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NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in GRIN2A encoding the GluN2A subunit are associated with a spectrum of disorders, ranging from mild speech and language delay to intractable neurodevelopmental disorders, including but not limited to developmental and epileptic encephalopathy. A de novo missense variant, p.Ser644Gly, was identified in a child with this disorder, and Grin2a knock-in mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibited altered hippocampal morphology at 2 weeks of age, and all homozygotes exhibited lethal tonic-clonic seizures by mid-third week. Heterozygous adults displayed susceptibility to induced generalized seizures, hyperactivity, repetitive and reduced anxiety behaviours, plus several unexpected features, including significant resistance to electrically-induced limbic seizures and to pentylenetetrazole induced tonic-clonic seizures. Multielectrode recordings of neuronal networks revealed hyperexcitability and altered bursting and synchronicity. In heterologous cells, mutant receptors had enhanced NMDA receptor agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. NMDA receptor-mediated synaptic currents in heterozygous hippocampal slices also showed a prolonged deactivation time course. Standard anti-epileptic drug monotherapy was ineffective in the patient. Introduction of NMDA receptor antagonists was correlated with a decrease in seizure burden. Chronic treatment of homozygous mouse pups with NMDA receptor antagonists significantly delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of using multiple experimental modalities to model and test therapies for severe neurodevelopmental disorders, while revealing significant biological complexities associated with GRIN2A developmental and epileptic encephalopathy.

No abstract

N-methyl-D-aspartate receptors (NMDARs), a subset of ligand-gated ionotropic glutamate receptors, are critical for learning, memory, and neuronal development. However, when NMDAR subunits are mutated, a host of neuropathological conditions can occur, including epilepsy. Recently, genetic variation within the GRIN2D gene, which encodes the GluN2D subunit of the NMDAR, has been associated with a set of early-onset neurological diseases, notably developmental and epileptic encephalopathy (DEE). Importantly, patients with GRIN2D variants are largely refractory to conventional anti-epileptic drug (AED) treatment, highlighting the need to further understand the distinctive characteristics of GluN2D in neurological and pathological functions. In this review, we first summarize GluN2D's unique spatial and temporal expression patterns, electrophyslogical profiles, and contributions to both pre-and postsynaptic signaling. Next, we review thirteen unique case studies from DEE patients harboring ten different causal GRIN2D variants. These patients are highly heterogenous, manifesting multiple seizure types, electroencephalographic recordings, and neurological and developmental outcomes. Lastly, this review concludes by highlighting the difficulty in treating patients with DEE-associated GRIN2D variants, and stresses the need for selective therapeutic agents delivered within a precise time window.

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic ligand-gated glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system (CNS). Several neurological disorders may involve NMDAR hypofunction, which has driven therapeutic interest in positive allosteric modulators (PAMs) of NMDAR function. Here we describe modest changes to the tetrahydroisoquinoline scaffold of GluN2C/ GluN2D-selective PAMs that expands activity to include GluN2A-and GluN2B-containing recombinant and synaptic NMDARs. These new analogues are distinct from GluN2C/GluN2D-selective compounds like (+)-(3chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ) by virtue of their subunit selectivity, molecular determinants of action, and allosteric regulation of agonist potency. The (S)-enantiomers of two analogues (EU1180-55, EU1180-154) showed activity at NMDARs containing all subunits (GluN2A, GluN2B, GluN2C, GluN2D), whereas the (R)enantiomers were primarily active at GluN2C-and GluN2D-containing NMDARs. Determination of the actions of enantiomers on triheteromeric receptors confirms their unique pharmacology, with greater activity of (S) enantiomers at GluN2A/GluN2D and GluN2B/GluN2D subunit combinations than (R) enantiomers. Evaluation of the (S)-EU1180-55 and EU1180-154 response of chimeric kainate/NMDA receptors revealed structural determinants of action within the pore-forming region and associated linkers. Scanning mutagenesis identified structural determinants within the GluN1 pre-M1 and M1 regions that alter the activity of (S)-EU1180-55 but not (R)-EU1180-55. By contrast, mutations in pre-M1 and M1 regions of GluN2D perturb the actions of only the (R)-EU1180-55 but not the (S) enantiomer. Molecular modeling supports the idea that the (S) and (R) enantiomers interact distinctly with GluN1 and GluN2 pre-M1 regions, suggesting that two distinct sites exist for these NMDAR PAMs, each of which has different functional effects.