The Molecular Mechanisms Of The Aggregation And Autophagic Degradation Of α-synuclein In An MPP~+-induced Cellular Model Of Parkinson's Disease

Part I The S-nitrosylation of protein-disulfide isomerase promotes the aggregation ofα-synuclein in an MPP+-induced cellular model of Parkinson's diseaseObjective we tried to set up a cellular Parkinson's disease model by using MPP+ and pheochromocytoma (PC12) cells. We investigated the role of oxidative stress in the pathology of Parkinson's disease in this model. It is well known that Protein-disufide isomerase (PDI) can inhibit the aggregation ofα-synuclein and S-nitrosylation of PDI causes loss of function of PDI. In this study, we examined the relationship between S-nitrosylation of PDI and the aggregation ofα-synuclein to explore the new therapeutic strategies of Parkinson's disease.Methods In this study, we used MPP+ as an inducer to treat PC12 cells and set up a cellular Parkinson's disease model. We applied the inhibitor of NOS and/or precursor of NO in this model, then we examined the level of NO and S-nitrosylated PDI level. In addition, we also detected the expression level ofα-synuclein by using Western blot to further analyze the relationship between S-nitrosylation of PDI and the aggregation ofα-synuclein.Results (1) We found that MPP+ treatment caused oxidative and nitrosative stress in PC12 cells. The production of NO was significantly increased and cell viability was markedly decreased after MPP+ treatment. In addition, the expression ofα-synuclein was increased by MPP+. (2) We detected the total PDI level after MPP+ treatment and found that there is no change for total level of PDI, but S-nitrosylated PDI level was markedly increased, suggesting that MPP+ induced S-nitrosylation of PDI. (3) The inhibitor of NOS reduced the production of NO and decreased S-nitrosylated PDI level at the same time. The expression ofα-synuclein was also reduced after the addition of the inhibitor of NOS, indicating the aggregation ofα-synuclein is related to S-nitrosylation of PDI. (4) L-arginine, a precursor of NO, blocked the effect of the inhibitor of NOS and led to diminished effect of the inhibitor of NOS on S-nitrosylation of PDI.Conclusion PDI has inhibitory effect on the aggregation ofα-synuclein in a cellular Parkinson's disease model; however, S-nitrosylation of PDI diminished the inhibitory effect of PDI. Nitrosative stress and NO overproduction contribute to S-nitrosylation of PDI. Inhibition of S-nitrosylation of PDI can prevent the aggregation ofα-synuclein and have a protective effect on cells.Part II Combination of regulating mTOR and IP3 pathways promotes the autophagic degradation ofα-synucleinObjective To study the effect of Lithium chloride (LiCl) on the degradation ofα-synuclein in a cellular Parkinson's disease model and explore the mechanisms for the degradation.Methods We treated PC12 cells with different concentrations of LiCl to find an optional concentration range that has no effect on cell survival. Selected concentration of LiCl was used in a cellular Parkinson's disease model induced by MPP+ in PC12 cells. After treatment of LiCl with/or without rapamycin in PC12 cells, cell viability was measured by MTT assay; IP3 level was detected by ELISA assay; the aggregation ofα-synuclein and auyophagic marker LC3 was examined by Western blot.Results We found that LiCl alone had no effects on the autophagic degradation ofα-synuclein in PC12 treated with MPP+; however, the combination of LiCl and rapamycin promoted the autophagic degradation ofα-synuclein and reduced the aggregation ofα-synuclein.Conclusion LiCl can inhibit glucose synthesis kinase-3β, an upstream of mTOR pathway, to block autophagy. At the same time, LiCl can reduce IP3 level to promote the autophagic degradation. Combination treatment with LiCl and rapamycin can regulate both mTOR and IP3 pathways to clear the aggregation ofα-synuclein. These findings provided a new clue for the clinical treatment of Parkinson's disease.Part III The protective effect of Dl-3n-butylphthalide on cell damage and the aggregation ofα-synuclein in a cellular Parkinson's disease modelObjective We explored the protective effect of Dl-3n-butylphthalide (NBP) on oxidative and nitrosative stress, and micondrial dysfunction. Further, we examined the effect of NBP on S-nitrosylation of Protein-disufide isomerase (PDI) and the aggregation ofα-synuclein.Methods In this study, MTT assay was used to measure cell viability induced by MPP+ in PC12 cells. Reactive oxidative species (ROS) and mitochondrial membrane potentials were examined by flow cytometry. Production of the nitric oxide derivative nitrite was determined by a nitrite detection kit. Autophagic degradation was examined under confocal microscopy.α-synuclein and total PDI was detected by Western blot. Biotin-labeling method was used to detect the S-nitrosylation of PDI.Results (1) NBP had protective effects on MPP+-induced cell death in PC12 cells and this protection was in a concentration-dependent manner. NBP at 10μM reached its maximal effects. (2) MPP+ treatment led to the increased ROS detected by flow cytometry, but NBP significant decreased cellular ROS. Glutathione (GSH) is an cellular anti-oxidative agent and was decreased after MPP+ treatment in PC12 cells. Cellular GSH was elevated by NBP treatment. (3) NBP also prevented MPP+ -induced mitochondrial dysfunction and mitophagy. (4) NBP reduced the overproduction of NO caused by MPP+ and decreased S-nitrosylation of PDI. The inhibitor of NOS blocked the overproduction of NO, which led to decreased S-nitrosylation of PDI and the aggregation ofα-synuclein. (5) NBP reduced the expression ofα-synuclein in MPP+-treated PC12 cells. Conclusion NBP has protective effects on cell damage in a cellular Parkinson's disease model in PC12 cells. NBP can reduce the production of oxidative and nitrosative stresses, prevent mitochondrial disfunction, inhibit the S-nitrosylation of PDI, and decrease the aggregation ofα-synuclein. Therefore, BNP will be a potential reagent for the treatment of Parkinson's disease.