Department of Neurology, Second Affiliated Hospital of Soochow University, Suzhou 215004, China.
Acta Pharmacol Sin. 2013 May;34(5):674-80. doi: 10.1038/aps.2013.29. Epub 2013 Apr 22.
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.
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.
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.
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.
越来越多的证据表明，α-突触核蛋白的积累可能在帕金森病的发病机制中起重要作用。本研究旨在探讨泛素蛋白酶体和自噬途径在 PC12 细胞中清除野生型和突变型α-突触核蛋白中的作用。
用蛋白酶体抑制剂环氧酶素、巨自噬抑制剂 3-MA 和巨自噬激活剂雷帕霉素单独或联合处理过表达野生型或 A30P 突变型α-突触核蛋白的 PC12 细胞。用 MTT 法评估细胞活力。用免疫荧光和 Western blot 分析检测细胞中α-突触核蛋白、LAMP-2A、E1 激活酶和 E2 连接酶的水平。用商业试剂盒测定糜蛋白酶样蛋白酶体活性。
当用环氧酶素和 3-MA 分别抑制野生型和突变型细胞中的蛋白酶体和巨自噬时，细胞活力明显下降，α-突触核蛋白水平升高。环氧酶素和 3-MA 均通过增加 CMA 限制酶 LAMP-2A 的水平激活伴侣介导的自噬（CMA）。此外，3-MA 或环氧酶素显著降低了糜蛋白酶样蛋白酶体活性。3-MA 或环氧酶素均未改变突变型或野生型细胞中 E1 激活酶的表达，但增加了 E2 连接酶的表达，尤其是两者联合使用时。巨自噬诱导剂雷帕霉素增加了细胞活力并减少了环氧酶素诱导的α-突触核蛋白积累。有趣的是，雷帕霉素也激活了 CMA。
我们的结果表明，在 PC12 细胞中清除α-突触核蛋白时，不同形式的自噬之间以及自噬与蛋白酶体途径之间存在复杂的相互作用。
