MOLECULAR GENETICS OF POLYGLUTAMINE INDUCED DEGENERATION

亨廷顿氏病（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重复扩增引起的。在HD和小鼠及细胞培养模型中，含有聚谷氨酰胺束的亨廷顿蛋白片段经历进行性核聚集。我们已经建立了一个果蝇HD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的神经元变性和死亡，以及扩展的重复包含蛋白的进行性核定位。人类和果蝇中聚谷氨酰胺扩增表型的相似性表明，聚谷氨酰胺诱导细胞死亡的分子机制，至少在一定程度上，从果蝇到人类是保守的。本研究的长期目标是利用我们的果蝇HD模型来揭示聚谷氨酰胺诱导细胞死亡的分子机制，以确定治疗靶点。将采用三管齐下的方法：通过检查各种表位标记构建体随时间的分布以及在核输出信号存在下表达这些构建体，将评估含聚谷氨酰胺蛋白的核聚集对体内细胞毒性的作用。我们还将研究聚谷氨酰胺扩增片段与病理和野生型重复长度的相互作用，这些重复长度表现为截断片段或在全长蛋白内。2. 通过在这些基因突变的背景纯合子中表达Q120转基因，可以检测已鉴定基因在果蝇中可能修饰聚谷氨酰胺诱导的神经元细胞死亡的作用。我们将通过在遗传马赛克背景下表达Q120构建体来评估潜在的疾病修饰基因，包括对感兴趣突变的纯合斑块。3. 对与Q120表达相关的光感受器变性的突变进行大规模的基因筛选，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和在紫外线选择测试中对野生型反应的评分来评分。突变将通过“局部跳跃”技术进行定位和克隆。抑制或增强将通过与Q120系和诱导细胞培养系统共表达这些突变来验证。

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.