Study Of The Role Of SUMOylation On Phenotype Of SCA3/MJD Drosophila Model And Its Mechanism

Background:Spinocerebellar ataxia type3(SCA3/MJD) is the most common subtype of hereditary spinocerebellar ataxia. It is caused by the CAG repeat amplification mutation in the3’end of MJD1encoding abnormal polyglutamine (polyglutamine, polyQ) in the carboxy-terminal of ataxin-3protein. Small ubiquitin-related modifier (SUMO) is a newly discovered protein family as important post-translational modification factor. SUMO family comprises at least four members SUMO-1,SUMO-2,SUMO-3and SUMO-4. Like ubiquitination, SUMOylation require SENP(Sentrin-specific proteases), E1enzyme (SAE1/SAE2),E2enzyme (Ubc9) and E3enzymes.The present studies found that SUMO-1play a role in Parkinson’s disease, Alzheimer’s disease,amyotrophic lateral sclerosis multiple system atrophy and polyglutamine diseases. Smt3is the homologous gene for SUMO in Drosophila, and lwr for Ubc9.Our previous study identified that the major SUMO-1binding site of ataxin-3was located on lysine166. SUMOylation did not influence the subcellular localization, ubiquitination or aggregates formation of mutant-type ataxin-3,but partially increased its stability and the cell apoptosis.Our findings revealed the role of ataxin-3SUMOylation in SCA3/MJD pathogenesis.Objective:We aim to further explore the role of SUMOylation on SCA3/MJD in vivo experiments using transgenic SCA3/MJD Drosophila model and study itspossible mechanismMethod:1.The role of de-SUMOylation of ataxin-3on phenotype of SCA3/MJD transgenetic Drosophila and its possible mechanism1)Recombinant genetic technology, site-directed mutagenesis were applied to construct Drosophila micro-injection plasmid pUAST-attB-SCA3-Q27, pUAST-attB-SCA3-Q27-K166R, pUAST-attB- SCA3-Q84and pUAST-attB-SCA3-Q84-K166R, which were used to micro-injection to get corresponding transgenetic SCA3Droso-phila. After that, tansgenetic Drosophila were picked out and were double balanced. The corresponding SCA3/MJD Drosophila were obtained.2)The effect of de-sumoylation of ataxin-3on phenotype of SCA3/MJD Drosophila, including eye phenotype,eclosion, the proportion of females to males,longevity, wings,crawling speed was evaluated.3)The effect of de-sumoylation of ataxin-3on the levels of ataxin-3and Smt3protein level was tested by Western blot.2.Down regulation of SUMOylation on phenotype of SCA3/MJD transgenetic Drosophila and its possible mechanism1)Western blot was applied to detect the Smt3levels of wild-type and polyQ expanded SCA3Drosophila.2)Balancers were applied to construct Drosophila expressing SCA3.fl-Q78only, and Drosophila that also expression interference RNA for Smt3or dominant negative lwr.3)After down regulating SUMOylation pathway, the effect on phenotype of the polyQ expanded SCA3Drosophila was evaluated, including eye phenotype,eclosion, the proportion of females to males,longevity, wings and crawling speed.4)After down regulating SUMOylation pathway, levels of ataxin-3protein were tested by Western blot. Results:1.De-SUMOylation of ataxin-3on phenotype of SCA3/MJD transgenetic Drosophila and its mechanism1)The UAS-SCA3-Q27, UAS-SCA3-Q27-K166R, UAS-SCA3-Q84and UAS-SCA3-Q84-K166transgenic Drosophila were successfully constructed.2)After expression of SCA3-Q27,SCA3-Q27-K166R, SCA3-Q84and SCA3-Q84-K166in the compound eyes,no obvious abnormalities were observed.After expressing SCA3-Q27or SCA3-Q27-K166R in the nervous system, the eclosion, the proportion of females to males,longevity, wings and crawling speed were not different from the normal controls.While expressing SCA3-Q84or SCA3-Q84-K-166R in the nervous system, compared with wild-type flies, decreased eclosion rate,increased proportion of females to males, shortened life expectancy, abnormal wings and slowed crawling speed were observed, and more obviously when SCA3-Q84-K166R was expressed.3)When SCA3-Q27or SCA3-Q27-K166R was expressed in nervous system, no SDS insoluble ataxin-3was observed and its sumoylation levels were the same as wild type Drosophila.While SCA3-Q84or SCA3-Q84-K166R was expressed, SDS insoluble ataxin-3was formed.When the polyQ expanded SCA3-Q84was mutated at K166to R,the Smt3level was decrease and the SDS insoluble ataxin-3was increased, which indicated that de-SUMOylation of SCA3-Q84increased its stability and ability to form SDS insoluble fraction.2.Down regulation of SUMOylation on phenotype of SCA3/MJD transgenetic Drosophila and its mechanism1)Smt3levels were increased when SCA3-Q78was expressed in the nervous system, but not in the instance when SCA3-Q27was expressed. The major increased Smt3protein was co-located with the SDS insoluble protein.2) After the down regulation of SUMOylation pathway, the eyes of gmr-GAL4/+;UAS-Q78/+Drosophila showed aggravated depigmen tation. While expressed in the nervous system, shortened life span, lower crawling speed were observed.3)After the down regulation of SUMOylation pathway, Western blot showed increased SDS insoluble ataxin-3,suggesting that its enhanced toxicity was relevant with increased stability and ability of ataxin-3to form SDS insoluble fraction.Conclusion:1.When the locus of K166was mutated to R, the phenotype of polyQ expanded SCA3/MJD transgenetic Drosophila was worsened, including decreased eclosion rate,increased proportion of females to males,shortened life expectancy, abnormal wings and slowed crawling speed, which indicated the protect effect of SUMOylation of ataxin-3.2.The enhanced toxicity of K166R mutant polyQ expanded ataxin-3was related to its increased stability and ability to form SDS insoluble fraction.3.The level of Smt3in Drosophila expressing polyQ expanded ataxin-3was increased which further confirmed that SUMOylation was involved in the regulation of the toxicity of ataxin-3.4.After the down regulation of SUMOylation pathway, Drosophila expressing polyQ expanded ataxin-3had more severe phenotype, shortened life span, decrease climing ability, which indicated that SUMOylation may have a protective effect.5.The worsend phenotype of SCA3/MJD Drosophila was related to the increased stability and ability of ataxin-3to form SDS insoluble fraction after down regulation of SUMOylation.