MECHANISMS OF DENDRITE MORPHOGENESIS BY THE ANAPHASE-PROMOTING COMPLEX

后期促进复合体的枝晶形态发生机制

• 批准号: 8424656
• 负责人: Albert Hong-Jae Kim
• 金额: $ 16.37万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2017-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8424656
• 关键词: 26S proteasome; Adult; Affect; Anaphase; Anatomy; Architecture; Binding; Binding Sites; Biochemical; Biochemistry; Bioinformatics; Biology; Brain; Brain Diseases; CSPG6 gene; Cell Cycle; Cell division; Cells; Cerebellar cortex structure; Cessation of life; Cognitive deficits; Collaborations; Complex; Coupled; Cytoplasmic Granules; DNA-Protein Interaction; Data; Dendrites; Dendritic Spines; Development; Development Plans; Disease; Doctor of Philosophy; Employee Strikes; Foundations; Future; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Genomics; Goals; Helix-Turn-Helix Motifs; Hippocampus (Brain); Human; Human Genetics; Image; Institution; International; Knockout Mice; Knowledge; Laboratories; Leadership; Limb structure; Link; Medical; Mental Retardation; Mentors; Methodology; Mitotic; Molecular; Monitor; Morphogenesis; Morphology; Mutation; Nerve Degeneration; Nervous system structure; Neurobiology; Neurodegenerative Disorders; Neurons; Neurosurgeon; Pathogenesis; Pathway interactions; Patients; Population; Process; Proliferating; Proteins; Psyche structure; Publishing; RNA Interference; Rattus; Regulation; Reporter; Reporting; Research; Research Personnel; Research Project Grants; Research Proposals; Roberts-SC phocomelia syndrome; Role; Signal Pathway; Sister Chromatid; Structure; Surveys; Synapses; Syndrome; Technology; Testing; Training; Transgenic Mice; Ubiquitin; Universities; Washington; anaphase-promoting complex; base; brain cell; career; career development; cdc Genes; cell type; chromatin immunoprecipitation; clinical phenotype; cohesin; cohesion; design; developmental disease; experience; genome-wide; in vivo; insight; multicatalytic endopeptidase complex; nervous system development; nervous system disorder; neural circuit; neuron development; next generation; novel; programs; research study; symposium; tool; ubiquitin ligase

DESCRIPTION (provided by applicant): The candidate is an academic neurosurgeon (MD, PhD), with a career scientific goal of understanding the molecular mechanisms of brain development and the pathological deregulation of those mechanisms in neurological diseases. The candidate has significant prior laboratory experience with a track record of successful, published research projects in developmental and excitotoxic neuronal death and neuronal morphogenesis. To prepare for the transition to successful independent investigator, the candidate's career development plan includes graduate-level coursework in bioinformatics, next-generation sequencing, and genomic analysis, as well as academic medical leadership and will be supplemented with seminars in Genetics, Anatomy and Neurobiology, and the Hope Center for Neurological Disorders, as well as presentation of the candidate's research at major national and international conferences. The proposed career development plan and scientific training will occur at Washington University in St. Louis, an institution with particular strengthsin neurobiology, genetics, and advanced genomic approaches, providing the candidate with important intellectual assistance and collaborations. The scientific training will be mentored by Dr. Jeff Milbrandt, whose laboratory focuses on elucidating mechanisms of gene regulation during nervous system development. His laboratory's expertise in the latest transgenic mouse technology, transcriptome analyses, and methodologies to study protein-DNA interactions, as well as his knowledge of cohesion biology will provide the candidate with the research tools needed to succeed as an independent investigator studying neuronal development and diseases that affect the human brain. Disturbances in neuronal dendrite morphology have been observed in diverse neurological disorders, raising the intriguing hypothesis that abnormalities in normal dendrite development contribute to human brain diseases. The candidate previously discovered that strikingly, major mitotic ubiquitin ligase Cdc20-Anaphase- Promoting Complex (Cdc20-APC) is required for dendrite morphogenesis in post-mitotic neurons of the brain. This research proposal will identify novel molecular mechanisms downstream of Cdc20-APC in the control of dendrite development, with direct relevance to human brain diseases. The first aim will define an exciting link between Cdc20-APC and the S5a subunit of the 26S proteasome, a multisubunit complex designed to destroy ubiquitinated substrates, in dendrite morphogenesis, suggesting the hypothesis that Cdc20-APC regulates proteasomal activity to drive dendrite elaboration. These experiments will use a rigorous RNA interference- based approach to determine the mechanism of S5a-driven dendrite morphogenesis and utilize a novel cellular fluorescent reporter to monitor Cdc20-APC regulation of proteasomal activity. The second aim will elucidate a Cdc20-APC signaling pathway to the cohesion complex in dendrite and dendritic spine morphogenesis. Human cohesinopathy syndromes are linked to mutations in cohesion genes and are characterized by mental retardation. This aim will test the hypothesis that dysregulation of a Cdc20-APC/cohesion dendrite morphogenesis pathway causes structural abnormalities in neurons, which may underlie the cognitive deficits seen in cohesinopathy patients. RNAi targeting the Cdc20-APC/cohesion pathway and transgenic mice carrying a conditional deletion of a core cohesion subunit will be extensively utilized for this aim. Direct downstream gene targets of cohesion in post-mitotic neurons will be identified through a genome-wide search for cohesion binding sites through chromatin immunoprecipitation coupled with next generation sequencing and correlated cohesion- dependent microarray analyses. The identification of novel Cdc20-APC downstream mechanisms in the control of dendrite morphogenesis will fill a significant gap in our understanding of cell-intrinsic mechanisms of neuronal connectivity and provide insights into the pathogenesis of the cognitive deficits observed in human cohesinopathies. PUBLIC HEALTH RELEVANCE: Dendrites represent the critical receiving end of communicating brain cells-or neurons, and disturbances in dendrite structure contribute to the cognitive deficits observed in neurological disorders, including mental retardation and adult neurodegenerative disorders. Therefore, identification of genes controlling dendrite development will reveal how these nervous system disorders occur, laying the foundation for potential future therapies for these patients. This project will identify genes that control dendrie structure using advanced imaging of dendrite architecture, biochemistry, and state-of-the-art genomic technologies.

