International Research Fellowship Program: Functional Analysis of the Glutamatergic Synapse Using Scalable RNAi Screening in Primary Hippocampal Cultures

0602050麦克拉伦国际研究奖学金计划使美国科学家和工程师能够在国外进行9到24个月的研究。该计划的奖项提供了联合研究的机会，并使用独特或互补的设施，专业知识和国外的实验条件。该奖项将支持由埃里克J麦克拉伦博士与博士24个月的研究奖学金。格兰特在英国Hinxton的Wellcome Trust桑格研究所进行的研究。该提案中概述的研究旨在从功能上描述突触蛋白质组的组成部分。NMDA受体复合物（NRC）是突触的主要成分，由185种蛋白质组成。这些蛋白质将通过RNA干扰（RNAi）靶向抑制它们在培养的海马神经元中的表达，以帮助阐明它们在突触传递中的功能。最终的目标是完善这种方法，使其能够扩大到整个小鼠蛋白质组。NRC中的合成短干扰RNA（siRNA）靶向基因将与载体肽连接以转染到原代海马神经元中。靶基因敲除的影响将通过三种方式进行分析：1）用微阵列进行全基因组转录谱分析，以确定靶基因参与的转录网络; 2）神经元的形态学分析，以确定突触数量、突起数量以及重要蛋白质的表达和分布的变化;以及3）将使用微电极阵列（MEA）分析由培养的海马细胞形成的网络中的电生理变化，包括自发放电率和模式。这些实验将证明哪些基因对正常神经元形态和突触功能至关重要，以及它们之间的关系。宿主团队在使用培养的海马神经元和MEA评估突触功能方面具有丰富的经验。此外，该小组在鉴定组成完整突触蛋白质组的蛋白质方面取得了进展，特别是NRC，这些知识对该项目将是非常宝贵的。最后，桑格研究所在人类基因组计划中的作用使其成为该项目基因组方面的专业知识和援助的极好来源。

0602050MacLarenThe International Research Fellowship Program enables U.S. scientists and engineers to conduct nine to twenty-four months of research abroad. The program's awards provide opportunities for joint research, and the use of unique or complementary facilities, expertise and experimental conditions abroad.This award will support a twenty-four-month research fellowship by Dr. Erik J. MacLaren to work with Dr. Seth G. Grant at Wellcome Trust Sanger Institute in Hinxton, in the United Kingdom.The research outlined in this proposal aims to functionally characterize the components of the synaptic proteome. The NMDA receptor complex (NRC) is a major component of glutamatergic synapses and is comprised of 185 proteins. These proteins will be targeted by RNA interference (RNAi) to inhibit their expression in cultured hippocampal neurons to help elucidate their function in synaptic transmission. The ultimate goal is to perfect this method so it can be scaled up to the entire mouse proteome. Synthetic short interfering RNAs (siRNAs) targeting genes in the NRC will be linked to a vector peptide for transfection into primary hippocampal neurons. The effects of the target gene's knock-down will be assayed in three ways: 1) genome-wide transcriptional profiling with microarrays to identify the transcriptional networks in which the target gene is involved; 2) morphological assays of the neurons to identify changes in the number of synapses, number of processes, and the expression and distribution of important proteins; and 3) electrophysiological changes in the networks formed by the hippocampal cells in culture, including spontaneous firing rates and patterns, will be assayed using microelectrode arrays (MEAs). These experiments will demonstrate which genes are essential for normal neuronal morphology and function at glutamatergic synapses and how they are related to one another. The host team has extensive experience in using cultured hippocampal neurons and MEAs to assess synaptic function. Additionally, the group has made strides in identifying the proteins comprising the complete synaptic proteome, particularly the NRC, and this knowledge would be invaluable to this project. Finally, the Sanger Institute's role in the Human Genome Project make it an excellent source of expertise and assistance for spearheading the genomic aspect of this project.