Mangold, Colleen A; Kuny, Chad V; Szpara, Moriah L

    2017-12-01

    Neuron-virus interactions that occur during herpes simplex virus (HSV) infection are not fully understood. Neurons are the site of lifelong latency and are a crucial target for long-term suppressive therapy or viral clearance. A reproducible neuronal model of human origin would facilitate studies of HSV and other neurotropic viruses. Current neuronal models in the herpesvirus field vary widely and have caveats, including incomplete differentiation, nonhuman origins, or the use of dividing cells that have neuropotential but lack neuronal morphology. In this study, we used a robust approach to differentiate human SH-SY5Y neuroblastoma cells over 2.5 weeks, producing a uniform population of mature human neuronal cells. We demonstrate that terminally differentiated SH-SY5Y cells have neuronal morphology and express proteins with subcellular localization indicative of mature neurons. These neuronal cells are able to support a productive HSV-1 infection, with kinetics and overall titers similar to those seen in undifferentiated SH-SY5Y cells and the related SK-N-SH cell line. However, terminally differentiated, neuronal SH-SY5Y cells release significantly less extracellular HSV-1 by 24 h postinfection (hpi), suggesting a unique neuronal response to viral infection. With this model, we are able to distinguish differences in neuronal spread between two strains of HSV-1. We also show expression of the antiviral protein cyclic GMP-AMP synthase (cGAS) in neuronal SH-SY5Y cells, which is the first demonstration of the presence of this protein in nonepithelial cells. These data provide a model for studying neuron-virus interactions at the single-cell level as well as via bulk biochemistry and will be advantageous for the study of neurotropic viruses in vitro IMPORTANCE Herpes simplex virus (HSV) affects millions of people worldwide, causing painful oral and genital lesions, in addition to a multitude of more severe symptoms such as eye disease, neonatal infection, and, in rare

  6. Bee venom protects SH-SY5Y human neuroblastoma cells from 1-methyl-4-phenylpyridinium-induced apoptotic cell death.

    Science.gov (United States)

    Doo, Ah-Reum; Kim, Seung-Nam; Kim, Seung-Tae; Park, Ji-Yeun; Chung, Sung-Hyun; Choe, Bo-Young; Chae, Younbyoung; Lee, Hyejung; Yin, Chang-Shik; Park, Hi-Joon

    2012-01-06

    Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by progressive selective loss of dopaminergic neurons in the substantia nigra. Recently, bee venom was reported to protect dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced mice PD model, however, the underlying mechanism is not fully understood. The objective of the present study is to investigate the neuroprotective mechanism of bee venom against Parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP(+)), in SH-SY5Y human neuroblastoma cells. Our results revealed that bee venom pretreatment (1-100 ng/ml) increased the cell viability and decreased apoptosis assessed by DNA fragmentation and caspase-3 activity assays in MPP(+)-induced cytotoxicity in SH-SY5Y cells. Bee venom increased the anti-apoptotic Bcl-2 expression and decreased the pro-apoptotic Bax, cleaved PARP expressions. In addition, bee venom prevented the MPP(+)-induced suppression of Akt phosphorylation, and the neuroprotective effect of bee venom against MPP(+)-induced cytotoxicity was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. These results suggest that the anti-apoptotic effect of bee venom is mediated by the cell survival signaling, the PI3K/Akt pathway. These results provide new evidence for elucidating the mechanism of neuroprotection of bee venom against PD. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. PPARbeta agonists trigger neuronal differentiation in the human neuroblastoma cell line SH-SY5Y.

    Science.gov (United States)

    Di Loreto, S; D'Angelo, B; D'Amico, M A; Benedetti, E; Cristiano, L; Cinque, B; Cifone, M G; Cerù, M P; Festuccia, C; Cimini, A

    2007-06-01

    Neuroblastomas are pediatric tumors originating from immature neuroblasts in the developing peripheral nervous system. Differentiation therapies could help lowering the high mortality due to rapid tumor progression to advanced stages. Oleic acid has been demonstrated to promote neuronal differentiation in neuronal cultures. Herein we report on the effects of oleic acid and of a specific synthetic PPARbeta agonist on cell growth, expression of differentiation markers and on parameters responsible for the malignancy such as adhesion, migration, invasiveness, BDNF, and TrkB expression of SH-SY5Y neuroblastoma cells. The results obtained demonstrate that many, but not all, oleic acid effects are mediated by PPARbeta and support a role for PPARbeta in neuronal differentiation strongly pointing towards PPAR ligands as new therapeutic strategies against progression and recurrences of neuroblastoma.

  8. BDNF and the maturation of posttranscriptional regulatory networks in human SH-SY5Y neuroblast differentiation

    Directory of Open Access Journals (Sweden)

    Belinda J Goldie

    2014-10-01

    Full Text Available The SH-SY5Y culture system is a convenient neuronal model with the potential to elaborate human/primate-specific transcription networks and pathways related to human cognitive disorders. While this system allows for the exploration of specialised features in the human genome, there is still significant debate about how this model should be implemented, and its appropriateness for answering complex functional questions related to human neural architecture. In view of these questions we sought to characterise the posttranscriptional regulatory structure of the two-stage ATRA differentiation, BDNF maturation protocol proposed by Encinas and colleagues (2010 using integrative whole-genome gene and microRNA (miRNA expression analysis. We report that ATRA-BDNF induced significant increases in expression of key synaptic genes, brain-specific miRNA and miRNA biogenesis machinery, and in AChE activity, compared with ATRA alone. Functional annotation clustering associated BDNF more significantly with neuronal terms, and with synaptic terms not found in ATRA-only clusters. While our results support use of SH-SY5Y as a neuronal model, we advocate considered selection of the differentiation agent/s relative to the system being modelled.

  9. BDNF and the maturation of posttranscriptional regulatory networks in human SH-SY5Y neuroblast differentiation.

    Science.gov (United States)

    Goldie, Belinda J; Barnett, Michelle M; Cairns, Murray J

    2014-01-01

    The SH-SY5Y culture system is a convenient neuronal model with the potential to elaborate human/primate-specific transcription networks and pathways related to human cognitive disorders. While this system allows for the exploration of specialized features in the human genome, there is still significant debate about how this model should be implemented, and its appropriateness for answering complex functional questions related to human neural architecture. In view of these questions we sought to characterize the posttranscriptional regulatory structure of the two-stage ATRA differentiation, BDNF maturation protocol proposed by Encinas et al. (2000) using integrative whole-genome gene and microRNA (miRNA) expression analysis. We report that ATRA-BDNF induced significant increases in expression of key synaptic genes, brain-specific miRNA and miRNA biogenesis machinery, and in AChE activity, compared with ATRA alone. Functional annotation clustering associated BDNF more significantly with neuronal terms, and with synaptic terms not found in ATRA-only clusters. While our results support use of SH-SY5Y as a neuronal model, we advocate considered selection of the differentiation agent/s relative to the system being modeled.

  10. Drp-1 dependent mitochondrial fragmentation and protective autophagy in dopaminergic SH-SY5Y cells overexpressing alpha-synuclein.

    Science.gov (United States)

    Martinez, Jimena Hebe; Alaimo, Agustina; Gorojod, Roxana Mayra; Porte Alcon, Soledad; Fuentes, Federico; Coluccio Leskow, Federico; Kotler, Mónica Lidia

    2018-04-01

    Parkinson's disease is a neurodegenerative movement disorder caused by the loss of dopaminergic neurons from substantia nigra. It is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. Additional common features of this disease are the mitochondrial dysfunction and the activation/inhibition of autophagy both events associated to the intracellular accumulation of α-synuclein. The mechanism by which these events contribute to neural degeneration remains unknown. In the present work we investigated the effect of α-synuclein on mitochondrial dynamics and autophagy/mitophagy in SH-SY5Y cells, an in vitro model of Parkinson disease. We demonstrated that overexpression of wild type α-synuclein causes moderated toxicity, ROS generation and mitochondrial dysfunction. In addition, α-synuclein induces the mitochondrial fragmentation on a Drp-1-dependent fashion. Overexpression of the fusion protein Opa-1 prevented both mitochondrial fragmentation and cytotoxicity. On the other hand, cells expressing α-synuclein showed activated autophagy and particularly mitophagy. Employing a genetic strategy we demonstrated that autophagy is triggered in order to protect cells from α-synuclein-induced cell death. Our results clarify the role of Opa-1 and Drp-1 in mitochondrial dynamics and cell survival, a controversial α-synuclein research issue. The findings presented point to the relevance of mitochondrial homeostasis and autophagy in the pathogenesis of PD. Better understanding of the molecular interaction between these processes could give rise to novel therapeutic methods for PD prevention and amelioration. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Neuroprotective Effect of Arctigenin via Upregulation of P-CREB in Mouse Primary Neurons and Human SH-SY5Y Neuroblastoma Cells

    Science.gov (United States)

    Zhang, Nan; Wen, Qingping; Ren, Lu; Liang, Wenbo; Xia, Yang; Zhang, Xiaodan; Zhao, Dan; Sun, Dong; Hu, Yv; Hao, Haiguang; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian; Kang, Tingguo

    2013-01-01

    Arctigenin (Arc) has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ) production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1) protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN) expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB) induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB. PMID:24025424

  12. Neuroprotective Effect of Arctigenin via Upregulation of P-CREB in Mouse Primary Neurons and Human SH-SY5Y Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Tingguo Kang

    2013-09-01

    Full Text Available Arctigenin (Arc has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1 protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB.

  13. Protective effects of TRH and its analogues against various cytotoxic agents in retinoic acid (RA)-differentiated human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Jaworska-Feil, L; Jantas, D; Leskiewicz, M; Budziszewska, B; Kubera, M; Basta-Kaim, A; Lipkowski, A W; Lason, W

    2010-12-01

    TRH (thyroliberin) and its analogues were reported to possess neuroprotective effects in cellular and animal experimental models of acute and chronic neurodegenerative diseases. In the present study we evaluated effects of TRH and its three stable analogues, montirelin (CG-3703), RGH-2202 and Z-TRH (N-(carbobenzyloxy)-pGlutamyl-Histydyl-Proline) on the neuronally differentiated human neuroblastoma SH-SY5Y cell line, which is widely accepted for studying potential neuroprotectants. We found that TRH and all the tested analogues at concentrations 0.1-50 μM attenuated cell damage induced by MPP(+) (2 mM), 3-nitropropionate (10 mM), hydrogen peroxide (0.5 mM), homocysteine (250 μM) and beta-amyloid (20μM) in retinoic acid differentiated SH-SY5Y cells. Furthermore, we demonstrated that TRH and its analogues decreased the staurosporine (0.5 μM)-induced LDH release, caspase-3 activity and DNA fragmentation, which indicate the anti-apoptotic proprieties of these peptides. The neuroprotective effects of TRH (10 μM) and RGH-2202 (10 μM) on St-induced cell death was attenuated by inhibitors of PI3-K pathway (wortmannin and LY294002), but not MAPK/ERK1/2 (PD98059 and U0126). Moreover, TRH and its analogues at neuroprotective concentrations (1 and 10 μM) increased expression of Bcl-2 protein, as confirmed by Western blot analysis. All in all, these results extend data on neuroprotective properties of TRH and its analogues and provide evidence that mechanism of anti-apoptotic effects of these peptides in SH-SY5Y cell line involves induction of PI3K/Akt pathway and Bcl-2. Furthermore, the data obtained on human cell line with a dopaminergic phenotype suggest potential utility of TRH and its analogues in the treatment of some neurodegenerative diseases including Parkinson's disease. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2-M17 Neuroblastoma Cell Lines upon Differentiation.

    Directory of Open Access Journals (Sweden)

    Roberta Filograna

    Full Text Available Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate on the human neuroblastoma SH-SY5Y and BE(2-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2-M17 represent two alternative cell models for the neuroscience field.

  15. Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation.

    Science.gov (United States)

    Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

    2015-01-01

    Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field.

  16. Human neuroblastoma SH-SY5Y cells show increased resistance to hyperthermic stress after differentiation, associated with elevated levels of Hsp72.

    Science.gov (United States)

    Cheng, Lesley; Smith, Danielle J; Anderson, Robin L; Nagley, Phillip

    2011-01-01

    Terminally differentiated neurones in the central nervous system need to be protected from stress. We ask here whether differentiation of progenitor cells to neurones is accompanied by up-regulation of Hsp72, with acquisition of enhanced thermotolerance. Human neuroblastoma SH-SY5Y cells were propagated in an undifferentiated form and subsequently differentiated into neurone-like cells. Thermotolerance tests were carried out by exposure of cells to various temperatures, monitoring nuclear morphology as index of cell death. Abundance of Hsp72 was measured in cell lysates by western immunoblotting. The differentiation of SH-SY5Y cells was accompanied by increased expression of Hsp72. Further, in both cell states, exposure to mild hyperthermic stress (43°C for 30 min) increased Hsp72 expression. After differentiation, SH-SY5Y cells were more resistant to hyperthermic stress compared to their undifferentiated state, correlating with levels of Hsp72. Stable exogenous expression of Hsp72 in SH-SY5Y cells (transfected line 5YHSP72.1, containing mildly elevated levels of Hsp72), led to enhanced resistance to hyperthermic stress. Hsp72 was found to be inducible in undifferentiated 5YHSP72.1 cells; such heat-treated cells displayed enhanced thermotolerance. Treatment of cells with KNK437, a suppressor of Hsp72 induction, resulted in acute thermosensitisation of all cell types tested here. Hsp72 has a major role in the enhanced hyperthermic resistance acquired during neuronal differentiation of SH-SY5Y cells. These findings model the requirement in intact organisms for highly differentiated neurones to be specially protected against thermal stress.

  17. Chrysotoxine, a novel bibenzyl compound selectively antagonizes MPP⁺, but not rotenone, neurotoxicity in dopaminergic SH-SY5Y cells.

    Science.gov (United States)

    Song, Ju-Xian; Shaw, Pang-Chui; Wong, Ngok-Shun; Sze, Cho-Wing; Yao, Xin-Sheng; Tang, Chi-Wai; Tong, Yao; Zhang, Yan-Bo

    2012-07-11

    Chrysotoxine is a naturally occurring bibenzyl compound found in medicinal Dendrobium species. We previously reported that chrysotoxine structure-specifically suppressed 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell death. Whether chrysotoxine and other structurally similar bibenzyl compounds could also inhibit the neurotoxicity of 1-methyl-4-phenyl pyridinium (MPP(+)) and rotenone has not been investigated. We showed herein that chrysotoxine inhibited MPP(+), but not rotenone, induced dopaminergic cell death in SH-SY5Y cells. The overproduction of reactive oxygen species (ROS), mitochondrial dysfunction as indexed by the decrease in membrane potential, increase in calcium concentration and NF-κB activation triggered by MPP(+) were blocked by chrysotoxine pretreatment. The imbalance between the pro-apoptotic signals (Bax, caspase-3, ERK and p38 MAPK) and the pro-survival signals (Akt/PI3K/GSK-3β) induced by MPP(+) was partially or totally rectified by chrysotoxine. The results indicated that ROS inhibition, mitochondria protection, NF-κB modulation and regulation of multiple signals determining cell survival and cell death were involved in the protective effects of chrysotoxine against MPP(+) toxicity in SH-SY5Y cells. Given the different toxic profiles of 6-OHDA and MPP(+) as compared to rotenone, our results also indicated that DAT inhibition may partially account for the neuroprotective effects of chrysotoxine. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  18. Coffee induces vascular endothelial growth factor (VEGF) expression in human neuroblastama SH-SY5Y cells.

    Science.gov (United States)

    Kakio, Shota; Funakoshi-Tago, Megumi; Kobata, Kenji; Tamura, Hiroomi

    2017-07-01

    Recent evidence indicates that hypoxia-inducible vascular endothelial growth factor (VEGF) has neurotrophic and neuroprotective effects on neuronal and glial cells. On the other hand, recent epidemiological studies showed that daily coffee consumption has been associated with a lower risk of several neuronal disorders. Therefore, we investigated the effect of coffee on VEGF expression in human neuroblastoma SH-SY5Y cells. We found that even low concentration of coffee (coffee was attributed to the coffee-dependent inhibition of prolyl hydroxylation of HIF1α, which is essential for proteolytic degradation of HIF-1α. However, no inhibition was observed at the catalytic activity in vitro. Coffee component(s) responsible for the activation of HIF-1α was not major constituents such as caffeine, caffeic acid, chlorogenic acid, and trigonelline, but was found to emerge during roasting process. The active component(s) was extractable with ethyl acetate. Our results suggest that daily consumption of coffee may induce VEGF expression in neuronal cells. This might be related to protective effect of coffee on neural disorders such as Alzheimer's disease and Parkinson's disease.

  19. Dichloroacetate stimulates changes in the mitochondrial network morphology via partial mitophagy in human SH-SY5Y neuroblastoma cells

    Czech Academy of Sciences Publication Activity Database

    Pajuelo-Reguera, David; Alán, Lukáš; Olejár, Tomáš; Ježek, Petr

    2015-01-01

    Roč. 46, č. 6 (2015), s. 2409-2418 ISSN 1019-6439 R&D Projects: GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : dichloroacetate * mitochondria * mitophagy * neuroblastoma SH-SY5Y cells * mitochondrial network Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.018, year: 2015

  20. The biologic role of ganglioside in neuronal differentiation--effects of GM1 ganglioside on human neuroblastoma SH-SY5Y cells.

    OpenAIRE

    Lee, M. C.; Lee, W. S.; Park, C. S.; Juhng, S. W.

    1994-01-01

    Human neuroblastoma SH-SY5Y cell is a cloned cell line which has many attractive features for the study of neuronal proliferation and neurite outgrowth, because it has receptors for insulin, IGF-I and PDGF. Gangliosides are sialic acid containing glycosphingolipids which form an integral part of the plasma membrane of many mammalian cells. They inhibit cell growth mediated by tyrosine kinase receptors and ligand-stimulated tyrosine kinase activity, and autophosphorylation of EGF(epidermal gro...

  1. Effect of toluene diisocyanate on homeostasis of intracellular-free calcium in human neuroblastoma SH-SY5Y Cells

    International Nuclear Information System (INIS)

    Liu, P.-S.; Chiung, Y.-M.; Kao, Y.-Y.

    2006-01-01

    The mechanisms of TDI (2,4-toluene diisocyanate)-induced occupational asthma are not fully established. Previous studies have indicated that TDI induces non-specific bronchial hyperreactivity to methacholine and induces contraction of smooth muscle tissue by activating 'capsaicin-sensitive' nerves resulting asthma. Cytosolic-free calcium ion concentrations ([Ca 2+ ] c ) are elevated when either capsaicin acts at vanilloid receptors, or methacholine at muscarinic receptors. This study therefore investigated the effects of TDI on Ca 2+ mobilization in human neuroblastoma SH-SY5Y cells. TDI was found to elevate [Ca 2+ ] c by releasing Ca 2+ from the intracellular stores and extracellular Ca 2+ influx. 500 μM TDI induced a net [Ca 2+ ] c increase of 112 ± 8 and 78 ± 6 nM in the presence and absence of extracellular Ca 2+ , respectively. In Ca 2+ -free buffer, TDI induced Ca 2+ release from internal stores to reduce their Ca 2+ content and this reduction was evidenced by a suppression occurring on the [Ca 2+ ] c rise induced by thapsigargin, ionomycin, and methacholine after TDI incubation. In the presence of extracellular Ca 2+ , simultaneous exposure to TDI and methacholine led a higher level of [Ca 2+ ] c compared to single methacholine stimulation, that might explain that TDI induces bronchial hyperreactivity to methacholine. We conclude that TDI is capable of interfering the [Ca 2+ ] c homeostasis including releasing Ca 2+ from internal stores and inducing extracellular Ca 2+ influx. The interaction of this novel character and bronchial hyperreactivity need further investigation

  2. Hydrogen peroxide modifies both activity and isoforms of acetylcholinesterase in human neuroblastoma SH-SY5Y cells

    Directory of Open Access Journals (Sweden)

    Alba Garcimartín

    2017-08-01

    Full Text Available The involvement of cholinergic system and the reactive oxygen species (ROS in the pathogenesis of some degenerative diseases has been widely reported; however, the specific impact of hydrogen peroxide (H2O2 on the acetylcholinesterase (AChE activity as well as AChE isoform levels has not been clearly established. Hence, the purpose of present study is to clarify whether H2O2 alters these parameters.Human neuroblastoma SH-SY5Y cells were treated with H2O2 (1–1000 µM for 24 h and AChE activity and AChE and cytochrome c levels were evaluated. AChE activity was strongly increased from 1 µM to 1000 µM of H2O2. The results of the kinetic study showed that H2O2 affected Vmax but not Km; and also that H2O2 changed the sigmoid kinetic observed in control samples to hyperbolic kinetic. Thus, results suggest that H2O2 acts as an allosteric activators. In addition, H2O2, (100–1000 µM reduced the total AChE content and modified its isoform profile (mainly 50-, 70-, and 132-kDa·H2O2 from 100 µM to 1000 µM induced cytochrome c release confirming cell death by apoptosis. All these results together suggest: a the involvement of oxidative stress in the imbalance of AChE; and b treatment with antioxidant agents may be a suitable strategy to protect cholinergic system alterations promoted by oxidative stress. Keywords: Acetylcholinesterase, Hydrogen peroxide, Alternative splicing, Cell culture, Cell death

  3. Palmitic acid induces neurotoxicity and gliatoxicity in SH-SY5Y human neuroblastoma and T98G human glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Yee-Wen Ng

    2018-04-01

    Full Text Available Background Obesity-related central nervous system (CNS pathologies like neuroinflammation and reactive gliosis are associated with high-fat diet (HFD related elevation of saturated fatty acids like palmitic acid (PA in neurons and astrocytes of the brain. Methods Human neuroblastoma cells SH-SY5Y (as a neuronal model and human glioblastoma cells T98G (as an astrocytic model, were treated with 100–500 µM PA, oleic acid (OA or lauric acid (LA for 24 h or 48 h, and their cell viability was assessed by 3-(4,5-dimetylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. The effects of stable overexpression of γ-synuclein (γ-syn, a neuronal protein recently recognized as a novel regulator of lipid handling in adipocytes, and transient overexpression of Parkinson’s disease (PD α-synuclein [α-syn; wild-type (wt and its pathogenic mutants A53T, A30P and E46K] in SH-SY5Y and T98G cells, were also evaluated. The effects of co-treatment of PA with paraquat (PQ, a Parkinsonian pesticide, and leptin, a hormone involved in the brain-adipose axis, were also assessed. Cell death mode and cell cycle were analyzed by Annexin V/PI flow cytometry. Reactive oxygen species (ROS level was determined using 2′,7′-dichlorofluorescien diacetate (DCFH-DA assay and lipid peroxidation level was determined using thiobarbituric acid reactive substances (TBARS assay. Results MTT assay revealed dose- and time-dependent PA cytotoxicity on SH-SY5Y and T98G cells, but not OA and LA. The cytotoxicity was significantly lower in SH-SY5Y-γ-syn cells, while transient overexpression of wt α-syn or its PD mutants (A30P and E46K, but not A53T modestly (but still significantly rescued the cytotoxicity of PA in SH-SY5Y and T98G cells. Co-treatment of increasing concentrations of PQ exacerbated PA’s neurotoxicity. Pre-treatment of leptin, an anti-apoptotic adipokine, did not successfully rescue SH-SY5Y cells from PA-induced cytotoxicity—suggesting a mechanism of PA

  4. Protective effect of Pycnogenol in human neuroblastoma SH-SY5Y cells following acrolein-induced cytotoxicity.

    Science.gov (United States)

    Ansari, Mubeen A; Keller, Jeffrey N; Scheff, Stephen W

    2008-12-01

    Oxidative stress is one of the hypotheses involved in the etiology of Alzheimer's disease (AD). Considerable attention has been focused on increasing the intracellular glutathione (GSH) levels in many neurodegenerative diseases, including AD. Pycnogenol (PYC) has antioxidant properties and stabilizes intracellular antioxidant defense systems including glutathione levels. The present study investigated the protective effects of PYC on acrolein-induced oxidative cell toxicity in cultured SH-SY5Y neuroblastoma cells. Decreased cell survival in SH-SY5Y cultures treated with acrolein correlated with oxidative stress, increased NADPH oxidase activity, free radical production, protein oxidation/nitration (protein carbonyl, 3-nitrotyrosine), and lipid peroxidation (4-hydroxy-2-nonenal). Pretreatment with PYC significantly attenuated acrolein-induced cytotoxicity, protein damage, lipid peroxidation, and cell death. A dose-response study suggested that PYC showed protective effects against acrolein toxicity by modulating oxidative stress and increasing GSH. These findings provide support that PYC may provide a promising approach for the treatment of oxidative stress-related neurodegenerative diseases such as AD.

  5. Inhibition of WNT signaling reduces differentiation and induces sensitivity to doxorubicin in human malignant neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Suebsoonthron, Junjira; Jaroonwitchawan, Thiranut; Yamabhai, Montarop; Noisa, Parinya

    2017-06-01

    Neuroblastoma is one of the most common cancers in infancy, arising from the neuroblasts during embryonic development. This cancer is difficult to treat and resistance to chemotherapy is often found; therefore, clinical trials of novel therapeutic approaches, such as targeted-cancer signaling, could be an alternative for a better treatment. WNT signaling plays significant roles in the survival, proliferation, and differentiation of human neuroblastoma. In this report, WNT signaling of a malignant human neuroblastoma cell line, SH-SY5Y cells, was inhibited by XAV939, a specific inhibitor of the Tankyrase enzyme. XAV939 treatment led to the reduction of β-catenin within the cells, confirming its inhibitory effect of WNT. The inhibition of WNT signaling by XAV939 did not affect cell morphology, survival, and proliferation; however, the differentiation and sensitivity to anticancer drugs of human neuroblastoma cells were altered. The treatment of XAV939 resulted in the downregulation of mature neuronal markers, including β-tubulin III, PHOX2A, and PHOX2B, whereas neural progenitor markers (PAX6, TFAP2α, and SLUG) were upregulated. In addition, the combination of XAV939 significantly enhanced the sensitivity of SH-SY5Y and IMR-32 cells to doxorubicin in both 2D and 3D culture systems. Microarray gene expression profiling suggested numbers of candidate target genes of WNT inhibition by XAV939, in particular, p21, p53, ubiquitin C, ZBED8, MDM2, CASP3, and FZD1, and this explained the enhanced sensitivity of SH-SY5Y cells to doxorubicin. Altogether, these results proposed that the altered differentiation of human malignant neuroblastoma cells by inhibiting WNT signaling sensitized the cells to anticancer drugs. This approach could thus serve as an effective treatment option for aggressive brain malignancy.

  6. Evaluation of the neurotoxic/neuroprotective role of organoselenides using differentiated human neuroblastoma SH-SY5Y cell line challenged with 6-hydroxydopamine.

    Science.gov (United States)

    Lopes, Fernanda Martins; Londero, Giovana Ferreira; de Medeiros, Liana Marengo; da Motta, Leonardo Lisbôa; Behr, Guilherme Antônio; de Oliveira, Valeska Aguiar; Ibrahim, Mohammad; Moreira, José Cláudio Fonseca; Porciúncula, Lisiane de Oliveira; da Rocha, João Batista Teixeira; Klamt, Fábio

    2012-08-01

    It is well established that oxidative stress plays a major role in several neurodegenerative conditions, like Parkinson disease (PD). Hence, there is an enormous effort for the development of new antioxidants compounds with therapeutic potential for the management of PD, such as synthetic organoselenides molecules. In this study, we selected between nine different synthetic organoselenides the most eligible ones for further neuroprotection assays, using the differentiated human neuroblastoma SH-SY5Y cell line as in vitro model. Neuronal differentiation of exponentially growing human neuroblastoma SH-SY5Y cells was triggered by cultivating cells with DMEM/F12 medium with 1% of fetal bovine serum (FBS) with the combination of 10 μM retinoic acid for 7 days. Differentiated cells were further incubated with different concentrations of nine organoselenides (0.1, 0.3, 3, 10, and 30 μM) for 24 h and cell viability, neurites densities and the immunocontent of neuronal markers were evaluated. Peroxyl radical scavenging potential of each compound was determined with TRAP assay. Three organoselenides tested presented low cytotoxicity and high antioxidant properties. Pre-treatment of cells with those compounds for 24 h lead to a significantly neuroprotection against 6-hydroxydopamine (6-OHDA) toxicity, which were directly related to their antioxidant properties. Neuroprotective activity of all three organoselenides was compared to diphenyl diselenide (PhSe)₂, the simplest of the diaryl diselenides tested. Our results demonstrate that differentiated human SH-SY5Y cells are suitable cellular model to evaluate neuroprotective/neurotoxic role of compounds, and support further evaluation of selected organoselenium molecules as potential pharmacological and therapeutic drugs in the treatment of PD.

  7. Specific pesticide-dependent increases in α-synuclein levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines.

    Science.gov (United States)

    Chorfa, Areski; Bétemps, Dominique; Morignat, Eric; Lazizzera, Corinne; Hogeveen, Kevin; Andrieu, Thibault; Baron, Thierry

    2013-06-01

    Epidemiological studies indicate a role of genetic and environmental factors in Parkinson's disease involving alterations of the neuronal α-synuclein (α-syn) protein. In particular, a relationship between Parkinson's disease and occupational exposure to pesticides has been repeatedly suggested. Our objective was to precisely assess changes in α-syn levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines following acute exposure to pesticides (rotenone, paraquat, maneb, and glyphosate) using Western blot and flow cytometry. These human cell lines express α-syn endogenously, and overexpression of α-syn (wild type or mutated A53T) can be obtained following recombinant adenoviral transduction. We found that endogenous α-syn levels in the SH-SY5Y neuroblastoma cell line were markedly increased by paraquat, and to a lesser extent by rotenone and maneb, but not by glyphosate. Rotenone also clearly increased endogenous α-syn levels in the SK-MEL-2 melanoma cell line. In the SH-SY5Y cell line, similar differences were observed in the α-syn adenovirus-transduced cells, with a higher increase of the A53T mutated protein. Paraquat markedly increased α-syn in the SK-MEL-2 adenovirus-transduced cell line, similarly for the wild-type or A53T proteins. The observed differences in the propensities of pesticides to increase α-syn levels are in agreement with numerous reports that indicate a potential role of exposure to certain pesticides in the development of Parkinson's disease. Our data support the hypothesis that pesticides can trigger some molecular events involved in this disease and also in malignant melanoma that consistently shows a significant but still unexplained association with Parkinson's disease.

  8. Protective Effects of Fisetin Against 6-OHDA-Induced Apoptosis by Activation of PI3K-Akt Signaling in Human Neuroblastoma SH-SY5Y Cells.

    Science.gov (United States)

    Watanabe, Ryoko; Kurose, Takumi; Morishige, Yuta; Fujimori, Ko

    2018-02-01

    6-Hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS) that are associated with various neurodegenerative diseases such as Parkinson's disease. 3,3',4',7-Tetrahydroxyflavone (fisetin), a plant flavonoid has a variety of physiological effects such as antioxidant activity. In this study, we investigated the molecular mechanism of the neuroprotective effects of fisetin against 6-OHDA-induced cell death in human neuroblastoma SH-SY5Y cells. 6-OHDA-mediated cell toxicity was reduced in a fisetin concentration-dependent manner. 6-OHDA-mediated elevation of the expression of the oxidative stress-related genes such as hemeoxygenase-1, NAD(P)H dehydrogenase quinone 1, NF-E2-related factor 2, and γ-glutamate-cysteine ligase modifier was suppressed by fisetin. Fisetin also lowered the ratio of the proapoptotic Bax protein and the antiapoptotic Bcl-2 protein in SH-SY5Y cells. Moreover, fisetin effectively suppressed 6-OHDA-mediated activation of caspase-3 and caspase-9, which leads to the cell death, while, 6-OHDA-induced caspase-3/7 activity was lowered. Furthermore, fisetin activated the PI3K-Akt signaling, which inhibits the caspase cascade, and fisetin-mediated inhibition of 6-OHDA-induced cell death was negated by the co-treatment with an Akt inhibitor. These results indicate that fisetin protects 6-OHDA-induced cell death by activating PI3K-Akt signaling in human neuronal SH-SY5Y cells. This is the first report that the PI3K-Akt signaling is involved in the fisetin-protected ROS-mediated neuronal cell death.

  9. A fluorescence assay for measuring acetylcholinesterase activity in rat blood and a human neuroblastoma cell line (SH-SY5Y).

    Science.gov (United States)

    Santillo, Michael F; Liu, Yitong

    2015-01-01

    Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of the neurotransmitter acetylcholine, and inhibition of AChE can have therapeutic applications (e.g., drugs for Alzheimer's disease) or neurotoxic consequences (e.g., pesticides). A common absorbance-based AChE activity assay that uses 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) can have limited sensitivity and be prone to interference. Therefore, an alternative assay was developed, in which AChE activity was determined by measuring fluorescence of resorufin produced from coupled enzyme reactions involving acetylcholine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). The Amplex Red assay was used for two separate applications. First, AChE activity was measured in rat whole blood, which is a biomarker for exposure to AChE inhibitor pesticides. Activity was quantified from a 10(5)-fold dilution of whole blood, and there was a linear correlation between Amplex Red and DTNB assays. For the second application, Amplex Red assay was used to measure AChE inhibition potency in a human neuroblastoma cell line (SH-SY5Y), which is important for assessing pharmacological and toxicological potential of AChE inhibitors including drugs, phytochemicals, and pesticides. Five known reversible inhibitors were evaluated (IC50, 7-225 nM), along with irreversible inhibitors chlorpyrifos-oxon (ki=1.01 nM(-1)h(-1)) and paraoxon (ki=0.16 nM(-1)h(-1)). Lastly, in addition to inhibition, AChE reactivation was measured in SH-SY5Y cells incubated with pralidoxime chloride (2-PAM). The Amplex Red assay is a sensitive, specific, and reliable fluorescence method for measuring AChE activity in both rat whole blood and cultured SH-SY5Y cells. Published by Elsevier Inc.

  10. Graphene Oxide–Silver Nanoparticles Nanocomposite Stimulates Differentiation in Human Neuroblastoma Cancer Cells (SH-SY5Y

    Directory of Open Access Journals (Sweden)

    Sangiliyandi Gurunathan

    2017-11-01

    Full Text Available Recently, graphene and graphene related nanocomposite receive much attention due to high surface-to-volume ratio, and unique physiochemical and biological properties. The combination of metallic nanoparticles with graphene-based materials offers a promising method to fabricate novel graphene–silver hybrid nanomaterials with unique functions in biomedical nanotechnology, and nanomedicine. Therefore, this study was designed to prepare graphene oxide (GO silver nanoparticles (AgNPs nanocomposite (GO-AgNPs containing two different nanomaterials in single platform with distinctive properties using luciferin as reducing agents. In addition, we investigated the effect of GO-AgNPs on differentiation in SH-SY5Y cells. The synthesized GO-AgNPs were characterized by ultraviolet-visible absorption spectroscopy (UV-vis, X-ray diffraction (XRD, scanning electron microscopy (SEM, transmission electron microscopy (TEM and Raman spectroscopy. The differentiation was confirmed by series of cellular and biochemical assays. The AgNPs were distributed uniformly on the surface of graphene oxide with an average size of 25 nm. As prepared GO-AgNPOs induces differentiation by increasing the expression of neuronal differentiation markers and decreasing the expression of stem cell markers. The results indicated that the redox biology involved the expression of various signaling molecules, which play an important role in differentiation. This study suggests that GO-AgNP nanocomposite could stimulate differentiation of SH-SY5Y cells. Furthermore, understanding the mechanisms of differentiation of neuroblastoma cells could provide new strategies for cancer and stem cell therapies. Therefore, these studies suggest that GO-AgNPs could target specific chemotherapy-resistant cells within a tumor.

  11. Mechanisms of CDDO-imidazolide-mediated cytoprotection against acrolein-induced neurocytotoxicity in SH-SY5Y cells and primary human astrocytes.

    Science.gov (United States)

    Speen, Adam; Jones, Colton; Patel, Ruby; Shah, Halley; Nallasamy, Palanisamy; Brooke, Elizabeth A S; Zhu, Hong; Li, Y Robert; Jia, Zhenquan

    2015-10-01

    Acrolein is a ubiquitous unsaturated aldehyde has been implicated in the pathogenesis of various neurological disorders. However, limited study has been conducted into potential therapeutic protection and underlying mechanism against acrolein-induced cytotoxicity via upregulation of cellular aldehyde-detoxification defenses. In this study we have utilized RA-differentiated human SH-SY5Y cells and primary human astrocytes to investigate the induction of glutathione (GSH) by the synthetic triterpenoid 2-cyano-3,12-dixooleana-1,9-dien-28-imidazolide (CDDO-Im) and the protective effects CDDO-Im-mediated antioxidant defenses on acrolein toxicity. Acrolein exposure to RA-differentiated SH-SY5Y cells resulted in a significant time dependent depletion of cellular GSH preceding a reduction in cell viability and LDH release. Further, we demonstrated the predominance of cellular GSH in protection against acrolein-induced cytotoxicity. Buthionine sulfoximine (BSO) at 25μM dramatically depleted GSH and significantly potentiated acrolein-induced cytotoxicity. Pretreatment of the cells with 100nM CDDO-Im afforded a dramatic protection against acrolein-induced cytotoxicity. Pretreatment of BSO and CDDO was found to prevent the CDDO-Im-mediated GSH induction and partially reversed the cytoprotective effects of CDDO-Im against acrolein cytotoxicity. Overall, this study represents for the first time the CDDO-Im mediated upregulation of GSH is a predominant mechanism against acrolein-induced neurotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. The SH-SY5Y cell line in Parkinson's disease research: a systematic review.

    Science.gov (United States)

    Xicoy, Helena; Wieringa, Bé; Martens, Gerard J M

    2017-01-24

    Parkinson's disease (PD) is a devastating and highly prevalent neurodegenerative disease for which only symptomatic treatment is available. In order to develop a truly effective disease-modifying therapy, improvement of our current understanding of the molecular and cellular mechanisms underlying PD pathogenesis and progression is crucial. For this purpose, standardization of research protocols and disease models is necessary. As human dopaminergic neurons, the cells mainly affected in PD, are difficult to obtain and maintain as primary cells, current PD research is mostly performed with permanently established neuronal cell models, in particular the neuroblastoma SH-SY5Y lineage. This cell line is frequently chosen because of its human origin, catecholaminergic (though not strictly dopaminergic) neuronal properties, and ease of maintenance. However, there is no consensus on many fundamental aspects that are associated with its use, such as the effects of culture media composition and of variations in differentiation protocols. Here we present the outcome of a systematic review of scientific articles that have used SH-SY5Y cells to explore PD. We describe the cell source, culture conditions, differentiation protocols, methods/approaches used to mimic PD and the preclinical validation of the SH-SY5Y findings by employing alternative cellular and animal models. Thus, this overview may help to standardize the use of the SH-SY5Y cell line in PD research and serve as a future user's guide.

  13. Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

    OpenAIRE

    Khwanraj, Kawinthra; Phruksaniyom, Chareerut; Madlah, Suriyat; Dharmasaroja, Permphan

    2015-01-01

    The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson's disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+ and evaluate the differential expression of tyrosine hydroxylase (TH) in undifferentiated and retinoic acid- (RA-) induced differentiated cells. The western blot results showed a gradual decre...

  14. Am80 induces neuronal differentiation via increased tropomyosin-related kinase B expression in a human neuroblastoma SH-SY5Y cell line.

    Science.gov (United States)

    Shiohira, Hideo; Kitaoka, Akira; Enjoji, Munechika; Uno, Tsukasa; Nakashima, Manabu

    2012-01-01

    Am80, a synthetic retinoid, has been used in differentiation therapy for acute promyelocytic leukemia (APL). All-trans retinoic acid (ATRA) as one of natural retinoid has been also used to treat APL. ATRA treatment causes neuronal differentiation by inducing tropomyosin-related kinase B (TrkB) expression and increasing the sensitivity to brain-derived neurotrophic factor (BDNF), a TrkB ligand. In the present study, we investigated the effects of Am80 on neuronal differentiation, BDNF sensitivity and TrkB expression in human neuroblastoma SH-SY5Y cells. Treatment with Am80 induced morphological differentiation of neurite outgrowth and increased the expression of GAP43 mRNA, a neuronal differentiation marker. Additionally, TrkB protein was also increased, and exogenous BDNF stimulation after treatment with Am80 induced greater neurite outgrowth than without BDNF treatment. These results suggest that Am80 induced neuronal differentiation by increasing TrkB expression and BDNF sensitivity.

  15. Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (. mu. ) and enkephalin (delta) binding sites

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    Kazmi, S.M.I.; Mishra, R.K.

    1986-06-13

    Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of ..mu.. and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of (/sup 3/H)-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin (DADLE) in the presence of 10/sup -5/M D-Pro/sup 4/-morphiceptin (to block the ..mu.. receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of (/sup 3/H)-dihydromorphine, together with the higher potency of morphine analogues to displace (/sup 3/H)-naloxone binding established the presence of ..mu.. sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of (/sup 3/H)-DADLE binding.

  16. Pre-exposure to 50 Hz magnetic fields modifies menadione-induced genotoxic effects in human SH-SY5Y neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Jukka Luukkonen

    Full Text Available BACKGROUND: Extremely low frequency (ELF magnetic fields (MF are generated by power lines and various electric appliances. They have been classified as possibly carcinogenic by the International Agency for Research on Cancer, but a mechanistic explanation for carcinogenic effects is lacking. A previous study in our laboratory showed that pre-exposure to ELF MF altered cancer-relevant cellular responses (cell cycle arrest, apoptosis to menadione-induced DNA damage, but it did not include endpoints measuring actual genetic damage. In the present study, we examined whether pre-exposure to ELF MF affects chemically induced DNA damage level, DNA repair rate, or micronucleus frequency in human SH-SY5Y neuroblastoma cells. METHODOLOGY/PRINCIPAL FINDINGS: Exposure to 50 Hz MF was conducted at 100 µT for 24 hours, followed by chemical exposure for 3 hours. The chemicals used for inducing DNA damage and subsequent micronucleus formation were menadione and methyl methanesulphonate (MMS. Pre-treatment with MF enhanced menadione-induced DNA damage, DNA repair rate, and micronucleus formation in human SH-SY5Y neuroblastoma cells. Although the results with MMS indicated similar effects, the differences were not statistically significant. No effects were observed after MF exposure alone. CONCLUSIONS: The results confirm our previous findings showing that pre-exposure to MFs as low as 100 µT alters cellular responses to menadione, and show that increased genotoxicity results from such interaction. The present findings also indicate that complementary data at several chronological points may be critical for understanding the MF effects on DNA damage, repair, and post-repair integrity of the genome.

  17. Pre-exposure to 50 Hz magnetic fields modifies menadione-induced genotoxic effects in human SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    Luukkonen, Jukka; Liimatainen, Anu; Höytö, Anne; Juutilainen, Jukka; Naarala, Jonne

    2011-03-23

    Extremely low frequency (ELF) magnetic fields (MF) are generated by power lines and various electric appliances. They have been classified as possibly carcinogenic by the International Agency for Research on Cancer, but a mechanistic explanation for carcinogenic effects is lacking. A previous study in our laboratory showed that pre-exposure to ELF MF altered cancer-relevant cellular responses (cell cycle arrest, apoptosis) to menadione-induced DNA damage, but it did not include endpoints measuring actual genetic damage. In the present study, we examined whether pre-exposure to ELF MF affects chemically induced DNA damage level, DNA repair rate, or micronucleus frequency in human SH-SY5Y neuroblastoma cells. Exposure to 50 Hz MF was conducted at 100 µT for 24 hours, followed by chemical exposure for 3 hours. The chemicals used for inducing DNA damage and subsequent micronucleus formation were menadione and methyl methanesulphonate (MMS). Pre-treatment with MF enhanced menadione-induced DNA damage, DNA repair rate, and micronucleus formation in human SH-SY5Y neuroblastoma cells. Although the results with MMS indicated similar effects, the differences were not statistically significant. No effects were observed after MF exposure alone. The results confirm our previous findings showing that pre-exposure to MFs as low as 100 µT alters cellular responses to menadione, and show that increased genotoxicity results from such interaction. The present findings also indicate that complementary data at several chronological points may be critical for understanding the MF effects on DNA damage, repair, and post-repair integrity of the genome.

  18. Heat shock protein 70 modulates neural progenitor cells dynamics in human neuroblastoma SH-SY5Y cells exposed to high glucose content.

    Science.gov (United States)

    Salimi, Leila; Rahbarghazi, Reza; Jafarian, Vahab; Biray Avci, Çıgır; Goker Bagca, Bakiye; Pinar Ozates, Neslihan; Khaksar, Majid; Nourazarian, Alireza

    2018-01-18

    In the current experiment, detrimental effects of high glucose condition were investigated on human neuroblastoma cells. Human neuroblastoma cell line SH-SY5Y were exposed to 5, 40, and 70 mM glucose over a period of 72 h. Survival rate and the proliferation of cells were analyzed by MTT and BrdU incorporation assays. Apoptosis was studied by the assays of flow cytometry and PCR array. In order to investigate the trans-differentiation capacity of the cell into mature neurons, we used immunofluorescence imaging to follow NeuN protein level. The transcription level of HSP70 was shown by real-time PCR analysis. MMP-2 and -9 activities were shown by gelatin Zymography. According to data from MTT and BrdU incorporation assay, 70 mM glucose reduced cell viability and proliferation rate as compared to control (5 mM glucose) and cells treated with 40 mM glucose (P Cell exposure to 70 mM glucose had potential to induced apoptosis after 72 h (P SH-SY5Y cells to detrimental effects of high glucose condition during trans-differentiation into mature neuron-like cells. Real-time PCR analysis confirmed the expression of HSP70 in cells under high content glucose levels, demonstrating the possible cell compensatory response to an insulting condition (p control vs 70 mM group  cells being exposed to 70 mM glucose. High glucose condition could abrogate the dynamics of neural progenitor cells. The intracellular level of HSP70 was proportional to cell damage in high glucose condition. © 2018 Wiley Periodicals, Inc.

  19. Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

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    Hossain, Md. Motarab [Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC (United States); Banik, Naren L. [Department of Neurosciences, Medical University of South Carolina, Charleston, SC (United States); Ray, Swapan K., E-mail: swapan.ray@uscmed.sc.edu [Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC (United States)

    2012-08-01

    Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (-)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-{kappa}B), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro

  20. Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells.

    Science.gov (United States)

    Hossain, Md Motarab; Banik, Naren L; Ray, Swapan K

    2012-08-01

    Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (-)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro

  1. The Human NADPH Oxidase, Nox4, Regulates Cytoskeletal Organization in Two Cancer Cell Lines, HepG2 and SH-SY5Y

    Directory of Open Access Journals (Sweden)

    Simon Auer

    2017-05-01

    Full Text Available NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase, where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors could be studied as a new target for cancer treatment, in particular for inhibition of metastasis.

  2. Enhancement of chemically induced reactive oxygen species production and DNA damage in human SH-SY5Y neuroblastoma cells by 872 MHz radiofrequency radiation

    Energy Technology Data Exchange (ETDEWEB)

    Luukkonen, Jukka [Department of Environmental Science, University of Kuopio, Bioteknia 2, P.O. Box 1627, FI-70211 Kuopio (Finland)], E-mail: Jukka.Luukkonen@uku.fi; Hakulinen, Pasi; Maeki-Paakkanen, Jorma [Department of Environmental Health, National Public Health Institute, P.O. Box 95, FI-70701 Kuopio (Finland); Juutilainen, Jukka; Naarala, Jonne [Department of Environmental Science, University of Kuopio, Bioteknia 2, P.O. Box 1627, FI-70211 Kuopio (Finland)

    2009-03-09

    The objective of the study was to investigate effects of 872 MHz radiofrequency (RF) radiation on intracellular reactive oxygen species (ROS) production and DNA damage at a relatively high SAR value (5 W/kg). The experiments also involved combined exposure to RF radiation and menadione, a chemical inducing intracellular ROS production and DNA damage. The production of ROS was measured using the fluorescent probe dichlorofluorescein and DNA damage was evaluated by the Comet assay. Human SH-SY5Y neuroblastoma cells were exposed to RF radiation for 1 h with or without menadione. Control cultures were sham exposed. Both continuous waves (CW) and a pulsed signal similar to that used in global system for mobile communications (GSM) mobile phones were used. Exposure to the CW RF radiation increased DNA breakage (p < 0.01) in comparison to the cells exposed only to menadione. Comparison of the same groups also showed that ROS level was higher in cells exposed to CW RF radiation at 30 and 60 min after the end of exposure (p < 0.05 and p < 0.01, respectively). No effects of the GSM signal were seen on either ROS production or DNA damage. The results of the present study suggest that 872 MHz CW RF radiation at 5 W/kg might enhance chemically induced ROS production and thus cause secondary DNA damage. However, there is no known mechanism that would explain such effects from CW RF radiation but not from GSM modulated RF radiation at identical SAR.

  3. Enhancement of chemically induced reactive oxygen species production and DNA damage in human SH-SY5Y neuroblastoma cells by 872 MHz radiofrequency radiation

    International Nuclear Information System (INIS)

    Luukkonen, Jukka; Hakulinen, Pasi; Maeki-Paakkanen, Jorma; Juutilainen, Jukka; Naarala, Jonne

    2009-01-01

    The objective of the study was to investigate effects of 872 MHz radiofrequency (RF) radiation on intracellular reactive oxygen species (ROS) production and DNA damage at a relatively high SAR value (5 W/kg). The experiments also involved combined exposure to RF radiation and menadione, a chemical inducing intracellular ROS production and DNA damage. The production of ROS was measured using the fluorescent probe dichlorofluorescein and DNA damage was evaluated by the Comet assay. Human SH-SY5Y neuroblastoma cells were exposed to RF radiation for 1 h with or without menadione. Control cultures were sham exposed. Both continuous waves (CW) and a pulsed signal similar to that used in global system for mobile communications (GSM) mobile phones were used. Exposure to the CW RF radiation increased DNA breakage (p < 0.01) in comparison to the cells exposed only to menadione. Comparison of the same groups also showed that ROS level was higher in cells exposed to CW RF radiation at 30 and 60 min after the end of exposure (p < 0.05 and p < 0.01, respectively). No effects of the GSM signal were seen on either ROS production or DNA damage. The results of the present study suggest that 872 MHz CW RF radiation at 5 W/kg might enhance chemically induced ROS production and thus cause secondary DNA damage. However, there is no known mechanism that would explain such effects from CW RF radiation but not from GSM modulated RF radiation at identical SAR

  4. Impact of haloperidol and quetiapine on the expression of genes encoding antioxidant enzymes in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Schmidt, Andreas Johannes; Hemmeter, Ulrich Michael; Krieg, Jürgen-Christian; Vedder, Helmut; Heiser, Philip

    2009-05-01

    Antipsychotics are known to alter antioxidant activities in vivo. Therefore, the aim of the present study was to examine in the human neuroblastoma SH-SY5Y cell line the impact of a typical (haloperidol) and an atypical (quetiapine) antipsychotic on the expression of genes encoding the key enzymes of the antioxidant metabolism (Cu, Zn superoxide dismutase; Mn superoxide dismutase; glutathione peroxidase; catalase) and enzymes of the glutathione metabolism (gamma-glutamyl cysteine synthetase, glutathione-S-transferase, gamma-glutamyltranspeptidase, glutathione reductase). The cells were incubated for 24h with 0.3, 3, 30 and 300microM haloperidol and quetiapine, respectively; mRNA levels were measured by polymerase chain reaction. In the present study, we observed mostly significant decreases of mRNA contents. With respect to the key pathways, we detected mainly effects on the mRNA levels of the hydrogen peroxide detoxifying enzymes. Among the enzymes of the glutathione metabolism, glutathione-S-transferase- and gamma-glutamyltranspeptidase-mRNA levels showed the most prominent effects. Taken together, our results demonstrate a significantly reduced expression of genes encoding for antioxidant enzymes after treatment with the antipsychotics, haloperidol and quetiapine.

  5. Impact of plant extracts tested in attention-deficit/hyperactivity disorder treatment on cell survival and energy metabolism in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Schmidt, Andreas Johannes; Krieg, Jürgen-Christian; Hemmeter, Ulrich Michael; Kircher, Tilo; Schulz, Eberhard; Clement, Hans-Willi; Heiser, Philip

    2010-10-01

    Plant extracts such as Hypericum perforatum and Pycnogenol have been tested as alternatives to the classical ADHD drugs. It has been possible to describe neuroprotective effects of such plant extracts. A reduction of ADHD symptoms could be shown in clinical studies after the application of Pycnogenol, which is a pine bark extract. The impacts of the standardized herbal extracts Hypericum perforatum, Pycnogenol and Enzogenol up to a concentration of 5000 ng/mL on cell survival and energy metabolism in human SH-SY5Y neuroblastoma cells has been investigated in the present examination. Hypericum perforatum significantly decreased the survival of cells after treatment with a concentration of 5000 ng/mL, whereas lower concentrations exerted no significant effects. Pycnogenol( induced a significant increase of cell survival after incubation with a concentration of 32.25 ng/mL and a concentration of 250 ng/mL. Other applied concentrations of Pycnogenol failed to exert significant effects. Treatment with Enzogenol did not lead to significant changes in cell survival.Concerning energy metabolism, the treatment of cells with a concentration of 5000 ng/mL Hypericum perforatum led to a significant increase of ATP levels, whereas treatment with a concentration of 500 ng/mL had no significant effect. Incubation of cells with Pycnogenol and Enzogenol exerted no significant effects.None of the tested substances caused any cytotoxic effect when used in therapeutically relevant concentrations. Copyright © 2010 John Wiley & Sons, Ltd.

  6. Cold Shock Induced Protein RBM3 but Not Mild Hypothermia Protects Human SH-SY5Y Neuroblastoma Cells From MPP+-Induced Neurotoxicity

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    Hai-Jie Yang

    2018-05-01

    Full Text Available The cold shock protein RBM3 can mediate mild hypothermia-related protection in neurodegeneration such as Alzheimer's disease. However, it remains unclear whether RBM3 and mild hypothermia provide same protection in model of Parkinson's disease (PD, the second most common neurodegenerative disorder. In this study, human SH-SY5Y neuroblastoma cells subjected to insult by 1-methyl-4-phenylpyridinium (MPP+ served as an in-vitro model of PD. Mild hypothermia (32°C aggravated MPP+-induced apoptosis, which was boosted when RBM3 was silenced by siRNA. In contrast, overexpression of RBM3 significantly reduced this apoptosis. MPP+ treatment downregulated the expression of RBM3 both endogenously and exogenously and suppressed its induction by mild hypothermia (32°C. In conclusion, our data suggest that cold shock protein RBM3 provides neuroprotection in a cell model of PD, suggesting that RBM3 induction may be a suitable strategy for PD therapy. However, mild hypothermia exacerbates MPP+-induced apoptosis even that RBM3 could be synthesized during mild hypothermia.

  7. N-Methyl-D aspartate receptor-mediated effect on glucose transporter-3 levels of high glucose exposed-SH-SY5Y dopaminergic neurons.

    Science.gov (United States)

    Engin, Ayse Basak; Engin, Evren Doruk; Karakus, Resul; Aral, Arzu; Gulbahar, Ozlem; Engin, Atilla

    2017-11-01

    High glucose and insulin lead to neuronal insulin resistance. Glucose transport into the neurons is achieved by regulatory induction of surface glucose transporter-3 (GLUT3) instead of the insulin. N-methyl-D aspartate (NMDA) receptor activity increases GLUT3 expression. This study explored whether an endogenous NMDA receptor antagonist, kynurenic acid (KynA) affects the neuronal cell viability at high glucose concentrations. SH-SY5Y neuroblastoma cells were exposed to 150-250 mg/dL glucose and 40 μU/mL insulin. In KynA and N-nitro-l-arginine methyl ester (L-NAME) supplemented cultures, oxidative stress, mitochondrial metabolic activity (MTT), nitric oxide as nitrite+nitrate (NOx) and GLUT3 were determined at the end of 24 and 48-h incubation periods. Viable cells were counted by trypan blue dye. High glucose-exposed SH-SY5Y cells showed two-times more GLUT3 expression at second 24-h period. While GLUT3-stimulated glucose transport and oxidative stress was increased, total mitochondrial metabolic activity was significantly reduced. Insulin supplementation to high glucose decreased NOx synthesis and GLUT3 levels, in contrast oxidative stress increased three-fold. KynA significantly reduced oxidative stress, and increased MTT by regulating NOx production and GLUT3 expression. KynA is a noteworthy compound, as an endogenous, specific NMDA receptor antagonist; it significantly reduces oxidative stress, while increasing cell viability at high glucose and insulin concentrations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line.

    Science.gov (United States)

    Graser, Stephanie; Mentrup, Birgit; Schneider, Doris; Klein-Hitpass, Ludger; Jakob, Franz; Hofmann, Christine

    2015-10-01

    Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5Y(TNAPhigh) neuroblastoma cells compared to SH-SY5Y(TNAPlow) cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5Y(TNAPhigh) cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5Y(TNAPhigh) cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5Y(TNAPhigh) cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Extreme sensitivity of gene expression in human SH-SY5Y neurocytes to ultra-low doses of Gelsemium sempervirens

    Science.gov (United States)

    2014-01-01

    Background Gelsemium sempervirens L. (Gelsemium s.) is a traditional medicinal plant, employed as an anxiolytic at ultra-low doses and animal models recently confirmed this activity. However the mechanisms by which it might operate on the nervous system are largely unknown. This work investigates the gene expression of a human neurocyte cell line treated with increasing dilutions of Gelsemium s. extract. Methods Starting from the crude extract, six 100 × (centesimal, c) dilutions of Gelsemium s. (2c, 3c, 4c, 5c, 9c and 30c) were prepared according to the French homeopathic pharmacopoeia. Human SH-SY5Y neuroblastoma cells were exposed for 24 h to test dilutions, and their transcriptome compared by microarray to that of cells treated with control vehicle solutions. Results Exposure to the Gelsemium s. 2c dilution (the highest dose employed, corresponding to a gelsemine concentration of 6.5 × 10-9 M) significantly changed the expression of 56 genes, of which 49 were down-regulated and 7 were overexpressed. Several of the down-regulated genes belonged to G-protein coupled receptor signaling pathways, calcium homeostasis, inflammatory response and neuropeptide receptors. Fisher exact test, applied to the group of 49 genes down-regulated by Gelsemium s. 2c, showed that the direction of effects was significantly maintained across the treatment with high homeopathic dilutions, even though the size of the differences was distributed in a small range. Conclusions The study shows that Gelsemium s., a medicinal plant used in traditional remedies and homeopathy, modulates a series of genes involved in neuronal function. A small, but statistically significant, response was detected even to very low doses/high dilutions (up to 30c), indicating that the human neurocyte genome is extremely sensitive to this regulation. PMID:24642002

  10. Arctigenin Confers Neuroprotection Against Mechanical Trauma Injury in Human Neuroblastoma SH-SY5Y Cells by Regulating miRNA-16 and miRNA-199a Expression to Alleviate Inflammation.

    Science.gov (United States)

    Song, Jie; Li, Na; Xia, Yang; Gao, Zhong; Zou, Sa-Feng; Yan, Yu-Hui; Li, Shao-Heng; Wang, Yue; Meng, Ya-Kun; Yang, Jing-Xian; Kang, Ting-Guo

    2016-09-01

    Mechanical trauma injury is a severe insult to neural cells. Subsequent secondary injury involves the release of inflammatory factors that have dramatic consequences for undamaged cells, leading to normal cell death after the initial injury. The present study investigated the capacity for arctigenin (ARC) to prevent secondary effects and evaluated the mechanism underlying the action of microRNA (miRNA)-199a and miRNA-16 in a mechanical trauma injury (MTI) model using SH-SY5Y cells in vitro. SH-SY5Y cells are often applied to in vitro models of neuronal function and differentiation. Recently, miRNAs have been demonstrated to play a crucial role in NF-κB and cholinergic signaling, which can regulate inflammation. The cell model was established by scratch-induced injury of human SH-SY5Y cells, which mimics the characteristics of MTI. A cell counting kit-8 (CCK-8), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunocytochemistry were used to measure cell viability. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the inflammatory cytokine and cholinesterase (CHE) content. The lactate dehydrogenase (LDH) content was measured to assess the degree of cell injury. The mRNA levels were measured by RT-PCR to analyze ARC's mechanism of action. miRNA inhibitors and mimics were used to inhibit and strengthen the expression of miRNAs. Protein expression was detected by western blotting analysis. ARC treatment reduced the TNF-α and IL-6 levels as well as the number of TUNEL+ apoptotic SH-SY5Y cells surrounding the scratch and increased the IL-10 level compared to the controls. ARC attenuated the increase of the cell damage degree and LDH content induced by scratching, indicating increased cell survival. Mechanistic studies showed that ARC upregulated the miRNA-16 and miRNA-199a levels to reduce upstream protein (IKKα and IKKβ) expression and inhibit NF-κB signaling pathway activity; moreover, the increased miRNA-199a suppresses

  11. Identification and classification of genes regulated by phosphatidylinositol 3-kinase- and TRKB-mediated signalling pathways during neuronal differentiation in two subtypes of the human neuroblastoma cell line SH-SY5Y

    OpenAIRE

    Nishida, Yuichiro; Adati, Naoki; Ozawa, Ritsuko; Maeda, Aasami; Sakaki, Yoshiyuki; Takeda, Tadayuki

    2008-01-01

    Abstract Background SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signalling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is unclear. To investigate this mechanism, we compared the gene expression profiles in SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which show a different phenotype during RA-mediated differentiation. Findings SH-SY5Y-A cells diffe...

  12. Interleukin-18 alters protein expressions of neurodegenerative diseases-linked proteins in human SH-SY5Y neuron-like cells

    Directory of Open Access Journals (Sweden)

    Elina M Sutinen

    2014-08-01

    Full Text Available Chronic inflammation and oxidative stress (OS are present in Alzheimer´s disease (AD brains in addition to neuronal loss, Amyloid-β (Aβ plaques and hyperphosphorylated tau-protein neurofibrillary tangles. Previously we showed that levels of the pro-inflammatory cytokine, interleukin-18 (IL-18, are elevated in post-mortem AD brains. IL-18 can modulate the tau kinases, Cdk5 and GSK3β, as well as Aβ-production. IL-18 levels are also increased in AD risk diseases, including type-2 diabetes and obesity. Here, we explored other IL-18 regulated proteins in neuron-like SH-SY5Y cells. Differentiated SH-SY5Y cells, incubated with IL-18 for 24, 48 or 72h, were analyzed by two-dimensional gel electrophoresis (2D-DIGE. Specific altered protein spots were chosen and identified with mass spectrometry and verified by western immunoblotting. IL-18 had time-dependent effects on the SH-SY5Y proteome, modulating numerous protein levels/modifications. We concentrated on those related to OS (DDAH2, peroxiredoxins 2, 3 and 6, DJ-1, BLVRA, Aβ-degradation (MMP14, TIMP2, Aβ-aggregation (Septin-2 and modifications of axon growth and guidance associated, collapsing response mediator protein 2 (CRMP2. IL-18 significantly increased antioxidative enzymes, indicative of OS, and altered levels of glycolytic α- and γ-enolase and multifunctional 14-3-3γ and -ε, commonly affected in neurodegenerative diseases. MMP14, TIMP2, α-enolase and 14-3-3ε, indirectly involved in Aβ metabolism, as well as Septin-2 showed changes that increase Aβ levels. Increased 14-3-3γ may contribute to GSK3β driven tau hyperphosphorylation and CRMP2 Thr514 and Ser522 phosphorylation with the Thr555-site, a target for Rho kinase, showing time-dependent changes. IL-18 also increased caspase-1 levels and vacuolization of the cells. Although our SH-SY5Y cells were not aged, as neurons in AD, our work suggests that heightened or prolonged IL-18 levels can drive protein changes of known

  13. An Involvement of PI3-K/Akt Activation and Inhibition of AIF Translocation in Neuroprotective Effects of Undecylenic Acid (UDA) Against Pro-Apoptotic Factors-Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells.