应聘者描述(由申请人提供)：应聘者是一名学术神经外科医生(医学博士)，其职业科学目标是了解大脑发育的分子机制以及神经系统疾病中这些机制的病理性放松。应聘者有丰富的实验室经验，在发育性和兴奋性毒性神经元死亡和神经元形态发生方面有成功的已发表研究项目的记录。为了准备过渡到成功的独立研究人员，候选人的职业发展计划包括生物信息学、下一代测序和基因组分析方面的研究生课程，以及学术医学领导力，并将辅之以遗传学、解剖学和神经生物学以及神经疾病希望中心的研讨会，以及在国内和国际主要会议上介绍候选人的研究。拟议的职业发展计划和科学培训将在圣路易斯的华盛顿大学进行，这是一所在神经生物学、遗传学和先进基因组方法方面具有特别优势的机构，为应聘者提供重要的智力援助和合作。这项科学培训将由杰夫·米尔布兰特博士指导，他的实验室专注于阐明神经系统发育过程中的基因调控机制。他的实验室在最新的转基因小鼠技术、转录组分析和研究蛋白质-DNA相互作用的方法方面的专业知识，以及他在凝聚力生物学方面的知识，将为候选人提供成功成为研究神经元发育和影响人类大脑的疾病的独立研究员所需的研究工具。在各种神经疾病中观察到神经元树突形态的紊乱，提出了一个耐人寻味的假说，即 正常的树突发育有助于人类大脑疾病。候选人先前发现，大脑有丝分裂后神经元的树突形态发生需要主要有丝分裂泛素连接酶CDC20-后期促进复合体(CDC20-APC)。这项研究计划将确定与人类大脑疾病直接相关的CDC20-APC下游控制树突发育的新分子机制。第一个目标将在树突形态发生中定义CDC20-APC和26S蛋白酶体的S5a亚单位之间的令人兴奋的联系，26S蛋白酶体是一个多亚单位复合体，旨在破坏泛素化的底物，这表明假设CDC20-APC调节蛋白酶体的活性来驱动树突的形成。这些实验将使用一种严格的基于RNA干扰的方法来确定S5a驱动的树突形态发生的机制，并利用一种新的细胞荧光报告程序来监测Cdc20-APC对蛋白酶体活性的调节。第二个目的将阐明在树突和树突棘的形态发生中，CDC20-APC信号通路通向凝聚复合体。人类粘连素病综合征与凝聚力基因突变有关，并以智力低下为特征。这一目标将检验一种假说，即CDC20-APC/凝聚力树突状细胞形态发生通路的失调会导致神经元的结构异常，这可能是粘附性脑病患者认知缺陷的基础。靶向CDC20-APC/Cohesion途径的RNAi和携带核心凝聚力亚单位有条件缺失的转基因小鼠将被广泛用于这一目的。有丝分裂后神经元凝聚力的直接下游基因靶标将通过染色质免疫沉淀结合下一代测序和相关的凝聚力依赖微阵列分析在全基因组范围内寻找凝聚力结合位点来识别。识别控制树突形态发生的新的CDC20-APC下游机制将填补我们对神经元连接的细胞内在机制的理解的一个重要空白，并为人类粘连疾病中观察到的认知缺陷的发病机制提供见解。 与公共卫生相关：树突是沟通脑细胞或神经元的关键接收端，树突结构的障碍导致了在神经疾病中观察到的认知缺陷，包括精神发育迟缓和成人神经退行性疾病。因此，识别控制树突发育的基因将揭示这些神经系统疾病是如何发生的，为这些患者未来的潜在治疗奠定基础。该项目将使用树枝状结构、生物化学和最先进的基因组技术的先进成像技术来识别控制树枝状结构的基因。