MOLECULAR GENETICS OF POLYGLUTAMINE INDUCED DEGENERATION

亨廷顿氏病（HD）是由亨廷顿蛋白（huntingtin）外显子1内的多态性CAG重复扩增引起的，亨廷顿蛋白是一种功能未知的基因。 在HD和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进行进行性核聚集。 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性神经元变性和死亡，以及扩展的含重复序列蛋白的渐进核定位。 在人类和苍蝇的聚谷氨酰胺扩大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的基础上，至少部分地，从果蝇到人保守。拟议的研究的长期目标是利用我们的果蝇模型HD解开聚谷氨酰胺诱导的细胞死亡的分子机制，努力确定治疗靶点。 将采取三管齐下的办法：1.将通过检查各种表位标记的构建体随时间的分布和通过在核输出信号存在下表达这些构建体来评估含聚谷氨酰胺蛋白的核聚集对体内细胞毒性的作用。 我们还将研究多聚谷氨酰胺扩增片段与病理性和野生型重复长度的相互作用，这些重复长度作为截短片段或在全长蛋白质内呈现。 2.通过在这些基因突变的背景纯合中表达Q120转基因，将检查在果蝇中鉴定的可能修饰聚谷氨酰胺诱导的神经元细胞死亡的基因的作用。 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修饰基因，包括感兴趣的突变纯合的补丁。 3.一个大规模的基因突变，改变与Q120表达相关的光感受器变性的筛选将被用来确定多聚谷氨酰胺诱导的细胞死亡的增强子和抑制子。诱变雄性将与携带Q120的雌性杂交。通过检查假瞳孔模式和通过在UV选择试验中对回复至野生型应答进行评分，对影响退化的突变进行评分。 使用“局部跳跃”技术定位和克隆突变。抑制或增强将通过与Q120细胞系和诱导型细胞培养系统共表达此类突变来验证。

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.