    Science.gov (United States)

    Jantas, Danuta; Piotrowski, Marek; Lason, Wladyslaw

    2015-12-01

    Undecylenic acid (UDA), a naturally occurring 11-carbon unsaturated fatty acid, has been used for several years as an economical antifungal agent and a nutritional supplement. Recently, the potential usefulness of UDA as a neuroprotective drug has been suggested based on the ability of this agent to inhibit μ-calpain activity. In order to verify neuroprotective potential of UDA, we tested protective efficacy of this compound against cell damage evoked by pro-apoptotic factors (staurosporine and doxorubicin) and oxidative stress (hydrogen peroxide) in human neuroblastoma SH-SY5Y cells. We showed that UDA partially protected SH-SY5Y cells against the staurosporine- and doxorubicin-evoked cell death; however, this effect was not connected with its influence on caspase-3 activity. UDA decreased the St-induced changes in mitochondrial and cytosolic AIF level, whereas in Dox-model it affected only the cytosolic AIF content. Moreover, UDA (1-40 μM) decreased the hydrogen peroxide-induced cell damage which was connected with attenuation of hydrogen peroxide-mediated necrotic (PI staining, ADP/ATP ratio) and apoptotic (mitochondrial membrane potential, caspase-3 activation, AIF translocation) changes. Finally, we demonstrated that an inhibitor of PI3-K/Akt (LY294002) but not MAPK/ERK1/2 (U0126) pathway blocked the protection mediated by UDA in all tested models of SH-SY5Y cell injury. These in vitro data point to UDA as potentially effective neuroprotectant the utility of which should be further validated in animal studies. © 2015 Wiley Periodicals, Inc.

  14. Neuroprotective effects of metabotropic glutamate receptor group II and III activators against MPP(+)-induced cell death in human neuroblastoma SH-SY5Y cells: the impact of cell differentiation state.

    Science.gov (United States)

    Jantas, D; Greda, A; Golda, S; Korostynski, M; Grygier, B; Roman, A; Pilc, A; Lason, W

    2014-08-01

    Recent studies have documented that metabotropic glutamate receptors from group II and III (mGluR II/III) are a potential target in the symptomatic treatment of Parkinson's disease (PD), however, the neuroprotective effects of particular mGluR II/III subtypes in relation to PD pathology are recognized only partially. In the present study, we investigated the effect of various mGluR II/III activators in the in vitro model of PD using human neuroblastoma SH-SY5Y cell line and mitochondrial neurotoxin MPP(+). We demonstrated that all tested mGluR ligands: mGluR II agonist - LY354740, mGluR III agonist - ACPT-I, mGluR4 PAM - VU0361737, mGluR8 agonist - (S)-3,4-DCPG, mGluR8 PAM - AZ12216052 and mGluR7 allosteric agonist - AMN082 were protective against MPP(+)-evoked cell damage in undifferentiated (UN-) SH-SY5Y cells with the highest neuroprotection mediated by mGluR8-specific agents. However, in retinoic acid- differentiated (RA-) SH-SY5Y cells we found protection mediated only by mGluR8 activators. We also demonstrated the cell proliferation stimulating effect for mGluR4 and mGluR8 PAMs. Next, we showed that the protection mediated by mGluR II/III activators in UN-SH-SY5Y was not accompanied by the modulation of caspase-3 activity, however, a decrease in the number of apoptotic nuclei was found. Finally, we showed that the inhibitor of necroptosis, necrostatin-1 blocked the mGluR III-mediated protection. Altogether our comparative in vitro data add a further proof to neuroprotective effects of mGluR agonists or PAMs and point to mGluR8 as a promising target for neuroprotective interventions in PD. The results also suggest the participation of necroptosis-related molecular pathways in neuroprotective effects of mGluR III activation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. The human VGF-derived bioactive peptide TLQP-21 binds heat shock 71 kDa protein 8 (HSPA8on the surface of SH-SY5Y cells.

    Directory of Open Access Journals (Sweden)

    Shamim Akhter

    Full Text Available VGF (non-acronymicis a secreted chromogranin/secretogranin that gives rise to a number of bioactive peptides by a complex proteolysis mechanism. VGF-derived peptides exert an extensive array of biological effects in energy metabolism, mood regulation, pain, gastric secretion function, reproduction and, perhaps, cancer. It is therefore surprising that very little is known about receptors and binding partners of VGF-derived peptides and their downstream molecular mechanisms of action. Here, using affinity chromatography and mass spectrometry-based protein identification, we have identified the heat shock cognate 71 kDa protein A8 (HSPA8as a binding partner of human TLQP-21 on the surface of human neuroblastomaSH-SY5Y cells. Binding of TLQP-21 to membrane associated HSPA8 in live SH-SY5Y cells was further supported by cross-linking to live cells. Interaction between HSPA8 and TLQP-21 was confirmed in vitro by label-free Dynamic Mass Redistribution (DMR studies. Furthermore, molecular modeling studies show that TLQP-21 can be docked into the HSPA8 peptide binding pocket. Identification of HSPA8 as a cell surface binding partner of TLQP-21 opens new avenues to explore the molecular mechanisms of its physiological actions, and of pharmacological modulation thereof.

  16. Vasoactive intestinal peptide-induced neurite remodeling in human neuroblastoma SH-SY5Y cells implicates the Cdc42 GTPase and is independent of Ras-ERK pathway

    International Nuclear Information System (INIS)

    Alleaume, Celine; Eychene, Alain; Harnois, Thomas; Bourmeyster, Nicolas; Constantin, Bruno; Caigneaux, Evelyne; Muller, Jean-Marc; Philippe, Michel

    2004-01-01

    Vasoactive intestinal peptide (VIP) is known to regulate proliferation or differentiation in normal and tumoral cells. SH-SY5Y is a differentiated cell subclone derived from the SK-N-SH human neuroblastoma cell line and possess all the components for an autocrine action of VIP. In the present study, we investigated the morphological changes and intracellular signaling pathways occurring upon VIP treatment of SH-SY5Y cells. VIP induced an early remodeling of cell projections: a branched neurite network spread out and prominent varicosities developed along neurites. Although activated by VIP, the Ras/ERK pathway was not required for the remodeling process. In contrast, pull-down experiments revealed a strong Cdc42 activation by VIP while expression of a dominant-negative Cdc42 prevented the VIP-induced neurite changes, suggesting an important role for this small GTPase in the process. These data provide the first evidence for a regulation of the activity of Rho family GTPases by VIP and bring new insights in the signaling pathways implicated in neurite remodeling process induced by VIP in neuroblastoma cells

  17. Neurotoxicity induced by dexamethasone in the human neuroblastoma SH-SY5Y cell line can be prevented by folic acid.

    Science.gov (United States)

    Budni, J; Romero, A; Molz, S; Martín-de-Saavedra, M D; Egea, J; Del Barrio, L; Tasca, C I; Rodrigues, A L S; López, M G

    2011-09-08

    Folic acid (folate) is a vitamin of the B-complex group that is essential for cell replication. Folate is a major determinant of one-carbon metabolism, in which S-adenosylmethionine donates methyl groups that are crucial for neurological function. Many roles for folic acid have been reported, including neuroprotective and antidepressant properties. On the other hand, increased concentrations of corticoids have proven neurotoxic effects and hypersecretion of glucocorticoids has been linked to different mood disorders. The purpose of this study was to investigate the potential protective effect of folic acid on dexamethasone-induced cellular death in SH-SY5Y neuroblastoma cell line and the possible intracellular signaling pathway involved in such effect. Exposure to 1 mM dexamethasone for 48 h caused a significant reduction of cell viability measured as 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) reduction. Exposure of SH-SY5Y cells for 72 h to increasing concentrations of folate (1-300 μM) was not cytotoxic. However, pretreatment with folate (10-300 μM) reduced dexamethasone-induced toxicity in a significant manner. To explore the putative intracellular signaling pathways implicated in the protective effect of folate we used different protein kinase inhibitors. The protective effect of folic acid on dexamethasone-induced neurotoxicity was reversed by the phosphatidylinositol-3 kinase/Akt (PI3K/Akt, LY294002), Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII, KN-93), and protein kinase A (PKA, H-89) inhibitors, but not the mitogen-activated protein/extracellular signal-regulated kinase (MEK1/2, PD98059) and protein kinase C (PKC, chelerythrine) inhibitors. In conclusion, the results of this study show that folic acid can protect against dexamethasone-induced neurotoxicity and its protective mechanism is related to a signaling pathway that involves PI3K/Akt, CaMKII, and PKA. Copyright © 2011. Published by Elsevier Ltd.

  18. The signaling cascades of Ganoderma lucidum extracts in stimulating non-amyloidogenic protein secretion in human neuroblastoma SH-SY5Y cell lines.

    Science.gov (United States)

    Pinweha, Sirinthorn; Wanikiat, Payong; Sanvarinda, Yupin; Supavilai, Porntip

    2008-12-19

    Ganoderma lucidum (GL) is a medicinal mushroom that possesses various pharmacological properties which are also documented in the ancient reports where GL is praised for its effects on the promotion of health and longevity. In this study, we have investigated the effect of GL mycelia extracts on the non-amyloidogenic protein secretion (sAPPalpha) and the amyloid precursor protein (APP) expression in SH-SY5Y neuroblastoma cells. In order to characterize the signaling pathway which mediates GL-enhanced sAPPalpha secretion, we used inhibitors of nerve growth factor (NGF) signaling pathways, phosphatidylinositol 3 kinase (PI3K), phospholipase Cgamma1 (PLCgamma1), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK1/2), to block GL-mediated sAPPalpha secretion as well as ERK1/2 and PKC activation by using Western blot analysis. Our results provided for the first time evidence that GL mycelia extracts increased APP expression and promoted sAPPalpha secretion. In addition, GL extracts activated ERK1/2 and PKC phosphorylation. The complex signaling cascades of PI3K and ERK may be responsible for GL-mediated sAPPalpha secretion.

  19. Lycopene protects human SH-SY5Y neuroblastoma cells against hydrogen peroxide-induced death via inhibition of oxidative stress and mitochondria-associated apoptotic pathways

    Science.gov (United States)

    FENG, CHUNSHENG; LUO, TIANFEI; ZHANG, SHUYAN; LIU, KAI; ZHANG, YANHONG; LUO, YINAN; GE, PENGFEI

    2016-01-01

    Oxidative stress, which is characterized by excessive production of reactive oxygen species (ROS), is a common pathway that results in neuronal injury or death due to various types of pathological stress. Although lycopene has been identified as a potent antioxidant, its effect on hydrogen peroxide (H2O2)-induced neuronal damage remains unclear. In the present study, pretreatment with lycopene was observed to protect SH-SY5Y neuroblastoma cells against H2O2-induced death via inhibition of apoptosis resulting from activation of caspase-3 and translocation of apoptosis inducing factor (AIF) to the nucleus. Furthermore, the over-produced ROS, as well as the reduced activities of anti-oxidative enzymes, superoxide dismutase and catalase, were demonstrated to be alleviated by lycopene. Additionally, lycopene counteracted H2O2-induced mitochondrial dysfunction, which was evidenced by suppression of mitochondrial permeability transition pore opening, attenuation of the decline of the mitochondrial membrane potential, and inhibition of the increase of Bax and decrease of Bcl-2 levels within the mitochondria. The release of cytochrome c and AIF from the mitochondria was also reduced. These results indicate that lycopene is a potent neuroprotectant against apoptosis, oxidative stress and mitochondrial dysfunction, and could be administered to prevent neuronal injury or death. PMID:27035331

  20. Identification and classification of genes regulated by phosphatidylinositol 3-kinase- and TRKB-mediated signalling pathways during neuronal differentiation in two subtypes of the human neuroblastoma cell line SH-SY5Y

    Directory of Open Access Journals (Sweden)

    Sakaki Yoshiyuki

    2008-10-01

    Full Text Available Abstract Background SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA, but the molecular mechanism of activation in the signalling pathway mediated by phosphatidylinositol 3-kinase (PI3K is unclear. To investigate this mechanism, we compared the gene expression profiles in SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E, each of which show a different phenotype during RA-mediated differentiation. Findings SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF for full differentiation. After exposing cells to a PI3K inhibitor, LY294002, we identified 386 genes and categorised these genes into two clusters dependent on the PI3K signalling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster, including 158 neural genes, was greatly reduced in SK-N-SH cells and partially impaired in SH-SY5Y-E cells, which is consistent with a defect in the neuronal phenotype of these cells. Additional stimulation with BDNF induced a set of neural genes that were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in differentiated SH-SY5Y-A cells. Conclusion We identified gene clusters controlled by PI3K- and TRKB-mediated signalling pathways during the differentiation of two subtypes of SH-SY5Y cells. The TRKB-mediated bypass pathway compensates for impaired neural function generated by defects in several signalling pathways, including PI3K in SH-SY5Y-E cells. Our expression profiling data will be useful for further elucidation of the signal transduction-transcriptional network involving PI3K or TRKB.

  1. Identification and classification of genes regulated by phosphatidylinositol 3-kinase- and TRKB-mediated signalling pathways during neuronal differentiation in two subtypes of the human neuroblastoma cell line SH-SY5Y.

    Science.gov (United States)

    Nishida, Yuichiro; Adati, Naoki; Ozawa, Ritsuko; Maeda, Aasami; Sakaki, Yoshiyuki; Takeda, Tadayuki

    2008-10-28

    SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signalling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is unclear. To investigate this mechanism, we compared the gene expression profiles in SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which show a different phenotype during RA-mediated differentiation. SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. After exposing cells to a PI3K inhibitor, LY294002, we identified 386 genes and categorised these genes into two clusters dependent on the PI3K signalling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster, including 158 neural genes, was greatly reduced in SK-N-SH cells and partially impaired in SH-SY5Y-E cells, which is consistent with a defect in the neuronal phenotype of these cells. Additional stimulation with BDNF induced a set of neural genes that were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in differentiated SH-SY5Y-A cells. We identified gene clusters controlled by PI3K- and TRKB-mediated signalling pathways during the differentiation of two subtypes of SH-SY5Y cells. The TRKB-mediated bypass pathway compensates for impaired neural function generated by defects in several signalling pathways, including PI3K in SH-SY5Y-E cells. Our expression profiling data will be useful for further elucidation of the signal transduction-transcriptional network involving PI3K or TRKB.

  2. SIRB, sans iron oxide rhodamine B, a novel cross-linked dextran nanoparticle, labels human neuroprogenitor and SH-SY5Y neuroblastoma cells and serves as a USPIO cell labeling control.

    Science.gov (United States)

    Shen, Wei-Bin; Vaccaro, Dennis E; Fishman, Paul S; Groman, Ernest V; Yarowsky, Paul

    2016-05-01

    This is the first report of the synthesis of a new nanoparticle, sans iron oxide rhodamine B (SIRB), an example of a new class of nanoparticles. SIRB is designed to provide all of the cell labeling properties of the ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle Molday ION Rhodamine B (MIRB) without containing the iron oxide core. MIRB was developed to label cells and allow them to be tracked by MRI or to be manipulated by magnetic gradients. SIRB possesses a similar size, charge and cross-linked dextran coating as MIRB. Of great interest is understanding the biological and physiological changes in cells after they are labeled with a USPIO. Whether these effects are due to the iron oxide buried within the nanoparticle or to the surface coating surrounding the iron oxide core has not been considered previously. MIRB and SIRB represent an ideal pairing of nanoparticles to identify nanoparticle anatomy responsible for post-labeling cytotoxicity. Here we report the effects of SIRB labeling on the SH-SY5Y neuroblastoma cell line and primary human neuroprogenitor cells (hNPCs). These effects are contrasted with the effects of labeling SH-SY5Y cells and hNPCs with MIRB. We find that SIRB labeling, like MIRB labeling, (i) occurs without the use of transfection reagents, (ii) is packaged within lysosomes distributed within cell cytoplasm, (iii) is retained within cells with no loss of label after cell storage, and (iv) does not alter cellular viability or proliferation, and (v) SIRB labeled hNPCs differentiate normally into neurons or astrocytes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol, Retinoic Acid and Cholesterol

    OpenAIRE

    Teppola, Heidi; Sarkanen, Jertta-Riina; Jalonen, Tuula O.; Linne, Marja-Leena

    2015-01-01

    Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was...

  4. Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol, Retinoic Acid and Cholesterol

    OpenAIRE

    Teppola, Heidi; Sarkanen, Jertta-Riina; Jalonen, Tuula; Linne, Marja-Leena

    2016-01-01

    Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was...

  5. Kalopanacis Cortex extract-capped gold nanoparticles activate NRF2 signaling and ameliorate damage in human neuronal SH-SY5Y cells exposed to oxygen–glucose deprivation and reoxygenation

    Directory of Open Access Journals (Sweden)

    Park SY

    2017-06-01

    Full Text Available Sun Young Park,1 Seon Yeong Chae,1,2 Jin Oh Park,2 Kyu Jin Lee,2 Geuntae Park1,2 1Bio-IT Fusion Technology Research Institute, 2Department of Nanofusion Technology, Graduate School, Pusan National University, Busan, Republic of Korea Abstract: Recently, environment-friendly synthesis of gold nanoparticles (GNPs has been extensively explored by biologists and chemists. However, significant research is still required to determine whether “eco-friendly” GNPs are beneficial to human health and to elucidate the molecular mechanisms of their effects on human cells. We used human neuronal SH-SY5Y cells to show that treatment with Kalopanacis Cortex extract-capped GNPs (KC-GNs, prepared via an eco-friendly, fast, one-pot synthetic route, protected neuronal cells against oxygen–glucose deprivation/reoxygenation (OGD/R-induced damage. To prepare GNPs, Kalopanacis Cortex was used without any chemical reducing and stabilizing agents. Ultraviolet–visible spectroscopy showed maximum absorbance at 526 nm owing to KC-GN surface plasmon resonance. Hydrodynamic size (54.02±2.19 nm and zeta potential (-20.3±0.04 mV were determined by dynamic light scattering. The average diameter (41.07±3.05 nm was determined by high-resolution transmission electron microscopy. Energy-dispersive X-ray diffraction spectroscopy and X-ray diffraction confirmed the presence of assembled GNPs. Fourier transform infrared analysis suggested that functional groups such as O–H, C–C, and C–N participated in KC-GN formation. Cell viability assays indicated that KC-GNs restored the viability of OGD/R-treated SH-SY5Y cells. Flow cytometry demonstrated that KC-GNs inhibited the OGD/R-induced reactive oxygen species production and mitochondrial membrane potential disruption. KC-GNs also inhibited the apoptosis of OGD/R-exposed cells. Western blot analysis indicated that the OGD/R-induced cellular apoptosis and simultaneous increases in the expression of cleaved caspase-3, p

  6. Survivin knockdown increased anti-cancer effects of (−)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

    International Nuclear Information System (INIS)

    Hossain, Md. Motarab; Banik, Naren L.; Ray, Swapan K.

    2012-01-01

    Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (−)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro

  7. Survivin knockdown increased anti-cancer effects of (−)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N- BE2 and SH-SY5Y cells

    Science.gov (United States)

    Hossain, Md. Motarab; Banik, Naren L.; Ray, Swapan K.

    2012-01-01

    Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (−)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in expression of NFP, NSE, and e-cadherin and also decreases in expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro network

  8. Neuroprotective effects of Salvia aristata Aucher ex Benth. on hydrogen peroxide induced apoptosis in SH-SY5Y neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    M. A. Esmaeili

    2015-08-01

    Full Text Available Background and objectives: Oxidative stress is implicated in the neuronal damage associated with Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotropic lateral sclerosis and cerebral ischemic stroke. The present work was designed to establish the neuroprotective effects of Salvia aristata extract on H2O2-induced apoptosis in human dopaminergic SH-SY5Y cells. Methods: The total phenol and flavonoids contents of the plant extracts were quantified by colorimetric methods. The antioxidant activity was assessed using DPPH free radicals scavenging activity assay, and the neuroprotective effect on H2O2-induced oxidative stress was also investigated using human dopaminergic SH-SY5Y cells by MTT assay and western blotting techniques. Results: The highest scavenging activity was found for methanol extract of S. aristata roots (85.28 ± 2.61 μg/mL, with the highest total phenolic and flavonoids content (90.28 mg total phenols as gallic acid and 250.12 mg total flavonoids as rutin, respectively. Our results also, showed that H2O2-induced cytotoxicity in SH-SY5Y cells was suppressed by treatment with S. aristata. Moreover, S. aristata root extract was effective in attenuating the disruption of mitochondrial membrane potential and apoptotic cell death has induced by H2O2.  S. aristata suppressed the down-regulation of Bcl-2, upregulation of Bax, and the release of mitochondrial cytochrome c to cytosol. In addition, S. aristata attenuated caspase-3, and -9 activation, and eventually protected the cells against H2O2-induced apoptosis. Conclusion: Theresults of the present study suggest that treatment of SH-SY5Y cells with S. aristata could block H2O2-induced apoptosis by regulating Bcl-2 family members and by suppressing caspase cascade activation.

  9. Paracrine Maturation and Migration of SH-SY5Y Cells by Dental Pulp Stem Cells.

    Science.gov (United States)

    Gervois, P; Wolfs, E; Dillen, Y; Hilkens, P; Ratajczak, J; Driesen, R B; Vangansewinkel, T; Bronckaers, A; Brône, B; Struys, T; Lambrichts, I

    2017-06-01

    Neurological disorders are characterized by neurodegeneration and/or loss of neuronal function, which cannot be adequately repaired by the host. Therefore, there is need for novel treatment options such as cell-based therapies that aim to salvage or reconstitute the lost tissue or that stimulate host repair. The present study aimed to evaluate the paracrine effects of human dental pulp stem cells (hDPSCs) on the migration and neural maturation of human SH-SY5Y neuroblastoma cells. The hDPSC secretome had a significant chemoattractive effect on SH-SY5Y cells as shown by a transwell assay. To evaluate neural maturation, SH-SY5Y cells were first induced toward neuronal cells, after which they were exposed to the hDPSC secretome. In addition, SH-SY5Y cells subjected to the hDPSC secretome showed increased neuritogenesis compared with nonexposed cells. Maturated cells were shown to increase immune reactivity for neuronal markers compared with controls. Ultrastructurally, retinoic acid (RA) signaling and subsequent exposure to the hDPSC secretome induced a gradual rise in metabolic activity and neuronal features such as multivesicular bodies and cytoskeletal elements associated with cellular communication. In addition, electrophysiological recordings of differentiating cells demonstrated a transition toward a neuronal electrophysiological profile based on the maximum tetrodotoxin (TTX)-sensitive, Na + current. Moreover, conditioned medium (CM)-hDPSC-maturated SH-SY5Y cells developed distinct features including, Cd 2+ -sensitive currents, which suggests that CM-hDPSC-maturated SH-SY5Y acquired voltage-gated Ca 2+ channels. The results reported in this study demonstrate the potential of hDPSCs to support differentiation and recruitment of cells with neuronal precursor characteristics in a paracrine manner. Moreover, this in vitro experimental design showed that the widely used SH-SY5Y cell line can improve and simplify the preclinical in vitro research on the molecular

  10. Ursodeoxycholic acid suppresses mitochondria-dependent programmed cell death induced by sodium nitroprusside in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Chun, Hong Sung; Low, Walter C.

    2012-01-01

    Although ursodeoxycholic acid (UDCA) and its highly water-soluble formula (Yoo's solution; YS) have been shown to prevent neuronal damage, the effects of UDCA or YS against Parkinson's disease (PD)-related dopaminergic cell death has not been studied. This study investigated the protective effects of UDCA and YS on sodium nitroprusside (SNP)-induced cytotoxicity in human dopaminergic SH-SY5Y cells. Both UDCA (50–200 μM) and YS (100–200 μM) dose-dependently prevented SNP (1 mM)-induced cell death. Results showed that both UDCA and YS effectively attenuated the production of total reactive oxygen species (ROS), peroxynitrite (ONOO − ) and nitric oxide (NO), and markedly inhibited the mitochondrial membrane potential (MMP) loss and intracellular reduced glutathione (GSH) depletion. SNP-induced programmed cell death events, such as nuclear fragmentation, caspase-3/7 and -9 activation, Bcl-2/Bax ratio decrease, and cytochrome c release, were significantly attenuated by both UDCA and YS. Furthermore, selective inhibitor of phosphatidylinositiol-3-kinase (PI3K), LY294002, and Akt/PKB inhibitor, triciribine, reversed the preventive effects of UDCA on the SNP-induced cytotoxicity and Bax translocation. These results suggest that UDCA can protect SH-SY5Y cells under programmed cell death process by regulating PI3K-Akt/PKB pathways.

  11. Differential Effects of Methyl-4-Phenylpyridinium Ion, Rotenone, and Paraquat on Differentiated SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    João Barbosa Martins

    2013-01-01

    Full Text Available Paraquat (PQ, a cationic nonselective bipyridyl herbicide, has been used as neurotoxicant to modulate Parkinson’s disease in laboratory settings. Other compounds like rotenone (ROT, a pesticide, and 1-methyl-4-phenylpyridinium ion (MPP+ have been widely used as neurotoxicants. We compared the toxicity of these three neurotoxicants using differentiated dopaminergic SH-SY5Y human cells, aiming to elucidate their differential effects. PQ-induced neurotoxicity was shown to be concentration and time dependent, being mitochondrial dysfunction followed by neuronal death. On the other hand, cells exposure to MPP+ induced mitochondrial dysfunction, but not cellular lyses. Meanwhile, ROT promoted both mitochondrial dysfunction and neuronal death, revealing a biphasic pattern. To further elucidate PQ neurotoxic mechanism, several protective agents were used. SH-SY5Y cells pretreatment with tiron (TIR and 2-hydroxybenzoic acid sodium salt (NaSAL, both antioxidants, and Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, a nitric oxide synthase inhibitor, partially protected against PQ-induced cell injury. Additionally, 1-(2-[bis(4-fluorophenylmethoxy]ethyl-4-(3-phenyl-propylpiperazine (GBR 12909, a dopamine transporter inhibitor, and cycloheximide (CHX, a protein synthesis inhibitor, also partially protected against PQ-induced cell injury. In conclusion, we demonstrated that PQ, MPP+, and ROT exerted differential toxic effects on dopaminergic cells. PQ neurotoxicity occurred through exacerbated oxidative stress, with involvement of uptake through the dopamine transporter and protein synthesis.

  12. SNJ-1945, a calpain inhibitor, protects SH-SY5Y cells against MPP+ and rotenone

    OpenAIRE

    Knaryan, Varduhi H.; Samantaray, Supriti; Sookyoung, Park; Azuma, Mitsuyoshi; Inoue, Jun; Banik, Naren L.

    2013-01-01

    Complex pathophysiology of Parkinson’s disease (PD) involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in PD and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. In order to unravel these mechanisms SH-SY5Y neuroblastoma cells were differentiated into dopaminergic and cholinergic phenotypes respectively and used as cell culture model following exposure to two...

  13. Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

    Directory of Open Access Journals (Sweden)

    Kawinthra Khwanraj

    2015-01-01

    Full Text Available The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson’s disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+ and evaluate the differential expression of tyrosine hydroxylase (TH in undifferentiated and retinoic acid- (RA- induced differentiated cells. The western blot results showed a gradual decrease in TH in undifferentiated cells and a gradual increase in TH in differentiated cells from days 4 to 10 after cell plating. Immunostaining revealed a gradual increase in TH along with neuritic outgrowth in differentiated cells on days 4 and 7 of RA treatment. For the study on cell susceptibility to MPP+ and the expression of apoptosis-related genes, MTT assay showed a decrease in cell viability to approximately 50% requiring 500 and 1000 μM of MPP+ for undifferentiated and RA-differentiated cells, respectively. Using real-time RT-PCR, treatment with 500 μM MPP+ led to significant increases in the Bax/Bcl-2 ratio, p53, and caspase-3 in undifferentiated cells but was without significance in differentiated cells. In conclusion, differentiated cells may be more suitable, and the shorter duration of RA differentiation may make the SH-SY5Y cell model more accessible.

  14. Protection against 1-methyl-4-phenyl pyridinium-induced neurotoxicity in human neuroblastoma SH-SY5Y cells by Soyasaponin I by the activation of the phosphoinositide 3-kinase/AKT/GSK3β pathway.

    Science.gov (United States)

    Guo, Zheng; Cao, Wei; Zhao, Shifeng; Han, Zengtai; Han, Boxiang

    2016-07-06

    Parkinson's disease (PD) can be ascribed to the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta, and thus molecules with neuroprotective ability may have therapeutic value against PD. In the current study, the neuroprotective effects and underlying mechanisms of Soyasaponin I (Soya-I), a naturally occurring triterpene extracted from a widely used ingredient in many foods, such as Glycine max (soybean), were evaluated in a widely used cellular PD model in which neurotoxicity was induced by 1-methyl-4-phenyl pyridinium (MPP) in cultured SH-SY5Y cells. We found that Soya-I at 10-40 μM considerably protected against MPP-induced neurotoxicity as evidenced by an increase in cell viability, a decrease in lactate dehydrogenase release, and a reduction in apoptotic nuclei. Moreover, Soya-I effectively inhibited the elevated intracellular accumulation of reactive oxygen species as well as the Bax/Bcl-2 ratio caused by MPP. Most importantly, Soya-I markedly reversed the inhibition of protein expression of phosphorylated AKT and phosphorylated GSK3β caused by MPP. LY294002, the specific inhibitor of phosphoinositide 3-kinase, significantly abrogated the upregulated phosphorylated AKT and phosphorylated GSK3β offered by Soya-I, suggesting that the neuroprotection of Soya-I was mainly dependent on the activation of the phosphoinositide 3-kinase/AKT/GSK3β signaling pathway. The results taken together indicate that Soya-I may be a potential candidate for further preclinical study aimed at the prevention and treatment of PD.

  15. The Secretome of Bone Marrow and Wharton Jelly Derived Mesenchymal Stem Cells Induces Differentiation and Neurite Outgrowth in SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    Ana O. Pires

    2014-01-01

    Full Text Available The goal of this study was to determine and compare the effects of the secretome of mesenchymal stem cells (MSCs isolated from human bone-marrow (BMSCs and the Wharton jelly surrounding the vein and arteries of the umbilical cord (human umbilical cord perivascular cells (HUCPVCs on the survival and differentiation of a human neuroblastoma cell line (SH-SY5Y. For this purpose, SH-SY5Y cells were differentiated with conditioned media (CM from the MSCs populations referred above. Retinoic acid cultured cells were used as control for neuronal differentiated SH-SY5Y cells. SH-SY5Y cells viability assessment revealed that the secretome of BMSCs and HUCPVCs, in the form of CM, was able to induce their survival. Moreover, immunocytochemical experiments showed that CM from both MSCs was capable of inducing neuronal differentiation of SH-SY5Y cells. Finally, neurite lengths assessment and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR analysis demonstrated that CM from BMSCs and HUCPVCs differently induced neurite outgrowth and mRNA levels of neuronal markers exhibited by SH-SY5Y cells. Overall, our results show that the secretome of both BMSCs and HUCPVCs was capable of supporting SH-SY5Y cells survival and promoting their differentiation towards a neuronal phenotype.

  16. Glutathione transferase-M2-2 secreted from glioblastoma cell protects SH-SY5Y cells from aminochrome neurotoxicity.

    Science.gov (United States)

    Cuevas, Carlos; Huenchuguala, Sandro; Muñoz, Patricia; Villa, Monica; Paris, Irmgard; Mannervik, Bengt; Segura-Aguilar, Juan

    2015-04-01

    U373MG cells are able to take up aminochrome that induces glutathione transferase M2-2 (GSTM2) expression in a concentration-dependent manner where 100 µM aminochrome increases GSTM2 expression by 2.1-fold (P protects SH-SY5Y cells incubated with 10 µM aminochrome. The significant protection provided by U373MG-conditioned medium in SH-SY5Y cells incubated with aminochrome was dependent on GSTM2 internalization into SH-SY5Y cells as evidenced by (i) uptake of (14)C-GSTM2 released from U373MG cells into SH-SY5Y cells, a process inhibited by anti-GSTM2 antiserum; (ii) lack of protection of U373MG-conditioned medium in the presence of anti-GSTM2 antiserum on SH-SY5Y cells treated with aminochrome; and (iii) lack of protection of conditioned medium from U373MGsiGST6 that expresses an siRNA directed against GSTM2 on SH-SY5Y cells treated with aminochrome. In conclusion, our results demonstrated that U373MG cells protect SH-SY5Y cells against aminochrome neurotoxicity by releasing GSTM2 into the conditioned medium and subsequent internalization of GSTM2 into SH-SY5Y cells. These results suggest a new mechanism of protection of dopaminergic neurons mediated by astrocytes by releasing GSTM2 into the intersynaptic space and subsequent internalization into dopaminergic neuron in order to protect these cells against aminochrome neurotoxicity.

  17. Gadd153 and NF-κB crosstalk regulates 27-hydroxycholesterol-induced increase in BACE1 and β-amyloid production in human neuroblastoma SH-SY5Y cells.

    Directory of Open Access Journals (Sweden)

    Gurdeep Marwarha

    Full Text Available β-amyloid (Aβ peptide, accumulation of which is a culprit for Alzheimer's disease (AD, is derived from the initial cleavage of amyloid precursor protein by the aspartyl protease BACE1. Identification of cellular mechanisms that regulate BACE1 production is of high relevance to the search for potential disease-modifying therapies that inhibit BACE1 to reduce Aβ accumulation and AD progression. In the present study, we show that the cholesterol oxidation product 27-hydroxycholesterol (27-OHC increases BACE1 and Aβ levels in human neuroblastoma SH-SY5Y cells. This increase in BACE1 involves a crosstalk between the two transcription factors NF-κB and the endoplasmic reticulum stress marker, the growth arrest and DNA damage induced gene-153 (gadd153, also called CHOP. We specifically show that 27-OHC induces a substantial increase in NF-κB binding to the BACE1 promoter and subsequent increase in BACE1 transcription and Aβ production. The NF-κB inhibitor, sc514, significantly attenuated the 27-OHC-induced increase in NF-κB-mediated BACE1 expression and Aβ genesis. We further show that the 27-OHC-induced NF-κB activation and increased NF-κB-mediated BACE1 expression is contingent on the increased activation of gadd153. Silencing gadd153 expression with siRNA alleviated the 27-OHC-induced increase in NF-κB activation, NF-κB binding to the BACE1 promoter, and subsequent increase in BACE1 transcription and Aβ production. We also show that increased levels of BACE1 in the triple transgenic mouse model for AD is preceded by gadd153 and NF-κB activation. In summary, our study demonstrates that gadd153 and NF-κB work in concert to regulate BACE1 expression. Agents that inhibit gadd153 activation and subsequent interaction with NF-κB might be promising targets to reduce BACE1 and Aβ overproduction and may ultimately serve as disease-modifying treatments for AD.

  18. Regulation of nicotinic receptor subtypes following chronic nicotinic agonist exposure in M10 and SH-SY5Y neuroblastoma cells

    DEFF Research Database (Denmark)

    Warpman, U; Friberg, L; Gillespie, A

    1998-01-01

    investigated in human neuroblastoma SH-SY5Y cells (expressing alpha3, alpha5, beta2, and beta4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines...

  19. Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol, Retinoic Acid and Cholesterol.

    Science.gov (United States)

    Teppola, Heidi; Sarkanen, Jertta-Riina; Jalonen, Tuula O; Linne, Marja-Leena

    2016-04-01

    Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E2) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E2 (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p differentiation.

  20. Paracrine Maturation and Migration of SH-SY5Y Cells by Dental Pulp Stem Cells

    OpenAIRE

    Gervois, Pascal; Wolfs, Esther; Dillen, Yörg; Hilkens, Petra; Ratajczak, Jessica; Driesen, Ronald; Vangansewinkel, Tim; Bronckaers, Annelies; Brône, Bert; Struys, Tom; Lambrichts, Ivo

    2017-01-01

    Neurological disorders are characterized by neurodegeneration and/or loss of neuronal function, which cannot be adequately repaired by the host. Therefore, there is need for novel treatment options such as cell-based therapies that aim to salvage or reconstitute the lost tissue or that stimulate host repair. The present study aimed to evaluate the paracrine effects of human dental pulp stem cells (hDPSCs) on the migration and neural maturation of human SH-SY5Y neuroblastoma cells. The hDPSC s...

  1. A natural product from Cannabis sativa subsp. sativa inhibits homeodomain-interacting protein kinase 2 (HIPK2), attenuating MPP+-induced apoptosis in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Wang, Guan; Zhu, Lingjuan; Zhao, Yuqian; Gao, Suyu; Sun, Dejuan; Yuan, Jingquan; Huang, Yuxin; Zhang, Xue; Yao, Xinsheng

    2017-06-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a conserved serine/threonine kinase, which regulate transcription, cell differentiation, proliferation and apoptosis. Previous evidences indicated that HIPK2 could be involved in the pathogenesis of neurodegenerative diseases, suggesting as a novel target for Parkinson's disease (PD) therapeutic development. Herein, gene microarray analysis was performed to verify the key regulatory function of HIPK2 in PD. (Z)-methylp-hydroxycinnamate (ZMHC, 7) with other eighteen compounds were isolated from Cannabis sativa subsp. sativa, growing in Bama Yao Autonomous County, one of the five largest longevity regions of the world. Intriguingly, ZMHC was identified to bind HIPK2 with high affinity through molecular modeling and molecular dynamics (MD) simulations. Moreover, cell morphology, flow cytometry and western blot assay suggested that ZMHC inhibited HIPK2, which attenuated MPP + -induced apoptosis in SH-SY5Y cells. In conclusion, these findings discovered a natural product that inhibited HIPK2, and highlighted that ZMHC could be a potential precursor agent for future PD therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. SNJ-1945, a calpain inhibitor, protects SH-SY5Y cells against MPP+ and rotenone

    Science.gov (United States)

    Knaryan, Varduhi H.; Samantaray, Supriti; Sookyoung, Park; Azuma, Mitsuyoshi; Inoue, Jun; Banik, Naren L.

    2014-01-01

    Complex pathophysiology of Parkinson’s disease (PD) involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in PD and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. In order to unravel these mechanisms SH-SY5Y neuroblastoma cells were differentiated into dopaminergic and cholinergic phenotypes respectively and used as cell culture model following exposure to two parkinsonian neurotoxicants MPP+ and rotenone. SNJ-1945, a cell-permeable calpain inhibitor was tested for its neuroprotective efficacy. MPP+ and rotenone dose-dependently elevated the levels of intracellular free Ca2+ and induced a concomitant rise in the levels of active calpain. SNJ-1945 pre-treatment significantly protected cell viability and preserved cellular morphology following MPP+ and rotenone exposure. The neurotoxicants elevated the levels of reactive oxygen species (ROS) more profoundly in SH-SY5Y cells differentiated into dopaminergic phenotype, and this effect could be attenuated with SNJ-1945 pre-treatment. In contrast, significant levels of inflammatory mediators (cyclooxygenase-2, Cox-2 and cleaved p10 fragment of caspase-1) were upregulated in the cholinergic phenotype, which could be dose-dependently attenuated by the calpain inhibitor. Overall, SNJ-1945 was efficacious against MPP+ or rotenone-induced ROS generation, inflammatory mediators, and proteolysis. A post-treatment regimen of SNJ-1945 was also examined in cells and partial protection was attained with calpain inhibitor administration 1–3 h after exposure to MPP+ or rotenone. Taken together these results indicate that calpain inhibition is a valid target for protection against parkinsonian neurotoxicants, and SNJ-1945 is an efficacious calpain inhibitor in this context. PMID:24341912

  3. The Secretome of Bone Marrow and Wharton Jelly Derived Mesenchymal Stem Cells Induces Differentiation and Neurite Outgrowth in SH-SY5Y Cells

    OpenAIRE

    Ana O. Pires; Andreia Neves-Carvalho; Nuno Sousa; António J. Salgado

    2014-01-01

    The goal of this study was to determine and compare the effects of the secretome of mesenchymal stem cells (MSCs) isolated from human bone-marrow (BMSCs) and the Wharton jelly surrounding the vein and arteries of the umbilical cord (human umbilical cord perivascular cells (HUCPVCs)) on the survival and differentiation of a human neuroblastoma cell line (SH-SY5Y). For this purpose, SH-SY5Y cells were differentiated with conditioned media (CM) from the MSCs populations referred above. Retinoic ...

  4. Damage of Neuroblastoma Cell SH-SY5Y Mediated by MPP+ Inhibits Proliferation of T-Cell Leukemia Jurkat by Co-Culture System

    Directory of Open Access Journals (Sweden)

    Fuli Wang

    2014-06-01

    Full Text Available The adaptive immune system has implications in pathology of Parkinson’s disease (PD. Research data demonstrated that the peripheral CD4+ T-cell population decreased in pathogenesis of PD. The effect of damaged dopaminergic neurons on peripheral T cells of PD is still unknown. In this study, we constructed a neuronal and glial cells co-culture model by using human neuroblastoma cells SH-SY5Y and gliomas cells U87. After the co-culture cells were treated with neurotoxin 1-methyl-4-phenylpyridinium (MPP+ for 24 h, the conditioned media was harvested and used to cultivate T-cell leukemia Jurkat cells for another 24 h. We then analyzed the cell proliferation, cell cycle and necrosis effect of Jurkat cells. The results showed that co-culture medium of SH-SY5Y and U87 cells with MPP+ treatment inhibited the proliferation of Jurkat cells compared to control medium without MPP+, even though the same concentration of MPP+ had very little toxicity to the Jurkat cell. Furthermore, co-culture medium with low concentration of MPP+ (100 µM arrested Jurkat cells cycle in G2/M phase through increasing cell cycle division 2 (CDC2 and CyclinB1 expression level, whereas co-culture medium with high concentration of MPP+ (500 µM induced Jurkat cell necrosis through cellular swelling and membrane breakage. Our data implies that damaged dopamine neurons with glial cells can lead to the reduced number or inhibited proliferation activity of peripheral T cells.

  5. Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, L.; Liang, H. [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan (China); Cao, W. [Department of Obstetrics, Qingdao Central Hospital, Qingdao (China); Xu, R.; Ju, X.L. [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan (China)

    2014-05-23

    Neuroblastoma is a solid tumor that occurs mainly in children. Malignant neuroblastomas have a poor prognosis because conventional chemotherapeutic agents are not very effective. Survivin, a member of the inhibitor of the apoptosis protein family, plays a significant role in cell division, inhibition of apoptosis, and promotion of cell proliferation and invasion. Previous studies found that survivin is highly expressed in some malignant neuroblastomas and is correlated with poor prognosis. The aim of this study was to investigate whether survivin could serve as a potential therapeutic target of human neuroblastoma. We employed RNA interference to reduce survivin expression in the human neuroblastoma SH-SY5Y cell line and analyzed the effect of RNA interference on cell proliferation and invasion in vitro and in vivo. RNA interference of survivin led to a significant decrease in invasiveness and proliferation and increased apoptosis in SH-SY5Y cells in vitro. RNA interference of survivin inhibited tumor growth in vivo by 68±13% (P=0.002) and increased the number of apoptotic cells by 9.8±1.2% (P=0.001) compared with negative small interfering RNA (siRNA) treatment controls. Moreover, RNA interference of survivin inhibited the formation of lung metastases by 92% (P=0.002) and reduced microvascular density by 60% (P=0.0003). Survivin siRNA resulted in significant downregulation of survivin mRNA and protein expression both in vitro and in vivo compared with negative siRNA treatment controls. RNA interference of survivin was found to be a potent inhibitor of SH-SY5Y tumor growth and metastasis formation. These results support further clinical development of RNA interference of survivin as a treatment of neuroblastoma and other cancer types.

  6. Emulsion-core and polyelectrolyte-shell nanocapsules: biocompatibility and neuroprotection against SH-SY5Y cells

    International Nuclear Information System (INIS)

    Piotrowski, Marek; Szczepanowicz, Krzysztof; Jantas, Danuta; Leśkiewicz, Monika; Lasoń, Władysław; Warszyński, Piotr

    2013-01-01

    The emulsion-core and polyelectrolyte-coated nanocapsules, designed as water-insoluble neuroprotective drug delivery system, were synthesized using layer-by-layer saturation method. The isopropyl myristate was used as oil phase and docusate sodium salt as emulsifier. For the polyelectrolyte shell preparation, synthetic polyelectrolytes, cationic (PDADMAC, PAH, and PLL) and anionic (PGA) were used. The particle size and zeta potential of nanocapsules were characterized by the dynamic light scattering. The average size of synthesized nanocapsules ranged from ∼80 to ∼100 nm. Zeta potential values ranged from less than approximately −30 mV for the polyanion layers to greater than approximately +30 mV for the polycation layers. Biocompatibilities of the synthesized nanocarriers were evaluated against SH-SY5Y human neuroblastoma cells using various biochemical assays. The results obtained show that synthesized nanocapsules coated with PLL and PGA were nontoxic to SH-SY5Y cells, and they were used as nanocarriers for model neuroprotective drug (a calpain inhibitor MDL 28170). The neuroprotective action of the encapsulated MDL 28170 against hydrogen peroxide-induced oxidative stress cytotoxicity was evaluated in the same cell line. The results showed that nanoencapsulated form of MDL 28170 were biocompatible and protected SH-SY5Y cells against the H 2 O 2 (0.5 mM/24 h)-induced damage in 20–40 times lower concentrations than those of the same drug added directly to the culture medium. These data suggest that the nanoscale carriers of neuroprotective drugs might serve as novel promising therapeutic agents for oxidative stress-related neurodegenerative processes

  7. Emulsion-core and polyelectrolyte-shell nanocapsules: biocompatibility and neuroprotection against SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Piotrowski, Marek, E-mail: ncpiotro@cyf-kr.edu.pl; Szczepanowicz, Krzysztof [Polish Academy of Sciences, Jerzy Haber Institute of Catalysis and Surface Chemistry (Poland); Jantas, Danuta; Leśkiewicz, Monika; Lasoń, Władysław [Polish Academy of Sciences, Institute of Pharmacology (Poland); Warszyński, Piotr [Polish Academy of Sciences, Jerzy Haber Institute of Catalysis and Surface Chemistry (Poland)

    2013-11-15

    The emulsion-core and polyelectrolyte-coated nanocapsules, designed as water-insoluble neuroprotective drug delivery system, were synthesized using layer-by-layer saturation method. The isopropyl myristate was used as oil phase and docusate sodium salt as emulsifier. For the polyelectrolyte shell preparation, synthetic polyelectrolytes, cationic (PDADMAC, PAH, and PLL) and anionic (PGA) were used. The particle size and zeta potential of nanocapsules were characterized by the dynamic light scattering. The average size of synthesized nanocapsules ranged from ∼80 to ∼100 nm. Zeta potential values ranged from less than approximately −30 mV for the polyanion layers to greater than approximately +30 mV for the polycation layers. Biocompatibilities of the synthesized nanocarriers were evaluated against SH-SY5Y human neuroblastoma cells using various biochemical assays. The results obtained show that synthesized nanocapsules coated with PLL and PGA were nontoxic to SH-SY5Y cells, and they were used as nanocarriers for model neuroprotective drug (a calpain inhibitor MDL 28170). The neuroprotective action of the encapsulated MDL 28170 against hydrogen peroxide-induced oxidative stress cytotoxicity was evaluated in the same cell line. The results showed that nanoencapsulated form of MDL 28170 were biocompatible and protected SH-SY5Y cells against the H{sub 2}O{sub 2} (0.5 mM/24 h)-induced damage in 20–40 times lower concentrations than those of the same drug added directly to the culture medium. These data suggest that the nanoscale carriers of neuroprotective drugs might serve as novel promising therapeutic agents for oxidative stress-related neurodegenerative processes.

  8. Effect of graphene oxide on undifferentiated and retinoic acid-differentiated SH-SY5Y cells line

    Science.gov (United States)

    Lv, Min; Zhang, Yujie; Liang, Le; Wei, Min; Hu, Wenbing; Li, Xiaoming; Huang, Qing

    2012-06-01

    Graphene oxide (GO), has created an unprecedented opportunity for development and application in biology, due to its abundant functional groups and well water solubility. Recently, the potential toxicity of GO in the environment and in humans has garnered more and more attention. In this paper, we systematically studied the cytotoxicity of GO nanosheets via examining the effect of GO on the morphology, viability and differentiation of a human neuroblastoma SH-SY5Y cell line, which was an ideal model used to study neuronal disease in vitro. The results suggested that GO had no obvious cytotoxicity at low concentration (cells exhibited dose- and time-dependent decreases at high concentration (>=80 μg mL-1). Moreover, GO did not induce apoptosis. Very interestingly, GO significantly enhanced the differentiation of SH-SY5Y induced-retinoic acid (RA) by evaluating neurite length and the expression of neuronal marker MAP2. These data provide a promising application for neurodegenerative diseases.

  9. FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation

    Science.gov (United States)

    Pirou, Caroline; Montazer-Torbati, Fatemeh; Jah, Nadège; Delmas, Elisabeth; Lasbleiz, Christelle; Mignotte, Bernard; Renaud, Flore

    2017-01-01

    Neuroblastoma, a sympathetic nervous system tumor, accounts for 15% of cancer deaths in children. In contrast to most human tumors, p53 is rarely mutated in human primary neuroblastoma, suggesting impaired p53 activation in neuroblastoma. Various studies have shown correlations between fgf1 expression levels and both prognosis severity and tumor chemoresistance. As we previously showed that fibroblast growth factor 1 (FGF1) inhibited p53-dependent apoptosis in neuron-like PC12 cells, we initiated the study of the interaction between the FGF1 and p53 pathways in neuroblastoma. We focused on the activity of either extracellular FGF1 by adding recombinant rFGF1 in media, or of intracellular FGF1 by overexpression in human SH-SY5Y and mouse N2a neuroblastoma cell lines. In both cell lines, the genotoxic drug etoposide induced a classical mitochondrial p53-dependent apoptosis. FGF1 was able to inhibit p53-dependent apoptosis upstream of mitochondrial events in SH-SY5Y cells by both extracellular and intracellular pathways. Both rFGF1 addition and etoposide treatment increased fgf1 expression in SH-SY5Y cells. Conversely, rFGF1 or overexpressed FGF1 had no effect on p53-dependent apoptosis and fgf1 expression in neuroblastoma N2a cells. Using different FGF1 mutants (that is, FGF1K132E, FGF1S130A and FGF1S130D), we further showed that the C-terminal domain and phosphorylation of FGF1 regulate its intracrine anti-apoptotic activity in neuroblastoma SH-SY5Y cells. This study provides the first evidence for a role of an intracrine growth factor pathway on p53-dependent apoptosis in neuroblastoma, and could lead to the identification of key regulators involved in neuroblastoma tumor progression and chemoresistance. PMID:29048426

  10. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Park, Jae Hyeon; Lee, Jeong Eun; Shin, In Chul; Koh, Hyun Chul

    2013-01-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  11. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jae Hyeon [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2013-04-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  12. Chronic, low-dose rotenone reproduces Lewy neurites found in early stages of Parkinson's disease, reduces mitochondrial movement and slowly kills differentiated SH-SY5Y neural cells

    Directory of Open Access Journals (Sweden)

    Liu Lei

    2008-12-01

    Full Text Available Abstract Background Parkinson's disease, the most common adult neurodegenerative movement disorder, demonstrates a brain-wide pathology that begins pre-clinically with alpha-synuclein aggregates ("Lewy neurites" in processes of gut enteric and vagal motor neurons. Rostral progression into substantia nigra with death of dopamine neurons produces the motor impairment phenotype that yields a clinical diagnosis. The vast majority of Parkinson's disease occurs sporadically, and current models of sporadic Parkinson's disease (sPD can utilize directly infused or systemic neurotoxins. Results We developed a differentiation protocol for human SH-SY5Y neuroblastoma that yielded non-dividing dopaminergic neural cells with long processes that we then exposed to 50 nM rotenone, a complex I inhibitor used in Parkinson's disease models. After 21 days of rotenone, ~60% of cells died. Their processes retracted and accumulated ASYN-(+ and UB-(+ aggregates that blocked organelle transport. Mitochondrial movement velocities were reduced by 8 days of rotenone and continued to decline over time. No cytoplasmic inclusions resembling Lewy bodies were observed. Gene microarray analyses showed that the majority of genes were under-expressed. qPCR analyses of 11 mtDNA-encoded and 10 nDNA-encoded mitochondrial electron transport chain RNAs' relative expressions revealed small increases in mtDNA-encoded genes and lesser regulation of nDNA-encoded ETC genes. Conclusion Subacute rotenone treatment of differentiated SH-SY5Y neuroblastoma cells causes process retraction and partial death over several weeks, slowed mitochondrial movement in processes and appears to reproduce the Lewy neuritic changes of early Parkinson's disease pathology but does not cause Lewy body inclusions. The overall pattern of transcriptional regulation is gene under-expression with minimal regulation of ETC genes in spite of rotenone's being a complex I toxin. This rotenone-SH-SY5Y model in a

  13. Caspase Activation of p21-Activated Kinase 2 Occurs During Cisplatin-Induced Apoptosis of SH-SY5Y Neuroblastoma Cells and in SH-SY5Y Cell Culture Models of Alzheimer’s and Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    Jerry W. Marlin

    2010-04-01

    Full Text Available p21-activated kinase 2 (PAK-2 appears to have a dual function in the regulation of cell survival and cell death. Activation of full-length PAK-2 by the p21 G-proteins Rac or Cdc42 stimulates cell survival. However, PAK-2 is unique among the PAK family because it is also activated through proteolytic cleavage by caspase 3 or similar caspases to generate the constitutively active PAK-2p34 fragment. Caspase activation of PAK-2 correlates with the induction of apoptosis in response to many stimuli and recombinant expression of PAK-2p34 has been shown to stimulate apoptosis in several human cell lines. Here, we show that caspase activation of PAK-2 also occurs during cisplatin-induced apoptosis of SH-SY5Y neuroblastoma cells as well as in SH-SY5Y cell culture models for Alzheimer’s and Parkinson’s disease. Inhibition of mitochondrial complex I or of ubiquitin/proteasome-mediated protein degradation, which both appear to be involved in Parkinson’s disease, induce apoptosis and caspase activation of PAK-2 in SH-SY5Y cells. Overexpression of the amyloid precursor protein, which results in accumulation and aggregation of β-amyloid peptide, the main component of β-amyloid plaques in Alzheimer’s disease, also induces apoptosis and caspase activation of PAK-2 in SH-SY5Y cells. Expression of the PAK-2 regulatory domain inhibits caspase-activated PAK-2p34 and prevents apoptosis in 293T human embryonic kidney cells, indicating that caspase activation of PAK-2 is directly involved in the apoptotic response. This is the first evidence that caspase activation of PAK-2 correlates with apoptosis in cell culture models of Alzheimer’s and Parkinson’s disease and that selective inhibition of caspase-activated PAK-2p34 could prevent apoptosis.

  14. Synthesis of reduced-size gold nanostars and internalization in SH-SY5Y cells

    KAUST Repository

    Dacarro, Giacomo

    2017-07-01

    The synthesis of large pentatwinned five-branched gold nanostars (GNS) has been modified so to obtain overall dimensions shrunk to 60% and a lower branches aspect ratio, leading to a dramatic blue shift of their two near-infrared (NIR) localized surface plasmon resonances (LSPR) absorptions but still maintaining one LSPR in the biotransparent NIR range. The interactions of polyethylene glycol (PEG) coated large and shrunk GNS with SH-SY5Y cells revealed that the large ones (DCI - diameter of the circumference in which GNS can be inscribed = 76 nm) are internalized more efficiently than the shrunk ones (DCI = 46 nm), correlating with a decreased cells surving fraction.

  15. Synthesis of reduced-size gold nanostars and internalization in SH-SY5Y cells

    KAUST Repository

    Dacarro, Giacomo; Pallavicini, Piersandro; Bertani, Serena Maria; Chirico, Giuseppe; D'Alfonso, Laura; Falqui, Andrea; Marchesi, Nicoletta; Pascale, Alessia; Sironi, Laura; Taglietti, Angelo; Zuddas, Efisio

    2017-01-01

    The synthesis of large pentatwinned five-branched gold nanostars (GNS) has been modified so to obtain overall dimensions shrunk to 60% and a lower branches aspect ratio, leading to a dramatic blue shift of their two near-infrared (NIR) localized surface plasmon resonances (LSPR) absorptions but still maintaining one LSPR in the biotransparent NIR range. The interactions of polyethylene glycol (PEG) coated large and shrunk GNS with SH-SY5Y cells revealed that the large ones (DCI - diameter of the circumference in which GNS can be inscribed = 76 nm) are internalized more efficiently than the shrunk ones (DCI = 46 nm), correlating with a decreased cells surving fraction.

  16. Pinocembrin Suppresses H2O2-Induced Mitochondrial Dysfunction by a Mechanism Dependent on the Nrf2/HO-1 Axis in SH-SY5Y Cells.

    Science.gov (United States)

    de Oliveira, Marcos Roberto; da Costa Ferreira, Gustavo; Brasil, Flávia Bittencourt; Peres, Alessandra

    2018-02-01

    Mitochondria are susceptible to redox impairment, which has been associated with neurodegeneration. These organelles are both a source and target of reactive species. In that context, there is increasing interest in finding natural compounds that modulate mitochondrial function and mitochondria-related signaling in order to prevent or to treat diseases involving mitochondrial impairment. Herein, we investigated whether and how pinocembrin (PB) would prevent mitochondrial dysfunction elicited by the exposure of human neuroblastoma SH-SY5Y cells to hydrogen peroxide (H 2 O 2 ). PB (25 μM) was administrated for 4 h before H 2 O 2 treatment (300 μM for 24 h). PB prevented H 2 O 2 -induced loss of cell viability mitochondrial depolarization in SH-SY5Y cells. PB also attenuated redox impairment in mitochondrial membranes. The production of superoxide anion radical (O 2 -• ) and nitric oxide (NO • ) was alleviated by PB in cells exposed to H 2 O 2 . PB suppressed the H 2 O 2 -induced inhibition of the tricarboxylic acid (TCA) cycle enzymes aconitase, α-ketoglutarate dehydrogenase, and succinate dehydrogenase. Furthermore, PB induced anti-inflammatory effects by abolishing the H 2 O 2 -dependent activation of the nuclear factor-κB (NF-κB) and upregulation of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). The PB-induced antioxidant and anti-inflammatory effects are dependent on the heme oxygenate-1 (HO-1) enzyme and on the activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), since HO-1 inhibition (with 0.5 μM ZnPP IX) or Nrf2 silencing (with small interfering RNA (siRNA)) abolished the effects of PB. Overall, PB afforded cytoprotection by the Nrf2/HO-1 axis in H 2 O 2 -treated SH-SY5Y cells.

  17. Neuroprotective effect of Demethoxycurcumin, a natural derivative of Curcumin on rotenone induced neurotoxicity in SH-SY 5Y Neuroblastoma cells.

    Science.gov (United States)

    Ramkumar, Muthu; Rajasankar, Srinivasagam; Gobi, Veerappan Venkatesh; Dhanalakshmi, Chinnasamy; Manivasagam, Thamilarasan; Justin Thenmozhi, Arokiasamy; Essa, Musthafa Mohamed; Kalandar, Ameer; Chidambaram, Ranganathan

    2017-04-18

    Mitochondrial dysfunction and oxidative stress are the main toxic events leading to dopaminergic neuronal death in Parkinson's disease (PD) and identified as vital objective for therapeutic intercession. This study investigated the neuro-protective effects of the demethoxycurcumin (DMC), a derivative of curcumin against rotenone induced neurotoxicity. SH-SY5Y neuroblastoma cells are divided into four experimental groups: untreated cells, cells incubated with rotenone (100 nM), cells treated with DMC (50 nM) + rotenone (100 nM) and DMC alone treated. 24 h after treatment with rotenone and 28 h after treatment with DMC, cell viability was assessed using the MTT assay, and levels of ROS and MMP, plus expression of apoptotic protein were analysed. Rotenone induced cell death in SH-SY5Y cells was significantly reduced by DMC pretreatment in a dose-dependent manner, indicating the potent neuroprotective effects of DMC. Rotenone treatment significantly increases the levels of ROS, loss of MMP, release of Cyt-c and expression of pro-apoptotic markers and decreases the expression of anti-apoptotic markers. Even though the results of the present study indicated that the DMC may serve as a potent therapeutic agent particularly for the treatment of neurodegenerative diseases like PD, further pre-clinical and clinical studies are required.

  18. Protective effect of Codium tomentosum alga on SH-SY5Y model of neurotoxicity induced by 6-hydroxydopamine (6 – OHDA

    Directory of Open Access Journals (Sweden)

    Joana Silva

    2014-06-01

    Full Text Available Parkinson Disease (PD is characterized by the loss of dopaminergic cell bodies in the substantia nigra and oxidative stress, mitochondrial dysfunction and apoptosis seem to be involved in the pathogenesis of the disease. The aim of this study was to evaluate the neuroprotective effect of different algae extracts on the cell death induced by by 6-OH-dopamine on a neuronal human cell model (SH- SY5Y. The neuroprotective effect of methanolic and dichloromethane algae extract (Padina pavonica, Sargassum muticum, Saccorhiza polyschides, Codium tomentosum, Ulva compressa in the presence of 6-OH-DA was assessed using the MTT method. The activity of caspase-3 and the quantification of hydrogen peroxide (extracellular environment and real time production production were studied in the presence and in the absence of the macroalgae extracts. The highest neuroprotective effect was exhibited by methanolic fractions (1000 µg/ml; 24 hours of S. muticum (115.80 ± 8.53% of viable cells, S. polyschides (106.51 ± 4.26 of viable cells, P. pavonica (95.98 ± 3.95 of viable cells and dichloromethane (1000 µg/ml; 24 hours fraction of C. tomentosum alga (102.22 ± 4.24 of viable cells, when compared with 6-OH-DA condition (100μM; 24 hours (67.40 ± 3.56 of viable cells. For the extracts that exhibited the highest neuroprotective effect was studied the possible alterations induced in the mechanisms of action previous selected. The dichloromethane fraction of Codium tomentosum alga (1000 µg/ml highly reduced the 6-OH-DA induced increase of caspase-3 activity, the less quantity of H2O2 in the extracellular environment and less production of H2O2 on real time (2.58 ± 1.77 UAF mg of protein-1 min-1; 0.0 ± 0.005 µM of H2O2, 54.07 ± 6.66 UAF min-1, respectively when compared with the 6-OH-DA condition at 100μM (4.5 ± 1:35 UAF mg of protein-1 min-1; ± 0.4 0.0019 µM of H2O2; 214.26 ± 8:46 UAF min-1, respectively. These results suggest that Codium tomentosum

  19. Alternatively Spliced Methionine Synthase in SH-SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal

    Directory of Open Access Journals (Sweden)

    Mostafa Waly

    2016-01-01

    Full Text Available The folate and cobalamin (Cbl- dependent enzyme methionine synthase (MS is highly sensitive to oxidation and its activity affects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient-derived fibroblasts. Here we show that MS mRNA in SH-SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl or the combination of hydroxocobalamin (OHCbl and S-adenosylmethionine (SAM. OHCbl-based activity was eliminated by depletion of the antioxidant glutathione (GSH but could be rescued by provision of either glutathionylcobalamin (GSCbl or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury-containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate-limiting for GSH synthesis. Thimerosal treatment decreased cellular levels of GSCbl and MeCbl. These findings indicate that the alternatively spliced form of MS expressed in SH-SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action.

