Role Of CHIP In Pathogenesis Of Spinocerebellar Ataxia Type 3

BackgroundSpinocerebellar ataxia type 3(SCA3),known as Machado-Joseph disease(MJD),is the most common type of SCA in Asia.SCA3 is also the most common PolyQ(Polyglutamine)disease following Huntington's disease(HD).Most SCA3 is autosomal dominant disease.Patients of SCA3 usually have cerebellar ataxia,dysarthria,dysphagia and dyskinesia.The disease is caused by the abnormal amplification of CAG repeats in ATXN3 gene on 14q32,1,which leads to the increase number of PolyQ in the carboxy-terminal of Ataxin3.Though it is known that SC A3 is caused by the mutant ATXN3,the pathogenic mechanism of SCA3 is still unclear.Therefore,revealing the pathogenesis of SCA3,finding novel methods for diagnose early-phase disease,and exploring the treatment strategies for SCA3 are still urgent issues of Neurology need to be solved.The main pathology of SC A3 is the accumulation of aggregates in neurons of cerebellum and brainstem,which also known as inclusion body.Most of the researches on SCA3 focus on protein degradation pathway,abnormal transcription regulation,mitochondrial DNA damage and energy support disorder.Recently,dysfunction of protein quality control system was considered the main reason of SCA3.Molecular chaperones and protein degradation systems,especially ubiquitin-proteasome pathway,are main factors for protein quality control system.Carboxyl terminus of Hsc70 interacting protein(CHIP)is one HSP70 interacting protein mainly expressed in skeletal muscle,heart,pancreas and brain.CHIP function as a bridge in the protein quality control system as its chaperone and E3 ubiquitin ligase activity.Previous study found significant down-regulation of CHIP protein in SCA3 transgenic mouse,while the mechanism of the down-regulation CHIP protein and the relationship between the reduction of CHIP protein and the pathogenic mechanism of SCA3 haven't been elucidated.Heat shock transcription factor 1(HSF1)is one stress-responsive transcription factor that protects cells from protein misfolding,aggregation,and cell apoptosis by transcriptional activating genes involved in stress adaptation and cell survival.HSF1 turns to trimer and binds to heat shock elements promoter of the target gene and activates the expression of HSP40,HSP70,HSP90 and other HSPs,when cells are stressed.Previous study showed that cells expressing HD-related proteins had insufficient HSP27,HSP70 and HSP90 when stressed.Previous study also suggested that stress intolerance may also exist in SCA3 model.While whether stress intolerance in SCA3 model is truly exist and whether the stress intolerance is related to the decreased CHIP protein need further study.As the symptom of SCA3 is heterogeneous,identifying it from HD,Amyotrophic lateral spinal sclerosis,Parkinson's syndrome,Spinal bulbar muscular atrophy,and other types of Spinocerebellar ataxia is difficult.Although sequencing is one accurate diagnostic method,sequencing is much expensive and time consuming,which is not suitable for initial screening of outpatients.Biomarkers are biochemical indicators that have been used in recent years to reveal the change of structure or function of systems,organs,tissues,cells,and subcellular cells,and can be also used for disease diagnosis and drug evaluation.Researches on SCA3 are mainly focused on central nervous system,few researches on biomarkers had been done.Therefore,finding biomarkers that can be used as a preliminary screening of clinical diseases for the diagnosis of SCA3,monitor the progress of SCA3,and evaluate the effect of treatment is much valued.Objectives1.Explore the mechanism of CHIP decreasing from transcription,translation,protein distribution,and protein degradation pathways by using SC A3 transgenic mice,SCA3 stable cell lines,SCA3 patients derived skin fibroblasts,and neural stem cells(NSCs)differentiated from Induced pluripotent stem cells(iPSCs).2.Further study whether CHIP is involved in stress intolerance in SCA3 and the related molecular mechanism.3.Comparing the serum CHIP level between the controls and SCA3 patients to analyze whether serum CHIP protein