Functional Dissection of Huntington's Disease Protein Huntingtin Using Drosophila

使用果蝇对亨廷顿病蛋白亨廷顿蛋白进行功能解剖

• 批准号: 8454828
• 负责人: Sheng Zhang
• 金额: $ 6.25万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454828
• 关键词: Adult; Affect; Affinity Chromatography; Alleles; Animal Model; Biochemical; Biological Assay; Biological Models; Brain; Brain Diseases; Caenorhabditis elegans; Cell physiology; Collaborations; Complement; DRPLA protein; Degenerative Disorder; Development; Disease; Disease Outcome; Dissection; Drosophila genus; Endocytosis; Evaluation; Event; Gene Expression; Gene Family; Genes; Genetic; Genetic Models; Genetic screening method; Genome; Goals; Homologous Gene; Human; Huntington Disease; Invertebrates; Lead; Maps; Methods; Modeling; Molecular; Mus; Nematoda; Nerve Degeneration; Neurodegenerative Disorders; Outcome; Outcomes Research; Pathogenesis; Phenotype; Physiological; Play; Prevalence; Prevention; Preventive; Protein Family; Proteins; RNA Interference; Reporting; Research; Risk; Role; Societies; System; Testing; Therapeutic; Tissues; Toxic effect; Transport Vesicles; Yeasts; age related; base; deletion analysis; expectation; genetic analysis; human Huntingtin protein; human disease; in vivo; innovation; insight; mature animal; mutant; neurodegenerative phenotype; neurotoxicity; parkin gene/protein; polyglutamine; public health relevance; tool

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.

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。 公共卫生关系：包括亨廷顿氏病（HD）在内的年龄相关性神经退行性疾病的患病率日益增加，正成为对现代社会福祉的日益严峻的挑战。HD是一种脑退行性疾病，仅在美国就影响了至少35，000人，另有50万人处于危险之中。目前没有预防或治疗HD的方法。最近的研究表明，疾病蛋白亨廷顿蛋白的正常功能的改变在疾病的发病机制中起着至关重要的作用，有效的治疗方法可能是保护野生型亨廷顿蛋白的正常活性。我们的研究重点是通过研究果蝇中亨廷顿蛋白的同源物来确定亨廷顿蛋白的正常功能，果蝇是一个简单但研究充分且功能强大的遗传模型系统。我们希望我们的研究结果将有助于我们了解正常的亨廷顿蛋白功能，这不仅有助于揭示HD发病机制的分子事件，而且有助于找到有效的预防和治疗这种可怕的脑部疾病。