  20. Protection against oxidant-induced apoptosis by mitochondrial thioredoxin in SH-SY5Y neuroblastoma cells

    International Nuclear Information System (INIS)

    Chen Yan; Yu Min; Jones, Dean P.; Greenamyre, J. Timothy; Cai Jiyang

    2006-01-01

    Mitochondrial oxidative stress plays important roles in aging and age-related degenerative disorders. The newly identified mitochondrial thioredoxin (mtTrx; Trx2) is a key component of the mitochondrial antioxidant system which is responsible for the clearance of reactive intermediates and repairs proteins with oxidative damage. Here, we show that in cultured SH-SY5Y human neuroblastoma 1cells, overexpression of mtTrx inhibited apoptosis and loss of mitochondrial membrane potential induced by a chemical oxidant, tert-butylhydroperoxide (tBH). The effects of calcium ionophore (Br-A23187) were not affected by mtTrx, suggesting the protection was specific against oxidative injury. The mitochondrial glutathione pool was oxidized by tBH, and this oxidation was not inhibited by increased mtTrx. Consequently, the antioxidant function of mtTrx is not redundant, but rather in addition, to that of GSH. Mutations of Cys90 and Cys93 to serines rendered mtTrx ineffective in protection against tBH-induced cytoxicity. These data indicate that mtTrx controls the mitochondrial redox status independently of GSH and is a key component of the defensive mechanism against oxidative stress in cultured neuronal cells

  1. Effect of Amaranthus on Advanced Glycation End-Products Induced Cytotoxicity and Proinflammatory Cytokine Gene Expression in SH-SY5Y Cells

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    Warisa Amornrit

    2015-09-01

    Full Text Available Amaranthus plants, or spinach, are used extensively as a vegetable and are known to possess medicinal properties. Neuroinflammation and oxidative stress play a major role in the pathogenesis of many neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. Advanced glycation end-products (AGEs cause cell toxicity in the human neuronal cell line, SH-SY5Y, through an increase in oxidative stress, as shown by reducing cell viability and increasing cell toxicity in a dose-dependent manner. We found that preincubation of SH-SY5Y cells with either petroleum ether, dichloromethane or methanol extracts of A. lividus and A. tricolor dose-dependently attenuated the neuron toxicity caused by AGEs treatment. Moreover, the results showed that A. lividus and A. tricolor extracts significantly downregulated the gene expression of the pro-inflammatory cytokines, TNF-α, IL-1 and IL-6 genes in AGEs-induced cells. We concluded that A. lividus and A. tricolor extracts not only have a neuroprotective effect against AGEs toxicity, but also have anti-inflammatory activity by reducing pro-inflammatory cytokine gene expression. This suggests that Amaranthus may be useful for treating chronic inflammation associated with neurodegenerative disorders.

  2. Oxidative stress response in SH-SY5Y cells exposed to short-term 1800 MHz radiofrequency radiation.

    Science.gov (United States)

    Marjanovic Cermak, Ana Marija; Pavicic, Ivan; Trosic, Ivancica

    2018-01-28

    The exact mechanism that could explain the effects of radiofrequency (RF) radiation exposure at non-thermal level is still unknown. Increasing evidence suggests a possible involvement of reactive oxygen species (ROS) and development of oxidative stress. To test the proposed hypothesis, human neuroblastoma cells (SH-SY5Y) were exposed to 1800 MHz short-term RF exposure for 10, 30 and 60 minutes. Electric field strength within Gigahertz Transverse Electromagnetic cell (GTEM) was 30 V m -1 and specific absorption rate (SAR) was calculated to be 1.6 W kg -1 . Cellular viability was measured by MTT assay and level of ROS was determined by fluorescent probe 2',7'-dichlorofluorescin diacetate. Concentrations of malondialdehyde and protein carbonyls were used to assess lipid and protein oxidative damage and antioxidant activity was evaluated by measuring concentrations of total glutathione (GSH). After radiation exposure, viability of irradiated cells remained within normal physiological values. Significantly higher ROS level was observed for every radiation exposure time. After 60 min of exposure, the applied radiation caused significant lipid and protein damage. The highest GSH concentration was detected after 10 minute-exposure. The results of our study showed enhanced susceptibility of SH-SY5Y cells for development of oxidative stress even after short-term RF exposure.

  3. Effects of Ginkgo biloba extract on the apoptosis of oxygen and glucose-deprived SH-SY5Y cells and its mechanism.

    Science.gov (United States)

    Ba, Xiao-Hong; Min, Lian-Qiu

    2015-01-01

    The aim was to observe the effects of the extract of Ginkgo biloba (EGb761) on the apoptosis of oxygen and glucose-deprived (OGD) human neuroblastoma cells (SH-SY5Y) cells and explore its mechanism. SH-SY5Y cells were divided into normal control group, OGD group, OGD for 4 h and EGb761-pretreated groups including very low-concentration (20 μg/ml), low-concentration group (25 μg/ml), moderate-concentration group (50 μg/ml) and high-concentration group (100 μg/ml). Twenty four hours after reoxygenation, cell viability was determined with 3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide assay, apoptosis rate was detected with annexin V-fluorescein isothiocyanate/propidium iodide double staining flow cytometry and the protein level of apoptosis-inducing factor (AIF) was observed with immunofluorescence technique in each group. Cell viability was significantly lower in OGD group than in EGb761-pretreated groups, especially in moderate-concentration group (50 μg/ml) (P cells probably through inhibiting AIF nuclear translocation. This study provides a theoretical basis for the application of EGb761 in clinical practice.

  4. Promotion of SH-SY5Y Cell Growth by Gold Nanoparticles Modified with 6-Mercaptopurine and a Neuron-Penetrating Peptide

    Science.gov (United States)

    Xiao, Yaruo; Zhang, Enqi; Fu, Ailing

    2017-12-01

    Much effort has been devoted to the discovery of effective biomaterials for nerve regeneration. Here, we reported a novel application of gold nanoparticles (AuNPs) modified with 6-mercaptopurine (6MP) and a neuron-penetrating peptide (RDP) as a neurophic agent to promote proliferation and neurite growth of human neuroblastoma (SH-SY5Y) cells. When the cells were treated with 6MP-AuNPs-RDP conjugates, they showed higher metabolic activity than the control. Moreover, SH-SY5Y cells were transplanted onto the surface coated with 6MP-AuNPs-RDP to examine the effect of neurite development. It can be concluded that 6MP-AuNPs-RDP attached to the cell surface and then internalized into cells, leading to a significant increase of neurite growth. Even though 6MP-AuNPs-RDP-treated cells were recovered from frozen storage, the cells still maintained constant growth, indicating that the cells have excellent tolerance to 6MP-AuNPs-RDP. The results suggested that the 6MP-AuNPs-RDP had promising potential to be developed as a neurophic nanomaterial for neuronal growth.

  5. Protective Effect of Edaravone Against Aβ25-35-Induced Mitochondrial Oxidative Damage in SH-SY5Y Cells.

    Science.gov (United States)

    Zhang, G-L; Zhang, L; Guo, Y-Y; Ma, Z-L; Wang, H-Y; Li, T; Liu, J; Du, Y; Yao, L; Li, T-T; Du, J-M

    2017-05-20

    Amyloid-β (Aβ)-induced oxidative stress plays an important role in the pathogenesis of Alzheimer's disease (AD). Recent studies show that Aβ accumulation may lead to mitochondrial oxidative damage. In the present study, we investigated the protective effect of edaravone on mitochondrial damage in SH-SY5Y cells treated with Aβ25-35. SH-SY5Y cells were pre-treated with 20, 40 or 80 μM edaravone before treatment with 25 μM Aβ25-35. After 24h cell culture, cellular apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), ATP levels and mitochondrial morphology were evaluated. SH-SY5Y cells exposed to Aβ25-35 had high levels of apoptosis and ROS; loss of ΔΨm, decreased ATP levels and presence of mitochondrial swelling. However, these effects were significantly inhibited by edaravone pre-treatment. These results indicate that edaravone prevents mitochondria oxidative damage caused by Aβ in SH-SY5Y cells, which suggests that it may have potential clinical application in AD therapy.

  6. Acetaldehyde Induces Cytotoxicity of SH-SY5Y Cells via Inhibition of Akt Activation and Induction of Oxidative Stress

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    Tingting Yan

    2016-01-01

    Full Text Available Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. It has been shown that heavy drinking is associated with an earlier onset of neurodegenerative diseases such as Alzheimer’s disease. Acetaldehyde, the most toxic metabolite of ethanol, is speculated to mediate the brain tissue damage and cognitive dysfunction induced by the chronic excessive consumption of alcohol. However, the exact mechanisms by which acetaldehyde induces neurotoxicity are not totally understood. In this study, we investigated the cytotoxic effects of acetaldehyde in SH-SY5Y cells and found that acetaldehyde induced apoptosis of SH-SY5Y cells by downregulating the expression of antiapoptotic Bcl-2 and Bcl-xL and upregulating the expression of proapoptotic Bax. Acetaldehyde treatment led to a significant decrease in the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB. In addition, acetaldehyde induced the activation of p38 mitogen-activated protein kinase (MAPK while inhibiting the activation of extracellular signal-regulated kinases (ERKs, p44/p42MAPK. Meanwhile, acetaldehyde treatment caused an increase in the production of reactive oxygen species and elevated the oxidative stress in SH-SY5Y cells. Therefore, acetaldehyde induces cytotoxicity of SH-SY5Y cells via promotion of apoptotic signaling, inhibition of cell survival pathway, and induction of oxidative stress.

  7. Involvement of activation of the Nrf2/ARE pathway in protection against 6-OHDA-induced SH-SY5Y cell death by α-iso-cubebenol.

    Science.gov (United States)

    Park, Sun Young; Kim, Do Yeon; Kang, Jong-Koo; Park, Geuntae; Choi, Young-Whan

    2014-09-01

    Free radical-mediated neurodegeneration is one of the many causes of Parkinson's disease (PD). As part of our ongoing studies on the identification of biologically active Schisandra chinensis components, we have isolated and structurally elucidated α-iso-cubebenol. This study was carried out in an attempt to clarify the neuroprotective effect of α-iso-cubebenol on toxin-insulted dopaminergic neuronal death using 6-hydroxy-dopamine (6-OHDA)-induced dopaminergic SH-SY5Y cells. α-iso-cubebenol significantly attenuated the loss of mitochondrial function (MTT assay) and membrane integrity (lactate dehydrogenase assay) associated with 6-OHDA-induced neurotoxicity. Pretreatment of the cells with α-iso-cubebenol diminished the intracellular accumulation of reactive oxygen species (ROS) and calcium in response to 6-OHDA. Moreover, α-iso-cubebenol protected against 6-OHDA-induced neurotoxicity through inhibition of SH-SY5Y cell apoptosis. In addition, JC-1 staining, which is a well-established measure of mitochondrial damage, was decreased after treatment with α-iso-cubebenol. Notably, α-iso-cubebenol inhibited the release of mitochondrial flavoprotein apoptosis inducing factor (AIF) from the mitochondria to the cytosol and nucleus following 6-OHDA treatment. In addition, α-iso-cubebenol reduced the 6-OHDA-induced phosphorylation of ERK and induced the phosphorylation of PKA, PKB, and CREB in a dose-dependent manner. Moreover, α-iso-cubebenol stimulated the activation of Nrf2, a downstream target of CREB. Furthermore, α-iso-cubebenol stimulated the expression of multiple antioxidant response genes (NQO-1 and HO-1). Finally, CREB and Nrf2 siRNA transfection diminished α-iso-cubebenol-mediated neuroprotection. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Polychlorinated Biphenyls Induce Mitochondrial Dysfunction in SH-SY5Y Neuroblastoma Cells.

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    Stefania Cocco

    Full Text Available Chronic exposure to polychlorinated biphenyls (PCBs, ubiquitous environmental contaminants, can adversely affect the development and function of the nervous system. Here we evaluated the effect of PCB exposure on mitochondrial function using the PCB mixture Aroclor-1254 (A1254 in SH-SY5Y neuroblastoma cells. A 6-hour exposure to A1254 (5 μg/ml reduced cellular ATP production by 45%±7, and mitochondrial membrane potential, detected by TMRE, by 49%±7. Consistently, A1254 significantly decreased oxidative phosphorylation and aerobic glycolysis measured by extracellular flux analyzer. Furthermore, the activity of mitochondrial protein complexes I, II, and IV, but not V (ATPase, measured by BN-PAGE technique, was significantly reduced after 6-hour exposure to A1254. The addition of pyruvic acid during exposure to A1254 significantly prevent A1254-induced cell injury, restoring resting mitochondrial membrane potential, ATP levels, oxidative phosphorylation and aerobic glycolysis. Furthermore, pyruvic acid significantly preserved the activity of mitochondrial complexes I, II and IV and increased basal activity of complex V. Collectively, the present results indicate that the neurotoxicity of A1254 depends on the impairment of oxidative phosphorylation, aerobic glycolysis, and mitochondrial complexes I, II, and IV activity and it was counteracted by pyruvic acid.

  9. Zinc oxide nanoparticles and SH-SY5Y cell line

    Science.gov (United States)

    Zheng, Jinghui

    The Arctic and sub-arctic regions are impacted by the growth of the global nanotechnology industry. Nanomaterials have unique chemical and physical properties that may lead to toxicological effects that interfere with normal cellular metabolism. Zinc oxide nanoparticles (ZnO NPs) are now very common and widely used in daily life. In industry, ZnO NPs are used to protect different materials from damage caused by UV exposure. The scientific literature suggests that ZnO NPs can have negative impacts on both living organisms and plants. However, there is a paucity of research on the mechanisms by which ZnO NPs may affect the neuronal cells. This study investigates how ZnO NPs interact with the neuroblastoma cell line SH-SY5Y. Using transmission electron microscopy, we observed that the ZnO NPs form 36 nm particles on average, and increase the level of vascular endothelial growth factor (VEGF) in extracellular fluid, as measured by an enzyme-linked immunosorbent assay (ELISA). Moreover, ZnO NPs, in presence of tumor necrosis factor-alpha (TNF-alpha), can also decrease the level of extracellular VEGF compared with TNF-alpha treatment alone. These findings suggest the basis for more studies on understanding the mechanism by which ZnO NPs impact cytokine signaling. Another direction is using ELISA technology to observe the interactions of NPs with different cell types such as neuronal stem cells.

  10. The effect of UV-filters on the viability of neuroblastoma (SH-SY5Y) cell line.

    Science.gov (United States)

    Broniowska, Żaneta; Pomierny, Bartosz; Smaga, Irena; Filip, Małgorzata; Budziszewska, Bogusława

    2016-05-01

    Topical application of cosmetic products, containing ultraviolet filters (UV filters) are recommended as a protection against sunburns and in order to reduce the risk of skin cancer. However, some UV filters can be absorbed through skin and by consuming contaminated food. Among the chemical UV filters, benzophenone-3 (BP-3), 3-(4-methylbenzylidene)camphor (4-MBC) and 2-ethylhexyl-4-methoxycinnamate (OMC) are absorbed through the skin to the greatest extent. So far, these lipophilic compounds were demonstrated to influence the gonadal and thyroid hormone function, but their effect on central nervous system cells has not been investigated, yet. In the present study, we investigated the effect of some UV filters on cell viability and caspase-3 activity in SH-SY5Y cells. It has been found that benzophenone-2 (BP-2), BP-3, 4-methylbenzophenone (4-MBP) and OMC present in the culture medium for 72h in high concentration (10(-5) and 10(-4)M) and 4-MBC only 10(-4)M produced a significant cytotoxic effect, as determined both by the MTT reduction test and LDH release assay. In contrast to necrotic changes, all tested UV filters increased caspase-3 activity in much lower concentrations (from 10(-8) to 10(-7)M). Proapoptotic properties of the test compounds were positively verified by Hoechst staining. The obtained results indicated that UV filters adversely affected the viability of nerve cells, most likely by enhancing the process of apoptosis. The most potent effect was exerted by BP-3 and 4-MBC and at concentrations that may be reached in vivo. Since human exposure to UV filters is significant these compound should be taken into consideration as one of the possible factors involved in pathogenesis of neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Force spectroscopy of membrane hardness of SH-SY5Y neuroblastoma cells before and after differentiation

    Science.gov (United States)

    Kwon, Sangwoo; Yang, Woochul; Choi, Yun Kyong; Park, Jung Keuck

    2014-05-01

    Atomic force microscopy (AFM) is utilized in many studies for measuring the structure and the physical characteristics of soft and bio materials. In particular, the force spectroscopy function in the AFM system allows us to explore the mechanical properties of bio cells. In this study, we probe the variation in the membrane hardness of human neuroblastoma SH-SY5Y cells (SH-cells) before and after differentiation by using force spectroscopy. The SH-cell, which is usually differentiated by using a chemical treatment with retinoic acid (RA), is a neuronal cell line employed widely as an in-vitro model for neuroscience research. In force spectroscopy, the force-distance curves are obtained from both the original and the RA-treated cells while the AFM tip approaches and pushes on the cell membranes. The slope deduced from linear region in the force-distance curve is the spring constant and corresponds to the hardness of the cell membrane. The spring constant of the RA-treated cells (0.597 ± 0.010 nN/nm) was smaller than that of the original cells (0.794 ± 0.010 nN/nm), reflecting a hardness decrease in the cells differentiated with the RA treatments. The results clearly demonstrated that the differentiated cells are softer than the original cells. The change in the elasticity of the differentiated cells might be caused by morphological modification during differentiation process. We suggest that force spectroscopy can be employed as a novel method to determine the degree of differentiation of stem cells into various functional cells.

  12. Modulation of chemotherapy-induced cytotoxicity in SH-SY5Y neuroblastoma cells by caffeine and chlorogenic acid.

    Science.gov (United States)

    Hall, Susan; Anoopkumar-Dukie, Shailendra; Grant, Gary D; Desbrow, Ben; Lai, Richard; Arora, Devinder; Hong, Yinna

    2017-06-01

    Chemotherapy is an important treatment modality for malignancy but is limited by significant toxicity and it susceptibility to numerous drug interactions. While the interacting effects with medications are well known, there is limited evidence on the interaction with commonly consumed food and natural products. The aim of this study was to evaluate the bioactive constituents of coffee (caffeine and chlorogenic acid) on the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in vitro. Pretreatment with caffeine (100 nM and 10 μM) sensitized SH-SY5Y cells to doxorubicin-induced toxicity and increased apoptosis and sensitized PC3 cells to gemcitabine-induced toxicity. Pretreatment with 10 μM caffeine decreased total cell reactive oxygen species (ROS) production but increased mitochondrial ROS production. In contrast, caffeine (10 nM and 10 μM) protected cells against gemcitabine-induced toxicity and apoptosis. Similarly, 1 μM and 10 μM caffeine protected cells against paclitaxel-induced toxicity and mitochondrial ROS production. Chlorogenic acid had no effect on chemotherapy-induced toxicity in SH-SY5Y cells. In conclusion, this study provides preliminary evidence that caffeine, not chlorogenic acid, modulates the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in SH-SY5Y cells via different mechanisms.

  13. Neurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells

    Science.gov (United States)

    Wen, Xianjie; Xu, Shiyuan; Liu, Hongzhen; Zhang, Quinguo; Liang, Hua; Yang, Chenxiang; Wang, Hanbing

    2013-01-01

    There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca2+ ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation. PMID:23658789

  14. Curcumin Attenuated Bupivacaine-Induced Neurotoxicity in SH-SY5Y Cells Via Activation of the Akt Signaling Pathway.

    Science.gov (United States)

    Fan, You-Ling; Li, Heng-Chang; Zhao, Wei; Peng, Hui-Hua; Huang, Fang; Jiang, Wei-Hang; Xu, Shi-Yuan

    2016-09-01

    Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.

  15. Neurotoxicity induced by bupivacaine via T-type calcium channels in SH-SY5Y cells.

    Directory of Open Access Journals (Sweden)

    Xianjie Wen

    Full Text Available There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca(2+ ([Ca2+]i, cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation.

  16. Role of D-Limonene in Autophagy Induced by Bergamot Essential Oil in SH-SY5Y Neuroblastoma Cells

    Science.gov (United States)

    Russo, Rossella; Cassiano, Maria Gilda Valentina; Ciociaro, Antonella; Adornetto, Annagrazia; Varano, Giuseppe Pasquale; Chiappini, Carlotta; Berliocchi, Laura; Tassorelli, Cristina; Bagetta, Giacinto; Corasaniti, Maria Tiziana

    2014-01-01

    Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a well characterized, widely used plant extract. BEO exerts anxiolytic, analgesic and neuroprotective activities in rodents through mechanisms that are only partly known and need to be further investigated. To gain more insight into the biological effects of this essential oil, we tested the ability of BEO (0.005–0.03%) to modulate autophagic pathways in human SH-SY5Y neuroblastoma cells. BEO-treated cells show increased LC3II levels and appearance of dot-like formations of endogenous LC3 protein that colocalize with the lysosome marker LAMP-1. Autophagic flux assay using bafilomycin A1 and degradation of the specific autophagy substrate p62 confirmed that the observed increase of LC3II levels in BEO-exposed cells is due to autophagy induction rather than to a decreased autophagosomal turnover. Induction of autophagy is an early and not cell-line specific response to BEO. Beside basal autophagy, BEO also enhanced autophagy triggered by serum starvation and rapamycin indicating that the underlying mechanism is mTOR independent. Accordingly, BEO did not affect the phosphorylation of ULK1 (Ser757) and p70S6K (Thr389), two downstream targets of mTOR. Furthermore, induction of autophagy by BEO is beclin-1 independent, occurs in a concentration-dependent manner and is unrelated to the ability of BEO to induce cell death. In order to identify the active constituents responsible for these effects, the two most abundant monoterpenes found in the essential oil, d-limonene (125–750 µM) and linalyl acetate (62.5–375 µM), were individually tested at concentrations comparable to those found in 0.005–0.03% BEO. The same features of stimulated autophagy elicited by BEO were reproduced by d-limonene, which rapidly increases LC3II and reduces p62 levels in a concentration-dependent manner. Linalyl acetate was ineffective in replicating BEO effects; however, it greatly enhanced LC3 lipidation triggered by d

  17. Curcumin I mediates neuroprotective effect through attenuation of quinoprotein formation, p-p38 MAPK expression, and caspase-3 activation in 6-hydroxydopamine treated SH-SY5Y cells.

    Science.gov (United States)

    Meesarapee, Benjawan; Thampithak, Anusorn; Jaisin, Yamaratee; Sanvarinda, Pimtip; Suksamrarn, Apichart; Tuchinda, Patoomratana; Morales, Noppawan Phumala; Sanvarinda, Yupin

    2014-04-01

    6-Hydroxydopamine (6-OHDA) selectively enters dopaminergic neurons and undergoes auto-oxidation resulting in the generation of reactive oxygen species and dopamine quinones, subsequently leading to apoptosis. This mechanism mimics the pathogenesis of Parkinson's disease and has been used to induce experimental Parkinsonism in both in vitro and in vivo systems. In this study, we investigated the effects of curcumin I (diferuloylmethane) purified from Curcuma longa on quinoprotein production, phosphorylation of p38 MAPK (p-p38), and caspase-3 activation in 6-OHDA-treated SH-SY5Y dopaminergic cells. Pretreatment of SH-SY5Y with curcumin I at concentrations of 1, 5, 10, and 20 μM, significantly decreased the formation of quinoprotein and reduced the levels of p-p38 and cleaved caspase-3 in a dose-dependent manner. Moreover, the levels of the dopaminergic neuron marker, phospho-tyrosine hydroxylase (p-TH), were also dose-dependently increased upon treatment with curcumin I. Our results clearly demonstrated that curcumin I protects neurons against oxidative damage, as shown by attenuation of p-p38 expression, caspase-3-activation, and toxic quinoprotein formation, together with the restoration of p-TH levels. This study provides evidence for the therapeutic potential of curcumin I in the chemoprevention of oxidative stress-related neurodegeneration. Copyright © 2013 John Wiley & Sons, Ltd.

  18. Neuroprotective Effect of Ginkgolide B on Bupivacaine-Induced Apoptosis in SH-SY5Y Cells

    Science.gov (United States)

    Li, Le; Zhang, Qing-guo; Lai, Lu-ying; Wen, Xian-jie; Zheng, Ting; Cheung, Chi-wai; Zhou, Shu-qin; Xu, Shi-yuan

    2013-01-01

    Local anesthetics are used routinely and effectively. However, many are also known to activate neurotoxic pathways. We tested the neuroprotective efficacy of ginkgolide B (GB), an active component of Ginkgo biloba, against ROS-mediated neurotoxicity caused by the local anesthetic bupivacaine. SH-SY5Y cells were treated with different concentrations of bupivacaine alone or following preincubation with GB. Pretreatment with GB increased SH-SY5Y cell viability and attenuated intracellular ROS accumulation, apoptosis, mitochondrial dysfunction, and ER stress. GB suppressed bupivacaine-induced mitochondrial depolarization and mitochondria complex I and III inhibition and increased cleaved caspase-3 and Htra2 expression, which was strongly indicative of activation of mitochondria-dependent apoptosis with concomitantly enhanced expressions of Grp78, caspase-12 mRNA, protein, and ER stress. GB also improved ultrastructural changes indicative of mitochondrial and ER damage induced by bupivacaine. These results implicate bupivacaine-induced ROS-dependent mitochondria, ER dysfunction, and apoptosis, which can be attenuated by GB through its antioxidant property. PMID:24228138

  19. Pharmacological Characterization of the Mechanisms Involved in Delayed Calcium Deregulation in SH-SY5Y Cells Challenged with Methadone

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    Sergio Perez-Alvarez

    2012-01-01

    Full Text Available Previously, we have shown that SH-SY5Y cells exposed to high concentrations of methadone died due to a necrotic-like cell death mechanism related to delayed calcium deregulation (DCD. In this study, we show that, in terms of their Ca2+ responses to 0.5 mM methadone, SH-SY5Y cells can be pooled into four different groups. In a broad pharmacological survey, the relevance of different Ca2+-related mechanisms on methadone-induced DCD was investigated including extracellular calcium, L-type Ca2+ channels, μ-opioid receptor, mitochondrial inner membrane potential, mitochondrial ATP synthesis, mitochondrial Ca2+/2Na+-exchanger, reactive oxygen species, and mitochondrial permeability transition. Only those compounds targeting mitochondria such as oligomycin, FCCP, CGP 37157, and cyclosporine A were able to amend methadone-induced Ca2+ dyshomeostasis suggesting that methadone induces DCD by modulating the ability of mitochondria to handle Ca2+. Consistently, mitochondria became dramatically shorter and rounder in the presence of methadone. Furthermore, analysis of oxygen uptake by isolated rat liver mitochondria suggested that methadone affected mitochondrial Ca2+ uptake in a respiratory substrate-dependent way. We conclude that methadone causes failure of intracellular Ca2+ homeostasis, and this effect is associated with morphological and functional changes of mitochondria. Likely, this mechanism contributes to degenerative side effects associated with methadone treatment.

  20. Agmatine Protects Against 6-OHDA-Induced Apoptosis, and ERK and Akt/GSK Disruption in SH-SY5Y Cells.

    Science.gov (United States)

    Amiri, Esmat; Ghasemi, Rasoul; Moosavi, Maryam

    2016-08-01

    6-Hydroxydopamine (6-OHDA), a metabolite of dopamine is known to induce dopaminergic cell toxicity which makes that a suitable agent inducing an experimental model of Parkinson's disease (PD). Agmatine has been shown to protect against some cellular and animal PD models. This study was aimed to assess whether agmatine prevents 6-OHDA-induced SH-SY5Y cell death and if yes, then how it affects Akt/glycogen synthesis kinase-3β (GSK-3β) and extracellular signal-regulated kinases (ERK) signals. The cells were treated with different drugs, and their viability was examined via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and morphological observation. Western blot studies were done to assess cleaved caspase-3, Akt/GSK-3β, and ERK proteins. 6-OHDA-induced cell death and caspase-3 cleavage, while agmatine prevented those changes. 6-OHDA also decreased the amount of phosphorylated Akt (pAkt)/Akt while increased GSK-3β activity which was prevented by agmatine. Additionally, this toxin increased pERK/ERK ratio which was averted again by agmatine. The PI3/Akt inhibitor, LY294002, impeded the changes induced by agmatine, while ERK inhibitor (PD98059) did not disturb the effects of agmatine, and by itself, it preserved the cells against 6-OHDA toxicity. This study revealed that agmatine is protective in 6-OHDA model of PD and affects Akt/GSK-3β and ERK pathways.

  1. Gallic Acid Protects 6-OHDA Induced Neurotoxicity by Attenuating Oxidative Stress in Human Dopaminergic Cell Line.

    Science.gov (United States)

    Chandrasekhar, Y; Phani Kumar, G; Ramya, E M; Anilakumar, K R

    2018-04-18

    Gallic acid is one of the most important polyphenolic compounds, which is considered an excellent free radical scavenger. 6-Hydroxydopamine (6-OHDA) is a neurotoxin, which has been implicated in mainly Parkinson's disease (PD). In this study, we investigated the molecular mechanism of the neuroprotective effects of gallic acid on 6-OHDA induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that 6-OHDA induced cytotoxicity in SH-SY5Y cells was suppressed by pre-treatment with gallic acid. The percentage of live cells (90%) was high in the pre-treatment of gallic acid when compared with 6-OHDA alone treated cell line. Moreover, gallic acid was very effective in attenuating the disruption of mitochondrial membrane potential, elevated levels of intracellular ROS and apoptotic cell death induced by 6-OHDA. Gallic acid also lowered the ratio of the pro-apoptotic Bax protein and the anti-apoptotic Bcl-2 protein in SH-SY5Y cells. 6-OHDA exposure was up-regulated caspase-3 and Keap-1 and, down-regulated Nrf2, BDNF and p-CREB, which were sufficiently reverted by gallic acid pre-treatment. These findings indicate that gallic acid is able to protect the neuronal cells against 6-OHDA induced injury and proved that gallic acid might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.

  2. The SH-SY5Y cell line in Parkinson's disease research: a systematic review

    OpenAIRE

    Xicoy, H; Wieringa, B; Martens, G.J.

    2017-01-01

    Parkinson?s disease (PD) is a devastating and highly prevalent neurodegenerative disease for which only symptomatic treatment is available. In order to develop a truly effective disease-modifying therapy, improvement of our current understanding of the molecular and cellular mechanisms underlying PD pathogenesis and progression is crucial. For this purpose, standardization of research protocols and disease models is necessary. As human dopaminergic neurons, the cells mainly affected in PD, ar...

  3. Cordyceps sinensis Oral Liquid Inhibits Damage Induced by Oxygen and Glucose Deprivation in SH-SY5Y Cells.

    Science.gov (United States)

    Zou, Ying-Xin; Liu, Yu-Xiang; Ruan, Ming-Hua; Zhou, Yi; Wang, Jia-Chun; Chu, Zhi-Yong

    2016-01-01

    Cordyceps sinensis has been used in traditional Chinese medicine for thousands of years. It has been demonstrated to have a variety of biological activities, and an extract of it has been demonstrated to possess a protective effect in occlusion-induced focal cerebral ischemia of the middle cerebral artery in rats. It could be explored as an agent for treatment of ischemic stroke, and the mechanisms need to be studied further. The study intended to investigate the protective effects of the Cordyceps sinensis oral liquid (CSOL) against damage induced by oxygen and glucose deprivation (OGD) in SH-SY5Y cells. DESIGN • The research team designed an in vitro study. The study occurred at the Naval Medical Research Institute in Shanghai, China. SH-SY5Y cells were exposed to CSOL in doses of 0.01, 0.03, 0.10, 0.30, and 1.00 mg/mL, creating 5 intervention groups. The OGD condition was induced by transfer of the cells from high-glucose Dulbecco's Modified Eagle's medium (DMEM) in a box gassed with air containing 5% CO2 to glucose-free DMEM in a box gassed with 94% N2, 5% CO2, and 1% O2. Like the cells for the interventions groups, the cells for a model group were cultured with high-glucose DMEM and were transferred to the OGD, but they received no dose of COSL. Cells in a control group were cultured with high-glucose DMEM, were not transferred to the OGD condition, and did not receive any dose of COSL. Cell viability was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The apoptosis and the mitochondrial membrane potential (MMP) were detected by flow cytometry, and the protein expression of caspase-3 was observed by western blot. After exposure to OGD, the cell viability of cells treated with 0.01, 0.03, 0.10, 0.30, and 1.00 mg/mL of CSOL increased in a dose-effect relationship. Compared with the cells in the model group, the treatment of CSOL at all the experimental concentrations significantly inhibited both the cell apoptosis

  4. Interleukin-24 induces neuroblastoma SH-SY5Y cell differentiation, growth inhibition, and apoptosis by promoting ROS production.

    Science.gov (United States)

    Li, Yuan; Zhang, Hongwei; Zhu, Xiaoyu; Feng, Dongchuan; Gong, Jinchao; Han, Tao

    2013-11-01

    Neuroblastoma is among the most aggressive tumors that occur in childhood and infancy. The clinical prognosis of children with advanced-stage neuroblastoma is still poor. Interleukin-24 (IL-24) is emerging as a new cytokine involved in tumor cellular proliferation, differentiation, and apoptosis and has been widely studied as a tumor inhibitor. However, little is known about this cytokine's role in neuroblastoma. In this study, we investigated the possible effects of IL-24 on inducing neuroblastoma cell differentiation, growth inhibition, and apoptosis in vitro. Our data show that IL-24 promotes neuroblastoma SH-SY5Y cell differentiation, growth inhibition, and apoptosis. Furthermore, we found that the differentiation- and apoptosis-inducing action of IL-24 depends on the accumulation of reactive oxygen species (ROS). These results suggest that IL-24 can induce neuroblastoma cell differentiation and apoptosis and may be a potential therapeutic agent for neuroblastoma.

  5. PDGF-mediated protection of SH-SY5Y cells against Tat toxin involves regulation of extracellular glutamate and intracellular calcium

    International Nuclear Information System (INIS)

    Zhu Xuhui; Yao Honghong; Peng Fuwang; Callen, Shannon; Buch, Shilpa

    2009-01-01

    The human immunodeficiency virus (HIV-1) protein Tat has been implicated in mediating neuronal apoptosis, one of the hallmark features of HIV-associated dementia (HAD). Mitigation of the toxic effects of Tat could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study we demonstrated that Tat-induced neurotoxicity was abolished by NMDA antagonist-MK801, suggesting the role of glutamate in this process. Furthermore, we also found that pretreatment of SH-SY5Y cells with PDGF exerted protection against Tat toxicity by decreasing extracellular glutamate levels. We also demonstrated that extracellular calcium chelator EGTA was able to abolish PDGF-mediated neuroprotection, thereby underscoring the role of calcium signaling in PDGF-mediated neuroprotection. We also showed that Erk signaling pathway was critical for PDGF-mediated protection of cells. Additionally, blocking calcium entry with EGTA resulted in suppression of PDGF-induced Erk activation. These findings thus underscore the role of PDGF-mediated calcium signaling and Erk phosphorylation in the protection of cells against HIV Tat toxicity.

  6. Protective effects of Arctium lappa L. roots against hydrogen peroxide-induced cell injury and potential mechanisms in SH-SY5Y cells.

    Science.gov (United States)

    Tian, Xing; Guo, Li-Ping; Hu, Xiao-Long; Huang, Jin; Fan, Yan-Hua; Ren, Tian-Shu; Zhao, Qing-Chun

    2015-04-01

    Accumulated evidence has shown that excessive reactive oxygen species (ROS) have been implicated in neuronal cell death related with various chronic neurodegenerative disorders. This study was designed to explore neuroprotective effects of ethyl acetate extract of Arctium lappa L. roots (EAL) on hydrogen peroxide (H2O2)-induced cell injury in human SH-SY5Y neuroblastoma cells. The cell viability was significantly decreased after exposure to 200 μM H2O2, whereas pretreatment with different concentrations of EAL attenuated the H2O2-induced cytotoxicity. Hoechst 33342 staining indicated that EAL reversed nuclear condensation in H2O2-treated cells. Meanwhile, TUNEL assay with DAPI staining showed that EAL attenuated apoptosis was induced by H2O2. Pretreatment with EAL also markedly elevated activities of antioxidant enzyme (GSH-Px and SOD), reduced lipid peroxidation (MDA) production, prevented ROS formation, and the decrease of mitochondrial membrane potential. In addition, EAL showed strong radical scavenging ability in 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assays. Furthermore, EAL inhibited H2O2-induced apoptosis by increases in the Bcl-2/Bax ratio, decreases in cytochrome c release, and attenuation of caspase-3, caspase-9 activities, and expressions. These findings suggest that EAL may be regarded as a potential antioxidant agent and possess potent neuroprotective activity against H2O2-induced injury.

  7. Some commonly used brominated flame retardants cause Ca2+-ATPase inhibition, beta-amyloid peptide release and apoptosis in SH-SY5Y neuronal cells.

    Directory of Open Access Journals (Sweden)

    Fawaz Al-Mousa

    Full Text Available Brominated flame retardants (BFRs are chemicals commonly used to reduce the flammability of consumer products and are considered pollutants since they have become widely dispersed throughout the environment and have also been shown to bio-accumulate within animals and man. This study investigated the cytotoxicity of some of the most commonly used groups of BFRs on SH-SY5Y human neuroblastoma cells. The results showed that of the BFRs tested, hexabromocyclododecane (HBCD, tetrabromobisphenol-A (TBBPA and decabromodiphenyl ether (DBPE, all are cytotoxic at low micromolar concentrations (LC(50 being 2.7 ± 0.7 µM, 15 ± 4 µM and 28 ± 7 µM, respectively. They induced cell death, at least in part, by apoptosis through activation of caspases. They also increased intracellular [Ca(2+] levels and reactive-oxygen-species within these neuronal cells. Furthermore, these BFRs also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells. Elevated intracellular [Ca(2+] levels appear to occur through a mechanism involving microsomal Ca(2+-ATPase inhibition and this maybe responsible for Ca(2+-induced mitochondrial dysfunction. In addition, µM levels of these BFRs caused β-amyloid peptide (Aβ-42 processing and release from these cells with a few hours of exposure. These results therefore shows that these pollutants are both neurotoxic and amyloidogenic in-vitro.

  8. Retinoic acid-induced differentiation increases the rate of oxygen consumption and enhances the spare respiratory capacity of mitochondria in SH-SY5Y cells.

    Science.gov (United States)

    Xun, Zhiyin; Lee, Do-Yup; Lim, James; Canaria, Christie A; Barnebey, Adam; Yanonne, Steven M; McMurray, Cynthia T

    2012-04-01

    Retinoic acid (RA) is used in differentiation therapy to treat a variety of cancers including neuroblastoma. The contributing factors for its therapeutic efficacy are poorly understood. However, mitochondria (MT) have been implicated as key effectors in RA-mediated differentiation process. Here we utilize the SH-SY5Y human neuroblastoma cell line as a model to examine how RA influences MT during the differentiation process. We find that RA confers an approximately sixfold increase in the oxygen consumption rate while the rate of glycolysis modestly increases. RA treatment does not increase the number of MT or cause measurable changes in the composition of the electron transport chain. Rather, RA treatment significantly increases the mitochondrial spare respiratory capacity. We propose a competition model for the therapeutic effects of RA. Specifically, the high metabolic rate in differentiated cells limits the availability of metabolic nutrients for use by the undifferentiated cells and suppresses their growth. Thus, RA treatment provides a selective advantage for the differentiated state. Published by Elsevier Ireland Ltd.

  9. A multidisciplinary approach to study the functional properties of neuron-like cell models constituting a living bio-hybrid system: SH-SY5Y cells adhering to PANI substrate

    Directory of Open Access Journals (Sweden)

    S. Caponi

    2016-11-01

    Full Text Available A living bio-hybrid system has been successfully implemented. It is constituted by neuroblastic cells, the SH-SY5Y human neuroblastoma cells, adhering to a poly-anyline (PANI a semiconductor polymer with memristive properties. By a multidisciplinary approach, the biocompatibility of the substrate has been analyzed and the functionality of the adhering cells has been investigated. We found that the PANI films can support the cell adhesion. Moreover, the SH-SY5Y cells were successfully differentiated into neuron-like cells for in vitro applications demonstrating that PANI can also promote cell differentiation. In order to deeply characterize the modifications of the bio-functionality induced by the cell-substrate interaction, the functional properties of the cells have been characterized by electrophysiology and Raman spectroscopy. Our results confirm that the PANI films do not strongly affect the general properties of the cells, ensuring their viability without toxic effects on their physiology. Ascribed to the adhesion process, however, a slight increase of the markers of the cell suffering has been evidenced by Raman spectroscopy and accordingly the electrophysiology shows a reduction at positive stimulations in the cells excitability.

  10. Low-dose/dose-rate γ radiation depresses neural differentiation and alters protein expression profiles in neuroblastoma SH-SY5Y cells and C17.2 neural stem cells.

    Science.gov (United States)

    Bajinskis, Ainars; Lindegren, Heléne; Johansson, Lotta; Harms-Ringdahl, Mats; Forsby, Anna

    2011-02-01

    The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose γ-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs γ rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate γ rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism.

  11. Alzheimer's disease presenilin-1 exon 9 deletion and L250S mutations sensitize SH-SY5Y neuroblastoma cells to hyperosmotic stress-induced apoptosis

    DEFF Research Database (Denmark)

    Tanii, H; Ankarcrona, M; Flood, F

    2000-01-01

    . In the present study, we determined whether PS1 mutations also sensitize cells to hyperosmotic stress-induced apoptosis. For this, we established SH-SY5Y neuroblastoma cell lines stably transfected with wild-type PS1 or either the PS1 exon 9 deletion (deltaE9) or PS1 L250S mutants. Cultured cells were exposed...

  12. Curcumin Rescues a PINK1 Knock Down SH-SY5Y Cellular Model of Parkinson's Disease from Mitochondrial Dysfunction and Cell Death.

    Science.gov (United States)

    van der Merwe, Celia; van Dyk, Hayley Christy; Engelbrecht, Lize; van der Westhuizen, Francois Hendrikus; Kinnear, Craig; Loos, Ben; Bardien, Soraya

    2017-05-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of dopaminergic neurons in the substantia nigra. Mutations in the PINK1 gene result in an autosomal recessive form of early-onset PD. PINK1 plays a vital role in mitochondrial quality control via the removal of dysfunctional mitochondria. The aim of the present study was to create a cellular model of PD using siRNA-mediated knock down of PINK1 in SH-SY5Y neuroblastoma cells The possible protective effects of curcumin, known for its many beneficial properties including antioxidant and anti-inflammatory effects, was tested on this model in the presence and absence of paraquat, an additional stressor. PINK1 siRNA and control cells were separated into four treatment groups: (i) untreated, (ii) treated with paraquat, (iii) pre-treated with curcumin then treated with paraquat, or (iv) treated with curcumin. Various parameters of cellular and mitochondrial function were then measured. The PINK1 siRNA cells exhibited significantly decreased cell viability, mitochondrial membrane potential (MMP), mitochondrial respiration and ATP production, and increased apoptosis. Paraquat-treated cells exhibited decreased cell viability, increased apoptosis, a more fragmented mitochondrial network and decreased MMP. Curcumin pre-treatment followed by paraquat exposure rescued cell viability and increased MMP and mitochondrial respiration in control cells, and significantly decreased apoptosis and increased MMP and maximal respiration in PINK1 siRNA cells. These results highlight a protective effect of curcumin against mitochondrial dysfunction and apoptosis in PINK1-deficient and paraquat-exposed cells. More studies are warranted to further elucidate the potential neuroprotective properties of curcumin.

  13. Antihypoxic effect of miR-24 in SH-SY5Y cells under hypoxia via downregulating expression of neurocan

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Xingyuan, E-mail: sunxingyuan@sina.com; Ren, Zhanjun; Pan, Yunzhi; Zhang, Chenxin

    2016-09-02

    Hypoxia-induced apoptosis-related mechanisms involved in the brain damage following cerebral ischemia injury. A subset of the small noncoding microRNA (miRNAs) is regulated by tissue oxygen levels, and miR-24 was found to be activated by hypoxic conditions. However, the roles of miR-24 and its target gene in neuron are not well understood. Here, we validated miRNA-24 is down-regulated in patients with cerebral infarction. Hypoxia suppressed the expression of miR-24, but increased the expression of neurocan in both mRNA and protein levels in SH-SY5Y cells. MiR-24 mimics reduced the expression of neurocan, suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. By luciferase reporter assay, neurocan is validated a direct target gene of miR-24. Furthermore, knockdown of neurocan suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. Taken together, miR-24 overexpression or silencing of neurocan shows an antihypoxic effect in SH-SY5Y cells. Therefore, miR-24 and neurocan play critical roles in neuron cell apoptosis and are potential therapeutic targets for ischemic brain disease. - Highlights: • miR-24 and neurocan play critical roles in neuron cell apoptosis. • miR-24 and neurocan are potential therapeutic targets for ischemic brain disease. • Antihypoxic effect of miR-24 and neurocan in SH-SY5Y cells.

  14. TNF-alpha-induced apoptosis is prevented by erythropoietin treatment on SH-SY5Y cells

    International Nuclear Information System (INIS)

    Pregi, Nicolas; Wenker, Shirley; Vittori, Daniela; Leiros, Claudia Perez; Nesse, Alcira

    2009-01-01

    The growth factor erythropoietin (Epo) has shown neuronal protective action in addition to its well known proerythroid activity. Furthermore, Epo has dealt with cellular inflammation by inhibiting the expression of several proinflammatory cytokines, such as IL-1 and TNF-α. The action of TNF can have both apoptotic and antiapoptotic consequences due to altered balance between different cell signalling pathways. This work has focused on the apoptotic effects of this cytokine and the potential protective action of Epo. The model we used was neuroblastoma SH-SY5Y cells cultured in the presence of 25 ng/ml TNF-α or pretreated with 25 U/ml Epo for 12 h before the addition of TNF-α. Apoptosis was evaluated by differential cell count after Hoechst staining, analysis of DNA ladder pattern, and measurement of caspase activity. Despite its ability to induce NF-κB nuclear translocation, TNF-α induced cell death, which was found to be associated to upregulation of TNF Receptor 1 expression. On the other hand, cells activated by Epo became resistant to cell death. Prevention of death receptor upregulation and caspase activation may explain this antiapoptotic effect of Epo, which may be also favoured by the induction of a higher expression of protective factors, such as Bcl-2 and NF-κB, through mechanisms involving Jak/STAT and PI3K signalling pathways

  15. Neurosupportive Role of Vanillin, a Natural Phenolic Compound, on Rotenone Induced Neurotoxicity in SH-SY5Y Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Chinnasamy Dhanalakshmi

    2015-01-01

    Full Text Available Vanillin, a phenolic compound, has been reported to offer neuroprotection against experimental Huntington’s disease and global ischemia by virtue of its antioxidant, anti-inflammatory, and antiapoptotic properties. The present study aims to elucidate the underlying neuroprotective mechanism of vanillin in rotenone induced neurotoxicity. Cell viability was assessed by exposing SH-SY5Y cells to various concentrations of rotenone (5–200 nM for 24 h. The therapeutic effectiveness of vanillin against rotenone was measured by pretreatment of vanillin at various concentrations (5–200 nM and then incubation with rotenone (100 nM. Using effective dose of vanillin (100 nM, mitochondrial membrane potential, levels of reactive oxygen species (ROS, and expression patterns of apoptotic markers were assessed. Toxicity of rotenone was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, release of cyt-c, and enhanced expressions of proapoptotic and downregulation of antiapoptotic indices via the upregulation of p38 and JNK-MAPK pathway proteins. Our results indicated that the pretreatment of vanillin attenuated rotenone induced mitochondrial dysfunction, oxidative stress, and apoptosis. Thus, vanillin may serve as a potent therapeutic agent in the future by virtue of its multiple pharmacological properties in the treatment of neurodegenerative diseases including PD.

  16. Activated cathepsin L is associated with the switch from autophagy to apoptotic death of SH-SY5Y cells exposed to 6-hydroxydopamine

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lingyun, E-mail: lingyunlee@126.com [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China); Experimental Center, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Gao, Luyan [Experimental Center, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Song, Yunzhen; Qin, Zheng-Hong [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China); Liang, Zhongqin, E-mail: liangzhongqin@suda.edu.cn [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China)

    2016-02-12

    Autophagy and apoptosis are common responses to pathological damage in the process of Parkinson's disease (PD), and lysosome dysfunction may contribute to the etiology of PD's neurodegenerative process. In this study, we demonstrated that the neurotoxin 6-hydroxydopamine (6-OHDA) increased autophagy in SH-SY5Y cells, as determined by detection of the lysosome marker lysosomal-associated membrane protein1, the autophagy protein light chain 3 (LC3)-II and the autophagy substrate P62 protein. Meanwhile, autophagy repression with 3-methyladenine accelerated the activation of caspase-3 and PARP and aggravated the cell apoptotic death induced by 6-OHDA. Furthermore, we found that 6-OHDA treatment resulted in a transient increase in the intracellular and nuclear expression of cathepsin L (CTSL). The CTSL inhibitor, Z-FY-CHO, could promote autophagy, decrease accumulation of P62, and block activation of caspase-3 and PARP. Taken together, these results suggest that activation of autophagy may primarily be a protective process in SH-SY5Y cell death induced by 6-OHDA, and the nuclear translocation of CTSL could enhance the cell apoptotic cascade via disturbing autophagy-apoptotic systems in SH-SY5Y cells. Our findings highlight the potential role of CTSL in the cross talk between autophagy and apoptosis, which might be considered a therapeutic strategy for treatment of pathologic conditions associated with neurodegeneration. - Highlights: • Inhibition of autophagy aggravated the cell apoptotic death in SH-SY5Y cells. • Activation of cathepsin L impaired the autophagy pathway. • Activation of cathepsin L enhanced the cell apoptotic cascade. • Cathepsin L involves in the cross talk between autophagy and apoptosis.

  17. Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency

    Directory of Open Access Journals (Sweden)

    Paola Maura Tricarico

    2017-03-01

    Full Text Available Background/Aims: Mevalonate Kinase Deficiency (MKD, is a hereditary disease due to mutations in mevalonate kinase gene (MVK. MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA, the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. Methods: SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. Results: MVK mutants’ over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. Conclusions: We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation.

  18. Lack of Prenylated Proteins, Autophagy Impairment and Apoptosis in SH-SY5Y Neuronal Cell Model of Mevalonate Kinase Deficiency.

    Science.gov (United States)

    Tricarico, Paola Maura; Romeo, Alessandra; Gratton, Rossella; Crovella, Sergio; Celsi, Fulvio

    2017-01-01

    Mevalonate Kinase Deficiency (MKD), is a hereditary disease due to mutations in mevalonate kinase gene (MVK). MKD has heterogeneous clinical phenotypes: the correlation between MVK mutations and MKD clinical phenotype is still to be fully elucidated. Deficiency of prenylated proteins has been hypothesized as possible MKD pathogenic mechanism. Based on this hypothesis and considering that neurologic impairment characterizes Mevalonic Aciduria (MA), the most severe form of MKD, we studied the effects of I268T and N301T MVK mutations on protein prenylation, autophagy and programmed cell death in SH-SY5Y neuroblastoma cell lines. SH-SY5Y cells were transiently transfected, with the pCMV-6 plasmid containing MVK wild type and the two mutated sequences. Protein prenylation levels were evaluated using GFP-RhoA-F to assess farnesylation, and GFP-RhoA to evaluate geranylgeranylation; autophagy was measured by evaluating LC3 and p62 protein levels, while Annexin V-FITC and Propidium Iodide staining allowed apoptosis detection. MVK mutants' over-expression causes decreased levels of farnesylation and geranylgeranylation, and also increased LC3 lipidation in SH-SY5Y, with concomitant p62 accumulation. Treatment with bafilomycin A1 (an inhibitor of vacuolar H+-ATPase, a late autophagy inhibitor) further increase LC3-II and p62 levels, suggesting that degradation of autophagolysosome could be impaired. SH-SY5Y, with both MVK mutants, showed apoptosis increase; the presence of N301T associated with augmented cell death. We hypothesize that mevalonate pathway impairment causes alteration of farnesylation and geranylgeranylation proteins and alteration of the autophagic flux; these changes can induce apoptosis, possibly more relevant in the presence of N301T mutation. © 2017 The Author(s)Published by S. Karger AG, Basel.

  19. JNK signaling pathway regulates sorbitol-induced Tau proteolysis and apoptosis in SH-SY5Y cells by targeting caspase-3.

    Science.gov (United States)

    Olivera Santa-Catalina, Marta; Caballero Bermejo, Montaña; Argent, Ricardo; Alonso, Juan C; Centeno, Francisco; Lorenzo, María J

    2017-12-15

    Growing evidence suggests that Diabetes Mellitus increases the risk of developing Alzheimer's disease. It is well known that hyperglycemia, a key feature of Diabetes Mellitus, may induce plasma osmolarity disturbances. Both hyperglycemia and hyperosmolarity promote the altered post-translational regulation of microtubule-associated protein Tau. Interestingly, abnormal hyperphosphorylation and cleavage of Tau have been proven to lead to the genesis of filamentous structures referred to as neurofibrillary tangles, the main pathological hallmark of Alzheimer's disease. We have previously described that hyperosmotic stress induced by sorbitol promotes Tau proteolysis and apoptosis in SH-SY5Y cells via caspase-3 activation. In order to gain insights into the regulatory mechanisms of such processes, in this work we explored the intracellular signaling pathways that regulate these events. We found that sorbitol treatment significantly enhanced the activation of conventional families of MAPK in SH-SY5Y cells. Tau proteolysis was completely prevented by JNK inhibition but not affected by either ERK1/2 or p38 MAPK blockade. Moreover, inhibition of JNK, but not ERK1/2 or p38 MAPK, efficiently prevented sorbitol-induced apoptosis and caspase-3 activation. In summary, we provide evidence that JNK signaling pathway is an upstream regulator of hyperosmotic stress-induced Tau cleavage and apoptosis in SH-SY5Y through the control of caspase-3 activation. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Short Chemical Ischemia Triggers Phosphorylation of eIF2α and Death of SH-SY5Y Cells but not Proteasome Stress and Heat Shock Protein Response in both SH-SY5Y and T98G Cells.

    Science.gov (United States)

    Klacanova, Katarina; Pilchova, Ivana; Klikova, Katarina; Racay, Peter

    2016-04-01

    Both translation arrest and proteasome stress associated with accumulation of ubiquitin-conjugated protein aggregates were considered as a cause of delayed neuronal death after transient global brain ischemia; however, exact mechanisms as well as possible relationships are not fully understood. The aim of this study was to compare the effect of chemical ischemia and proteasome stress on cellular stress responses and viability of neuroblastoma SH-SY5Y and glioblastoma T98G cells. Chemical ischemia was induced by transient treatment of the cells with sodium azide in combination with 2-deoxyglucose. Proteasome stress was induced by treatment of the cells with bortezomib. Treatment of SH-SY5Y cells with sodium azide/2-deoxyglucose for 15 min was associated with cell death observed 24 h after treatment, while glioblastoma T98G cells were resistant to the same treatment. Treatment of both SH-SY5Y and T98G cells with bortezomib was associated with cell death, accumulation of ubiquitin-conjugated proteins, and increased expression of Hsp70. These typical cellular responses to proteasome stress, observed also after transient global brain ischemia, were not observed after chemical ischemia. Finally, chemical ischemia, but not proteasome stress, was in SH-SY5Y cells associated with increased phosphorylation of eIF2α, another typical cellular response triggered after transient global brain ischemia. Our results showed that short chemical ischemia of SH-SY5Y cells is not sufficient to induce both proteasome stress associated with accumulation of ubiquitin-conjugated proteins and stress response at the level of heat shock proteins despite induction of cell death and eIF2α phosphorylation.

  1. Fentanyl activates hypoxia-inducible factor 1 in neuronal SH-SY5Y cells and mice under non-hypoxic conditions in a μ-opioid receptor-dependent manner.

    Science.gov (United States)

    Daijo, Hiroki; Kai, Shinichi; Tanaka, Tomoharu; Wakamatsu, Takuhiko; Kishimoto, Shun; Suzuki, Kengo; Harada, Hiroshi; Takabuchi, Satoshi; Adachi, Takehiko; Fukuda, Kazuhiko; Hirota, Kiichi

    2011-09-30

    Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor responsible for hypoxia-induced gene expression. Perioperative drugs including anesthetics have been reported to affect HIF-1 activity. However, the effect of fentanyl on HIF-1 activity is not well documented. In this study, we investigated the effect of fentanyl and other opioids on HIF-1 activity in human SH-SY5Y neuroblastoma cells, hepatoma Hep3B cells, lung adenocarcinoma A549 cells and mice. Cells were exposed to fentanyl, and HIF-1 protein expression was examined by Western blot analysis using anti-HIF-1α and β antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time reverse transcriptase (RT)-PCR (qRT-PCR) and luciferase assay. Furthermore, fentanyl was administered intraperitoneally and HIF-1-dependent gene expression was investigated by qRT-PCR in the brains and kidneys of mice. A 10-μM concentration of fentanyl and other opioids, including 1 μM morphine and 4 μM remifentanil, induced HIF-1α protein expression and HIF-1 target gene expression in an opioid receptor-dependent manner in SH-SY5Y cells with activity peaking at 24h. Fentanyl did not augment HIF-1α expression during hypoxia-induced induction. HIF-1α stabilization assays and experiments with cycloheximide revealed that fentanyl increased translation from HIF-1α mRNA but did not stabilize the HIF-1α protein. Furthermore, fentanyl induced HIF-1 target gene expression in the brains of mice but not in their kidneys in a naloxone-sensitive manner. In this report, we describe for the first time that fentanyl, both in vitro and in vivo, induces HIF-1 activation under non-hypoxic conditions, leading to increases in expression of genes associated with adaptation to hypoxia. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Dose-dependent folic acid and memantine treatments promote synergistic or additive protection against Aβ(25-35) peptide-induced apoptosis in SH-SY5Y cells mediated by mitochondria stress-associated death signals.

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    Chen, Ta-Fu; Tang, Ming-Chi; Chou, Chia-Hui; Chiu, Ming-Jang; Huang, R-F S

    2013-12-01

    Increased dietary folic acid (FA) is associated with reduced risks of Alzheimer's disease (AD). The AD drug memantine (Mn) has had limited therapeutic effects for the treatment of patients with moderate to severe AD. This study investigated whether and the underlying mechanisms by which the combination of Mn and FA may have synergistic or additive effects in protecting against amyloid-β(25-35) peptide (Aβ)-induced neurocytotoxicity. Aβ treatment of human neuroblastoma SH-SY5Y cells significantly induced a 6-fold increase of apoptotic cells compared with the Aβ-untreated group. Preincubation of Aβ-exposed cells with FA (500 μM) or Mn (20 μM) caused a 22% and 10% reduction of apoptotic cells, respectively, whereas the combo-treatments at such doses synergistically alleviated Aβ-induced apoptosis by 60% (P<0.05). The apoptotic protection by the combo-treatments coincided with attenuating Aβ-elicited mitochondrial (mt) membrane depolarization and abolishing Aβ-induced mt cytochrome c release to the cytosol. Increased levels of FA at 1000 μM in combination with 20 μM Mn exerted an additive protection against Aβ(25-35)-induced-apoptosis as compared to the isolate Mn group (P<0.05). The combo-treatments reversed Aβ-elicited mt membrane depolarization, attenuated Aβ-elicited mt cytochrome c release to the cytosol, and diminished Aβ-promoted superoxide generation. The apoptotic-protection by such combo-treatments was partially abolished by carbonyl cyanide 3-chlorophenylhydrazone (mt membrane potential uncoupler) and sodium azide (mt cytochrome c oxidase inhibitor). Taken together, the data demonstrated that dose-dependent FA and Mn synergistically or additively protected SH-SY5Y cells against Aβ-induced apoptosis, which was partially, if not completely, mediated by mt stress-associated death signals. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Identification of novel microRNAs in post-transcriptional control of Nrf2 expression and redox homeostasis in neuronal, SH-SY5Y cells.

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    Madhusudhanan Narasimhan

    Full Text Available Nuclear factor-erythroid 2-related factor 2 (Nrf2/NFE2L2, a redox-sensitive transcription factor plays a critical role in adaptation to cellular stress and affords cellular defense by initiating transcription of antioxidative and detoxification genes. While a protein can be regulated at multiple levels, control of Nrf2 has been largely studied at post-translational regulation points by Keap1. Importantly, post-transcriptional/translational based regulation of Nrf2 is less understood and to date there are no reports on such mechanisms in neuronal systems. In this context, studies involving the role of microRNAs (miRs which are normally considered as fine tuning regulators of protein production through translation repression and/or post-transcriptional alterations, are in place. In the current study, based on in-silico analysis followed by immunoblotting and real time analysis, we have identified and validated for the first time that human NFE2L2 could be targeted by miR153/miR27a/miR142-5p/miR144 in neuronal, SH-SY5Y cells. Co-transfection studies with individual miR mimics along with either WT 3' UTR of human Nrf2 or mutated miRNA targeting seed sequence within Nrf2 3' UTR, demonstrated that Nrf2 is a direct regulatory target of these miRs. In addition, ectopic expression of miR153/miR27a/miR142-5p/miR144 affected Nrf2 mRNA abundance and nucleo-cytoplasmic concentration of Nrf2 in a Keap1 independent manner resulting in inefficient transactivating ability of Nrf2. Furthermore, forced expression of miRs diminished GCLC and GSR expression resulting in alteration of Nrf2 dependent redox homeostasis. Finally, bioinformatics based miRNA-disease network analysis (MDN along with extended computational network analysis of Nrf2 associated pathologic processes suggests that if in a particular cellular scenario where any of these miR153/miR27a/miR142-5p/miR144 either individually or as a group is altered, it could affect Nrf2 thus triggering and

  4. Pretreatment of MQA, a caffeoylquinic acid derivative compound, protects against H2O2-induced oxidative stress in SH-SY5Y cells.

