Functional Dissection of Huntington's Disease Protein Huntingtin Using Drosophila

描述（由申请人提供）：亨廷顿病（HD）是由亨廷顿蛋白（Htt）内聚谷氨酰胺（polyQ）通道异常扩张引起的。最近的研究表明，Htt蛋白的正常功能在决定最终的疾病结局中起着关键作用。然而，野生型Htt的功能研究一直受到小鼠Htt突变模型的早期致命性和Htt蛋白异常大尺寸的阻碍。尽管发现了大量的Htt相互作用蛋白（HIPs），但Htt在正常细胞中的作用仍然不明确，这成为研究HD发病机制和开发合理治疗这种毁灭性疾病的主要障碍。在一个简单的，遗传上可处理的系统中表征Htt同源物将补充已建立的哺乳动物模型。与秀丽隐杆线虫或酵母不同，在果蝇中存在一个单一的Htt同源物（dhtt），使我们能够在这个研究得很好的遗传模型系统中表征这个Htt家族蛋白。在初步研究中，我们建立了dhtt的零突变体，这是无脊椎动物模式生物中Htt家族基因的第一个突变等位基因。我们发现，与早期基于rnai的研究结果相反，dhtt对果蝇的发育是必不可少的，但去除内源性dhtt可以显著加速与polyq扩展Htt毒性果蝇模型相关的神经退行性表型，支持Htt的正常功能对HD发病至关重要；此外，dhtt是维持成年动物的活动能力和长期生存所必需的，它的缺失会影响成年大脑轴突末端的复杂性。这些研究使我们能够利用果蝇强大的遗传系统和丰富的实验工具，对dhtt零突变体进行更详细的表征，并对dhtt和HIPs同源物之间潜在的功能相互作用进行系统的评估。这项研究的结果将为Htt的正常功能和最终的HD机制提供重要的见解。在这项应用中，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dhtt相关表型，并使用在果蝇体内建立的实验来直接测试Htt在轴突囊泡运输和内噬作用中的细胞作用；(2)利用基因组标记方法建立dHtt蛋白体内分析的多功能工具箱，并进行缺失研究，绘制dHtt蛋白的功能域；(3)通过检测果蝇同源物与dhtt之间的遗传相互作用，评估哺乳动物HIPs的生理相关性，并采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. PUBLIC HEALTH RELEVANCE: The increasing prevalence of age-related neurodegenerative disorders, including Huntington's disease (HD), is becoming a mounting challenge to the well being of modern society. HD is a devastative brain degenerative disorder that affects at least 35,000 people in the U.S. alone, with another half a million at risk. Currently there is no preventive method or cure against HD. Recent studies suggest that alterations in the normal functions of disease protein Huntingtin play a critical role in the disease pathogenesis, and an effective therapeutic approach against HD might be to protect the normal activity of wildtype Huntingtin. Our research focuses on defining the normal functions of Huntingtin by studying its homologue in Drosophila, a simpler but well-studied and powerful genetic model system. We expect that the outcome of our research will contribute to our understanding of normal Huntingtin functions, which will not only help to uncover the molecular events underlying HD pathogenesis, but also to find effective prevention and treatments against this dreadful brain disease.