Biochemistry and Cell Biology of alpha-Synucleinopathies

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Parkinson disease, dementia with Lewy bodies and multiple system atrophy are neurodegenerative disorders referred to as synucleinopathies. They are characterized by accumulation of alpha-synuclein (alpha-SN) as fibrillar inclusions in selectively vulnerable neurons and gila. Our understanding of the cellular mechanisms underlying the formation of alpha-SN inclusions and the toxicity of alpha-SN is poorly understood. In order to better understand the process of alpha-SN aggregation, our laboratory has used human neuronal [BE(2)-M17D] cells and the tetracycline-off (TetOff) inducible mechanism to generate cells overexpressing wild-type human alpha-SN. After 7 days of induction, sarkosyl-insoluble alpha-SN aggregates are detected in these cells. Exposure of our cell model to rotenone, a potent mitochondrial complex I inhibitor, led to the formation of RIPA buffer-insoluble, higher molecular weight sarkosyl-soluble alpha-SN species (consistent with oligomers) and sarkosyl-insoluble alpha-SN within five days. The oligomers were detected on Western blots as discrete bands containing truncated alpha-SN, and they exhibited no immunoreactivity with antibodies to alpha-SN phosphorylated at Ser129 or ubiquitin. Such oligomers, which were barely detectable in cells without rotenone treatment, may serve as seeds for alpha-SN polymerization or may be toxic to cells when they reach a critical concentration. In the studies proposed below, we plan to characterize alpha-SN oligomers further, focusing on determining (i) whether similar oligomers are produced in cells exposed to other neurotoxins relevant to PD and can be found in brains of familial and sporadic synucleinopathies, (ii) the mechanisms involved in their formation, and (iii) their role in alpha-SN assembly and neurotoxicity. In addition, we will determine (iv) whether overexpressing alpha-SN alters the gene expression profile in neuronal cells. These studies will contribute to our understanding of the formation of alpha-SN inclusions and their selective neurotoxicity for neurons.