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    Tian, Xing; Gao, Lingyue; An, Li; Jiang, Xiaowen; Bai, Junpeng; Huang, Jian; Meng, Weihong; Zhao, Qingchun

    2016-12-01

    Compound MQA (1,5-O-dicaffeoyl-3-O-[4-malic acid methyl ester]-quinic acid) is a natural caffeoylquinic acid derivative isolated from Arctium lappa L. roots. This study aims to explore the neuroprotective effects of MQA against hydrogen peroxide (H 2 O 2 )-induced oxidative stress in SH-SY5Y neuroblastoma cells. The SH-SY5Y cells were divided into four groups, including control, 20 μM MQA, 200 μM H2O2, 200 μM H2O2 + 20 μM MQA groups. The effects of MQA on H 2 O 2 -induced cell death were measured by MTT and LDH assays. Hoechst 33342 and Annexin V-PI double staining were used to observed H2O2-induced apoptosis. Also, the effects of MQA on antioxidant system and mitochondrial pathway were explored. Further, steady-state phosphorylation levels of ERK1/2, Akt and GSK-3β were examined by Western blot analysis. Pretreatment with MQA prevented cell death in SH-SY5Y cells exposed to 200 μM H2O2 for 3 h. Meanwhile, Hoechst 33342 and Annexin V-PI double staining showed that MQA attenuated H 2 O 2 -induced apoptosis. These changes are related to elevation in SOD activity, reduction in MDA production and ROS formation, and increases in mitochondrial membrane potential (MMP). In addition, the potential mechanisms of MQA against H 2 O 2 -induced apoptosis are associated with increases in the Bcl-2/Bax ratio, decreases in cytochrome c release, caspase-3 and caspase-9 expressions, phosphorylation of ERK1/2, and dephosphorylation of AKT and GSK-3β. These findings suggest that protective effects of MQA against H 2 O 2 -induced apoptosis might be associated with mitochondrial apoptosis, ERK1/2 and AKT/GSK-3β pathway.

  5. N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents.

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    Mazzoccoli, Carmela; Ruggieri, Vitalba; Tataranni, Tiziana; Agriesti, Francesca; Laurenzana, Ilaria; Fratello, Angelo; Capitanio, Nazzareno; Piccoli, Claudia

    2016-05-03

    Neuroblastoma is the most commonly extra-cranial solid tumor of childhood frequently diagnosed. The nervous system-specific metabolite N-acetylaspartate (NAA) is synthesized from aspartate and acetyl-CoA in neurons, it is among the most abundant metabolites present in the central nervous system (CNS) and appears to be involved in many CNS disorders. The functional significance of the high NAA concentration in the brain remains uncertain, but it confers to NAA a unique clinical significance exploited in magnetic resonance spectroscopy. In the current study, we show that treatment of SH-SY5Y neuroblastoma-derived cell line with sub-cytotoxic physiological concentrations of NAA inhibits cell growth. This effect is partly due to enhanced apoptosis, shown by decrease of the anti-apoptotic factors survivin and Bcl-xL, and partly to arrest of the cell-cycle progression, linked to enhanced expression of the cyclin-inhibitors p53, p21Cip1/Waf1 and p27Kip1. Moreover, NAA-treated SH-SY5Y cells exhibited morphological changes accompanied with increase of the neurogenic markers TH and MAP2 and down-regulation of the pluripotency markers OCT4 and CXCR4/CD184. Finally, NAA-pre-treated SH-SY5Y cells resulted more sensitive to the cytotoxic effect of the chemotherapeutic drugs Cisplatin and 5-fluorouracil.To our knowledge, this is the first study demonstrating the neuronal differentiating effects of NAA in neuroblastoma cells. NAA may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment.

  6. The anti-inflammatory effect of melatonin in SH-SY5Y neuroblastoma cells exposed to sublethal dose of hydrogen peroxide.

    Science.gov (United States)

    Nopparat, Chutikorn; Chantadul, Varunya; Permpoonputtana, Kannika; Govitrapong, Piyarat

    2017-06-01

    Brain inflammaging is considered as one of the underlying factors of neurodegenerative diseases. The present study aimed to investigate the effects of melatonin, an endogenous indoleamine mainly synthesized by the pineal gland, on hydrogen peroxide (H 2 O 2 )-induced inflammaging state in SH-SY5Y cells. Our data showed that p21 Cip1 and p16 INK4a , cell cycle arrest markers, and the number of senescence-associated β-galactosidase (SA-βgal) staining increased significantly in H 2 O 2 -treated cells. Melatonin treatment could reverse this effect. Flow cytometry analysis showed a significantly higher percentage in the G0/G1 phase and a lower proportion in the S phase of H 2 O 2 treated cells. Cells pretreated with H 2 O 2 showed a dramatic decrease in the formation of Ki67 immunoactivity while the treatment with melatonin increased Ki67-positive cell. Both mRNA and protein expression levels of the pro-inflammatory cytokines, interleukin-1β (IL-1β), IL-6 and, tumor necrosis factor-α (TNF-α) which were increased after induction with H 2 O 2 , could be attenuated by melatonin. In addition, melatonin decreased the phospho-nuclear factor kappa B (pNF-κB) expression and prevented its nuclear translocation, as well as abrogated the reduction of nuclear factor erythroid 2-related factor 2 (Nrf2) in SH-SY5Y cells exposed to H 2 O 2 . The present data suggested the importance of melatonin on ameliorating inflammation in SH-SY5Y cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Insulin-like Growth Factor-I Mediates Neuroprotection in Proteasome Inhibition-Induced Cytotoxicity in SH-SY5Y Cells

    Science.gov (United States)

    Cheng, Benxu; Maffi, Shivani Kaushal; Martinez, Alex Anthony; Acosta, Yolanda P Villarreal; Morales, Liza D; Roberts, James L

    2011-01-01

    The proteasome is an enzyme complex responsible for targeted intracellular proteolysis. Alterations in proteasome-mediated protein clearance have been implicated in the pathogenesis of aging, Alzheimer's disease (AD) and Parkinson's disease (PD). In such diseases, proteasome inhibition may contribute to formation of abnormal protein aggregates, which in turn activate intracellular unfolded protein responses that cause oxidative stress and apoptosis. In this study, we investigated the protective effect of Insulin-like Growth Factor-I (IGF-1) for neural SH-SY5Y cells treated with the proteasomal inhibitor, Epoxomicin, In SH-SY5Y cells, Epoxomicin treatment results in accumulation of intracellular ubiquitinated proteins and cytochrome c release from damaged mitochondria, leading to cell death, in Epoxomicin time- and dose-dependent manner. In cells treated with small amounts of IGF-1, the same dosages of Epoxomicin reduced both mitochondrial damage (cytochrome c release) and reduced caspase-3 activation and PARP cleavage, both of which are markers of apoptosis. Notably, however, IGF-1-treated SH-SY5Y cells still contained ubiquitinated protein aggregates. This result indicates that IGF-1 blocks the downstream apoptotic consequences of Epoxomicin treatment leading to decreased proteasome function. Clues as to the mechanism for this protective effect come from (a) increased AKT phosphorylation observed in IGF-1-protected cells, vs. cells exposed to Epoxomicin without IGF-1, and (b) reduction of IGF-1 protection by pretreatment of the cells with LY294002 (an inhibitor of PI3-kinase). Together these findings suggest that activation of PI3/AKT pathways by IGF-1 is involved in IGF-1 neuroprotection against apoptosis following proteasome inhibition. PMID:21545837

  8. An Extract from Shrimp Processing By-Products Protects SH-SY5Y Cells from Neurotoxicity Induced by Aβ25–35

    Directory of Open Access Journals (Sweden)

    Yongping Zhang

    2017-03-01

    Full Text Available Increased evidence suggests that marine unsaturated fatty acids (FAs can protect neurons from amyloid-β (Aβ-induced neurodegeneration. Nuclear magnetic resonance (NMR, high performance liquid chromatography (HPLC and gas chromatography (GC assays showed that the acetone extract 4-2A obtained from shrimp Pandalus borealis industry processing wastes contained 67.19% monounsaturated FAs and 16.84% polyunsaturated FAs. The present study evaluated the anti-oxidative and anti-inflammatory effects of 4-2A in Aβ25–35-insulted differentiated SH-SY5Y cells. Cell viability and cytotoxicity were measured by using 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT and lactate dehydrogenase (LDH assays. Quantitative PCR and Western blotting were used to study the expression of neurotrophins, pro-inflammatory cytokines and apoptosis-related genes. Administration of 20 μM Aβ25–35 significantly reduced SH-SY5Y cell viability, the expression of nerve growth factor (NGF and its tyrosine kinase TrkA receptor, as well as the level of glutathione, while increased reactive oxygen species (ROS, nitric oxide, tumor necrosis factor (TNF-α, brain derived neurotrophic factor (BDNF and its TrkB receptor. Aβ25–35 also increased the Bax/Bcl-2 ratio and Caspase-3 expression. Treatment with 4-2A significantly attenuated the Aβ25–35-induced changes in cell viability, ROS, GSH, NGF, TrkA, TNF-α, the Bax/Bcl-2 ratio and Caspase-3, except for nitric oxide, BDNF and TrKB. In conclusion, 4-2A effectively protected SH-SY5Y cells against Aβ-induced neuronal apoptosis/death by suppressing inflammation and oxidative stress and up-regulating NGF and TrKA expression.

  9. Neuronal-like differentiated SH-SY5Y cells adaptation to a mild and transient H2 O2 -induced oxidative stress.

    Science.gov (United States)

    Akki, Rachid; Siracusa, Rosalba; Morabito, Rossana; Remigante, Alessia; Campolo, Michela; Errami, Mohammed; La Spada, Giuseppina; Cuzzocrea, Salvatore; Marino, Angela

    2018-03-01

    Preconditioning (PC) is a cell adaptive response to oxidative stress and, with regard to neurons, can be considered as a neuroprotective strategy. The aim of the present study was to verify how neuronal-like differentiated SH-SY5Y cells adapt to a mild and transient H 2 O 2 -induced oxidative stress and, hence, whether may be considered as more sensitive cell model to study PC pathways. A first screening allowed to define H 2 O 2 concentrations for PC (10μM-50μM), applied before damage(100μM H 2 O 2 ). Cell viability measured 24 hours after 100μM H 2 O 2 -induced damage was ameliorated by 24-hour pre-exposure to low-concentration H 2 O 2 (10μM-30μM) with cell size as well restored. Markers for apoptosis (Bcl-2 and Bad), inflammation (iNOS), and redox system (MnSOD) were also determined, showing that, in cells pre-exposed to 10μM H 2 O 2 and then submitted to 100μM H 2 O 2 , Bcl-2 levels were higher, Bad and iNOS levels were lower than those observed in damaged cells, and MnSOD levels were unchanged. Such findings show that (1) neuronal-like differentiated SH-SY5Y cells are a suitable model to investigate PC response and more sensitive to the effect of a mild and transient H 2 O 2 -induced oxidative stress with respect to other neuronal cells; (2) 10μM H 2 O 2 -induced PC is mediated by apoptotic and inflammatory pathways, unlike antioxidant system; (3) such neuroprotective strategy and underlying signals proven in neuronal-like differentiated SH-SY5Y cells may contribute to understand in vivo PC mechanisms and to define a window for pharmacological intervention, namely, related to ischemic brain damage. Neuronal-like differentiated SH-SY5Y cells are a suitable model to investigate PC, an endogenous neuroprotective response to a mild and transient H 2 O 2 -induced oxidative stress, elicited by 24-hour exposure to very low H 2 O 2 concentrations and mediated by both apoptotic and inflammatory pathways. This model reflects in vivo PC mechanisms occurring

  10. Inhibition of amyloid β aggregation and protective effect on SH-SY5Y cells by triterpenoid saponins from the cactus Polaskia chichipe.

    Science.gov (United States)

    Fujihara, Koji; Koike, Shin; Ogasawara, Yuki; Takahashi, Kunio; Koyama, Kiyotaka; Kinoshita, Kaoru

    2017-07-01

    Alzheimer's disease (AD) destroys brain function, especially in the hippocampus, and is a social problem worldwide. A major pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) peptides, resulting in neuronal cell death in the brain. Here, we isolated four saponins (1-4) and elucidated their structures from 1D and 2D NMR and HRFABMS spectral data. The structures of 1 and 2 were determined as new saponins which have cochalic acid as the aglycon, and 3 was determined as a new saponin with oleanolic acid as the aglycon. Compound 4 was confirmed as the known saponin chikusetsusaponin V (=ginsenoside R 0 ). Isolated saponins (1-4) and six previously reported saponins (5-10) were tested for their inhibitory effects of Aβ aggregation and their protective effects on SH-SY5Y cells against Aβ-associated toxicity. As the results, compounds 3 and 4 showed inhibitory effect of Aβ aggregation and compounds 5-8 exerted the protective effects on SH-SY5Y cells against Aβ-associated toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Protection against MPP(+)-induced neurotoxicity in SH-SY5Y cells by tormentic acid via the activation of PI3-K/Akt/GSK3β pathway.

    Science.gov (United States)

    Zhao, Qing; Ye, Junli; Wei, Na; Fong, Chichun; Dong, Xiaoli

    2016-07-01

    The cause of Parkinson's disease (PD) could be ascribed to the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta, and thus molecules with neuroprotective ability may have therapeutic value against PD. In the current study, the neuroprotective effects and underlying mechanisms of tormentic acid (TA), a naturally occurring triterpene extracted from medicinal plants such as Rosa rugosa and Potentilla chinensis, were evaluated in a widely used cellular PD model in which neurotoxicity was induced by MPP(+) in cultured SH-SY5Y cells. We found that TA at 1-30 μM substantially protected against MPP(+)-induced neurotoxicity, as evidenced by the increase in cell viability, decrease in lactate dehydrogenase release and the reduction in apoptotic nuclei. Moreover, TA effectively inhibited the elevated intracellular accumulation of reactive oxygen species as well as Bax/Bcl-2 ratio caused by MPP(+). Most importantly, TA markedly reversed the inhibition of protein expression of phosphorylated Akt (Ser 473) and phosphorylated GSK3β (Ser 9) caused by MPP(+). LY294002, the specific inhibitor of PI3-K, significantly abrogated the up-regulated phosphorylated Akt and phosphorylated GSK3β offered by TA, suggesting that the neuroprotection of TA was mainly dependent on the activation of PI3-K/Akt/GSK3β signaling pathway. The results taken together indicate that TA may be a potential candidate for further preclinical study aimed at the prevention and treatment of PD. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells.

    Science.gov (United States)

    Talman, Virpi; Amadio, Marialaura; Osera, Cecilia; Sorvari, Salla; Boije Af Gennäs, Gustav; Yli-Kauhaluoma, Jari; Rossi, Daniela; Govoni, Stefano; Collina, Simona; Ekokoski, Elina; Tuominen, Raimo K; Pascale, Alessia

    2013-07-01

    Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20μM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10μM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that

  13. Rosiglitazone inhibits chlorpyrifos-induced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Park, Jae Hyeon; Jang, Sea Jeong [Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2014-07-15

    Oxidative stress can lead to expression of inflammatory transcription factors, which are important regulatory elements in the induction of inflammatory responses. One of the transcription factors, nuclear transcription factor kappa-B (NF-κB) plays a significant role in the inflammation regulatory process. Inflammatory cell death has been implicated in neuronal cell death in some neurodegenerative disorders such as Parkinson's disease (PD). In this study, we investigated the molecular mechanisms underlying apoptosis initiated by chlorpyrifos (CPF)-mediated oxidative stress. Based on the cytotoxic mechanism of CPF, we examined the neuroprotective effects of rosiglitazone (RGZ), a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, against CPF-induced neuronal cell death. The treatment of SH-SY5Y cells with CPF induced oxidative stress. In addition, CPF activated the p38, JNK and ERK mitogen-activated protein kinases (MAPKs), and induced increases in the inflammatory genes such as COX-2 and TNF-α. CPF also induced nuclear translocation of NF-κB and inhibitors of NF-κB abolished the CPF-induced COX-2 expression. Pretreatment with RGZ significantly reduced ROS generation and enhanced HO-1 expression in CPF-exposed cells. RGZ blocked the activation of both p38 and JNK signaling, while ERK activation was strengthened. RGZ also attenuated CPF-induced cell death through the reduction of NF-κB-mediated proinflammatory factors. Results from this study suggest that RGZ may exert an anti-apoptotic effect against CPF-induced cytotoxicity by attenuation of oxidative stress as well as inhibition of the inflammatory cascade via inactivation of signaling by p38 and JNK, and NF-κB. - Highlights: • CPF induces apoptotic cell death in SH-SY5Y cells • ROS involved in CPF-mediated apoptotic cell death • Inflammation involved in CPF-mediated apoptotic cell death • Rosiglitazone modulates ROS and inflammatory response in CPF-treated cells.

  14. A proteomic analysis of the interactions between poly(L-lactic acid nanofibers and SH-SY5Y neuronal-like cells

    Directory of Open Access Journals (Sweden)

    Ana Marote

    2016-11-01

    Full Text Available Poly (L-lactic acid (PLLA is a biodegradable and biocompatible polymer that has been put forward as a promising material for therapeutic approaches aiming to restore neuronal function. The topographic cues present in PLLA-based scaffolds, defined by the technique used in their preparation, have been shown to play a role on the cellular behavior of adherent cells. Even though this interaction has been shown to influence the regenerative output of the scaffold, there is a lack of studies addressing this response at the proteomic level. Hence, this work focuses on the effect of electrospun PLLA-based nanofibers on the proteome, cellular processes and signaling pathways of SH-SY5Y neuroblastoma cells. It also further explores how these molecular mediators might influence cell proliferation and differentiation upon in vitro culture. For that, mass spectrometry followed by bioinformatics analysis was firstly performed and further complemented with Western blot, cell viability and imaging assays. Results show that PLLA nanofibers differentially activate and inhibit specific cellular functions and signaling pathways related to cell division, apoptosis, actin remodeling, among others. These ultimately block cellular proliferation and induce morphological rearrangements through cytoskeleton remodeling, adaptations that turn cells more prone to differentiate. In synthesis, PLLA nanofibers shift the SH-SY5Y cells proteome towards a state more responsive to differentiation-inductive cues such as the retinoic acid. Unveiling cells responses to nanomaterials is an important step to increase the tools available for their manipulation and potentiate their use in neural tissue engineering. Further studies should be performed to compare the effects of other topographic cues on cellular behavior.

  15. PACAP Protects Against Ethanol and Nicotine Toxicity in SH-SY5Y Cells: Implications for Drinking-Smoking Co-morbidity.

    Science.gov (United States)

    Manavalan, Sridharan; Getachew, Bruk; Manaye, Kebreten F; Khundmiri, Syed J; Csoka, Antonei B; McKinley, Raechel; Tamas, Andrea; Reglodi, Dora; Tizabi, Yousef

    2017-07-01

    The detrimental effects of heavy drinking and smoking are multiplied when the two are combined. Treatment modalities for each and especially for the combination are very limited. Although in low concentration, alcohol and nicotine, each may have beneficial effects including neuroprotection, their combination, instead of providing additive protection, may actually lead to toxicity in cell cultures. Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous 38 amino-acid peptide with demonstrated protection against neuronal injury, trauma as well as various endogenous and exogenous toxic agents. The aim of this study was to investigate whether PACAP may also protect against toxicity induced by high alcohol, high nicotine, or the combination of low alcohol and nicotine concentrations, and if so, whether this effect was mediated via PAC1 receptor. We used the neuroblastoma-derived SH-SY5Y cells and applied various colorimetric assays for determination of cell viability or toxicity. Results indicate that PACAP blocks toxicity induced by high alcohol and high nicotine as well as their combination at low concentrations. The effects of PACAP in turn were blocked by the PACAP antagonist (PACAP 6-38), indicating involvement of the PACAP receptor PAC1 and possibly vasoactive intestinal peptide (VIP) receptors in PACAP's protection. Moreover, no combined toxicity of low alcohol and low nicotine could be detected in calcium-free medium. These findings suggest possible beneficial effects of PACAP in preventing alcohol and nicotine toxicity and that calcium contributes to the damage induced by combination of low alcohol and nicotine in SH-SY5Y cells.

  16. IGF-1 protects SH-SY5Y cells against MPP+-induced apoptosis via PI3K/PDK-1/Akt pathway.

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    Kim, Chanyang; Park, Seungjoon

    2018-03-01

    Insulin-like growth factor (IGF)-1 is a well-known anti-apoptotic pro-survival factor and phosphatidylinositol-3-kinase (PI3K)/Akt pathway is linked to cell survival induced by IGF-1. It is also reported that Akt signaling is modulated by 3-phosphoinositide-dependent kinase-1 (PDK1). In the current study, we investigated whether the anti-apoptotic effect of IGF-1 in SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP + ) is associated with the activity of PI3K/PDK1/Akt pathway. Treatment of cells with IGF-1 inhibited MPP + -induced apoptotic cell death. IGF-1-induced activation of Akt and the protective effect of IGF-1 on MPP + -induced apoptosis were abolished by chemical inhibition of PDK1 (GSK2334470) or PI3K (LY294002). The phosphorylated levels of Akt and PDK1 were significantly suppressed after MPP + exposure, while IGF-1 treatment completely restored MPP+-induced reductions in phosphorylation. IGF-1 protected cells from MPP + insult by suppressing intracellular reactive oxygen species (ROS) production and malondialdehyde levels and increasing superoxide dismutase activity. Mitochondrial ROS levels were also increased during MPP + exposure, which were attenuated by IGF-1 treatment. In addition, IGF-1-treated cells showed increased activities of succinate dehydrogenase and citrate synthase, stabilization of mitochondrial transmembrane potential, increased ratio of Bcl-2 to Bax, prevention of cytochrome c release and inhibition of caspase-3 activation with PARP cleavage. Furthermore, the protective effects of IGF-1 on oxidative stress and mitochondrial dysfunction were attenuated when cells were preincubated with GSK2334470 or LY294002. Our data suggest that IGF-1 protects SH-SY5Y cells against MPP + -associated oxidative stress by preserving mitochondrial integrity and inhibiting mitochondrial apoptotic cascades via the activation of PI3K/PDK1/Akt pathway. © 2018 The authors.

  17. Pharmacognostical Analysis and Protective Effect of Standardized Extract and Rizonic Acid from Erythrina velutina against 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells

    Science.gov (United States)

    Silva, Aline H.; Fonseca, Francisco Noé; Pimenta, Antônia T. A.; Lima, MaryAnne S.; Silveira, Edilberto Rocha; Viana, Glauce S. B.; Vasconcelos, Silvânia M. M.; Leal, Luzia Kalyne A. M.

    2016-01-01

    Background: Erythrina velutina is a tree common in the northeast of Brazil extensively used by traditional medicine for the treatment of central nervous system disorders. Objective: To develop a standardized ethanol extract of E. velutina (EEEV) and to investigate the neuroprotective potential of the extract and rizonic acid (RA) from E. velutina on neuronal cells. Materials and methods: The plant drug of E. velutina previously characterized was used for the production of EEEV. Three methods were evaluated in order to obtain an extract with higher content of phenols. The neuroprotective effect of standardized EEEV (HPLC-PDA) and RA was investigated on SH-SY5Y cell exposure to the neurotoxin 6-hydroxydopamine (6-OHDA). Results: The powder of the plant drug was classified as moderately coarse and several quality control parameters were determined. EEEV produced by percolation gave the highest phenol content when related to others extractive methods, and its HPLC-PDA analysis allowed to identify four flavonoids and RA, some reported for the first time for the species. EEEV and RA reduced significantly the neurotoxicity induced by 6-OHDA in SH-SY5Y cells determined by the MTT assay and the nitrite concentration. EEEV also showed a free radical scavenging activity. Conclusion: This is the first pharmacological study about E. velutina which used a controlled standardized extract since the preparation of the herbal drug. This extract and RA, acting as an antioxidant, presents a neuroprotective effect suggesting that they have potential for future development as a therapeutic agent in neurodegenerative disease as Parkinson. SUMMARY The powder of Erythrina velutina was classified as moderately coarse and several quality-control parameters were determined.Ethanolic extract from E. velutina (EEEV) produced by percolation gave the highest phenol content when related to others extractive methods and its HPLC–PDA analysis of EEEV allowed to identify four flavonoids and rizonic

  18. In vitro neuroprotective potential of four medicinal plants against rotenone-induced toxicity in SH-SY5Y neuroblastoma cells.

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    Seoposengwe, Keabetswe; van Tonder, Jacob John; Steenkamp, Vanessa

    2013-12-12

    Lannea schweinfurthii, Zanthoxylum capense, Scadoxus puniceus and Crinum bulbispermum are used traditionally to treat neurological disorders. The aim of this study was to evaluate the cytoprotective potential of the four plants, after induction of toxicity using rotenone, in SH-SY5Y neuroblastoma cells. Cytotoxicity of the plant extracts and rotenone was assessed using the sulforhodamine B (SRB) assay. Fluorometry was used to measure intracellular redox state (reactive oxygen species (ROS) and intracellular glutathione content), mitochondrial membrane potential (MMP) and caspase-3 activity, as a marker of apoptotic cell death. Of the tested plants, the methanol extract of Z. capense was the least cytotoxic; LC50 121.3 ± 6.97 μg/ml, while S. puniceus methanol extract was the most cytotoxic; LC50 20.75 ± 1.47 μg/ml. Rotenone reduced intracellular ROS levels after 24 h exposure. Pre-treating cells with S. puniceus and C. bulbispermum extracts reversed the effects of rotenone on intracellular ROS levels. Rotenone exposure also decreased intracellular glutathione levels, which was counteracted by pre-treatment with any one of the extracts. MMP was reduced by rotenone, which was neutralized by pre-treatment with C. bulbispermum ethyl acetate extract. All extracts inhibited rotenone-induced activation of caspase-3. The studied plants demonstrated anti-apoptotic activity and restored intracellular glutathione content following rotenone treatment, suggesting that they may possess neuroprotective properties.

  19. OGG1 Involvement in High Glucose-Mediated Enhancement of Bupivacaine-Induced Oxidative DNA Damage in SH-SY5Y Cells

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    Liu, Zhong-Jie; Zhao, Wei; Zhang, Qing-Guo; Li, Le; Lai, Lu-Ying; Jiang, Shan; Xu, Shi-Yuan

    2015-01-01

    Hyperglycemia can inhibit expression of the 8-oxoG-DNA glycosylase (OGG1) which is one of the key repair enzymes for DNA oxidative damage. The effect of hyperglycemia on OGG1 expression in response to local anesthetics-induced DNA damage is unknown. This study was designed to determine whether high glucose inhibits OGG1 expression and aggravates bupivacaine-induced DNA damage via reactive oxygen species (ROS). SH-SY5Y cells were cultured with or without 50 mM glucose for 8 days before they were treated with 1.5 mM bupivacaine for 24 h. OGG1 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. ROS was estimated using the redox-sensitive fluorescent dye DCFH-DA. DNA damage was investigated with immunostaining for 8-oxodG and comet assays. OGG1 expression was inhibited in cells exposed to high glucose with concomitant increase in ROS production and more severe DNA damage as compared to control culture conditions, and these changes were further exacerbated by bupivacaine. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) prevented high glucose and bupivacaine mediated increase in ROS production and restored functional expression of OGG1, which lead to attenuated high glucose-mediated exacerbation of bupivacaine neurotoxicity. Our findings indicate that subjects with diabetes may experience more detrimental effects following bupivacaine use. PMID:26161242

  20. Impact of lithium alone or in combination with haloperidol on oxidative stress parameters and cell viability in SH-SY5Y cell culture.

    Science.gov (United States)

    Gawlik-Kotelnicka, Oliwia; Mielicki, Wojciech; Rabe-Jabłońska, Jolanta; Lazarek, Jerry; Strzelecki, Dominik

    2016-02-01

    It has been reported that lithium may inhibit lipid peroxidation and protein oxidation. Lithium salts also appear to stimulate cell proliferation, increase neurogenesis, and delay cell death. Oxidative stress and neurodegeneration may play an important role in the pathophysiology of bipolar disorder and the disease course thereof. The aim of this research is to estimate the influence of lithium (alone and in combination with haloperidol) on the parameters of oxidative stress and viability of SH-SY5Y cell lines in neutral and pro-oxidative conditions. The evaluated oxidative stress parameter was lipid peroxidation. The viability of the cell lines was measured utilising the MTT test. In neutral conditions, higher levels of thiobarbituric acid reactive substances were observed in those samples which contained both haloperidol and lithium than in other samples. However, these differences were not statistically significant. Cell viability was significantly higher in therapeutic lithium samples than in the controls; samples of haloperidol alone as well as those of haloperidol with lithium did not differ from controls. The results of our study may indicate that lithium possess neuroprotective properties that may be partly due to antioxidative effects. The combination of lithium and haloperidol may generate increased oxidative stress.

  1. OGG1 Involvement in High Glucose-Mediated Enhancement of Bupivacaine-Induced Oxidative DNA Damage in SH-SY5Y Cells

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    Zhong-Jie Liu

    2015-01-01

    Full Text Available Hyperglycemia can inhibit expression of the 8-oxoG-DNA glycosylase (OGG1 which is one of the key repair enzymes for DNA oxidative damage. The effect of hyperglycemia on OGG1 expression in response to local anesthetics-induced DNA damage is unknown. This study was designed to determine whether high glucose inhibits OGG1 expression and aggravates bupivacaine-induced DNA damage via reactive oxygen species (ROS. SH-SY5Y cells were cultured with or without 50 mM glucose for 8 days before they were treated with 1.5 mM bupivacaine for 24 h. OGG1 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR and western blot. ROS was estimated using the redox-sensitive fluorescent dye DCFH-DA. DNA damage was investigated with immunostaining for 8-oxodG and comet assays. OGG1 expression was inhibited in cells exposed to high glucose with concomitant increase in ROS production and more severe DNA damage as compared to control culture conditions, and these changes were further exacerbated by bupivacaine. Treatment with the antioxidant N-acetyl-L-cysteine (NAC prevented high glucose and bupivacaine mediated increase in ROS production and restored functional expression of OGG1, which lead to attenuated high glucose-mediated exacerbation of bupivacaine neurotoxicity. Our findings indicate that subjects with diabetes may experience more detrimental effects following bupivacaine use.

  2. Microtubule proteins and their post-translational forms in the cerebrospinal fluid of patients with paraparesis associated with HTLV-I infection and in SH-SY5Y cells: An in vitro model of HTLV-I-induced disease

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    HORACIO MALDONADO

    2008-01-01

    Full Text Available HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP is characterized by axonal degeneration of the corticospinal tracts. The specific requirements for transport of proteins and organelles to the distal part of the long axon are crucial in the corticospinal tracts. Microtubule dysfunction could be involved in this disease, configuring an axonal transport disease. We measured tubulin and its post-translational modified forms (acetylated and tyrosinated in CSF of patients and controls, as well as tau and its phosphorylated forms. There were no significant differences in the contents of tubulin and acetyl-tubulin between patients and controls; tyrosyl-tubulin was not detected. In HAM/TSP, tau levéis were significantly reduced, while the ratio of pT181/total tau was higher in patients than in controls, this being completely different from what is reported in other neurodegenerative diseases. Phosphorylation at T181 was also confirmed by Mass Spectrometry analysis. Western Blotting with monospecific polyclonal antibodies against pS199, pT205, pT231, pS262, pS356, pS396, pS404 and pS422 did not show differences in phosphorylation in these residues between patients and controls. Treating human SH-SY5Y neuroblastoma cells, a well-known in vitro neurite retraction model, with culture supernatant of MT-2 cells (HTLV-I infected cell line that secretes the viral Tax protein we observed neurite retraction and an increase in tau phosphorylation at T181. A disruption of normal phosphorylation of tau protein in T181 could result in its dysfunction, contributing to axonal damage.

  3. [Pt(O,O'-acac)(γ-acac)(DMS)] alters SH-SY5Y cell migration and invasion by the inhibition of Na+/H+ exchanger isoform 1 occurring through a PKC-ε/ERK/mTOR Pathway.

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Calabriso, Nadia; Cossa, Luca Giulio; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2014-01-01

    We previously showed that [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibits cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.

  4. Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells.

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    Ahmad, Waqas; Li, Yingying; Guo, Yidi; Wang, Xinyu; Duan, Ming; Guan, Zhenhong; Liu, Zengshan; Zhang, Maolin

    2017-06-01

    Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID 50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.

  5. The enzyme lecithin-cholesterol acyltransferase esterifies cerebrosterol and limits the toxic effect of this oxysterol on SH-SY5Y cells.

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    La Marca, Valeria; Spagnuolo, Maria Stefania; Cigliano, Luisa; Marasco, Daniela; Abrescia, Paolo

    2014-07-01

    Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)-hydroxycholesterol, 24(S)OH-C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin-cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH-C. Proteoliposomes containing apolipoprotein A-I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A-I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH-C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH-C esterification. When highly neurotoxic 24(S)OH-C was treated with enzyme and proteoliposomes before incubation with differentiated SH-SY5Y cells, the neuron survival improved. The esters of 24(S)OH-C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH-C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH-C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol-induced neurotoxic injuries to neurons in culture. 24-hydroxycholesterol (24(S)OH-C) is neurotoxic. The enzyme lecithin-cholesterol acyltransferase (LCAT) synthesizes monoesters of 24(S)OH-C in reaction mixtures with proteoliposomes containing phospholipids and apolipoprotein A-I or apolipoprotein E. The esters, also produced by incubation of cerebrospinal fluid only with tritiated 24(S)OH-C, are embedded into lipoproteins that do not enter neurons in culture. The enzyme activity limits the toxicity of 24-hydroxycholesterol in neuron culture. © 2014

  6. 17β-estradiol-induced regulation of the novel 5-HT1A-related transcription factors NUDR and Freud-1 in SH SY5Y cells.

    Science.gov (United States)

    Adeosun, Samuel O; Albert, Paul R; Austin, Mark C; Iyo, Abiye H

    2012-05-01

    Nuclear deformed epidermal autoregulatory factor-1 (NUDR/Deaf-1) and five prime repressor element under dual repression (Freud-1) are novel transcriptional regulators of the 5-HT(1A) receptor, a receptor that has been implicated in the pathophysiology of various psychiatric illnesses. The antidepressant effect of 17β-Estradiol (17βE(2)) is purported to involve the downregulation of this receptor. We investigated the possible role of NUDR and Freud-1 in 17βE(2)-induced downregulation of the 5-HT(1A) receptor in the neuroblastoma cell line SH SY5Y. Cells were treated with 10 nM of 17βE(2) for 3 or 48 h, followed by a 24-h withdrawal period. Proteins were isolated and analyzed by western blotting. 17βE(2) treatment increased NUDR immunoreactivity while Freud-1 and the 5-HT(1A) receptor showed significant decreases. Upon withdrawal of 17βE(2), protein expression returned to control levels, except for NUDR, which remained significantly elevated in the 3-h treatment. Taken together, these data support a non-genomic downregulation of 5-HT(1A) receptor protein by 17βE(2), which does not involve NUDR and Freud-1. Rather, changes in both transcription factors seem to be compensatory/homeostatic responses to changes in 5-HT(1A) receptor induced by 17βE(2). These observations further highlight the importance of NUDR and Freud-1 in regulating 5-HT(1A) receptor expression.

  7. A novel compound derived from danshensu inhibits apoptosis via upregulation of heme oxygenase-1 expression in SH-SY5Y cells.

    Science.gov (United States)

    Pan, Li-Long; Liu, Xin-Hua; Jia, Yao-Ling; Wu, Dan; Xiong, Qing-Hui; Gong, Qi-Hai; Wang, Yang; Zhu, Yi-Zhun

    2013-04-01

    Heme oxygenase-1 (HO-1) has potential anti-apoptotic properties. A novel compound [4-(2-acetoxy-3-((R)-3-(benzylthio)-1-methoxy-1-oxopropan-2- ylamino)-3-oxopropyl)-1,2-phenylene diacetate (DSC)] was synthesized by joining danshensu and cysteine through an appropriate linker. This study investigated if the cytoprotective properties of DSC involved the induction of HO-1. We evaluated the cytoprotective effects of DSC on H2O2-induced cell damage, apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) loss, and apoptosis-related proteins expression and its underlying mechanisms. DSC concentration-dependently attenuated cell death, lactate dehydrogenase release, intracellular and mitochondrial ROS production, and ΔΨm collapse, modulated apoptosis-related proteins (Bcl-2, Bax, caspase-3, p53, and cleaved PARP) expression, and inhibited phosphorylation of extracellular signal-regulated kinase 1/2 in SH-SY5Y cells induced by H2O2. In addition, DSC concentration-dependently induced HO-1 expression associated with nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf-2), while the effect of DSC was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Furthermore, the protective effect of DSC on H2O2-induced cell death was abolished by HO-1 inhibitor ZnPP, but was mimicked by carbon monoxide-releasing moiety CORM-3 or HO-1 by-product bilirubin. Finally, DSC inhibited H2O2-induced changes of Bcl-2, Bax, and caspase-3 expression, and all of these effects were reversed by HO-1 silencing. Induction of HO-1 may be, at least in part, responsible for the anti-apoptotic property of DSC, an effect that involved the activation of PI3K/Akt/Nrf-2 axis. DSC might have the potential for beneficial therapeutic interventions for neurodegenerative diseases. Copyright © 2013. Published by Elsevier B.V.

  8. The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria, within the context of a tumour stem cell-like phenotype.

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    Pierdomenico Ruggeri

    Full Text Available The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs, correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.

  9. Early LC3 lipidation induced by d-limonene does not rely on mTOR inhibition, ERK activation and ROS production and it is associated with reduced clonogenic capacity of SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    Berliocchi, Laura; Chiappini, Carlotta; Adornetto, Annagrazia; Gentile, Debora; Cerri, Silvia; Russo, Rossella; Bagetta, Giacinto; Corasaniti, Maria Tiziana

    2018-02-01

    d-Limonene is a natural monoterpene abundant in Citrus essential oils. It is endowed with several biological activities, including inhibition of carcinogenesis and promotion of tumour regression. Recently, d-limonene has been shown to modulate autophagic markers in vitro at concentrations found in vivo, in clinical pharmacokinetic studies. Autophagy is an intracellular catabolic process serving as both an adaptive metabolic response and a quality control mechanism. Because autophagy defects have been linked to a wide range of human pathologies, including neurodegeneration and cancer, there is a need for new pharmacological tools to control deregulated autophagy. To better understand the effects of d-limonene on autophagy, to identify the molecular mechanisms through which this monoterpene rapidly triggers LC3 lipidation and to evaluate the role for autophagy in long-term effects of d-limonene. Human SH-SY5Y neuroblastoma, HepG2 hepatocellular carcinoma and MCF7 breast cancer cells were used. Endogenous LC3-II levels were evaluated by western blotting. Autophagic flux assay was performed using bafilomycin A1 and chloroquine. Intracellular distribution of LC3 protein was studied by confocal microscopy analysis of LC3B-GFP transduced cells. Expression of lysosomal-membrane protein LAMP-1 was assessed by immunofluorescence analysis. Phosphorylated levels of downstream substrates of mTOR kinase (p70S6 kinase, 4E-BP1, and ULK1) and ERK were analyzed by western blotting. Production of reactive oxygen species (ROS) was assessed by live confocal microscopy of cells loaded with CellROX ® Green Reagent. Clonogenic assay was used to evaluate the ability of treated cells to proliferate and form colonies. LC3 lipidation promoted by d-limonene correlates with autophagosome formation and stimulation of basal autophagy. LC3 lipidation does not rely on inhibition of mTOR kinase, which instead appears to be transiently activated. In addition, d-limonene rapidly activates ERK and

  10. [Pt(O,O’-acac)(γ-acac)(DMS)] Alters SH-SY5Y Cell Migration and Invasion by the Inhibition of Na+/H+ Exchanger Isoform 1 Occurring through a PKC-ε/ERK/mTOR Pathway

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Calabriso, Nadia; Cossa, Luca Giulio; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2014-01-01

    We previously showed that [Pt(O,O’-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibites cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation. PMID:25372487

  11. Ginsenoside Rb1 Attenuates Oxygen-Glucose Deprivation-Induced Apoptosis in SH-SY5Y Cells via Protection of Mitochondria and Inhibition of AIF and Cytochrome c Release

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    Pengfei Ge

    2013-10-01

    Full Text Available To investigate the role of mitochondria in the protective effects of ginsenoside Rb1 on cellular apoptosis caused by oxygen-glucose deprivation, in this study, MTT assay, TUNEL staining, flow cytometry, immunocytochemistry and western blotting were used to examine the cellular viability, apoptosis, ROS level, mitochondrial membrane potential, and the distribution of apoptosis inducing factor, cytochrome c, Bax and Bcl-2 in nucleus, mitochondria and cytoplasm. We found that pretreatment with GRb1 improved the cellular viability damaged by OGD. Moreover, GRb1 inhibited apoptosis in SH-SY5Y cells induced by OGD. Further studies showed that the elevation of cellular reactive oxygen species levels and the reduction of mitochondrial membrane potential caused by OGD were both counteracted by GRb1. Additionally, GRb1 not only suppressed the translocation of apoptosis inducing factor into nucleus and cytochrome c into cytoplasm, but also inhibited the increase of Bax within mitochondria and alleviated the decrease of mitochondrial Bcl-2. Our study indicates that the protection of GRb1 on OGD-induced apoptosis in SH-SY5Y cells is associated with its protection on mitochondrial function and inhibition of release of AIF and cytochrome c.

  12. Melatonin reverses H2 O2 -induced senescence in SH-SY5Y cells by enhancing autophagy via sirtuin 1 deacetylation of the RelA/p65 subunit of NF-κB.

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    Nopparat, Chutikorn; Sinjanakhom, Puritat; Govitrapong, Piyarat

    2017-08-01

    Autophagy, a degradation mechanism that plays a major role in maintaining cellular homeostasis and diminishes in aging, is considered an aging characteristic. Melatonin is an important hormone that plays a wide range of physiological functions, including the anti-aging effect, potentially via the regulation of the Sirtuin1 (SIRT1) pathway. The deacetylation ability of SIRT1 is important for controlling the function of several transcription factors, including nuclear factor kappa B (NF-ĸB). Apart from inflammation, NF-ĸB can regulate autophagy by inhibiting Beclin1, an initiator of autophagy. Although numerous studies have revealed the role of melatonin in regulating autophagy, very limited experiments have shown that melatonin can increase autophagic activity via SIRT1 in a senescent model. This study focuses on the effect of melatonin on autophagy via the deacetylation activity of SIRT1 on RelA/p65, a subunit of NF-ĸB, to determine whether melatonin can attenuate the aging condition. SH-SY5Y cells were treated with H 2 O 2 to induce the senescent state. These results demonstrated that melatonin reduced a number of beta-galactosidase (SA-βgal)-positive cells, a senescent marker. In addition, melatonin increased the protein levels of SIRT1, Beclin1, and LC3-II, a hallmark protein of autophagy, and reduced the levels of acetylated-Lys310 in the p65 subunit of NF-ĸB in SH-SY5Y cells treated with H 2 O 2 . Furthermore, in the presence of SIRT1 inhibitor, melatonin failed to increase autophagic markers. The present data indicate that melatonin enhances autophagic activity via the SIRT1 signaling pathway. Taken together, we propose that in modulating autophagy, melatonin may provide a therapeutically beneficial role in the anti-aging processes. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Gastrodin Protects Apoptotic Dopaminergic Neurons in a Toxin-Induced Parkinson’s Disease Model

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    Hemant Kumar

    2013-01-01

    Full Text Available Gastrodia elata (GE Blume is one of the most important traditional plants in Oriental countries and has been used for centuries to improve various conditions. The phenolic glucoside gastrodin is an active constituent of GE. The aim of this study was to investigate the neuroprotective role of gastrodin in 1-methyl-4-phenylpyridinium (MPP+/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP induced human dopaminergic SH-SY5Y cells and mouse model of Parkinson’s disease (PD, respectively. Gastrodin significantly and dose dependently protected dopaminergic neurons against neurotoxicity through regulating free radicals, Bax/Bcl-2 mRNA, caspase-3, and cleaved poly(ADP-ribose polymerase (PARP in SH-SY5Y cells stressed with MPP+. Gastrodin also showed neuroprotective effects in the subchronic MPTP mouse PD model by ameliorating bradykinesia and motor impairment in the pole and rotarod tests, respectively. Consistent with this finding, gastrodin prevented dopamine depletion and reduced reactive astrogliosis caused by MPTP as assessed by immunohistochemistry and immunoblotting in the substantiae nigrae and striatata of mice. Moreover, gastrodin was also effective in preventing neuronal apoptosis by attenuating antioxidant and antiapoptotic activities in these brain areas. These results strongly suggest that gastrodin has protective effects in experimental PD models and that it may be developed as a clinical candidate to ameliorate PD symptoms.

  14. Hydrogen sulfide inhibits A2A adenosine receptor agonist induced β-amyloid production in SH-SY5Y neuroblastoma cells via a cAMP dependent pathway.

    Directory of Open Access Journals (Sweden)

    Bhushan Vijay Nagpure

    Full Text Available Alzheimer's disease (AD is the leading cause of senile dementia in today's society. Its debilitating symptoms are manifested by disturbances in many important brain functions, which are influenced by adenosine. Hence, adenosinergic system is considered as a potential therapeutic target in AD treatment. In the present study, we found that sodium hydrosulfide (NaHS, an H2S donor, 100 µM attenuated HENECA (a selective A2A receptor agonist, 10-200 nM induced β-amyloid (1-42 (Aβ42 production in SH-SY5Y cells. NaHS also interfered with HENECA-stimulated production and post-translational modification of amyloid precursor protein (APP by inhibiting its maturation. Measurement of the C-terminal APP fragments generated from its enzymatic cleavage by β-site amyloid precursor protein cleaving enzyme 1 (BACE1 showed that NaHS did not have any significant effect on β-secretase activity. However, the direct measurements of HENECA-elevated γ-secretase activity and mRNA expressions of presenilins suggested that the suppression of Aβ42 production in NaHS pretreated cells was mediated by inhibiting γ-secretase. NaHS induced reductions were accompanied by similar decreases in intracellular cAMP levels and phosphorylation of cAMP responsive element binding protein (CREB. NaHS significantly reduced the elevated cAMP and Aβ42 production caused by forskolin (an adenylyl cyclase, AC agonist alone or forskolin in combination with IBMX (a phosphodiesterase inhibitor, but had no effect on those caused by IBMX alone. Moreover, pretreatment with NaHS significantly attenuated HENECA-elevated AC activity and mRNA expressions of various AC isoforms. These data suggest that NaHS may preferentially suppress AC activity when it was stimulated. In conclusion, H2S attenuated HENECA induced Aβ42 production in SH-SY5Y neuroblastoma cells through inhibiting γ-secretase via a cAMP dependent pathway.

  15. Luteolin Isolated from the Medicinal Plant Elsholtzia rugulosa (Labiatae Prevents Copper-Mediated Toxicity in β-Amyloid Precursor Protein Swedish Mutation Overexpressing SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    Guanhua Du

    2011-03-01

    Full Text Available Luteolin, a 3’,4’,5,7-tetrahydroxyflavone, is a plant flavonoid and pharmacologically active agent that has been isolated from several plant species. In the present study, the effects of luteolin obtained from the medicinal plant Elsholtzia rugulosa and the related mechanisms were examined in an Alzheimer's disease (AD cell model. In this model, copper was used to exacerbate the neurotoxicity in β-amyloid precursor protein Swedish mutation stably overexpressed SH-SY5Y cells (named “APPsw cells” for short. Based on this model, we demonstrated that luteolin increased cell viability, reduced intracellular ROS generation, enhanced the activity of SOD and reversed mitochondrial membrane potential dissipation. Inhibition of caspase-related apoptosis was consistently involved in the neuroprotection afforded by luteolin. Furthermore, it down-regulated the expression of AβPP and lowered the secretion of Aβ1-42. These results indicated that luteolin from the Elsholtzia rugulosa exerted neroprotective effects through mechanisms that decrease AβPP expression, lower Aβ secretion, regulate the redox imbalance, preserve mitochondrial function, and depress the caspase family-related apoptosis.

  16. Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aβ(1-42) secretion in SH-SY5Y neural cells.

    Science.gov (United States)

    Khan, Mahfuz B; Lang, Michelle J; Huang, Ming-Bo; Raymond, Andrea; Bond, Vincent C; Shiramizu, Bruce; Powell, Michael D

    2016-04-01

    In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aβ) and Aβ peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND.

  17. Organic solvent-induced changes in membrane geometry in human SH-SY5Y neuroblastoma cells - a common narcotic effect?

    NARCIS (Netherlands)

    Meulenberg, C.J.W.; de Groot, A.; Westerink, R.H.S.; Vijverberg, H.P.M.

    2016-01-01

    Exposure to organic solvents may cause narcotic effects. At the cellular level, these narcotic effects have been associated with a reduction in neuronal excitability caused by changes in membrane structure and function. In order to critically test whether changes in membrane geometry contribute to

  18. Germinated Brown Rice Alters Aβ(1-42 Aggregation and Modulates Alzheimer’s Disease-Related Genes in Differentiated Human SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    Nur Hanisah Azmi

    2015-01-01

    Full Text Available The pathogenesis of Alzheimer’s disease involves complex etiological factors, of which the deposition of beta-amyloid (Aβ protein and oxidative stress have been strongly implicated. We explored the effects of H2O2, which is a precursor for highly reactive hydroxyl radicals, on neurotoxicity and genes related to AD on neuronal cells. Candidate bioactive compounds responsible for the effects were quantified using HPLC-DAD. Additionally, the effects of germinated brown rice (GBR on the morphology of Aβ(1-42 were assessed by Transmission Electron Microscopy and its regulatory effects on gene expressions were explored. The results showed that GBR extract had several phenolic compounds and γ-oryzanol and altered the structure of Aβ(1-42 suggesting an antiamyloidogenic effect. GBR was also able to attenuate the oxidative effects of H2O2 as implied by reduced LDH release and intracellular ROS generation. Furthermore, gene expression analyses showed that the neuroprotective effects of GBR were partly mediated through transcriptional regulation of multiple genes including Presenilins, APP, BACE1, BACE2, ADAM10, Neprilysin, and LRP1. Our findings showed that GBR exhibited neuroprotective properties via transcriptional regulation of APP metabolism with potential impact on Aβ aggregation. These findings can have important implications for the management of neurodegenerative diseases like AD and are worth exploring further.

  19. Melandrii Herba Extract Attenuates H2O2-Induced Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells and Scopolamine-Induced Memory Impairment in Mice

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    Kwang Min Lee

    2017-09-01

    Full Text Available Oxidative stress plays a significant role in the etiology of a variety of neurodegenerative diseases. In this study, we found that Melandrii Herba extract (ME attenuated oxidative-induced damage in cells. Mechanistically, ME exhibited protection from H2O2-induced neurotoxicity via caspase-3 inactivation, Bcl-2 downregulation, Bax upregulation, and MAPK activation (ERK 1/2, JNK 1/2, and p38 MAPK in vitro. Moreover, our in vivo data showed that ME was able to attenuate scopolamine-induced cognitive impairment. These results provide in vitro and in vivo evidence that ME exhibits neuroprotective properties against oxidative stress, which suggests that ME is worthy of further investigation as a complementary, or even as an alternative, product for preventing and treating neurodegenerative disorders.

  20. The neuroprotective effects of α-iso-cubebene on dopaminergic cell death: involvement of CREB/Nrf2 signaling.

    Science.gov (United States)

    Park, Sun Young; Son, Beung Gu; Park, Young Hoon; Kim, Cheol-Min; Park, Geuntae; Choi, Young-Whan

    2014-09-01

    As a part of ongoing studies to elucidate pharmacologically active components of Schisandra chinensis, we isolated and studied α-iso-cubebene. The neuroprotective mechanisms of α-iso-cubebene in human neuroblastoma SH-SY5Y cells were investigated. α-Iso-cubebene significantly inhibited cytotoxicity and apoptosis due to 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in dopaminergic SH-SY5Y cells. Pretreatment of cells with α-iso-cubebene reduced intracellular accumulation of ROS and calcium in response to 6-OHDA. The neuroprotective effects of α-iso-cubebene were found to result from protecting the mitochondrial membrane potential. Notably, α-iso-cubebene inhibited the release of apoptosis-inducing factor from the mitochondria into the cytosol and nucleus after 6-OHDA treatment. α-Iso-cubebene also induced the activation of PKA/PKB/CREB/Nrf2 and suppressed 6-OHDA-induced neurotoxicity. α-Iso-cubebene was found to induce phosphorylation of PKA and PKB and activate Nrf2 and CREB signaling pathways in a dose-dependent manner. Additionally, α-iso-cubebene stimulated the expression of the antioxidant response genes NQO1 and HO-1. Finally, α-iso-cubebene-mediated neuroprotective effects were found to be reversible after transfection with CREB and Nrf2 small interfering RNAs.

  1. TrkAIII Promotes Microtubule Nucleation and Assembly at the Centrosome in SH-SY5Y Neuroblastoma Cells, Contributing to an Undifferentiated Anaplastic Phenotype

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    Antonietta R. Farina

    2013-01-01

    Full Text Available The alternative TrkAIII splice variant is expressed by advanced stage human neuroblastomas (NBs and exhibits oncogenic activity in NB models. In the present study, employing stable transfected cell lines and assays of indirect immunofluorescence, immunoprecipitation, Western blotting, microtubule regrowth, tubulin kinase, and tubulin polymerisation, we report that TrkAIII binds α-tubulin and promotes MT nucleation and assembly at the centrosome. This effect depends upon spontaneous TrkAIII activity, TrkAIII localisation to the centrosome and pericentrosomal area, and the capacity of TrkAIII to bind, phosphorylate, and polymerise tubulin. We propose that this novel role for TrkAIII contributes to MT involvement in the promotion and maintenance of an undifferentiated anaplastic NB cell morphology by restricting and augmenting MT nucleation and assembly at the centrosomal MTOC.

  2. Physically disconnected non-diffusible cell-to-cell communication between neuroblastoma SH-SY5Y and DRG primary sensory neurons.

    Science.gov (United States)

    Chaban, Victor V; Cho, Taehoon; Reid, Christopher B; Norris, Keith C

    2013-01-01

    Cell-cell communication occurs via a variety of mechanisms, including long distances (hormonal), short distances (paracrine and synaptic) or direct coupling via gap junctions, antigen presentation, or ligand-receptor interactions. We evaluated the possibility of neuro-hormonal independent, non-diffusible, physically disconnected pathways for cell-cell communication using dorsal root ganglion (DRG) neurons. We assessed intracellular calcium ([Ca(2+)]) in primary culture DRG neurons that express ATP-sensitive P2X3, capsaicinsensitive TRPV1 receptors modulated by estradiol. Physically disconnected (dish-in-dish system; inner chamber enclosed) mouse DRG were cultured for 12 hours near: a) media alone (control 1), b) mouse DRG (control 2), c) human neuroblastoma SHSY-5Y cells (cancer intervention), or d) mouse DRG treated with KCl (apoptosis intervention). Chemosensitive receptors [Ca(2+)](i) signaling did not differ between control 1 and 2. ATP (10 μM) and capsaicin (100nM) increased [Ca(2+)](i) transients to 425.86 + 49.5 nM, and 399.21 ± 44.5 nM, respectively. 17β-estradiol (100 nM) exposure reduced ATP (171.17 ± 48.9 nM) and capsaicin (175.01±34.8 nM) [Ca(2+)](i) transients. The presence of cancer cells reduced ATP- and capsaicin-induced [Ca(2+)](i) by >50% (pcommunication.

  3. Histone deacetylase 4 promotes ubiquitin-dependent proteasomal degradation of Sp3 in SH-SY5Y cells treated with di(2-ethylhexyl)phthalate (DEHP), determining neuronal death

    International Nuclear Information System (INIS)

    Guida, Natascia; Laudati, Giusy; Galgani, Mario; Santopaolo, Marianna; Montuori, Paolo; Triassi, Maria; Di Renzo, Gianfranco; Canzoniero, Lorella M.T.; Formisano, Luigi

    2014-01-01

    Phthalates, phthalic acid esters, are widely used as plasticizers to produce polymeric materials in industrial production of plastics and daily consumable products. Animal studies have shown that di(2-ethylhexyl)phthalate (DEHP) may cause toxic effects in the rat brain. In the present study, chronic exposure to DEHP (0.1–100 μM) caused dose-dependent cell death via the activation of caspase-3 in neuroblastoma cells. Intriguingly, this harmful effect was prevented by the pan-histone deacetylase (HDAC) inhibitor trichostatin A, by the class II HDAC inhibitor MC-1568, but not by the class I HDAC inhibitor MS-275. Furthermore, DEHP reduced specificity protein 3 (Sp3) gene expression, but not Sp3 mRNA, after 24 and 48 h exposures. However, Sp3 protein reduction was prevented by pre-treatment with MC-1568, suggesting the involvement of class II HDACs in causing this effect. Then, we investigated the possible relationship between DEHP-induced neuronal death and the post-translational mechanisms responsible for the down-regulation of Sp3. Interestingly, DEHP-induced Sp3 reduction was associated to its deacetylation and polyubiquitination. Co-immunoprecipitation studies showed that Sp3 physically interacted with HDAC4 after DEHP exposure, while HDAC4 inhibition by antisense oligodeoxynucleotide reverted the DEHP-induced degradation of Sp3. Notably, Sp3 overexpression was able to counteract the detrimental effect induced by DEHP. Taken together, these results suggest that DEHP exerts its toxic effect by inducing deacetylation of Sp3 via HDAC4, and afterwards, Sp3-polyubiquitination. - Highlights: • Di(2-ethylhexyl)phthalate (DEHP) is cytotoxic to SH-SY5Y cells and cortical neurons. • DEHP-induced cytotoxicity is mediated by apoptosis. • DEHP-induced apoptotic cell death is inhibited by class II HDAC MC-1568. • DEHP neurotoxicity is caused by HDAC4-mediated Sp3 degradation by ubiquitin

  4. Histone deacetylase 4 promotes ubiquitin-dependent proteasomal degradation of Sp3 in SH-SY5Y cells treated with di(2-ethylhexyl)phthalate (DEHP), determining neuronal death

    Energy Technology Data Exchange (ETDEWEB)

    Guida, Natascia; Laudati, Giusy [Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatologic Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini 5, 80131 Naples (Italy); Galgani, Mario; Santopaolo, Marianna [Laboratorio di Immunologia, Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), Napoli (Italy); Montuori, Paolo; Triassi, Maria [Department of Preventive Medical Sciences, University Federico II, Via Pansini 5, 80131 Naples (Italy); Di Renzo, Gianfranco [Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatologic Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini 5, 80131 Naples (Italy); Canzoniero, Lorella M.T., E-mail: canzon@unisannio.it [Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatologic Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini 5, 80131 Naples (Italy); Division of Pharmacology, Department of Science and Technology, University of Sannio, Via Port' Arsa 11, 82100 Benevento (Italy); Formisano, Luigi, E-mail: cformisa@unisannio.it [Division of Pharmacology, Department of Neuroscience, Reproductive and Odontostomatologic Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini 5, 80131 Naples (Italy); Division of Pharmacology, Department of Science and Technology, University of Sannio, Via Port' Arsa 11, 82100 Benevento (Italy)

    2014-10-01

    Phthalates, phthalic acid esters, are widely used as plasticizers to produce polymeric materials in industrial production of plastics and daily consumable products. Animal studies have shown that di(2-ethylhexyl)phthalate (DEHP) may cause toxic effects in the rat brain. In the present study, chronic exposure to DEHP (0.1–100 μM) caused dose-dependent cell death via the activation of caspase-3 in neuroblastoma cells. Intriguingly, this harmful effect was prevented by the pan-histone deacetylase (HDAC) inhibitor trichostatin A, by the class II HDAC inhibitor MC-1568, but not by the class I HDAC inhibitor MS-275. Furthermore, DEHP reduced specificity protein 3 (Sp3) gene expression, but not Sp3 mRNA, after 24 and 48 h exposures. However, Sp3 protein reduction was prevented by pre-treatment with MC-1568, suggesting the involvement of class II HDACs in causing this effect. Then, we investigated the possible relationship between DEHP-induced neuronal death and the post-translational mechanisms responsible for the down-regulation of Sp3. Interestingly, DEHP-induced Sp3 reduction was associated to its deacetylation and polyubiquitination. Co-immunoprecipitation studies showed that Sp3 physically interacted with HDAC4 after DEHP exposure, while HDAC4 inhibition by antisense oligodeoxynucleotide reverted the DEHP-induced degradation of Sp3. Notably, Sp3 overexpression was able to counteract the detrimental effect induced by DEHP. Taken together, these results suggest that DEHP exerts its toxic effect by inducing deacetylation of Sp3 via HDAC4, and afterwards, Sp3-polyubiquitination. - Highlights: • Di(2-ethylhexyl)phthalate (DEHP) is cytotoxic to SH-SY5Y cells and cortical neurons. • DEHP-induced cytotoxicity is mediated by apoptosis. • DEHP-induced apoptotic cell death is inhibited by class II HDAC MC-1568. • DEHP neurotoxicity is caused by HDAC4-mediated Sp3 degradation by ubiquitin.

  5. Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

    Science.gov (United States)

    Tong, Zhi-Bin; Hogberg, Helena; Kuo, David; Sakamuru, Srilatha; Xia, Menghang; Smirnova, Lena; Hartung, Thomas; Gerhold, David

    2017-02-01

    More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Caracterização da diferenciação neural induzida por ácido retinóico da linhagem de neuroblastoma humano SH-SY5Y e seu uso como ferramenta para pesquisa em neurociências

    OpenAIRE

    Fernanda Martins Lopes

    2012-01-01

    Os mecanismos moleculares que levam ao dano da via nigroestriatal durante a progressão da Doença de Parkinson (DP) ainda não estão totalmente elucidados. Dessa forma, existe a necessidade de desenvolver modelos experimentais adequados para o estudo desse distúrbio neurodegenerativo. A linhagem de neuroblastoma humano SH-SY5Y tratada com neurotoxinas indutoras deste distúrbio (ex.: 6-hidroxidopamina - 6-OHDA) é amplamente utilizada como modelo in vitro da DP. Muitos estudos mostram que esta li...

  7. Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)2] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38MAPK/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment.

    Science.gov (United States)

    Filomeni, Giuseppe; Cardaci, Simone; Da Costa Ferreira, Ana Maria; Rotilio, Giuseppe; Ciriolo, Maria Rosa

    2011-08-01

    We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N']copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment. © The Authors Journal compilation © 2011 Biochemical Society

  8. Neuroprotective Effects of Erucin against 6-Hydroxydopamine-Induced Oxidative Damage in a Dopaminergic-like Neuroblastoma Cell Line

    Directory of Open Access Journals (Sweden)

    Giorgio Cantelli-Forti

    2012-08-01

    Full Text Available Oxidative stress (OS contributes to the cascade leading to the dysfunction or death of dopaminergic neurons during Parkinson’s disease (PD. A strategy to prevent the OS of dopaminergic neurons may be the use of phytochemicals as inducers of endogenous antioxidants and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic-like neuroblastoma SH-SY5Y cell line with isothiocyanate erucin (ER, a compound of cruciferous vegetables, resulted in significant increases of both total glutathione (GSH levels and total antioxidant capacity at the cytosolic level. The increase of GSH levels was associated with an increase in the resistance of SH-SY5Y cells to neuronal death, in terms of apoptosis, induced by 6-hydroxydopamine (6-OHDA. The pretreatment of SH-SY5Y cells with ER was also shown to prevent the redox status impairment, in terms of intracellular ROS and O2•− formation, and loss of mitochondrial membrane potential, early events that are initiators of the apoptotic process, induced by 6-OHDA. Last, the antiapoptotic and antioxidant effects of ER were abolished by buthionine sulfoximine, supporting the main role of GSH in the neuroprotective effects recorded by ER. These results suggest that ER may prevent the oxidative damage induced by 6-OHDA.

