Loss-of-function mechanisms in Huntington's disease

亨廷顿病的功能丧失机制

基本信息

批准号：
7194241
负责人：
Scott Zeitlin
金额：
$ 30万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-01 至 2008-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7194241
关键词：
22q Alleles Apoptotic Biological Assay CAG repeat Caspase Corpus striatum structure Cultured Cells Cytoskeleton DLG4 gene Data Disease Disease model Dominant-Negative Mutation ES Cell Line Epitopes Exhibits Exons Gene Targeting Genes Genetic Glutamine Homologous Gene Human Huntington Disease Huntington gene Immune Immunoprecipitation In Vitro Knock-in Mouse Knock-out Lead Length Link Male Sterility Mediating Modeling Mus N-Methyl-D-Aspartate Receptors N-Methylaspartate N-terminal Nervous System Heredodegenerative Disorders Neurodegenerative Disorders Neurons Pathogenesis Phenotype Play Process Property Prosencephalon Proteins Proteolysis Proteolytic Processing Range Rate Resistance Role Scaffolding Protein Severities Site Specificity Stimulus Stretching System Testing Testis Testis Brain Therapeutic Thinking Transgenic Organisms Trinucleotide Repeats Work base cell type design disease phenotype embryonic stem cell gain of function gain of function mutation gene therapy human Huntingtin protein in vivo loss of function mouse model mutant novel polyglutamine polypeptide postnatal presynaptic density protein 95 progressive neurodegeneration promoter receptor function research study

项目摘要

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.

描述（由申请人提供）：亨廷顿氏病（HD）是一种主导的遗传神经退行性疾病，是由编码大型蛋白质（Huntingtin; htt）在未知功能的HD基因内扩展引起的。 HD被认为是有害功能获得的结果，该功能是由CAG重复序列编码的多谷氨酰胺扩展所赋予的。 HTT在疾病过程中的正常功能的作用尚不清楚，但是我们最近的工作和其他工作表明，正常HTT功能的丧失也可能导致发病机理。我们的长期目标是使用遗传方法使用细胞培养和小鼠模型来了解HTT正常功能在HD发病机理中的作用。为了实现这一目标，我们提出了三个互补的特定目的，旨在测试HD中不同功能丧失机制的潜在贡献。第四个目标旨在测试基于HD小鼠模型中正常HTT功能的潜在治疗策略。（1）为了检验HTT突变HTT通过聚谷氨酰胺延伸隔离野生型HTT的能力而发生的假设，我们将产生小鼠HD基因同源物（HDH-DELTAQ）的表位标准等位基因，该等位基因缺乏鼠HD基因同源物（HDH-DELTAQ），而这些等位基因缺乏即将到来的Party party the Party the多氯丁胺拉伸。将在细胞培养中评估这种修饰版本的HTT版本抗性HTT隔离的能力。此外，为了测试HTT是否能够通过与自身的相互作用来参与潜在的显性阴性相互作用，将在每个HDH等位基因中具有针对性插入不同表位标签的ES细胞系列，以用于免疫沉淀下拉分析。（2）测试HTT功能丧失是否通过突变HTT激活caspase介导的蛋白水解的能力以及HTT的蛋白水解切割是HD发病机理中的速率限制一步突变htt。两种突变蛋白均在内源性HDH启动子的控制下表达，从而可以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与蛋白质伴侣的相互作用的显性阴性干扰发生。为了在体内检验这一假设，我们将表征失去HTT与突触后密度95蛋白的影响，这可能导致在HDH条件敲除小鼠模型中改变N-甲基-D-天冬氨酸（NMDA）受体功能。（4）最后，我们将尝试通过过度表达前表调节的前脑中的HTT的抗优势抗性形式来挽救HD小鼠模型中的表型。