帕金森病细胞模型中分子伴侣介导自噬对α-突触核蛋白低聚体水平的影响

Effects of chaperone-mediated autophagy on the level of α-synuclein oligomers in Parkinson's disease cell model

目的·探讨在泛素蛋白酶体系统(ubiquitin proteasome system,UPS)受损的帕金森病(Parkinson's disease,PD)细胞模型中分子伴侣介导自噬(chaperone-mediated autophagy,CMA)对α-突触核蛋白低聚体水平的影响。方法·培养稳转野生型α-突触核蛋白的人神经母细胞瘤SK-N-SH细胞系,加入蛋白酶体抑制剂lactacystin构建PD细胞模型。通过Western blotting检测α-突触核蛋白低聚体、溶酶体相关膜蛋白2A型(lysosome-associated membrane protein type 2A,LAMP2A)和相对分子质量为70 000的热休克同源蛋白(heat-shock cognate protein of 70 kDa,HSC70)水平;使用LAMP2A siRNA抑制CMA功能,检测对α-突触核蛋白低聚体水平及细胞存活率的影响;通过免疫共沉淀观察LAMP2A与α-突触核蛋白低聚体的相互作用。结果·PD细胞模型中α-突触核蛋白低聚体表达水平升高,与CMA功能密切相关的蛋白LAMP2A和HSC70水平应激性增加;抑制LAMP2A表达,α-突触核蛋白低聚体水平进一步升高,细胞存活率降低,且通过免疫共沉淀检测到LAMP2A与α-突触核蛋白低聚体存在相互作用。结论·PD细胞模型中,CMA是调节α-突触核蛋白低聚体水平的途径之一,抑制其功能可进一步增加α-突触核蛋白低聚体水平及细胞毒性作用。

Objective · To investigate the effects of chaperone-mediated autophagy (CMA) on α-synuclein oligomers level in the Parkinson's disease (PD) cell model with impaired ubiquitin proteasome system (UPS). Methods · The PD cell model was established by adding the proteasome inhibitor lactacystin in the SK-N-SH cell line stably transfected with wild type α-synuclein. The levels of α-synuclein oligomers, lysosome-associated membrane protein type 2A (LAMP2A) and 70 kDa heat shock homologous protein (HSC70) were detected using Western blotting. CMA function was inhibited with LAMP2A siRNA, and its effects on α-synuclein oligomers and cell viability were detected. Furthermore, the interaction of LAMP2A with α-synuclein oligomers was detected by immunoprecipitation. Results · In the PD cell model, the levels of α-synuclein oligomers, and CMA related proteins, i.e. LAMP2A and HSC70, were increased. Inhibiting the expression of LAMP2A further increased α-synuclein oligomers level, while it decreased cell viability. Furthermore, LAMP2A could interact with α-synuclein oligomers. Conclusion · In the PD cell model, CMA is one of the pathways regulating α-synuclein oligomers level. Inhibiting CMA function can further increase the α-synuclein oligomers level and deteriorate cell survival.