Relationship Between Polyglutamine Disease Protein Ataxin-3 And Parkin-mediated Mitophagy

Objective:To investigate the effect of polyglutamine disease protein Ataxin-3 on parkin-mediated mitophagy.Methods:The plasmids containing the target genes(Ataxin-3-20Q-EGFP,Ataxin-3-80Q-EGFP,Ataxin-3-130Q-EGFP,Flag-parkin,Flag-parkin K161N,Flag-parkin T240R,Flag-parkin C418R,Flag-parkin C431F,Flag-parkin P437L)were amplified and stored at-20℃ until further use.Human embryonic kidney epithelial cells(HEK293 cells)were transfected with the amplified plasmids.Antimycin A and Oligomycin were used to create a mitochondrial damage model.Immunofluorescence was used to detect the localization and aggregation of Ataxin-3 and parkin and their mutants on mitochondria and mitochondrial clearance.Results:We found that Ataxin-3-20Q-EGFP,Ataxin-3-80Q-EGFP and Ataxin-3-130Q-EGFP,three proteins expressed after mitochondrial damage in transfected HEK293 cells,containing different lengths of glutamine repeats,could accumulate on damaged mitochondria in a punctate pattern in cells within a short period of time and colocalize with parkin.Also,Ataxin-3 is critical in mitochondria clearance in long-term A/O treatment.The effects of different pathological mutants of Parkin in mitophagy were also found,in which the RINGO region mutant K161N partially slowed the transport to mitochondria,the RING1 region mutant T240R could transport to mitochondria but could not recruit ataxin-3,and the RING2 region mutants C418R,C431F,and P437L completely failed to localize to mitochondria.Conclusion:Ataxin-3 protein and pakin can participate in mitophagy.Pathological mutations in different sites of parkin lead to impairment of its function in regulating mitophagy.