The Role Of Parkinson’s Disease-associated Protein Parkin On Synucleinopathies Induced By β-synuclein P123H

Parkin/PARK2is an E3ubiquitin ligase associated with autosomal-recessive Parkinsonism. Themolecular weight of Parkin protein is52kD. Parkin protein is nearly dispersed in the cytoplasm. Parkinprotects against a variety of cytotoxic stress and plays an important role in the protection of Parkinsonism.Lysosome-like inclusions can be formed by β-synP123H in cells, resulting in cell degeneration.At home and abroad, there were many articles reported that Parkin could mediate mitophagy. Butthe roles of Parkin in the process of β-synP123H synucleinopathies and autophagy are not clear. Thisproject will focus on the mechanism and regulation of Parkin and its mutants on synuclein degradation andautophagy in the process of β-synP123H synucleinopathies and autophagy.First, we obtained Parkin wild-type (ParkinWT) and its mutants with HA-tagged by two-stepPCR, and then constructed the plasmid. Next we transiently transfected ParkinWT and mutants in Helacells to ensure the plasmid can be expressed. Then we explored the distribution of ParkinWT and itsmutants in Hela cells by cell climbing slice and immunofluorescence, we found that ParkinWT is nearlydispersed in the cytoplasm, while the mutants take the shape of small granular structures that formperinuclear inclusions. Then we analyzed the effects of ParkinWT and mutants on cell survival and toxicityby WST-1and LDH assay, and we found that Parkin mutants have cellular toxicity.Secondly, we constructed and screened ParkinWT and its mutants stable cells. In Parkin stablecells, we researched the roles of ParkinWT and its mutants on mitochondria by immunofluorescence stain.We found that Parkin mutants caused mitochondrial fragmentation and aggregation. After treatment in thestable cells by carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP), ParkinWT can be recruited on the impaired mitochondria, but Parkin mutants lost this function.Finally, in ParkinWT and mutant ParkinT240R stable cells, we transiently transfected pCEP4,β-synWT and β-synP123H, and researched the roles of ParkinWT and ParkinT240R on autophagy andβ-synP123H degradation by immunofluorescence and Western Blot. We found that ParkinWT can promoteβ-synP123H degradation by autophagy lysosomal pathway, and inhibit β-synP123H accumulation in cells,but Parkin mutants have the opposite roles in cells. We transiently transfected pTRE2hyg, ParkinWT andfour mutants in β-synP123H stable cells, and we found that the phosphorylation level of mTOR, p70S6K,4EBP-1and the relative expression of Atg5, β-syn, Beclin-1also verified this point.Conclusion: The distribution of ParkinWT and mutants is different in cells. Parkin mutants leadto mitochondrial fragmentation and aggregation which couldn’t be recruited on the impaired mitochondria,and have cellular toxicity, reduce cell viability, then induce cell degradation. ParkinWT regulates synucleinautophagy via mTOR pathway, promotes β-synP123H into autophagolysosome and further degradation,and plays a protective role in synucleinopathy cells. Disease-causing mutations in Parkin increase theaccumulation of β-syn P123H protein and enhance neurodegeneration associated with dementia with lewybodies.