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

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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.