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Hepcidin Decreases Rotenone-Induced ?-Synuclein Accumulation via Autophagy in SH-SY5Y Cells.

ABSTRACT: Parkinson's disease (PD) is a neurodegenerative disorder, and the hallmarks of this disease include iron deposition and ?-synuclein (?-syn) aggregation. Hepcidin could reduce iron in the central and peripheral nervous systems. Here, we hypothesized that hepcidin could further decrease ?-syn accumulation via reducing iron. Therefore, rotenone or ?-syn was introduced into human neuroblastoma SH-SY5Y cells to imitate the pathological progress of PD in vitro. This study investigated the clearance effects of hepcidin on ?-syn induced by a relatively low concentration of rotenone exposure or ?-syn overexpression to elucidate the potential clearance pathway involved in this process. We demonstrated that SH-SY5Y cell viability was impaired after rotenone treatment in a dose-dependent manner. ?-syn expression and iron content increased under a low concentration rotenone (25 nM for 3 days) treatment in SH-SY5Y cells. Pre-treatment with hepcidin peptide suppressed the abovementioned effects of rotenone. However, hepcidin did not affect treatment with rotenone under high iron conditions. Hepcidin also played a role in reducing ?-syn accumulation in rotenone and ?-syn overexpression conditions. We identified that the probable clearance effect of hepcidin on ?-syn was mediated by the autophagy pathway using pretreatment with autophagy inhibitors (3-MA and CQ) and detection of autophagy protein markers (LC3II/I and p62). In conclusion, hepcidin eliminated ?-syn expression via the autophagy pathway in rotenone-treated and ?-syn overexpression SH-SY5Y cells. This study highlights that hepcidin may offer a potential therapeutic perspective in ?-syn accumulation diseases.

SUBMITTER: Li M

PROVIDER: S-EPMC7596286 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Hepcidin Decreases Rotenone-Induced α-Synuclein Accumulation via Autophagy in SH-SY5Y Cells.

Li Meiqi M Hu Jianan J Yuan Xiaoyu X Shen Lihua L Zhu Li L Luo Qianqian Q

Frontiers in molecular neuroscience 20201016

Parkinson's disease (PD) is a neurodegenerative disorder, and the hallmarks of this disease include iron deposition and α-synuclein (α-syn) aggregation. Hepcidin could reduce iron in the central and peripheral nervous systems. Here, we hypothesized that hepcidin could further decrease α-syn accumulation via reducing iron. Therefore, rotenone or α-syn was introduced into human neuroblastoma SH-SY5Y cells to imitate the pathological progress of PD in vitro. This study investigated th ...[more]

PMID: 33177988

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Project description:Parkinson's disease (PD) is characterized by selective degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). α-synuclein (α-syn) is known to regulate mitochondrial function and both PINK1 and Parkin have been shown to eliminate damaged mitochondria in PD. Mechanistic target of rapamycin (mTOR) is expressed in several distinct subcellular compartments and mediates the effects of nutrients, growth factors, and stress on cell growth. However, the contributions of these various regulators to DAergic cell death have been demonstrated mainly in culture with serum, which is known to dramatically influence endogenous growth rate and toxin susceptibility through nutrient and growth factor signaling. Therefore, we compared neurotoxicity induced by the mitochondrial inhibitor rotenone (ROT, 5 or 10 μM for 24 h) in SH-SY5Y cells cultured with 10% fetal bovine serum (FBS), 1% FBS, or 1% bovine serum albumin (BSA, serum-free). In addition, C57BL/6J mice were injected with 12 μg ROT into the right striatum, and brains examined by histology and Western blotting 2 weeks later for evidence of DAergic cell death and the underlying signaling mechanisms. ROT dose-dependently reduced SH-SY5Y cell viability in all serum groups without a significant effect of serum concentration. ROT injection also significantly reduced immunoreactivity for the DAergic cell marker tyrosine hydroxylase (TH) in both the mouse striatum and SNpc. Western blotting revealed that ROT inhibited TH and Parkin expression while increasing α-syn and PINK1 expression in both SH-SY5Y cells and injected mice, consistent with disruption of mitochondrial function. Moreover, expression levels of the mTOR signaling pathway components mTORC, AMP-activated protein kinase (AMPK), ULK1, and ATG13 were altered in ROT-induced PD. Further, serum level influenced mTOR signaling in the absence of ROT and the changes in response to ROT. Signs of endoplasmic reticulum (ER) stress and altered expression of tethering proteins mediating mitochondria-associated ER contacts (MAMs) were also altered concomitant with ROT-induced neurodegeneration. Taken together, this study demonstrates that complex mechanism involving mitochondrial dysfunction, altered mTOR nutrient-sensing pathways, ER stress, and disrupted MAM protein dynamics are involved in DAergic neurodegeneration in response to ROT.

Project description:Complex pathophysiology of Parkinson's disease involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in Parkinson's disease and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. 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 Ca(2+) 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 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 up-regulated in the cholinergic phenotype, which could be dose-dependently attenuated by the calpain inhibitor. Overall, SNJ-1945 was efficacious against MPP(+) or rotenone-induced reactive oxygen species 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. SH-SY5Y cells, differentiated as dopaminergic (TH positive) and cholinergic (ChAT positive), were used as in vitro models for Parkinson's disease. MPP+ and rotenone induced up-regulation of calpain, expression, and activity as a common mechanism of neurodegeneration. SNJ-1945, a novel calpain inhibitor, protected both the cell phenotypes against MPP+ and rotenone.

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Hepcidin Decreases Rotenone-Induced ?-Synuclein Accumulation via Autophagy in SH-SY5Y Cells.

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Hepcidin Decreases Rotenone-Induced α-Synuclein Accumulation <i>via</i> Autophagy in SH-SY5Y Cells.

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