Mechanisms to suppress polyglutamine-dependent toxicity in Machado-Joseph Disease

 描述（由申请方提供）：Machado-Joseph病（MJD）是一种显性遗传性神经退行性疾病，其特征为肌肉协调丧失，目前仅存在对症治疗。MJD是CAG/多聚谷氨酰胺疾病，其中患者在ATXN 3基因中具有扩增的CAG核苷酸重复。这导致异常长的多聚谷氨酰胺束，以及共济失调蛋白-3蛋白的错误折叠和聚集。由于降低疾病蛋白水平可改善多聚谷氨酰胺疾病动物模型的病理学，因此降低致病性共济失调蛋白-3水平可能是MJD的可行疗法。我们最近证明，ataxin-3不需要泛素化被降解。相反，它的营业额是由其N端的泛素结合位点2（UbS 2）调节的。共济失调蛋白-3是 通过UbS 2与蛋白酶体相关蛋白Rad 23 A/B相互作用而稳定。突变UbS 2以破坏共济失调蛋白-3和Rad 23 A/B之间的相互作用，降低了培养的哺乳动物细胞中共济失调蛋白-3的蛋白水平。这些发现使我们得出结论，UbS 2可能是一个潜在的目标，通过它来增强共济失调蛋白-3降解MJD治疗。为了验证这一假设，首先，我们建议确定共济失调蛋白-3在体外降解的分子机制。为了实现这一点，我们将用野生型或突变型共济失调蛋白-3、蛋白酶体和Rad 23 A/B的混合物进行一组体外反应。我们将通过免疫共沉淀和分子排阻色谱法检测哺乳动物细胞中的分子复合物，证实我们在体外重组试验中的发现。其次，我们将测试我们的假设，在体内的ataxin-3降解产生转基因果蝇线，表达ataxin-3与其所有的结构域完整，或ataxin-3与UbS 2突变。我们将 通过我们构建体的组织特异性表达确定Rad 23和UbS 2在共济失调蛋白-3依赖性毒性中的作用。我们将研究这些苍蝇的致死率、寿命、形态学、组织学和运动性。最后，我们将进行一个基于发现的筛选，以寻找新的抑制剂。 利用我们在实验室开发的方法，在体内观察共济失调蛋白-3依赖性变性，该方法通过使用膜结合的GFP报告了完整果蝇的视网膜完整性。这项拟议研究的目标与国家神经疾病和中风研究所的使命一致，因为这项研究的结果可以转化为对MJD患者的有效治疗。

 DESCRIPTION (provided by applicant): Machado-Joseph Disease (MJD) is a dominantly inherited neurodegenerative disease characterized by a loss of muscle coordination, for which currently only symptomatic treatments exist. MJD is a CAG/polyglutamine disorder, in which patients possess expanded CAG nucleotide repeats in the ATXN3 gene. This results in an abnormally long polyglutamine tract, as well as misfolding and aggregation of the ataxin-3 protein. Since reducing levels of disease proteins improves pathology in animal models of polyglutamine diseases, reducing levels of pathogenic ataxin-3 could be a viable therapy for MJD. We recently demonstrated that ataxin-3 does not require ubiquitination to be degraded. Rather, its turnover is regulated by ubiquitin-binding site 2 (UbS2) on its N terminus. Ataxin-3 is stabilized by its interaction with the proteasome-associated proteins Rad23A/B through UbS2. Mutating UbS2 to disrupt the interaction between ataxin-3 and Rad23A/B decreases ataxin-3 protein levels in cultured mammalian cells. These findings led us to conclude that UbS2 could be a potential target through which to enhance ataxin-3 degradation for MJD therapy. To test this hypothesis, first, we propose to determine the molecular mechanism by which ataxin-3 is degraded in vitro. To accomplish this, we will carry out a panel of in vitro reactions with differet combinations of wild type or mutated ataxin-3, proteasomes, and Rad23A/B. We will confirm our findings from in vitro recombinant assays by examining molecular complexes in mammalian cells through co-immunoprecipitations and size-exclusion chromatography. Secondly, we will test our hypothesis for ataxin-3 degradation in vivo by generating transgenic Drosophila lines that express either ataxin-3 with all of its domains intact, or ataxin-3 with UbS2 mutated. We will determine the role of Rad23 and UbS2 in ataxin-3-dependent toxicity through tissue-specific expression of our constructs. We will examine lethality, longevity, morphology, histology, and motility in these flies. Finally, we will conduct a discovery-based screen for novel suppressors of ataxin-3 dependent degeneration in vivo, utilizing a method that we developed in our laboratory, which reports retinal integrity in intact flies by using membrane-bound GFP. The goals of this proposed research coincide with the mission of the National Institute of Neurological Disorders and Stroke, as the results from this research can be translated into efficacious therapies for patients suffering from MJD.