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.

亨廷顿氏病（HD）是由亨廷顿（Huntingtin）的外显蛋白（Exon 1）重复膨胀（unknwon功能的基因）引起的。 在HD，小鼠和细胞培养模型中，包含聚谷氨酰胺道的Huntingtin片段经历了进行性核聚集。 我们已经建立了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的神经元变性和死亡，以及扩展的重复蛋白的逐步核定核定位。 人类和苍蝇中多谷氨酸膨胀的表型之间的相似性表明，多谷氨酰胺诱导的细胞死亡的分子机制至少部分是从果蝇到人的一部分。拟议研究的长期目标是利用我们的果蝇模型来揭示多谷氨酰胺诱导的细胞死亡的分子机制，以识别治疗靶标。 将使用一种三管齐下的方法：1。将通过检查随着时间的推移和表达各种表位标记的构建体的分布来评估含聚氟烷明蛋白在体内细胞毒性中的核聚集的作用。 我们还将检查多谷氨酰胺扩展的片段的相互作用与以截断片段或全长蛋白质内的病理和野生型重复长度相互作用。 2。可以通过在这些基因中突变的纯合子中表达Q120转基因来检查果蝇中确定的基因在果蝇中的作用。 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改良基因，包括纯合子的贴片以供感兴趣的突变。 3。用于改变与Q120表达相关的光感受器变性的突变的大规模遗传筛选将用于识别多谷氨酰胺诱导的细胞死亡的增强子和抑制器。诱变的雄性将被交叉至Q120含有Q120的女性。影响变性的突变将通过检查伪吡啶模式并在UV选择测试中对野生型反应进行评分来评估。 突变将通过“当地跳跃”技术进行局部和克隆。 通过与Q120系和可诱导细胞培养系统共同表达此类突变，可以验证抑制或增强。