MPP~+-induced Autophagic Stress In Cellular Injury And Its Regulation Mechanisms

Part I MPP~+ induced autophagic stress and caused the autophagic clearance impairment ofα-synuclein in pheochromocytoma cellsObjective To determine the role of autophagic stress induced by 1-methyl- 4- phenylpyridinium (MPP~+) in pheochromocytoma (PC12) cell injury, the mechanism of autophagic clearance impairment and abnormal aggregation ofα-synuclein, and the relationship between different effects of autophagic stress and injury in PC12 cells.Methods Before and after treatment of MPP~+, we measured cell viability through the MTT assay, and detected the expression ofα-synuclein and microtubule-associated protein light chain 3-Ⅱ(LC3-Ⅱ) in the level of protein through western blot. In addition, MDC staining and immunofluorescence microscopy were performed to observe the alterations of autophagy and the signals forα-synuclein, LC3-Ⅱ, lysosomal-associated membrane protein 1 (LAMP-1) and their co-localization in PC12 and A30P cells. Meantime, after treatment of different concentrations of MPP~+, effects of different levels of autophagic stress on the expression ofα-synuclein, LC3 and dynein in the level of protein and apoptosis.Results After treatment of MPP~+ for 24 h, cell viability decreased in PC12 + MPP~+ group (A=0.748±0.067, P<0.001) and A30P + MPP~+ group (A=0.347±0.043, P<0.01). The increase inα-synuclein was significant in the level of protein in PC12 + MPP~+ group ( P<0.01) and A30P + MPP~+ group (P<0.001), especially in A30P + MPP~+ group. In MPP~+-treated groups, LC3-Ⅱp rotein (PC12 + MPP~+: 0.666±0.078, P<0.05; A30P + MPP~+: 1.688±0.214, P<0.05) and the average number of late autophagic vacuoles (L-AVs) per cell (all P <0.001) increased compared with untreated ones. Besides, The signals for LC3-Ⅱandα-synuclein and the extent of their co-localization increased in MPP~+-treated groups; Though the signals for LAMP-1 that labeles lysosome increased, but the extent of co-localization between LAMP-1 and LC3-Ⅱwas reduced after treated with MPP~+ for 24 h. In addition, different concentrations of MPP~+-induced autophagic stress had different effects on PC12 cells. The level of autophagic stress was significantly increased with the concentration of MPP~+, and cell viability was decreased, apoptotic cells were increased, the expression of aggregation -prone proteinα-synuclein increased, the expression of key motor protein dynein was reduced, which mediated the fusion of autophagosomes with lysosomes.Conclusion These results suggested that MPP~+-induced autophagic stress closely related with impairedα-synuclein autophagic clearance caused by dynein dysfunction, which further indicated that dynein play an important role in cell injury, and dynein may be a key regulation target ofα-synuclein autophagic degradation.Part II Dynein:a key regulation target of autophagic stressObjective To investigate dynein-mediatedα-synuclein degradation dysfunction in the formation of autophagic stress and its mechanisms, and to provide new treatment strategies and regulatory targets for the prevention and treatment of PD.Methods siRNA was used to construct cell model of knock-down dynein, and then detectedα-synuclein expression and changes in the level of autophagy through immunocytochemistry and Western blot methods, and the ultrastructure changes of cells as well as morphological changes of autophagy were observed by transmission electron microscopy(TEM). Western blot was used to observe the changes of dynein,α-synuclein and autophagy in rotenone-induced PD rat striatum.Results Dynein disfunction by siRNA technology caused obviousα-synuclein aggregation (P <0.01). Treated by rotenone, the expression of dynein was decreased (P <0.01) andα-synuclein was overexpressed and aggregated (P <0.001), LC3-Ⅱexpression was increased (ie, autophagosomes were increased) (P <0.05), phosphorylated mTOR level was decreased (ie, mTOR inhibition upregulate autophagy) (P <0.05), which showed that rotenone treatment caused dynein dysfunction leading to accumulatedα-synuclein as well as the increased autophagosomes (ie, increase in the level of autophagic stress), mTOR participated in induction of autophagy.Conclusion These results indicated that dynein mediated autophagic degradation ofα-synuclein and its dysfunction resulted inα-synuclein aggregation and accumulation of autophagosomes, so dynein regulated autophagic degradation ofα-synuclein through autophagy and may be a potential regulation target for prevention and treatment of PD.