Abstract
Aim:
A growing body of evidence suggests that α-synuclein accumulation may play an important role in the pathogenesis of Parkinson's disease. The aim of this study was to investigate the roles of the proteasome and autophagy pathways in the clearance of wild-type and mutant α-synuclein in PC12 cells.
Methods:
PC12 cells overexpressing either wild-type or A30P mutant α-synuclein were treated with the proteasome inhibitor epoxomicin, the macroautophagy inhibitor 3-MA and the macroautophagy activator rapamycin alone or in combination. The cell viability was assessed using MTT assay. Immunofluorescence and Western blot analysis were used to detect the level of α-synuclein, LAMP-2A, E1 activase, and E2 ligase in the cells. Chymotrypsin-like proteasomal activity was measured using a commercial kit.
Results:
When the proteasome and macroautophagy in the wild-type and mutant cells were inhibited with epoxomicin and 3-MA, respectively, the cell viability was significantly decreased, and the α-synuclein level was increased. Both epoxomicin and 3-MA activated the chaperone-mediated autophagy (CMA) by increasing the level of the CMA-limiting enzyme LAMP-2A. Furthermore, 3-MA or epoxomicin significantly decreased chymotrypsin-like proteasomal activity. 3-MA or epoxomicin did not change E1 activase expression in either mutant or wild-type cells, but increased E2 ligase expression, especially when used together. Macroautophagy inducer rapamycin increased the cell viability and reduced epoxomicin-induced α-synuclein accumulation. Interestingly, CMA was also activated by rapamycin.
Conclusion:
Our results demonstrate the existence of complex crosstalk between different forms of autophagy and between autophagy and the proteasome pathway in the clearance of α-synuclein in PC12 cells.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (No 81171213) and the Natural Science Foundation of the Jiangsu Province of China (No BK2010228). We are grateful to Professor IC Bruce for critical reading of the manuscript.
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Yang, F., Yang, Yp., Mao, Cj. et al. Crosstalk between the proteasome system and autophagy in the clearance of α-synuclein. Acta Pharmacol Sin 34, 674–680 (2013). https://doi.org/10.1038/aps.2013.29
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DOI: https://doi.org/10.1038/aps.2013.29
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