The Role Of Mitochondria-associated Proteins In Regulating Synucleinopathies

PINK1 is a mitochondrial serine/threonine kinase, which protects mitochondria from cellular stress and regulates neuronal differentiation. Mutations in PINK1(PARK6) are cause of autosomal recessive early-onset Parkinson’s disease(PD). β-synuclein(β-syn) is homologous to α-synuclein(α-syn), its missense mutation(P123H) has been linked to familial dementia with Lewy bodies(DLBs). β-syn P123 H formed eosinophilic inclusion bodies in cells and colocalized with LAMP-2, ATP13A2, cathepsin as well as other lysosomal markers. The aggregation of β-syn P123 H in cytoplasm induce apoptosis of cells and it can produce toxicity which will accelerate neurodegeneration. PINK1 maintains mitochondrial homeostasis, and plays a key role in the pathological process of PD, but the mechanism of PINK1 mediated synucleinopathies processes by mitochondria is unclear. Using the HeLa cells stably expressing Parkin and the B103 neuroblastoma cells stably expressing β-syn P123 H, this project will explore the protective effect of PINK1/Parkin on mitophagy, and investigate the role and molecular mechanism of PINK1 on β-syn P123 H autophagic degradation.In this paper the wild-type PINK1(PINK1 WT) and the eukaryotic expression plasmid PINK1 mutant(Q126P, E240 K, G309 D, L347 P, P498L) were constructed through PCR and two-step PCR. Next we transiently transfected PINK1 WT and mutants in HeLa cells to ensure the plasmid highly expressed. WST-1 cell activity assay and LDH cytotoxicity assay were conducted to analysis the effects of PINK1 WT and mutants on cell survival. The results showed that PINK1 mutations caused cell activity reduction and toxicity. Compared to controls, no significant difference was observed in HeLa cells transfected with wild PINK1.Immunocytochemical stain was performed to observe the localization of PINK1 WT and mutants, as well as the distribution of Parkin T240 R and R275 W in HeLa cells. Using mitochondrial TMRE fluorescence labeling technology, FCCP group as a positive control, the effect of Parkin T240 R and R275 W on mitochondrial membrane potential was investigated. In oder to study the effect of PINK1 and Parkin gene on mitochondrial dynamics, fusion and fission protein were analyzed by liposome transfection and Western Blot. The results of immunofluorescence cytochemistry showed that: PINK1 mutations induce mitochondrial rupture and aggregation. Parkin mutants aggregate in HeLa cells and lead to reduction of mitochondrial membrane potential. Western blot showed that: The dynamic balance between mitochondrial fusion and fission was destroyed in cells with PINK1 and Parkin mutant. Wild-type PINK1 and Parkin are involved in mitochondrial quality control, and they can promote the β-syn P123 H autophagic degradation.Immunofluorescence experiments show that β-syn P123 H and Parkin aggregates were degraded through the autophagy-lysosome pathway, and Parkin T240 R increase β-syn P123 H aggregates formation. To investigate the effects of autophagy on cell apoptosis induced by β-synP123 H and ParkinT240 R, autophagy inducer Rapamycin and inhibitors 3-MA, NH4 Cl were used in TUNEL assay. The percentage of TUNEL positive cells was calculated under a fluorescent microscope, and the statistic results indicated that: compared with the control group with DMSO, the dead cells treated with the autophagy inhibitors 3-MA and NH4 Cl increased, but the number of rapamycin group decreased obviously.In summary, PINK1 mutations induce mitochondrial rupture and aggregation, and result in cell activity reduction and toxicity. Parkin mutants aggregate in cells and lead to reduction of mitochondrial membrane potential. Parkin and β-syn P123 H aggregates are degraded by autophagy-lysosome pathway, and Parkin mutants increase β-syn P123 H aggregates formation. Wild-type PINK1 and Parkin are involved in mitochondrial quality control, and they can promote the β-syn P123 H autophagic degradation. Mitochondrial autophagy and fission/fusion were mediated by mitochondrial-associated protein PINK1 and Parkin, and they can play a protective action in synucleinopathies induced by β-synP123 H.