Functional Dissection of Huntington's Disease Protein Huntingtin Using Drosophila

描述（由申请人提供）：亨廷顿病（HD）是由亨廷顿（Htt）蛋白内多聚谷氨酰胺（polyQ）片段的异常扩张引起的。最近的研究表明，Htt蛋白的正常功能在决定疾病的最终结果中起着关键作用。然而，对野生型Htt的功能研究受到小鼠Htt突变模型的早期致死性和Htt蛋白的不寻常的大尺寸的阻碍。尽管Htt相互作用蛋白（Htt interacting proteins，HIP）已被大量发现，但Htt在正常细胞中的作用仍不明确，这成为研究HD发病机制和开发合理治疗方法的主要障碍。在一个简单的，遗传上易于处理的系统中表征Htt同源物将补充已建立的哺乳动物模型。不像C。线虫或酵母中，一个单一的Htt同源物存在于果蝇（dhtt），使我们能够在这个研究充分的遗传模型系统中表征这个Htt家族蛋白。在初步研究中，我们已经建立了一个空突变的dhtt，第一个突变等位基因的Htt家族基因在无脊椎动物模式生物。我们发现，与早期基于RNAi的研究结果相反，dhtt对果蝇发育是不利的，但去除内源性dhtt可以显著加速与polyQ扩展的Htt毒性的果蝇模型相关的神经退行性表型，支持Htt的正常功能对于HD发病机制是重要的;此外，dhtt是维持成年动物的活动性和长期生存所必需的，它的缺失会影响成年大脑中轴突末端的复杂性。这些研究使我们能够利用强大的遗传系统和丰富的实验工具，在果蝇进行更详细的表征dhtt无效突变体和dhtt和HIP同源物之间的潜在功能相互作用进行系统的评估。这项研究的结果将为Htt的正常功能以及最终HD的潜在机制提供重要的见解。在本研究中，我们提出了以下具体目标：（1）通过超微结构和基因表达分析来表征dtt相关的表型，并使用已建立的果蝇体内实验来直接测试Htt在轴突囊泡运输和内吞中的细胞作用;（2）利用基因组标签技术建立dHtt蛋白体内分析的通用工具箱，（3）通过检测果蝇同源基因与dhtt之间的遗传相互作用来评估哺乳动物HIPs的生理相关性，并利用基于串联亲和纯化（TAP）的方法直接分离果蝇HIPs。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is caused by an abnormal expansion of a polyglutamine (polyQ) tract within the Huntingtin (Htt) protein. Recent studies have demonstrated that normal functions of Htt protein play a critical role in determining the final disease outcome. However, functional studies on wildtype Htt have been hampered by the early lethality of murine Htt mutant models and by the unusual large size of Htt protein. Despite the identification of large number of Htt interacting proteins (HIPs), the normal cellular roles of Htt remain poorly defined, which is becoming a major obstacle in studying the pathogenesis of HD and developing rational therapies to treat this devastating disease. Characterizing a Htt homolog in a simple, genetically tractable system will complement the established mammalian models. Unlike in C. elegans or yeast, a single Htt homolog exists in Drosophila (dhtt), allowing us to characterize this Htt family protein in this well-studied genetic model system. In preliminary studies, we have established a null-mutant for dhtt, the first mutant allele for a Htt family gene in an invertebrate model organism. We found that contrary to the results from an earlier RNAi-based study, dhtt is dispensable for Drosophila development, but removing endogenous dhtt can significantly accelerate the neurodegenerative phenotypes associated with a Drosophila model of polyQ-expanded Htt toxicity, supporting that normal function of Htt is important for HD pathogenesis; Furthermore, dhtt is required for maintaining the mobility and long-term survival of adult animals, and its absence affects axonal terminal complexity in the adult brain. These studies allow us to use the powerful genetic system and abundant experimental tools in Drosophila to carry out more detailed characterization of the dhtt null mutant and perform systematic evaluation of potential functional interactions between dhtt and HIPs homologues. Outcome of this research will provide critical insights into the normal function of Htt and ultimately the mechanisms underlying HD. In this application, we propose the following Specific Aims: (1) Characterize dhtt-associated phenotypes by ultrastructural and gene expression analyses, and use established in vivo assays in Drosophila to directly test proposed cellular roles of Htt in axonal vesicle transport and endocytosis; (2) Use a genome-tagging approach to establish a versatile toolbox for in vivo analysis of dHtt protein, and perform deletion study to map the functional domains in dHtt protein; (3) Assess the physiological relevance of mammalian HIPs by testing genetic interactions between their Drosophila homologues and dhtt, and use the Tandem Affinity Purification (TAP)-based approach to directly isolate Drosophila HIPs.