Yang Fang, Yang Ya-Ping, Mao Cheng-Jie, Cao Bi-Yin, Cai Zheng-Lin, Shi Ji-Jun, Huang Jin-Zhong, Zhang Ping, Liu Chun-Feng
Department of Neurology, Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou 215004, China; Department of Neurology, Nanjing Jinling Hospital, Nanjing 210002, China.
Neurosci Lett. 2009 May 1;454(3):203-8. doi: 10.1016/j.neulet.2009.03.027. Epub 2009 Mar 14.
Parkinson's disease is a common neurodegenerative disease in the elderly. Its causes and mechanisms are not clearly understood. To explore the specific role of autophagy and the ubiquitin-proteasome pathway in apoptosis, a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate pheochromocytoma (PC12) cell lines transfected with human mutant (A30P) and wild-type (WT) alpha-synuclein. The apoptosis ratio was assessed by flow cytometry. LC3, heat shock protein 70 (hsp70) and caspase-3 expression in cell culture were determined by Western blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy. Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P and WT cells was significantly higher after treatment with inhibitors of the proteasome and macroautophagy. The results of Western blots for caspase-3 expression were similar to those of flow cytometry; hsp70 protein was significantly higher in the proteasome inhibitor group than in control, but in the autophagy inhibitor and stimulator groups, hsp70 was similar to control. These findings show that inhibition of the proteasome and autophagy promotes apoptosis, and the macroautophagy stimulator rapamycin reduces the apoptosis ratio. And inhibiting or stimulating autophagy has less impact on hsp70 than the proteasome pathway.
帕金森病是老年人常见的神经退行性疾病。其病因和发病机制尚不清楚。为了探究自噬和泛素 - 蛋白酶体途径在细胞凋亡中的具体作用，选用了一种特异性蛋白酶体抑制剂、自噬抑制剂和自噬刺激剂，对转染了人类突变型（A30P）和野生型（WT）α-突触核蛋白的嗜铬细胞瘤（PC12）细胞系进行研究。通过流式细胞术评估细胞凋亡率。通过蛋白质免疫印迹法测定细胞培养物中微管相关蛋白1轻链3（LC3）、热休克蛋白70（hsp70）和半胱天冬酶 - 3的表达。用透射电子显微镜评估细胞凋亡和自噬的特征。与对照组或雷帕霉素（自噬刺激剂）组相比，蛋白酶体和自噬抑制剂处理后，A30P和WT细胞的凋亡率显著更高。半胱天冬酶 - 3表达的蛋白质免疫印迹结果与流式细胞术结果相似；蛋白酶体抑制剂组中的hsp70蛋白显著高于对照组，但在自噬抑制剂和自噬刺激剂组中，hsp70与对照组相似。这些研究结果表明，蛋白酶体和自噬的抑制促进细胞凋亡，自噬刺激剂雷帕霉素降低凋亡率。并且抑制或刺激自噬对hsp70的影响小于蛋白酶体途径。
