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: 8669171
• 负责人: Sheng Zhang
• 金额: $ 38.02万
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8669171
• 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; 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.

描述（由申请人提供）：亨廷顿病（HD）是由亨廷顿蛋白（Htt）内的聚谷氨酰胺（polyQ）束异常扩张引起的。最近的研究表明，Htt 蛋白的正常功能在决定最终疾病结果方面发挥着关键作用。然而，对野生型 Htt 的功能研究因鼠 Htt 突变模型的早期致死性和 Htt 蛋白异常大的尺寸而受到阻碍。尽管鉴定了大量的 Htt 相互作用蛋白 (HIP)，但 Htt 的正常细胞作用仍不清楚，这正成为研究 HD 发病机制和开发治疗这种破坏性疾病的合理疗法的主要障碍。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。与线虫或酵母不同，果蝇 (dhtt) 中存在单个 Htt 同源物，使我们能够在这个经过充分研究的遗传模型系统中表征该 Htt 家族蛋白。在初步研究中，我们已经建立了 dhtt 的无效突变体，这是无脊椎动物模型生物中 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对于果蝇发育来说是可有可无的，但去除内源性dhtt可以显着加速与polyQ扩展Htt毒性果蝇模型相关的神经退行性表型，支持Htt的正常功能对于HD发病机制很重要；此外，dhtt 是维持成年动物的活动能力和长期生存所必需的，它的缺乏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果蝇强大的遗传系统和丰富的实验工具对dhtt无效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评估。这项研究的结果将为 Htt 的正常功能以及最终 HD 的机制提供重要的见解。在本申请中，我们提出以下具体目标：（1）通过超微结构和基因表达分析来表征 dhtt 相关表型，并使用果蝇体内已建立的测定法来直接测试 Htt 在轴突囊泡运输和内吞作用中的细胞作用； （2）利用基因组标记方法建立dHtt蛋白体内分析的通用工具箱，并进行缺失研究以绘制dHtt蛋白的功能域； (3) 通过测试果蝇同源物与 dhtt 之间的遗传相互作用来评估哺乳动物 HIP 的生理相关性，并使用基于串联亲和纯化 (TAP) 的方法直接分离果蝇 HIP。