Huntingtin interacting proteins as modifiers of Huntington's disease

描述(申请人提供)：亨廷顿病(HD)是一种进行性致命的神经退行性疾病，由亨廷顿蛋白基因中编码CAG区的聚谷氨酰胺扩张引起。亨廷顿蛋白的正常功能还不是很清楚。多谷氨酰胺毒性的确切性质以及它作用于导致细胞功能障碍的关键靶点也仍有待阐明。为了更好地了解亨廷顿蛋白的正常和致病功能，我们利用酵母双杂交和基于质谱学的方法进行了一次大规模的筛选，以发现亨廷顿蛋白相互作用蛋白(HIPS)。在使用数值和统计方法进行数据分析后，确定了234个髋关节的高置信度组。为了测试这些基因与HD病理的相关性，在HD的果蝇模型中，测试了编码60个相互作用蛋白的直系物的基因对毒性多谷氨酰胺表型的修改能力。这些被测试的基因中有80%是HD毒性的修饰物，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有关。到目前为止，对果蝇的遗传分析已经确定了25个HD毒性的功能丧失抑制因子。这些结果表明，在我们的研究中发现的髋关节整体富含在HD细胞病理中起直接作用的蛋白质。这些结果还表明，HIPS可能同样富含可以改变人类HD的蛋白质。这项研究的主要目的是测试在我们的高通量研究中确定的HIP在哺乳动物细胞和HD小鼠模型中修改HD表型的能力。我们将使用siRNAs在突变HD毒性的人和小鼠神经细胞模型中下调这些基因的表达。在特定情况下，还将评估基因表达下调对亨廷顿蛋白的新陈代谢、定位和/或翻译后修饰的影响。在这些检测中显示效果的候选蛋白质将被优先用于更广泛的HD小鼠模型研究。修改亨廷顿蛋白毒性和/或生化特性的HIPS将在转基因HD小鼠脑中与亨廷顿蛋白共同定位。HIPS的表达减少可以在基于细胞的分析中抑制毒性(和/或果蝇)，将在HD的小鼠模型中进行抑制测试。这将通过构建适当的shRNA表达转基因小鼠品系，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HIP shRNA病毒转染来研究HD小鼠HIP击倒的效果。这些研究的最终目的是在基于细胞和/或果蝇的分析中确定HD表型的修饰因素，确定它们的作用机制，并在HD的小鼠模型中验证它们。我们预计这些研究将为HD的病理本质提供有用的见解，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.