Experimental Study Of The New Degradation Pathway Of α-synuclein And The Molecular Mechanism Of Neuroprotective Role Of Parkin

PARTⅠ The new degradation pathway of α-synucleinAim: α-synuclein is considered to be important in the pathogenesis of Parkinson’s disease(PD). Abnormal accumulation of α-synuclein is the main pathological characteristics of PD. Therefore, the clearance of α-synucelin effectively has been became the potential target for the treatment of PD. This study is thought to discuss whether α-synuclein is degraded by mitophagy.Methods: Overexpression of EGFP-α-synuclein and EGFP-α-synuclein-A53 T in SH-SY5 Y and He La cells. Mitochondrial isolate method as well as Western blot were performed to detect the accumulation of EGFP-α-synuclein and EGFP-α-synuclein-A53 T in mitochondria. Treat the cells with mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone(CCCP), autophagy enhancer rapamycin or autolysosome inhibitor Bafilomycin A1, fluorescence microscope was used to observe the changes in the green fluorescence of EGFP-α-synuclein and EGFP-α-synuclein-A53 T, Western blot was applied to detect protein level of EGFP-α-synuclein, EGFP-α-synuclein-A53 T, TOMM20 and COXⅣ. Besides, He La cells were co-transfected with Flag or Flag-Parkin along with EGFP-α-synuclein or EGFP-α-synuclein-A53 T. Fluorescence and Western blot analysis were applied to detect EGFP-α-synuclein and EGFP-α-synuclein-A53 T levels after treatment with CCCP. α-synuclein-A53 T transgenic mice were fed with food which contain CCCP for 8 months. Western blot analysis was used to determine the expression levels of α-synuclein in midbrain.Results: Mitochondrial isolate assay suggested that EGFP-α-synuclein and EGFP-α-synuclein-A53 T could accumulation in mitochondria. CCCP induced mitophagy significantly reduced EGFP-α-synuclein and EGFP-α-synuclein-A53 T fluorescence intensity and protein level in SH-SY5 Y or He La cells, but rapamycin could not. However, the autolysosome inhibitor Bafilomycin A1 could inhibit CCCP induced mitophagy. The decreased protein levels of EGFP-α-synuclein-A53 T were blocked by exposure to CCCP and Bafilomycin A1 at the same time. In He La cells, CCCP and rapamycin could not bring any changes on protein level of EGFP-α-synuclein and EGFP-α-synuclein-A53 T. CCCP induced mitophagy reduced the fluorescence intensity and protein level of EGFP-α-synuclein and EGFP-α-synuclein-A53 T after overexpression of Parkin in He La cells. Animal results showed that the expression levels of α-synuclein in midbrain dramatically reduced after feeding food which contained CCCP.Conclusion: α-synuclein and α-synuclein-A53 T could degradation by CCCP induced mitophagy. This may be become a new pathsway for α-synuclein degradation. PARTⅡ Parkin stabilizes p62 to repress 6-OHDA induced cell deathAim:Loss of function mutations in the PARK2 gene are associated with the occurance of Parkinson’s disease. This study is thought to explore the molecular mechanism of the protective effect of parkin on neurons.Methods: Immunoprecipitation assays were performed to identify the ubiquitination of p62 and interaction between parkin and p62. The cells were subjected to propidium iodide(PI) staining to identify the cell death. SH-SY5 Y cells that were transfected with p62 or parkin were treated wit h or without 6-OHDA. The activation of ERK was detected with Western blot. Overexpression of parkin in the He La cells which with or without p62, the levels of p-ERK1/2 and p62 were performed for Western blot assays.Results: Parkin interacts with p62 and ubiquitinates p62. Western blot showed that overexpression of parkin increases p62 protein level and represses ERK1/2 activation, however, parkin fails to inhibit ERK1/2 activation in p62 knockdown cells. These results suggest that parkin influences the activation of ERK1/2 is dependent on p62. Both parkin and p62 repress 6-OHDA-induced ERK1/2 activation. Furthermore, overexpression of parkin inhibits 6-OHDA-induced dopaminergic(DA) neuronal death..Conclusion: Parkin ubiquitinates and stabilizes p62 to reduce ERK1/2 activation induced by 6-OHDA and protects neuronal cells against 6-OHDA-induced cell death.