Sequence Context-Specific Modifiers of Huntingtin Aggregation in C.elegans

描述(由申请人提供)：这里提出的实验是为了解决HD中的一个基本问题：亨廷顿(Htt)中扩展的聚谷氨酰胺(PolyQ)的序列上下文是否对毒性机制有显著贡献。换句话说，突变体Htt中多Q扩展的毒性在多大程度上受到不同片段或全长Htt中不同侧翼序列的存在的影响？为了解决这个问题，我们将产生一个新的转基因线虫集合，表达各种N-末端蛋白水解性切割片段和全长Htt，每个都包含短(Q15)、中(Q40)和长(Q128)多聚Q扩展。这将允许在一个单一的动物模型系统中，比较神经元中表达的不同Htt蛋白片段的生物和生化表型。由于我们的实验室以前已经建立了线虫对多聚Q单独的行为、细胞和遗传反应，这些研究应该揭示侧翼Htt序列的贡献在多大程度上影响细胞毒性。由于毒性无疑代表了多个分子缺陷的整合，我们将进行全基因组RNAi筛选以确定遗传修饰物。如果对表达不同长度Htt片段的转基因株系鉴定出相同的修饰物，我们将得出结论，Htt毒性的分子基础不受不同序列背景的影响。或者，如果这些遗传筛选揭示了不同类别的修饰物，我们将得出结论，Htt侧翼序列对细胞机制有很强的影响。这项研究的结果可能对分析和解释目前许多关于Htt在其他转基因模型系统中的表达的研究具有重要意义。与公共健康相关：这里提出的研究与公共健康相关，因为它直接检查了多谷氨酰胺疾病的遗传/分子机制，多谷氨酰胺疾病是衰老神经退行性疾病的一个子集。通过了解疾病的分子基础，我们将更好地准备开发治疗策略来对抗受影响患者的疾病症状。

DESCRIPTION (provided by applicant): The experiments proposed here are to address a fundamental question in HD; whether the sequence context of the expanded polyglutamine (polyQ) in Huntingtin (Htt) contributes significantly to the mechanisms of toxicity. In other words, to what extent is the toxicity of the polyQ expansion in mutant Htt influenced by the presence of different flanking sequences in the various fragments or full-length Htt? To address this, we will generate a new collection of transgenic C. elegans expressing various N-terminal proteolytic cleavage fragments and full-length Htt, each containing short (Q15), medium (Q40), and long (Q128) polyQ expansions. This will allow for comparison, in a single animal model system, of the biological and biochemical phenotypes of different Htt protein fragments expressed in neurons. Because our laboratory has previously established the C. elegans behavioral, cellular and genetic responses to polyQ alone, these studies should reveal to what extent cellular toxicity is influenced by contributions of the flanking Htt sequences. As toxicity undoubtedly represents the integration of multiple molecular defects, we will perform genome-wide RNAi screens to identify genetic modifiers. If the same modifiers are identified for transgenic lines expressing different length Htt fragments, we will conclude that the molecular basis for Htt toxicity is not influenced by varying sequence context. Alternatively, if distinct classes of modifiers are revealed by these genetic screens, we will conclude that the Htt flanking sequences have a strong influence on the cellular machinery. The results of this study are likely to have important implications for the analysis and interpretation of many current studies on Htt expression in other transgenic model systems. RELEVANCE TO PUBLIC HEALTH: The research proposed here is relevant to public health in that it directly examines the genetic/molecular mechanisms of polyglutamine diseases, a subset of neurodegenerative diseases of aging. By understanding the molecular underpinings of disease, we will be better poised to develop therapeutic strategies to combat disease symptoms in affected patients.