MECHANISMS OF DENDRITE MORPHOGENESIS BY THE ANAPHASE-PROMOTING COMPLEX

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。候选人具有重要的实验室经验，在发育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者，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课程，以及学术医学领导，并将补充遗传学，解剖学和神经生物学研讨会，以及神经疾病的希望中心，以及在主要的国家和国际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，该大学在神经生物学、遗传学和先进的基因组方法方面具有特别的优势，为候选人提供重要的智力援助和合作。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，以及他对凝聚力生物学的了解，将为候选人提供成功研究神经元发育和影响人类大脑的疾病所需的研究工具。在各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，即神经元树突形态的异常， 正常的树突发育有助于人类大脑疾病。该候选人先前发现，引人注目的是，主要有丝分裂泛素连接酶Cdc 20-后期促进复合物（Cdc 20-APC）是脑有丝分裂后神经元中树突形态发生所需的。这项研究计划将确定Cdc 20-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。第一个目标将定义一个令人兴奋的联系Cdc 20-APC和S5 a亚基的26 S蛋白酶体，一个多亚基复合物，旨在破坏泛素化底物，在树突形态发生，这表明假设Cdc 20-APC调节蛋白酶体活性，驱动树突的制定。这些实验将使用严格的基于RNA干扰的方法来确定S5 a驱动的树突形态发生的机制，并利用新的细胞荧光报告基因来监测Cdc 20-APC对蛋白酶体活性的调节。第二个目标将阐明树突和树突棘形态发生中的Cdc 20-APC信号通路。人类粘着蛋白病综合征与粘着基因的突变有关，其特征是智力迟钝。这一目标将测试的假设，失调的Cdc 20-APC/凝聚树突形态发生途径导致神经元的结构异常，这可能是认知缺陷的基础上看到的cohesinopathy患者。靶向Cdc 20-APC/凝聚力途径的RNAi和携带核心凝聚力亚基的条件性缺失的转基因小鼠将被广泛用于这一目的。有丝分裂后神经元中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凝聚力依赖性微阵列分析，通过全基因组搜索凝聚力结合位点来鉴定。新的Cdc 20-APC下游机制的树突形态发生控制的鉴定将填补我们的理解神经元连接的细胞内在机制的显着差距，并提供洞察人类cohesinopathies中观察到的认知缺陷的发病机制。 公共卫生相关性：树突代表了沟通脑细胞或神经元的关键接收端，树突结构的紊乱有助于在神经系统疾病中观察到的认知缺陷，包括精神发育迟滞和成人神经退行性疾病。因此，控制树突发育的基因的鉴定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的先进成像、生物化学和最先进的基因组技术来识别控制树枝结构的基因。

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.