PROJECT SUMMARY Recent evidence from genetics, neuroimaging and postmortem studies collectively points to synaptic pathways as a locus of pathophysiologic mechanisms of schizophrenia. In particular, dysregulation of glutamatergic signaling has been among the most highly implicated for synaptic pathology of the illness. Previously, we proposed Src kinase (Src) as one of the points of convergence for multitudes of molecular alterations in glutamatergic complexes in the postsynaptic density (PSD) in schizophrenia. Our scientific premise is that Src kinase and its aberrant associations with its binding partners form a pathophysiologic subnetwork underlying altered PSD/glutamatergic signaling and thus represent potential therapeutic targets. This premise is predicated on a series of observations we have made including 1) NMDA receptor (NMDAR) hypoactivity in the postmortem dorsolateral prefrontal cortex (DLPFC) of schizophrenia is associated with decreased tyrosine phosphorylation of GluN2 in NMDAR complexes. 2) NMDAR hypoactivity is driven by hypoactivity of Src kinase, which results from aberrant protein-protein interaction (PPI) of Src with its signaling partners. 3) Molecular and behavioral phenotypes of NMDAR hypoactivity can be rescued by modifying altered Src-PSD-95 association using a peptide, called Src activating PSD-95 inhibiting peptide (SAPIP). Synaptic alterations lead to disrupted circuit activity via aberrant interactions between pyramidal neurons (PNs) and interneurons (INs). The goal of this project is to determine Src mediated NMDAR signaling alterations distinctly in PNs vs. INs and their differential responses to potential therapeutic interventions. Previous postmortem assessments of glutamatergic signaling, by our lab and others, were conducted in bulk tissues, not offering a cell type specific resolution. To address this, we will employ two recently devised methods. The first is sequential immunoprecipitation (IP), in which the receptor complexes in each cell type are captured first by GluN1, common to both cell types, followed by another round of IP using a bait protein that is expressed exclusively in the cell type of interest and yet integral to the protein complexes of interest. The second is multi-channel confocal microscopic analysis of synaptosomes combined with cell type specific labeling and the proximity ligation assay (PLA). Employing these complementary methods, we will examine postmortem DLPFC and test if Src kinase activity and their downstream signaling are altered in PNs vs. INs in schizophrenia (Aim 1). In addition, we will examine how Src-NMDAR signaling is modulated by SAPIP and other agents in each cell type (Aim 2). The results of this study will be the first to address signaling and PPIs in postmortem brains in specific cell types and will inform the distinct constellation of glutamatergic receptor complexes and signaling in pyramidal vs. interneurons.