Genetic variants in the genes GRIN1, GRIN2A, GRIN2B, and GRIN2D, which encode subunits of the N-methyl-D-aspartate receptor (NMDAR), have been associated with severe and heterogeneous neurologic and neurodevelopmental disorders, including early onset epilepsy, developmental and epileptic encephalopathy, intellectual disability, and autism spectrum disorders. Missense variants in these genes can result in gain or loss of the NMDAR function, requiring opposite therapeutic treatments. Computational methods that predict pathogenicity and molecular functional effects of missense variants are therefore crucial for therapeutic applications. We assembled 223 missense variants from patients, 631 control variants from the general population, and 160 missense variants characterized by electrophysiological readouts that show whether they can enhance or reduce the function of the receptor. This includes new functional data from 33 variants reported here, for the first time. By mapping these variants onto the NMDAR protein structures, we found that pathogenic/benign variants and variants that increase/decrease the channel function were distributed unevenly on the protein structure, with spatial proximity to ligands bound to the agonist and antagonist binding sites being a key predictive feature for both variant pathogenicity and molecular functional consequences. Leveraging distances from ligands, we developed two machine-learning based predictors for NMDA variants: a pathogenicity predictor which outperforms currently available predictors and the first molecular function (increase/decrease) predictor. Our findings can have direct application to patient care by improving diagnostic yield for genetic neurodevelopmental disorders and by guiding personalized treatment informed by the knowledge of the molecular disease mechanism.

Genetic variants in genes GRIN1 , GRIN2A , GRIN2B , and GRIN2D , which encode subunits of the N-methyl-D-aspartate receptor (NMDAR), have been associated with severe and heterogeneous neurologic diseases. Missense variants in these genes can result in gain or loss of the NMDAR function, requiring opposite therapeutic treatments. Computational methods that predict pathogenicity and molecular functional effects are therefore crucial for accurate diagnosis and therapeutic applications. We assembled missense variants: 201 from patients, 631 from general population, and 159 characterized by electrophysiological readouts showing whether they can enhance or reduce the receptor function. This includes new functional data from 47 variants reported here, for the first time. We found that pathogenic/benign variants and variants that increase/decrease the channel function were distributed unevenly on the protein structure, with spatial proximity to ligands bound to the agonist and antagonist binding sites being key predictive features. Leveraging distances from ligands, we developed two independent machine learning-based predictors for NMDAR missense variants: a pathogenicity predictor which outperforms currently available predictors (AUC=0.945, MCC=0.726), and the first binary predictor of molecular function (increase or decrease) (AUC=0.809, MCC=0.523). Using these, we reclassified variants of uncertain significance in the ClinVar database and refined a previous genome-informed epidemiological model to estimate the birth incidence of molecular mechanism-defined GRIN disorders. Our findings demonstrate that distance from ligands is an important feature in NMDARs that can enhance variant pathogenicity prediction and enable functional prediction. Further studies with larger numbers of phenotypically and functionally characterized variants will enhance the potential clinical utility of this method.### Competing Interest StatementH.Y. and S.F.T. are co-inventors of Emory-owned intellectual property. S.F.T. is a member of the SAB for Sage Therapeutics, Eumentis Therapeutics, Neurocrine, the GRIN2B Foundation, the CureGRIN Foundation, and CombinedBrain. S.F.T. is a consultant for GRIN Therapeutics. H.Y. is the PI on a research grant from Sage Therapeutics and GRIN Therapeutics to Emory. S.F.T. is PI on a research grant from GRIN Therapeutics to Emory. S.F.T. is cofounder of NeurOp, Inc. and Agrithera. DL recieved funds from the Simons Foundation and GRIN Therapeutics.### Funding StatementThis work was supported by NS111619 (ST), HD082373 (HY), and AG072142 (SJM).### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present work are contained in the manuscript

