Leveraging the dysbindin-1 - Src pathway to restore GluN2A function in schizophrenia - Abstract: Functional variants in GRIN2A, which encodes the GluN2A subunit of the N-methyl-D-aspartate receptor (NMDAR), have been found in patients with Schizophrenia (Scz) and have been linked to impaired working memory (WM). The consequences of GluN2A dysfunction on neural activity and behavior are poorly understood and there are no methods to restore impaired GluN2A function in Scz. Sarcoma Tyrosine Kinase (Src) regulates NMDAR current via GluN2A phosphorylation. Src serves as a hub upon which several proteins dysregulated in Scz interact to influence NMDAR function, including dysbindin-1 (dys-1), suggesting that a Src pathway influences glutamate function via GluN2A. Src activity is reduced in prefrontal cortex (PFC), a region critical for WM, in Scz patients. Indeed, reduction of Src activity in mice impairs trace fear conditioning (TFC), which relies on retention of a cue for 20 seconds, indicative of WM. Thus, the Src pathway is a promising mechanism to explain WM deficits across multiple etiologies, and a provides a means to restore GluN2A function across a range of molecular and genetic conditions. The current proposal will assess the role of the dys-1à Srcà GluN2A pathway on molecular (synaptosomal NMDAR co-immunoprecipitation), single cell (patch clamp), in vivo network function (multi-electrode arrays) and WM (TFC), using a cell-circuit-behavior approach to determine how dysfunctions at each level interact to impair outcomes. The synapse specific activator of Src (TAT-SAPIP), which increases Src availability, will be used to assess the extent to which increasing Src activity restores GluN2A function. Aim 1 will assess the effects of dys-1 removal on cellular, circuit, and behavioral measures related to WM. Dys-1 knockout (KO) increases NMDAR channel decay constant, gamma excitatory/inhibitory (E/I) balance, and TFC. Further, dys-1 KO reduces NMDAR EPSCs, which is restored by increasing Src activity. We propose that decreased dys-1 in KO mice impairs NMDAR activity by reducing GluN2A phosphorylation via Src, and that administration of TAT-SAPIP will restore GluN2A function, thereby restoring cellular, circuit, and behavioral outcomes. Aim 2 will assess the effects of reduced Src on cellular, circuit, and behavioral measures related to WM. Reduction of Src impairs TFC and reduces TFC-dependent GluN2A phosphorylation, reduces NMDAR EPSCs and disrupts E/I balance. Preliminary data indicate that TAT- SAPIP selectively enhances Src in the synapse, facilitates NMDAR EPSCs in wild type (WT) but not Src KO mice, and that chronic TAT-SAPIP restores TFC in Src het mice. We hypothesize that enhancing Src activity will restore normal GluN2A phosphorylation and function, thereby restoring each outcome. Aim 3 will assess the effects of reduced GluN2A availability on cellular, circuit, and behavioral measures related to WM. Reduction of GluN2A function (Grin2A het mice) slows receptor kinetics, will disrupt network gamma E/I balance, and TFC. TAT-SAPIP is expected to restore GluN2A function. This proposal advances a novel mechanistic approach to treating Scz that builds upon previous genetic, post-mortem and preclinical studies.
