Excitotoxic neuronal damage or death following stroke is believed to be mediated by elevated Ca2+ influx through the N-methyl-D-aspartate receptor (NMDAR) class of glutamate receptors. The GluN2 subunit subtype (2A vs 2B) of the receptor determines its biophysical and signaling properties. GluN2B-containing NMDARs are the primary mediators of excitotoxicity, but how they are regulated is poorly understood. Based on extensive preliminary data, I will test the hypothesis that direct physical coupling of GluN2B to the actin cytoskeleton is critical for its synaptic activity. I propose that GluN2B and actin interact via two biochemical interfaces. First, phosphorylation of GluN2B tyrosine Y1252 creates a binding site for the Nck2 adaptor protein, a key regulator of actin polymerization by the Arp2/3 complex. Our prior work indicates that disrupting this interaction in neurons reduces GluN2B function. Second, I present preliminary data that a segment of the GluN2B cytoplasmic tail (C-tail) itself binds saturably and with high affinity to actin filaments. In my first aim, I will use cell type and brain region-selective conditional inactivation of Nck2 in mice to study how its disruption impacts of NMDAR activity. I will study whether and how loss of Nck2 function impacts NMDAR localization or gating using high resolution microscopy and electrophysiology methods. Given the central role of Nck2 in regulating actin polymerization, I will also use immunofluorescence and fluorescence recovery after photobleaching of GFP-actin in live cultured neurons to determine how disruption of Nck2 function impacts actin levels and actin dynamics in dendritic spines. In Aim 2, I will elucidate whether and how direct binding of actin to GluN2B impacts receptor function. First, I will identify specific residues on C-tail of the GluN2B that mediate binding to actin. Next, I will use electrophysiology to measure the activity and gating properties of NMDAR-containing C-tail mutations that disrupt actin binding to test how this actin-binding region impacts GluN2B function. Understanding how interactions with Nck2 and actin controls GluN2B-containing NMDAR activity will reveal how disruptions of these functions contribute to cognitive deficits and could suggest therapeutic strategies for more selective targeting of the receptor for treatment in stroke.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||01/01/2020 → 12/31/2021|