Role of CPEB in dendritic CaMKlla mRNA transport and translation

描述（由申请人提供）：树突蛋白的合成对于多种形式的长期突触可塑性至关重要，这些可塑性被认为是学习和记忆的基础。然而，调控活性诱导的树突翻译的分子机制尚不清楚。一种假设是，活性可能调节mRNA结合蛋白（RBP），控制特定mRNA的树突定位和翻译。本研究旨在探讨RBP细胞质多聚腺苷化元件结合蛋白1 （CPEB1）在树突mRNA靶向和局部翻译中的作用。总体假设是CPEB1与多蛋白树突状复合物相关，该复合物调节已知CPEB1靶mRNA CaMKIIa的活性诱导转运和翻译。本研究的第一个目的是研究1)与野生型相比，CPEB1缺失神经元中多蛋白复合物的树突定位；2)突触中介导CPEB1复合物活性调节的分子机制。为此提出的两种方法是培养海马神经元的高分辨率定量免疫荧光和突触组分的生化分析。第二个目的是研究CPEB1复合物是否调节CaMKIIa mRNA的基础和活性诱导的树突定位。CaMKIIa mRNA的定位将在CPEB1缺失的神经元和慢病毒介导的CPEB1复合体蛋白的敲低中使用原位杂交和标记mRNA结构的实时成像进行检测。第三个目的是利用荧光mRNA报告细胞的活细胞成像结合慢病毒介导的CPEB1复合物组分的操作来评估CPEB1复合物是否调节树突camkla mRNA的翻译。与NINDS“追求对神经元正常活动的理解”的使命一致，该提案研究了神经元活动可能调节新蛋白质局部合成的潜在机制。这项工作有助于为突触功能障碍的有效疗法的发展提供基础知识。总结：学习和记忆缺陷是各种神经系统疾病的症状，如智力迟钝、自闭症、创伤性脑损伤和阿尔茨海默病。这项拟议的研究调查了神经元连接的经验依赖性修改的调节-被认为是学习和记忆的基础的生物过程。掌握这些基本知识对于理解这些广泛传播的疾病的病理以及建立有用的治疗方法至关重要。

DESCRIPTION (provided by applicant): Dendritic protein synthesis is essential for several forms of long-term synaptic plasticity that are thought to underlie learning and memory. However, the molecular mechanisms that regulate activity-induced dendritic translation remain unclear. One hypothesis is that activity may regulate mRNA binding proteins (RBP) that control the dendritic localization and translation of specific mRNAs. This proposal investigates the role of the RBP cytoplasmic polyadenylation element binding protein 1 (CPEB1) in dendritic mRNA targeting and local translation. The overall hypothesis is that CPEB1 associates with a multi-protein dendritic complex that regulates activity-induced transport and translation of known CPEB1 target mRNA CaMKIIa. The first aim of this proposal examines 1) dendritic localization of the multi-protein complex in CPEB1 null neurons compared to wild type and 2) the molecular mechanisms mediating activity regulation of the CPEB1 complex at synapses. The two proposed approaches for this aim are high-resolution quantitative immunofluorescence in cultured hippocampal neurons and biochemical analysis of synaptic fractions. The second aim addresses whether the CPEB1 complex regulates basal and activity-induced dendritic localization of CaMKIIa mRNA. CaMKIIa mRNA localization will be assayed in CPEB1 null neurons and following lentiviral-mediated knockdown of the CPEB1 complex proteins using in situ hybridization and live imaging of a tagged mRNA construct. The third aim evaluates whether the CPEB1 complex regulates dendritic CamKlla mRNA translation using live-cell imaging of a fluorescent mRNA reporter combined with lentiviral-mediated manipulation of the CPEB1 complex components. Consistent with the mission of NINDS to "pursue an understanding of the normal activities" of neurons, this proposal examines a potential mechanism by which activity might regulate the local synthesis of new proteins. This work contributes to the basic knowledge that founds the development of useful therapies for disorders of synaptic function. Lay summary: Deficits in learning and memory are symptoms of varied neurological conditions such as mental retardation, autism, traumatic brain injury and Alzheimer's disease. The proposed study investigates the regulation of experience-dependent modification of neuronal connections - the biological process that is thought to underlie learning and memory. Acquiring this basic knowledge is critical for understanding the pathology of these wide-spread disorders as well as the establishment of useful therapies.