Loss-of-function mechanisms in Huntington's disease

描述(申请人提供)：亨廷顿病(HD)是一种显性遗传性神经退行性疾病，由HD基因内一段CAG重复序列的扩展引起，该基因编码一种功能未知的大蛋白质(Huntingtin；HTT)。HD被认为是由CAG重复序列编码的聚谷氨酰胺的扩展延伸而产生的有害的功能获得的结果。HTT的正常功能在疾病过程中的作用尚不清楚，但我们和其他人最近的工作表明，正常HTT功能的丧失也可能参与发病机制。我们的长期目标是利用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，旨在测试不同的功能丧失机制在HD中的潜在贡献。第四个目标是测试一种潜在的治疗策略，即在HD小鼠模型中恢复正常的HTT功能。(1)为了验证HD功能丧失可能是由于突变体HTT通过聚谷氨酰胺延伸隔离野生型HTT的能力而发生的，我们将产生小鼠HD基因同源基因的表位标记等位基因(HDH-deltaQ)，该等位基因正好缺少聚谷氨酰胺延伸。这种改良版本的HTT抵抗突变的HTT的能力将在细胞培养中进行评估。此外，为了测试HTT是否能够通过与自身相互作用参与潜在的显性-负性相互作用，将产生一个在每个HDH等位基因中定向插入不同表位标签的ES细胞系，用于免疫沉淀下拉分析。(2)为了测试HTT功能丧失是否可能通过突变型HTT激活caspase介导的蛋白分解而发生，以及如果HTT的蛋白水解性切割是HD发病机制中的限速步骤，我们将比较两种Huckin HD小鼠模型的表型的发生和发展：第一种表达全长突变的HTT，第二种表达突变的HTT的截短版本。这两种突变蛋白都是在内源性HDH启动子的控制下表达的，从而能够在两种模型之间进行直接比较。(3)HTT功能丧失也可能通过野生型HTT与蛋白质伴侣相互作用的显性-负性干扰发生。为了在体内验证这一假说，我们将在HDH条件性基因敲除小鼠模型中表征失去HTT与突触后密度95蛋白相互作用的影响，这可能导致N-甲基-D-天冬氨酸(NMDA)受体功能改变。(4)最后，我们将试图通过在前脑过度表达一种受时间调控的显性-负性抵抗形式的HTT来挽救HD小鼠模型的表型。

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.