Ubiquitin And SUMO Involved In α-syn Degradation In PD Model Mice

Objective: To provide a theoretical basis for the pathogenesis of PD,we investigate the changes of α-syn modification by ubiquitin and SUMO after a-syn aggregation caused by Parkinson’s disease(PD)and discuss the ubiquitin and SUMO upstream and downstream-related molecular expression changes in PD mice model,and explore the changes in autophagy pathway-related protein expression.Methods: Forty 2-month-old C57BL/6 mice were randomly and evenly divided into control group and Parkinson’s model group.We used MPTP to construct an acute PD mice model.The mice were received 7d of administrations,and then were subjected to behavioral examination on the 30 th day after administration.Firstly,we examined the behavioral changes of PD mice by step pitch and rotarod tests,and detected the representative molecular changes in PD pathology by immunohistochemistry to verify the establishment of PD model.Secondly,immunoblotting and immunofluorescence assays were used to detect the expression changes of ubiquitin and SUMO-1 in various parts of the brain,and the changes in participation of α-syn modification.Lastly,immunofluorescence was used to detect the co-localization change of α-syn,proteasome and lysosomes,and the function of proteasome and lysosome.The immunoblotting was used to detect the expression change of autophagy-related proteins in PD model.Results: Firstly,via step pitch and rotarod tests of PD model and the immunohistochemical detection of α-syn and TH,we found that PD model mice’s behavioral abilities were decreased,while α-syn aggregated,TH was decreased,which indicated success modeling of PD mice.Main lesion of PD is located in substantia nigra site.By immunoblotting and immunofluorescence methods,we found that ubiquitin expression in substantia nigra of PD rats was significantly lower than that of control group,and SUMO-1 expression was significantly higher than that of control group.Through co-staining with α-syn,the correlation coefficient between α-syn and ubiquitin or SUMO-1 was calculated,and we found that the co-localization degree of α-syn and ubiquitin in substantia nigra of PD model mice decreased,while co-localization degree of α-syn and SUMO-1 increased.The expression change of E1,E2 and E3 ubiquitin ligases in PD mice was detected by immunoblotting.The results showed that expressions of UBE2E1,Hip-2 and Parkin in the substantia nigra of PD rats were decreased.In addition,Hsp70 and Hsp90,as two regulatory factors for downstream of ubiquitin and SUMO-1,their immunoblotting results showed that these two proteins were elevated in PD mice.Subsequently,we found that more α-syn in the PD model mice were located in the lysosome,different from the main location of proteasome in control group.In addition,by detecting the activity of 20 S proteasome and the activity of hydrolase cathepsin B in lysosomes,we found that MPTP injection did not significantly affect the activity of 26 S proteasome,but decreased the activity of lysosomes.Finally,we examined the expression changes of autophagy-associated proteins Beclin-1,P62,LC3 and p-mTOR.The results of immunoblotting showed that the autophagy-related proteins in the PD model mice were significantly increased,indicating that the level of autophagy in the PD model was increased.Conclusion: 1.The ubiquitination in PD rats is increased after α-syn aggregation,and SUMO-induced modification is increased,and more of them are degradated by lysosomal pathway.2.The level of autophagy in the PD model is increased,The lysosomal activity in the PD model is reduced and the proteasome activity is not affected.In addition,ubiquitin and SUMO upstream and downstream-related molecules have abnormal expressions in the PD model.