MOLECULAR GENETICS OF POLYGLUTAMINE INDUCED DEGENERATION

亨廷顿病(HD)是由亨廷顿蛋白基因外显子1内CAG重复序列的多态扩大引起的，亨廷顿蛋白是一种功能未知的基因。在HD、小鼠和细胞培养模型中，含有聚谷氨酰胺束的亨廷顿蛋白片段经历了渐进性的核聚集。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，包括依赖于年龄和重复长度的神经元退化和死亡，以及扩展的包含重复的蛋白的进行性核定位。在人类和果蝇中，多谷氨酰胺扩大的表型之间的相似性表明，多谷氨酰胺诱导细胞死亡的分子机制至少在一定程度上是从果蝇到人类保守的。这项拟议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，以努力确定治疗靶点。将使用三管齐下的方法：1.通过检测不同表位标记的构建体随时间的分布以及在核输出信号存在的情况下表达这些构建体来评估含有聚谷氨酰胺的蛋白的核聚集在体内对细胞毒性的作用。我们还将研究聚谷氨酰胺扩展片段与病理和野生型重复长度的相互作用，这些重复长度以截断片段的形式出现或在全长蛋白质中出现。2.在果蝇中识别的基因可能改变多谷氨酰胺诱导的神经细胞死亡的作用将通过在这些基因突变纯合的背景中表达Q120转基因来检验。我们将通过在基因马赛克背景中表达Q120结构来评估潜在的疾病修饰基因，包括对感兴趣的突变纯合的斑块。3.对与Q120表达相关的改变光感受器退化的突变的大规模基因筛查将用于识别多谷氨酰胺诱导的细胞死亡的增强子和抑制子。突变的雄性将与携带Q120的雌性杂交。影响变性的突变将通过检查假性皮下模式和在紫外线选择测试中恢复到野生型反应来评分。突变将使用“局部跳跃”技术进行定位和克隆。抑制或增强将通过与Q120品系和可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.