The Study Of The Pathogenesis Of The PINK1/parkin Mediated Autophagy Dysfunction In SCA3/MJD

Objective: To investigate whether ataxin-3is involved in theregulation of mitochondrial autophagy on PINK1/Parkin mediated; clearof wild-type and mutant ataxin-3mitochondria PINK1/Parkin mediatedautophagy; elaborate PINK1/Parkin mediated mitochondrial autophagydysfunction in the pathogenesis of SCA3/MJD.Methods: in vitro cultured cells as a model, by the target plasmidtransfection, related drugs and other interventions, immunofluorescence-laser confocal clear ataxin-3is involved in the degradation ofmitochondrial PINK1/parkin mediated autophagy pathway, werstem blotstudies of wild-type ataxin-3PINK1protein and mitochondrial proteindegradation; polyQ expansion mutant ataxin-3the PINK1protein as wellas the mitochondrial protein degradation. Inhibit autophagic-lysosomalpathway, wild-type and polyQ expansion the PINK1protein degradationin mutant ataxin-3; inhibit the proteasome pathway, wild-type as well aspolyQ expansion, PINK1protein degradation in mutant ataxin-3.Results: The results show an immunofluorescence-laser confocal: analogmitochondrial injury under the conditions, the the PINK1proteinaggregates in the intracellular distribution has been changed, but PINK1 completely can not be transferred to the nucleus, mitochondria partialaggregation, different degrees of PINK1colocalization. In the wild-typeataxin-3intervention groups, PINK1protein aggregates localized in thenucleus; Poly Q expanded mutant ataxin-3group of PINK1aggregateslocalized in the nucleus. Wild-type ataxin-3the J functional areasmutations in the intervention group, analog mitochondrial damage,PINK1and mitochondrial proteins were found to cluster both appearsignificantly colocalization; expanded polyQ mutation in the J functionalareas mutant ataxin-3intervention groups group of PINK1protein andmitochondrial protein aggregation, both partial co-localization.Separately transfected with wild-type ataxin-324Q and Poly Q expansionmutant ataxin-384Q, regardless of CCCP,3-MA, MG-132treatment,wild-type ataxin-324Q protein aggregates are always located in thenucleus. Inhibition of autophagy group of wild-type ataxin-324Q-lysosomal pathway, wild-type ataxin-324Q and mitochondrial proteinsignificantly clustered perinuclear, showing colocalization; inhibition ofubiquitin-proteasome pathway simulation mitochondria damage,wild-type ataxin-324protein aggregates significantly reduced. Poly Qexpanded mutant ataxin-384Q group, regardless of CCCP,3-MA,MG-132treatment, Poly Q expansion mutant ataxin-384Q proteinaggregates always localized in the cytoplasm in the inhibition ofautophagy-dissolved enzyme pathway simulation mitochondrial damage, polyQ expansion mutant ataxin-384Q protein aggregatessignificantly reduced; mitochondrial damage in the inhibition of theubiquitin-proteasome pathway simulation mutant ataxin-384Q proteinaggregates and mitochondria, polyQ expansion protein gathered morethan before the co-localization of both. Western blot Show:non-pharmacological intervention condition, compared with theindividual expression of PINK1, ataxin-3intervention groups PINK1,mitochondrial protein were significantly reduced, particularly in theataxin-378Q group PINK1protein, mitochondrial protein wassignificantly reduced. Analog mitochondrial injury, PINK1protein,mitochondrial protein was significantly reduced compared with beforeintervention; while in ataxin-378Q group, PINK1, mitochondrial proteindecreased more obvious. Inhibition of autophagy-lysosomal pathway,and non-ataxin-3intervention group compared to both the wild-typeataxin-324Q polyQ expanded group or the mutant ataxin-378Q groupcompared with PINK1protein ataxin-3group than the non-reduced,polyQ expansion mutant ataxin-378Q group less is more obvious; in theinhibition of autophagy-lysosomes, the non-ataxin-3groupsignificantly increased mitochondria, ataxin-3treated group wassignificantly reduced compared with the previous mitochondrial proteinsbetween the two groups without difference.Inhibition of ubiquitin-proteasome pathway, and inhibition of proteasome pathway than before, regardless of the control group or the ataxin-3intervention groups,PINK1protein compared with the previous increase, particularly in theataxin-324Q group increased significantly. Inhibition of ubiquitin-proteasome pathway, PINK1group was significantly reduced comparedwith the previous mitochondria; whereas the wild-type ataxin-3intervention groups had no significant changes in mitochondrial protein,while the polyQ expansion mutant ataxin-3group of mitochondrialprotein increased significantly.Conclusions: PINK1protein and mitochondrial protein metabolismbasically the same trend. Either wild-type or mutant polyQ expandedataxin-3can affect the PINK1protein via the ubiquitin-proteasomepathway and autophagy-lysosomal pathway metabolism. Whenautophagy was inhibited, either wild-type or mutant polyQ expandedataxin-3group PINK1are available through the ubiquitin-proteasomepathway metabolism. When the inhibition of ubiquitin-proteasomepathway, the wild-type ataxin-3group PINK1not pass autophagy-lysosome pathway metabolites; polyQ expansion mutant ataxin-3groupPINK1via autophagy-lysosomal pathway metabolism, mitochondrialproteins not through autophagy-lysosomal pathway metabolism. Sinceautophagy-different lysosomal pathway metabolism, which may be thepathogenesis in SCA3/MJD.