could be used as a biomarker for SCA3.Methods1.LV-cells expressing empty vectors,LV-Q28 cells expressing wild-type Ataxin3,and LV-Q84 cells expressing mutant Ataxin3 were constructed by infecting N2a cells with lentivirus.The skin of normal and SCA3 patients were taken and skin primary fibroblasts were cultured.2.Detected the cell viability and apoptosis level of the above cells at 24h,48h and 72h after culturing.3.The proteins of the stable cell lines,fibroblasts and different age of SCA3 transgenic mice were extracted.The levels of CHIP protein were detected,and the relationship between the age of SCA3 mouse and CHIP level was analyzed.4.Total RNA from brain and cerebellum of the above cells and 40w transgenic mice were extracted,the changes of mRNA in CHIP and Ataxin3 at the transcription level were tested.5.The above stable cell lines,skin fibroblasts and 40w transgenic mice were used to detect the levels of CHIP protein in soluble and insoluble proteins.Cellular immunofluorescence was used to detect the colocalization of Ataxin3 and CHIP proteins and the presence of inclusion bodies.The distribution of Ataxin3 and CHIP proteins in the brainstem and cerebellum of 24w and 40w SCA3 mice and the inclusion bodies were detected.The differences of the size and number of inclusion bodies between 24 and 40w mice were compared.6.10?M proteasome inhibitor(MG 132)was used to inhibit the proteasome pathway in LV-,LV-Q28 and LV-Q84 cells.The ubiquitination level of CHIP in each group were analyzed;MG132 and PI3K autophagy inhibitors(3-MA)were used to inhibit the proteasome pathway and autophagolysosomal pathway of LV-,LV-Q28 and LV-Q84 cells,respectively.Western blot was used to test the CHIP protein of each cell line before and after treatment.Incremental differences of each cell group before and after treatment were analyzed to reveal the protein degradation changes on the CHIP protein in SC A3.7.Control and SCA3 patients-derived skin fibroblasts were used to reprogram into iPSCs and further induce differentiation into NSCs.CHIP level in NSC cells were further detected.8.LV-,LV-Q28 and LV-Q84 cells and the fibroblasts were placed in incubator at 42? for 4 hours,the levels of stress-related proteins were detected by non-denaturing gel electrophoresis,western blot and immunofluorescence.The interaction between Ataxin3 and stress-related proteins was detected by Co-immunoprecipitation.Stress intolerance in SC A3 cells were finally analyzed.9.LV-Q84 cells and the above-extracted SCA3 fibroblasts were infected with CHIP expressing lentivirus.Cells were placed in incubator at 42? for 4 hours,and continue to be cultured.CCK8 were used to test the cell viability after 24 hours,48 hours,and 72 hours of culture.The number of inclusion bodies in cells were detected by immunofluorescence.Expression of heat shock related proteins were detected by western blot.Finally,role of protection of CHIP was analyzed.10.As SCA3 is rare genetic disease,Exploratory case-control study was performed,a total of 80 SCA3 patients and matched control were collected in this study.ELISA were used to measure serum CHIP protein levels,and Student's t test were used to analyze serum CHIP protein levels between controls and SCA3 patients.Pearson correlation test was used to explore the association between serum CHIP protein levels and age,age of onset,duration of disease,SARA and ICARS scores.Multiple regression models were used to analyze the influence factors of serum CHIP level.Results1.LV-,LV-Q28,and LV-Q84 cell lines were successfully constructed.The skin fibroblasts of two SC A3 patients and one normal control with the similar age and gender were successfully isolated and cultured.2.After 24h,48h and 72h of culture,the viability of LV-Q84 cells were significantly lower than that of LV-Q28 cells.P<0.05,the difference was statistically significant.Similar results showed in fibroblasts.3.The level of CHIP protein in LV-Q84 cells was significantly lower than that in LV-Q28 cells,P<0.05,the difference was statistically significant.Similar results appeared in fibroblasts from SCA3 patients.The level of CHIP protein in 40w SCA3 