Objective: To elucidate the role of α-synuclein, parkin and mitochondria in a cell-based model of neurodegeneration relevant to Parkinson’s disease pathogenesis.

Background: The suggested mechanisms of PD pathogenesis have been deciphered using various experimental models,mainly the toxin-based models,which have questionable relevance in explaining the pathogenesis of sporadic PD. Thus, a model of neurodegeneration in cultured cells relevant to PD employing toxic effects of endogenous molecules like dopamine (DA) and iron would be relevant. 

Methods: SHSY5Y neuroblastoma cells were treated with varying concentrations of iron (20-100 µM) and dopamine (10 – 50 µM) for a variable period of time. Cell death was assessed by trypan blue dye-exclusion test and lactate dehydrogenase release assays. The nature of cell death was analyzed by examining nuclear morphology after PI and Hoechst staining. Mitochondrial parameters were analyzed using JC-1 dye and ATP synthesis assays. α-Synuclein, parkin and Bax expressions were analyzed by Western blotting and qRT-PCR for mRNA. α-synuclein and parkin knockdown was carried out by using specific siRNA. 

Results: Both DA and iron causes dose-dependent and time-dependent cell death.10 µM DA over a period of 96 h produces nearly 40% cell death along with decreases of mitochondrial membrane potential and ATP synthesis but the degree of cell death by iron was much lower. However, similar intra-cellular accumulation of  α-synuclein took place in both conditions. When parkin expression levels were compared between DA-treated and iron-treated cells, a significant increase of parkin expression were noticed after iron, but not DA exposure suggesting a protective action of parkin against dysfunctional mitochondria. We are currently verifying this protective role of parkin by knock-down experiments. The nature of cell death appears to be apoptosis and secondary necrosis.The involvement of Bax in DA or iron mediated cell death will also be explored. 

Conclusions: The results strongly suggest that oxidative stress mediated by DA or iron can initiate neurodegeneration through the involvement of α-synuclein and mitochondria. Parkin may play a protective role against neurodegeneration by preventing dysfunction of mitochondria. An increased accumulation of α-synuclein and downregulation of parkin in sporadic PD brain have been documented.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/alpha-synucleinopathy-and-mitochondrial-dysfunction-in-a-cell-based-model-of-neurodegeneration-implications-in-the-pathogenesis-of-sporadic-parkinsons-disease/