Loss-of-function mechanisms in Huntington's disease

描述（由申请人提供）：亨廷顿舞蹈病（HD）是一种显性遗传性神经退行性疾病，是由亨廷顿舞蹈病基因中CAG重复序列的扩增引起的，该基因编码一种功能未知的大蛋白（亨廷顿舞蹈症；htt）。HD被认为是由CAG重复序列编码的聚谷氨酰胺的扩展所带来的有害的功能获得的结果。htt正常功能在疾病过程中的作用尚不清楚，但我们最近的工作和其他人的研究表明，正常htt功能的丧失也可能有助于发病机制。我们的长期目标是通过细胞培养和小鼠模型，利用遗传学方法了解htt的正常功能在HD发病机制中的作用。为了实现这一目标，我们提出了三个互补的具体目标，旨在测试HD中不同功能丧失机制的潜在贡献。第四个目标是在HD小鼠模型中测试一种基于恢复正常htt功能的潜在治疗策略。(1)为了验证突变体htt通过聚谷氨酰胺片段隔离野生型htt的能力可能导致HD功能丧失的假设，我们将生成一个小鼠HD基因同源物（Hdh-deltaQ）的表位标记等位基因，该等位基因正是缺乏聚谷氨酰胺片段。这个改良版的htt抵抗突变体htt隔离的能力将在细胞培养中进行评估。此外，为了测试htt是否能够通过自身相互作用参与潜在的显性-负相互作用，将生成一个在每个Hdh等位基因中靶向插入不同表位标签的ES细胞系，用于免疫沉淀下拉试验。(2)为了检验htt的功能丧失是否可能通过突变体htt激活caspase介导的蛋白水解的能力而发生，以及htt的蛋白水解裂解是否为HD发病的限速步骤，我们将比较两种敲入HD小鼠模型所表现出的表型的发生和进展：第一种表达全长突变体htt，第二种表达突变体htt的截断版本。两种突变蛋白都在内源性Hdh启动子的控制下表达，从而可以直接比较两种模型。(3)野生型Htt与蛋白伴侣相互作用的显性负干扰也可能导致Htt功能丧失。为了在体内验证这一假设，我们将在Hdh条件敲除小鼠模型中描述失去htt与突触后密度95蛋白相互作用可能导致n -甲基- d -天冬氨酸（NMDA）受体功能改变的影响。(4)最后，我们将尝试通过在前脑过度表达一种暂时受调控的显性负抗性htt形式来挽救HD小鼠模型中的表型。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a dominant hereditary neurodegenerative disease that is caused by the expansion of a stretch of CAG repeats within the HD gene that encodes a large protein (huntingtin; htt) of unknown function. HD is thought to be the consequence of a deleterious gain-of-function that is conferred by the expanded stretch of polyglutamine encoded by the CAG repeats. The role of the normal function of htt in the disease process is unknown, but our recent work and that of others suggests that loss of normal htt function may also contribute to pathogenesis. Our long-term objective is to use genetic approaches to understand the role of htt's normal functions in HD pathogenesis using both cell culture and mouse models. To accomplish this objective, we propose three complementary specific aims that are designed to test the potential contribution of different loss-of-function mechanisms in HD. A fourth aim is designed to test a potential therapeutic strategy based on restoring normal htt function in HD mouse models. (1) To test the hypothesis that loss-of-function in HD may occur through mutant htt's ability to sequester wild-type htt via the polyglutamine stretch, we will generate an epitope-tagged allele of the mouse HD gene homologue (Hdh-deltaQ) that lacks precisely the polyglutamine stretch. The ability of this modified version of htt to resist sequestration by mutant htt will be assessed in cell culture. In addition, in order to test if htt is capable of participating in potential dominant-negative interactions by interacting with itself, an ES cell line with targeted insertion of different epitope tags in each Hdh allele will be generated for use in immunoprecipitation pull-down assays. (2) To test if htt loss-of-function may occur through mutant htt's ability to activate caspase-mediated proteolysis, and if proteolytic cleavage of htt is a rate-limiting step in HD pathogenesis, we will compare the onset and progression of phenotypes exhibited by two knockin HD mouse models: the first expressing a full-length mutant htt, and the second expressing a truncated version of mutant htt. Both mutant proteins are expressed under the control of the endogenous Hdh promoter, enabling a direct comparison between the two models. (3) Htt loss-of-function may also occur via dominant-negative interference of wild-type htt interactions with protein partners. To test this hypothesis in vivo, we will characterize the impact of losing htt interactions with the postsynaptic density 95 protein that could lead to altered N-methyl-D-aspartate (NMDA) receptor function in an Hdh conditional knockout mouse model. (4) Finally, we will attempt to rescue phenotypes in an HD mouse model by over-expressing a temporally regulated dominant-negative resistant form of htt in the forebrain.