Elucidating the mechanistic function of Fragile X Mental Retardation Protein (FMRP) in neurons is a critical goal in understanding the basis of Fragile X Syndrome. This proposal will test the hypothesis that FMRP is required for the glutamatergic regulation of mRNA transport in dendrites and its subsynaptic translation. Recent identification of mRNAs that are bound by FMRP now makes it possible to investigate whether these RNA-protein interactions occur in dendrites and at synapses. An inherent difficulty in studying RNA-protein interactions in dendrites has been the lack of suitable high resolution microscopic technology to visualize mRNA transport and identify sites of local translation. A new view of FMRP function is made possible by utilizing novel microscopic and imaging technology to visualize mRNP complexes in live neurons. We have recently shown that FMRP is localized in the form of RNA granules that exhibit dynamic and activity-dependent movements in dendrites and spines. Experiments in Specific Aim 1 will apply high resolution fluorescence in situ hybridization methods and quantitative digital imaging analysis to determine whether specific mRNAs have altered localization and regulation in hippocampal cultures from Fmr1 knockout mice. We will determine whether FMRP binding elements function as zipcodes to localize FMRP target mRNAs using transfection of reporter constructs. Experiments in Specific Aim 2 will use live cell imaging technology to determine whether glutamatergic signaling and synaptic activity regulates the dynamic trafficking of FMRP and bound mRNAs in dendrites and spines. Experiments in Specific Aim 3 will use combined imaging and biochemical methods to elucidate a role for FMRP in the glutamatergic regulation of dendritic and synaptic protein synthesis. These studies will provide new insight into the molecular and cellular basis for altered synaptic plasticity in Fragile X Syndrome.
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