Role of CPEB in dendritic CaMKlla mRNA transport and translation

描述(申请人提供)：树突状蛋白合成对于几种形式的长期突触可塑性是必不可少的，这些形式被认为是学习和记忆的基础。然而，调节活性诱导的树突状细胞转化的分子机制仍不清楚。一种假设是，活性可能调节mRNA结合蛋白(RBP)，RBP控制特定mRNAs的树突状定位和翻译。本研究旨在研究RBP胞浆多聚腺苷酸化元件结合蛋白1(CPEB1)在树突状mRNA靶向和局部翻译中的作用。总体假设是CPEB1与一个多蛋白树突复合体结合，该复合体调节已知的CPEB1靶基因CaMKIIa的活性诱导的转运和翻译。该方案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体的树突定位；2)介导CPEB1复合体在突触处活性调节的分子机制。为此目的提出的两种方法是培养的海马神经元的高分辨率定量免疫荧光和突触部分的生化分析。第二个目的是研究CPEB1复合体是否调节CaMKIIa mRNA的基础和活性诱导的树突状定位。在慢病毒介导的CPEB1复合蛋白的敲除之后，将使用原位杂交和标记的mRNA构建的实时成像来检测CaMKIIa在CPEB1缺失神经元中的mRNA定位。第三个目的是通过荧光mRNA报告的活细胞成像和慢病毒介导的CPEB1复合体成分的操纵，评估CPEB1复合体是否调节树突状CamKlla mRNA的翻译。与NINDS“了解神经元的正常活动”的使命一致，这项提议研究了一种潜在的机制，通过这种机制，活动可能调节新蛋白质的局部合成。这项工作有助于为开发有效的突触功能障碍治疗方法提供基础知识。总结：学习和记忆缺陷是各种神经疾病的症状，如智力低下、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经验依赖的神经元连接修饰的调节--这一生物过程被认为是学习和记忆的基础。获得这些基础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.