The goal of this proposal is to secure three years of postdoctoral research training support in order to structurally define in detail both GluN1-2A-2B and GluN1-2B-3A N-methyl-D-aspartate receptors (NMDARs) and to identify modulators that will specifically target these tri-heteromeric receptors without targeting di- heteromeric receptors. Reagents which specifically recognize and modulate the activity of GluN1-2A-2B and GluN1-2B-3A have the potential to boost cognition, and may lead to promising therapeutics to treat mental health disorders including depression, memory loss, and addiction. I have experience in electrophysiology with a background in ion channels and membrane protein biochemistry, and the proposed research and career development activities that I plan to pursue during this fellowship will provide the advanced training necessary to achieve my long-term career objective to be an independent researcher in the field of neuropharmacology. NMDARs belong to a large family of ionotropic glutamate receptors which are the prevailing regulators of neural communication, and their misregulation has been linked to numerous mental health disorders. Within the glutamate receptor family, heterotetrameric NMDARs are integral mediators of electrical and chemical signaling by sensing neurotransmitter release and transmembrane voltage potential at sites of neurotransmission known as synapses. Repeated activation of NMDARs results in synaptic plasticity in the forms of long term potentiation (LTP) and long term depression (LTD), which directly influence brain development and function including learning and memory. Changes in LTP and LTD are correlated with the specific activation of two NMDAR subtypes, the tri-heteromeric GluN1-2A-2B and GluN1-2B-3A receptors. However, how the activation of GluN1-2A-2B or GluN1-2B-3A correlates with changes in synaptic plasticity has been difficult to discern due to limited structural characterization of tri-heteromeric NMDARs and due to overlapping pharmacology profiles of these receptors with di-heteromeric NMDARs.
In Aim 1, I will determine the structures of the human orthologues of GluN1-2A-2B and GluN1-2B-3A by cryo-electron microscopy, and in Aim 2 I will identify and test nanobody modulators that specifically target tri-heteromeric NMDARs using yeast display. The Furukawa Lab at Cold Spring Harbor Laboratory (CSHL) has vast experience working with NMDARs and is uniquely positioned to support my proposed aims using optimized NMDAR expression strategies, electrophysiology setups, and structural biology expertise. Additionally, I will have immediate access to a state-of-the-art cryo-EM facility as well as an on-campus Antibody and Display facility, which are both managed by highly regarded technical staff. The research proposed here will allow me to gain tremendous experience in modern structural biology techniques, display strategies used to develop novel immunotherapies, and has the potential to uncover subtype-specific NMDAR modulators that can address the effects of debilitating mental health disorders that I wish to pursue as an independent investigator.
Activation of brain expressed NMDA receptors results in synaptic plasticity, which causes broad neurological changes in cognitive capacity, memory retention, and learning. Two NMDA receptor subtypes, GluN1-2A-2B and GluN1-2B-3A, have been shown to be the primary determinants of synaptic plasticity, and compounds which specifically target these receptors have the potential to augment cognition and counteract debilitating mental health conditions like memory loss and substance addiction. This proposal seeks to characterize the 3D-structures of these two receptors and to identify nanobodies that specifically target and modulate them, which, combined, will reveal their role in synaptic plasticity.
