1-甲基-4-苯基吡啶离子诱导嗜铬细胞瘤细胞自噬应激并导致α-突触核蛋白的自噬性清除障碍

1-methyl-4-phenylpyridinium ion induced autophagic stress and caused autophagic clearance impairment of a-synuclein in pheochromocytoma cells

目的探讨1-甲基-4-苯基吡啶离子（MPP^＋）所诱导的自噬应激在嗜铬细胞瘤（PCI2）细胞损伤中的作用以及MPP^＋导致α-突触核蛋白自噬性清除障碍及其异常聚集的可能机制。方法在MPP^＋处理细胞24h后，采用四甲基偶氮盐法检测细胞活力，Westernblot检测α-突触核蛋白及微管相关蛋白1轻链3-Ⅱ（LC3-Ⅱ）在蛋白水平表达的变化，并用MDC染色和免疫荧光标记观察自噬水平的变化及仪-突触核蛋白、LC3-Ⅱ和溶酶体相关膜蛋白-1（LAMP-1）在细胞内的共定位情况。结果MPP^＋处理后细胞的活力明显下降；与未处理组相比（PCI2组：0．20±0．08；A30P组：0．76±0．09），PCI2＋MPP^＋组（0．66±0．07，t=5．7271，P=0．0023）和A30P＋MPP^＋组（1．71±0．40，t=8．6100，P=0．0005）α-突触核蛋白表达增加；LC3-β蛋白的表达水平及自噬泡的平均数目均增高。另外，MPP^＋处理后，α-突触核蛋白和LC3-Ⅱ的荧光信号及其共定位增加，尽管LAMP-1标记的溶酶体荧光信号增加，但与LC3-H标记的自噬体共定位程度却减小。结论MPP^＋导致自噬体与溶酶体的融合出现障碍，引起α-突触核蛋白的自噬性清除障碍与自噬应激的出现，最终导致细胞的损伤甚至死亡。

Objective To investigate the role of autophagie stress induced by 1-methyl-4- phenylpyridinium （MPP^＋） in pheoehromocytoma （PC12） cell injury, and the mechanism of autophagic elearance impairment and abnormal aggregation of α-synuclein. Methods Before and after treatment of MPP^＋, the viability was measured through the MTT assay, and the expression of α-synuclein and microtubule-associated protein light chain 3-Ⅱ （LC3-Ⅱ） in the level of protein was detected through Western blot. In addition, MDC staining and immunofluorescence microscopy were performed to observe the alterations of autophagy and the signals for α-synuclein, LC3-Ⅱ, lysosomal-assoeiated membrane protein 1 （ LAMP-1 ） and their co-localization in PC12 and A30P cells. Results After treatment of MPP^＋ for 24 h, cell viability decreased in PC12 ＋ MPP^＋ group and A30P ＋ MPP^＋ group. The increase in α-synuclein was significant in the level of protein in PC12 ＋ MPP^＋ group （0.66 ± 0.07,t =5.7271,P =0.0023） and A30P ＋ MPP^＋ group （ 1.71 ± 0. 40, t = 8. 6100, P = 0. 0005 ）, especially in A30P ＋ MPP^＋ group, compared with the untreated groups （ PC12 group： 0. 20 ± 0. 08 ; A30P： 0. 76 ± 0.09）. In MPP^＋ -treated groups, LC3-Ⅱ protein and the average number of late autophagic vacuoles per cell increased compared with untreated ones. Besides, the signals for LC3-Ⅱ and α-synuclein and the extent of their co-localization increased in MPP^＋ -treated groups. Though the signals for LAMP-1 that labeles lysosome increased, the extent of co-localization between LAMP-1 and LC3-Ⅱ was reduced after treated with MPP^＋ for 24 h. Conclusions MPP^＋ impaires the fusion of autophagosomes and lysosomes, leading to the autophagic clearance impairment of α-synuclein and autophagic stress, and finally causes the cell damage even death.