Activity-dependent variation in synaptic AMPA receptor (AMPAR) content, referred to as ‘synaptic plasticity’, is a mechanism whereby information is stored in neural networks that give rise to higher order cognitive skills such as learning and memory. During long-term potentiation (LTP), a widely studied form of synaptic plasticity, extrasynaptic AMPARs are recruited from nearby reserve pools, including perisynaptic regions on the cell surface and intracellular compartments, and subsequent anchored with the postsynaptic density (PSD). A large body of evidence spanning decades of investigation has established mechanisms by which AMPARs are anchored within the PSD. In contrast, the molecular mechanisms that govern AMPAR synaptic targeting to establish reserve pools of extrasynaptic receptors are largely unknown. Given that recruitment of reserve pools of extrasynaptic AMPARs underlies the rapid strengthening of synapses that occurs during LTP, the molecular mechanisms that establish such reserve pools are critical to our understanding of synaptic plasticity and represent a major gap in our knowledge. SynDIG (Synapse Differentiation Induced Gene) defines a family of four genes (SynDIG1-4) that encode brain- specific transmembrane proteins. Here we will determine the function of SynDIG4 (SD4), also known as Prrt1 (Proline-rich transmembrane protein 1) in the regulation of the reserve pool of AMPARs. Proteomic studies indicate that SD4 is a component of AMPAR complexes; however, SD4 is not enriched in the PSD, but instead colocalizes with GluA1-containing AMPARs at non-synaptic sites. Remarkably, tetanus-induced LTP, which is dependent on GluA1, is abolished in acute hippocampal slices from SD4 knockout (KO) while theta-burst stimulation LTP (TBS-LTP), which is independent of GluA1, is not impaired. Furthermore, SD4 KO mice exhibit profound deficits in two independent cognitive assays (Morris water maze, novel object recognition), demonstrating a critical role for SD4 in hippocampal-dependent learning and memory. Moreover, extrasynaptic AMPARs are reduced in SD4 KO compared with wild-type (WT) neurons. Given that reserve pools of extrasynaptic AMPARs are critical for synaptic plasticity, we hypothesize that SD4 maintains such reserve pools of extrasynaptic GluA1-containing AMPARs that are deployed during tetanus-induced LTP. In this collaborative dual-PI application we propose a comprehensive multidisciplinary approach to investigate SD4-dependent regulation of extrasynaptic GluA1-containing AMPARs with molecular, cellular, and electrophysiological methods. In Aim 1 we will define the mechanism by which SD4 maintains extrasynaptic GluA1-containing AMPARs. In Aim 2 we will determine whether synaptic targeting of reserve pools of GluA1- containing AMPARs during plasticity requires SD4. In Aim 3 we will test whether homomeric GluA1-dependent synapse plasticity mechanisms require SD4 ex vivo at multiple ages. The results of these studies will provide molec...