N-methyl- d -aspartate receptors (NMDARs) are members of the glutamate receptor family and participate in excitatory postsynaptic transmission throughout the central nervous system. Genetic variants in GRIN genes encoding NMDAR subunits are associated with a spectrum of neurological disorders. The M3 transmembrane helices of the NMDAR couple directly to the agonist-binding domains and form a helical bundle crossing in the closed receptors that occludes the pore. The M3 functions as a transduction element whose conformational change couples ligand binding to opening of an ion conducting pore. In this study, we report the functional consequences of 48 de novo missense variants in GRIN1 , GRIN2A , and GRIN2B that alter residues in the M3 transmembrane helix. These de novo variants were identified in children with neurological and neuropsychiatric disorders including epilepsy, developmental delay, intellectual disability, hypotonia and attention deficit hyperactivity disorder. All 48 variants in M3 for which comprehensive testing was completed produce a gain-of-function (28/48) compared to loss-of-function (9/48); 11 variants had an indeterminant phenotype. This supports the idea that a key structural feature of the M3 gate exists to stabilize the closed state so that agonist binding can drive channel opening. Given that most M3 variants enhance channel gating, we assessed the potency of FDA-approved NMDAR channel blockers on these variant receptors. These data provide new insight into the structure–function relationship of the NMDAR gate, and suggest that variants within the M3 transmembrane helix produce a gain-of-function.

The short pre-M1 helix within the S1-M1 linker (also referred to as the pre-M1 linker) between the agonist-binding domain (ABD, S1) and the M1 transmembrane helix of the NMDA receptor (NMDAR) is devoid of missense variants within the healthy population but is a locus for de novo pathogenic variants associated with neurological disorders. Several de novo variants within this helix have been identified in patients presenting early in life with intellectual disability, developmental delay, and/or epilepsy. In this study, we evaluated functional properties for twenty variants within the pre-M1 linker in GRIN1, GRIN2A, and GRIN2B genes, including six novel missense variants. The effects of pre-M1 variants on agonist potency, sensitivity to endogenous allosteric modulators, response time course, channel open probability, and surface expression were assessed. Our data indicated that virtually all of the variants evaluated altered channel function, and multiple variants had profound functional consequences, which may contribute to the neurological conditions in the patients harboring the variants in this region. These data strongly suggest that the residues within the pre-M1 helix play a key role in channel gating and are highly intolerant to genetic variation.

Advances in sequencing technology have generated a large amount of genetic data from patients with neurological conditions. These data have provided diagnosis of many rare diseases, including a number of pathogenic de novo missense variants in GRIN genes encoding N-methyl-d-aspartate receptors (NMDARs). To understand the ramifications for neurons and brain circuits affected by rare patient variants, functional analysis of the variant receptor is necessary in model systems. For NMDARs, this functional analysis needs to assess multiple properties in order to understand how variants could impact receptor function in neurons. One can then use these data to determine whether the overall actions will increase or decrease NMDAR-mediated charge transfer. Here, we describe an analytical and comprehensive framework by which to categorize GRIN variants as either gain-of-function (GoF) or loss-of-function (LoF) and apply this approach to GRIN2B variants identified in patients and the general population. This framework draws on results from six different assays that assess the impact of the variant on NMDAR sensitivity to agonists and endogenous modulators, trafficking to the plasma membrane, response time course and channel open probability. We propose to integrate data from multiple in vitro assays to arrive at a variant classification, and suggest threshold levels that guide confidence. The data supporting GoF and LoF determination are essential to assessing pathogenicity and patient stratification for clinical trials as personalized pharmacological and genetic agents that can enhance or reduce receptor function are advanced. This approach to functional variant classification can generalize to other disorders associated with missense variants.

BACKGROUND:Mutations in the NMDA receptor are known to disrupt glutamatergic signaling crucial for early neurodevelopment, often leading to severe global developmental delay/intellectual disability, epileptic encephalopathy, and cerebral palsy phenotypes. Both seizures and movement disorders can be highly treatment-refractory.RESULTS:We describe a targeted ABA n-of-1 treatment trial with intrathecal MgSO4, rationally designed based on the electrophysiologic properties of this gain of function mutation in the GRIN1 NMDA subunit.CONCLUSION:Although the invasive nature of the trial necessitated a short-term, non-randomized, unblinded intervention, quantitative longitudinal neurophysiologic monitoring indicated benefit, providing class II evidence in support of intrathecal MgSO4 for select forms of GRIN disorders.

