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

 描述(申请人提供)：马查多-约瑟夫病(MJD)是一种以肌肉协调性丧失为特征的主要遗传性神经退行性疾病，目前仅有对症治疗。MJD是一种CAG/多谷氨酰胺疾病，患者在ATXN3基因上存在CAG核苷酸重复序列。这会导致异常长的聚谷氨酰胺链，以及ataxin-3蛋白的错误折叠和聚集。由于降低疾病蛋白水平改善了聚谷氨酰胺疾病动物模型的病理，因此降低致病性ataxin-3水平可能是治疗MJD的可行方法。我们最近证明了ataxin-3不需要泛素化就能被降解。相反，它的周转受其N端的泛素结合位点2(UbS2)的调节。Aaxin-3是 通过UbS2与蛋白酶体相关蛋白Rad23A/B相互作用稳定。突变UbS2以破坏ataxin-3和Rad23A/B之间的相互作用会降低培养的哺乳动物细胞中ataxin-3的蛋白水平。这些发现使我们得出结论，UbS2可能是一个潜在的靶点，通过它来促进MJD治疗中ataxin-3的降解。为了验证这一假设，首先，我们建议确定在体外降解ataxin-3的分子机制。为了实现这一点，我们将对野生型或突变的ataxin-3、蛋白酶体和Rad23A/B的不同组合进行一系列体外反应。我们将通过免疫共沉淀和尺寸排除层析检查哺乳动物细胞中的分子复合体，以确认我们在体外重组试验中的发现。其次，我们将通过产生表达ataxin-3的转基因果蝇株系来验证我们的假说在体内降解ataxin-3，或者表达ataxin-3的所有结构域完整，或者表达ataxin-3的UbS2突变。我们会 通过我们的构建物的组织特异性表达，确定RAD23和UbS2在ataxin-3依赖毒性中的作用。我们将检查这些苍蝇的致死率、寿命、形态、组织学和运动性。最后，我们将进行一次基于发现的筛选，寻找新的抑制者 体内依赖于Aaxin-3的退化，利用我们实验室开发的一种方法，通过使用膜结合GFP来报告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不谋而合，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.