MOLECULAR GENETICS OF POLYGLUTAMINE INDUCED DEGENERATION

聚谷氨酰胺诱导变性的分子遗传学

• 批准号: 6187732
• 负责人: GEORGE R JACKSON
• 金额: $ 10.56万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-05 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6187732
• 关键词: Drosophilidae; Huntington's disease; cell death; cell nucleus; cytotoxicity; disease /disorder model; epitope mapping; genetically modified animals; glutamine; homopeptide; immunocytochemistry; intracellular transport; molecular genetics; mutant; neural degeneration; neurogenetics; nucleic acid repetitive sequence; phenotype; protein transport; tissue /cell culture

Huntington's disease (HD) is caused by expansion of a polymorphic CAG repeat within exon 1 of huntingtin, a gene of unknwon function. In HD and mouse and cell culture models, huntingtin fragments containing the polyglutamine tract undergo progressive nuclear aggregation. We have established a Drosophila model of HD that shares important features of the human phenotype, including age- and repeat-length-dependent neuronal degeneration and death, as well progressive nuclear localization of expanded repeat-containing protein. The similarities between the polyglutamine-expanded phenotype in humans and flies suggest that the molecular mechanisms underlying polyglutamine-induced cell death are, at least in part, conserved from Drosophila to man. The long-term objective of the proposed research is to utilize our Drosophila model of HD to unravel the molecular mechanisms of polyglutamine-induced cell death in an effort to identify therapeutic targets. A three-pronged approach will be used: 1. The role of nuclear aggregation of polygluytamine-containing protein on cytotoxicity in vivo will be assessed by examining the distribution of various epitope-tagged constructs over time and by expressing these constructs in the presence of a nuclear export signal. We will also examine the interaction of polyglutamine-expanded fragments with both pathologic and wild type repeat lengths presented as truncated fragments or within the full length protein. 2. The role of identified genes in Drosophila that may modify polyglutamine-induced neuronal cell death will be examined by expressing the Q120 transgene in a background homozygous for mutations in these genes. We will assess potential disease-modifying genes by expressing the Q120 construct in a genetic mosaic background, including patches homozygous for the mutation of interest. 3. A large-scale genetic screen for mutations that alter the photoreceptor degeneration associated with Q120 expression will be used to identify enhancers and suppressors of polyglutamine-induced cell death. Mutagenized males will be crossed to Q120-bearing females. Mutations affecting degeneration will be scored by examining the pseudopupil pattern and by scoring reversion to the wild type response in a UV choice test. Mutations will be localized and cloned using the technizue of "local hopping." Suppression or enhancement will be verified by co-expressing such mutations with Q120 lines and in inducible cell culture systems.

亨廷顿病 (HD) 是由亨廷顿蛋白（一种功能未知的基因）外显子 1 内多态性 CAG 重复序列的扩增引起的。 在 HD 以及小鼠和细胞培养模型中，含有多聚谷氨酰胺束的亨廷顿蛋白片段经历渐进性核聚集。 我们建立了 HD 的果蝇模型，该模型具有人类表型的重要特征，包括年龄和重复长度依赖性神经元变性和死亡，以及扩展的含重复蛋白的渐进核定位。 人类和果蝇中聚谷氨酰胺扩增表型之间的相似性表明，聚谷氨酰胺诱导的细胞死亡的分子机制至少部分地从果蝇到人类是保守的。本研究的长期目标是利用我们的 HD 果蝇模型来揭示多聚谷氨酰胺诱导的细胞死亡的分子机制，从而确定治疗靶点。 将使用三管齐下的方法： 1. 含聚谷氨酰胺蛋白的核聚集对体内细胞毒性的作用将通过检查各种表位标记构建体随时间的分布并通过在存在核输出信号。 我们还将检查聚谷氨酰胺扩增片段与以截短片段或全长蛋白质内呈现的病理和野生型重复长度之间的相互作用。 2.通过在这些基因突变纯合的背景中表达Q120转基因，将检查果蝇中可能改变多聚谷氨酰胺诱导的神经元细胞死亡的已鉴定基因的作用。 我们将通过在遗传镶嵌背景中表达 Q120 构建体来评估潜在的疾病缓解基因，包括目标突变纯合的补丁。 3. 对改变与 Q120 表达相关的光感受器变性的突变进行大规模遗传筛选，将用于鉴定多聚谷氨酰胺诱导的细胞死亡的增强子和抑制子。诱变的雄性将与携带 Q120 的雌性杂交。影响变性的突变将通过检查假瞳孔模式并通过在紫外线选择测试中对恢复到野生型反应进行评分来评分。 突变将使用“局部跳跃”技术进行本地化和克隆。 通过与 Q120 系和诱导细胞培养系统共表达此类突变来验证抑制或增强。