N-methyl-D-aspartate receptors (NMDARs) play vital roles in normal brain functions (i.e., learning, memory, and neuronal development) and various neuropathological conditions, such as epilepsy, autism, Parkinson's disease, Alzheimer's disease, and traumatic brain injury. Endogenous neuroactive steroids such as 24(S)-hydroxycholesterol (24(S)-HC) have been shown to influence NMDAR activity, and positive allosteric modulators (PAMs) derived from 24(S)-hydroxycholesterol scaffold can also enhance NMDAR function. This study describes the structural determinants and mechanism of action for 24(S)-hydroxycholesterol and two novel synthetic analogs (SGE-550 and SGE-301) on NMDAR function. We also show that these agents can mitigate the altered function caused by a set of loss-of-function missense variants in NMDAR GluN subunit-encoding GRIN genes associated with neurological and neuropsychiatric disorders. We anticipate that the evaluation of novel neuroactive steroid NMDAR PAMs may catalyze the development of new treatment strategies for GRIN-related neuropsychiatric conditions.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 μM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 μM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

BACKGROUND:Malformations of cortical development (MCDs) have been reported in a subset of patients with pathogenic heterozygous variants in GRIN1 or GRIN2B, genes which encode for subunits of the N-methyl-D-aspartate receptor (NMDAR). The aim of this study was to further define the phenotypic spectrum of NMDAR-related MCDs.METHODS:We report the clinical, radiological and molecular features of 7 new patients and review data on 18 previously reported individuals with NMDAR-related MCDs. Neuropathological findings for two individuals with heterozygous variants in GRIN1 are presented. We report the clinical and neuropathological features of one additional individual with homozygous pathogenic variants in GRIN1.RESULTS:Heterozygous variants in GRIN1 and GRIN2B were associated with overlapping severe clinical and imaging features, including global developmental delay, epilepsy, diffuse dysgyria, dysmorphic basal ganglia and hippocampi. Neuropathological examination in two fetuses with heterozygous GRIN1 variants suggests that proliferation as well as radial and tangential neuronal migration are impaired. In addition, we show that neuronal migration is also impaired by homozygous GRIN1 variants in an individual with microcephaly with simplified gyral pattern.CONCLUSION:These findings expand our understanding of the clinical and imaging features of the 'NMDARopathy' spectrum and contribute to our understanding of the likely underlying pathogenic mechanisms leading to MCD in these patients.

N-methyl-D-aspartate receptors (NMDARs) are tetrameric assemblies of two glutamate N-methyl-D-aspartate receptor subunits, GluN1 and two GluN2, that mediate excitatory synaptic transmission in the central nervous system. Four genes (GRIN2A-D) encode four distinct GluN2 subunits (GluN2A-D). Thus, NMDARs can be diheteromeric assemblies of two GluN1 plus two identical GluN2 subunits, or triheteromeric assemblies of two GluN1 subunits plus two different GluN2 subunits. An increasing number of de novo GRIN variants have been identified in patients with neurologic conditions and with GRIN2A and GRIN2B harboring the vast majority (> 80%) of variants in these cases. These variants produce a wide range of effects on NMDAR function depending upon its subunit subdomain location and additionally on the subunit composition of diheteromeric versus triheteromeric NMDARs. Increasing evidence implicates triheteromeric GluN1/GluN2A/GluN2B receptors as a major component of the NMDAR pool in the adult cortex and hippocampus. Here, we explore the ability of GluN2A- and GluN2B-selective inhibitors to reduce excess current flow through triheteromeric GluN1/GluN2A/GluN2B receptors that contain one copy of GRIN2A or GRIN2B gain-of-function variants. Our data reveal a broad range of sensitivities for variant-containing triheteromeric receptors to subunit-selective inhibitors, with some variants still showing strong sensitivity to inhibitors, whereas others are relatively insensitive. Most variants, however, retain sensitivity to non-selective channel blockers and the competitive antagonist D-(-)-2-amino-5-phosphonopentanoic acid. These results suggest that with comprehensive analysis, certain disease-related GRIN2A and GRIN2B variants can be identified as potential targets for subunit-selective modulation and potential therapeutic gain. SIGNIFICANCE STATEMENT Triheteromeric NMDA receptors that contain one copy each of the GluN2A and GluN2B subunits show intermediate sensitivity to GluN2A- and GluN2B-selective inhibitors, making these compounds candidates for attenuating overactive, GRIN variant-containing NMDA receptors associated with neurological conditions. We show that functional evaluation of variant properties with inhibitor pharmacology can support selection of a subset of variants for which GluN2 subunit-selective agents remain effective inhibitors of variant-containing triheteromeric NMDA receptors.

