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: 8722051
• 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 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8722051
• 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 Chips; 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 sequencing; 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.

描述（由申请人提供）：候选人是一名学术神经外科医生（MD、PhD），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失调。该候选人之前拥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拥有成功、已发表的研究项目的记录。为了准备向成功的独立研究者过​​渡，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，并将辅以遗传学、解剖学和神经生物学研讨会和希望神经疾病中心，以及候选人在主要国内和国际会议上的研究报告。拟议的职业发展计划和科学培训将在圣路易斯华盛顿大学进行，该机构在神经生物学、遗传学和先进基因组方法方面具有特殊优势，为候选人提供重要的智力帮助和合作。科学培训将由 Jeff Milbrandt 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 相互作用的方法学方面的专业知识，以及他在凝聚生物学方面的知识将为候选人提供成功作为研究神经元发育和疾病的独立研究者所需的研究工具影响人类大脑。在多种神经系统疾病中观察到神经元树突形态的紊乱，提出了一个有趣的假设： 正常的树突发育会导致人类大脑疾病。该候选人之前惊人地发现，主要的有丝分裂泛素连接酶 Cdc20-Anaphase-Promoting Complex (Cdc20-APC) 是大脑有丝分裂后神经元树突形态发生所必需的。该研究计划将确定 Cdc20-APC 下游控制树突发育的新分子机制，与人类大脑疾病直接相关。第一个目标将确定 Cdc20-APC 和 26S 蛋白酶体的 S5a 亚基之间的令人兴奋的联系，26S 蛋白酶体是一种多亚基复合物，旨在破坏树突形态发生中的泛素化底物，这表明 Cdc20-APC 调节蛋白酶体活性以驱动树突精细化的假设。这些实验将使用严格的基于 RNA 干扰的方法来确定 S5a 驱动的树突形态发生的机制，并利用新型细胞荧光报告基因来监测 Cdc20-APC 对蛋白酶体活性的调节。第二个目标是阐明树突和树突棘形态发生中的内聚复合体的 Cdc20-APC 信号通路。人类粘连病综合征与粘连基因突变有关，其特点是智力低下。这一目标将检验以下假设：Cdc20-APC/粘连树突形态发生途径的失调会导致神经元的结构异常，这可能是粘连病患者认知缺陷的基础。针对 Cdc20-APC/凝聚通路的 RNAi 和携带有条件删除核心凝聚亚基的转基因小鼠将被广泛用于此目的。有丝分裂后神经元内聚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内聚依赖性微阵列分析在全基因组范围内搜索内聚结合位点来鉴定。控制树突形态发生的新型 Cdc20-APC 下游机制的鉴定将填补我们对神经元连接细胞内在机制理解的重大空白，并为人类粘连病中观察到的认知缺陷的发病机制提供见解。