  9. Blood brain barrier permeability of (−-epigallocatechin gallate, its proliferation-enhancing activity of human neuroblastoma SH-SY5Y cells, and its preventive effect on age-related cognitive dysfunction in mice

    Directory of Open Access Journals (Sweden)

    Monira Pervin

    2017-03-01

    Conclusion: Cognitive dysfunction in mice is suppressed after ingesting GTCs when a low concentration of EGCG is incorporated into the brain parenchyma via the BBB. Nerve cell proliferation/differentiation was enhanced by a low concentration of EGCG. Furthermore, the additive effect of EGC and GA suggests that EGCG sustains a preventive effect after the hydrolysis to EGC and GA.

  10. Familial CJD Associated PrP Mutants within Transmembrane Region Induced Ctm-PrP Retention in ER and Triggered Apoptosis by ER Stress in SH-SY5Y Cells

    Science.gov (United States)

    Wang, Xin; Shi, Qi; Xu, Kun; Gao, Chen; Chen, Cao; Li, Xiao-Li; Wang, Gui-Rong; Tian, Chan; Han, Jun; Dong, Xiao-Ping

    2011-01-01

    Background Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrPC) to the pathogenic one (PrPSc). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. Methodology/Principal Findings To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and capase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed. Conclusions/Significance The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants

  11. Familial CJD associated PrP mutants within transmembrane region induced Ctm-PrP retention in ER and triggered apoptosis by ER stress in SH-SY5Y cells.

    Directory of Open Access Journals (Sweden)

    Xin Wang

    Full Text Available BACKGROUND: Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP gene that are presumed to favor conversion of the cellular isoform of PrP (PrP(C to the pathogenic one (PrP(Sc. The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K into the cultured cells in order to explore the pathogenic mechanism of familial prion disease. METHODOLOGY/PRINCIPAL FINDINGS: To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER, the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL and PrP with three amino acids exchange in transmembrane region (PrP-3AV were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and caspase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K failed. CONCLUSIONS/SIGNIFICANCE: The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them

  12. Protection of dopaminergic neurons by 5-lipoxygenase inhibitor.

    Science.gov (United States)

    Kang, Kai-Hsiang; Liou, Horng-Hui; Hour, Mann-Jen; Liou, Houng-Chi; Fu, Wen-Mei

    2013-10-01

    Neuroinflammation and oxidative stress are important factors that induce neurodegeneration in age-related neurological disorders. 5-Lipoxygenase (5-LOX) is the enzyme responsible for catalysing the synthesis of leukotriene or 5-HETE from arachidonic acid. 5-LOX is expressed in the central nervous system and may cause neurodegenerative disease. In this study, we investigated the effect of the pharmacological inhibition of 5-lipoxygenase on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/MPP(+)-induced dopaminergic neuronal death in midbrain neuron-glia co-cultures and in mice. It was found that 5-LOX was over-expressed in astrocytes after the injection of MPTP into C57BL6 mice. MK-886, a specific inhibitor of 5-LOX activating protein (FLAP), significantly increased [(3)H]-dopamine uptake, a functional indicator of the integrity of dopaminergic neurons, in midbrain cultures or the SH-SY5Y human dopaminergic cell line following MPP(+) treatment. In addition, LTB₄, one of 5-LOX's downstream products, was increased in the striatum and substantia nigra following MPTP injection in mice. LTB₄ but not LTD₄ and 5-HETE enhanced MPP(+)-induced neurotoxicity in primary midbrain cultures. MK-886 administration increased the number of tyrosine hydroxylase-positive neurons in the substantia nigra and the dopamine content in the striatum in MPTP-induced parkinsonian mice. Furthermore, the MPTP-induced upregulation of LTB₄ in the striatum and substantia nigra was antagonised by MK-886. These results suggest that 5-LOX inhibitors may be developed as novel neuroprotective agents and LTB₄ may play an important pathological role in Parkinson's disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Effects of 1950 MHz radiofrequency electromagnetic fields on Aβ processing in human neuroblastoma and mouse hippocampal neuronal cells

    International Nuclear Information System (INIS)

    Park, Jeongyeon; Kwon, Jong Hwa; Kim, Nam; Song, Kiwon

    2018-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disease leading to progressive loss of memory and other cognitive functions. One of the well-known pathological markers of AD is the accumulation of amyloid-beta protein (Aβ), and its plaques, in the brain. Recent studies using Tg-5XFAD mice as a model of AD have reported that exposure to radiofrequency electromagnetic fields (RF-EMF) from cellular phones reduced Aβ plaques in the brain and showed beneficial effects on AD. In this study, we examined whether exposure to 1950 MHz RF-EMF affects Aβ processing in neural cells. We exposed HT22 mouse hippocampal neuronal cells and SH-SY5Y human neuroblastoma cells to RF-EMF (SAR 6 W/kg) for 2 h per day for 3 days, and analyzed the mRNA and protein expression of the key genes related to Aβ processing. When exposed to RF-EMF, mRNA levels of APP, BACE1, ADAM10 and PSEN1 were decreased in HT22, but the mRNA level of APP was not changed in SH-SY5Y cells. The protein expression of APP and BACE1, as well as the secreted Aβ peptide, was not significantly different between RF-EMF–exposed 7w-PSML, HT22 and SH-SY5Y cells and the unexposed controls. These observations suggest that RF-EMF exposure may not have a significant physiological effect on Aβ processing of neural cells in the short term. However, considering that we only exposed HT22 and SH-SY5Y cells to RF-EMF for 2 h per day for 3 days, we cannot exclude the possibility that 1950 MHz RF-EMF induces physiological change in Aβ processing with long-term and continuous exposure.

  14. Silencing of PINK1 expression affects mitochondrial DNA and oxidative phosphorylation in dopaminergic cells.

    Directory of Open Access Journals (Sweden)

    Matthew E Gegg

    Full Text Available Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD. Impairment of the mitochondrial electron transport chain (ETC and an increased frequency in deletions of mitochondrial DNA (mtDNA, which encodes some of the subunits of the ETC, have been reported in the substantia nigra of PD brains. The identification of mutations in the PINK1 gene, which cause an autosomal recessive form of PD, has supported mitochondrial involvement in PD. The PINK1 protein is a serine/threonine kinase localized in mitochondria and the cytosol. Its precise function is unknown, but it is involved in neuroprotection against a variety of stress signalling pathways.In this report we have investigated the effect of silencing PINK1 expression in human dopaminergic SH-SY5Y cells by siRNA on mtDNA synthesis and ETC function. Loss of PINK1 expression resulted in a decrease in mtDNA levels and mtDNA synthesis. We also report a concomitant loss of mitochondrial membrane potential and decreased mitochondrial ATP synthesis, with the activity of complex IV of the ETC most affected. This mitochondrial dysfunction resulted in increased markers of oxidative stress under basal conditions and increased cell death following treatment with the free radical generator paraquat.This report highlights a novel function of PINK1 in mitochondrial biogenesis and a role in maintaining mitochondrial ETC activity. Dysfunction of both has been implicated in sporadic forms of PD suggesting that these may be key pathways in the development of the disease.

  15. Evaluation of neuroprotective properties of two synthetic prenylated ...

    African Journals Online (AJOL)

    Human neuroblastoma SH-SY5Y cell, 6-Hydroxydopamine. Tropical Journal of ... in vivo and in vitro settings. 6-OHDA ... dopaminergic neurons and reestablishes a new ... cell death as seen in Parkinson's disease [3]. ... and 6- hydroxydopamine in a dopaminergic cell line - ..... previous study demonstrated in rodent model.

  16. Caspase-1 Deficiency Alleviates Dopaminergic Neuronal Death via Inhibiting Caspase-7/AIF Pathway in MPTP/p Mouse Model of Parkinson's Disease.

    Science.gov (United States)

    Qiao, Chen; Zhang, Lin-Xia; Sun, Xi-Yang; Ding, Jian-Hua; Lu, Ming; Hu, Gang

    2017-08-01

    Caspase family has been recognized to be involved in dopaminergic (DA) neuronal death and to exert an unfavorable role in Parkinson's disease (PD) pathology. Our previous study has revealed that caspase-1, as an important component of NLRP3 inflammasome, induces microglia-mediated neuroinflammation in the pathogenesis of PD. However, the role of caspase-1 in DA neuronal degeneration in the onset of PD remains unclear. Here, we showed that caspase-1 knockout ameliorated DA neuronal loss and dyskinesia in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine/probenecid (MPTP/p)-induced PD model mice. We further found that caspase-1 knockout decreased MPTP/p-induced caspase-7 cleavage, subsequently inhibited nuclear translocation of poly (ADP-ribose) polymerase 1 (PARP1), and reduced the release of apoptosis-inducing factor (AIF). Consistently, we demonstrated that caspase-1 inhibitor suppressed caspase-7/PARP1/AIF-mediated apoptosis pathway by 1-methyl-4-phenylpyridinium ion (MPP + ) stimulation in SH-SY5Y cells. Caspase-7 overexpression reduced the protective effects of caspase-1 inhibitor on SH-SY5Y cell apoptosis. Collectively, our results have revealed that caspase-1 regulates DA neuronal death in the pathogenesis of PD in mice via caspase-7/PARP1/AIF pathway. These findings will shed new insight into the potential of caspase-1 as a target for PD therapy.

  17. Amentoflavone protects dopaminergic neurons in MPTP-induced Parkinson's disease model mice through PI3K/Akt and ERK signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Qin; Qin, Liyue; Huang, Fei, E-mail: Fei_H@hotmail.com; Wang, Xiaoshuang; Yang, Liu; Shi, Hailian; Wu, Hui; Zhang, Beibei; Chen, Ziyu; Wu, Xiaojun, E-mail: xiaojunwu320@126.com

    2017-03-15

    Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc). Mitochondrial dysfunction and cell apoptosis are suggested to be actively involved in the pathogenesis of PD. In the present study, the neuroprotective effect of amentoflavone (AF), a naturally occurring biflavonoid from Selaginella tamariscina, was examined in PD models both in vitro and in vivo. On SH-SY5Y cells, AF treatment dose-dependently reduced 1-methyl-4-phenylpyridinium (MPP{sup +})-induced nuclear condensation and loss of cell viability without obvious cytotoxicity. It inhibited the activation of caspase-3 and p21 but increased the Bcl-2/Bax ratio. Further study disclosed that AF enhanced the phosphorylation of PI3K, Akt and ERK1/2 down-regulated by MPP{sup +} in SH-SY5Y cells, the effect of which could be blocked by LY294002, the inhibitor of PI3K. Consistently, AF alleviated the behavioral deterioration in pole and traction tests and rescued the loss of dopaminergic neurons in SNpc and fibers in striatum in methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced mice. It also could enhance the activation of PI3K and Akt as well as Bcl-2/Bax ratio in SN. Moreover, AF alleviated gliosis as well as the gene expression levels of IL-1β and iNOS in SN. Collectively, these results suggested that AF protected dopaminergic neurons against MPTP/MPP{sup +}-induced neurotoxicity, which might be mediated through activation of PI3K/Akt and ERK signaling pathways in dopaminergic neurons and attenuation of neuroinflammation. - Highlights: • AF protected dopaminergic neurons against MPTP/MPP{sup +}-induced neurotoxicity. • AF modulated PI3K/Akt and ERK signaling pathways. • AF could alleviate neuroinflammation in SN.

  18. Amentoflavone protects dopaminergic neurons in MPTP-induced Parkinson's disease model mice through PI3K/Akt and ERK signaling pathways

    International Nuclear Information System (INIS)

    Cao, Qin; Qin, Liyue; Huang, Fei; Wang, Xiaoshuang; Yang, Liu; Shi, Hailian; Wu, Hui; Zhang, Beibei; Chen, Ziyu; Wu, Xiaojun

    2017-01-01

    Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc). Mitochondrial dysfunction and cell apoptosis are suggested to be actively involved in the pathogenesis of PD. In the present study, the neuroprotective effect of amentoflavone (AF), a naturally occurring biflavonoid from Selaginella tamariscina, was examined in PD models both in vitro and in vivo. On SH-SY5Y cells, AF treatment dose-dependently reduced 1-methyl-4-phenylpyridinium (MPP + )-induced nuclear condensation and loss of cell viability without obvious cytotoxicity. It inhibited the activation of caspase-3 and p21 but increased the Bcl-2/Bax ratio. Further study disclosed that AF enhanced the phosphorylation of PI3K, Akt and ERK1/2 down-regulated by MPP + in SH-SY5Y cells, the effect of which could be blocked by LY294002, the inhibitor of PI3K. Consistently, AF alleviated the behavioral deterioration in pole and traction tests and rescued the loss of dopaminergic neurons in SNpc and fibers in striatum in methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced mice. It also could enhance the activation of PI3K and Akt as well as Bcl-2/Bax ratio in SN. Moreover, AF alleviated gliosis as well as the gene expression levels of IL-1β and iNOS in SN. Collectively, these results suggested that AF protected dopaminergic neurons against MPTP/MPP + -induced neurotoxicity, which might be mediated through activation of PI3K/Akt and ERK signaling pathways in dopaminergic neurons and attenuation of neuroinflammation. - Highlights: • AF protected dopaminergic neurons against MPTP/MPP + -induced neurotoxicity. • AF modulated PI3K/Akt and ERK signaling pathways. • AF could alleviate neuroinflammation in SN.

  19. Functional coupling between heterologously expressed dopamine D(2) receptors and KCNQ channels

    DEFF Research Database (Denmark)

    Ljungstrom, Trine; Grunnet, Morten; Jensen, Bo Skaaning

    2003-01-01

    protein of the G(alphai/o) subtype. Cells of the human neuroblastoma line SH-SY5Y were co-transfected transiently with KCNQ4 and D(2L) receptors. Stimulation of D(2L) receptors increased the KCNQ4 current ( n=6) as determined in whole-cell patch-clamp recordings. The specificity of the dopaminergic...

  20. PARIS reprograms glucose metabolism by HIF-1α induction in dopaminergic neurodegeneration.

    Science.gov (United States)

    Kang, Hojin; Jo, Areum; Kim, Hyein; Khang, Rin; Lee, Ji-Yeong; Kim, Hanna; Park, Chi-Hu; Choi, Jeong-Yun; Lee, Yunjong; Shin, Joo-Ho

    2018-01-22

    Our previous study found that PARIS (ZNF746) transcriptionally suppressed transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) in the substantia nigra (SN) of AAV-PARIS injected mice. In this study, we revealed that PARIS overexpression reprogrammed glucose metabolic pathway, leading to the increment of glycolytic proteins along with TKT reduction in the SN of AAV-PARIS injected mice. Knock-down of TKT in differentiated SH-SY5Y cells led to an increase of glycolytic enzymes and decrease of PPP-related enzymes whereas overexpression of TKT restored PARIS-mediated glucose metabolic shift, suggesting that glucose metabolic alteration by PARIS is TKT-dependent. Inhibition of PPP by either PARIS overexpression or TKT knock-down elevated the level of H 2 O 2 , and diminished NADPH and GSH levels, ultimately triggering the induction of HIF-1α, a master activator of glycolysis. In addition, TKT inhibition by stereotaxic injection of oxythiamine demonstrated slight decrement of dopaminergic neurons (DNs) in SN but not cortical neurons in the cortex, suggesting that TKT might be a survival factor of DNs. In differentiated SH-SY5Y, cell toxicity by GFP-PARIS was partially restored by introduction of Flag-TKT and siRNA-HIF-1α. We also observed the increase of HIF-1α and glycolytic hexokinase 2 in the SN of Parkinson's disease patients. Taken together, these results suggest that PARIS accumulation might distort the balance of glucose metabolism, providing clues for understanding mechanism underlying selective DNs death by PARIS. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells

    International Nuclear Information System (INIS)

    Hwang, Yong Pil; Jeong, Hye Gwang

    2010-01-01

    Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a popular traditional herbal medicine. Ginsenoside Rb1 (Rb1), an active component commonly found in ginseng root, is a phytoestrogen that exerts estrogen-like activity. In this study, we demonstrate that the phytoestrogen Rb1 inhibits 6-hydroxydopamine (6-OHDA)-induced oxidative injury via an ER-dependent Gβ1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of SH-SY5Y cells with Rb1 significantly reduced 6-OHDA-induced caspase-3 activation and subsequent cell death. Rb1 also up-regulated HO-1 expression, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, Rb1 induced both Nrf2 nuclear translocation, which is upstream of HO-1 expression and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Also, Rb1-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that Rb1 augments the cellular antioxidant defenses through ER-dependent HO-1 induction via the Gβ1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Thus our study indicates that Rb1 has a partial cytoprotective role in dopaminergic cell culture systems.

  2. Human neuronal cell based assay: A new in vitro model for toxicity evaluation of ciguatoxin.

    Science.gov (United States)

    Coccini, Teresa; Caloni, Francesca; De Simone, Uliana

    2017-06-01

    Ciguatoxins (CTXs) are emerging marine neurotoxins representing the main cause of ciguatera fish poisoning, an intoxication syndrome which configures a health emergency and constitutes an evolving issue constantly changing due to new vectors and derivatives of CTXs, as well as their presence in new non-endemic areas. The study applied the neuroblastoma cell model of human origin (SH-SY5Y) to evaluate species-specific mechanistic information on CTX toxicity. Metabolic functionality, cell morphology, cytosolic Ca 2+ i responses, neuronal cell growth and proliferation were assessed after short- (4-24h) and long-term exposure (10days) to P-CTX-3C. In SH-SY5Y, P-CTX-3C displayed a powerful cytotoxicity requiring the presence of both Veratridine and Ouabain. SH-SY5Y were very sensitive to Ouabain: 10 and 0.25nM appeared the optimal concentrations, for short- and long-term toxicity studies, respectively, to be used in co-incubation with Veratridine (25μM), simulating the physiological and pathological endogenous Ouabain levels in humans. P-CTX-3C cytotoxic effect, on human neurons co-incubated with OV (Ouabain+Veratridine) mix, was expressed starting from 100pM after short- and 25pM after long-term exposure. Notably, P-CTX-3C alone at 25nM induced cytotoxicity after 24h and prolonged exposure. This human brain-derived cell line appears a suitable cell-based-model to evaluate cytotoxicity of CTX present in marine food contaminated at low toxic levels and to characterize the toxicological profile of other/new congeners. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Differentiation-associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells

    International Nuclear Information System (INIS)

    Heikkilae, J.E.; Scott, J.G.; Suominen, L.A.; Akerman, K.E.O.

    1987-01-01

    Muscarinic receptor-linked increases in intracellular free Ca 2+ as measured with quin-2 and Ca 2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca 2+ and stimulation of Ca 2+ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3 H-NMS, by a non-labelled agonist was studied

  4. Kidins220/ARMS depletion is associated with the neural-to Schwann-like transition in a human neuroblastoma cell line model.

    Science.gov (United States)

    Rogers, Danny A; Schor, Nina F

    2013-03-10

    Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Analysis of Epidermal Growth Factor Receptor Related Gene Expression Changes in a Cellular and Animal Model of Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    In-Su Kim

    2017-02-01

    Full Text Available We employed transcriptome analysis of epidermal growth factor receptor related gene expression changes in cellular and animal models of Parkinson’s disease (PD. We used a well-known Parkinsonian toxin 1-methyl-4-phenylpyridine (MPP+ to induce neuronal apoptosis in the human neuroblastoma SH-SY5Y cell line. The MPP+-treatment of SH-SY5Y cells was capable of inducing neuro-apoptosis, but it remains unclear what kinds of transcriptional genes are affected by MPP+ toxicity. Therefore the pathways that were significantly perturbed in MPP+ treated human neuroblastoma SH-SY5Y cells were identified based on genome-wide gene expression data at two time points (24 and 48 h. We found that the Epidermal Growth Factor Receptor (EGFR pathway-related genes showed significantly differential expression at all time points. The EGFR pathway has been linked to diverse cellular events such as proliferation, differentiation, and apoptosis. Further, to evaluate the functional significance of the altered EGFR related gene expression observed in MPP+-treated SH-SY5Y cells, the EGFR related GJB2 (Cx26 gene expression was analyzed in an MPP+-intoxicated animal PD model. Our findings identify that the EGFR signaling pathway and its related genes, such as Cx26, might play a significant role in dopaminergic (DAergic neuronal cell death during the process of neuro-apoptosis and therefore can be focused on as potential targets for therapeutic intervention.

  6. Neuronal effects of 4-t-Butylcatechol: A model for catechol-containing antioxidants

    International Nuclear Information System (INIS)

    Lo, Y.-C.; Liu Yuxin; Lin, Y.-C.; Shih, Y.-T.; Liu, C.-M.; Burka, Leo T.

    2008-01-01

    Many herbal medicines and dietary supplements sold as aids to improve memory or treat neurodegenerative diseases or have other favorable effects on the CNS contain a catechol or similar 1,2-dihydroxy aromatic moiety in their structure. As an approach to isolate and examine the neuroprotective properties of catechols, a simple catechol 4-t-Butylcatechol (TBC) has been used as a model. In this study, we investigated the effects of TBC on lipopolysaccharide (LPS)-activated microglial-induced neurotoxicity by using the in vitro model of coculture murine microglial-like cell line HAPI with the neuronal-like human neuroblastoma cell line SH-SY5Y. We also examined the effects of TBC on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. TBC at concentrations from 0.1-10 μM had no toxic effect on HAPI cells and SH-SY5Y cells, and it inhibited LPS (100 ng/ml)-induced increases of superoxide, intracellular ROS, gp91 Phox , iNOS and a decrease of HO-1 in HAPI cells. Under coculture condition, TBC significantly reduced LPS-activated microglia-induced dopaminergic SH-SY5Y cells death. Moreover, TBC (0.1-10 μM) inhibited 6-OHDA-induced increases of intracellular ROS, iNOS, nNOS, and a decrease of mitochondria membrane potential, and cell death in SH-SY5Y cells. However, the neurotoxic effects of TBC (100 μM) on SH-SY5Y cells were also observed including the decrease in mitochondria membrane potential and the increase in COX-2 expression and cell death. TBC-induced SH-SY5Y cell death was attenuated by pretreatment with NS-398, a selective COX-2 inhibitor. In conclusion, this study suggests that TBC might possess protective effects on inflammation- and oxidative stress-related neurodegenerative disorders. However, the high concentration of TBC might be toxic, at least in part, for increasing COX-2 expression

  7. Mesocortical dopaminergic function and human cognition

    International Nuclear Information System (INIS)

    Weinberger, D.R.; Berman, K.F.; Chase, T.N.

    1988-01-01

    In summary, we have reviewed rCBF data in humans that suggest that mesoprefrontal dopaminergic activity is involved in human cognition. In patients with Parkinson's disease and possibly in patients with schizophrenia, prefrontal physiological activation during a cognitive task that appears to depend on prefrontal neural systems correlates positively with cognitive performance on the task and with clinical signs of dopaminergic function. It may be possible in the future to examine prefrontal dopamine metabolism directly during prefrontal cognition using positron emission tomography and tracers such as F-18 DOPA. 21 references

  8. Controversial Effects of D-Amino Acid Oxidase Activator (DAOA)/G72 on D-Amino Acid Oxidase (DAO) Activity in Human Neuronal, Astrocyte and Kidney Cell Lines: The N-methyl D-aspartate (NMDA) Receptor Hypofunction Point of View.

    Science.gov (United States)

    Jagannath, Vinita; Brotzakis, Zacharias Faidon; Parrinello, Michele; Walitza, Susanne; Grünblatt, Edna

    2017-01-01

    Dysfunction of D-amino acid oxidase ( DAO ) and DAO activator ( DAOA )/ G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA) receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y), astrocyte-like (1321N1) and kidney-like (HEK293) human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO) showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD)] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD), which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.

  9. Controversial Effects of D-Amino Acid Oxidase Activator (DAOA/G72 on D-Amino Acid Oxidase (DAO Activity in Human Neuronal, Astrocyte and Kidney Cell Lines: The N-methyl D-aspartate (NMDA Receptor Hypofunction Point of View

    Directory of Open Access Journals (Sweden)

    Vinita Jagannath

    2017-10-01

    Full Text Available Dysfunction of D-amino acid oxidase (DAO and DAO activator (DAOA/G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y, astrocyte-like (1321N1 and kidney-like (HEK293 human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD, which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.

  10. The role of alpha-synuclein in melanin synthesis in melanoma and dopaminergic neuronal cells.

    Directory of Open Access Journals (Sweden)

    Tianhong Pan

    Full Text Available The relatively high co-occurrence of Parkinson's disease (PD and melanoma has been established by a large number of epidemiological studies. However, a clear biological explanation for this finding is still lacking. Ultra-violet radiation (UVR-induced skin melanin synthesis is a defense mechanism against UVR-induced damage relevant to the initiation of melanoma, whereas, increased neuromelanin (NM, the melanin synthesized in dopaminergic neurons, may enhance the susceptibility to oxidative stress-induced neuronal injury relevant to PD. SNCA is a PD-causing gene coding for alpha-Synuclein (α-Syn that expresses not only in brain, but also in skin as well as in tumors, such as melanoma. The findings that α-Syn can interact with tyrosinase (TYR and inhibit tyrosine hydroxylase (TH, both of which are enzymes involved in the biosynthesis of melanin and dopamine (DA, led us to propose that α-Syn may participate in the regulation of melanin synthesis. In this study, by applying ultraviolet B (UVB light, a physiologically relevant stimulus of melanogenesis, we detected melanin synthesis in A375 and SK-MEL-28 melanoma cells and in SH-SY5Y and PC12 dopaminergic neuronal cells and determined effects of α-Syn on melanin synthesis. Our results showed that UVB light exposure increased melanin synthesis in all 4 cell lines. However, we found that α-Syn expression reduced UVB light-induced increase of melanin synthesis and that melanin content was lower when melanoma cells were expressed with α-Syn, indicating that α-Syn may have inhibitory effects on melanin synthesis in melanoma cells. Different from melanoma cells, the melanin content was higher in α-Syn-over-expressed dopaminergic neuronal SH-SY5Y and PC12 cells, cellular models of PD, than that in non-α-Syn-expressed control cells. We concluded that α-Syn could be one of the points responsible for the positive association between PD and melanoma via its differential roles in melanin synthesis in

  11. Impact of psychostimulants and atomoxetine on the expression of 8-hydroxyguanine glycosylase 1 in human cells.

    Science.gov (United States)

    Schmidt, Andreas Johannes; Clement, Hans-Willi; Gebhardt, Stefan; Hemmeter, Ulrich Michael; Schulz, Eberhard; Krieg, Jürgen-Christian; Kircher, Tilo; Heiser, Philip

    2010-06-01

    Oxidative DNA damage as one sign of reactive oxygen species induced oxidative stress is an important factor in the pathogenesis of various psychiatric disorders. Altered levels of DNA base damage products as well as the expression of the main repair enzyme 8-hydroxyguanine glycosylase 1 have been described. The aim of the present study was to examine the effects of drugs (amphetamine, methylphenidate and atomoxetine) used in the treatment of attention deficit-hyperactivity disorder on the expression of this enzyme via reverse transcriptase-polymerase chain reaction in human neuroblastoma SH-SY5Y and human monocytic U-937 cells at concentrations of 50, 500 and 5,000 ng/ml. We observed decreased expression of this enzyme for all applied substances. In U-937 cells, the significance level was reached after treatment with 5,000 ng/ml amphetamine as well as after treatment with 50, 500 and 5,000 ng/ml atomoxetine. Incubation of SH-SY5Y cells with 50 and 5,000 ng/ml amphetamine and 5,000 ng/ml methylphenidate led to significant decreases of 8-hydroxyguanine glycosylase 1. As a positive correlation between the expression of 8-hydroxyguanine glycosylase 1 and the level of oxidative DNA damage products has been described, we accordingly consider these substances (amphetamine, methylphenidate and atomoxetine) to possibly play a protective role in this process.

  12. Evidence of vanillin binding to CAMKIV explains the anti-cancer mechanism in human hepatic carcinoma and neuroblastoma cells.

    Science.gov (United States)

    Naz, Huma; Tarique, Mohd; Khan, Parvez; Luqman, Suaib; Ahamad, Shahzaib; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2018-01-01

    Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr kinase family, and is associated with different types of cancer and neurodegenerative diseases. Vanillin is a natural compound, a primary component of the extract of the vanilla bean which possesses varieties of pharmacological features including anti-oxidant, anti-inflammatory, anti-bacterial and anti-tumor. Here, we have investigated the binding mechanism and affinity of vanillin to the CAMKIV which is being considered as a potential drug target for cancer and neurodegenerative diseases. We found that vanillin binds strongly to the active site cavity of CAMKIV and stabilized by a large number of non-covalent interactions. We explored the utility of vanillin as anti-cancer agent and found that it inhibits the proliferation of human hepatocyte carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cells in a dose-dependent manner. Furthermore, vanillin treatment resulted into the significant reduction in the mitochondrial membrane depolarization and ROS production that eventually leads to apoptosis in HepG2 and SH-SY5Y cancer cells. These findings may offer a novel therapeutic approach by targeting the CAMKIV using natural product and its derivative with a minimal side effect.

  13. A preliminary investigation into the impact of a pesticide combination on human neuronal and glial cell lines in vitro.

    Directory of Open Access Journals (Sweden)

    Michael D Coleman

    Full Text Available Many pesticides are used increasingly in combinations during crop protection and their stability ensures the presence of such combinations in foodstuffs. The effects of three fungicides, pyrimethanil, cyprodinil and fludioxonil, were investigated together and separately on U251 and SH-SY5Y cells, which can be representative of human CNS glial and neuronal cells respectively. Over 48h, all three agents showed significant reductions in cellular ATP, at concentrations that were more than tenfold lower than those which significantly impaired cellular viability. The effects on energy metabolism were reflected in their marked toxic effects on mitochondrial membrane potential. In addition, evidence of oxidative stress was seen in terms of a fall in cellular thiols coupled with increases in the expression of enzymes associated with reactive species formation, such as GSH peroxidase and superoxide dismutase. The glial cell line showed significant responsiveness to the toxin challenge in terms of changes in antioxidant gene expression, although the neuronal SH-SY5Y line exhibited greater vulnerability to toxicity, which was reflected in significant increases in caspase-3 expression, which is indicative of the initiation of apoptosis. Cyprodinil was the most toxic agent individually, although oxidative stress-related enzyme gene expression increases appeared to demonstrate some degree of synergy in the presence of the combination of agents. This report suggests that the impact of some pesticides, both individually and in combinations, merits further study in terms of their impact on human cellular health.

  14. The M1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

    International Nuclear Information System (INIS)

    Mei, Lin.

    1989-01-01

    The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated [ 3 H]IP 1 accumulation in the SH-SY5Y cells was decreased in the presence of 1μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M 1 mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m 1 gene. The transfected B82 cells (cTB10) showed specific [ 3 H](-)QNB binding activity. The mAChRs in these cells are of the M 1 type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M 1 mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M 1 mAChR densities in these cells characterized by [ 3 H](-)MQNB binding ranged from 12 fmol/10 6 cells in LK3-1 cells to 260 fmol/10 6 cells in the LK3-8 cells

  15. The structural proteins of epidemic and historical strains of Zika virus differ in their ability to initiate viral infection in human host cells.

    Science.gov (United States)

    Bos, Sandra; Viranaicken, Wildriss; Turpin, Jonathan; El-Kalamouni, Chaker; Roche, Marjolaine; Krejbich-Trotot, Pascale; Desprès, Philippe; Gadea, Gilles

    2018-03-01

    Mosquito-borne Zika virus (ZIKV) recently emerged in South Pacific islands and Americas where large epidemics were documented. In the present study, we investigated the contribution of the structural proteins C, prM and E in the permissiveness of human host cells to epidemic strains of ZIKV. To this end, we evaluated the capacity of the epidemic strain BeH819015 to infect epithelial A549 and neuronal SH-SY5Y cells in comparison to the African historical MR766 strain. For that purpose, we generated a molecular clone of BeH819015 and a chimeric clone of MR766 which contains the BeH819015 structural protein region. We showed that ZIKV containing BeH819015 structural proteins was much less efficient in cell-attachment leading to a reduced susceptibility of A549 and SH-SY5Y cells to viral infection. Our data illustrate a previously underrated role for C, prM, and E in ZIKV epidemic strain ability to initiate viral infection in human host cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Acute Consumption of Bordo Grape Juice and Wine Improves Serum Antioxidant Status in Healthy Individuals and Inhibits Reactive Oxygen Species Production in Human Neuron-Like Cells.

    Science.gov (United States)

    Copetti, Cristiane; Franco, Fernanda Wouters; Machado, Eduarda da Rosa; Soquetta, Marcela Bromberger; Quatrin, Andréia; Ramos, Vitor de Miranda; Moreira, José Cláudio Fonseca; Emanuelli, Tatiana; Sautter, Cláudia Kaehler; Penna, Neidi Garcia

    2018-01-01

    Few studies investigated the biological effects of American grape cultivars. We investigated the metabolic response after acute consumption of grape juice or wine from Bordo grapes ( Vitis labrusca ) in a placebo-controlled crossover study with fifteen healthy volunteers. Blood samples were collected 1 hour after the intake of 100 mL of water, juice, or wine to measure TBARS, ABTS, FRAP, glucose, and uric acid levels. To evaluate differences in cellular response, intracellular reactive species production (DCFH-DA) and metabolic mitochondrial viability (MTT) were assessed after exposure of human neuron-like cells (SH-SY5Y) to juice or wine. Glycemia was reduced after juice or wine consumption, whereas blood levels of uric acid were reduced after juice consumption but increased after wine consumption. Juice and wine consumption reduced plasma lipid peroxidation and increased plasma antioxidant capacity (ABTS and FRAP assays). Furthermore, juice inhibited H 2 O 2 -induced intracellular production of reactive species (RS) and increased the viability of SH-SY5Y cells. In contrast, wine (dealcoholized) exhibited a per se effect by inducing the production of RS and reducing cell viability. These results indicate a positive impact of acute consumption of Bordo juice and wine on human oxidative status, whereas only juice had protective effects against oxidative stress-induced cytotoxicity.

  17. Acute Consumption of Bordo Grape Juice and Wine Improves Serum Antioxidant Status in Healthy Individuals and Inhibits Reactive Oxygen Species Production in Human Neuron-Like Cells

    Directory of Open Access Journals (Sweden)

    Cristiane Copetti

    2018-01-01

    Full Text Available Few studies investigated the biological effects of American grape cultivars. We investigated the metabolic response after acute consumption of grape juice or wine from Bordo grapes (Vitis labrusca in a placebo-controlled crossover study with fifteen healthy volunteers. Blood samples were collected 1 hour after the intake of 100 mL of water, juice, or wine to measure TBARS, ABTS, FRAP, glucose, and uric acid levels. To evaluate differences in cellular response, intracellular reactive species production (DCFH-DA and metabolic mitochondrial viability (MTT were assessed after exposure of human neuron-like cells (SH-SY5Y to juice or wine. Glycemia was reduced after juice or wine consumption, whereas blood levels of uric acid were reduced after juice consumption but increased after wine consumption. Juice and wine consumption reduced plasma lipid peroxidation and increased plasma antioxidant capacity (ABTS and FRAP assays. Furthermore, juice inhibited H2O2-induced intracellular production of reactive species (RS and increased the viability of SH-SY5Y cells. In contrast, wine (dealcoholized exhibited a per se effect by inducing the production of RS and reducing cell viability. These results indicate a positive impact of acute consumption of Bordo juice and wine on human oxidative status, whereas only juice had protective effects against oxidative stress-induced cytotoxicity.

  18. Effect of four medicinal plants on amyloid-beta induced neurotoxicity in SH-SY5Y Cells

    CSIR Research Space (South Africa)

    Adewusi, EA

    2013-01-01

    Full Text Available Amyloid-beta peptide (Aß) is implicated in the pathogenesis of Alzheimer’s disease (AD), a neurodegenerative disorder. This study was designed to determine the effect of four medicinal plants used to treat neurodegenerative diseases on Aß...

  19. Transduced PEP-1-PON1 proteins regulate microglial activation and dopaminergic neuronal death in a Parkinson's disease model.

    Science.gov (United States)

    Kim, Mi Jin; Park, Meeyoung; Kim, Dae Won; Shin, Min Jea; Son, Ora; Jo, Hyo Sang; Yeo, Hyeon Ji; Cho, Su Bin; Park, Jung Hwan; Lee, Chi Hern; Kim, Duk-Soo; Kwon, Oh-Shin; Kim, Joon; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2015-09-01

    Parkinson's disease (PD) is an oxidative stress-mediated neurodegenerative disorder caused by selective dopaminergic neuronal death in the midbrain substantia nigra. Paraoxonase 1 (PON1) is a potent inhibitor of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) against oxidation by destroying biologically active phospholipids with potential protective effects against oxidative stress-induced inflammatory disorders. In a previous study, we constructed protein transduction domain (PTD) fusion PEP-1-PON1 protein to transduce PON1 into cells and tissue. In this study, we examined the role of transduced PEP-1-PON1 protein in repressing oxidative stress-mediated inflammatory response in microglial BV2 cells after exposure to lipopolysaccharide (LPS). Moreover, we identified the functions of transduced PEP-1-PON1 proteins which include, mitigating mitochondrial damage, decreasing reactive oxidative species (ROS) production, matrix metalloproteinase-9 (MMP-9) expression and protecting against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in SH-SY5Y cells. Furthermore, transduced PEP-1-PON1 protein reduced MMP-9 expression and protected against dopaminergic neuronal cell death in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model. Taken together, these results suggest a promising therapeutic application of PEP-1-PON1 proteins against PD and other inflammation and oxidative stress-related neuronal diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Neurotrophic effects of growth/differentiation factor 5 in a neuronal cell line.

    Science.gov (United States)

    Toulouse, André; Collins, Grace C; Sullivan, Aideen M

    2012-04-01

    The neurotrophin growth/differentiation factor 5 (GDF5) is studied as a potential therapeutic agent for Parkinson's disease as it is believed to play a role in the development and maintenance of the nigrostriatal system. Progress in understanding the effects of GDF5 on dopaminergic neurones has been hindered by the use of mixed cell populations derived from primary cultures or in vivo experiments, making it difficult to differentiate between direct and indirect effects of GDF5 treatment on neurones. In an attempt to establish an useful model to study the direct neuronal influence of GDF5, we have characterised the effects of GDF5 on a human neuronal cell line, SH-SY5Y. Our results show that GDF5 has the capability to promote neuronal but not dopaminergic differentiation. We also show that it promotes neuronal survival in vitro following a 6-hydroxydopamine insult. Our results show that application of GDF5 to SH-SY5Y cultures induces the SMAD pathway which could potentially be implicated in the intracellular transmission of GDF5's neurotrophic effects. Overall, our study shows that the SH-SY5Y neuroblastoma cell line provides an excellent neuronal model to study the neurotrophic effects of GDF5.

  1. Expression of caspase-3 gene in apoptotic HL-60 cell and different human tumor cell lines

    International Nuclear Information System (INIS)

    Li Xiaoming; Song Tianbao

    1999-01-01

    Objective: To research the expression of caspase-3 gene in the apoptotic and the control HL-60 cells and in the different human tumor cell lines. Methods: Caspase-3 mRNA in the control and γ-radiation-induced apoptotic HL-60 cells, and in the 6 types of human tumor cell lines, was analysed by Northern blot. Results: The caspase-3 gene transcript was more highly expressed in leukemia cells HL-60, CEM, K562 and neuroblastoma SH-SY5Y than in cervical adenocarcinoma HeLa and breast carcinoma MCF7, and more highly in the radiation-induced apoptotic HL-60 than in the control HL-60 cells. Conclusion: The high level of expression of caspase-3 may aid the efforts to understand the tumor cell sensitivity to radiation, apoptosis and its inherent ability to survive

  2. Physiological characterisation of human iPS-derived dopaminergic neurons.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Hartfield

    Full Text Available Human induced pluripotent stem cells (hiPSCs offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease, such as Parkinson's disease (PD, in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis, release, and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH and G protein-activated inward rectifier potassium channel 2 (Kir3.2, henceforth referred to as GIRK2, representative of the A9 population of substantia nigra pars compacta (SNc neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (<10 Hz and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3 receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites, and in the soma. Finally, neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+ which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models.

  3. Comparison of the amyloid pore forming properties of rat and human Alzheimer’s beta-amyloid peptide 1-42: Calcium imaging data

    Directory of Open Access Journals (Sweden)

    Coralie Di Scala

    2016-03-01

    Full Text Available The data here consists of calcium imaging of human neuroblastoma SH-SY5Y cells treated with the calcium-sensitive dye Fluo-4AM and then incubated with nanomolar concentrations of either human or rat Alzheimer’s β-amyloid peptide Aβ1-42. These data are both of a qualitative (fluorescence micrographs and semi-quantitative nature (estimation of intracellular calcium concentrations of cells probed by Aβ1-42 peptides vs. control untreated cells. Since rat Aβ1-42 differs from its human counterpart at only three amino acid positions, this comparative study is a good assessment of the specificity of the amyloid pore forming assay. The interpretation of this dataset is presented in the accompanying study “Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides” [1].

  4. Overexpression of DJ-1/PARK7, the Parkinson's disease-related protein, improves mitochondrial function via Akt phosphorylation on threonine 308 in dopaminergic neuron-like cells.

    Science.gov (United States)

    Zhang, Yi; Gong, Xiao-Gang; Wang, Zhen-Zhen; Sun, Hong-Mei; Guo, Zhen-Yu; Hu, Jing-Hong; Ma, Ling; Li, Ping; Chen, Nai-Hong

    2016-05-01

    DJ-1/PARK7, the Parkinson's disease-related protein, plays an important role in mitochondrial function. However, the mechanisms by which DJ-1 affects mitochondrial function are not fully understood. Akt is a promoter of neuron survival and is partly involved in the neurodegenerative process. This research aimed at investigating a possible relationship between DJ-1 and Akt signalling in regulating mitochondrial function in the dopaminergic neuron-like cells SH-SY5Y and PC-12. Overexpression of DJ-1 was firstly validated at both the transcriptional and translational levels after transit transfection with plasmid pcDNA3-Flag-DJ-1. Confocal fluorescence microscopy demonstrated that overexpression of DJ-1 increased the mitochondrial mass, but did not disrupt the mitochondrial morphology. In addition, mitochondrial complex I activity was raised in DJ-1-overexpressing cells, and this rise occurred with an increase in cellular adenosine 5'-triphosphate content. Moreover, immunoblotting demonstrated that the levels of phosphoinositide 3-kinase and the total Akt were not altered in DJ-1-overexpressing cells, and nor was the Akt phosphorylation on serine 473 changed. By contrast, Akt phosphorylation on threonine 308 was significantly augmented by overexpression of DJ-1, and the expression of glycogen synthase kinase-3beta, a downstream effector of Akt, was suppressed. In summary, these results suggest that overexpression of DJ-1 improves the mitochondrial function, at least in part, through a mechanism involving Akt phosphorylation on threonine 308. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  5. Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.

    Science.gov (United States)

    Branca, Jacopo J V; Morucci, Gabriele; Malentacchi, Francesca; Gelmini, Stefania; Ruggiero, Marco; Pacini, Stefania

    2015-09-01

    The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia. © 2015 Wiley Periodicals, Inc.

  6. C282Y-HFE gene variant affects cholesterol metabolism in human neuroblastoma cells.

    Science.gov (United States)

    Ali-Rahmani, Fatima; Huang, Michael A; Schengrund, C-L; Connor, James R; Lee, Sang Y

    2014-01-01

    Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells.

  7. Protection by polyphenol extract from olive stones against apoptosis produced by oxidative stress in human neuroblastoma cells

    Science.gov (United States)

    Cortés-Castell, Ernesto; Veciana-Galindo, Carmen; Torró-Montell, Luis; Palazón-Bru, Antonio; Sirvent-Segura, Elia; Gil-Guillén, Vicente; Rizo-Baeza, Mercedes

    2016-02-16

    We evaluated the protective activity of an extract from a by-product such as olive stones, through its ability to inhibit H202 induced apoptosis in the SH-SY5Y human neuroblastoma cell line. To such end, 20,000 cells/well were cultivated and differentiation with retinoic acid was initiated. Once the cells were differentiated, apoptosis was induced with and without H2O2 extract. Finally, cDNA extraction was performed, and pro-apoptotic genes Bax and anti-apoptotic genes Bcl-2 were analyzed. Quantification of the gene expression was performed using the GAPDH gene marker. Cell viability with the extract is 97.6% (SD 5.7) with 10 mg/l and 62.8% (SD 1.2) to 50 mg/l, using 10 mg/l for the biomarker assay. The retinoic acid differentiated SH-S cell line (10 μM) shows a clear apoptosis when treated with H2O2 150 μM, with a Bax/Bcl-2 ratio of 3.75 (SD 0.80) in contrast to the differentiated control cells subjected to H2O2 and with extract, which have the same ratio of 1.02 (SD 0.01-0.03). The olive stone extract shows anti-apoptotic activity in the provoked cell death of SH-SY5Y human neuroblastoma cells in their normal state, defending them from oxidative stress which produces a significant increase in the apoptotic gene ratio in contrast to anti-apoptotic genes (Bax/Bcl-2).

  8. Δ⁹-tetrahydrocannabinol (Δ⁹-THC) exerts a direct neuroprotective effect in a human cell culture model of Parkinson's disease.

    Science.gov (United States)

    Carroll, C B; Zeissler, M-L; Hanemann, C O; Zajicek, J P

    2012-10-01

    Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is neuroprotective in models of Parkinson's disease (PD). Although CB1 receptors are increased within the basal ganglia of PD patients and animal models, current evidence suggests a role for CB1 receptor-independent mechanisms. Here, we utilized a human neuronal cell culture PD model to further investigate the protective properties of Δ⁹-THC. Differentiated SH-SY5Y neuroblastoma cells were exposed to PD-relevant toxins: 1-methyl-4-phenylpyridinium (MPP+), lactacystin and paraquat. Changes in CB1 receptor level were determined by quantitative polymerase chain reaction and Western blotting. Cannabinoids and modulatory compounds were co-administered with toxins for 48 h and the effects on cell death, viability, apoptosis and oxidative stress assessed. We found CB1 receptor up-regulation in response to MPP+, lactacystin and paraquat and a protective effect of Δ⁹-THC against all three toxins. This neuroprotective effect was not reproduced by the CB1 receptor agonist WIN55,212-2 or blocked by the CB1 antagonist AM251. Furthermore, the antioxidants α-tocopherol and butylhydroxytoluene as well as the antioxidant cannabinoids, nabilone and cannabidiol were unable to elicit the same neuroprotection as Δ⁹-THC. However, the peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist T0070907 dose-dependently blocked the neuroprotective, antioxidant and anti-apoptotic effects of Δ⁹-THC, while the PPARγ agonist pioglitazone resulted in protection from MPP+-induced neurotoxicity. Furthermore, Δ⁹-THC increased PPARγ expression in MPP+-treated SH-SY5Y cells, another indicator of PPARγ activation. We have demonstrated up-regulation of the CB1 receptor in direct response to neuronal injury in a human PD cell culture model, and a direct neuronal protective effect of Δ⁹-THC that may be mediated through PPARγ activation. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological

  9. Dose-dependent effect of Curcuma longa for the treatment of Parkinson's disease.

    Science.gov (United States)

    Ma, Xiao-Wei; Guo, Rui-You

    2017-05-01

    Curcuma longa is a plant that belongs to the ginger family, Zingiberaceae. It has been used in Siddha medicine for thousands of years in Asia. Parkinson's disease (PD) is a degenerative disorder of the central nervous system that affects the motor system of the brain. Death of dopamine-producing cells in the substantia nigra leads to PD. Exposure to salsolinol, which is an endogenous neurotoxin, has been associated with damage to dopamine-producing cells. The present study assessed the toxicity of salsolinol in SH-SY5Y human neuroblastoma cells and subsequently investigated the neuroprotective potential of C. longa extract in salsolinol-induced toxic conditions in SH-SY5Y cells. Sulphorhodamine-B assay showed the protective effect of the anti-apoptotic effect of treated SH-SY5Y cells. Fluorescence microscopy and confocal laser scanning microscope analysis indicated the anti-apoptotic impact of the C. longa extract. Mitochondria-derived reactive oxygen species were reduced in C. longa extract-treated SH-SY5Y cells. Downregulated mRNA expression levels of p53, Bcl-2-associated X protein and caspase 3 were observed in the C. longa extract-treated SH-SY5Y cells. Caspase 3 activity was reduced in the C. longa extract-treated SH-SY5Y cells. In conclusion, the present findings demonstrated that solsolinol is neurotoxic to SH-SY5Y cells, and C. longa extract may be useful in the treatment of PD.

  10. Positive transcriptional regulation of the human micro opioid receptor gene by poly(ADP-ribose) polymerase-1 and increase of its DNA binding affinity based on polymorphism of G-172 -> T.

    Science.gov (United States)

    Ono, Takeshi; Kaneda, Toshio; Muto, Akihiro; Yoshida, Tadashi

    2009-07-24

    Micro opioid receptor (MOR) agonists such as morphine are applied widely in clinical practice as pain therapy. The effects of morphine through MOR, such as analgesia and development of tolerance and dependence, are influenced by individual specificity. Recently, we analyzed single nucleotide polymorphisms on the human MOR gene to investigate the factors that contribute to individual specificity. In process of single nucleotide polymorphisms analysis, we found that specific nuclear proteins bound to G(-172) --> T region in exon 1 in MOR gene, and its affinity to DNA was increased by base substitution from G(-172) to T(-172). The isolated protein was identified by mass spectrometry and was confirmed by Western blotting to be poly(ADP-ribose) polymerase-1 (PARP-1). The overexpressed PARP-1 bound to G(-172) --> T and enhanced the transcription of reporter vectors containing G(-172) and T(-172). Furthermore, PARP-1 inhibitor (benzamide) decreased PARP-1 binding to G(-172) --> T without affecting mRNA or protein expression level of PARP-1 and down-regulated the subsequent MOR gene expression in SH-SY5Y cells. Moreover, we found that tumor necrosis factor-alpha enhanced MOR gene expression as well as increased PARP-1 binding to the G(-172) --> T region and G(-172) --> T-dependent transcription in SH-SY5Y cells. These effects were also inhibited by benzamide. In this study, our data suggest that PARP-1 positively regulates MOR gene transcription via G(-172) --> T, which might influence individual specificity in therapeutic opioid effects.

  11. The epigenetic regulation of HIF-1α by SIRT1 in MPP{sup +} treated SH-SY5Y cells

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    Dong, Su-Yan; Guo, Yan-Jie; Feng, Ya; Cui, Xin-Xin [Department of Neurology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080 (China); Kuo, Sheng-Han [Department of Neurology, College of Physicians and Surgeons, Columbia University, New York (United States); Liu, Te, E-mail: liute1979@126.com [Shanghai Geriatric Institute of Chinese Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200031 (China); Wu, Yun-Cheng, E-mail: yunchw@medmail.com.cn [Department of Neurology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080 (China)

    2016-02-05

    Both silent information regulator 1 (SIRT1) and hypoxia inducible factor 1 (HIF-1) have been found to play important roles in the pathophysiology of Parkinson's disease (PD). However, their mechanisms and their relationship still require further study. In the present study, we focused on the change and relationship of SIRT1 and HIF-1α in PD. PD cell models were established by using methyl-4-phenylpyridinium (MPP{sup +}), which induced inhibition of cell proliferation, cell cycle arrest and apoptosis. We found that the expression of HIF-1α and its target genes VEGFA and LDHA increased and that SIRT1 expression was inhibited in MPP{sup +} treated cells. With further analysis, we found that the acetylation of H3K14 combined with the HIF-1α promoter was dramatically increased in cells treated with MPP{sup +}, which resulted in the transcriptional activation of HIF-1α. Moreover, the acetylation of H3K14 and the expression of HIF-1α increased when SIRT1 was knocked down, suggesting that SIRT1 was involved in the epigenetic regulation of HIF-1α. At last, phenformin, another mitochondrial complex1 inhibitor, was used to testify that the increased HIF-1a was not due to off target effects of MPP{sup +}. Therefore, our results support a link between PD and SIRT1/HIF-1α signaling, which may serve as a clue for understanding PD.

  12. La infección con el virus del dengue induce apoptosis en células del neuroblastoma humano SH-SY5Y

    Directory of Open Access Journals (Sweden)

    Jaime E. Castellanos

    2016-08-01

    Conclusión. Estos resultados sugieren, en su conjunto, que la regulación positiva del TNF-α podría hacer parte del proceso que induce daño y muerte celular durante el desarrollo de la encefalitis por dengue.

  13. Bradykinin-potentiating PEPTIDE-10C, an argininosuccinate synthetase activator, protects against H2O2-induced oxidative stress in SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    Querobino, Samyr Machado; Ribeiro, César Augusto João; Alberto-Silva, Carlos

    2018-05-01

    Bradykinin-potentiating peptides (BPPs - 5a, 7a, 9a, 10c, 11e, and 12b) of Bothrops jararaca (Bj) were described as argininosuccinate synthase (AsS) activators, improving l-arginine availability. Agmatine and polyamines, which are l-arginine metabolism products, have neuroprotective properties. Here, we investigated the neuroprotective effects of low molecular mass fraction from Bj venom (LMMF) and two synthetic BPPs (BPP-10c, BPP-12b, BPP-10c showed higher protective capacity than BPP-12b. LMMF pretreatment was unable to prevent the reduction of cell viability caused by H 2 O 2 . The neuroprotective mechanism of BPP-10c against oxidative stress was investigated. BPP-10c reduced ROS generation and lipid peroxidation in relation to cells treated only with H 2 O 2 . BBP-10c increased AsS expression and was not neuroprotective in the presence of MDLA, a specific inhibitor of AsS. BPP-10c reduced iNOS expression and nitrate levels but decreased NF-kB expression. Furthermore, BPP-10c protected the mitochondrial membrane against oxidation. Overall, we demonstrated for the first time neuroprotective mechanisms of BPPs against oxidative stress, opening new perspectives to the study and application of these peptides for the treatment of neurodegenerative diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. The cyclophilin D/Drp1 axis regulates mitochondrial fission contributing to oxidative stress-induced mitochondrial dysfunctions in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Xiao, Anqi; Gan, Xueqi; Chen, Ruiqi; Ren, Yanming; Yu, Haiyang; You, Chao

    2017-01-01

    Oxidative stress plays a central role in the pathogenesis of various neurodegenerative diseases. Increasing evidences have demonstrated that structural abnormalities in mitochondria are involved in oxidative stress related nerve cell damage. And Drp1 plays a critical role in mitochondrial dynamic imbalance insulted by oxidative stress-derived mitochondria. However, the status of mitochondrial fusion and fission pathway and its relationship with mitochondrial properties such as mitochondrial membrane permeability transition pore (mPTP) have not been fully elucidated. Here, we demonstrated for the first time the role of Cyclophilin D (CypD), a crucial component for mPTP formation, in the regulation of mitochondrial dynamics in oxidative stress treated nerve cell. We observed that CypD-mediated phosphorylation of Drp1 and subsequently augmented Drp1 recruitment to mitochondria and shifts mitochondrial dynamics toward excessive fission, which contributes to the mitochondrial structural and functional dysfunctions in oxidative stress-treated nerve cells. CypD depletion or over expression accompanies mitochondrial dynamics/functions recovery or aggravation separately. We also demonstrated first time the link between the CypD to mitochondrial dynamics. Our data offer new insights into the mechanism of mitochondrial dynamics which contribute to the mitochondrial dysfunctions, specifically the role of CypD in Drp1-mediated mitochondrial fission. The protective effect of CsA, or other molecules affecting the function of CypD hold promise as a potential novel therapeutic strategy for governing oxidative stress pathology via mitochondrial pathways. - Highlights: • Demonstrated first time the link between the mPTP to mitochondrial dynamics. • The role of Cyclophilin D in the regulation of Drp1-mediated mitochondrial fission. • CsA as a potential target for governing oxidative stress related neuropathology.

  15. Human neuromelanin: an endogenous microglial activator for dopaminergic neuron death

    OpenAIRE

    Zhang, Wei; Zecca, Luigi; Wilson, Belinda; Ren, RW; Wang, Yong-jun; Wang, Xiao-min; Hong, Jau-Shyong

    2013-01-01

    Substantial evidence indicates that neuroinflammation caused by over-activation of microglial in the substantia nigra is critical in the pathogenesis of dopaminergic neurodegeneration in Parkinson’s disease (PD). Increasing data demonstrates that environmental factors such as rotenone, paraquat play pivotal roles in the death of dopaminergic neurons. Here, potential role and mechanism of neuromelanin (NM), a major endogenous component in dopaminergic neurons of the substantia nigra, on microg...

  16. Caspase 8/10 are not mediating apoptosis in neuroblastoma cells treated with CDK inhibitory drugs

    OpenAIRE

    Ribas i Fortuny, Judit; Gómez Arbonés, Javier; Boix Torras, Jacint

    2005-01-01

    Olomoucine and Roscovitine are pharmacological inhibitors of cyclin-dependent kinases (CDK) displaying a promising profile as anticancer agents. Both compounds are effective inductors of apoptosis in a human neuroblastoma cell line, SH-SY5Y. The characterization of this process had suggested the involvement of an extrinsic pathway [Ribas, J., Boix, J., 2004. Cell differentiation, Caspase inhibition, and macromolecular synthesis blockage, but not Bcl-2 or Bcl-XL proteins, protect SH-SY5Y cells...

  17. 3-(Fur-2-yl)-10-(2-phenylethyl)-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one, a novel adenosine receptor antagonist with A(2A)-mediated neuroprotective effects.

    Science.gov (United States)

    Scatena, Alessia; Fornai, Francesco; Trincavelli, Maria Letizia; Taliani, Sabrina; Daniele, Simona; Pugliesi, Isabella; Cosconati, Sandro; Martini, Claudia; Da Settimo, Federico

    2011-09-21

    In this study, compound FTBI (3-(2-furyl)-10-(2-phenylethyl)[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one) was selected from a small library of triazinobenzimidazole derivatives as a potent A(2A) adenosine receptor (AR) antagonist and tested for its neuroprotective effects against two different kinds of dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) and methamphetamine (METH), in rat PC12 and in human neuroblastoma SH-SY5Y cell lines. FTBI, in a concentration range corresponding to its affinity for A(2A) AR subtype, significantly increased the number of viable PC12 cells after their exposure to METH and, to a similar extent, to MPP+, as demonstrated in both trypan blue exclusion assay and in cytological staining. These neuroprotective effects were also observed with a classical A(2A) AR antagonist, ZM241385, and appeared to be completely counteracted by the AR agonist, NECA, supporting A(2A) ARs are directly involved in FTBI-mediated effects. Similarly, in human SH-SY5Y cells, FTBI was able to prevent cell toxicity induced by MPP+ and METH, showing that this A(2A) AR antagonist has a neuroprotective effect independently by the specific cell model. Altogether these results demonstrate that the A(2A) AR blockade mediates cell protection against neurotoxicity induced by dopaminergic neurotoxins in dopamine containing cells, supporting the potential use of A(2A) AR antagonists in dopaminergic degenerative diseases including Parkinson's disease.

  18. HPRT deficiency coordinately dysregulates canonical Wnt and presenilin-1 signaling: a neuro-developmental regulatory role for a housekeeping gene?

    Directory of Open Access Journals (Sweden)

    Tae Hyuk Kang

    2011-01-01

    Full Text Available We have used microarray-based methods of global gene expression together with quantitative PCR and Western blot analysis to identify dysregulation of genes and aberrant cellular processes in human fibroblasts and in SH-SY5Y neuroblastoma cells made HPRT-deficient by transduction with a retrovirus stably expressing an shRNA targeted against HPRT. Analysis of the microarray expression data by Gene ontology (GO and Gene Set Enrichment Analysis (GSEA as well as significant pathway analysis by GeneSpring GX10 and Panther Classification System reveal that HPRT deficiency is accompanied by aberrations in a variety of pathways known to regulate neurogenesis or to be implicated in neurodegenerative disease, including the canonical Wnt/β-catenin and the Alzheimer's disease/presenilin signaling pathways. Dysregulation of the Wnt/β-catenin pathway is confirmed by Western blot demonstration of cytosolic sequestration of β-catenin during in vitro differentiation of the SH-SY5Y cells toward the neuronal phenotype. We also demonstrate that two key transcription factor genes known to be regulated by Wnt signaling and to be vital for the generation and function of dopaminergic neurons; i.e., Lmx1a and Engrailed 1, are down-regulated in the HPRT knockdown SH-SY5Y cells. In addition to the Wnt signaling aberration, we found that expression of presenilin-1 shows severely aberrant expression in HPRT-deficient SH-SY5Y cells, reflected by marked deficiency of the 23 kDa C-terminal fragment of presenilin-1 in knockdown cells. Western blot analysis of primary fibroblast cultures from two LND patients also shows dysregulated presenilin-1 expression, including aberrant proteolytic processing of presenilin-1. These demonstrations of dysregulated Wnt signaling and presenilin-1 expression together with impaired expression of dopaminergic transcription factors reveal broad pleitropic neuro-regulatory defects played by HPRT expression and suggest new directions for

  19. Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: modulation by the Nrf2/Trx axis.

    Science.gov (United States)

    Niso-Santano, Mireia; González-Polo, Rosa A; Bravo-San Pedro, José M; Gómez-Sánchez, Rubén; Lastres-Becker, Isabel; Ortiz-Ortiz, Miguel A; Soler, Germán; Morán, José M; Cuadrado, Antonio; Fuentes, José M

    2010-05-15

    Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases. Copyright 2010 Elsevier Inc. All rights reserved.

  20. Dopaminergic modulation of the human reward system: a placebo-controlled dopamine depletion fMRI study

    NARCIS (Netherlands)

    da Silva Alves, Fabiana; Schmitz, Nicole; Figee, Martijn; Abeling, Nico; Hasler, Gregor; van der Meer, Johan; Nederveen, Aart; de Haan, Lieuwe; Linszen, Don; van Amelsvoort, Therese

    2011-01-01

    Reward related behaviour is linked to dopaminergic neurotransmission. Our aim was to gain insight into dopaminergic involvement in the human reward system. Combining functional magnetic resonance imaging with dopaminergic depletion by α-methylparatyrosine we measured dopamine-related brain activity

  1. Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

    Directory of Open Access Journals (Sweden)

    Sofie Ignoul

    Full Text Available BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432 have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y, endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4 and not with late endosomal/lysosomal markers (LAMP-1, Rab7. Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6

  2. Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells

    DEFF Research Database (Denmark)

    Dreyer-Andersen, Nanna; Almeida, Ana Sofia; Jensen, Pia

    2018-01-01

    Exploratory studies using human fetal tissue have suggested that intrastriatal transplantation of dopaminergic neurons may become a future treatment for patients with Parkinson's disease. However, the use of human fetal tissue is compromised by ethical, regulatory and practical concerns. Human stem...... cells constitute an alternative source of cells for transplantation in Parkinson's disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting...... in Parkinson's disease....

  3. Monocrotophos Induces the Expression of Xenobiotic Metabolizing Cytochrome P450s (CYP2C8 and CYP3A4) and Neurotoxicity in Human Brain Cells.

