Common synaptic phenotypes arising from diverse mutations in the human NMDA receptor subunit GluN2A

Abstract

Dominant mutations in the human gene GRIN2A, encoding NMDA receptor (NMDAR) subunit GluN2A, make a significant and growing contribution to the catalogue of published single-gene epilepsies. Understanding the disease mechanism in these epilepsy patients is complicated by the surprising diversity of effects that the mutations have on NMDARs. Here we have examined the cell-autonomous effect of five GluN2A mutations, 3 loss-of-function and 2 gain-of-function, on evoked NMDAR-mediated synaptic currents (NMDA-EPSCs) in CA1 pyramidal neurons in cultured hippocampal slices. Despite the mutants differing in their functional incorporation at synapses, prolonged NMDA-EPSC current decays (with only marginal changes in charge transfer) were a common effect for both gain- and loss-of-function mutants. Modelling NMDA-EPSCs with mutant properties in a CA1 neuron revealed that the effect of GRIN2A mutations can lead to abnormal temporal integration and spine calcium dynamics during trains of concerted synaptic activity. Investigations beyond establishing the molecular defects of GluN2A mutants are much needed to understand their impact on synaptic transmission.

Synaptic Dysfunction by Mutations in GRIN2B: Influence of Triheteromeric NMDA Receptors on Gain-of-Function and Loss-of-Function Mutant Classification

“…We also provide results highlighting the importance of the identity of the subunit that is mutated (rather than the mutation itself) for its effect on NMDA-EPSCs. Similar to our findings for GluN2A [ 40 ], we also demonstrate partial haploinsufficiency for neurons that are heterozygous for LoF alleles of Grin2b .…”

“…All mice were conventionally housed and were in a 12-12 light–dark cycle throughout. Genotype was confirmed prior to experimental use via PCR, as described previously [ 40 ]. In accordance with the Animals Scientific Procedures Act 1986 (amended in 2012), neonatal pups were culled for use in experiments by trained and approved personnel via cervical dislocation, an appropriate Schedule 1 procedure.…”

“…CA1 pyramidal neurons in organotypic slice cultures were transfected by single cell electroporation between 6–8 days in vitro (DIV) [ 40 , 50 ]. Neurons were electroporated with plasmids expressing pCMV Cre-GFP (3 ng/μL 0.58 nM) with or without WT or mutant (R540H, R696H, C456Y, C461F and C436R) pCMV GRIN2B cDNA (10 ng/μL, 1.59 nM).…”

“…Recording protocols for pharmacologically isolated NMDA-EPSCs are exactly as described previously [ 40 ]. AMPA-EPSCs were measured at a holding potential of −100 mV.…”

“…Recently, we used molecular replacement experiments in cultured hippocampal slices to evaluate how GRIN2A mutations can affect basal synaptic function [ 40 ]. A surprising finding from this work was that completely different, i.e., gain-of-function (GoF) and loss-of-function (LoF), mutations led to surprisingly similar defects in synaptic transmission.…”