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AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

ABSTRACT: Therapeutic strategies are needed to protect dopaminergic neurons in Parkinson's disease (PD) patients. Oxidative stress caused by dopamine may play an important role in PD pathogenesis. Selective autophagy of mitochondria (mitophagy), mainly regulated by PINK1 and PARKIN, plays an important role in the maintenance of cell homeostasis. Mutations in those genes cause accumulation of damaged mitochondria, leading to nigral degeneration and early-onset PD. AMBRA1ActA is a fusion protein specifically expressed at the mitochondria, and whose expression has been shown to induce a powerful mitophagy in mammalian cells. Most importantly, the pro-autophagy factor AMBRA1 is sufficient to restore mitophagy in fibroblasts of PD patients carrying PINK1 and PARKIN mutations. In this study, we investigated the potential neuroprotective effect of AMBRA1-induced mitophagy against 6-hydroxydopamine (6-OHDA)- and rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We demonstrated that AMBRA1ActA overexpression was sufficient to induce mitochondrial clearance in SH-SY5Y cells. We found that apoptosis induced by 6-OHDA and rotenone was reversed by AMBRA1-induced mitophagy. Finally, transfection of SH-SY5Y cells with a vector encoding AMBRA1ActA significantly reduced 6-OHDA and rotenone-induced generation of reactive oxygen species (ROS). Altogether, our results indicate that AMBRA1ActA is able to induce mitophagy in SH-SY5Y cells in order to suppress oxidative stress and apoptosis induced by both 6-OHDA and rotenone. These results strongly suggest that AMBRA1 may have promising neuroprotective properties with an important role in limiting ROS-induced dopaminergic cell death, and the utmost potential to prevent PD or other neurodegenerative diseases associated with mitochondrial oxidative stress.

SUBMITTER: Di Rita A

PROVIDER: S-EPMC5932353 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

Di Rita Anthea A D'Acunzo Pasquale P Simula Luca L Campello Silvia S Strappazzon Flavie F Cecconi Francesco F

Frontiers in cellular neuroscience 20180418

Therapeutic strategies are needed to protect dopaminergic neurons in Parkinson's disease (PD) patients. Oxidative stress caused by dopamine may play an important role in PD pathogenesis. Selective autophagy of mitochondria (mitophagy), mainly regulated by PINK1 and PARKIN, plays an important role in the maintenance of cell homeostasis. Mutations in those genes cause accumulation of damaged mitochondria, leading to nigral degeneration and early-onset PD. AMBRA1<sup>ActA</sup> is a fusion protein ...[more]

PMID: 29755319

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Project description:BackgroundObesity-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.MethodsHuman 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.ResultsMTT 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-induced leptin resistance. Annexin V/PI flow cytometry analysis revealed PA-induced increase in percentages of cells in annexin V-positive/PI-negative quadrant (early apoptosis) and subG0-G1 fraction, accompanied by a decrease in G2-M phase cells. The PA-induced ROS production and lipid peroxidation was at greater extent in T98G as compared to that in SH-SY5Y.DiscussionIn conclusion, PA induces apoptosis by increasing oxidative stress in neurons and astrocytes. Taken together, the results suggest that HFD may cause neuronal and astrocytic damage, which indirectly proposes that CNS pathologies involving neuroinflammation and reactive gliosis could be prevented via the diet regimen.

Dataset Information

AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

Publications

AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

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