Low Expression Of BAG5 Protein In Skin Tissue Of PD Patients With PINK1^R492X Mutation And Its Feedback Regulation

Background:Parkinson's disease?PD?is a common neurodegenerative disorder.PD is common in the elderly.In recent years,its has a tendency in young and middle-aged ones.About 15%PD patients have a family history,the typical symptoms of clinical manifestation are resting tremor,muscle rigidity,balance and posture to reduce obstacles to movement.A number of inherited genetic loci related to PD have been found so far,and the R492X mutation in PINK1 gene?PTEN-induced putative kinase 1?has been shown to be associated with the onset of autosomal recessive early-oneset parkinsonism?AREP?.Bcl-2-associated athanogene?BAG?-family proteins can interact with the anti-apoptotic protein Bcl-2.BAG5 protein,the only one that contains multiple BAG domains,has been found be expressed in the cerebral cortex,hippocampus and substantial nigra in animals.While the degradation pathway of PINK1 protein is mainly the ubiquitin-proteasome pathway?UPP?.The mutation of PINK1 gene may lead to dysfunction of the ubiquitin-proteasome system?UPS?and induce the occurrence of PD.Previous studies on PD were mostly based on cell culture and animal models.However,there are few reports about abnormal regulation of mutant proteins associated with PD pathogenesis in vivo in early-onset PD patients.Objective:Skin specimen was taken on PINK1^R492XPD patients by skin biopsy technique.The expression changes of BAG5 protein and ubiquitin related marker Ub in the skin of PINK1^R492XPD patients were detected.And on the basis of this,the role of BAG5 protein in pathogenesis and neuropathology of early-onset Parkinson's disease was preliminarily studied from eukaryotic cell level.Methods:1.Clinical date of the PD patients with PINK1^R492XPD mutation were collected,and graded by Hoehn&Yahr scales.The study samples inclueded 6 PD patients with PINK1^R492XPD mutation and 6 normal controls.Skin specimens were collected by skin biopsy.Immunofluorescence?Immunhistochemistry and Western blotting were used to detect the expression of BAG5 proten and ubiqutin.2.Transfecting the recombinant plasmid EGFP-BAG5,HA-PINK1?R492X?into HEK-293 cells with the eukaryotic cell transfection technique.By using the electrophysiological method,we examined colocalization between PINK1^R492X and BAG5.The effect of BAG5 protein on the degradation of PINK1^R492X protein was detected by plasmid transfection,RNA interference technique and chase-time experiment.3.Statistical analysis was performed using the SPSS 17.0.Date were shown as mean±SD.A P value less than 0.05 was regarded for statistical significance.Results:1.Compared with control group,the express of BAG5 and ubiquitin in the skin dermal layer of PINK1^R492X PD patients with the mutation were decreased?P<0.05?.2.The subcellular co-localization of PINK1^R492X protein and BAG5 protein were detected by immunocytochemistry labeling.3.Overexpression of BAG5 protein downregulated the expression of PINK1^R492X?P<0.05?,and interfered with endogenous BAG5,which raised the expression level of PINK1^R492X?P<0.01?.4.BAG5 protein promotes the degradation of PINK1^R492X protein.Conclusion:1.The expression of BAG5 and ubiquitin in the skin tissue of patients with PINK1^R492XPD were significantly down-regulated.BAG5 may regulate PINK1^R492X through the ubiquitin proteasome system,which may be involved in the pathogenesis of early-onset PD.2.BAG5 expression level changes can be used as one of the early diagnostic indicators of PD.