homozygous mice was significantly lower than that of heterozygous and wild type mice,P<0.05.The level of CHIP protein in the cerebellum of SCA3 mice gradually decreased with the progress of the disease.Ranking as 1w wild mice,1w homozygous mutant SCA3 mice,24w homozygous mutant SCA3 mice,40w homozygous mutant SCA3 mice,P<0.05.The difference was statistically significant4.No obvious change of CHIP mRNA level was observed in the above SCA3 stable cell model and 40w SCA3 transgenic mice.The 40w homozygous SCA3 mice had significantly higher Ataxin3 mRNA level in the brain than the heterozygous SC A3 mice,and the mRNA of Ataxin3 in heterozygous SCA3 mouse was significantly higher than that of wild type,P<0.05,the difference was statistically significant.5.The level of CHIP in soluble protein of LV-Q84 cells was significantly lower than that of LV-Q28 cells,but the level of CHIP in insoluble protein of LV-Q84 was significantly higher than that of LV-Q28 cells,P<0.05,the difference was statistically significant.Similar results showed in fibroblasts.Comparing the level of CHIP in soluble protein showed that homozygous SCA3 mice of 40w were significantly lower than heterozygous SCA3 mice of the same age,and much lower in wild-type mice.Opposite results were detected in insoluble proteins.P<0.05,the difference was statistically significant.Colocalization of Ataxin3 and CHIP protein in aggregates was found in LV-Q84 cells,SCA3 patient derived fibroblasts,and SCA3 heterozygous and homozygous transgenic mice.Inclusion bodies were detected in the cerebellum and brainstem of 24w and 40w SCA3 mice,and the number of inclusion bodies in 24w mice was significantly less than that of 40w SC A3 mice of the same genotype,P<0.05,the difference was statistically significant.6.Ubiquitination analysis showed that the ubiquitin CHIP in LV-Q84 cells was lower than that in LV-Q28 cells.There was no significant difference of CHIP increment between LV-Q28 and LV-Q84 cells no matter treated with MG132 or 3-MA.7.The levels of CHIP protein in NSC cells derived from SCA3 patients were significantly higher than those in normal people,and no inclusion bodies were detected in SCA3 patient derived NSC cells.8.Non-denaturing gel electrophoresis showed that the trimeric HSF1 level in LV-Q84 cells was significantly lower than that in LV-Q28 cells after heat stress.The elevated level of stress-related protein DNAJB1 in LV-Q84 cells is significantly lower than that in LV-Q28 cells after heat stress,and the level of DNAJB1 mRNA in LV-Q84 cells was also lower than that in LU-Q28 cells,P<0.05,the difference statistically significant,similar results appeared in fibroblasts.Co-IP showed that the mutant Ataxin3 protein interacted with DNAJB1,while the interaction of wild-type Ataxin3 protein with DNAJB1 was not detected.9.The number of inclusion bodies in CHIP overexpressing LV-Q84 cells was significantly lower than that of LV-Q84 cells.After heat stress,the cell viability of CHIP overexpressed LV-Q84 cells were higher than that of LV-Q84 cells,and the stress-related protein DNAJB1 was also upregulated.Significantly increased at high levels,P<0.05.10.The serum CHIP protein levels of SCA3 patients and normal people respectively were(80.93±28.68)ng/mL and(40.37±18.55)ng/mL,and statistical analysis showed that P<0.05.Correlation analysis found that age,age of onset,and duration of disease were withdrawn from the model.SARA and ICARS scores were related to the serum CHIP protein level(SARA:?=0.500,P=0.034;ICARS:?=0.561,P=0.015).Conclusion1.A large amount of CHIP protein was isolated in inclusion bodies,which caused abnormal distribution of CHIP protein.2.The activated trimeric HSF1 was reduced in SC A3 cell model,which resulting in insufficient transcriptional activation of the DNAJB1.And the cells finally suffered stress intolerance.Overexpression CHIP in SCA3 cell model could significantly improve its stress intolerance.3.Compared with the normal controls,the serum CHIP protein level of SCA3 patients was significantly higher.And the serum CHIP protein level was related to the severity of the disease,which may be a promising biomarker of SCA3.