Parkinson’s Disease-associated Protein DJ-1Modulates The Experimental Synucleinopathies Induced By β-synuclein P123H

DJ-1/PARK7(Parkinson protein7) gene deletion or mutation is a causative reason for recessiveearly-onset Parkinson’s disease (PD). However, the cause induced by DJ-1mutants in Parkinson’s diseasehas not been understood. The β-Syn P123H accumulation in cytoplasm accelerate the neurodegeneration.In such process of degeneration the relation of DJ-1and β-Syn P123H is still unclear, so our purpose wasto investigate the role of DJ-1on synuclein metablism and explore the interaction of β-Syn P123H andDJ-1mutants in stable expression239T cell lines.First we reconstructed the pCDNA3.1DJ-1wild type and mutants plasmids, in which DJ-1mutationL166P, D149A, A104T was prepared by two-step PCR method for three mutated eukaryotic expressionplasmids. Full-length plasmids were determined by sequence. To establish the stable expressing synucleincell line, we transfected β-Syn P123H plasmid in293T cells using200μg/mL of Hygromycin B asresistance screening.Secondly, to determine the expression of transfected plasmids, the immunoblotting andimmunofluorescence stain were used to detect the tagged protein C-Myc of DJ-1and β-Syn P123H, andReal-time PCR to detect the DJ-1and β-Syn mRNA levels in stable cell lines.To explore the mitochondrial oxidative stress in stable expressing β-Syn P123H transfected DJ-1cells,mitochondrial were labeled by fluorescence after treatment of Carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP,10μg/mL). The cell viability and death rate were analyzed by WST-1, lactatedehydrogenase (LDH) release assay and TUNEL stain.Finally, we examined the expression of Atg-5, Beclin-1, LC3on mammalian autophagy pathway. Thephospho level of mTOR, p70-S6K,4E-BP1in mTOR pathway was probed by immunoblotting method.Lysosomal autophagy and proteasome inhibitors were used to identify the degradation pathway of β-SynP123H and reconstructing DJ-1protein.Conclusion: In β-Syn P123H overexpressing cells synuclein was prone to accumulation and aggregateto form the lysosomal inclusions. β-Syn P123H in cytoplasm causes mitochondrial depolarization, whileDJ-1WT might partly recover mitochondrial depolarization and relieve the effect of β-Syn P123H protein toxic aggregate by WST-1and LDH assay. Wild-type DJ-1dispersed in cytoplasm and nucleus, andenhanced β-Syn P123H degradation by lysosomal autophagy pathway. DJ-1mutants gathered inmitochondrial and was degraded by the proteasome. DJ-1mutants impaired the autophagy pathway andincrease the autophagic cell death. These results suggest DJ-1modulate synuclein metabolism inParkinson’s disease and other neurodegenerative disorders.