It is now clear that protein synthesis is required for animals to establish long-term memories. Misregulation of synaptic tranmission and protein synthesis plays an important role in many diseases. Until recently, it was assumed that all of the proteins required for all neuronal function were made in the cell body. The discovery of polyribosomes at the base of neuronal synapses suggested the possibility that proteins might be synthesized in dendrites in response to synaptic activity. In the previous grant period, we focussed our attention on the development of imaging techniques to visualize protein synthesis in dendrites and discovered several forms of plasticity that are implemented by local protein synthesis. In this proposal we will examine the signaling mechanisms that couple miniature synaptic transmission (minis) to the protein translation machinery. We previously discovered that minis tonically inhibit the dendritic protein synthesis machinery. Loss of minis leads to an upregulation of translation and a rapid homeostatic response. We wish to examine which intracellular signaling pathways couple neurotransmitter receptor activation to the protein synthesis machinery. We will also determine whether there is stimulation-dependent assembly and trafficking of ribosomes in dendrites. Previous observations of ribosomes in dendrites of hippocampal neurons suggest that the translational capacity of synapses in spines is limited by the number of ribosomes available in the dendritic pool. Using biochemical approaches and dynamic time-lapse imaging, we will examine whether polyribosomes might be assembled locally in spines or trafficked to spines. One of big unanswered questions concerns the relative contributions of somatically vs. dendritic synthesized proteins to synaptic function and plasticity. Recently we have developed a technique that can be used to identify the constituents of the dendritically proteome. Building on this technology, we will modify our procedure for labelling proteins in lysates to include fluorescent labelling of proteins in intact cells and tissue slices. We will develop multiple fluorescent tags to separately track proteins made in the cell body and the dendrites.The fate of proteins synthesized in these two compartments will be analyzed over time to address the fractional contribution of somatic vs. dendritic protein synthesis to the synaptic protein population and how these contributions change with synaptic activity and plasticity.
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