    Science.gov (United States)

    Tripathi, Vinay Kumar; Kumar, Vivek; Pandey, Ankita; Vatsa, Pankhi; Dhasmana, Anupam; Singh, Rajat Pratap; Appikonda, Sri Hari Chandan; Hwang, Inho; Lohani, Mohtashim

    2017-07-01

    Expression of various cytochrome P450s (CYPs) in mammalian brain cells is well documented. However, such studies are hampered in neural/glial cells of human origin due to nonavailability of human brain cells. To address this issue, we investigated the expression and inducibility of CYP2C8 and CYP3A4 and their responsiveness against cyclophosphamide (CPA) and organophosphorus pesticide monocrotophos (MCP), a known developmental neurotoxicant in human neural (SH-SY5Y) and glial (U373-MG) cell lines. CPA induced significant expression of CYP2C8 and CYP3A4 in both types of cells in a time-dependent manner. Neural cell line exhibited relatively higher constitutive and inducible expression of CYPs than the glial cell line. MCP exposure alone could not induce the significant expression of CYPs, whereas the cells preexposed to CPA showed a significant response to MCP. Similar to the case of CPA induced expressions, neural cells were found to be more vulnerable than glial cells. Our data indicate differential expressions of CYPs in cultured human neural and glial cell lines. The findings were synchronized with protein ligand docking studies, which showed a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR and PXR. Similarly, the known CYP inducer CPA has also shown significant high docking scores with the two studied CYP regulators. We also observed a significant induction in reactive oxygen species (ROS), lipid peroxides (LPO), micronucleus (MN), chromosomal aberration (CA), and reduction in reduced glutathione (GSH) and catalase following the exposure of MCP. Moreover, the expressions of apoptotic markers such as caspase-3, caspase-9, Bax, and p53 were significantly upregulated, whereas the levels of antiapoptotic marker, Bcl2, was downregulated after the exposure of MCP in both cell lines. These findings confirm the involvement of ROS-mediated oxidative stress, which subsequently triggers apoptosis pathways in both human neural (SH-SY5Y

  4. Synthesis of new heterocyclic compounds based on pyrazolopyridine scaffold and evaluation of their neuroprotective potential in MPP+-induced neurodegeneration.

    Science.gov (United States)

    Jouha, Jabrane; Loubidi, Mohammed; Bouali, Jamila; Hamri, Salha; Hafid, Abderrafia; Suzenet, Franck; Guillaumet, Gérald; Dagcı, Taner; Khouili, Mostafa; Aydın, Fadime; Saso, Luciano; Armagan, Güliz

    2017-03-31

    Neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and Huntington's disease affect millions of people in the world. Thus several new approaches to treat brain disorders are under development. The aim of the present study is to synthesize potential neuroprotective heterocyclic compounds based on pyrazolopyridine derivatives and then to evaluate their effects in MPP + -induced neurodegeneration in human neuroblastoma cell line (SH-SY5Y cells). The effects of the compounds on cell viability were measured by MTT assay and the changes in apoptosis-related proteins including bax, Bcl-2, Bcl-xl and caspase-3 were investigated by western blot technique. Based on the cell viability results obtained by MTT assay, the percentage of neuroprotection-induced by compounds against MPP + -induced neurotoxicity in SH-SY5Y cells was between 20% and 30% at 5 μM concentrations of all synthesized compounds. Moreover, the downregulation in pro-apoptotic proteins including bax and caspase-3 were found following the novel synthesized compounds treatments and these effects were observed in a dose-dependent manner. Our results provide an evidence that these heterocyclic compounds based on pyrazolopyridine derivatives may have a role on dopaminergic neuroprotection via antiapoptotic pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. L-Dopa decarboxylase expression profile in human cancer cells.

    Science.gov (United States)

    Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

    2011-02-01

    L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

  6. Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

    DEFF Research Database (Denmark)

    Anwar, Mohammad Raffaqat; Andreasen, Christian Maaløv; Lippert, Solvej Kølvraa

    2008-01-01

    differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co......Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic...

  7. Molecular mechanism of action of opioids in human neuroblastoma cells

    International Nuclear Information System (INIS)

    Yu, V.C.K.

    1987-01-01

    A series of human neuroblastoma cell lines was screened for the presence of opioid receptor sites. Of these cell lines, SK-N-SH was found to express approximately 50,000 μ and 10,000 δ opioid receptor sites/cell. In vitro characterization revealed that the binding properties of these receptor sites closely resembled those of human and rodent brain. Phosphatidylinositol turnover as a potential second messenger system for the μ receptor was examined in SK-N-SH cells. Neurotransmitter receptor systems were determined in the three sub-clones of SK-N-SH cells. Cells of the SH-SY5Y line, a phenotypically stable subclone of SK-N-SH cells, were induced to differentiate by treatment with various inducing agents, and changes of several neurotransmitter receptor systems were determined. Nerve growth factor (NGF) and retinoic acid (RA) up-regulated, while dBcAMP down-regulated opioid receptor sites. [ 3 H]Dopamine uptake was slightly enhanced only in RA-treated cells. Strikingly, the efficacy of PGE 1 -stimulated accumulation of cAMP was enhanced by 15- to 30-fold upon RA treatment

  8. Identification of proteins sensitive to thermal stress in human neuroblastoma and glioma cell lines.

    Directory of Open Access Journals (Sweden)

    Guilian Xu

    Full Text Available Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y and glial (CCF-STTG1 lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48 residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.

  9. Stress-induced localization of HSPA6 (HSP70B') and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells.

    Science.gov (United States)

    Khalouei, Sam; Chow, Ari M; Brown, Ian R

    2014-05-01

    The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B') and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.

  10. β-N-Methylamino-L-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling.

    Science.gov (United States)

    Engskog, Mikael K R; Ersson, Lisa; Haglöf, Jakob; Arvidsson, Torbjörn; Pettersson, Curt; Brittebo, Eva

    2017-05-01

    β-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC-MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

  11. A Novel Perspective on Dopaminergic Processing of Human Addiction.

    Science.gov (United States)

    Badgaiyan, Rajendra D

    2013-01-01

    Converging evidence from clinical, animal, and neuroimaging experiments suggests that the addictive behavior is associated with dysregulated dopamine neurotransmission. The precise role of dopamine in establishment and maintenance of addiction however is unclear. In this context animal studies on the brain reward system and the associative memory processing provide a novel insight. It was shown that both processing involve dopamine neurotransmission and both are disrupted in addiction. These findings indicate that dysregulated dopamine neurotransmission alters the brain processing of not only the reward system but also that of the memory of association between an addictive substance and reward. These alterations lead to maladaptive motivational behavior leading to chemical dependency. This concept however is based mostly on the data obtained in laboratory animals because of the paucity of human data. Due to lack of a reliable technique to study neurotransmission in the live human brain, it has been a problem to study the role of dopamine in human volunteers. A recently developed dynamic molecular imaging technique however, provides an opportunity to study these concepts in human volunteers because the technique allows detection, mapping and measurement of dopamine released in the live human brain during task performance.

  12. Amperometric Microsensors Monitoring Glutamate-Evoked In Situ Responses of Nitric Oxide and Carbon Monoxide from Live Human Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Yejin Ha

    2017-07-01

    Full Text Available In the brain, nitric oxide (NO and carbon monoxide (CO are important signaling gases which have multifaceted roles, such as neurotransmitters, neuromodulators, and vasodilators. Even though it is difficult to measure NO and CO in a living system due to their high diffusibility and extremely low release levels, electrochemical sensors are promising tools to measure in vivo and in vitro NO and CO gases. In this paper, using amperometric dual and septuple NO/CO microsensors, real-time NO and CO changes evoked by glutamate were monitored simultaneously for human neuroblastoma (SH-SY5Y cells. In cultures, the cells were differentiated and matured into functional neurons by retinoic acid and brain-derived neurotrophic factor. When glutamate was administrated to the cells, both NO and CO increases and subsequent decreases returning to the basal levels were observed with a dual NO/CO microsensor. In order to facilitate sensor’s measurement, a flower-type septuple NO/CO microsensor was newly developed and confirmed in terms of the sensitivity and selectivity. The septuple microsensor was employed for the measurements of NO and CO changes as a function of distances from the position of glutamate injection. Our sensor measurements revealed that only functionally differentiated cells responded to glutamate and released NO and CO.

  13. Characterization and uptake of radiolabelled meta-iodobenzylguanidine (MIBG) in a human neuroblastoma heterotransplant model in athymic rats

    International Nuclear Information System (INIS)

    Nilsson, S.; Paahlman, S.; Arnberg, H.; Letocha, H.; Westlin, J.E.

    1993-01-01

    Cells from an established human neuroblastoma cell line, SH-SY5Y, were demonstrated to grow and form solid tumours in nude rats. This cell line, which is an adrenergic subclone of the SK-N-SH cell line, has previously been used in differentiation model studies. The tumours retained the neuronal phenotype of the cultured cells, as evidenced by the expression of neuron-specific enolase (NSE) and chromogranin A + B. The transcription factor Isl-1, a protein expressed in subsets of neurons and endocrine cells as well as in neuroblastoma cells, was also expressed in the transplanted tumours, thus further verifying the retained phenotype of the cells under in vivo conditions. At scintigraphy utilizing 123 I-MIBG the optimal tumour/background ratio was obtained 20 h after injection. The assessment of tissue/serum ratios showed the highest uptake in the spleen (0.067% per gram of inj. activity), neuroblastoma tumours (0.067% per gram of inj. activity) and in the adrenals (0.065% per gram of inj. activity). (orig.)

  14. Characterization and uptake of radiolabelled meta-iodobenzylguanidine (MIBG) in a human neuroblastoma heterotransplant model in athymic rats

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson, S. (Dept. of Oncology, Univ. Hospital, Uppsala (Sweden)); Paahlman, S. (Dept. of Pathology, Univ. Hospital, Uppsala (Sweden)); Arnberg, H. (Dept. of Oncology, Univ. Hospital, Uppsala (Sweden)); Letocha, H. (Dept. of Oncology, Univ. Hospital, Uppsala (Sweden)); Westlin, J.E. (Dept. of Oncology, Univ. Hospital, Uppsala (Sweden))

    1993-01-01

    Cells from an established human neuroblastoma cell line, SH-SY5Y, were demonstrated to grow and form solid tumours in nude rats. This cell line, which is an adrenergic subclone of the SK-N-SH cell line, has previously been used in differentiation model studies. The tumours retained the neuronal phenotype of the cultured cells, as evidenced by the expression of neuron-specific enolase (NSE) and chromogranin A + B. The transcription factor Isl-1, a protein expressed in subsets of neurons and endocrine cells as well as in neuroblastoma cells, was also expressed in the transplanted tumours, thus further verifying the retained phenotype of the cells under in vivo conditions. At scintigraphy utilizing [sup 123]I-MIBG the optimal tumour/background ratio was obtained 20 h after injection. The assessment of tissue/serum ratios showed the highest uptake in the spleen (0.067% per gram of inj. activity), neuroblastoma tumours (0.067% per gram of inj. activity) and in the adrenals (0.065% per gram of inj. activity). (orig.).

  15. Lycopene attenuates Aβ1-42 secretion and its toxicity in human cell and Caenorhabditis elegans models of Alzheimer disease.

    Science.gov (United States)

    Chen, Wei; Mao, Liuqun; Xing, Huanhuan; Xu, Lei; Fu, Xiang; Huang, Liyingzi; Huang, Dongling; Pu, Zhijun; Li, Qinghua

    2015-11-03

    Growing evidence suggests concentration of lycopene was reduced in plasma of patients with Alzheimer disease (AD). Lycopene, a member of the carotenoid family, has been identified as an antioxidant to attenuate oxidative damage and has neuroprotective role in several AD models. However, whether lycopene is involved in the pathogenesis of AD and molecular underpinnings are elusive. In this study, we found that lycopene can significantly delay paralysis in the Aβ1-42-transgenic Caenorhabditis elegans strain GMC101. Lycopene treatment reduced Aβ1-42 secretion in SH-SY5Y cells overexpressing the Swedish mutant form of human β-amyloid precursor protein (APPsw). Next, we found lycopene can down-regulate expression level of β-amyloid precursor protein(APP) in APPsw cells. Moreover, lycopene treatment can not change endogenous reactive oxygen species level and apoptosis in APPsw cells. However, lycopene treatment protected against H2O2-induced oxidative stress and copper-induced damage in APPsw cells. Collectively, our data support that elevated lycopene contributes to the lower pathogenesis of AD. Our findings suggest that increasing lycopene in neurons may be a novel approach to attenuate onset and development of AD. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis in human neuronal cell line SHSY5Y.

    Science.gov (United States)

    Vidau, Cyril; González-Polo, Rosa A; Niso-Santano, Mireia; Gómez-Sánchez, Rubén; Bravo-San Pedro, José M; Pizarro-Estrella, Elisa; Blasco, Rafael; Brunet, Jean-Luc; Belzunces, Luc P; Fuentes, José M

    2011-12-01

    Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Regulation of proteolytic cleavage of brain-derived neurotrophic factor precursor by antidepressants in human neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Lin PY

    2015-10-01

    Full Text Available Pao-Yen Lin1,2 1Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 2Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan Abstract: Evidence has supported the role of brain-derived neurotrophic factor (BDNF in antidepressant effect. The precursor of BDNF (proBDNF often exerts opposing biological effects on mature BDNF (mBDNF. Hence, the balance between proBDNF and mBDNF might be critical in total neurotrophic effects, leading to susceptibility to or recovery from depression. In the current study, we measured the protein expression levels of proBDNF, and its proteolytic products, truncated BDNF, and mBDNF, in human SH-SY5Y cells treated with different antidepressants. We found that the treatment significantly increased the production of mBDNF, but decreased the production of truncated BDNF and proBDNF. These results support that antidepressants can promote proBDNF cleavage. Further studies are needed to clarify whether proBDNF cleavage plays a role in antidepressant mechanisms. Keywords: antidepressant, mature BDNF, neurotrophic effect, proBDNF cleavage 

  18. Ligands for the peroxisome proliferator-activated receptor-γ have inhibitory effects on growth of human neuroblastoma cells in vitro

    International Nuclear Information System (INIS)

    Valentiner, Ursula; Carlsson, Margarita; Erttmann, Rudolf; Hildebrandt, Herbert; Schumacher, Udo

    2005-01-01

    The thiazolidinedione (TZD) or glitazone class of peroxisome proliferator-activated-γ (PPAR-γ) ligands not only induce adipocyte differentiation and increase insulin sensitivity, but also exert growth inhibitory effects on several carcinoma cell lines in vitro as well as in vivo. In the current study the in vitro effect of four PPAR-γ agonists (ciglitazone, pioglitazone, troglitazone, rosiglitazone) on the cell growth of seven human neuroblastoma cell lines (Kelly, LAN-1, LAN-5, LS, IMR-32, SK-N-SH, SH-SY5Y) was investigated. Growth rates were assessed by a colorimetric XTT-based assay kit. Expression of PPAR-γ protein was examined by immunohistochemistry and Western blot analysis. All glitazones inhibited in vitro growth and viability of the human neuroblastoma cell lines in a dose-dependent manner showing considerable effects only at high concentrations (10 μM and 100 μM). Effectiveness of the glitazones on neuroblastoma cell growth differed depending on the cell line and the agent. The presence of PPAR-γ protein was demonstrated in all cell lines. Our findings indicate that ligands for PPAR-γ may be useful therapeutic agents for the treatment of neuroblastoma. Thus the effect of glitazones on the growth of neuroblastoma should now be investigated in an in vivo animal model

  19. Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells

    DEFF Research Database (Denmark)

    Dreyer-Andersen, Nanna; Almeida, Ana Sofia; Jensen, Pia

    2018-01-01

    cells constitute an alternative source of cells for transplantation in Parkinson's disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting...... in both protection and generation of reactive oxygen species. The present study investigated the effect of CO produced by a novel CO-releasing molecule on dopaminergic differentiation of human neural stem cells. Short-term exposure to 25 ppm CO at days 0 and 4 significantly increased the relative content...... of β-tubulin III-immunoreactive immature neurons and tyrosine hydroxylase expressing catecholaminergic neurons, as assessed 6 days after differentiation. Also the number of microtubule associated protein 2-positive mature neurons had increased significantly. Moreover, the content of apoptotic cells...

  20. Novelty-Sensitive Dopaminergic Neurons in the Human Substantia Nigra Predict Success of Declarative Memory Formation.

    Science.gov (United States)

    Kamiński, Jan; Mamelak, Adam N; Birch, Kurtis; Mosher, Clayton P; Tagliati, Michele; Rutishauser, Ueli

    2018-04-12

    The encoding of information into long-term declarative memory is facilitated by dopamine. This process depends on hippocampal novelty signals, but it remains unknown how midbrain dopaminergic neurons are modulated by declarative-memory-based information. We recorded individual substantia nigra (SN) neurons and cortical field potentials in human patients performing a recognition memory task. We found that 25% of SN neurons were modulated by stimulus novelty. Extracellular waveform shape and anatomical location indicated that these memory-selective neurons were putatively dopaminergic. The responses of memory-selective neurons appeared 527 ms after stimulus onset, changed after a single trial, and were indicative of recognition accuracy. SN neurons phase locked to frontal cortical theta-frequency oscillations, and the extent of this coordination predicted successful memory formation. These data reveal that dopaminergic neurons in the human SN are modulated by memory signals and demonstrate a progression of information flow in the hippocampal-basal ganglia-frontal cortex loop for memory encoding. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  1. Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Zhenqiang Zhao

    2016-12-01

    Full Text Available Mouse embryonic fibroblasts (MEFs and human foreskin fibroblasts (HFFs are used for the culture of human embryonic stem cells (hESCs. MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs =1:1 and HFFs feeder respectively, and then were differentiated into DA neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR (qRT-PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of TH positive cells and expressed higher levels of FOXA2, PITX3, NURR1 and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons.

  2. SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila.

    Science.gov (United States)

    Zanon, Alessandra; Kalvakuri, Sreehari; Rakovic, Aleksandar; Foco, Luisa; Guida, Marianna; Schwienbacher, Christine; Serafin, Alice; Rudolph, Franziska; Trilck, Michaela; Grünewald, Anne; Stanslowsky, Nancy; Wegner, Florian; Giorgio, Valentina; Lavdas, Alexandros A; Bodmer, Rolf; Pramstaller, Peter P; Klein, Christine; Hicks, Andrew A; Pichler, Irene; Seibler, Philip

    2017-07-01

    Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  3. Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

    Science.gov (United States)

    Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

    2016-02-01

    We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

  4. Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

    International Nuclear Information System (INIS)

    Hong, Seokheon; Kim, Joo Yeon; Hwang, Joohyun; Shin, Ki Soon; Kang, Shin Jung

    2013-01-01

    Highlights: •Heptachlor inhibited mitochondrial electron transport chain complex III activity. •Heptachlor promoted generation of reactive oxygen species. •Heptachlor induced Bax activation. •Heptachlor induced mitochondria-mediated and caspase-dependent apoptosis. -- Abstract: Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD

  5. Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Seokheon; Kim, Joo Yeon; Hwang, Joohyun [Department of Molecular Biology, Sejong University, Seoul 143-747 (Korea, Republic of); Shin, Ki Soon [Department of Biology, Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kang, Shin Jung, E-mail: sjkang@sejong.ac.kr [Department of Molecular Biology, Sejong University, Seoul 143-747 (Korea, Republic of)

    2013-08-09

    Highlights: •Heptachlor inhibited mitochondrial electron transport chain complex III activity. •Heptachlor promoted generation of reactive oxygen species. •Heptachlor induced Bax activation. •Heptachlor induced mitochondria-mediated and caspase-dependent apoptosis. -- Abstract: Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.

  6. PINK1 is necessary for long term survival and mitochondrial function in human dopaminergic neurons.

    Directory of Open Access Journals (Sweden)

    Alison Wood-Kaczmar

    2008-06-01

    Full Text Available Parkinson's disease (PD is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD.

  7. Modulation of DNA base excision repair during neuronal differentiation

    DEFF Research Database (Denmark)

    Sykora, Peter; Yang, Jenq-Lin; Ferrarelli, Leslie K

    2013-01-01

    DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because...

  8. Oxytocin gene polymorphisms influence human dopaminergic function in a sex-dependent manner.

    Science.gov (United States)

    Love, Tiffany M; Enoch, Mary-Anne; Hodgkinson, Colin A; Peciña, Marta; Mickey, Brian; Koeppe, Robert A; Stohler, Christian S; Goldman, David; Zubieta, Jon-Kar

    2012-08-01

    Oxytocin, classically involved in social and reproductive activities, is increasingly recognized as an antinociceptive and anxiolytic agent, effects which may be mediated via oxytocin's interactions with the dopamine system. Thus, genetic variation within the oxytocin gene (OXT) is likely to explain variability in dopamine-related stress responses. As such, we examined how OXT variation is associated with stress-induced dopaminergic neurotransmission in a healthy human sample. Fifty-five young healthy volunteers were scanned using [¹¹C]raclopride positron emission tomography while they underwent a standardized physical and emotional stressor that consisted of moderate levels of experimental sustained deep muscle pain, and a baseline, control state. Four haplotype tagging single nucleotide polymorphisms located in regions near OXT were genotyped. Measures of pain, affect, anxiety, well-being and interpersonal attachment were also assessed. Female rs4813625 C allele carriers demonstrated greater stress-induced dopamine release, measured as reductions in receptor availability from baseline to the pain-stress condition relative to female GG homozygotes. No significant differences were detected among males. We also observed that female rs4813625 C allele carriers exhibited higher attachment anxiety, higher trait anxiety and lower emotional well-being scores. In addition, greater stress-induced dopamine release was associated with lower emotional well-being scores in female rs4813625 C allele carriers. Our results suggest that variability within the oxytocin gene appear to explain interindividual differences in dopaminergic responses to stress, which are shown to be associated with anxiety traits, including those linked to attachment style, as well as emotional well-being in women. Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  9. Alternative polyadenylation and miR-34 family members regulate tau expression

    DEFF Research Database (Denmark)

    Dickson, John R; Kruse, Carla; Montagna, Daniel R

    2013-01-01

    . Tau expresses two 3'-UTR isoforms, long and short, as a result of alternative polyadenylation. Using luciferase reporter constructs, we found that expression from these isoforms is differentially controlled in human neuroblastoma cell lines M17D and SH-SY5Y. Several microRNAs were computationally...

  10. Expression and regulation of CYP17A1 and 3β-hydroxysteroid dehydrogenase in cells of the nervous system

    DEFF Research Database (Denmark)

    Emanuelsson, I; Almokhtar, M; Wikvall, K

    2018-01-01

    investigation we studied the expression of these enzymes in cultured primary rat astrocytes, in neuron-enriched cells from rat cerebral cortex and in human neuroblastoma SH-SY5Y cells, a cell line often used as an in vitro model of neuronal function and differentiation. As part of this study we also examined...

  11. Human erythrocytes and neuroblastoma cells are affected in vitro by Au(III) ions

    International Nuclear Information System (INIS)

    Suwalsky, Mario; Gonzalez, Raquel; Villena, Fernando; Aguilar, Luis F.; Sotomayor, Carlos P.; Bolognin, Silvia; Zatta, Paolo

    2010-01-01

    Gold compounds are well known for their neurological and nephrotoxic implications. However, haematological toxicity is one of the most serious toxic and less studied effects. The lack of information on these aspects of Au(III) prompted us to study the structural effects induced on cell membranes, particularly that of human erythrocytes. AuCl 3 was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of multibilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence that Au(III) interacts with red cell membranes as follows: (a) in scanning electron microscopy studies on human erythrocytes it was observed that Au(III) induced shape changes at a concentration as low as 0.01 μM; (b) in isolated unsealed human erythrocyte membranes Au(III) induced a decrease in the molecular dynamics and/or water content at the glycerol backbone level of the lipid bilayer polar groups in a 5-50 μM concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 μm-1 mM range induced increasing structural perturbation only to dimyristoylphosphatidylcholine bilayers. Additional experiments were performed in human neuroblastoma cells SH-SY5Y. A statistically significant decrease of cell viability was observed with Au(III) ranging from 0.1 μM to 100 μM.

  12. Effects of the Absorption Behaviour of ZnO Nanoparticles on Cytotoxicity Measurements

    Directory of Open Access Journals (Sweden)

    Nigar Najim

    2014-01-01

    Full Text Available ZnO absorbs certain wavelengths of light and this behavior is more pronounced for nanoparticles of ZnO. As many toxicity measurements rely on measuring light transmission in cell lines, it is essential to determine how far this light absorption influences experimental toxicity measurements. The main objective was to study the ZnO absorption and how this influenced the cytotoxicity measurements. The cytotoxicity of differently sized ZnO nanoparticles in normal and cancer cell lines derived from lung tissue (Hs888Lu, neuron-phenotypic cells (SH-SY5Y, neuroblastoma (SH-SY5Y, human histiocytic lymphoma (U937, and lung cancer (A549 was investigated. Our results demonstrate that the presence of ZnO affected the cytotoxicity measurements due to the absorption characteristic of ZnO nanoparticles. The data revealed that the ZnO nanoparticles with an average particle size of around 85.7 nm and 190 nm showed cytotoxicity towards U937, SH-SY5Y, differentiated SH-SY5Y, and Hs888Lu cell lines. No effect on the A549 cells was observed. It was also found that the cytotoxicity of ZnO was particle size, concentration, and time dependent. These studies are the first to quantify the influence of ZnO nanoparticles on cytotoxicity assays. Corrections for absorption effects were carried out which gave an accurate estimation of the concentrations that produce the cytotoxic effects.

  13. Age-Dependent Effects of Methylphenidate on the Human Dopaminergic System in Young vs Adult Patients With Attention-Deficit/Hyperactivity Disorder: A Randomized Clincal Trial

    NARCIS (Netherlands)

    Schrantee, A.; Tamminga, H.G.H.; Bouziane, C.; Bottelier, M.A.; Bron, E.E.; Mutsaerts, H.-J.M.M.; Zwinderman, A.H.; Groote, I.R.; Rombouts, S.A.R.B.; Lindauer, R.J.L.; Klein, S.; Niessen, W.J.; Opmeer, B.C.; Boer, F.; Lucassen, P.J.; Andersen, S.L.; Geurts, H.M.; Reneman, L.

    2016-01-01

    Importance: Although numerous children receive methylphenidate hydrochloride for the treatment of attention-deficit/hyperactivity disorder (ADHD), little is known about age-dependent and possibly lasting effects of methylphenidate on the human dopaminergic system. Objectives: To determine whether

  14. Age-dependent effects of methylphenidate on the human dopaminergic system in young vs adult patients with attention-deficit/hyperactivity disorder: A randomized clinical trial

    NARCIS (Netherlands)

    Schrantee, A. (Anouk); Tamminga, H.G.H. (Hyke G. H.); C. Bouziane (Cheima); Bottelier, M.A. (Marco A.); E.E. Bron (Esther); H.J.M.M. Mutsaerts (Henri J. M.); A.H. Zwinderman (Ailko); Groote, I.R. (Inge R.); S.A.R.B. Rombouts (Serge); Lindauer, R.J.L. (Ramon J. L.); S. Klein (Stefan); W.J. Niessen (Wiro); B.C. Opmeer (Brent); Boer, F. (Frits); P.J. Lucassen; Andersen, S.L. (Susan L.); H.M. Geurts (Hilde ); L. Reneman (Liesbeth)

    2016-01-01

    textabstractIMPORTANCE Although numerous children receivemethylphenidate hydrochloride for the treatment of attention-deficit/hyperactivity disorder (ADHD), little is known about age-dependent and possibly lasting effects of methylphenidate on the human dopaminergic system. OBJECTIVES To determine

  15. Age-Dependent Effects of Methylphenidate on the Human Dopaminergic System in Young vs Adult Patients With Attention-Deficit/Hyperactivity Disorder: A Randomized Clinical Trial

    NARCIS (Netherlands)

    Schrantee, Anouk; Tamminga, Hyke G. H.; Bouziane, Cheima; Bottelier, Marco A.; Bron, Esther E.; Mutsaerts, Henk-Jan M. M.; Zwinderman, Aeilko H.; Groote, Inge R.; Rombouts, Serge A. R. B.; Lindauer, Ramon J. L.; Klein, Stefan; Niessen, Wiro J.; Opmeer, Brent C.; Boer, Frits; Lucassen, Paul J.; Andersen, Susan L.; Geurts, Hilde M.; Reneman, Liesbeth

    2016-01-01

    Although numerous children receive methylphenidate hydrochloride for the treatment of attention-deficit/hyperactivity disorder (ADHD), little is known about age-dependent and possibly lasting effects of methylphenidate on the human dopaminergic system. To determine whether the effects of

  16. Monocrotophos induces the expression and activity of xenobiotic metabolizing enzymes in pre-sensitized cultured human brain cells.

    Directory of Open Access Journals (Sweden)

    Vinay K Tripathi

    Full Text Available The expression and metabolic profile of cytochrome P450s (CYPs is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y and glial (U373-MG cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC, cyclophosphamide (CPA, ethanol and known neurotoxicant- monocrotophos (MCP, a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against

  17. Lateralization and gender differences in the dopaminergic response to unpredictable reward in the human ventral striatum.

    Science.gov (United States)

    Martin-Soelch, Chantal; Szczepanik, Joanna; Nugent, Allison; Barhaghi, Krystle; Rallis, Denise; Herscovitch, Peter; Carson, Richard E; Drevets, Wayne C

    2011-05-01

    Electrophysiological studies have shown that mesostriatal dopamine (DA) neurons increase activity in response to unpredicted rewards. With respect to other functions of the mesostriatal dopaminergic system, dopamine's actions show prominent laterality effects. Whether changes in DA transmission elicited by rewards also are lateralized, however, has not been investigated. Using [¹¹C]raclopride-PET to assess the striatal DA response to unpredictable monetary rewards, we hypothesized that such rewards would induce an asymmetric reduction in [¹¹C]raclopride binding in the ventral striatum, reflecting lateralization of endogenous dopamine release. In 24 healthy volunteers, differences in the regional D₂/₃ receptor binding potential (ΔBP) between an unpredictable reward condition and a sensorimotor control condition were measured using the bolus-plus-constant-infusion [¹¹C]raclopride method. During the reward condition subjects randomly received monetary awards while performing a 'slot-machine' task. The ΔBP between conditions was assessed in striatal regions-of-interest and compared between left and right sides. We found a significant condition × lateralization interaction in the ventral striatum. A significant reduction in binding potential (BP(ND) ) in the reward condition vs. the control condition was found only in the right ventral striatum, and the ΔBP was greater in the right than the left ventral striatum. Unexpectedly, these laterality effects appeared to be partly accounted for by gender differences, as our data showed a significant bilateral BP(ND) reduction in women while in men the reduction reached significance only in the right ventral striatum. These data suggest that DA release in response to unpredictable reward is lateralized in the human ventral striatum, particularly in males. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  18. Down-regulation of glutaminase C in human hepatocarcinoma cell by diphenylarsinic acid, a degradation product of chemical warfare agents

    International Nuclear Information System (INIS)

    Kita, Kayoko; Suzuki, Toshihide; Ochi, Takafumi

    2007-01-01

    In a poisonous incident in Kamisu, Japan, it is understood that diphenylarsinic acid (DPAA) was a critical contaminant of ground water. Most patients showed dysfunction of the central nervous system. To understand the overall mechanism of DPAA toxicity and to gain some insight into the application of a remedy specific for intoxication, the molecular target must be clarified. As an approach, a high throughput analysis of cell proteins in cultured human hepatocarcinoma HpG2 exposed to DPAA was performed by two-dimensional electrophoresis (2-DE). Four proteins, which were up- and down-regulated by exposure of cultured HepG2 cells to DPAA, were identified. They were chaperonin containing TCP-1 (CCT) beta subunit, aldehyde dehydrogenase 1 (ALDH1), ribosomal protein P0 and glutaminase C (GAC). Of these, GAC was the only protein that was down-regulated by DPAA exposure, and cellular expression levels were reduced by DPAA in a concentration- and time-dependent manner. Decrease in cellular GAC levels was accompanied by decreased activity of the enzyme, phosphate-activated glutaminase (PAG). Decreased expression of GAC by DPAA was also observed in human cervical carcinoma HeLa and neuroblastoma SH-SY5Y cells. By contrast, no significant changes in GAC protein expression were observed when cells were incubated with arsenite [iAs (III)] and trivalent dimethylarsinous acid [DMA (III)]. In the central nervous system, GAC plays a role in the production of the neurotransmitter glutamic acid. Selective inhibition of GAC expression by DPAA may be a cause of dysfunction of glutamatergic neuronal transmission and the resultant neurological impairments

  19. Down-regulation of glutaminase C in human hepatocarcinoma cell by diphenylarsinic acid, a degradation product of chemical warfare agents

    Energy Technology Data Exchange (ETDEWEB)

    Kita, Kayoko [Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Sagamiko-chou, Sagamihara, Kanagawa 229-0195 (Japan); Suzuki, Toshihide [Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Sagamiko-chou, Sagamihara, Kanagawa 229-0195 (Japan); Ochi, Takafumi [Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Sagamiko-chou, Sagamihara, Kanagawa 229-0195 (Japan)

    2007-05-01

    In a poisonous incident in Kamisu, Japan, it is understood that diphenylarsinic acid (DPAA) was a critical contaminant of ground water. Most patients showed dysfunction of the central nervous system. To understand the overall mechanism of DPAA toxicity and to gain some insight into the application of a remedy specific for intoxication, the molecular target must be clarified. As an approach, a high throughput analysis of cell proteins in cultured human hepatocarcinoma HpG2 exposed to DPAA was performed by two-dimensional electrophoresis (2-DE). Four proteins, which were up- and down-regulated by exposure of cultured HepG2 cells to DPAA, were identified. They were chaperonin containing TCP-1 (CCT) beta subunit, aldehyde dehydrogenase 1 (ALDH1), ribosomal protein P0 and glutaminase C (GAC). Of these, GAC was the only protein that was down-regulated by DPAA exposure, and cellular expression levels were reduced by DPAA in a concentration- and time-dependent manner. Decrease in cellular GAC levels was accompanied by decreased activity of the enzyme, phosphate-activated glutaminase (PAG). Decreased expression of GAC by DPAA was also observed in human cervical carcinoma HeLa and neuroblastoma SH-SY5Y cells. By contrast, no significant changes in GAC protein expression were observed when cells were incubated with arsenite [iAs (III)] and trivalent dimethylarsinous acid [DMA (III)]. In the central nervous system, GAC plays a role in the production of the neurotransmitter glutamic acid. Selective inhibition of GAC expression by DPAA may be a cause of dysfunction of glutamatergic neuronal transmission and the resultant neurological impairments.

  20. Calpain activation by ROS mediates human ether-a-go-go-related gene protein degradation by intermittent hypoxia.

    Science.gov (United States)

    Wang, N; Kang, H S; Ahmmed, G; Khan, S A; Makarenko, V V; Prabhakar, N R; Nanduri, J

    2016-03-01

    Human ether-a-go-go-related gene (hERG) channels conduct delayed rectifier K(+) current. However, little information is available on physiological situations affecting hERG channel protein and function. In the present study we examined the effects of intermittent hypoxia (IH), which is a hallmark manifestation of sleep apnea, on hERG channel protein and function. Experiments were performed on SH-SY5Y neuroblastoma cells, which express hERG protein. Cells were exposed to IH consisting of alternating cycles of 30 s of hypoxia (1.5% O2) and 5 min of 20% O2. IH decreased hERG protein expression in a stimulus-dependent manner. A similar reduction in hERG protein was also seen in adrenal medullary chromaffin cells from IH-exposed neonatal rats. The decreased hERG protein was associated with attenuated hERG K(+) current. IH-evoked hERG protein degradation was not due to reduced transcription or increased proteosome/lysomal degradation. Rather it was mediated by calcium-activated calpain proteases. Both COOH- and NH2-terminal sequences of the hERG protein were the targets of calpain-dependent degradation. IH increased reactive oxygen species (ROS) levels, intracellular Ca(2+) concentration ([Ca(2+)]i), calpain enzyme activity, and hERG protein degradation, and all these effects were prevented by manganese-(111)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride, a membrane-permeable ROS scavenger. These results demonstrate that activation of calpains by ROS-dependent elevation of [Ca(2+)]i mediates hERG protein degradation by IH. Copyright © 2016 the American Physiological Society.

  1. Exosomes from dental pulp stem cells rescue human dopaminergic neurons from 6-hydroxy-dopamine-induced apoptosis.

    Science.gov (United States)

    Jarmalavičiūtė, Akvilė; Tunaitis, Virginijus; Pivoraitė, Ugnė; Venalis, Algirdas; Pivoriūnas, Augustas

    2015-07-01

    Stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) have unique neurogenic properties that could be potentially exploited for therapeutic use. The importance of paracrine SHED signaling for neuro-regeneration has been recognized, but the exact mechanisms behind these effects are presently unknown. In the present study, we investigated the neuro-protective potential of exosomes and micro-vesicles derived from SHEDs on human dopaminergic neurons during oxidative stress-induced by 6-hydroxy-dopamine (6-OHDA). ReNcell VM human neural stem cells were differentiated into dopaminergic neurons and treated with 100 μmol/L of 6-OHDA alone or in combination with exosomes or micro-vesicles purified by ultracentrifugation from SHEDs cultivated in serum-free medium under two conditions: in standard two-dimensional culture flasks or on laminin-coated micro-carriers in a bioreactor. Real-time monitoring of apoptosis was performed with the use of time-lapse confocal microscopy and the CellEvent Caspase-3/7 green detection reagent. Exosomes but not micro-vesicles derived from SHEDs grown on the laminin-coated three-dimensional alginate micro-carriers suppressed 6-OHDA-induced apoptosis in dopaminergic neurons by approximately 80% throughout the culture period. Strikingly, no such effects were observed for the exosomes derived from SHEDs grown under standard culture conditions. Our results suggest that exosomes derived from SHEDs are considered as new potential therapeutic tool in the treatment of Parkinson's disease. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  2. Dynamics of the association of heat shock protein HSPA6 (Hsp70B') and HSPA1A (Hsp70-1) with stress-sensitive cytoplasmic and nuclear structures in differentiated human neuronal cells.

    Science.gov (United States)

    Shorbagi, Sadek; Brown, Ian R

    2016-11-01

    Heat shock proteins (Hsps) are cellular repair agents that counter the effects of protein misfolding that is a characteristic feature of neurodegenerative diseases. HSPA1A (Hsp70-1) is a widely studied member of the HSPA (Hsp70) family. The little-studied HSPA6 (Hsp70B') is present in the human genome and absent in mouse and rat; hence, it is missing in current animal models of neurodegenerative diseases. Differentiated human neuronal SH-SY5Y cells were employed to compare the dynamics of the association of YFP-tagged HSPA6 and HSPA1A with stress-sensitive cytoplasmic and nuclear structures. Following thermal stress, live-imaging confocal microscopy and Fluorescence Recovery After Photobleaching (FRAP) demonstrated that HSPA6 displayed a prolonged and more dynamic association, compared to HSPA1A, with centrioles that play critical roles in neuronal polarity and migration. HSPA6 and HSPA1A also targeted nuclear speckles, rich in RNA splicing factors, and the granular component of the nucleolus that is involved in rRNA processing and ribosomal subunit assembly. HSPA6 and HSPA1A displayed similar FRAP kinetics in their interaction with nuclear speckles and the nucleolus. Subsequently, during the recovery from neuronal stress, HSPA6, but not HSPA1A, localized with the periphery of nuclear speckles (perispeckles) that have been characterized as transcription sites. The stress-induced association of HSPA6 with perispeckles displayed the greatest dynamism compared to the interaction of HSPA6 or HSPA1A with other stress-sensitive cytoplasmic and nuclear structures. This suggests involvement of HSPA6 in transcriptional recovery of human neurons from cellular stress that is not apparent for HSPA1A.

  3. Silica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro

    Science.gov (United States)

    Phukan, Geetika; Shin, Tae Hwan; Shim, Jeom Soon; Paik, Man Jeong; Lee, Jin-Kyu; Choi, Sangdun; Kim, Yong Man; Kang, Seong Ho; Kim, Hyung Sik; Kang, Yup; Lee, Soo Hwan; Mouradian, M. Maral; Lee, Gwang

    2016-01-01

    The potential toxicity of nanoparticles, particularly to neurons, is a major concern. In this study, we assessed the cytotoxicity of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye (MNPs@SiO2(RITC)) in HEK293 cells, SH-SY5Y cells, and rat primary cortical and dopaminergic neurons. In cells treated with 1.0 μg/μl MNPs@SiO2(RITC), the expression of several genes related to the proteasome pathway was altered, and proteasome activity was significantly reduced, compared with control and with 0.1 μg/μl MNPs@SiO2(RITC)-treated cells. Due to the reduction of proteasome activity, formation of cytoplasmic inclusions increased significantly in HEK293 cells over-expressing the α–synuclein interacting protein synphilin-1 as well as in primary cortical and dopaminergic neurons. Primary neurons, particularly dopaminergic neurons, were more vulnerable to MNPs@SiO2(RITC) than SH-SY5Y cells. Cellular polyamines, which are associated with protein aggregation, were significantly altered in SH-SY5Y cells treated with MNPs@SiO2(RITC). These findings highlight the mechanisms of neurotoxicity incurred by nanoparticles. PMID:27378605

  4. Diphenyl diselenide protects against methylmercury-induced inhibition of thioredoxin reductase and glutathione peroxidase in human neuroblastoma cells: a comparison with ebselen.

    Science.gov (United States)

    Meinerz, Daiane F; Branco, Vasco; Aschner, Michael; Carvalho, Cristina; Rocha, João Batista T

    2017-09-01

    Exposure to methylmercury (MeHg), an important environmental toxicant, may lead to serious health risks, damaging various organs and predominantly affecting the brain function. The toxicity of MeHg can be related to the inhibition of important selenoenzymes, such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Experimental studies have shown that selenocompounds play an important role as cellular detoxifiers and protective agents against the harmful effects of mercury. The present study investigated the mechanisms by which diphenyl diselenide [(PhSe) 2 ] and ebselen interfered with the interaction of mercury (MeHg) and selenoenzymes (TrxR and GPx) in an in vitro experimental model of cultured human neuroblastoma cells (SH-SY5Y). Our results established that (PhSe) 2 and ebselen increased the activity and expression of TrxR. In contrast, MeHg inhibited TrxR activity even at low doses (0.5 μm). Coexposure to selenocompounds and MeHg showed a protective effect of (PhSe) 2 on both the activity and expression of TrxR. When selenoenzyme GPx was evaluated, selenocompounds did not alter its activity or expression significantly, whereas MeHg inhibited the activity of GPx (from 1 μm). Among the selenocompounds only (PhSe) 2 significantly protected against the effects of MeHg on GPx activity. Taken together, these results indicate a potential use for ebselen and (PhSe) 2 against MeHg toxicity. Furthermore, for the first time, we have demonstrated that (PhSe) 2 caused a more pronounced upregulation of TrxR than ebselen in neuroblastoma cells, likely reflecting an important molecular mechanism involved in the antioxidant properties of this compound. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  5. Morphogenetic and neuronal characterization of human neuroblastoma multicellular spheroids cultured under undifferentiated and all-trans-retinoic acid-differentiated conditions

    Directory of Open Access Journals (Sweden)

    Gwon-Soo Jung

    2013-05-01

    Full Text Available In this study, we aimed to compare the morphogenetic andneuronal characteristics between monolayer cells andspheroids. For this purpose, we established spheroid formationby growing SH-SY5Y cells on the hydrophobic surfaces ofthermally-collapsed elastin-like polypeptide. After 4 days ofculture, the relative proliferation of the cells within spheroidswas approximately 92% of the values for monolayer cultures.As measured by quantitative assays for mRNA and proteinexpressions, the production of synaptophysin and neuronspecificenolase (NSE as well as the contents of cell adhesionmolecules (CAMs and extracellular matrix (ECM proteins aremuch higher in spheroids than in monolayer cells. Under theall-trans-retinoic acid (RA-induced differentiation condition,spheroids extended neurites and further up-regulated theexpression of synaptophysin, NSE, CAMs, and ECM proteins.Our data indicate that RA-differentiated SH-SY5Y neurospheroidsare functionally matured neuronal architectures. [BMBReports 2013; 46(5: 276-281

  6. IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Daniela Lehnen

    2017-10-01

    Full Text Available Human pluripotent stem cell (hPSC-derived mesencephalic dopaminergic (mesDA neurons can relieve motor deficits in animal models of Parkinson's disease (PD. Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP+ mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA neurons in vitro. Intrastriatal transplantation of IAP+ cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP+ mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies.

  7. IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

    Science.gov (United States)

    Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

    2017-10-10

    Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP + mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP + cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP + mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

  8. Melatonin pre-treatment mitigates SHSY-5Y cells against oxaliplatin induced mitochondrial stress and apoptotic cell death

    Science.gov (United States)

    Choudhury, Arnab; Kar, Sudeshna; Tabassum, Heena

    2017-01-01

    Oxaliplatin (Oxa) treatment to SH-SY5Y human neuroblastoma cells has been shown by previous studies to induce oxidative stress, which in turn modulates intracellular signaling cascades resulting in cell death. While this phenomenon of Oxa-induced neurotoxicity is known, the underlying mechanisms involved in this cell death cascade must be clarified. Moreover, there is still little known regarding the roles of neuronal mitochondria and cytosolic compartments in mediating Oxa-induced neurotoxicity. With a better grasp of the mechanisms driving neurotoxicity in Oxa-treated SH-SY5Y cells, we can then identify certain pathways to target in protecting against neurotoxic cell damage. Therefore, the purpose of this study was to determine whether one such agent, melatonin (Mel), could confer protection against Oxa-induced neurotoxicity in SH-SY5Y cells. Results from the present study found Oxa to significantly reduce SH-SY5Y cell viability in a dose-dependent manner. Alternatively, we found Mel pre-treatment to SH-SY5Y cells to attenuate Oxa-induced toxicity, resulting in a markedly increased cell viability. Mel exerted its protective effects by regulating reactive oxygen species (ROS) production and reducing superoxide radicals inside Oxa-exposed. In addition, we observed pre-treatment with Mel to rescue Oxa-treated cells by protecting mitochondria. As Oxa-treatment alone decreases mitochondrial membrane potential (Δψm), resulting in an altered Bcl-2/Bax ratio and release of sequestered cytochrome c, so Mel was shown to inhibit these pathways. Mel was also found to inhibit proteolytic activation of caspase 3, inactivation of Poly (ADP Ribose) polymerase, and DNA damage, thereby allowing SH-SY5Y cells to resist apoptotic cell death. Collectively, our results suggest a role for melatonin in reducing Oxa induced neurotoxicity. Further studies exploring melatonin’s protective effects may prove successful in eliciting pathways to further alter the neurotoxic pathways of

  9. Melatonin pre-treatment mitigates SHSY-5Y cells against oxaliplatin induced mitochondrial stress and apoptotic cell death.

    Directory of Open Access Journals (Sweden)

    Mohammad Waseem

    Full Text Available Oxaliplatin (Oxa treatment to SH-SY5Y human neuroblastoma cells has been shown by previous studies to induce oxidative stress, which in turn modulates intracellular signaling cascades resulting in cell death. While this phenomenon of Oxa-induced neurotoxicity is known, the underlying mechanisms involved in this cell death cascade must be clarified. Moreover, there is still little known regarding the roles of neuronal mitochondria and cytosolic compartments in mediating Oxa-induced neurotoxicity. With a better grasp of the mechanisms driving neurotoxicity in Oxa-treated SH-SY5Y cells, we can then identify certain pathways to target in protecting against neurotoxic cell damage. Therefore, the purpose of this study was to determine whether one such agent, melatonin (Mel, could confer protection against Oxa-induced neurotoxicity in SH-SY5Y cells. Results from the present study found Oxa to significantly reduce SH-SY5Y cell viability in a dose-dependent manner. Alternatively, we found Mel pre-treatment to SH-SY5Y cells to attenuate Oxa-induced toxicity, resulting in a markedly increased cell viability. Mel exerted its protective effects by regulating reactive oxygen species (ROS production and reducing superoxide radicals inside Oxa-exposed. In addition, we observed pre-treatment with Mel to rescue Oxa-treated cells by protecting mitochondria. As Oxa-treatment alone decreases mitochondrial membrane potential (Δψm, resulting in an altered Bcl-2/Bax ratio and release of sequestered cytochrome c, so Mel was shown to inhibit these pathways. Mel was also found to inhibit proteolytic activation of caspase 3, inactivation of Poly (ADP Ribose polymerase, and DNA damage, thereby allowing SH-SY5Y cells to resist apoptotic cell death. Collectively, our results suggest a role for melatonin in reducing Oxa induced neurotoxicity. Further studies exploring melatonin's protective effects may prove successful in eliciting pathways to further alter the neurotoxic

  10. Cholinergic and dopaminergic neuronal differentiation of human adipose tissue derived mesenchymal stem cells.

    Science.gov (United States)

    Marei, Hany El Sayed; El-Gamal, Aya; Althani, Asma; Afifi, Nahla; Abd-Elmaksoud, Ahmed; Farag, Amany; Cenciarelli, Carlo; Thomas, Caceci; Anwarul, Hasan

    2018-02-01

    Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various cell types such as cartilage, bone, and fat cells. Recent studies have shown that induction of MSCs in vitro by growth factors including epidermal growth factor (EGF) and fibroblast growth factor (FGF2) causes them to differentiate into neural like cells. These cultures also express ChAT, a cholinergic marker; and TH, a dopaminergic marker for neural cells. To establish a protocol with maximum differentiation potential, we examined MSCs under three experimental culture conditions using neural induction media containing FGF2, EGF, BMP-9, retinoic acid, and heparin. Adipose-derived MSCs were extracted and expanded in vitro for 3 passages after reaching >80% confluency, for a total duration of 9 days. Cells were then characterized by flow cytometry for CD markers as CD44 positive and CD45 negative. MSCs were then treated with neural induction media and were characterized by morphological changes and Q-PCR. Differentiated MSCs expressed markers for immature and mature neurons; β Tubulin III (TUBB3) and MAP2, respectively, showing the neural potential of these cells to differentiate into functional neurons. Improved protocols for MSCs induction will facilitate and ensure the reproducibility and standard production of MSCs for therapeutic applications in neurodegenerative diseases. © 2017 Wiley Periodicals, Inc.

  11. Proneural transcription factor Atoh1 drives highly efficient differentiation of human pluripotent stem cells into dopaminergic neurons.

    Science.gov (United States)

    Sagal, Jonathan; Zhan, Xiping; Xu, Jinchong; Tilghman, Jessica; Karuppagounder, Senthilkumar S; Chen, Li; Dawson, Valina L; Dawson, Ted M; Laterra, John; Ying, Mingyao

    2014-08-01

    Human pluripotent stem cells (PSCs) are a promising cell resource for various applications in regenerative medicine. Highly efficient approaches that differentiate human PSCs into functional lineage-specific neurons are critical for modeling neurological disorders and testing potential therapies. Proneural transcription factors are crucial drivers of neuron development and hold promise for driving highly efficient neuronal conversion in PSCs. Here, we study the functions of proneural transcription factor Atoh1 in the neuronal differentiation of PSCs. We show that Atoh1 is induced during the neuronal conversion of PSCs and that ectopic Atoh1 expression is sufficient to drive PSCs into neurons with high efficiency. Atoh1 induction, in combination with cell extrinsic factors, differentiates PSCs into functional dopaminergic (DA) neurons with >80% purity. Atoh1-induced DA neurons recapitulate key biochemical and electrophysiological features of midbrain DA neurons, the degeneration of which is responsible for clinical symptoms in Parkinson's disease (PD). Atoh1-induced DA neurons provide a reliable disease model for studying PD pathogenesis, such as neurotoxin-induced neurodegeneration in PD. Overall, our results determine the role of Atoh1 in regulating neuronal differentiation and neuron subtype specification of human PSCs. Our Atoh1-mediated differentiation approach will enable large-scale applications of PD patient-derived midbrain DA neurons in mechanistic studies and drug screening for both familial and sporadic PD. ©AlphaMed Press.

  12. Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation

    Directory of Open Access Journals (Sweden)

    Daisuke Doi

    2014-03-01

    Full Text Available Human induced pluripotent stem cells (iPSCs can provide a promising source of midbrain dopaminergic (DA neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.

  13. Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension

    DEFF Research Database (Denmark)

    Krabbe, Christina; Courtois, Elise; Jensen, Pia

    2009-01-01

    Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x(L) and oxygen tension on dopaminergic different......Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x(L) and oxygen tension on dopaminergic...... days at 20% oxygen, hVMbcl-x(L) cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 +/- 0.8% to 17.2 +/- 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q...

  14. Metformin inhibition of neuroblastoma cell proliferation is differently modulated by cell differentiation induced by retinoic acid or overexpression of NDM29 non-coding RNA

    OpenAIRE

    Costa, Delfina; Gigoni, Arianna; Würth, Roberto; Cancedda, Ranieri; Florio, Tullio; Pagano, Aldo

    2014-01-01

    Background Metformin is a widely used oral hypoglycemizing agent recently proposed as potential anti-cancer drug. In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential. Methods Antiproliferative and cytotoxic effects of metformin were tested in human SKNBE2 and SH-SY5Y neuroblastoma cell lines and in SKNBE2 cells in whi...

  15. The "highs and lows" of the human brain on dopaminergics: Evidence from neuropharmacology.

    Science.gov (United States)

    Martins, Daniel; Mehta, Mitul A; Prata, Diana

    2017-09-01

    Rewards are appetitive events that elicit approach. Ground-breaking findings from neurophysiological experiments in animals, alongside neuropharmacology and neuroimaging research in human samples have identified dopamine as the main neurochemical messenger of global reward processing in the brain. However, dopamine's contribution to the different components of reward processing remains to be precisely defined. To facilitate the informed design and interpretation of reward studies in humans, we have systematically reviewed all existing human pharmacological studies investigating how drug manipulation of the dopamine system affects reward-related behaviour and its neural correlates. Pharmacological experiments in humans face methodological challenges in terms of the: 1) specificity and safety of the available drugs for administration in humans, 2) uncertainties about pre- or post-synaptic modes of action, and 3) possible interactions with inter-individual neuropsychological or genotypic variables. In order to circumvent some of these limitations, future research should rely on the combination of different levels of observation, in integrative pharmaco-genetics-neurobehavioral approaches, to more completely characterize dopamine's role in both general and modality-specific processing of reward. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. MicroRNA-138 is a potential regulator of memory performance in humans

    Directory of Open Access Journals (Sweden)

    Julia eSchröder

    2014-07-01

    Full Text Available Genetic factors underlie a substantial proportion of individual differences in cognitive functions in humans, including processes related to episodic and working memory. While genetic association studies have proposed several candidate memory genes, these currently explain only a minor fraction of the phenotypic variance. Here, we performed genome-wide screening on 13 episodic and working memory phenotypes in 1,318 participants of the Berlin Aging Study II aged 60 years or older. The analyses highlight a number of novel single nucleotide polymorphisms (SNPs associated with memory performance, including one located in a putative regulatory region of microRNA (miRNA hsa-mir-138-5p (rs9882688, P-value = 7.8x10-9. Expression quantitative trait locus analyses on next-generation RNA-sequencing data revealed that rs9882688 genotypes show a significant correlation with the expression levels of this miRNA in 309 human lymphoblastoid cell lines (P-value = 5x10-4. In silico modeling of other top-ranking GWAS signals identified an additional memory-associated SNP in the 3' untranslated region (3'UTR of DCP1B, a gene encoding a core component of the mRNA decapping complex in humans, predicted to interfere with hsa-mir-138-5p binding. This prediction was confirmed in vitro by luciferase assays showing differential binding of hsa-mir-138-5p to 3'UTR reporter constructs in two human cell lines (HEK293: P-value = 0.0470; SH-SY5Y: P-value = 0.0866. Finally, expression profiling of hsa-mir-138-5p and DCP1B mRNA in human post-mortem brain tissue revealed that both molecules are expressed simultaneously in frontal cortex and hippocampus, suggesting that the proposed interaction between hsa-mir-138-5p and DCP1B may also take place in vivo. In summary, by combining unbiased genome-wide screening with extensive in silico modeling, in vitro functional assays, and gene expression profiling, our study identified miRNA-138 as a potential molecular regulator of human memory

  17. Adiponectin is protective against oxidative stress induced cytotoxicity in amyloid-beta neurotoxicity.

    Directory of Open Access Journals (Sweden)

    Koon-Ho Chan

    Full Text Available Beta-amyloid (Aβ neurotoxicity is important in Alzheimer's disease (AD pathogenesis. Aβ neurotoxicity causes oxidative stress, inflammation and mitochondrial damage resulting in neuronal degeneration and death. Oxidative stress, inflammation and mitochondrial failure are also pathophysiological mechanisms of type 2 diabetes (T(2DM which is characterized by insulin resistance. Interestingly, T(2DM increases risk to develop AD which is associated with reduced neuronal insulin sensitivity (central insulin resistance. We studied the potential protective effect of adiponectin (an adipokine with insulin-sensitizing, anti-inflammatory and anti-oxidant properties against Aβ neurotoxicity in human neuroblastoma cells (SH-SY5Y transfected with the Swedish amyloid precursor protein (Sw-APP mutant, which overproduced Aβ with abnormal intracellular Aβ accumulation. Cytotoxicity was measured by assay for lactate dehydrogenase (LDH released upon cell death and lysis. Our results revealed that Sw-APP transfected SH-SY5Y cells expressed both adiponectin receptor 1 and 2, and had increased AMP-activated protein kinase (AMPK activation and enhanced nuclear factor-kappa B (NF-κB activation compared to control empty-vector transfected SH-SY5Y cells. Importantly, adiponectin at physiological concentration of 10 µg/ml protected Sw-APP transfected SH-SY5Y cells against cytotoxicity under oxidative stress induced by hydrogen peroxide. This neuroprotective action of adiponectin against Aβ neurotoxicity-induced cytotoxicity under oxidative stress involved 1 AMPK activation mediated via the endosomal adaptor protein APPL1 (adaptor protein with phosphotyrosine binding, pleckstrin homology domains and leucine zipper motif and possibly 2 suppression of NF-κB activation. This raises the possibility of novel therapies for AD such as adiponectin receptor agonists.

  18. The neuroprotective action of the mood stabilizing drugs lithium chloride and sodium valproate is mediated through the up-regulation of the homeodomain protein Six1

    International Nuclear Information System (INIS)

    Plant, Kathryn E.; Anderson, Elizabeth; Simecek, Nicole; Brown, Richard; Forster, Sam; Spinks, Jenny; Toms, Nick; Gibson, G. Gordon; Lyon, Jon; Plant, Nick

    2009-01-01

    The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimer's or Parkinsonism

  19. Population PKPD modeling of BACE1 inhibitor-induced reduction in Aβ levels in vivo and correlation to in vitro potency in primary cortical neurons from mouse and guinea pig.

    Science.gov (United States)

    Janson, Juliette; Eketjäll, Susanna; Tunblad, Karin; Jeppsson, Fredrik; Von Berg, Stefan; Niva, Camilla; Radesäter, Ann-Cathrin; Fälting, Johanna; Visser, Sandra A G

    2014-03-01

    The aims were to quantify the in vivo time-course between the oral dose, the plasma and brain exposure and the inhibitory effect on Amyloid β (Aβ) in brain and cerebrospinal fluid, and to establish the correlation between in vitro and in vivo potency of novel β-secretase (BACE1) inhibitors. BACE1-mediated inhibition of Aβ was quantified in in vivo dose- and/or time-response studies and in vitro in SH-SY5Y cells, N2A cells, and primary cortical neurons (PCN). An indirect response model with inhibition on Aβ production rate was used to estimate unbound in vivo IC 50 in a population pharmacokinetic-pharmacodynamic modeling approach. Estimated in vivo inhibitory potencies varied between 1 and 1,000 nM. The turnover half-life of Aβ40 in brain was predicted to be 0.5 h in mouse and 1 h in guinea pig. An excellent correlation between PCN and in vivo potency was observed. Moreover, a strong correlation in potency was found between human SH-SY5Y cells and mouse PCN, being 4.5-fold larger in SH-SY5Y cells. The strong in vivo-in vitro correlation increased the confidence in using human cell lines for screening and optimization of BACE1 inhibitors. This can optimize the design and reduce the number of preclinical in vivo effect studies.

  20. Actions of piperidine alkaloid teratogens at fetal nicotinic acetylcholine receptors.

    Science.gov (United States)

    Green, Benedict T; Lee, Stephen T; Panter, Kip E; Welch, Kevin D; Cook, Daniel; Pfister, James A; Kem, William R

    2010-01-01

    Teratogenic alkaloids are found in many species of plants including Conium maculatum L., Nicotiana glauca, Nicotiana tabaccum, and multiple Lupinus spp. Fetal musculoskeletal defects produced by alkaloids from these plants include arthrogyropisis, scoliosis, torticollis, kyposis, lordosis, and cleft palate. A pharmacodynamic comparison of the alkaloids ammodendrine, anabasine, anabaseine, anagyrine, and coniine in SH-SY5Y cells and TE-671 cells was made. These alkaloids and their enantiomers were more effective in depolarizing TE-671 cells which express the human fetal-muscle type nicotinic acetylcholine receptor (nAChR) relative to SH-SY5Y cells which predominately express autonomic nAChRs. The rank order of potency in TE-671 cells was: anabaseine>(+)-anabasine>(-)-anabasine > (+/-)-anabasine>anagyrine>(-)-coniine > (+/-)-coniine>(+)-coniine>(+/-)-ammodendrine>(+)-ammodendrine. The rank order potency in SH-SY5Y cells was: anabaseine>(+)-anabasine>(-)-coniine>(+)-coniine>(+)-ammodendrine>anagyrine>(-)-anabasine>(+/-)-coniine>(+/-)-anabasine>(-)-ammodendrine. The actions of these alkaloids at nAChRs in both cell lines could be distinguished by their maximum effects in depolarizing cell membrane potential. The teratogenic action of these compounds may be related to their ability to activate and subsequently desensitize nAChRs.

  1. Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Preeti Singh

    2017-01-01

    Full Text Available Sirtuins are highly conserved lysine deacetylases involved in ageing, energy production, and lifespan extension. The mammalian SIRT2 has been implicated in Parkinson’s disease (PD where studies suggest SIRT2 promotes neurodegeneration. We therefore evaluated the effects of SIRT2 manipulation in toxin treated SH-SY5Y cells and determined the expression and activity of SIRT2 in postmortem brain tissue from patients with PD. SH-SY5Y viability in response to oxidative stress induced by diquat or rotenone was measured following SIRT2 overexpression or inhibition of deacetylase activity, along with α-synuclein aggregation. SIRT2 in human tissues was evaluated using Western blotting, immunohistochemistry, and fluorometric activity assays. In SH-SY5Y cells, elevated SIRT2 protected cells from rotenone or diquat induced cell death and enzymatic inhibition of SIRT2 enhanced cell death. SIRT2 protection was mediated, in part, through elevated SOD2 expression. SIRT2 reduced the formation of α-synuclein aggregates but showed minimal colocalisation with α-synuclein. In postmortem PD brain tissue, SIRT2 activity was elevated compared to controls but also elevated in other neurodegenerative disorders. Results from both in vitro work and brain tissue suggest that SIRT2 is necessary for protection against oxidative stress and higher SIRT2 activity in PD brain may be a compensatory mechanism to combat neuronal stress.

