The amino acid glutamate mediates excitatory neurotransmission in the brain, through the activation of a recently identified family of receptors which are concentrated on dendrites at sites opposite excitatory nerve terminals. The processes that control the development of excitatory synapses, including the aggregation of postsynaptic receptors, are likely to be key to understanding plasticity inherent in learning, memory, and regeneration in the adult brain. In addition, the degeneration of excitatory synapses seen in Alzheimer's disease, and the glutamate-mediated toxicity seen in ALS may be examples of alterations in those mechanisms. Our present understanding of excitatory synaptogenesis is rudimentary. In cultured rat spinal neurons, AMPA type glutamate receptors are initially expressed throughout the dendritic surface, but over time, become concentrated at synapses, and disappear from non-synaptic sites. This alteration in distribution coincides with the synthesis of the AMPA receptor subunits GluR2 and/or GluR3 (hereafter referred to as GluR2/3). In addition, overexpression of the intracellular C-terminus shared by GluR2 and GluR3 disrupts the accumulation of synaptic AMPA receptors. In this grant, I outline an approach to understanding the mechanisms and molecules involved in the targeting of glutamate receptors to excitatory synapses. Using a sensitive, high resolution, in situ hybridization technique, the role of GluR2/3 in regulating the synaptic aggregation of AMPA receptors will be investigated by determining whether the delayed synaptic appearance of GluR2/3 is due solely to a delay in the synthesis of these subunits, or to a tight regulation of their distribution at the protein level. In addition, the synthesis of GluR2/3 will be examined in isolated neurons, revealing any inductive effect of synapse formation on the synthesis of these subunits. Once synthesized, GluR2 is transported to and stabilized at excitatory synapses through signals present in its C-terminus. To identify the precise sites necessary for the synaptic targeting of AMPA receptors, a mutagenesis strategy will be employed in which epitope tagged GluR2, cloned into an expression vector, will be subjected to both deletion and point mutations, and the resultant product transfected into cultured spinal neurons. The ability of the transfected receptor, as well as chimeric molecules created between GluR2 and non-synaptic molecules such as CD8, to target to synapses will be assessed. Finally, we will investigate the role of a novel immediate early gene NARP (neuronal activity regulated pentraxin) in the development and stabilization of excitatory synapses in cultured spinal neurons. NARP is expressed exclusively at excitatory synapses where it is secreted by the presynaptic terminal. In HEK-293 cells, NARP induces the aggregation of coexpressed AMPA receptor subunits through a direct interaction. Moreover, NARP expressing 293 cells can aggregate AMPA receptors in co-cultured spinal neurons.
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