Broadening access to neuroscience by understanding regulation of extrasynaptic AMPA receptors by transmembrane auxiliary proteins

About This Grant

ABSTRACT 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). AMPARs are associated with a diverse suite of transmembrane auxiliary proteins that regulate trafficking and biophysical properties. 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 maintain AMPARs as 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. In the hippocampus, the predominant AMPAR auxiliary factors are transmembrane AMPA receptor regulatory protein gamma 8 (TARP8), cornichon-2 (CNIH-2), and synapse differentiation induced gene 4 (SynDIG4; SD4), also known as Prrt1 (Proline-rich transmembrane protein 1). Previously, we found that extrasynaptic GluA1-AMPAR reserve pools are reduced in SD4 knockout (KO) hippocampal neurons compared to wild-type (WT). SD4 preferentially associates with affinity-purified GluA1-AMPARs over AMPAR complexes without GluA1. Furthermore, acute hippocampal slices from SD4 KO mice do not undergo GluA1-dependent tetanus- induced LTP, while GluA1-independent theta-burst stimulation LTP (TBS-LTP) remains unimpaired. Analyses of native hippocampal GluA1-AMPARs showed that SD4 is positioned at the interface of GluA1 and CNIH-2, consistent with SD4’s putative role in GluA1-AMPAR trafficking and localization. We hypothesize that SD4 establishes reserve pools of GluA1-AMPARs at perisynaptic sites that are targeted to synapses during LTP. In this application we propose a comprehensive multidisciplinary approach to investigate SD4-dependent regulation of GluA1-containing AMPARs with molecular, cellular, and electrophysiological methods. In Aim 1 we test the prediction that SD4 interaction with GluA1-AMPARs is required for synaptic plasticity. In Aim 2 we test the prediction that SD4 promotes TARP8-dependent synaptic targeting of GluA1-AMPARs during LTP. Completion of these studies will address a major gap in our understanding of the mechanisms by which AMPAR complexes are maintained in reserve pools outside of the PSD in hippocampal neurons and address the role played by SD4 in synaptic targeting of GluA1-AMPARs during plasticity. Through these studies to understand mechanisms by which AMPAR complexes are maintained in reserve pools for plasticity, we will simultaneously diversify the neuroscience workforce by training and mentoring individuals from diverse backgrounds in the proposed research project (Aim 3). We hypothesize that SD4 establishes reserve pools of GluA1-AMPARs at perisynaptic sites that are targeted to synapses during LTP. We further hypothesize that by providing structured mentoring and training for undergraduate students from marginalized backgrounds in the proposed research, we expect to broaden participation, promote retention, and diversity the neuroscience workforce. The experiments and activities proposed in the three aims test predictions of these hypotheses.