  2. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

    International Nuclear Information System (INIS)

    Kim, Dae Hong; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Hwang, Jae Sam; Seok, Heon; Choi, Hyemin; Lee, Dong Gun; Kim, Jae Il; Kim, Ho

    2014-01-01

    Highlights: • 11-mer peptide Lumbricusin, a defensin like peptide, is isolated from earthworm. • We here demonstrated that Lumbricusin has neurotropic and neuroprotective effects. • p27 degradation by Lumbricusin mediates effects of Lumbricusin on neuronal cells. - Abstract: We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH 2 -RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson’s disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27 Kip1 protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27 Kip1 significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27 Kip1 degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin

  3. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dae Hong; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng [Department of Life Science, College of Natural Science, Daejin University, Pocheon, Gyeonggido 487-711 (Korea, Republic of); Hwang, Jae Sam [Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441-707 (Korea, Republic of); Seok, Heon [Department of Biomedical Engineering, Jungwon University, Goesan, Chungcheongbukdo 367-700 (Korea, Republic of); Choi, Hyemin; Lee, Dong Gun [School of Life Sciences, KNU Creative Bioresearch Group (BK21 Plus Program), College of Natural Sciences, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu 702-701 (Korea, Republic of); Kim, Jae Il [School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of); Kim, Ho, E-mail: hokim@daejin.ac.kr [Department of Life Science, College of Natural Science, Daejin University, Pocheon, Gyeonggido 487-711 (Korea, Republic of)

    2014-06-06

    Highlights: • 11-mer peptide Lumbricusin, a defensin like peptide, is isolated from earthworm. • We here demonstrated that Lumbricusin has neurotropic and neuroprotective effects. • p27 degradation by Lumbricusin mediates effects of Lumbricusin on neuronal cells. - Abstract: We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH{sub 2}-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson’s disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27{sup Kip1} protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27{sup Kip1} significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27{sup Kip1} degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.

  4. Gold nanoparticles as scaffolds for poor water soluble and difficult to vehiculate antiparkinson codrugs

    Science.gov (United States)

    Di Crescenzo, A.; Cacciatore, I.; Petrini, M.; D'Alessandro, M.; Petragnani, N.; Del Boccio, P.; Di Profio, P.; Boncompagni, S.; Spoto, G.; Turkez, H.; Ballerini, P.; Di Stefano, A.; Fontana, A.

    2017-01-01

    We report the facile and non-covalent preparation of gold nanoparticles (AuNPs) stabilized by an antiparkinson codrug based on lipoic acid (LA). The obtained AuNPs appear stable in both dimethyl sulfoxide and fetal bovine serum and able to load an amount of codrug double the weight of gold. These NPs were demonstrated to be safe and biocompatible towards primary human blood cells and human neuroblastoma cells, one of the most widely used cellular models to study dopaminergic neural cells, therefore are ideal drug carriers for difficult to solubilize molecules. Very interestingly, the codrug-stabilized AuNPs were shown to reduce the accumulation of reactive oxygen species in SH-SY5Y cells treated with LD and did not change total oxidant status levels in cultured human blood cells, thus confirming the antioxidant role of LA although bound to AuNPs. The characterization of AuNPs in terms of loading and stability paves the way for their use in biomedical and pharmacological applications.

  5. A novel therapeutic approach to 6-OHDA-induced Parkinson's disease in rats via supplementation of PTD-conjugated tyrosine hydroxylase.

    Science.gov (United States)

    Wu, Shao Ping; Fu, Ai Ling; Wang, Yu Xia; Yu, Lei Ping; Jia, Pei Yuan; Li, Qian; Jin, Guo Zhang; Sun, Man Ji

    2006-07-21

    The present study aimed to evaluate whether the protein transduction domain (PTD)-conjugated human tyrosine hydroxylase (TH) fusion protein was effective on the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model rats. An expression vector pET-PTD-TH harbouring the PTD-TH gene was constructed and transformed to the Escherichia coli BL21 cells for expression. The expressed recombinant PTD-TH with a molecular weight of 61 kD was successfully transduced (1 microM) into the dopaminergic SH-sy5y human neuroblastoma cells in vitro and visualized by immunohistochemical assay. An in vivo experiment in rats showed that the iv administered PTD-TH protein (8 mg/kg) permeated across the blood-brain barrier, penetrated into the striatum and midbrain, and peaked at 5-8 h after the injection. The behavioral effects of PTD-TH on the apomorphine-induced rotations in the PD model rats 8 weeks after the 6-OHDA lesion showed that a single bolus of PTD-TH (8 mg/kg) iv injection caused a decrement of 60% of the contralateral turns on day 1 and 40% on days 5-17. The results imply that iv delivery of PTD-TH is therapeutically effective on the 6-OHDA-induced PD in rats, the PTD-mediated human TH treatment opening a promising therapeutic direction in treatment of PD.

  6. A novel therapeutic approach to 6-OHDA-induced Parkinson's disease in rats via supplementation of PTD-conjugated tyrosine hydroxylase

    International Nuclear Information System (INIS)

    Wu Shaoping; Fu Ailing; Wang Yuxia; Yu Leiping; Jia Peiyuan; Li Qian; Jin Guozhang; Sun Manji

    2006-01-01

    The present study aimed to evaluate whether the protein transduction domain (PTD)-conjugated human tyrosine hydroxylase (TH) fusion protein was effective on the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model rats. An expression vector pET-PTD-TH harbouring the PTD-TH gene was constructed and transformed to the Escherichia coli BL21 cells for expression. The expressed recombinant PTD-TH with a molecular weight of 61 kD was successfully transduced (1 μM) into the dopaminergic SH-sy5y human neuroblastoma cells in vitro and visualized by immunohistochemical assay. An in vivo experiment in rats showed that the iv administered PTD-TH protein (8 mg/kg) permeated across the blood-brain barrier, penetrated into the striatum and midbrain, and peaked at 5-8 h after the injection. The behavioral effects of PTD-TH on the apomorphine-induced rotations in the PD model rats 8 weeks after the 6-OHDA lesion showed that a single bolus of PTD-TH (8 mg/kg) iv injection caused a decrement of 60% of the contralateral turns on day 1 and 40% on days 5-17. The results imply that iv delivery of PTD-TH is therapeutically effective on the 6-OHDA-induced PD in rats, the PTD-mediated human TH treatment opening a promising therapeutic direction in treatment of PD

  7. Experiment list: SRX757029 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available s; ChIP-Seq source_name=Cultured SH-SY5Y cells || cell line=SH-SY5Y || infection=control shRNA || neuronal m... Diagnosis=Neuroblastoma 26745539,88.4,7.1,1085 GSM1542254: Sample H3K9me2 Input SH-SY5Y B3 I19; Homo sapien

  8. Upregulation of CRABP1 in human neuroblastoma cells overproducing the Alzheimer-typical Aβ42 reduces their differentiation potential

    Directory of Open Access Journals (Sweden)

    Weninger Annette

    2008-12-01

    conclude that increasing the Aβ42/Aβ40 ratio up-regulates CRABP1, which in turn reduces the differentiation potential of the human neuroblastoma cell line SH-SY5Y, but increases cell proliferation. This work might contribute to the better understanding of AD neurogenesis, currently a controversial topic.

  9. Coptis chinensis Franch. exhibits neuroprotective properties against oxidative stress in human neuroblastoma cells.

    Science.gov (United States)

    Friedemann, Thomas; Otto, Benjamin; Klätschke, Kristin; Schumacher, Udo; Tao, Yi; Leung, Alexander Kai-Man; Efferth, Thomas; Schröder, Sven

    2014-08-08

    The dried rhizome of Coptis chinensis Franch. (family Ranunculaceae) is traditionally used in Chinese medicine for the treatment of inflammatory diseases and diabetes. Recent studies showed a variety of activities of Coptis chinensis Franch. alkaloids, including neuroprotective, neuroregenerative, anti-diabetic, anti-oxidative and anti-inflammatory effects. However, there is no report on the neuroprotective effect of Coptis chinensis Franch. watery extract against tert-butylhydroperoxide (t-BOOH) induced oxidative damage. The aim of the study is to investigate neuroprotective properties of Coptis chinensis Franch. rhizome watery extract (CRE) and to evaluate its potential mechanism of action. Neuroprotective properties on t-BOOH induced oxidative stress were investigated in SH-SY5Y human neuroblastoma cells. Cells were pretreated with CRE for 2 h or 24 h followed by 2 h of treatment with t-BOOH. To evaluate the neuroprotective effect of CRE, cell viability, cellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the apoptotic rate were determined and microarray analyses, as well as qRT-PCR analyses were conducted. Two hours of exposure to 100 µM t-BOOH resulted in a significant reduction of cell viability, increased apoptotic rate, declined mitochondrial membrane potential (MMP) and increased ROS production. Reduction of cell viability, increased apoptotic rate and declined mitochondrial membrane potential (MMP) could be significantly reduced in cells pretreated with CRE (100 µg/ml) for 2h or 24h ahead of t-BOOH exposure with the greatest effect after 24h of pretreatment; however ROS production was not changed significantly. Furthermore, microarray analyses revealed that the expressions of 2 genes; thioredoxin-interacting protein (TXNIP) and mitochondrially encoded NADH dehydrogenase 1, were significantly regulated. Down regulation of TXNIP was confirmed by qRT-PCR. Due to its neuroprotective properties CRE might be a potential

  10. Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lei [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Department of Physiology, Nankai University School of Medicine, Tianjin 300071 (China); Carr, Aprell L. [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556 (United States); Li, Ping; Lee, Jessica; McGregor, Mary [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Li, Lei, E-mail: Li.78@nd.edu [Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (United States); Center for Zebrafish Research, University of Notre Dame, Notre Dame, IN 46556 (United States)

    2014-07-11

    Highlights: • Stil is a human oncogene that is conserved in vertebrate species. • Stil functions in the Shh pathway in mammalian cells. • The expression of Stil is required for mammalian dopaminergic cell proliferation. - Abstract: The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in all vertebrate species. In humans, the expression of Stil is involved in cancer cell survival, apoptosis and proliferation. In this research, we investigated the roles of Stil expression in cell proliferation of mammalian dopaminergic (DA) PC12 cells. Stil functions through the Sonic hedgehog (Shh) signal transduction pathway. Co-immunoprecipitation tests revealed that STIL interacts with Shh downstream components, which include SUFU and GLI1. By examining the expression of Stil, Gli1, CyclinD2 (cell-cycle marker) and PCNA (proliferating cell nuclear antigen), we found that up-regulation of Stil expression (transfection with overexpression plasmids) increased Shh signaling transduction and PC12 cell proliferation, whereas down-regulation of Stil expression (by shRNA) inhibited Shh signaling transduction, and thereby decreased PC12 cell proliferation. Transient transfection of PC12 cells with Stil knockdown or overexpression plasmids did not affect PC12 cell neural differentiation, further indicating the specific roles of Stil in cell proliferation. The results from this research suggest that Stil may serve as a bio-marker for neurological diseases involved in DA neurons, such as Parkinson’s disease.

  11. Endoplasmic reticulum stress-mediated neuronal apoptosis by acrylamide exposure

    Energy Technology Data Exchange (ETDEWEB)

    Komoike, Yuta, E-mail: komoike@research.twmu.ac.jp; Matsuoka, Masato, E-mail: matsuoka@research.twmu.ac.jp

    2016-11-01

    Acrylamide (AA) is a well-known neurotoxic compound in humans and experimental animals. However, intracellular stress signaling pathways responsible for the neurotoxicity of AA are still not clear. In this study, we explored the involvement of the endoplasmic reticulum (ER) stress response in AA-induced neuronal damage in vitro and in vivo. Exposure of SH-SY5Y human neuroblastoma cells to AA increased the levels of phosphorylated form of eukaryotic translation initiation factor 2α (eIF2α) and its downstream effector, activating transcription factor 4 (ATF4), indicating the induction of the unfolded protein response (UPR) by AA exposure. Furthermore, AA exposure increased the mRNA level of c/EBP homologous protein (CHOP), the ER stress-dependent apoptotic factor, and caused the accumulation of reactive oxygen species (ROS) in SH-SY5Y cells. Treatments of SH-SY5Y cells with the chemical chaperone, 4-phenylbutyric acid and the ROS scavenger, N-acetyl-cysteine reduced the AA-induced expression of ATF4 protein and CHOP mRNA, and resulted in the suppression of apoptosis. In addition, AA-induced eIF2α phosphorylation was also suppressed by NAC treatment. In consistent with in vitro study, exposure of zebrafish larvae at 6-day post fertilization to AA induced the expression of chop mRNA and apoptotic cell death in the brain, and also caused the disruption of brain structure. These findings suggest that AA exposure induces apoptotic neuronal cell death through the ER stress and subsequent eIF2α–ATF4–CHOP signaling cascade. The accumulation of ROS by AA exposure appears to be responsible for this ER stress-mediated apoptotic pathway. - Highlights: • Exposure of SH-SY5Y cells to AA activates the eIF2α–ATF4 pathway of the UPR. • Exposure of SH-SY5Y cells to AA induces the CHOP expression and apoptosis. • Exposure of zebrafish to AA induces the chop expression and apoptosis in the brain. • AA possibly induces apoptotic neuronal cell death through the ER

  12. Ropinirole and Pramipexole Promote Structural Plasticity in Human iPSC-Derived Dopaminergic Neurons via BDNF and mTOR Signaling

    Directory of Open Access Journals (Sweden)

    Ginetta Collo

    2018-01-01

    Full Text Available The antiparkinsonian ropinirole and pramipexole are D3 receptor- (D3R- preferring dopaminergic (DA agonists used as adjunctive therapeutics for the treatment resistant depression (TRD. While the exact antidepressant mechanism of action remains uncertain, a role for D3R in the restoration of impaired neuroplasticity occurring in TRD has been proposed. Since D3R agonists are highly expressed on DA neurons in humans, we studied the effect of ropinirole and pramipexole on structural plasticity using a translational model of human-inducible pluripotent stem cells (hiPSCs. Two hiPSC clones from healthy donors were differentiated into midbrain DA neurons. Ropinirole and pramipexole produced dose-dependent increases of dendritic arborization and soma size after 3 days of culture, effects antagonized by the selective D3R antagonists SB277011-A and S33084 and by the mTOR pathway kinase inhibitors LY294002 and rapamycin. All treatments were also effective in attenuating the D3R-dependent increase of p70S6-kinase phosphorylation. Immunoneutralisation of BDNF, inhibition of TrkB receptors, and blockade of MEK-ERK signaling likewise prevented ropinirole-induced structural plasticity, suggesting a critical interaction between BDNF and D3R signaling pathways. The highly similar profiles of data acquired with DA neurons derived from two hiPSC clones underpin their reliability for characterization of pharmacological agents acting via dopaminergic mechanisms.

  13. Curcumin Regulates Low-Linear Energy Transfer γ-Radiation-Induced NFκB-Dependent Telomerase Activity in Human Neuroblastoma Cells

    International Nuclear Information System (INIS)

    Aravindan, Natarajan; Veeraraghavan, Jamunarani; Madhusoodhanan, Rakhesh; Herman, Terence S.; Natarajan, Mohan

    2011-01-01

    Purpose: We recently reported that curcumin attenuates ionizing radiation (IR)-induced survival signaling and proliferation in human neuroblastoma cells. Also, in the endothelial system, we have demonstrated that NFκB regulates IR-induced telomerase activity (TA). Accordingly, we investigated the effect of curcumin in inhibiting IR-induced NFκB-dependent hTERT transcription, TA, and cell survival in neuroblastoma cells. Methods and Materials: SK-N-MC or SH-SY5Y cells exposed to IR and treated with curcumin (10-100 nM) with or without IR were harvested after 1 h through 24 h. NFκB-dependent regulation was investigated either by luciferase reporter assays using pNFκB-, pGL3-354-, pGL3-347-, or pUSE-IκBα-Luc, p50/p65, or RelA siRNA-transfected cells. NFκB activity was analyzed using an electrophoretic mobility shift assay and hTERT expression using the quantitative polymerase chain reaction. TA was determined using the telomerase repeat amplification protocol assay and cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide and clonogenic assay. Results: Curcumin profoundly inhibited IR-induced NFκB. Consequently, curcumin significantly inhibited IR-induced TA and hTERT mRNA at all points investigated. Furthermore, IR-induced TA is regulated at the transcriptional level by triggering telomerase reverse transcriptase (TERT) promoter activation. Moreover, NFκB becomes functionally activated after IR and mediates TA upregulation by binding to the κB-binding region in the promoter region of the TERT gene. Consistently, elimination of the NFκB-recognition site on the telomerase promoter or inhibition of NFκB by the IκBα mutant compromises IR-induced telomerase promoter activation. Significantly, curcumin inhibited IR-induced TERT transcription. Consequently, curcumin inhibited hTERT mRNA and TA in NFκB overexpressed cells. Furthermore, curcumin enhanced the IR-induced inhibition of cell survival. Conclusions: These results

  14. Anti-cancer stemness and anti-invasive activity of bitter taste receptors, TAS2R8 and TAS2R10, in human neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Yoona Seo

    Full Text Available Neuroblastoma (NB originates from immature neuronal cells and currently has a poor clinical outcome. NB cells possess cancer stem cells (CSCs characteristics that facilitate the initiation of a tumor, as well as its metastasis. Human bitter taste receptors, referred to as TAS2Rs, are one of five types of basic taste receptors and they belong to a family of G-protein coupled receptors. The recent finding that taste receptors are expressed in non-gustatory tissues suggest that they mediate additional functions distinct from taste perception. While it is generally admitted that the recognition of bitter tastes may be associated with a self-defense system to prevent the ingestion of poisonous food compounds, this recognition may also serve as a disease-related function in the human body. In particular, the anti-cancer stemness and invasion effects of TAS2Rs on NB cells remain poorly understood. In the present study, endogenous expression of TAS2R8 and TAS2R10 in SK-N-BE(2C and SH-SY5Y cells was examined. In addition, higher levels of TAS2R8 and TAS2R10 expression were investigated in more differentiated SY5Y cells. Both TAS2Rs were up-regulated following the induction of neuronal cell differentiation by retinoic acid. In addition, ectopic transfection of the two TAS2Rs induced neurite elongation in the BE(2C cells, and down-regulated CSCs markers (including DLK1, CD133, Notch1, and Sox2, and suppressed self-renewal characteristics. In particular, TAS2RS inhibited tumorigenicity. Furthermore, when TAS2Rs was over-expressed, cell migration, cell invasion, and matrix metalloproteinases activity were inhibited. Expression levels of hypoxia-inducible factor-1α, a well-known regulator of tumor metastasis, as well as its downstream targets, vascular endothelial growth factor and glucose transporter-1, were also suppressed by TAS2Rs. Taken together, these novel findings suggest that TAS2Rs targets CSCs by suppressing cancer stemness characteristics and NB

  15. A well-refined in vitro model derived from human embryonic stem cell for screening phytochemicals with midbrain dopaminergic differentiation-boosting potential for improving Parkinson's disease.

    Science.gov (United States)

    Hsieh, Wen-Ting; Chiang, Been-Huang

    2014-07-09

    Stimulation of endogenous neurogenesis is a potential approach to compensate for loss of dopaminergic neurons of substantia nigra compacta nigra (SNpc) in patients with Parkinson's disease (PD). This objective was to establish an in vitro model by differentiating pluripotent human embryonic stem cells (hESCs) into midbrain dopaminergic (mDA) neurons for screening phytochemicals with mDA neurogenesis-boosting potentials. Consequently, a five-stage differentiation process was developed. The derived cells expressed many mDA markers including tyrosine hydroxylase (TH), β-III tubulin, and dopamine transporter (DAT). The voltage-gated ion channels and dopamine release were also examined for verifying neuron function, and the dopamine receptor agonists bromocriptine and 7-hydroxy-2-(dipropylamino)tetralin (7-OH-DPAT) were used to validate our model. Then, several potential phytochemicals including green tea catechins and ginsenosides were tested using the model. Finally, ginsenoside Rb1 was identified as the most potent phytochemical which is capable of upregulating neurotrophin expression and inducing mDA differentiation.

  16. Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models.

    Science.gov (United States)

    Liu, Jing; Hu, Rui; Liu, Jianwei; Zhang, Butian; Wang, Yucheng; Liu, Xin; Law, Wing-Cheung; Liu, Liwei; Ye, Ling; Yong, Ken-Tye

    2015-12-01

    The toxicity of quantum dots (QDs) has been extensively studied over the past decade. Some common factors that originate the QD toxicity include releasing of heavy metal ions from degraded QDs and the generation of reactive oxygen species on the QD surface. In addition to these factors, we should also carefully examine other potential QD toxicity causes that will play crucial roles in impacting the overall biological system. In this contribution, we have performed cytotoxicity assessment of four types of QD formulations in two different human cancer cell models. The four types of QD formulations, namely, mercaptopropionic acid modified CdSe/CdS/ZnS QDs (CdSe-MPA), PEGylated phospholipid encapsulated CdSe/CdS/ZnS QDs (CdSe-Phos), PEGylated phospholipid encapsulated InP/ZnS QDs (InP-Phos) and Pluronic F127 encapsulated CdTe/ZnS QDs (CdTe-F127), are representatives for the commonly used QD formulations in biomedical applications. Both the core materials and the surface modifications have been taken into consideration as the key factors for the cytotoxicity assessment. Through side-by-side comparison and careful evaluations, we have found that the toxicity of QDs does not solely depend on a single factor in initiating the toxicity in biological system but rather it depends on a combination of elements from the particle formulations. More importantly, our toxicity assessment shows different cytotoxicity trend for all the prepared formulations tested on gastric adenocarcinoma (BGC-823) and neuroblastoma (SH-SY5Y) cell lines. We have further proposed that the cellular uptake of these nanocrystals plays an important role in determining the final faith of the toxicity impact of the formulation. The result here suggests that the toxicity of QDs is rather complex and it cannot be generalized under a few assumptions reported previously. We suggest that one have to evaluate the QD toxicity on a case to case basis and this indicates that standard procedures and comprehensive

  17. Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models

    International Nuclear Information System (INIS)

    Liu, Jing; Hu, Rui; Liu, Jianwei; Zhang, Butian; Wang, Yucheng; Liu, Xin; Law, Wing-Cheung; Liu, Liwei; Ye, Ling; Yong, Ken-Tye

    2015-01-01

    The toxicity of quantum dots (QDs) has been extensively studied over the past decade. Some common factors that originate the QD toxicity include releasing of heavy metal ions from degraded QDs and the generation of reactive oxygen species on the QD surface. In addition to these factors, we should also carefully examine other potential QD toxicity causes that will play crucial roles in impacting the overall biological system. In this contribution, we have performed cytotoxicity assessment of four types of QD formulations in two different human cancer cell models. The four types of QD formulations, namely, mercaptopropionic acid modified CdSe/CdS/ZnS QDs (CdSe-MPA), PEGylated phospholipid encapsulated CdSe/CdS/ZnS QDs (CdSe-Phos), PEGylated phospholipid encapsulated InP/ZnS QDs (InP-Phos) and Pluronic F127 encapsulated CdTe/ZnS QDs (CdTe-F127), are representatives for the commonly used QD formulations in biomedical applications. Both the core materials and the surface modifications have been taken into consideration as the key factors for the cytotoxicity assessment. Through side-by-side comparison and careful evaluations, we have found that the toxicity of QDs does not solely depend on a single factor in initiating the toxicity in biological system but rather it depends on a combination of elements from the particle formulations. More importantly, our toxicity assessment shows different cytotoxicity trend for all the prepared formulations tested on gastric adenocarcinoma (BGC-823) and neuroblastoma (SH-SY5Y) cell lines. We have further proposed that the cellular uptake of these nanocrystals plays an important role in determining the final faith of the toxicity impact of the formulation. The result here suggests that the toxicity of QDs is rather complex and it cannot be generalized under a few assumptions reported previously. We suggest that one have to evaluate the QD toxicity on a case to case basis and this indicates that standard procedures and comprehensive

  18. Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jing [Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853 (China); Hu, Rui [School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Liu, Jianwei [Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853 (China); Zhang, Butian; Wang, Yucheng [School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Liu, Xin [Lawrence Berkeley National Laboratory, Berkeley, CA (United States); Law, Wing-Cheung [Department of Industrial and System Engineering, The Hang Kong Polytechnic University, Hung Hom (Hong Kong); Liu, Liwei [School of Science, Changchun University of Science and Technology, Changchun 130022 (China); Ye, Ling, E-mail: lye_301@163.com [Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853 (China); Yong, Ken-Tye, E-mail: ktyong@ntu.edu.sg [School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore)

    2015-12-01

    The toxicity of quantum dots (QDs) has been extensively studied over the past decade. Some common factors that originate the QD toxicity include releasing of heavy metal ions from degraded QDs and the generation of reactive oxygen species on the QD surface. In addition to these factors, we should also carefully examine other potential QD toxicity causes that will play crucial roles in impacting the overall biological system. In this contribution, we have performed cytotoxicity assessment of four types of QD formulations in two different human cancer cell models. The four types of QD formulations, namely, mercaptopropionic acid modified CdSe/CdS/ZnS QDs (CdSe-MPA), PEGylated phospholipid encapsulated CdSe/CdS/ZnS QDs (CdSe-Phos), PEGylated phospholipid encapsulated InP/ZnS QDs (InP-Phos) and Pluronic F127 encapsulated CdTe/ZnS QDs (CdTe-F127), are representatives for the commonly used QD formulations in biomedical applications. Both the core materials and the surface modifications have been taken into consideration as the key factors for the cytotoxicity assessment. Through side-by-side comparison and careful evaluations, we have found that the toxicity of QDs does not solely depend on a single factor in initiating the toxicity in biological system but rather it depends on a combination of elements from the particle formulations. More importantly, our toxicity assessment shows different cytotoxicity trend for all the prepared formulations tested on gastric adenocarcinoma (BGC-823) and neuroblastoma (SH-SY5Y) cell lines. We have further proposed that the cellular uptake of these nanocrystals plays an important role in determining the final faith of the toxicity impact of the formulation. The result here suggests that the toxicity of QDs is rather complex and it cannot be generalized under a few assumptions reported previously. We suggest that one have to evaluate the QD toxicity on a case to case basis and this indicates that standard procedures and comprehensive

  19. Dopaminergic agonists for hepatic encephalopathy

    DEFF Research Database (Denmark)

    Als-Nielsen, B; Gluud, L L; Gluud, C

    2004-01-01

    Hepatic encephalopathy may be associated with an impairment of the dopaminergic neurotransmission. Dopaminergic agonists may therefore have a beneficial effect on patients with hepatic encephalopathy.......Hepatic encephalopathy may be associated with an impairment of the dopaminergic neurotransmission. Dopaminergic agonists may therefore have a beneficial effect on patients with hepatic encephalopathy....

  20. A Human Neural Crest Stem Cell-Derived Dopaminergic Neuronal Model Recapitulates Biochemical Abnormalities in GBA1 Mutation Carriers

    Directory of Open Access Journals (Sweden)

    Shi-Yu Yang

    2017-03-01

    Full Text Available Numerically the most important risk factor for the development of Parkinson's disease (PD is the presence of mutations in the glucocerebrosidase GBA1 gene. In vitro and in vivo studies show that GBA1 mutations reduce glucocerebrosidase (GCase activity and are associated with increased α-synuclein levels, reflecting similar changes seen in idiopathic PD brain. We have developed a neural crest stem cell-derived dopaminergic neuronal model that recapitulates biochemical abnormalities in GBA1 mutation-associated PD. Cells showed reduced GCase protein and activity, impaired macroautophagy, and increased α-synuclein levels. Advantages of this approach include easy access to stem cells, no requirement to reprogram, and retention of the intact host genome. Treatment with a GCase chaperone increased GCase protein levels and activity, rescued the autophagic defects, and decreased α-synuclein levels. These results provide the basis for further investigation of GCase chaperones or similar drugs to slow the progression of PD.

  1. Vitamin D regulates tyrosine hydroxylase expression: N-cadherin a possible mediator.

    Science.gov (United States)

    Cui, X; Pertile, R; Liu, P; Eyles, D W

    2015-09-24

    Vitamin D is a neuroactive steroid. Its genomic actions are mediated via the active form of vitamin D, 1,25(OH)2D3, binding to the vitamin D receptor (VDR). The VDR emerges in the rat mesencephalon at embryonic day 12, representing the peak period of dopaminergic cell birth. Our prior studies reveal that developmental vitamin D (DVD)-deficiency alters the ontogeny of dopaminergic neurons in the developing mesencephalon. There is also consistent evidence from others that 1,25(OH)2D3 promotes the survival of dopaminergic neurons in models of dopaminergic toxicity. In both developmental and toxicological studies it has been proposed that 1,25(OH)2D3 may modulate the differentiation and maturation of dopaminergic neurons; however, to date there is lack of direct evidence. The aim of the current study is to investigate this both in vitro using a human SH-SY5Y cell line transfected with rodent VDR and in vivo using a DVD-deficient model. Here we show that in VDR-expressing SH-SY5Y cells, 1,25(OH)2D3 significantly increased production of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. This effect was dose- and time-dependent, but was not due to an increase in TH-positive cell number, nor was it due to the production of trophic survival factors for dopamine neurons such as glial-derived neurotrophic factor (GDNF). In accordance with 1,25(OH)2D3's anti-proliferative actions in the brain, 1,25(OH)2D3 reduced the percentage of dividing cells from approximately 15-10%. Given the recently reported role of N-cadherin in the direct differentiation of dopaminergic neurons, we examined here whether it may be elevated by 1,25(OH)2D3. We confirmed this in vitro and more importantly, we showed DVD-deficiency decreases N-cadherin expression in the embryonic mesencephalon. In summary, in our in vitro model we have shown 1,25(OH)2D3 increases TH expression, decreases proliferation and elevates N-cadherin, a potential factor that mediates these processes

  2. Naringenin protects against 6-OHDA-induced neurotoxicity via activation of the Nrf2/ARE signaling pathway.

    Science.gov (United States)

    Lou, Haiyan; Jing, Xu; Wei, Xinbing; Shi, Huanying; Ren, Dongmei; Zhang, Xiumei

    2014-04-01

    There is increasing evidence that oxidative stress is critically involved in the pathogenesis of Parkinson's disease (PD), suggesting that pharmacological targeting of the antioxidant machinery may have therapeutic value. Naringenin, a natural flavonoid compound, has been reported to possess neuroprotective effect against PD related pathology; however the mechanisms underlying its beneficial effects are poorly defined. Thus, the purpose of the present study was to investigate the potential neuroprotective role of naringenin and to delineate its mechanism of action against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in models of PD both in vitro and in vivo. Naringenin treatment resulted in an increase in nuclear factor E2-related factor 2 (Nrf2) protein levels and subsequent activation of antioxidant response element (ARE) pathway genes in SH-SY5Y cells and in mice. Exposure of SH-SY5Y cells to naringenin provided protection against 6-OHDA-induced oxidative insults that was dependent on Nrf2, since treatment with Nrf2 siRNA failed to block against 6-OHDA neurotoxicity or induce Nrf2-dependent cytoprotective genes in SH-SY5Y cells. In mice, oral administration of naringenin resulted in significant protection against 6-OHDA-induced nigrostriatal dopaminergic neurodegeneration and oxidative damage. Our results indicate that activation of Nrf2/ARE signaling by naringenin is strongly associated with its neuroprotective effects against 6-OHDA neurotoxicity and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in PD. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Zebrafish GDNF and its co-receptor GFRα1 activate the human RET receptor and promote the survival of dopaminergic neurons in vitro.

    Directory of Open Access Journals (Sweden)

    Tuulia Saarenpää

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF is a ligand that activates, through co-receptor GDNF family receptor alpha-1 (GFRα1 and receptor tyrosine kinase "RET", several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRα1 and GDNF, and Drosophila melanogaster RET and two isoforms of co-receptor GDNF receptor-like. Our results report high-level insect cell expression of post-translationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRα1 and GDNF are comparably active as mammalian cell-produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFRα1-GDNF binding RET is 5.9 μM. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRα1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRα1 and GDNF cannot bind and activate non-mammalian RET and vice versa.

  4. Nrf2 signaling contributes to the neuroprotective effects of urate against 6-OHDA toxicity.

    Directory of Open Access Journals (Sweden)

    Ning Zhang

    Full Text Available BACKGROUND: Mounting evidence shows that urate may become a biomarker of Parkinson's disease (PD diagnosis and prognosis and a neuroprotectant candidate for PD therapy. However, the cellular and molecular mechanisms underlying its neuroprotective actions remain poorly understood. RESULTS: In this study, we showed that urate pretreatment protected dopaminergic cell line (SH-SY5Y and MES23.5 against 6-hydroxydopamine (6-OHDA- and hydrogen peroxide- induced cell damage. Urate was found to be accumulated into SH-SY5Y cells after 30 min treatment. Moreover, urate induced NF-E2-related factor 2 (Nrf2 accumulation by inhibiting its ubiquitinationa and degradation, and also promoted its nuclear translocation; however, it did not modulate Nrf2 mRNA level or Kelch-like ECH-associated protein 1 (Keap1 expression. In addition, urate markedly up-regulated the transcription and protein expression of γ-glutamate-cysteine ligase catalytic subunit (γ-GCLC and heme oxygenase-1 (HO-1, both of which are controlled by Nrf2 activity. Furthermore, Nrf2 knockdown by siRNA abolished the intracellular glutathione augmentation and the protection exerted by urate pretreatment. CONCLUSION: Our findings demonstrated that urate treatment may result in Nrf2-targeted anti-oxidant genes transcription and expression by reducing Nrf2 ubiquitination and degradation and promoting its nuclear translocation, and thus offer neuroprotection on dopaminergic cells against oxidative stresses.

  5. Sulfuretin Attenuates MPP+-Induced Neurotoxicity through Akt/GSK3β and ERK Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Ramesh Pariyar

    2017-12-01

    Full Text Available Parkinson’s disease (PD is the second most common neurodegenerative disease. It is caused by the death of dopaminergic neurons in the substantia nigra pars compacta. Oxidative stress and mitochondrial dysfunction contribute to the loss of dopaminergic neurons in PD. Sulfuretin is a potent antioxidant that is reported to be beneficial in the treatment of neurodegenerative diseases. In this study, we examined the protective effect of sulfuretin against 1-methyl-4-phenyl pyridinium (MPP+-induced cell model of PD in SH-SY5Y cells and the underlying molecular mechanisms. Sulfuretin significantly decreased MPP+-induced apoptotic cell death, accompanied by a reduction in caspase 3 activity and polyADP-ribose polymerase (PARP cleavage. Furthermore, it attenuated MPP+-induced production of intracellular reactive oxygen species (ROS and disruption of mitochondrial membrane potential (MMP. Consistently, sulfuretin decreased p53 expression and the Bax/Bcl-2 ratio. Moreover, sulfuretin significantly increased the phosphorylation of Akt, GSK3β, and ERK. Pharmacological inhibitors of PI3K/Akt and ERK abolished the cytoprotective effects of sulfuretin against MPP+. An inhibitor of GSK3β mimicked sulfuretin-induced protection against MPP+. Taken together, these results suggest that sulfuretin significantly attenuates MPP+-induced neurotoxicity through Akt/GSK3β and ERK signaling pathways in SH-SY5Y cells. Our findings suggest that sulfuretin might be one of the potential candidates for the treatment of PD.

  6. Validation of the reference tissue model for estimation of dopaminergic D{sub 2}-like receptor binding with [{sup 18}F](N-methyl)benperidol in humans

    Energy Technology Data Exchange (ETDEWEB)

    Antenor-Dorsey, Jo Ann V. [Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO (United States); Markham, Joanne; Moerlein, Stephen M. [Department of Radiology, Washington University School of Medicine, St. Louis, MO (United States); Videen, Tom O. [Department of Radiology, Washington University School of Medicine, St. Louis, MO (United States); Department of Neurology, Washington University School of Medicine, St. Louis, MO (United States); Perlmutter, Joel S. [Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO (United States); Department of Radiology, Washington University School of Medicine, St. Louis, MO (United States); Department of Neurology, Washington University School of Medicine, St. Louis, MO (United States); Program in Physical Therapy, Washington University School of Medicine, St. Louis, MO (United States)], E-mail: joel@npg.wustl.edu

    2008-04-15

    Positron emission tomography measurements of dopaminergic D{sub 2}-like receptors may provide important insights into disorders such as Parkinson's disease, schizophrenia, dystonia and Tourette's syndrome. The positron emission tomography (PET) radioligand [{sup 18}F](N-methyl)benperidol ([{sup 18}F]NMB) has high affinity and selectivity for D{sub 2}-like receptors and is not displaced by endogenous dopamine. The goal of this study is to evaluate the use of a graphical method utilizing a reference tissue region for [{sup 18}F]-NMB PET analysis by comparisons to an explicit three-compartment tracer kinetic model and graphical method that use arterial blood measurements. We estimated binding potential (BP) in the caudate and putamen using all three methods in 16 humans and found that the three-compartment tracer kinetic method provided the highest BP estimates while the graphical method using a reference region yielded the lowest estimates (P<.0001 by repeated-measures ANOVA). However, the three methods yielded highly correlated BP estimates for the two regions of interest. We conclude that the graphical method using a reference region still provides a useful estimate of BP comparable to methods using arterial blood sampling, especially since the reference region method is less invasive and computationally more straightforward, thereby simplifying these measurements.

  7. Validation of the reference tissue model for estimation of dopaminergic D2-like receptor binding with [18F](N-methyl)benperidol in humans

    International Nuclear Information System (INIS)

    Antenor-Dorsey, Jo Ann V.; Markham, Joanne; Moerlein, Stephen M.; Videen, Tom O.; Perlmutter, Joel S.

    2008-01-01

    Positron emission tomography measurements of dopaminergic D 2 -like receptors may provide important insights into disorders such as Parkinson's disease, schizophrenia, dystonia and Tourette's syndrome. The positron emission tomography (PET) radioligand [ 18 F](N-methyl)benperidol ([ 18 F]NMB) has high affinity and selectivity for D 2 -like receptors and is not displaced by endogenous dopamine. The goal of this study is to evaluate the use of a graphical method utilizing a reference tissue region for [ 18 F]-NMB PET analysis by comparisons to an explicit three-compartment tracer kinetic model and graphical method that use arterial blood measurements. We estimated binding potential (BP) in the caudate and putamen using all three methods in 16 humans and found that the three-compartment tracer kinetic method provided the highest BP estimates while the graphical method using a reference region yielded the lowest estimates (P<.0001 by repeated-measures ANOVA). However, the three methods yielded highly correlated BP estimates for the two regions of interest. We conclude that the graphical method using a reference region still provides a useful estimate of BP comparable to methods using arterial blood sampling, especially since the reference region method is less invasive and computationally more straightforward, thereby simplifying these measurements

  8. Brain serotonergic and dopaminergic modulators, perceptual responses and endurance exercise performance following caffeine co-ingested with a high fat meal in trained humans

    Directory of Open Access Journals (Sweden)

    Kilduff Liam P

    2010-05-01

    Full Text Available Abstract Background The present study examined putative modulators and indices of brain serotonergic and dopaminergic function, perceptual responses, and endurance exercise performance following caffeine co-ingested with a high fat meal. Methods Trained humans (n = 10 performed three constant-load cycling tests at 73% of maximal oxygen uptake (VO2max until exhaustion at 10°C remove space throughout. Prior to the first test, subjects consumed a 90% carbohydrate meal (Control trial and for the remaining two tests, a 90% fat meal with (FC trial and without (F trial caffeine. Results Time to exhaustion was not different between the F and FC trials (P > 0.05; [Control trial: 116(88-145 min; F trial: 122(96-144 min; FC trial: 127(107-176 min]. However, leg muscular discomfort during exercise was significantly lower on the FC relative to F trial (P P > 0.05 with the exception of plasma free-Trp:LNAA ratio which was higher at 90 min and at exhaustion during the FC trial (P Conclusions Neither brain 5-HT nor DA systems would appear to be implicated in the fatigue process when exercise is performed without significant thermoregulatory stress, thus indicating fatigue development during exercise in relatively cold temperatures to occur predominantly due to glycogen depletion.

  9. [F-18]fluoro-meta-L-tyrosine is a better PET tracer than [F-18]fluoro-L-dopa for the delineation of dopaminergic structures in the human brain

    International Nuclear Information System (INIS)

    Firnau, G.; Chirakal, R.; Nahmias, C.; Garnett, E.S.

    1990-01-01

    Fluorine-18 labelled fluoro-m-L-tyrosine (FmLtyr) and fluoro-L-Dopa (F-Dopa) have been synthesized, and the utility of FmLtyr for PET investigations of dopaminergic brain regions has been compared to that of F-dopa. Experimental results from both monkey and human studies indicate that FmLtyr gives better delineation of striatum, and is a better PET tracer than F-dopa

  10. Cellular response of human neuroblastoma cells to α-synuclein fibrils, the main constituent of Lewy bodies.

    Science.gov (United States)

    Pieri, Laura; Chafey, Philippe; Le Gall, Morgane; Clary, Guilhem; Melki, Ronald; Redeker, Virginie

    2016-01-01

    α-Synuclein (α-Syn) fibrils are the main constituent of Lewy bodies and a neuropathological hallmark of Parkinson's disease (PD). The propagation of α-Syn assemblies from cell to cell suggests that they are involved in PD progression. We previously showed that α-Syn fibrils are toxic because of their ability to bind and permeabilize cell membranes. Here, we document the cellular response in terms of proteome changes of SH-SY5Y cells exposed to exogenous α-Syn fibrils. We compare the proteomes of cells of neuronal origin exposed or not either to oligomeric or fibrillar α-Syn using two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Only α-Syn fibrils induce significant changes in the proteome of SH-SY5Y cells. In addition to proteins associated to apoptosis and toxicity, or proteins previously linked to neurodegenerative diseases, we report an overexpression of proteins involved in intracellular vesicle trafficking. We also report a remarkable increase in fibrillar α-Syn heterogeneity, mainly due to C-terminal truncations. Our results show that cells of neuronal origin adapt their proteome to exogenous α-Syn fibrils and actively modify those assemblies. Cells of neuronal origin adapt their proteome to exogenous toxic α-Syn fibrils and actively modify those assemblies. Our results bring insights into the cellular response and clearance events the cells implement to face the propagation of α-Syn assemblies associated to pathology.

  11. Curcumin attenuates paraquat-induced cell death in human neuroblastoma cells through modulating oxidative stress and autophagy.

    Science.gov (United States)

    Jaroonwitchawan, Thiranut; Chaicharoenaudomrung, Nipha; Namkaew, Jirapat; Noisa, Parinya

    2017-01-01

    Paraquat is a neurotoxic agent, and oxidative stress plays an important role in neuronal cell death after paraquat exposure. In this study, we assessed the neuroprotective effect of curcumin against paraquat and explored the underlying mechanisms of curcumin in vitro. Curcumin treatment prevented paraquat-induced reactive oxygen species (ROS) and apoptotic cell death. Curcumin also exerted a neuroprotective effect by increasing the expression of anti-apoptotic and antioxidant genes. The pretreatment of curcumin significantly decreased gene expression and protein production of amyloid precursor protein. The activation of autophagy process was found defective in paraquat-induced cells, indicated by the accumulation and reduction of LC3I/II. Noteworthy, curcumin restored LC3I/II expression after the pretreatment. Collectively, curcumin demonstrated as a prominent suppressor of ROS, and could reverse autophagy induction in SH-SY5Y cells. The consequences of this were the reduction of APP production and prevention of SH-SY5Y cells from apoptosis. Altogether, curcumin potentially serves as a therapeutic agent of neurodegenerative diseases, associated with ROS overproduction and autophagy dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Biodistribution and human dosimetry estimation of fluoro-L-DOPA as PET imaging agent of dopaminergic nerve transmitter systems

    International Nuclear Information System (INIS)

    Tang Ganghua; Wang Mingfang; Luo Lei; Gan Manquan; Tang Xiaolan; Zhang Lan; Wang Yongxian

    2002-01-01

    Objective: To investigate the biodistribution and human dosimetry estimation of 6-[ 18 F] Fluoro-L-DOPA (FDOPA). Methods: Biodistribution of FDOPA in normal rats and brain of hemi-Parkinsonism rats were determined. Human dosimetry estimation was performed by MIRD method based on the rats biodistribution data. Results: Biodistributions in normal rats showed high uptake in kidney, blood, striatum and hippocampi, fast clearance of radioactivity from kidney and blood, longer retain time in striatum and hippocampi, and higher striatum to cerebellum and striatum to cortex ratio. FDOPA uptake, striatum to cerebellum and striatum to cortex ratio in the lesioned side of hemi-Parkinsonism rats (P 2 to 2.3 x 10 -2 mGy/MBq and the effective dose in humans was estimated to be 2.05 x 10 -2 mSv/MBq after injection of FDOPA based on rats biodistribution data, which were consistent with those reported by literature on the whole. Conclusion: Human radiation dosimetry of FDOPA and other PET tracers can be estimated based on animals biodistribution data. The synthetic FDOPA is safe and efficient and can be used in animals, human and PD patients PET studies

  13. Evaluating the relationship between amyloid-β and α-synuclein phosphorylated at Ser129 in dementia with Lewy bodies and Parkinson’s disease

    OpenAIRE

    Swirski, Marta; Miners, J Scott; de Silva, Rohan; Lashley, Tammaryn; Ling, Helen; Holton, Janice; Revesz, Tamas; Love, Seth

    2014-01-01

    Introduction Lewy body and Alzheimer-type pathologies often co-exist. Several studies suggest a synergistic relationship between amyloid-β (Aβ) and α-synuclein (α-syn) accumulation. We have explored the relationship between Aβ accumulation and the phosphorylation of α-syn at serine-129 (pSer129 α-syn), in post-mortem human brain tissue and in SH-SY5Y neuroblastoma cells transfected to overexpress human α-syn. Methods We measured levels of Aβ40, Aβ42, α-syn and pSer129 α-syn by sandwich enzyme...

  14. Synergistic efficacy of a novel combination therapy controls growth of Bcl-x(L) bountiful neuroblastoma cells by increasing differentiation and apoptosis.

    Science.gov (United States)

    Mohan, Nishant; Banik, Naren L; Ray, Swapan K

    2011-11-01

    Neuroblastoma is the most prevalent extracranial solid tumor mainly in pediatric patients. We explored the efficacy of the combination of 2[(3-[2,3-dichlorophenoxy]propyl)amino]ethanol (2,3-DCPE, a small molecule inhibitor of the anti-apoptotic protein Bcl-x(L)) and N-(4-hydroxyphenyl) retinamide (4-HPR, a synthetic retinoid) in inducing differentiation and apoptosis in human malignant neuroblastoma cells. Immunofluorescence confocal microscopy and flow cytometry showed that the highest level of Bcl-x(L) expression occurred in SK-N-DZ cells followed by SH-SY5Y and IMR-32 cells. Combination of 20 μM 2,3-DCPE and 1 μM 4-HPR acted synergistically in decreasing viability of SK-N-DZ and SH-SY5Y cells. In situ methylene blue staining and protein gel blotting showed the efficacy of this combination of drugs in inducing neuronal differentiation morphologically and also biochemically with upregulation of the neuronal markers such as neurofilament protein (NFP) and neuron specific enolase (NSE) and downregulation of the differentiation inhibiting molecules such as N-Myc and Notch-1 in SK-N-DZ and SH-SY5Y cells. Annexin V-FITC/PI staining showed the synergistic action of this combination therapy in increasing apoptosis in both cell lines. Protein gel blotting manifested that combination therapy increased apoptosis with downregulation of the anti-apoptotic proteins Bcl-x(L), Bcl-2 and Mcl-1 and upregulation of the pro-apoptotic proteins Bax, p53, Puma (p53 upregulated modulator of apoptosis), and Noxa, ultimately causing activation of caspase-3. In conclusion, our results appeared highly encouraging in advocating the use of 2,3-DCPE and 4-HPR as a novel combination therapy for increasing both differentiation and apoptosis in human malignant neuroblastoma cells having Bcl-x(L) overexpression.

  15. Vorinostat increases expression of functional norepinephrine transporter in neuroblastoma in vitro and in vivo model systems

    Science.gov (United States)

    More, Swati S.; Itsara, Melissa; Yang, Xiaodong; Geier, Ethan G.; Tadano, Michelle K.; Seo, Youngho; VanBrocklin, Henry F.; Weiss, William A.; Mueller, Sabine; Haas-Kogan, Daphne A.; DuBois, Steven G.; Matthay, Katherine K.; Giacomini, Kathleen M.

    2011-01-01

    Purpose Histone deacetylase (HDAC) inhibition causes transcriptional activation or repression of several genes that in turn can influence the biodistribution of other chemotherapeutic agents. Here, we hypothesize that the combination of vorinostat, a HDAC inhibitor, with 131I-metaiodobenzylguanidine (MIBG) would lead to preferential accumulation of the latter in neuroblastoma (NB) tumors via increased expression of the human norepinephrine transporter (NET). Experimental Design In vitro and in vivo experiments examined the effect of vorinostat on the expression of NET, an uptake transporter for 131I-MIBG. Human NB cell lines (Kelly and SH-SY-5Y) and NB1691luc mouse xenografts were employed. The upregulated NET protein was characterized for its effect on 123I-MIBG biodistribution. Results Preincubation of NB cell lines, Kelly and SH-SY-5Y, with vorinostat caused dose-dependent increases in NET mRNA and protein levels. Accompanying this was a corresponding dose-dependent increase in MIBG uptake in NB cell lines. Four-fold and 2.5 fold increases were observed in Kelly and SH-SY-5Y cells, respectively, pre-treated with vorinostat in comparison to untreated cells. Similarly, NB xenografts, created by intravenous tail vein injection of NB1691-luc, and harvested from nude mice livers treated with vorinostat (150 mg/kg i.p.) showed substantial increases in NET protein expression. Maximal effect of vorinostat pretreatment in NB xenografts on 123I-MIBG biodistribution was observed in tumors that exhibited enhanced uptake in vorinostat treated (0.062 ± 0.011 μCi/(mg tissue-dose injected)) versus untreated mice (0.022 ± 0.003 μCi/(mg tissue-dose injected); p vorinostat treatment can enhance NB therapy with 131I-MIBG. PMID:21421857

  16. The thiopurine nucleoside analogue 6-methylmercaptopurine riboside (6MMPr) effectively blocks Zika virus replication.

    Science.gov (United States)

    de Carvalho, Otavio Valério; Félix, Daniele Mendes; de Mendonça, Leila Rodrigues; de Araújo, Catarina Maria Cataldi Sabino; de Oliveira Franca, Rafael Freitas; Cordeiro, Marli Tenório; Silva Júnior, Abelardo; Pena, Lindomar José

    2017-12-01

    Since the emergence of Zika virus (ZIKV) in Brazil in 2015, 48 countries and territories in the Americas have confirmed autochthonous cases of disease caused by the virus. ZIKV-associated neurological manifestations and congenital defects make the development of safe and effective antivirals against ZIKV of utmost importance. Here we evaluated the antiviral activity of 6-methylmercaptopurine riboside (6MMPr), a thiopurine nucleoside analogue derived from the prodrug azathioprine, against the epidemic ZIKV strain circulating in Brazil. In all of the assays, an epithelial (Vero) and a human neuronal (SH-SY5Y) cell line were used to evaluate the cytotoxicity and effective concentrations of 6MMPr against ZIKV. Levels of ZIKV-RNA, viral infectious titre and the percentage of infected cells in the presence or absence of 6MMPr were used to determine antiviral efficacy. 6MMPr decreased ZIKV production by >99% in both cell lines in a dose- and time-dependent manner. Interestingly, 6MMPr was 1.6 times less toxic to SH-SY5Y cells compared with Vero cells, presenting a 50% cytotoxic concentrations (CC 50 ) of 460.3 µM and 291 µM, respectively. The selectivity index of 6MMPr for Vero and SH-SY5Y cells was 11.9 and 22.7, respectively, highlighting the safety profile of the drug to neuronal cells. Taken together, these results identify, for the first time, the thiopurine nucleoside analogue 6MMPr as a promising antiviral candidate against ZIKV that warrants further in vivo evaluation. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  17. Moringa isothiocyanate complexed with α-cyclodextrin: a new perspective in neuroblastoma treatment.

    Science.gov (United States)

    Giacoppo, Sabrina; Iori, Renato; Rollin, Patrick; Bramanti, Placido; Mazzon, Emanuela

    2017-07-14

    Several lines of evidence suggest the consume of natural products for cancer prevention or treatment. In particular, isothiocyanates (ITCs) exerting anti-cancer properties, have received great interest as potential chemotherapeutic agents. This study was designed to assess the anti-proliferative activities of a new preparation of Moringa oleifera-derived 4-(α-L-rhamnopyranosyloxy)benzyl ITC (moringin) complexed with alpha-cyclodextrin (moringin + α-CD; MAC) on SH-SY5Y human neuroblastoma cells. This new formulation arises in the attempt to overcome the poor solubility and stability of moringin alone in aqueous media. SH-SY5Y cells were cultured and exposed to increasing concentrations of MAC (1.0, 2.5 and 5.0 μg). Cell proliferation was examined by MTT and cell count assays. The cytotoxic activity of the MAC complex was assessed by lactate dehydrogenase (LDH) assay and trypan blue exclusion test. In addition, western blotting analyses for the main apoptosis-related proteins were performed. Treatment of SH-SY5Y cells with the MAC complex reduced cell growth in concentration dependent manner. Specifically, MAC exhibited a potent action in inhibiting the PI3K/Akt/mTOR pathway, whose aberrant activation was found in many types of cancer. MAC was also found to induce the nuclear factor-κB (NF-κB) p65 activation by phosphorylation and its translocation into the nucleus. Moreover, treatment with MAC was able to down-regulate MAPK pathway (results focused on JNK and p38 expression). Finally, MAC was found to trigger apoptotic death pathway (based on expression levels of cleaved-caspase 3, Bax/Bcl-2 balance, p53 and p21). These findings suggest that use of MAC complex may open novel perspectives to improve the poor prognosis of patients with neuroblastoma.

  18. Polyelectrolyte-coated nanocapsules containing undecylenic acid: Synthesis, biocompatibility and neuroprotective properties.

    Science.gov (United States)

    Piotrowski, Marek; Jantas, Danuta; Szczepanowicz, Krzysztof; Łukasiewicz, Sylwia; Lasoń, Władysław; Warszyński, Piotr

    2015-11-01

    The main objectives of the present study were to investigate the biocompatibility of polyelectrolyte-coated nanocapsules and to evaluate the neuroprotective action of the nanoencapsulated water-insoluble neuroprotective drug-undecylenic acid (UDA), in vitro. Core-shell nanocapsules were synthesized using nanoemulsification and the layer-by-layer (LbL) technique (by saturation method). The average size of synthesized nanocapsules was around 80 nm and the concentration was 2.5 × 10(10) particles/ml. Their zeta potential values ranged from less than -30 mV for the ones with external polyanion layers through -4 mV for the PEG-ylated layers to more than 30 mV for the polycation layers. Biocompatibility of synthesized nanocarriers was evaluated in the SH-SY5Y human neuroblastoma cell line using cell viability/toxicity assays (MTT reduction, LDH release). The results obtained showed that synthesized nanocapsules coated with PLL and PGA (also PEG-ylated) were non-toxic to SH-SY5Y cells, therefore, they were used as nanocarriers for UDA. Moreover, studies with ROD/FITC-labeled polyelectrolytes demonstrated approximately 20% cellular uptake of synthetized nanocapsules. Further studies showed that nanoencapsulated form of UDA was biocompatible and protected SH-SY5Y cells against the staurosporine-induced damage in lower concentrations than those of the same drug added directly to the culture medium. These data suggest that designed nanocapsules might serve as novel, promising delivery systems for neuroprotective agents. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Perforin Promotes Amyloid Beta Internalisation in Neurons.

    Science.gov (United States)

    Lana, Erica; Khanbolouki, Mahbod; Degavre, Charline; Samuelsson, Eva-Britt; Åkesson, Elisabet; Winblad, Bengt; Alici, Evren; Lithner, Christina Unger; Behbahani, Homira

    2017-03-01

    Studies on the mechanisms of neuronal amyloid-β (Aβ) internalisation are crucial for understanding the neuropathological progression of Alzheimer's disease (AD). We here investigated how extracellular Aβ peptides are internalised and focused on three different pathways: (i) via endocytic mechanisms, (ii) via the receptor for advanced glycation end products (RAGE) and (iii) via the pore-forming protein perforin. Both Aβ 40 and Aβ 42 were internalised in retinoic acid differentiated neuroblastoma (RA-SH-SY5Y) cells. A higher concentration was required for Aβ 40 (250 nM) compared with Aβ 42 (100 nM). The internalised Aβ 40 showed a dot-like pattern of distribution whereas Aβ 42 accumulated in larger and distinct formations. By confocal microscopy, we showed that Aβ 40 and Aβ 42 co-localised with mitochondria, endoplasmic reticulum (ER) and lysosomes. Aβ treatment of human primary cortical neurons (hPCN) confirmed our findings in RA-SH-SY5Y cells, but hPCN were less sensitive to Aβ; therefore, a 20 (Aβ 40 ) and 50 (Aβ 42 ) times higher concentration was needed for inducing uptake. The blocking of endocytosis completely inhibited the internalisation of Aβ peptides in RA-SH-SY5Y cells and hPCN, indicating that this is a major pathway by which Aβ enters the cells. In addition, the internalisation of Aβ 42 , but not Aβ 40 , was reduced by 55 % by blocking RAGE. Finally, for the first time we showed that pore formation in cell membranes by perforin led to Aβ internalisation in hPCN. Understanding how Aβ is internalised sheds light on the pathological role of Aβ and provides further ideas of inhibitory strategies for preventing Aβ internalisation and the spreading of neurodegeneration in AD.

  20. Dehydroepiandrosterone protects male and female hippocampal neurons and neuroblastoma cells from glucose deprivation.

    Science.gov (United States)

    Vieira-Marques, Claudia; Arbo, Bruno Dutra; Ruiz-Palmero, Isabel; Ortiz-Rodriguez, Ana; Ghorbanpoor, Samar; Kucharski, Luiz Carlos; Arevalo, Maria A; Garcia-Segura, Luis Miguel; Ribeiro, Maria Flávia M

    2016-08-01

    Dehydroepiandrosterone (DHEA) modulates neurogenesis, neuronal function, neuronal survival and metabolism, enhancing mitochondrial oxidative capacity. Glucose deprivation and hypometabolism have been implicated in the mechanisms that mediate neuronal damage in neurological disorders, and some studies have shown that these mechanisms are sexually dimorphic. It was also demonstrated that DHEA is able to attenuate the hypometabolism that is related to some neurodegenerative diseases, eliciting neuroprotective effects in different experimental models of neurodegeneration. The aim of this study was to evaluate the effect of DHEA on the viability of male and female hippocampal neurons and SH-SY5Y neuroblastoma cells exposed to glucose deprivation. It was observed that after 12h of pre-treatment, DHEA was able to protect SH-SY5Y cells from glucose deprivation for 6h (DHEA 10(-12), 10(-8) and 10(-6)M) and 8h (DHEA 10(-8)M). In contrast, DHEA was not neuroprotective against glucose deprivation for 12 or 24h. DHEA (10(-8)M) also protected SH-SY5Y cells when added together or even 1h after the beginning of glucose deprivation (6h). Furthermore, DHEA (10(-8)M) also protected primary neurons from both sexes against glucose deprivation. In summary, our findings indicate that DHEA is neuroprotective against glucose deprivation in human neuroblastoma cells and in male and female mouse hippocampal neurons. These results suggest that DHEA could be a promising candidate to be used in clinical studies aiming to reduce neuronal damage in people from both sexes. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Metallothionein-III protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a PI3K and ERK/Nrf2-dependent manner

    International Nuclear Information System (INIS)

    Hwang, Yong Pil; Kim, Hyung Gyun; Han, Eun Hee; Jeong, Hye Gwang

    2008-01-01

    The zinc-binding protein metallothionein-III (MT-III) is associated with resistance to neuronal injury. However, the underlying mechanism for its effects is unclear. In this study, we demonstrate that MT-III prevents the accumulation of reactive oxygen species (ROS) in dopaminergic SH-SY5Y cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves phosphatidylinositol 3-kinase (PI3K) and ERK kinase/NF-E2-related factor 2 (Nrf2) dependent induction of the stress response protein heme oxygenase-1 (HO-1). Pretreatment of SH-SY5Y cells with MT-III significantly reduced 6-OHDA-induced generation of ROS, caspase-3 activation, and subsequent cell death. Also, MT-III up-regulates HO-1 expression and this expression confers neuroprotection against oxidative injury induced by 6-OHDA. Moreover, MT-III induces Nrf2 nuclear translocation, which is upstream of MT-III-induced HO-1 expression, and PI3K and ERK1/2 activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. Taken together, these results suggest that the PI3K and ERK/Nrf2 signaling pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1

  2. Identification of the endogenous key substrates of the human organic cation transporter OCT2 and their implication in function of dopaminergic neurons.

    Directory of Open Access Journals (Sweden)

    Dirk Taubert

    Full Text Available BACKGROUND: The etiology of neurodegenerative disorders, such as the accelerated loss of dopaminergic neurons in Parkinson's disease, is unclear. Current hypotheses suggest an abnormal function of the neuronal sodium-dependent dopamine transporter DAT to contribute to cell death in the dopaminergic system, but it has not been investigated whether sodium-independent amine transporters are implicated in the pathogenesis of Parkinson's disease. METHODOLOGY/PRINCIPAL FINDINGS: By the use of a novel tandem-mass spectrometry-based substrate search technique, we have shown that the dopaminergic neuromodulators histidyl-proline diketopiperazine (cyclo(his-pro and salsolinol were the endogenous key substrates of the sodium-independent organic cation transporter OCT2. Quantitative real-time mRNA expression analysis revealed that OCT2 in contrast to its related transporters was preferentially expressed in the dopaminergic regions of the substantia nigra where it colocalized with DAT and tyrosine hydroxylase. By assessing cell viability with the MTT reduction assay, we found that salsolinol exhibited a selective toxicity toward OCT2-expressing cells that was prevented by cyclo(his-pro. A frequent genetic variant of OCT2 with the amino acid substitution R400C reduced the transport efficiency for the cytoprotective cyclo(his-pro and thereby increased the susceptibility to salsolinol-induced cell death. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that the OCT2-regulated interplay between cyclo(his-pro and salsolinol is crucial for nigral cell integrity and that a shift in transport efficiency may impact the risk of Parkinson's disease.

  3. Towards a Non-Human Primate Model of Alpha-Synucleinopathy for Development of Therapeutics for Parkinson's Disease: Optimization of AAV1/2 Delivery Parameters to Drive Sustained Expression of Alpha Synuclein and Dopaminergic Degeneration in Macaque.

