Transgenic C. elegans as Amyloid Disease Model

描述（由申请人提供）：尽管有令人信服的证据表明-淀粉样肽(A)在阿尔茨海默病的病理中起着核心作用，但A的毒性机制及其特定靶点仍未得到解决。本项目是我们使用秀丽隐杆线虫作为模型研究（人类）a¿毒性基础的研究的延续。我们之前已经证明，转基因蠕虫表达人类A¿1-42，复制阿尔茨海默病病理的某些方面。通过分子和遗传方法的结合，我们现在已经确定了一组在这些转基因蠕虫模型中调节a¿毒性的保守基因。此外，对表达单氨基酸变体的转基因蠕虫的分析导致鉴定出一种在体内基本上无毒的A¿变体。本提案的目标是综合这些发现来确定：1)有毒A¿物种的身份，2)影响有毒物种形成的细胞途径，以及3)A¿的特定细胞靶点。本提案的具体目的是：1)通过构建和表征表达变异A¿肽的转基因蠕虫，对A¿毒性进行体内结构/功能分析。这些研究将直接测试“有毒A¿寡聚物”模型，2)确定进化保守修饰基因改变A¿毒性的分子机制，以及3)验证我们通过哺乳动物细胞培养和原代神经元培养确定的A¿毒性和保护基因的建议机制。我们提出的研究对阿尔茨海默病的诊断和治疗有直接的相关性。确定A¿的主要毒性形式可能对设计防止其形成或毒性活性的药物至关重要。表征影响A¿毒性的基因可能对阿尔茨海默病和其他神经退行性疾病的风险评估和有效干预措施的发展都很重要。公共卫生启示：有令人信服的证据表明，在阿尔茨海默病中发现的一种特定蛋白质——淀粉样肽——的积累是大脑病理的基础，阿尔茨海默病影响着约500万美国人。提出的研究旨在确定-淀粉样肽毒性的细胞和分子基础。了解这种蛋白的毒性机制对于开发阿尔茨海默病的治疗方法至关重要。

DESCRIPTION (provided by applicant): Although there is compelling evidence that the ¿-amyloid peptide (A¿) is centrally involved in Alzheimer's disease pathology, the mechanisms of A¿ toxicity and its specific targets remain unresolved. This project is a continuation of our studies using the intensely studied nematode worm Caenorhabditis elegans as a model to investigate the basis of (human) A¿ toxicity. We have previously shown that transgenic worms engineered to express human A¿ 1-42, replicate some aspects of Alzheimer's disease pathology. By a combination of molecular and genetic approaches, we have now identified a set of conserved genes that modulate A¿ toxicity in these transgenic worm models. In addition, analysis of transgenic worms expressing single amino acid variants has led to the identification of an A¿ variant that is substantially non-toxic in vivo. The goal of this proposal is to synthesize these findings to establish: 1) the identity of the toxic A¿ species, 2) the cellular pathways that influence the formation of the toxic species, and 3) the specific cellular targets of A¿. The Specific Aims of this proposal are to: 1) perform an in vivo structure/function analysis of A¿ toxicity by constructing and characterizing transgenic worms expressing variant A¿ peptides. These studies will directly test the "toxic A¿ oligomer" model, 2) determine the molecular mechanisms by which evolutionarily conserved modifier genes alter A¿ toxicity, and 3) validate the proposed mechanisms for both A¿ toxicity and the protective genes we have identified using mammalian cell culture and primary neuronal cultures. Our proposed studies have direct relevance for the diagnosis and treatment of Alzheimer's disease. The identity of the key toxic form(s) of A¿ may be critical for designing drugs that prevent its formation or toxic activity. Characterization of genes that affect the toxicity of A¿ may be important for both risk assessment and the development of effective interventions for Alzheimer's and other neurodegenerative diseases. PUBLIC HEALTH REVELANCE: There is compelling evidence that accumulation of a specific protein, the ¿- amyloid peptide, underlies the brain pathology found in Alzheimer's disease, which affects ~5,000,000 people in the US. The proposed study seeks to determine the cellular and molecular basis of ¿-amyloid peptide toxicity. Understanding the toxic mechanism of this protein make be critical for developing therapeutics for Alzheimer's disease.