Integrin class adhesion receptors exert potent influences over trophic signaling and ion channels in various nonneuronal cells. Research in this program has shown that integrins are concentrated at synapses and influence neurotrophin expression, and glutamate receptor and ion channel properties in mature forebrain neurons. Specifically, the soluble integrin ligand peptide GRGDSP was found to up-regulate neurotrophin expression in an NMDA receptor (NMDAR)-dependent fashion. This led to the further discovery that GRGDSP treatment increases synaptic currents mediated by AMPA- and NMDA-class glutamate receptors and phosphorylation of the glutamate receptor themselves. The proposed work builds on these results and will test the hypothesis that synaptic integrins control local signaling cascades that regulate the functional properties of AMPA and NMDA receptors. There are 5 Specific Aims. In immunocytochemical studies of cultured hippocampal neurons, Aim 1 will identify integrin subunits and integrin signaling elements located in glutamatergic spine synapses. Studies will test if spines contain multiple integrins, if specific integrins and glutamate receptor proteins are co-distributed, and if the integrin signaling proteins FAK and Pyk2 are differentially distributed, in association with specific integrins, across glutamatergic spine synapses. In parallel studies to be conducted in acute hippocampal slices and synaptoneurosomes, Aim 2 will test the hypothesis that GRGDSP and native matrix ligands (fibronectin, vitronectin, laminin) activate synaptic integrin signaling leading to phosphorylation of AMPA and NMDA receptor proteins. Cotreatment with disintegrins and integrin neutralizing antisera will verify that effects are integrin-mediated and will identify the specific integrins involved.
Aim 3 will test the hypothesis that native integrin ligands potentiate NMDAR- and AMPAR-mediated synaptic responses in electrophysiological studies of acute hippocampal slices; integrin function blocking antibodies will be used to identify integrins mediating these effects and to test if integrin effects are tonic or only occur with new ligand presentation.
Aim 4 will test the hypothesis that treatment with integrin ligands alters specific AMPAR-scaffold associations and increases AMPAR surface expression in studies of acute hippocampal slices and cultured hippocampal neurons, respectively. Finally, Aim 5 will test the hypothesis that integrin signaling regulates neuronal gene expression through serial effects on NMDAR and VSCC function. Specifically, studies will test if integrin ligands trigger NMDAR-dependent phosphorylation of L-type VSCCs, will identify the specific integrins involved, and will use function blocking antibodies to test if integrin effects on neuronal gene expression are tonic or episodic (i.e., occur only with new ligand presentation). These studies will further characterize and identify novel interactions between adhesion and neurotransmitter receptors that influence both the potency of synaptic transmission and the regulation of neurotrophic factor expression in adult brain. As a consequence these findings are important for understanding basic mechanisms of neuronal communication and survival in the adult CNS.
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