    Directory of Open Access Journals (Sweden)

    James B Koprich

    Full Text Available Recent failures in clinical trials for disease modification in Parkinson's disease have highlighted the need for a non-human primate model of the synucleinopathy underpinning dopaminergic neuron degeneration. The present study was defined to begin the development of such a model in cynomolgus macaque. We have validated surgical and vector parameters to define a means to provide a robust over-expression of alpha-synuclein which is associated with Lewy-like pathology and robust degeneration of the nigrostriatal pathway. Thus, an AAV1/2 vector incorporating strong transcription and transduction regulatory elements was used to deliver the gene for the human A53T mutation of alpha-synuclein. When injected into 4 sites within each substantia nigra (7 μl per site, 1.7 x 1012 gp/ml, this vector provided expression lasting at least 4 months, and a 50% loss of nigral dopaminergic neurons and a 60% reduction in striatal dopamine. Further studies will be required to develop this methodology into a validated model of value as a drug development platform.

  4. Representation of spontaneous movement by dopaminergic neurons is cell-type selective and disrupted in parkinsonism

    DEFF Research Database (Denmark)

    Dodson, Paul D.; Dreyer, Jakob K.; Jennings, Katie Ann

    2016-01-01

    receptor expressed by striatal neurons. Importantly, in aged mice harboring a genetic burden relevant for human Parkinson's disease, the precise movement-related firing of SNc dopaminergic neurons and the resultant striatal dopamine signaling were lost. These data show that distinct dopaminergic cell types......Midbrain dopaminergic neurons are essential for appropriate voluntary movement, as epitomized by the cardinal motor impairments arising in Parkinson's disease. Understanding the basis of such motor control requires understanding how the firing of different types of dopaminergic neuron relates...... of these dopaminergic neurons can manifest as rapid and robust fluctuations in striatal dopamine concentration and receptor activity. The exact nature of the movement-related signaling in the striatum depended on the type of dopaminergic neuron providing inputs, the striatal region innervated, and the type of dopamine...

  5. Anti-Proliferative Activity of Meroditerpenoids Isolated from the Brown Alga Stypopodium flabelliforme against Several Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Patricia Valentao

    2011-05-01

    Full Text Available The sea constitutes one of the most promising sources of novel compounds with potential application in human therapeutics. In particular, algae have proved to be an interesting source of new bioactive compounds. In this work, six meroditerpenoids (epitaondiol, epitaondiol diacetate, epitaondiol monoacetate, stypotriol triacetate, 14-ketostypodiol diacetate and stypodiol isolated from the brown alga Stypopodium flabelliforme were tested for their cell proliferation inhibitory activity in five cell lines. Cell lines tested included human colon adenocarcinoma (Caco-2, human neuroblastoma (SH-SY5Y, rat basophilic leukemia (RBL-2H3, murine macrophages (RAW.267 and Chinese hamster fibroblasts (V79. Antimicrobial activity of the compounds was also evaluated against Staphylococcus aureus, Salmonella typhimurium, Proteus mirabilis, Bacillus cereus, Enterococcus faecalis and Micrococcus luteus. Overall, the compounds showed good activity against all cell lines, with SH-SY5Y and RAW.267 being the most susceptible. Antimicrobial capacity was observed for epitaondiol monoacetate, stypotriol triacetate and stypodiol, with the first being the most active. The results suggest that these molecules deserve further studies in order to evaluate their potential as therapeutic agents.

  6. Nitro-Oxidative Stress after Neuronal Ischemia Induces Protein Nitrotyrosination and Cell Death

    Directory of Open Access Journals (Sweden)

    Marta Tajes

    2013-01-01

    Full Text Available Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y, a murine glial (BV2, a human endothelial cell line (HUVEC, and in primary cultures of human cerebral myocytes (HC-VSMCs after experimental ischemia in vitro. Neuronal, endothelial, and inducible NO synthase (NOS expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells with H2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia.

  7. Bace1 activity impairs neuronal glucose metabolism: rescue by beta-hydroxybutyrate and lipoic acid

    Directory of Open Access Journals (Sweden)

    John A Findlay

    2015-10-01

    Full Text Available Glucose hypometabolism and impaired mitochondrial function in neurons have been suggested to play early and perhaps causative roles in Alzheimer’s disease (AD pathogenesis. Activity of the aspartic acid protease, beta-site amyloid precursor protein (APP cleaving enzyme 1 (BACE1, responsible for beta amyloid peptide generation, has recently been demonstrated to modify glucose metabolism. We therefore examined, using a human neuroblastoma (SH-SY5Y cell line, whether increased BACE1 activity is responsible for a reduction in cellular glucose metabolism. Overexpression of active BACE1, but not a protease-dead mutant BACE1, protein in SH-SY5Y cells reduced glucose oxidation and the basal oxygen consumption rate, which was associated with a compensatory increase in glycolysis. Increased BACE1 activity had no effect on the mitochondrial electron transfer process but was found to diminish substrate delivery to the mitochondria by inhibition of key mitochondrial decarboxylation reaction enzymes. This BACE1 activity-dependent deficit in glucose oxidation was alleviated by the presence of beta hydroxybutyrate or α-lipoic acid. Consequently our data indicate that raised cellular BACE1 activity drives reduced glucose oxidation in a human neuronal cell line through impairments in the activity of specific tricarboxylic acid cycle enzymes. Because this bioenergetic deficit is recoverable by neutraceutical compounds we suggest that such agents, perhaps in conjunction with BACE1 inhibitors, may be an effective therapeutic strategy in the early-stage management or treatment of AD.

  8. PC-3 prostate carcinoma cells release signal substances that influence the migratory activity of cells in the tumor's microenvironment

    Directory of Open Access Journals (Sweden)

    Zänker Kurt S

    2010-07-01

    Full Text Available Abstract Background Tumor cells interact with the cells of the microenvironment not only by cell-cell-contacts but also by the release of signal substances. These substances are known to induce tumor vascularization, especially under hypoxic conditions, but are also supposed to provoke other processes such as tumor innervation and inflammatory conditions. Inflammation is mediated by two organ systems, the neuroendocrine system and the immune system. Therefore, we investigated the influence of substances released by PC-3 human prostate carcinoma cells on SH-SY5Y neuroblastoma cells as well as neutrophil granulocytes and cytotoxic T lymphocytes, especially with regard to their migratory activity. Results PC-3 cells express several cytokines and growth factors including vascular endothelial growth factors, fibroblast growth factors, interleukins and neurotrophic factors. SH-SY5Y cells are impaired in their migratory activity by PC-3 cell culture supernatant, but orientate chemotactically towards the source. Neutrophil granulocytes increase their locomotory activity only in response to cell culture supernantant of hypoxic but not of normoxic PC-3 cells. In contrast, cytotoxic T lymphocytes do not change their migratory activity in response to either culture supernatant, but increase their cytotoxicity, whereas supernatant of normoxic PC-3 cells leads to a stronger increase than that of hypoxic PC-3 cells. Conclusions PC-3 cells release several signal substances that influence the behavior of the cells in the tumor's microenvironment, whereas no clear pattern towards proinflammatory or immunosuppressive conditions can be seen.

  9. Altered sensitivity to ellagic acid in neuroblastoma cells undergoing differentiation with 12-O-tetradecanoylphorbol-13-acetate and all-trans retinoic acid.

    Science.gov (United States)

    Alfredsson, Christina Fjæraa; Rendel, Filip; Liang, Qui-Li; Sundström, Birgitta E; Nånberg, Eewa

    2015-12-01

    Ellagic acid has previously been reported to induce reduced proliferation and activation of apoptosis in several tumor cell lines including our own previous data from non-differentiated human neuroblastoma SH-SY5Y cells. The aim of this study was now to investigate if in vitro differentiation with the phorbol ester 12-O- tetradecanoylphorbol-13-acetate or the vitamin A derivative all-trans retinoic acid altered the sensitivity to ellagic acid in SH-SY5Y cells. The methods used were cell counting and LDH-assay for evaluation of cell number and cell death, flow cytometric analysis of SubG1- and TUNEL-analysis for apoptosis and western blot for expression of apoptosis-associated proteins. In vitro differentiation was shown to reduce the sensitivity to ellagic acid with respect to cell detachment, loss of viability and activation of apoptosis. The protective effect was phenotype-specific and most prominent in all-trans retinoic acid-differentiated cultures. Differentiation-dependent up-regulation of Bcl-2 and integrin expression is introduced as possible protective mechanisms. The presented data also point to a positive correlation between proliferative activity and sensitivity to ellagic-acid-induced cell detachment. In conclusion, the presented data emphasize the need to consider degree of neuronal differentiation and phenotype of neuroblastoma cells when discussing a potential pharmaceutical application of ellagic acid in tumor treatment. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  10. Coriandrum sativum and Lavandula angustifolia Essential Oils: Chemical Composition and Activity on Central Nervous System.

    Science.gov (United States)

    Caputo, Lucia; Souza, Lucéia Fátima; Alloisio, Susanna; Cornara, Laura; De Feo, Vincenzo

    2016-11-30

    The aims of this study are to determine the chemical composition of Lavandula angustifolia Mill. and Coriandrum sativum L. essential oils, to evaluate their cytotoxic effects in SH-SY5Y human neuroblastoma cells, to investigate whether an alteration of adenylate cyclase 1 (ADCY1) and of extracellular signal-regulated kinase (ERK) expression can take part in the molecular mechanisms of the essential oils, and to study their possible neuronal electrophysiological effects. The essential oils were obtained by hydrodistillation, and studied by GC and GC-MS. In the oils from L. angustifolia and C. sativum , linalool was the main component (33.1% and 67.8%, respectively). SH-SY5Y cells were incubated with different concentrations of essential oils and of linalool. Cell viability and effects on ADCY1 and ERK expression were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT and Western blotting, respectively. Variation in cellular electrophysiology was studied in primary cultures of rat cortical neurons with a multi-electrode array (MEA)-based approach. The essential oils and linalool revealed different cytotoxic activities. Linalool inhibited ADCY1 and ERK expression. Neuronal networks subjected to L. angustifolia and C. sativum essential oils showed a concentration-dependent inhibition of spontaneous electrical activity.

  11. SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson's disease.

    Science.gov (United States)

    Singh, Preeti; Hanson, Peter S; Morris, Christopher M

    2017-06-02

    Sirtuins (SIRTs) are NAD + dependent lysine deacetylases which are conserved from bacteria to humans and have been associated with longevity and lifespan extension. SIRT1, the best studied mammalian SIRT is involved in many physiological and pathological processes and changes in SIRT1 have been implicated in neurodegenerative disorders, with SIRT1 having a suggested protective role in Parkinson's disease. In this study, we determined the effect of SIRT1 on cell survival and α-synuclein aggregate formation in SH-SY5Y cells following oxidative stress. Over-expression of SIRT1 protected SH-SY5Y cells from toxin induced cell death and the protection conferred by SIRT1 was partially independent of its deacetylase activity, which was associated with the repression of NF-кB and cPARP expression. SIRT1 reduced the formation of α-synuclein aggregates but showed minimal co-localisation with α-synuclein. In post-mortem brain tissue obtained from patients with Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies and Alzheimer's disease, the activity of SIRT1 was observed to be down-regulated. These findings suggests a negative effect of oxidative stress in neurodegenerative disorders and possibly explain the reduced activity of SIRT1 in neurodegenerative disorders. Our study shows that SIRT1 is a pro-survival protein that is downregulated under cellular stress.

  12. Improved cell therapy protocols for Parkinson's disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons

    DEFF Research Database (Denmark)

    Sundberg, Maria; Bogetofte, Helle; Lawson, Tristan

    2013-01-01

    of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations......The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and h......ESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation...

  13. Managing Parkinson's disease with continuous dopaminergic stimulation

    NARCIS (Netherlands)

    Wolters, Erik; Lees, Andrew J.; Volkmann, Jens; van Laar, Teus; Hovestadt, Ad

    The pathophysiology of Parkinson's disease is marked by the loss of dopaminergic neurons, which leads to striatal dopaminergic deficiency. This causes resting tremor, hypokinesia, rigidity, bradykinesia, and loss of postural reflexes. Most current treatments for Parkinson's disease aim to restore

  14. Imaging of dopaminergic system in movement disorders

    International Nuclear Information System (INIS)

    Kim, Yu Kyeong; Kim, Sang Eun

    2007-01-01

    Parkinson's disease is a common neurodegenerative disorder that is mainly caused by dopaminergic neuron loss in the substantia nigra. Several radiopharmaceutics have been developed to evaluated the integrity of dopaminergic neuronal system. In vivo PET and SPECT imaging of presynaptic dopamine imaging are already applied to Parkinson's disease and other parkinsonism, and can demonstrate the dopaminergic dysfunction. This review summarized the use of the presynaptic dopaminergic imaging in PD as biomarkers in evaluation of disease progression as well as in diagnosis of PD

  15. The dopaminergic system and aggression in laying hens

    Science.gov (United States)

    The dopaminergic system regulates aggression in humans and other mammals. To investigate if birds with genetic propensity for high and low aggressiveness may exhibit distinctly different aggressive mediation via dopamine (DA) D1 and D2 receptor pathways, two high aggressive (DXL and LGPS) and one lo...

  16. LncRNA NEAT1 promotes autophagy in MPTP-induced Parkinson's disease through stabilizing PINK1 protein.

    Science.gov (United States)

    Yan, Wang; Chen, Zhao-Ying; Chen, Jia-Qi; Chen, Hui-Min

    2018-02-19

    Long non-coding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) was found to be closely related to the pathological changes in brain and nervous system. However, the role of NEAT1 and its potential mechanism in Parkinson's disease (PD) largely remain uncharacterized. In this study, PD mouse model was established by intraperitoneal injection of MPTP. The numbers of TH + neurons, NEAT1 expression and the level of PINK1, LC3-II, LC3-I protein were assessed in PD mice. SH-SY5Y cells were treated with MPP + as PD cell model. RNA pull-down assay was used to identify the interaction between NEAT1 and PINK1 in vitro. The endogenous expression of NEAT1 was modified by lentiviral vector carrying interference sequence for NEAT1 in vivo. The numbers of TH + neurons significantly decreased in PD mice compared with the control. The expressions of NEAT1, PINK1 protein and LC3-II/LC3-I level were increased by MPTP in vitro and in vivo. Moreover, NEAT1 positively regulated the protein level of PINK1 through inhibition of PINK1 protein degradation. And NEAT1 mediated the effects of MPP + on SH-SY5Y cells through stabilization of PINK1 protein. The results of in vivo experiments revealed that NEAT1 knockdown could effectively suppress MPTP-induced autophagy in vivo that alleviated dopaminergic neuronal injury. LncRNA NEAT1 promoted the MPTP-induced autophagy in PD through stabilization of PINK1 protein. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.

    Science.gov (United States)

    Sancho, Rosa M; Law, Bernard M H; Harvey, Kirsten

    2009-10-15

    Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2-DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2-DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2-DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease.

  18. Asiatic acid attenuates methamphetamine-induced neuroinflammation and neurotoxicity through blocking of NF-kB/STAT3/ERK and mitochondria-mediated apoptosis pathway.

    Science.gov (United States)

    Park, Ji-Hyun; Seo, Young Ho; Jang, Jung-Hee; Jeong, Chul-Ho; Lee, Sooyeun; Park, Byoungduck

    2017-12-11

    Methamphetamine (METH) is a commonly abused drug that may result in neurotoxic effects. Recent studies have suggested that involvement of neuroinflammatory processes in brain dysfunction is induced by misuse of this drug. However, the mechanism underlying METH-induced inflammation and neurotoxicity in neurons is still unclear. In this study, we investigated whether asiatic acid (AA) effected METH-mediated neuroinflammation and neurotoxicity in dopaminergic neuronal cells. And we further determined whether the effect involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK) pathway. We used the human dopaminergic neuroblastoma SH-SY5Y cell line, murine microglial BV2 cell line, and primary culture of rat embryo mesencephalic neurons. Pro-inflammatory cytokine production was monitored by ELISA and RT/real-time PCR. The cell cycle distribution and mitochondrial membrane integrity was analyzed by flow cytometry. We used immunoblotting, DNA-binding activity, and immunofluorescence staining to analyze the effect of AA on activation of the NF-κB, STAT3, MAPK-ERK, and apoptosis signaling pathways. METH induced TNF receptor (TNFR) expression and led to morphological changes of cells. Additionally, this drug increased pro-inflammatory cytokine (TNFα and IL-6) expression. AA significantly suppressed METH-induced TNFR expression in concentration dependent. Increased secretion of TNFα and IL-6 was inhibited in METH-stimulated neuronal cells by AA administration. AA showed significant protection against METH-induced translocation of NF-κB/STAT3 and ERK phosphorylation. AA inhibited METH-induced proteolytic fragmentation of caspase-3 and PARP. The pro-apoptotic protein Bax was significantly decreased, while the anti-apoptotic protein Bcl-xL was increased by AA treatment in METH-stimulated cells. A similar protective effect of AA on

  19. Induced dopaminergic neurons: A new promise for Parkinson’s disease

    Directory of Open Access Journals (Sweden)

    Zhimin Xu

    2017-04-01

    Full Text Available Motor symptoms that define Parkinson’s disease (PD are caused by the selective loss of nigral dopaminergic (DA neurons. Cell replacement therapy for PD has been focused on midbrain DA neurons derived from human fetal mesencephalic tissue, human embryonic stem cells (hESC or human induced pluripotent stem cells (iPSC. Recent development in the direct conversion of human fibroblasts to induced dopaminergic (iDA neurons offers new opportunities for transplantation study and disease modeling in PD. The iDA neurons are generated directly from human fibroblasts in a short period of time, bypassing lengthy differentiation process from human pluripotent stem cells and the concern for potentially tumorigenic mitotic cells. They exhibit functional dopaminergic neurotransmission and relieve locomotor symptoms in animal models of Parkinson’s disease. In this review, we will discuss this recent development and its implications to Parkinson’s disease research and therapy.

  20. Anti-human α-synuclein N-terminal peptide antibody protects against dopaminergic cell death and ameliorates behavioral deficits in an AAV-α-synuclein rat model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Md Shahaduzzaman

    Full Text Available The protein α-synuclein (α-Syn has a central role in the pathogenesis of Parkinson's disease (PD and immunotherapeutic approaches targeting this molecule have shown promising results. In this study, novel antibodies were generated against specific peptides from full length human α-Syn and evaluated for effectiveness in ameliorating α-Syn-induced cell death and behavioral deficits in an AAV-α-Syn expressing rat model of PD. Fisher 344 rats were injected with rAAV vector into the right substantia nigra (SN, while control rats received an AAV vector expressing green fluorescent protein (GFP. Beginning one week after injection of the AAV-α-Syn vectors, rats were treated intraperitoneally with either control IgG or antibodies against the N-terminal (AB1, or central region (AB2 of α-Syn. An unbiased stereological estimation of TH+, NeuN+, and OX6 (MHC-II immunostaining revealed that the α-Syn peptide antibodies (AB1 and AB2 significantly inhibited α-Syn-induced dopaminergic cell (DA and NeuN+ cell loss (one-way ANOVA (F (3, 30 = 5.8, p = 0.002 and (F (3, 29 = 7.92, p = 0.002 respectively, as well as decreasing the number of activated microglia in the ipsilateral SN (one-way ANOVA F = 14.09; p = 0.0003. Antibody treated animals also had lower levels of α-Syn in the ipsilateral SN (one-way ANOVA F (7, 37 = 9.786; p = 0.0001 and demonstrated a partial intermediate improvement of the behavioral deficits. Our data suggest that, in particular, an α-Syn peptide antibody against the N-terminal region of the protein can protect against DA neuron loss and, to some extent behavioral deficits. As such, these results may be a potential therapeutic strategy for halting the progression of PD.

  1. Aβ-Induced Insulin Resistance and the Effects of Insulin on the Cholesterol Synthesis Pathway and Aβ Secretion in Neural Cells.

    Science.gov (United States)

    Najem, Dema; Bamji-Mirza, Michelle; Yang, Ze; Zhang, Wandong

    2016-06-01

    Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) toxicity, tau pathology, insulin resistance, neuroinflammation, and dysregulation of cholesterol homeostasis, all of which play roles in neurodegeneration. Insulin has polytrophic effects on neurons and may be at the center of these pathophysiological changes. In this study, we investigated possible relationships among insulin signaling and cholesterol biosynthesis, along with the effects of Aβ42 on these pathways in vitro. We found that neuroblastoma 2a (N2a) cells transfected with the human gene encoding amyloid-β protein precursor (AβPP) (N2a-AβPP) produced Aβ and exhibited insulin resistance by reduced p-Akt and a suppressed cholesterol-synthesis pathway following insulin treatment, and by increased phosphorylation of insulin receptor subunit-1 at serine 612 (p-IRS-S612) as compared to parental N2a cells. Treatment of human neuroblastoma SH-SY5Y cells with Aβ42 also increased p-IRS-S612, suggesting that Aβ42 is responsible for insulin resistance. The insulin resistance was alleviated when N2a-AβPP cells were treated with higher insulin concentrations. Insulin increased Aβ release from N2a-AβPP cells, by which it may promote Aβ clearance. Insulin increased cholesterol-synthesis gene expression in SH-SY5Y and N2a cells, including 24-dehydrocholesterol reductase (DHCR24) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) through sterol-regulatory element-binding protein-2 (SREBP2). While Aβ42-treated SH-SY5Y cells exhibited increased HMGCR expression and c-Jun phosphorylation as pro-inflammatory responses, they also showed down-regulation of neuro-protective/anti-inflammatory DHCR24. These results suggest that Aβ42 may cause insulin resistance, activate JNK for c-Jun phosphorylation, and lead to dysregulation of cholesterol homeostasis, and that enhancing insulin signaling may relieve the insulin-resistant phenotype and the dysregulated cholesterol-synthesis pathway to promote A

  2. Activation of transglutaminase 2 by nerve growth factor in differentiating neuroblastoma cells: A role in cell survival and neurite outgrowth.

    Science.gov (United States)

    Algarni, Alanood S; Hargreaves, Alan J; Dickenson, John M

    2018-02-05

    NGF (nerve growth factor) and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 transamidase activity by NGF in retinoic acid-induced differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. The role of TG2 in NGF-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with NGF in N2a and SH-SY5Y cells. NGF mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON (Z-ZON-Val-Pro-Leu-OMe; Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-l-valinyl-l-prolinyl-l-leucinmethylester) and R283 (1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride) and by pharmacological inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB) and protein kinase C (PKC), and removal of extracellular Ca 2+ . Fluorescence microscopy demonstrated NGF induced in situ TG2 activity. TG2 inhibition blocked NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth in both cell lines. Together, these results demonstrate that NGF stimulates TG2 transamidase activity via a ERK1/2, PKB and PKC-dependent pathway in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. Furthermore, NGF-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results suggest a novel and important role of TG2 in the cellular functions of NGF. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Cosmosiin Increases ADAM10 Expression via Mechanisms Involving 5’UTR and PI3K Signaling

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    Zhuo Min

    2018-06-01

    Full Text Available The α-secretase “a disintegrin and metalloproteinase domain-containing protein” (ADAM10 is involved in the processing of amyloid precursor protein (APP. Upregulation of ADAM10 precludes the generation of neurotoxic β-amyloid protein (Aβ and represents a plausible therapeutic strategy for Alzheimer’s disease (AD. In this study, we explored compounds that can potentially promote the expression of ADAM10. Therefore, we performed high-throughput small-molecule screening in SH-SY5Y (human neuroblastoma cells that stably express a luciferase reporter gene driven by the ADAM10 promoter, including a portion of its 5’-untranslated region (5’UTR. This has led to the discovery of cosmosiin (apigenin 7-O-β-glucoside. Here, we report that in human cell lines (SH-SY5Y and HEK293, cosmosiin proportionally increased the levels of the immature and mature forms of the ADAM10 protein without altering its mRNA level. This effect was attenuated by translation inhibitors or by deleting the 5’UTR of ADAM10, suggesting that a translational mechanism was responsible for the increased levels of ADAM10. Luciferase deletion assays revealed that the first 144 nucleotides of the 5’UTR were necessary for mediating the cosmosiin-induced enhancement of ADAM10 expression in SH-SY5Y cells. Cosmosiin failed to increase the levels of the ADAM10 protein in murine cells, which lack native expression of the ADAM10 transcript containing the identified 5’UTR element. The potential signaling pathway may involve phosphatidylinositide 3-kinase (PI3K because pharmacological inhibition of PI3K attenuated the effect of cosmosiin on the expression of the ADAM10 protein. Finally, cosmosiin attenuated Aβ generation because the levels of Aβ40/42 in HEK-APP cells were significantly reduced after cosmosiin treatment. Collectively, we found that the first 144 nucleotides of the ADAM10 5’UTR, and PI3K signaling, are involved in cosmosiin-induced enhancement of the expression

  4. Serotonergic and dopaminergic modulation of attentional processes.

    Science.gov (United States)

    Boulougouris, Vasileios; Tsaltas, Eleftheria

    2008-01-01

    Disturbances in attentional processes are a common feature of several psychiatric disorders such as schizophrenia, attention deficit/hyperactivity disorder and Huntington's disease. The use of animal models has been useful in defining various candidate neural systems thus enabling us to translate basic laboratory science to the clinic and vice-versa. In this chapter, a comparative and integrated account is provided on the neuroanatomical and neurochemical modulation of basic behavioural operations such as selective attention, vigilance, set-shifting and executive control focusing on the comparative functions of the serotonin and dopamine systems in the cognitive control exerted by the prefrontal cortex. Specifically, we have reviewed evidence emerging from several behavioural paradigms in experimental animals and humans each of which centres on a different aspect of the attentional function. These paradigms offering both human and animal variants include the five-choice serial reaction time task (5CSRTT), attentional set-shifting and stop-signal reaction time task. In each case, the types of operation that are measured by the given paradigm and their neural correlates are defined. Then, the role of the ascending dopaminergic and serotonergic systems in the neurochemical modulation of its behavioural output are examined, and reference is made to clinical implications for neurological and neuropsychiatric disorders which exhibit deficits in these cognitive tests.

  5. Quercetin-mediated synthesis of graphene oxide–silver nanoparticle nanocomposites: a suitable alternative nanotherapy for neuroblastoma

    Directory of Open Access Journals (Sweden)

    Yuan YG

    2017-08-01

    Full Text Available Yu-Guo Yuan,1 Yan-Hong Wang,1 Hui-Hui Xing,1 Sangiliyandi Gurunathan2 1Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, People’s Republic of China; 2Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Republic of Korea Background: Graphene and graphene-related materials have gained substantial interest from both academia and industry for the development of unique nanomaterials for biomedical applications. Graphene oxide (GO and silver nanoparticles (AgNPs are a valuable platform for the development of nanocomposites, permitting the combination of nanomaterials with different physical and chemical properties to generate novel materials with improved and effective functionalities in a single platform. Therefore, this study was conducted to synthesize a graphene oxide–silver nanoparticle (GO-AgNPs nanocomposite using the biomolecule quercetin and evaluate the potential cytotoxicity and mechanism of GO-AgNPs in human neuroblastoma cancer cells (SH-SY5Y.Methods: The synthesized GO-AgNPs were characterized using various analytical techniques. The potential toxicities of GO-AgNPs were evaluated using a series of biochemical and cellular assays. The expression of apoptotic and anti-apoptotic genes was measured by quantitative real-time reverse transcription polymerase chain reaction. Further, apoptosis was confirmed by caspase-9/3 activity and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and GO-AgNPs-induced autophagy was also confirmed by transmission electron microscopy.Results: The prepared GO-AgNPs exhibited significantly higher cytotoxicity toward SH-SY5Y cells than GO. GO-AgNPs induced significant cytotoxicity in SH-SY5Y cells by the loss of cell viability, inhibition of cell proliferation, increased leakage of lactate dehydrogenase, decreased level of

  6. Interferon-γ increases neuronal death in response to amyloid-β1-42

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    Williams Alun

    2006-03-01

    Full Text Available Abstract Background Alzheimer's disease is a neurodegenerative disorder characterized by a progressive cognitive impairment, the consequence of neuronal dysfunction and ultimately the death of neurons. The amyloid hypothesis proposes that neuronal damage results from the accumulation of insoluble, hydrophobic, fibrillar peptides such as amyloid-β1-42. These peptides activate enzymes resulting in a cascade of second messengers including prostaglandins and platelet-activating factor. Apoptosis of neurons is thought to follow as a consequence of the uncontrolled release of second messengers. Biochemical, histopathological and genetic studies suggest that pro-inflammatory cytokines play a role in neurodegeneration during Alzheimer's disease. In the current study we examined the effects of interferon (IFN-γ, tumour necrosis factor (TNFα, interleukin (IL-1β and IL-6 on neurons. Methods Primary murine cortical or cerebellar neurons, or human SH-SY5Y neuroblastoma cells, were grown in vitro. Neurons were treated with cytokines prior to incubation with different neuronal insults. Cell survival, caspase-3 activity (a measure of apoptosis and prostaglandin production were measured. Immunoblots were used to determine the effects of cytokines on the levels of cytoplasmic phospholipase A2 or phospholipase C γ-1. Results While none of the cytokines tested were directly neurotoxic, pre-treatment with IFN-γ sensitised neurons to the toxic effects of amyloid-β1-42 or HuPrP82-146 (a neurotoxic peptide found in prion diseases. The effects of IFN-γ were seen on cortical and cerebellar neurons, and on SH-SY5Y neuroblastoma cells. However, pre-treatment with IFN-γ did not affect the sensitivity to neurons treated with staurosporine or hydrogen peroxide. Pre-treatment with IFN-γ increased the levels of cytoplasmic phospholipase A2 in SH-SY5Y cells and increased prostaglandin E2 production in response to amyloid-β1-42. Conclusion Treatment of neuronal cells

  7. Brain dopaminergic systems : imaging with positron tomography

    Energy Technology Data Exchange (ETDEWEB)

    Baron, J C [University of Caen/INSERM U, Caen (France). CYCERON; Comar, D [E.E.C. Concerted Action on P.E.T. Investigations of Cellular Regeneration and Degeneration, Orsay (France) CEA, 91 - Orsay (France). Service Hospitalier Frederic Joliot; Farde, L [Karolinska Sjukhuset, Stockholm (Sweden); Martinot, J L; Mazoyer, B [CEA, 91 - Orsay (France). Service Hospitalier Frederic Joliot Paris-

    1991-01-01

    Imaging of the dopaminergic system in the human brain with the in vivo use of Positron Emission Tomography emerged in the late 1980s as a tool of major importance in clinical neurosciences and pharmacology. The last few years have witnessed rapid development of new radiotracers specific to receptors, reuptake sites and enzymes of the dopamine system; the application of these radiotracers has led to major break-troughs in the pathophysiology and therapy of movement disorders and schizophrenic-like psychoses. This book is the first to collect, in a single volume, state-of-the-art contributions to the various aspects of this research. Its contents address methodological issues related to the design, labelling, quantitative imaging and compartmental modeli-sation of radioligands of the post-synaptic, pre-synaptic and enzyme sites of the dopamine system and to their use in clinical research in the fields of Parkinson's disease as well as other movement disorders, psychoses and neuroleptic receptor occupancy. The chapters were written by leading European scientists in the field of PET, gathered together in Caen (France, November 1990) under the aegis of the EEC Concerted Action on PET Investigations of Cellular Regeneration and Degeneration. This book provides a current and comprehensive overview on PET studies of the brain dopamine system which should aid and interest neurologists , psychiatrists, pharmacologists and medical imaging scientists. (author). refs.; figs.; tabs.

  8. Distinct effects of rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on cellular bioenergetics and cell death.

    Directory of Open Access Journals (Sweden)

    Samantha Giordano

    Full Text Available Parkinson's disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson's like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+, and 6-hydroxydopamine (6-OHDA, have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson's disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP(+ exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP(+, unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP(+ at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.

  9. Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride.

    Science.gov (United States)

    Brütting, Christine; Narasimhan, Harini; Hoffmann, Frank; Kornhuber, Malte E; Staege, Martin S; Emmer, Alexander

    2018-01-01

    Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS.

  10. Peptidomic analysis of human cell lines

    Science.gov (United States)

    Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.

    2011-01-01

    Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. PMID:21204522

  11. [Similarity of cycloprolylglycine to piracetam in antihypoxic and neuroprotective effects].

    Science.gov (United States)

    Kolisnikova, K N; Gudasheva, T A; Nazarova, G A; Antipov, T A; Voronina, T A; Seredenin, S B

    2012-01-01

    The antihypoxic activity of the endogenous cyclic dipeptide cycloprolylglycine (CPG) has been studied on a model of normobaric hypoxia with hypercapnia and its neuroprotective activity has been studied on a model of human neuroblastoma SH-SY5Y cell damage by 6-hydroxydopamine. It is established that CPG exhibits the antihypoxic activity at doses of 0.5 and 1.0 mg/kg (i.p.) on outbred and BALB/c mice, but not on C57B1/6 mice. The neuroprotective activity of CPG was detected in 10(-5) - 10(-8) M concentration range only when the treatment was carried out 24h before toxin introduction. The obtained data confirm the hypothesis that piracetam is a mimetic of the endogenous CPG neuropeptide.

  12. Detection of Temperature Difference in Neuronal Cells.

    Science.gov (United States)

    Tanimoto, Ryuichi; Hiraiwa, Takumi; Nakai, Yuichiro; Shindo, Yutaka; Oka, Kotaro; Hiroi, Noriko; Funahashi, Akira

    2016-03-01

    For a better understanding of the mechanisms behind cellular functions, quantification of the heterogeneity in an organism or cells is essential. Recently, the importance of quantifying temperature has been highlighted, as it correlates with biochemical reaction rates. Several methods for detecting intracellular temperature have recently been established. Here we develop a novel method for sensing temperature in living cells based on the imaging technique of fluorescence of quantum dots. We apply the method to quantify the temperature difference in a human derived neuronal cell line, SH-SY5Y. Our results show that temperatures in the cell body and neurites are different and thus suggest that inhomogeneous heat production and dissipation happen in a cell. We estimate that heterogeneous heat dissipation results from the characteristic shape of neuronal cells, which consist of several compartments formed with different surface-volume ratios. Inhomogeneous heat production is attributable to the localization of specific organelles as the heat source.

  13. Dopaminergic Dysregulation, Artistic Expressiveness, and Parkinson's Disease

    Science.gov (United States)

    López-Pousa, S.; Lombardía-Fernández, C.; Olmo, J. Garre; Monserrat-Vila, S.; Vilalta-Franch, J.; Calvó-Perxas, L.

    2012-01-01

    Background The most frequent behavioral manifestations in Parkinson's disease (PD) are attributed to the dopaminergic dysregulation syndrome (DDS), which is considered to be secondary to the iatrogenic effects of the drugs that replace dopamine. Over the past few years some cases of patients improving their creative abilities after starting treatment with dopaminergic pharmaceuticals have been reported. These effects have not been clearly associated to DDS, but a relationship has been pointed out. Methods Case study of a patient with PD. The evolution of her paintings along medication changes and disease advance has been analyzed. Results The patient showed a compulsive increase of pictorial production after the diagnosis of PD was made. She made her best paintings when treated with cabergolide, and while painting, she reported a feeling of well-being, with loss of awareness of the disease and reduction of physical limitations. Conclusions Dopaminergic antagonists (DA) trigger a dopaminergic dysfunction that alters artistic creativity in patients having a predisposition for it. The development of these skills might be due to the dopaminergic overstimulation due to the therapy with DA, which causes a neurophysiological alteration that globally determines DDS. PMID:23185168

  14. The Biological Effects of Bilirubin Photoisomers

    Science.gov (United States)

    Jasprova, Jana; Dal Ben, Matteo; Vianello, Eleonora; Goncharova, Iryna; Urbanova, Marie; Vyroubalova, Karolina; Gazzin, Silvia; Tiribelli, Claudio; Sticha, Martin; Cerna, Marcela; Vitek, Libor

    2016-01-01

    Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells. PMID:26829016

  15. Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide

    International Nuclear Information System (INIS)

    Ohno, Kazuki; Okuda, Kosaku; Uehara, Takashi

    2015-01-01

    Highlights: • PTEN is S-sulfhydrated endogenously in SH-SY5Y human neuroblastoma cells. • Preventing this modification by knocking down CBS renders PTEN sensitive to NO. • pAkt levels are increased significantly in CBS siRNA-transfected cells. • H 2 S functions as an endogenous regulator of PTEN in neuronal cells. - Abstract: Hydrogen sulfide (H 2 S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H 2 S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H 2 S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H 2 S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H 2 S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions

  16. Analgesic treatment of ciguatoxin-induced cold allodynia.

    Science.gov (United States)

    Zimmermann, Katharina; Deuis, Jennifer R; Inserra, Marco C; Collins, Lindon S; Namer, Barbara; Cabot, Peter J; Reeh, Peter W; Lewis, Richard J; Vetter, Irina

    2013-10-01

    Ciguatera, the most common form of nonbacterial ichthyosarcotoxism, is caused by consumption of fish that have bioaccumulated the polyether sodium channel activator ciguatoxin. The neurological symptoms of ciguatera include distressing, often persistent sensory disturbances such as paraesthesias and the pathognomonic symptom of cold allodynia. We show that intracutaneous administration of ciguatoxin in humans elicits a pronounced axon-reflex flare and replicates cold allodynia. To identify compounds able to inhibit ciguatoxin-induced Nav responses, we developed a novel in vitro ciguatoxin assay using the human neuroblastoma cell line SH-SY5Y. Pharmacological characterisation of this assay demonstrated a major contribution of Nav1.2 and Nav1.3, but not Nav1.7, to ciguatoxin-induced Ca2+ responses. Clinically available Nav inhibitors, as well as the Kv7 agonist flupirtine, inhibited tetrodotoxin-sensitive ciguatoxin-evoked responses. To establish their in vivo efficacy, we used a novel animal model of ciguatoxin-induced cold allodynia. However, differences in the efficacy of these compounds to reverse ciguatoxin-induced cold allodynia did not correlate with their potency to inhibit ciguatoxin-induced responses in SH-SY5Y cells or at heterologously expressed Nav1.3, Nav1.6, Nav1.7, or Nav1.8, indicating cold allodynia might be more complex than simple activation of Nav channels. These findings highlight the need for suitable animal models to guide the empiric choice of analgesics, and suggest that lamotrigine and flupirtine could be potentially useful for the treatment of ciguatera. Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  17. Roles of thioredoxin in nitric oxide-dependent preconditioning-induced tolerance against MPTP neurotoxin

    International Nuclear Information System (INIS)

    Chiueh, C.C.; Andoh, Tsugunobu; Chock, P. Boon

    2005-01-01

    Hormesis, a stress tolerance, can be induced by ischemic preconditioning stress. In addition to preconditioning, it may be induced by other means, such as gas anesthetics. Preconditioning mechanisms, which may be mediated by reprogramming survival genes and proteins, are obscure. A known neurotoxicant, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes less neurotoxicity in the mice that are preconditioned. Pharmacological evidences suggest that the signaling pathway of ·NO-cGMP-PKG (protein kinase G) may mediate preconditioning phenomenon. We developed a human SH-SY5Y cell model for investigating · NO-mediated signaling pathway, gene regulation, and protein expression following a sublethal preconditioning stress caused by a brief 2-h serum deprivation. Preconditioned human SH-SY5Y cells are more resistant against severe oxidative stress and apoptosis caused by lethal serum deprivation and 1-mehtyl-4-phenylpyridinium (MPP + ). Both sublethal and lethal oxidative stress caused by serum withdrawal increased neuronal nitric oxide synthase (nNOS/NOS1) expression and · NO levels to a similar extent. In addition to free radical scavengers, inhibition of nNOS, guanylyl cyclase, and PKG blocks hormesis induced by preconditioning. S-nitrosothiols and 6-Br-cGMP produce a cytoprotection mimicking the action of preconditioning tolerance. There are two distinct cGMP-mediated survival pathways: (i) the up-regulation of a redox protein thioredoxin (Trx) for elevating mitochondrial levels of antioxidant protein Mn superoxide dismutase (MnSOD) and antiapoptotic protein Bcl-2, and (ii) the activation of mitochondrial ATP-sensitive potassium channels [K(ATP)]. Preconditioning induction of Trx increased tolerance against MPP + , which was blocked by Trx mRNA antisense oligonucleotide and Trx reductase inhibitor. It is concluded that Trx plays a pivotal role in · NO-dependent preconditioning hormesis against MPTP/MPP +

  18. Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide

    Energy Technology Data Exchange (ETDEWEB)

    Ohno, Kazuki; Okuda, Kosaku; Uehara, Takashi, E-mail: uehara@pharm.okayama-u.ac.jp

    2015-01-02

    Highlights: • PTEN is S-sulfhydrated endogenously in SH-SY5Y human neuroblastoma cells. • Preventing this modification by knocking down CBS renders PTEN sensitive to NO. • pAkt levels are increased significantly in CBS siRNA-transfected cells. • H{sub 2}S functions as an endogenous regulator of PTEN in neuronal cells. - Abstract: Hydrogen sulfide (H{sub 2}S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H{sub 2}S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H{sub 2}S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H{sub 2}S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H{sub 2}S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.

  19. Protolichesterinic acid enhances doxorubicin-induced apoptosis in HeLa cells in vitro.

    Science.gov (United States)

    Brisdelli, Fabrizia; Perilli, Mariagrazia; Sellitri, Doriana; Bellio, Pierangelo; Bozzi, Argante; Amicosante, Gianfranco; Nicoletti, Marcello; Piovano, Marisa; Celenza, Giuseppe

    2016-08-01

    The aim of this study was to investigate the effect of protolichesterinic acid, a lichen secondary metabolite, on anti-proliferative activity of doxorubicin in three human cancer cell lines, HeLa, SH-SY5Y and K562 cells. The data obtained from MTT assays, performed on cells treated with protolichesterinic acid and doxorubicin alone and in combination, were analysed by the median-effect method as proposed by Chou and Talalay and the Bliss independence model. Apoptosis rate was evaluated by fluorescence microscopy, caspase-3, 8 and 9 activities were detected by spectrofluorimetric analysis and protein expression of Bim, Bid, Bax and Mcl-2 was analysed by Western blotting. The interaction of protolichesterinic acid with thioesterase domain of human fatty acid synthase (hFAS) was investigated by a molecular docking study. The in vitro activity of doxorubicin against HeLa cancer cell line, but not against SH-SY5Y and K562 cells, was synergically increased by protolichesterinic acid. The increased cytotoxicity caused by protolichesterinic acid in HeLa cells was due to a pro-apoptotic effect and was associated to caspase-3, 8 and 9 activation. The simultaneous treatment for 24h with protolichesterinic acid plus doxorubicin caused an increase of Bim protein expression and the appearance of cleaved form of Bid protein. The molecular modelling analysis showed that protolichesterinic acid seemed to behave as a competitive inhibitor of hFAS. These results suggest that protolichesterinic acid could be envisaged as an useful tool against certain types of tumor cells in combination with anticancer drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Evidence of dopaminergic processing of executive inhibition.

    Directory of Open Access Journals (Sweden)

    Rajendra D Badgaiyan

    Full Text Available Inhibition of unwanted response is an important function of the executive system. Since the inhibitory system is impaired in patients with dysregulated dopamine system, we examined dopamine neurotransmission in the human brain during processing of a task of executive inhibition. The experiment used a recently developed dynamic molecular imaging technique to detect and map dopamine released during performance of a modified Eriksen's flanker task. In this study, young healthy volunteers received an intravenous injection of a dopamine receptor ligand ((11C-raclopride after they were positioned in the PET camera. After the injection, volunteers performed the flanker task under Congruent and Incongruent conditions in a single scan session. They were required to inhibit competing options to select an appropriate response in the Incongruent but not in the Congruent condition. The PET data were dynamically acquired during the experiment and analyzed using two variants of the simplified reference region model. The analysis included estimation of a number of receptor kinetic parameters before and after initiation of the Incongruent condition. We found increase in the rate of ligand displacement (from receptor sites and decrease in the ligand binding potential in the Incongruent condition, suggesting dopamine release during task performance. These changes were observed in small areas of the putamen and caudate bilaterally but were most significant on the dorsal aspect of the body of left caudate. The results provide evidence of dopaminergic processing of executive inhibition and demonstrate that neurochemical changes associated with cognitive processing can be detected and mapped in a single scan session using dynamic molecular imaging.

  1. [In vitro early detection of amyloid plaques in Alzheimer's disease by Pittsburgh compound B-modified magnetic nanoparticles].

    Science.gov (United States)

    Zeng, J Q; Wu, J Q; Li, M H; Wang, P J

    2017-11-07

    Objective: To construct magnetic nanoparticles targeting β-amyloid (Aβ) plaques, the pathological biomarker of Alzheimer's disease (AD) and to study their binding capability in vitro . Methods: Superparamagnetic nanoparticles Mn(0.6)Zn(0.4)Fe(2)O(4) (MZF) were coated with amphiphilic star-block copolymeric micelles and modified with Aβ-specific probe Pittsburgh compound B (PiB) to construct a novel magnetic nanoparticle MZF-PiB, which specifically targeted amyloid plaques. Transmission electron microscope was used to study the morphological features of MZF-PiB. Superparamagnetism of MZF-PiB was assessed by its r(2) relaxation rate by using 3.0 T MRI scanner. Cytotoxic test was applied to determine biosafety of MZF-PiB nanoparticles in differentiated human neuroblastoma cells (SH-SY5Y) and Madin-Darby canine kidney (MDCK). In vitro binding tests were conducted via immunohistochemistry on 6-month old AD mice brain sections. Differences of cell viability between groups were compared with one-way analysis of variance. Results: MZF-PiB nanoparticles were successfully constructed. Transmission electron microscope images showed that the nanoparticles were about 100 nm in size. The r(2) relaxation rate was 163.11 mMS(-1). No differences were found in cell viability of SH-SY5Y and MDCK incubated with MZF-PiB suspension for 24 h or 48 h when compared with those of untreated cells ( F =2.336, 2.539, 0.293, 1.493, all P >0.05). In vitro binding tests indicated that the MZF-PiB were specifically bound to amyloid plaques. The smallest size of detected plaques was 27 μm. Conclusion: PiB-modified nanoparticles targeting Aβ are biologically safe and highly superparamagnetic, possessing the capability to detect amyloid plaques early in vitro and the potential for early diagnosis of AD.

  2. Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis

    International Nuclear Information System (INIS)

    Nam, Seung Taek; Kim, Dae Hong; Lee, Min Bum; Nam, Hyo Jung; Kang, Jin Ku; Park, Mi Jung; Lee, Ik Hwan; Seok, Heon; Lee, Dong Gun; Hwang, Jae Sam; Kim, Ho

    2013-01-01

    Highlights: •CopA3 peptide isolated from the Korean dung beetle has antimicrobial activity. •Our study reported that CopA3 has anticancer and immunosuppressive effects. •We here demonstrated that CopA3 has neurotropic and neuroprotective effects. •CopA3 degrades p27Kip1 protein and this mediates effects of CopA3 on neuronal cells. -- Abstract: We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer’s disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects

  3. The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion

    Science.gov (United States)

    Nalvarte, Ivan; Damdimopoulos, Anastasios E.; Rüegg, Joëlle; Spyrou, Giannis

    2015-01-01

    The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis. It transfers electrons from NADPH to a large variety of substrates, particularly to those containing redox-active cysteines. Previously, we reported that the classical form of cytosolic TrxR1 (TXNRD1_v1), when overexpressed in human embryonic kidney cells (HEK-293), prompted the cells to undergo differentiation [Nalvarte et al. (2004) J. Biol. Chem. 279, 54510–54517]. In the present study, we show that several genes associated with differentiation and adhesion are differentially expressed in HEK-293 cells stably overexpressing TXNRD1_v1 compared with cells expressing its splice variant TXNRD1_v2. Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity. Furthermore, differentiation of the neuroblastoma cell line SH-SY5Y induced by all-trans retinoic acid (ATRA) increased both TXNRD1_v1 and TXNRD1_v2 expressions along with several of the identified genes associated with differentiation and adhesion. Selenium supplementation in the SH-SY5Y cells also induced a differentiated morphology and changed expression of the adhesion protein fibronectin 1 and the differentiation marker cadherin 11, as well as different temporal expression of the studied TXNRD1 variants. These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1. PMID:26464515

  4. Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study

    Directory of Open Access Journals (Sweden)

    Hermanova Marketa

    2010-05-01

    Full Text Available Abstract Background We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2 and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA and inhibitors of lipoxygenases (LOX and cyclooxygenases (COX. This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Methods Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Results Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2 or SH-SY5Y after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX in combination with ATRA in both cell lines. Conclusions Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

  5. Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study.

    Science.gov (United States)

    Chlapek, Petr; Redova, Martina; Zitterbart, Karel; Hermanova, Marketa; Sterba, Jaroslav; Veselska, Renata

    2010-05-11

    We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines. Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

  6. DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

    Energy Technology Data Exchange (ETDEWEB)

    Lafaye, Céline; Barbier, Ewa; Miscioscia, Audrey; Saint-Pierre, Christine [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Kraut, Alexandra; Couté, Yohann [Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S_1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France); Plo, Isabelle [INSERM, U1009, Institut Gustave Roussy, Université Paris 11, 114 rue Edouard Vaillant, Villejuif F-94805 (France); Gasparutto, Didier; Ravanat, Jean-Luc [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Breton, Jean, E-mail: jean.breton@cea.fr [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France)

    2014-03-28

    Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.

  7. SuHeXiang Wan essential oil alleviates amyloid beta induced memory impairment through inhibition of tau protein phosphorylation in mice.

    Science.gov (United States)

    Jeon, Songhee; Hur, Jinyoung; Jeong, Ha Jin; Koo, Byung-Soo; Pak, Sok Cheon

    2011-01-01

    SuHeXiang Wan (SHXW), a traditional Chinese medicine, has been used orally for the treatment of seizures, infantile convulsions and stroke. Previously, we reported the effects of a modified SHXW essential oil in terms of sedative effect, anticonvulsant activity and antioxidative activity. The purpose of this study was to evaluate the potential beneficial effects of SHXW essential oil in neurodegenerative diseases such as Alzheimer's disease (AD). SHXW essential oil was extracted from nine herbs. The mouse AD model was induced by a single injection of amyloid β protein (Aβ(1-42)) into the hippocampus. The animals were divided into four groups, the negative control group injected with Aβ(42-1), the Aβ group injected with Aβ(1-42), the SHXW group inhaled SHXW essential oil and received Aβ(1-42) injection, and the positive control group administered with docosahexaenoic acid (DHA, 10 mg/kg) and with subsequent Aβ(1-42) injection. Mice were analyzed by behavioral tests and immunological examination in the hippocampus. An additional in vitro investigation was performed to examine whether SHXW essential oil inhibits Aβ(1-42) induced neurotoxicity in a human neuroblastoma cell line, SH-SY5Y cells. Pre-inhalation of SHXW essential oil improved the Aβ(1-42) induced memory impairment and suppressed Aβ(1-42) induced JNK, p38 and Tau phosphorylation in the hippocampus. SHXW essential oil suppressed Aβ-induced apoptosis and ROS production via an up-regulation of HO-1 and Nrf2 expression in SH-SY5Y cells. The present study suggests that SHXW essential oil may have potential as a therapeutic inhalation drug for the prevention and treatment of AD.

  8. H2O2 attenuates IGF-1R tyrosine phosphorylation and its survival signaling properties in neuronal cells via NR2B containing NMDA receptor.

    Science.gov (United States)

    Zeng, Zhiwen; Wang, Dejun; Gaur, Uma; Rifang, Liao; Wang, Haitao; Zheng, Wenhua

    2017-09-12

    Impairment of insulin-like growth factor I (IGF-I) signaling plays an important role in the development of neurodegeneration. In the present study, we investigated the effect of H 2 O 2 on the survival signaling of IGF-1 and its underlying mechanisms in human neuronal cells SH-SY5Y. Our results showed that IGF-1 promoted cell survival and stimulated phosphorylation of IGF-1R as well as its downstream targets like AKT and ERK1/2 in these cells. Meanwhile, these effects of IGF-1 were abolished by H 2 O 2 at 200μM concentration which did not cause any significant toxicity to cells itself in our experiments. Moreover, studies using various glutamate receptor subtype antagonists displayed that N-methyl-D -aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) blocked the effects of H 2 O 2 , whereas other glutamate receptor subtype antagonists, such as non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), metabolic glutamate receptor antagonists LY341495 and CPCCOEt, had no effect. Further studies revealed that NR2B-containing NMDARs are responsible for these effects as its effects were blocked by pharmacological inhibitor Ro25-698 or specific siRNA for NR2B, but not NR2A. Finally, our data also showed that Ca 2+ influx contributes to the effects of H 2 O 2 . Similar results were obtained in primary cultured cortical neurons. Taken together, the results from the present study suggested that H 2 O 2 attenuated IGF-1R tyrosine phosphorylation and its survival signaling properties via NR2B containing NMDA receptors and Ca 2+ influx in SH-SY5Y cells. Therefore, NMDAR antagonists, especially NR2B-selective ones, combined with IGF-1 may serve as an alternative therapeutic agent for oxidative stress related neurodegenerative disease.

  9. DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

    International Nuclear Information System (INIS)

    3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Lafaye, Céline; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Barbier, Ewa; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Miscioscia, Audrey; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Saint-Pierre, Christine; 1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" >Kraut, Alexandra; 1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" >Couté, Yohann; Plo, Isabelle; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Gasparutto, Didier; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Ravanat, Jean-Luc; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Breton, Jean

    2014-01-01

    Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis

  10. In vitro study of the neuropathic potential of the organophosphorus compounds fenamiphos and profenofos: Comparison with mipafox and paraoxon.

    Science.gov (United States)

    Emerick, Guilherme L; Fernandes, Laís S; de Paula, Eloísa Silva; Barbosa, Fernando; dos Santos, Neife Aparecida Guinaim; dos Santos, Antonio Cardozo

    2015-08-01

    Organophosphorus-induced delayed neuropathy (OPIDN) is a central-peripheral distal axonopathy that develops 8-14 days after poisoning by a neuropathic organophosphorus compound (OP). Several OPs that caused OPIDN were withdrawn from the agricultural market due to induction of serious delayed effects. Therefore, the development of in vitro screenings able to differentiate neuropathic from non-neuropathic OPs is of crucial importance. Thus, the aim of this study was to evaluate the differences in the neurotoxic effects of mipafox (neuropathic OP) and paraoxon (non-neuropathic OP) in SH-SY5Y human neuroblastoma cells, using the inhibition and aging of neuropathy target esterase (NTE), inhibition of acetylcholinesterase (AChE), activation of calpain, neurite outgrowth, cytotoxicity and intracellular calcium as indicators. Additionally, the potential of fenamiphos and profenofos to cause acute and/or delayed effects was also evaluated. Mipafox had the lowest IC50 and induced the highest percentage of aging of NTE among the OPs evaluated. Only mipafox was able to cause calpain activation after 24 h of incubation. Concentrations of mipafox and fenamiphos which inhibited at least 70% of NTE were also able to reduce neurite outgrowth. Cytotoxicity was higher in non-neuropathic than in neuropathic OPs while the intracellular calcium levels were higher in neuropathic than in non-neuropathic OPs. In conclusion, the SH-SY5Y cellular model was selective to differentiate neuropathic from non-neuropathic OPs; fenamiphos, but not profenofos presented results compatible with the induction of OPIDN. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Encapsulation of curcumin in polyelectrolyte nanocapsules and their neuroprotective activity

    Science.gov (United States)

    Szczepanowicz, Krzysztof; Jantas, Danuta; Piotrowski, Marek; Staroń, Jakub; Leśkiewicz, Monika; Regulska, Magdalena; Lasoń, Władysław; Warszyński, Piotr

    2016-09-01

    Poor water solubility and low bioavailability of lipophilic drugs can be potentially improved with the use of delivery systems. In this study, encapsulation of nanoemulsion droplets was utilized to prepare curcumin nanocarriers. Nanosize droplets containing the drug were encapsulated in polyelectrolyte shells formed by the layer-by-layer (LbL) adsorption of biocompatible polyelectrolytes: poly-L-lysine (PLL) and poly-L-glutamic acid (PGA). The size of synthesized nanocapsules was around 100 nm. Their biocompatibility and neuroprotective effects were evaluated on the SH-SY5Y human neuroblastoma cell line using cell viability/toxicity assays (MTT reduction, LDH release). Statistically significant toxic effect was clearly observed for PLL coated nanocapsules (reduction in cell viability about 20%-60%), while nanocapsules with PLL/PGA coating did not evoke any detrimental effects on SH-SY5Y cells. Curcumin encapsulated in PLL/PGA showed similar neuroprotective activity against hydrogen peroxide (H2O2)-induced cell damage, as did 5 μM curcumin pre-dissolved in DMSO (about 16% of protection). Determination of concentration of curcumin in cell lysate confirmed that curcumin in nanocapsules has cell protective effect in lower concentrations (at least 20 times) than when given alone. Intracellular mechanisms of encapsulated curcumin-mediated protection engaged the prevention of the H2O2-induced decrease in mitochondrial membrane potential (MMP) but did not attenuate Reactive Oxygen Species (ROS) formation. The obtained results indicate the utility of PLL/PGA shell nanocapsules as a promising, alternative way of curcumin delivery for neuroprotective purposes with improved efficiency and reduced toxicity.

  12. Apoptotic DNA Degradation into Oligonucleosomal Fragments, but Not Apoptotic Nuclear Morphology, Relies on a Cytosolic Pool of DFF40/CAD Endonuclease*

    Science.gov (United States)

    Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Gabernet, Gisela; García-Belinchón, Mercè; Sánchez-Osuna, María; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.

    2012-01-01

    Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. PMID:22253444

  13. Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Seung Taek; Kim, Dae Hong; Lee, Min Bum; Nam, Hyo Jung; Kang, Jin Ku; Park, Mi Jung; Lee, Ik Hwan [Department of Life Science, College of Natural Science, Daejin University, Pocheon, Gyeonggido 487-711 (Korea, Republic of); Seok, Heon [Department of Biomedical Science, Jungwon University, Goesan, Chungcheongbukdo 367-700 (Korea, Republic of); Lee, Dong Gun [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Hwang, Jae Sam [Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441-707 (Korea, Republic of); Kim, Ho, E-mail: hokim@daejin.ac.kr [Department of Life Science, College of Natural Science, Daejin University, Pocheon, Gyeonggido 487-711 (Korea, Republic of)

    2013-07-19

    Highlights: •CopA3 peptide isolated from the Korean dung beetle has antimicrobial activity. •Our study reported that CopA3 has anticancer and immunosuppressive effects. •We here demonstrated that CopA3 has neurotropic and neuroprotective effects. •CopA3 degrades p27Kip1 protein and this mediates effects of CopA3 on neuronal cells. -- Abstract: We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer’s disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.

  14. DNA Methylation Analysis of BRD1 Promoter Regions and the Schizophrenia rs138880 Risk Allele.

    Directory of Open Access Journals (Sweden)

    Mads Dyrvig

    Full Text Available The bromodomain containing 1 gene, BRD1 is essential for embryogenesis and CNS development. It encodes a protein that participates in histone modifying complexes and thereby regulates the expression of a large number of genes. Genetic variants in the BRD1 locus show association with schizophrenia and bipolar disorder and risk alleles in the promoter region correlate with reduced BRD1 expression. Insights into the transcriptional regulation of BRD1 and the pathogenic mechanisms associated with BRD1 risk variants, however, remain sparse. By studying transcripts in human HeLa and SH-SY5Y cells we provide evidence for differences in relative expression of BRD1 transcripts with three alternative 5' UTRs (exon 1C, 1B, and 1A. We further show that expression of these transcript variants covaries negatively with DNA methylation proportions in their upstream promoter regions suggesting that promoter usage might be regulated by DNA methylation. In line with findings that the risk allele of the rs138880 SNP in the BRD1 promoter region correlates with reduced BRD1 expression, we find that it is also associated with moderate regional BRD1 promoter hypermethylation in both adipose tissue and blood. Importantly, we demonstrate by inspecting available DNA methylation and expression data that these regions undergo changes in methylation during fetal brain development and that differences in their methylation proportions in fetal compared to postnatal frontal cortex correlate significantly with BRD1 expression. These findings suggest that BRD1 may be dysregulated in both the developing and mature brain of risk allele carriers. Finally, we demonstrate that commonly used mood stabilizers Lithium, Valproate, and Carbamazepine affect the expression of BRD1 in SH-SY5Y cells. Altogether this study indicates a link between genetic risk and epigenetic dysregulation of BRD1 which raises interesting perspectives for targeting the mechanisms pharmacologically.

  15. Redundant dopaminergic activity may enable compensatory axonal sprouting in Parkinson disease.

    Science.gov (United States)

    Arkadir, David; Bergman, Hagai; Fahn, Stanley

    2014-03-25

    Neurodegenerative diseases become clinically apparent only after a substantial population of neurons is lost. This raises the possibility of compensatory mechanisms in the early phase of these diseases. The importance of understanding these mechanisms cannot be underestimated because it may guide future disease-modifying strategies. Because the anatomy and physiology of the nigrostriatal dopaminergic pathways have been well described, the study of Parkinson disease can offer insight into these early compensatory mechanisms. Collateral axonal sprouting of dopaminergic terminals into the denervated striatum is the most studied compensatory mechanism in animal (almost exclusively rodent) models of Parkinson disease and is correlated with behavioral recovery after partial lesions. This sprouting, however, does not respect the normal anatomy of the original nigrostriatal pathways and leads to aberrant neuronal networks. We suggest here that the unique physiologic property of the dopaminergic innervation of the striatum, namely redundancy of information encoding, is crucial to the efficacy of compensatory axonal sprouting in the presence of aberrant anatomical connections. Redundant information encoding results from the similarity of representation of salient and rewarding events by many dopaminergic neurons, from the wide axonal field of a single dopaminergic neuron in the striatum, and from the nonspecific spatial effect of dopamine on striatal neurons (volume conductance). Finally, we discuss the relevance of these findings in animal models to human patients with Parkinson disease.

  16. Dopaminergic Polymorphisms, Academic Achievement, and Violent Delinquency.

    Science.gov (United States)

    Yun, Ilhong; Lee, Julak; Kim, Seung-Gon

    2015-12-01

    Recent research in the field of educational psychology points to the salience of self-control in accounting for the variance in students' report card grades. At the same time, a novel empirical study from molecular genetics drawing on the National Longitudinal Study of Adolescent Health (Add Health) data has revealed that polymorphisms in three dopaminergic genes (dopamine transporter [DAT1], dopamine D2 receptor [DRD2], and dopamine D4 receptor [DRD4]) are also linked to adolescents' grade point averages (GPAs). Juxtaposing these two lines of research, the current study reanalyzed the Add Health genetic subsample to assess the relative effects of these dopaminergic genes and self-control on GPAs. The results showed that the effects of the latter were far stronger than those of the former. The interaction effects between the dopaminergic genes and a set of environmental factors on academic performance were also examined, producing findings that are aligned with the "social push hypothesis" in behavioral genetics. Finally, based on the criminological literature on the link between academic performance and delinquency, we tested whether dopaminergic effects on violent delinquency were mediated by GPAs. The results demonstrated that academic performance fully mediated the linkage between these genes and violent delinquency. © The Author(s) 2014.

  17. Non-genomic actions of retinoic acid induce pi3k signaling pathway and phosphorylation of nuclear proteins

    OpenAIRE

    Laserna Mendieta, Emilio J.; Masiá, Susana; Barettino, Domingo

    2007-01-01

    Retinoic acid (RA), the active form of vitamin A, induces neuroblastoma cells SH-SY5Y to differentiate. In addition to its classical transcriptional actions regulating the expression of specific genes, RA acts in an extra-genomic way, modulating the activity of relevant signalling cascades. In particular, RA treatment of