Huntingtin interacting proteins as modifiers of Huntington's disease

描述（由申请人提供）：亨廷顿氏病（HD）是一种进行性致死性神经退行性疾病，由编码亨廷顿基因CAG通道的聚谷氨酰胺扩增引起。亨廷顿蛋白的正常功能尚不清楚。聚谷氨酰胺毒性的确切性质及其作用于细胞功能障碍的关键靶点也有待阐明。为了更深入地了解亨廷顿蛋白的正常和致病功能，我们采用酵母双杂交和基于质谱的方法进行了大规模筛选，以发现亨廷顿蛋白相互作用蛋白（HIPs）。通过数值和统计方法对数据进行分析，确定了234个HIPs的高置信度组。为了测试这些基因与HD病理的相关性，我们在果蝇HD模型中测试了60个相互作用蛋白的同源基因编码基因对毒性聚谷氨酰胺表型的修饰能力。这些测试的基因中有80%作为HD毒性的修饰因子，这表明蛋白质与亨廷顿蛋白物理相互作用的能力与其在表型分析中显示遗传相互作用的能力相关。迄今为止，果蝇的遗传分析已经确定了25种HD毒性的功能丧失抑制因子。这些结果表明，我们研究中鉴定的HIPs集合富含在HD细胞病理中起直接作用的蛋白质。这些结果还表明，HIPs可能同样富集了可以修饰人类HD的蛋白质。本研究的主要目的是测试我们在高通量研究中鉴定的HIPs在哺乳动物细胞和HD小鼠模型中修饰HD表型的能力。我们将使用sirna在突变HD毒性的人和小鼠神经细胞模型中敲除这些基因的表达。在特定情况下，基因表达敲低也将被评估对代谢、定位和/或翻译后修饰的影响。在这些试验中显示出效果的候选蛋白将优先用于HD小鼠模型的更广泛研究。修饰亨廷顿蛋白毒性和/或生化特性的HIPs将在转基因HD小鼠脑中与亨廷顿蛋白共定位进行测试。在基于细胞的实验（和/或果蝇）中，HIPs的表达降低可以抑制毒性，将在HD小鼠模型中进行抑制测试。这将通过构建合适的表达shrna的转基因小鼠系，将其杂交到HD小鼠模型中，并研究对小鼠HD表型的影响来完成。我们还将使用aav介导的病毒转染HIP shRNA来研究HIP敲除对HD小鼠的影响。这些研究的最终目的是在细胞和/或果蝇实验中确定HD表型的修饰因子，确定其作用机制，并在HD小鼠模型中验证这些修饰因子。我们预计这些研究将为HD的病理性质提供有用的见解，并为HD的治疗药物发现提供新的候选靶点。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a progressive fatal neurodegenerative disease caused by expansion in a polyglutamine encoding CAG tract in the huntingtin gene. The normal function of the huntingtin protein is not well understood. The precise nature of polyglutamine toxicity and the key targets that it acts upon to cause cellular dysfunction also remain to be elucidated. In order to provide greater insight into the normal and pathogenic functions of huntingtin we undertook a large-scale screen to discover huntingtin interacting proteins (HIPs) using yeast two-hybrid and mass spectrometry-based methods. After data analysis using numerical and statistical methods, a high confidence group of 234 HIPs was identified. In order to test these for relevance to the HD pathology, genes encoding orthologs of 60 interacting proteins were tested for their ability to modify a toxic polyglutamine phenotype in a Drosophila model of HD. 80% of those genes tested acted as modifiers of the HD toxicity indicating that the ability of a protein to physically interact with huntingtin correlated with its ability to show genetic interaction in a phenotypic assay. Genetic analysis in Drosophila has thus far identified 25 loss-of-function suppressors of HD toxicity. These results demonstrate that the ensemble of HIPs identified in our study are enriched for proteins that play a direct role in the cellular pathology of HD. These results also suggest that HIPs may be similarly enriched for proteins that can modify HD in human. The primary goal of this study is to test HIPs identified in our high-throughput studies for their ability modify HD phenotypes in mammalian cells and HD mouse models. We will use siRNAs to knock-down expression of each of these genes in human and mouse neuronal cell models of mutant HD toxicity. In specific cases, gene expression knock-downs will also be evaluated for effects on the metabolism, localization and/or post-translational modification of the huntingtin protein. Candidate proteins showing effects in these assays will be prioritized for more extensive studies in mouse models of HD. HIPs that modify the toxicity and/or biochemical properties of huntingtin will be tested for co-localization with huntingtin in transgenic HD mouse brain. HIPs whose reduced expression can suppress toxicity in cell-based assays (and/or Drosophila) will be tested for suppression in mouse models of HD. This will be done by constructing the appropriate shRNA-expressing transgenic mouse lines, crossing these into HD mouse models, and studying effects on the mouse HD phenotypes. We will also use AAV-mediated viral transfection of HIP shRNA to study effects of HIP knock-down in HD mice. The ultimate purpose of these studies is to identify modifiers of HD phenotypes in cell-based and/or Drosophila assays, determine their mechanisms of action and validate these in mouse models of HD. We anticipate that these studies will provide useful insight into the nature of HD pathology and also provide novel candidate targets for therapeutic drug discovery in HD.