Crosstalk Between Autophagy And Proteasome Degradation Pathways In PC12 Cells Overexpressing Human α-synuclein

PartⅠRole of autophagy and proteasome degradation pathways in apoptosis of PC12 cells overexpressing humanα-synucleinObjective Parkinson's disease is a common neurodegenerative disease in the aged people. Its causative agent and mechanisms are not clearly understood by now. Ubiquitin-proteasome and autophagy pathways play important roles in the pathology of Parkinson's disease. We aimed to study the different roles of the proteasome and autophagy pathways in apoptosis.Methods In this study, a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate pheochromocytoma (PC12) cell lines transfected with human mutant (A30P and A53T) and wild-type (WT)α-synuclein. Cell viability was measured by means of MTT methods. The apoptosis ratio was assessed by flow cytometry. Caspase 3 expression in cell culture were determined by western blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy.Results We found that the proteasome inhibitor epoxomicin and the macroautophagy inhibitor 3-MA significantly impaired cell viability. Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P, A53T 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; The hallmarks of apoptosis and autophagy in our cell lines using electron microscopy after 24 h drug treatment also showed that the proteasome inhibitor epoxomicin increased apoptosis and autophagy and the macroautophagy inhibitor also increased apoptosis.Conclusion These findings show that inhibition of the proteasome and autophagy promotes apoptosis, and the macroautophagy stimulator rapamycin reduces the apoptosis ratio.PartⅡCrosstalk between proteasome system and autophagy in PC12 cells overexpressing humanα-synucleinObjective Parkinson's disease (PD) is a common degenerative disorder of the central nervous system; the pathology includes the loss and degeneration of dopaminergic neurons and the formation of Lewy bodies in neurons. Recent studies found thatα-synuclein is the main component of Lewy bodies. Ubiquitin-proteasome and autophagy pathways play important roles in the degradation ofα-synuclein. So we aimed to study the different roles of the proteasome and autophagy pathways in the degradation ofα-synuclein, and the crosstalk between these proteasome and autophagy pathways.Methods In this study, PC12 cells, in which exogenous human wild-type or A30P, A53Tα-synuclein were overexpressed, were treated with a proteasome inhibitor and/or a autophagy inhibitor or a stimulator.Results We measured an increase inα-synuclein levels after treatment with the proteasome or autophagy inhibitors in all cell lines. The combination of epoxomicin with rapamycin decreased theα-synuclein accumulation induced by epoxomicin alone. In the presence of proteasome inhibitors, levels of LC3-II in cells were higher. We confirmed that the increase in lamp-2a was related to the inhibition of proteasome or macroautophagy. Interestingly, compared with the control, cells grown in medium with rapamycin also had an increased lamp-2a/β-actin ratio. The macroautophagy inhibitor 3-MA decreased chymotrypsin-like proteasome activity compared with control, and rapamycin tended to increase chymotrypsin-like proteasome activity. E1 enzyme expression did not change in both types of cells. 3-MA and epoxomicin increased E2 enzyme expression, especially when used together.Conclusion Proteasomal inhibitor and macroautophagy inhibitor decreaseα-synuclein clearance, and proteasomal inhibition results in compensatory activation of macroautophagy and chaperone-mediated autophagy(CMA). Our findings also suggest that macroautophagy inhibitor increase the expression of lamp-2a, then CMA is likely activated by inhibition of macroautophagy. In addition, in cells with macroautophagy inhibited, proteasome activity was down-regulated. Our results demonstrate the existence of crosstalk between different forms of autophagy and between autophagy and the proteasome pathway.PartⅢOxidative stress in PC12 cells overexpressing human wild type, A30P and A53Tα-synucleinObjective To study the changes of oxidative stress in cells subjected to impairment of the proteasome pathway and macroautophagy.Methods At 24h after drug treatment, the supernatant was collected and NO and SOD activity measured with a NO or SOD determination kits. And, western blot determined hsp70 and catalase expression.Results We found that the proteasome inhibitor epoxomicin increased NO levels in A30P and A53T cells, but the levels in WT cells did not changed with proteasome and autophagy drugs. And we also found that the proteasome inhibitor epoxomicin increased SOD levels in A30P, A53T and WT cells. High levels of hsp70 protein expression occurred in groups of cells treated with the proteasome inhibitor epoxomicin for 24 h. And the proteasome inhibitor epoxomicin increased the levels of CAT in cells. We did not find a significant change of CAT and hsp70 in cells after treatment with a macroautophagy inhibitor and a stimulator, but the effect of macroautophagy inhibition and stimulation slightly differed with the type of cell.Conclusion All these data indicate that the proteasome system is more tightly related to oxidative stress than autophagy. Oxidative stress as a mechanisms of PD during inhibition of the proteasome was more important than it during inhibition of autophagy.