The Role Of Autophagy-lysosome Pathway Mediated By Deubiquitinase USP8 In Parkinson's Disease

Aim:Parkinson's disease(PD)is the second most common neurodegenerative disease.However,the exact pathogenesis of PD remains elusive.Our study aims to identify the potential key factors which might participate in the PD pathogenesis and investigate their role on the regulation of autophagy.Methods:We obtained gene expression profiles of PD postmortem brains from GEO database to identify differentially expressed genes in PD compared to normal individuals.Based on these genes,we performed the functional annotation analysis to identify the key regulators and studied their biological functions.MES23.5 cells treated with MPP+ were used as the cellular model of PD.In this model,Western blot was performed to detect the protein levels of USP8 in control group and MPP+group.MES23.5 cells were transfected with Flag-USP8 and then the protein levels of cleaved caspase-3 were detected.To explore the effect of USP8 on autophagy,USP8 was silenced by transfecting siRNAs,and then the protein levels of LC3 and p62 were detected.USP8 was knocked down in HEK293 cells and then the cells were treated with autophagy inhibitor Bafilomycin A1(Baf A1)to detect the protein level of LC3.USP8 was knocked down in HEK293 cells that transfected with mCherry-EGFP-LC3 to observe LC3 puncta by immunofluorescence assays.To further explore the effect of USP8 on lysosome,USP8 were knocked down in MES23.5 cells to detect the protein levels and mRNA levels of TFEB?LAMP1 by Western blot and Real-time qPCR.USP8 was knocked down in HEK293 cells and then the cells were treated with a lysosomal acidity indicator Lysotracker to observe the fluorescence intensity.Finally,GeneMANIA was used to predict the proteins interacting with USP8,and GO functional annotation was performed on these proteins.And USP8 were knocked down in MES23.5 cells to detect the protein levels of CHMP1B.Results:We obtained five microarray datasets of PD postmortem brains from GEO database,and performed the differential expression analysis.252 genes were significantly down-regulated and 22 genes were significantly up-regulated in PD patients compared to normal individuals.The functional annotation analysis demonstrates that the proteasome pathway and the lysosome pathway were significantly down-regulated in PD patients,which indicated the protein degradation process was inhibited.The mRNA levels of USP8 were significantly down-regulated in PD patients compared to normal individuals.And the protein levels of USP8 were also down-regulated in PD cellular model.Overexpression of Flag-USP8 decreased the protein levels of cleaved caspase-3 in MPP+treatment group,indicating that USP8 has a protective effect in PD cellular model.The protein levels of LC3-? and p62 were increased when USP8 was knocked down in MES23.5 and HEK293 cells.USP8 was knocked down in HEK293 cells transfected with mCherry-EGFP-LC3,and we found that the yellow fluorescence of LC3 puncta was enhanced and the red fluorescence was weakened;however,there was no significant difference in yellow fluorescence when the cells were treated with Baf A1,indicating that the deficiency of USP8 results in block of the autophagy flux.The protein levels of LAMP1 were decreased when USP8 was knocked down in MES23.5 cells,while the protein levels of TFEB were no change.And the knockdown of USP8 significantly reduced the fluorescence intensity of lysosomes.GO functional annotation of the proteins interacting with USP8 indicated that these proteins play a role in endosomal transport.Moreover,the protein levels of CHMP1B were decreased when USP8 was knocked down,indicating that the deficiency of USP8 may affected the biosynthesis of lysosomes.Conclusion:Our analysis of gene expression profiles demonstrates that the expression of USP8 was significantly down-regulated in PD patients.And USP8 has a neuroprotective effect in vitro model of PD.Our study further indicates that the deficiency of USP8 results in the block of the autophagy flux due to the defect of lysosomal biosynthesis.In conclusion,our study demonstrates that the block of autophagy-lysosome pathway mediated by USP8 might participate in the PD pathogenesis.