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

基本信息

批准号：
8462704
负责人：
Sheng Zhang
金额：
$ 37.06万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
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/8462704
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

项目摘要

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）：亨廷顿氏病（HD）是由亨廷顿蛋白（HTT）蛋白质中聚谷氨酰胺（Polyq）裂片异常扩张引起的。最近的研究表明，HTT蛋白的正常功能在确定最终疾病结果中起关键作用。然而，关于野生型HTT的功能研究已受到鼠HTT突变模型的早期致死性和异常大尺寸的HTT蛋白的影响。尽管鉴定出大量的HTT相互作用蛋白（臀部），但HTT的正常细胞作用仍然很差，这在研究HD发病机理和发展有理理性疗法以治疗这种毁灭性疾病的情况下成为主要障碍。在简单的遗传处理系统中表征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，果蝇（DHTT）中存在单个HTT同源物，使我们能够在这个精心研究的遗传模型系统中表征该HTT家族蛋白。在初步研究中，我们为DHTT建立了无效的DHTT，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，与较早的基于RNAi的研究的结果相反，DHTT对于果蝇的发育是可支配的，但是去除内源性DHTT可以显着加速与果蝇模型相关的神经退行性表型，该模型与Polyq expaded Htt毒性相关的HTT毒性，支持HTT的正常功能，以支持HTT的正常功能，以实现HDTT的正常功能；此外，DHTT是维持成年动物的迁移率和长期存活所必需的，其缺失会影响成人大脑的轴突终端复杂性。这些研究使我们能够使用果蝇中强大的遗传系统和丰富的实验工具来对DHTT无效突变体进行更详细的表征，并对DHTT和HIPS同源物之间的潜在功能相互作用进行系统的评估。这项研究的结果将提供对HTT正常功能以及最终HD基础机制的正常功能的关键见解。在此应用中，我们提出了以下特定目的：（1）通过超微结构和基因表达分析表征与DHTT相关的表型，并在果蝇中使用体内测定中确定的在体内分析中使用以直接测试HTT在轴突囊泡运输和内侧病中HTT的细胞作用；（2）使用一种基因组标记方法来建立用于DHTT蛋白体内分析的多功能工具箱，并执行缺失研究以绘制DHTT蛋白中的功能域；（3）通过测试其果蝇同源物和DHTT之间的遗传相互作用，评估哺乳动物臀部的生理相关性，并使用基于串联的亲和力纯化（TAP）的方法直接分离果蝇臀部。