We report on an 8-year-old girl with severe developmental and epileptic encephalopathy due to the compound heterozygous null variants p.(Gln661*) and p.(Leu830Profs*2) in GRIN2A resulting in a knockout of the human GluN2A subunit of the N-methyl-D-aspartate receptor. Both parents had less severe GRIN2A-related phenotypes and were heterozygous carriers of the respective null variant. Functional investigations of both variants suggested a loss-of-function effect. This is the first description of an autosomal recessive, biallelic type of GRIN2A-related disorder. Nonetheless, there are marked parallels to two previously published families with severe epileptic encephalopathy due to homozygous null variants in GRIN1 as well as various knockout animal models. Compared to heterozygous null variants, biallelic knockout of either GluN1 or GluN2A is associated with markedly more severe phenotypes in both humans and mice. Furthermore, recent findings enable a potential precision medicine approach targeting GRIN-related disorders due to null variants.

N-Methyl-D-aspartate receptors (NMDARs) are highly expressed in brain and play important roles in neurodevelopment and various neuropathologic conditions. Here, we describe a new phenotype in an individual associated with a novel de novo deleterious variant in GRIN1 (c.1595C>A, p.Pro532His). The clinical phenotype is characterized with developmental encephalopathy, striking stimulus-sensitive myoclonus, and frontal lobe and frontal white matter hypoplasia, with no apparent seizures detected. NMDARs that contained the P532H within the glycine-binding domain of GluN1 with either the GluN2A or GluN2B subunits were evaluated for changes in their pharmacological and biophysical properties, which surprisingly revealed only modest changes in glycine potency but a significant decrease in glutamate potency, an increase in sensitivity to endogenous zinc inhibition, a decrease in response to maximally effective concentrations of agonists, a shortened synaptic-like response time course, a decreased channel open probability, and a reduced receptor cell surface expression. Molecule dynamics simulations suggested that the variant can lead to additional interactions across the dimer interface in the agonist-binding domains, resulting in a more open GluN2 agonist-binding domain cleft, which was also confirmed by single-molecule fluorescence resonance energy transfer measurements. Based on the functional deficits identified, several positive modulators were evaluated to explore potential rescue pharmacology.

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.

Significance Standard antidepressant treatments require weeks to show effectiveness. A single subanesthetic dose of ketamine rapidly attenuates many clinical signs and symptoms of depression; however, ketamine treatment also has many adverse effects, including dissociation and potential for abuse, which are mediated by NMDA glutamate receptor (NMDAR) inhibition. Previous work has revealed that the ketamine metabolite (2 R ,6 R )-hydroxynorketamine (HNK) induces antidepressant-like responses in rodents while minimizing the adverse effects observed with ketamine. The results of this study, using a multitude of experimental approaches, confirm that antidepressant-relevant concentrations of (2 R ,6 R )-HNK are not sufficient to block NMDARs. This provides a basis for work directed at alternative molecular targets and toward novel drugs that exert rapid antidepressant effects independent of NMDAR inhibition and NMDAR-mediated adverse effects.