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.

描述（由申请人提供）：候选人是一种学术神经外科医生（MD，PhD），其职业科学目标是理解脑发育的分子机制以及对神经系统疾病中这些机制的病理放松管制。该候选人具有先前的实验室经验，并具有成功，发表的研究项目的往绩记录，从而发育和兴奋性神经元死亡和神经元形态发生。为了准备向成功的独立研究者的过渡，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的研究生级课程，以及学术医学领导力，并将补充遗传学，解剖学和神经生物学的研讨会，以及希望神经学疾病的候选人及其国际候选人的候选人及其候选人的研究中心。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举行，这是一个具有特殊优势神经生物学，遗传学和先进基因组方法的机构，为候选人提供了重要的知识援助和合作。科学培训将由Jeff Milbrandt博士进行指导，Jeff Milbrandt博士的实验室着重于在神经系统发育过程中阐明基因调节的机制。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-DNA相互作用的方法方面的专业知识，以及他对凝聚生物学的了解，将为候选人提供成功的研究工具，以成功地作为研究影响人脑的神经元发展和疾病的独立研究人员。在各种神经系统疾病中已经观察到了神经元树突形态的障碍，这提出了一个有趣的假设，即异常 正常的树突发育有助于人脑疾病。候选人先前发现，在大脑后有丝质神经元中，降解的主要有丝分裂泛素连接酶CDC20-促进复合酶（CDC20-APC）是必需的。该研究建议将确定CDC20-APC下游的新型分子机制，以控制树突发育，与人脑疾病直接相关。第一个目的将定义CDC20-APC与26S ProteAsome的S5A亚基之间的令人兴奋的联系，这是一种旨在破坏泛素化底物的多生育综合体，在树突形态发生中，表明CDC20-APC调节蛋白酶体活性以驱动蛋白酶体的蛋白酶体可驱动树枝状列石精心仪。这些实验将使用严格的基于RNA的干扰方法来确定S5A驱动的树突形态发生的机制，并利用新型的细胞荧光报告基因监测蛋白酶体活性的CDC20-APC调节。第二个目的将阐明树突和树突状脊柱形态发生中凝聚复合物的CDC20-APC信号传导途径。人粘着蛋白病综合征与内聚力基因的突变有关，并以智力迟缓为特征。该目的将检验以下假设：Cdc20-APC/凝聚的树突形态发生途径的失调会导致神经元的结构异常，这可能是粘炎患者看到的认知缺陷的基础。针对CDC20-APC/内聚途径的RNAi和带有条件缺失的核心内聚力亚基的转基因小鼠将被广泛用于此目的。通过染色质免疫沉淀与下一代测序和相关的凝聚力依赖性微阵列分析，将通过全基因组搜索凝聚结合位点搜索凝聚力结合位点的直接下游基因靶标。在控制树突形态发生中，新型CDC20-APC下游机制的鉴定将填补我们对神经元连接性细胞中性机制的显着空白，并为人类粘粘膜中观察到的认知缺陷的发病机理提供了见解。 公共卫生相关性：树突代表了传达脑细胞或神经元的关键接收端，树突结构的障碍有助于神经系统疾病中观察到的认知缺陷，包括智力障碍和成人神经退行性疾病。因此，鉴定控制树突发育的基因将揭示这些神经系统疾病的发生，为这些患者的潜在未来疗法奠定了基础。该项目将使用对树突结构，生物化学和最先进的基因组技术的高级成像来识别控制树突结构的基因。