The Role Of Dynein In The Autophagic Clearance Of α-synuclein

PartⅠMPP+ impairs autophagic clearance of alpha-synuclein by impairing the activity of dyneinObjective: To determine whether MPP+ (1-methyl-4-phenylpyridinium) induces the up-regulation/aggregation ofα-synuclein by interfering the activity of dynein, leading to the impairment of autophagosome-lysosome fusion and autophagic clearance ofα-synuclein.Methods: Before and after treatment of MPP+, we measured cell viability through the MTT method, assessed the rate of apoptosis by Flow Cytometry, and detected the expression levels ofα-synuclein, microtubule-associated protein light chain 3(LC3-Ⅱ) and dynein through Western Blot and the expression levels ofα-synuclein and LC3 through RT-PCR. And immunofluorescence microscopy was performed to observe the signals of immunofluorescence forα-synuclein, LC3, dynein and lysosomal-associated membrane protein 1 (LAMP1) and their co-localization in PC12 and stably over-expressing human A30Pα-synuclein PC12 cell lines. In addition, we labelled the autophagic vacuoles (AVs) by MDC staining.Results: After treated with MPP+ for 24 h, cells became shrinked and round, many cells divorced from plate wall, some processes shortened and were broken. Cell viability significantly decreased and the rate of apoptosis increased in MPP+-treated groups, especially in MPP+ treated stably over-expressing human A30Pα-synuclein PC12 cell lines group. The expression ofα-synuclein, LC3 and dynein was obviously increased in the level of protein and mRNA after treatment of MPP+, compared with untreated group, while dynein was decreased in the level of protein. The immunofluorescent results showed that the signals forα-synuclein, LC3 and their co-localization increased in MPP+-treated PC12 cells, on the contrary, it is reduced for dynein and LAMP1, and their co-localization with α-synuclein and LC3. Besides, MDC staining indicated increase in both the average number of L-AVs per cell and the distribution size of L-AVs in MPP+ treated PC12 and stably over-expressing human A30Pα-synuclein PC12 cell lines group, especially in MPP+ treated stably over-expressing human A30Pα-synuclein PC12 cell lines group. Forthermore, integrated optical density (IOD) of MPP+-treated groups was also higher than untreated groups.Conclusion: These data demonstrated that MPP+ could impair autophagic clearance ofα-synuclein by interfering the activity of dynein, inducing the up-regulation/aggregation ofα-synuclein, which may be deleterious to cells. Accordingly, we consider that dynein is a vinculum between environmental factor(because MPP+ can be regarded as an environmental factor,which is associated with PD) and Parkinson's disease(PD), and it may be a potential therapeutic target of neurodegenerative diseases.PartⅡThe role of dynein activity in the autophagic clearance ofα-synucleinObjective: To determine whether ATP and EHNA interfere the activity of dynein, leading to the impairment of autophagosome-lysosome fusion and autophagic clearance ofα-synuclein.Methods: In this study, we use MPP+(0.5mM),EHNA(0.2mM),ATP(10um) to investigate pheochromocytoma (PC12) cell lines. Cell viability was measured by means of MTT methods. LC3-Ⅱ,α-synuclein,dynein expressions in cell culture were determined by Western Blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy.Results: After treated with MPP+ and EHNA for 24 h, the expression ofα-synuclein and LC3-II were obviously increased, but the level of dynein decreased. After treated with ATP again, this trend has been reversed. The results of immunofluorescent were similar to those of Western Blot. We found that the integrated optical density (IOD) ofα-synuclein and LC3-II increased in MPP+ and EHNA treated group. After adding ATP the IOD of dynein significantly increased, thereby reducing the IOD ofα-synuclein and LC3. The hallmarks of apoptosis and autophagy in our cell lines using electron microscopy after 24 h drug treatment also showed that MPP+ and EHNA increased apoptosis and autophagy, and ATP promotes autophagosome and lysosome fusion. Overexpressed dynein upregulated its function and promotedα-synuclein autophagic clearance (P <0.01).Conclusion: MPP+ and EHNA inhibited dynein activity, which hinders its transport function, leading to a decline in the degree of autophagosome and lysosome fusion, impairing the autophagic clearance ofα-synuclein. ATP reverses this pathological change by increasing the activity of dynein, and increase the autophagic clearance ofα-synuclein. From the perspective of increaseing dynein activity to treat Parkinson's disease is an important potential target.