The experiments of this proposal will define the mechanisms and functional role of mRNA localization and local translation at synaptic sites on neuronal dendrites. Local synthesis of critical proteins at synapses is necessary for long-lasting forms of synaptic plasticity including long-term potentiation (LTP) and long-term depression (LTD), and for the consolidation of memory. Recent studies indicate that the core neurological defect in a common neurological disorder Fragile X Mental Retardation Syndrome (FXS), may involve a disruption of protein synthesis at synapses due to the loss of Fragile X Mental Retardation Protein, the product of the Fmr1 gene which is defective in FXS. Thus, studies of mRNA transport, localization and translation may reveal new targets for therapy for this important and prevalent neurological disorder. Our previous studies have revealed aspects of the mechanisms underlying the selective targeting of mRNA to active synapses through studies of the unique immediate early gene (IEG) Arc, activity-regulated cytoskeleton-associated protein. Arc is strongly induced by physiological activity and its mRNA is rapidly delivered throughout dendrites based on a targeting signal in the mRNA sequence. Arc mRNA localizes selectively at active synapses, and mediates a local synthesis of Arc protein. Both the transcription of Arc mRNA in the nucleus, and the targeting of the newly synthesized transcript to active synapses are triggered by NMDA receptor activation of ERK1/2 and the MAP kinase pathway. Our studies have also revealed that other mRNAs are localized in different ways. The experiments of the present proposal will continue to identify and characterize mRNAs that are localized in dendrites, and further characterize the mechanisms underlying the docking of mRNAs at active synapses using in vivo and in vitro preparations. We will test the hypothesis that the docking mechanism involves a molecular scaffold beneath synapses or within the postsynaptic specialization that is modified by signals generated by intense synaptic activity. We will test the hypothesis that Arc mRNA contains sequences that determine that the mRNA will be transported into dendrites (a dendritic transport sequence) and sequence(s) that cause the mRNA to dock selectively at active synapses (a synaptic targeting sequence) by assessing targeting of exogenously expressed transcripts containing portions of Arc mRNA. Our previous studies indicate that other dendritic mRNAs do not re-localize to active synapses, and so we will determine whether these mRNAs are immobile in dendrites or instead lack the necessary targeting sequences. Together, our studies will reveal key features of the mechanism through which protein synthesis at synapses is regulated, thus providing insights into the fundamental mechanisms that underlie protein synthesis-dependent synaptic modifications, including the mechanisms that are disrupted in FXS and other neurological disorders that affect mRNA transport and localization. This research program will define biological mechanisms that make it possible for nerve cells to modify their connections with one-another (synapses) by controlling the synthesis of the protein building blocks of synapses at the synaptic contact site. These studies will provide novel insights into the basic mechanisms underlying information storage in the brain and neurological disorders that disrupt this fundamental mechanism, including Fragile-X Mental Retardation Syndrome.
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