Loss-of-function mechanisms in Huntington's disease

亨廷顿病的功能丧失机制

• 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
• 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

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，该基因编码功能未知的大蛋白（亨廷顿蛋白；htt）。 HD 被认为是由 CAG 重复序列编码的聚谷氨酰胺延伸段所赋予的有害功能获得的结果。 htt 正常功能在疾病过程中的作用尚不清楚，但我们和其他人最近的工作表明，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，利用遗传学方法来了解 htt 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，旨在测试不同功能丧失机制在 HD 中的潜在贡献。第四个目标是测试一种基于在 HD 小鼠模型中恢复正常 htt 功能的潜在治疗策略。 (1) 为了检验 HD 功能丧失可能是由于突变体 htt 通过聚谷氨酰胺延伸段隔离野生型 htt 的能力而发生的假设，我们将生成小鼠 HD 基因同源物 (Hdh-deltaQ) 的表位标记等位基因，该基因缺乏精确的聚谷氨酰胺延伸段。 htt 的这种修饰版本抵抗突变体 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失活相互作用，将生成在每个 Hdh 等位基因中定向插入不同表位标签的 ES 细胞系，用于免疫沉淀下拉测定。 (2) 为了测试 htt 功能丧失是否可能通过突变 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是 HD 发病机制中的限速步骤，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：第一个表达全长突变体 htt，第二个表达截短版本的 htt 突变体 htt。两种突变蛋白均在内源性 Hdh 启动子的控制下表达，从而可以直接比较两种模型。 (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失活干扰而发生。为了在体内测试这一假设，我们将描述失去 htt 与突触后密度 95 蛋白相互作用的影响，这可能导致 Hdh 条件敲除小鼠模型中 N-甲基-D-天冬氨酸 (NMDA) 受体功能改变。 (4)最后，我们将尝试通过在前脑中过度表达 htt 的时间调节显性失活抗性形式来挽救 HD 小鼠模型中的表型。