Delineating the GRIN1 phenotypic spectrum: a distinct genetic NMDA receptor encephalopathy

Objective:To determine the phenotypic spectrum caused by mutations in GRIN1 encoding the NMDA receptor subunit GluN1 and to investigate their underlying functional pathophysiology.Methods:We collected molecular and clinical data from several diagnostic and research cohorts. Functional consequences of GRIN1 mutations were investigated in Xenopus laevis oocytes.Results:We identified heterozygous de novo GRIN1 mutations in 14 individuals and reviewed the phenotypes of all 9 previously reported patients. These 23 individuals presented with a distinct phenotype of profound developmental delay, severe intellectual disability with absent speech, muscular hypotonia, hyperkinetic movement disorder, oculogyric crises, cortical blindness, generalized cerebral atrophy, and epilepsy. Mutations cluster within transmembrane segments and result in loss of channel function of varying severity with a dominant-negative effect. In addition, we describe 2 homozygous GRIN1 mutations (1 missense, 1 truncation), each segregating with severe neurodevelopmental phenotypes in consanguineous families.Conclusions:De novo GRIN1 mutations are associated with severe intellectual disability with cortical visual impairment as well as oculomotor and movement disorders being discriminating phenotypic features. Loss of NMDA receptor function appears to be the underlying disease mechanism. The identification of both heterozygous and homozygous mutations blurs the borders of dominant and recessive inheritance of GRIN1-associated disorders.Johannes R. Lemke (32EP30_136042/1) and Peter De Jonghe (G.A.136.11.N and FWO/ESF-ECRP) received financial support within the EuroEPINOMICS-RES network (www.euroepinomics.org) within the Eurocores framework of the European Science Foundation (ESF). Saskia Biskup and Henrike Heyne received financial support from the German Federal Ministry for Education and Research (BMBF IonNeurONet: 01 GM1105A and FKZ: 01EO1501). Katia Hardies is a PhD fellow of the Institute for Science and Technology (IWT) Flanders. Ingo Helbig was supported by intramural funds of the University of Kiel, by a grant from the German Research Foundation (HE5415/3-1) within the EuroEPINOMICS framework of the European Science Foundation, and additional grants of the German Research Foundation (DFG, HE5415/5-1, HE 5415/6-1), German Ministry for Education and Research (01DH12033, MAR 10/012), and grant by the German chapter of the International League against Epilepsy (DGfE). The project also received infrastructural support through the Institute of Clinical Molecular Biology in Kiel, supported in part by DFG Cluster of Excellence "Inflammation at Interfaces" and "Future Ocean." The project was also supported by the popgen 2.0 network (P2N) through a grant from the German Ministry for Education and Research (01EY1103) and by the International Coordination Action (ICA) grant G0E8614N. Christel Depienne, Caroline Nava, and Delphine Heron received financial support for exome analyses by the Centre National de Genotypage (CNG, Evry, France)

Ionotropic glutamate receptor dysfunction in pediatric neurodevelopment

Der N-methyl-D-aspartate (NMDA) Rezeptor ist ein ligandengesteuerter Ionenkanal und gehört zur Familie der ionotropen Glutamatrezeptoren (iGluR). Aufgrund seiner hohen Ca2+-Leitfähigkeit und seiner heterotetrameren Assemblierung spielt der NMDA Rezeptor innerhalb der iGluRs eine besondere Rolle. Für die Funktion sowie in der Entwicklung des zentralen Nervensystem, spielen diese Rezeptoren eine zentrale Rolle. Weiterhin sind sie an Prozessen wie Lernen und Gedächtnisbildung beteiligt. Eine Schädigung von NMDA Rezeptoren wird mit einer Reihe von neurologischen Erkrankungen in Verbindung gebracht und aufgrund ihrer wichtigen Rolle für die Gehirnentwicklung sind sie von großem Interesse in pathologischen Zusammenhängen. NMDA Rezeptoren sind aus zwei obligaten GluN1 und zwei GluN2(A-D) oder GluN3(A,B) Untereinheiten aufgebaut. Jede dieser Untereinheiten weist einen modularen Aufbau mit verschiedenen Domänen auf, welche für unterschiedliche Funktionen zuständig sind. Die extrazellulär gelegene N-terminale Domäne (NTD) hat eine modulierende Funktion und besitzt Bindestellen für allosterische Modulatoren, wie beispielsweise Zink. Die Liganden-Bindungs-Domäne (LBD) beinhaltet die Agonisten-Bindestellen und ist mit den Transmembrandomänen (TMD) verbunden. Die drei Transmembrandomänen (M1, M2, M3) und die Wiedereintrittsschleife (P-Loop) bilden die Ionenkanalpore. Die Pore wird durch die QRN Stelle verengt, welche entscheidend für die Permeationseigenschaften des Rezeptors ist. Ein Ziel der Arbeit war die Analyse des NMDA Rezeptors in pathologischen Zusammenhängen. Dabei wurde der Einfluss der allosterischen Modulation, welcher über die NTD vermittelt wird, untersucht. Weiterhin wurde der Einfluss der Porenregion bezüglich des Mg2+-Blocks und der Ca2+-Permeabilität analysiert. In Zusammenarbeit mit klinischen Arbeitsgruppen erhielten wir Informationen über Mutationen in verschiedenen NMDA Rezeptor Untereinheiten, aus Patienten mit verschiedenen Epilepsie-Syndromen. Für die Analyse wurden einzelne Mutationen in der NTD und der Ionenkanalregion ausgewählt und mittels zielgerichteter Mutagenese eingeführt. Anschließend erfolgte die funktionale Charakterisierung der ausgewählten Mutationen mittels der Zwei-Elektroden-Spannungsklemme (Two-electrode-voltage-clamp, TEVC) an Xenopus laevis Oozyten. Die elektrophysiologischen Analysen der verschiedenen Mutationen in der NTD und der Kanalpore zeigten eine Verstärkung (gain of function) der Ionenkanalfunktion durch eine Verminderung verschiedener Inhibitionsmechansimen. Die Mutation GluN2A p.Ala243Val in der NTD führte zu einem Verlust der Zn2+-Inhibition. Die Mutationen in der Kanalregion führten zu einer starken Verringerung des spannungsabhängigen Mg2+-Blocks sowie höheren, relativen Ca2+-Permeabilitäten. Diese Verstärkung (gain of function) der Ionenkanalfunktion könnte ein molekulares Korrelat für die Epilepsie-Syndrome darstellen, wodurch es zu einer Überaktivierung der Rezeptoren kommt. In neuronalen Netzen könnte dies zu einer Übererregung (Hyperexzitabilität) der exzitatorischen Neurotransmission führen. Dies würde mit der Hypothese übereinstimmen, dass Epilepsie durch Übererregung ausgelöst werden kann. Aus diesem Grund sind GluN1/GluN2A,B NMDA Rezeptor Antagonisten für die Behandlung neurologischer Erkrankungen, welche durch eine Übererregung ausgelöst werden, wie beispielsweise. Epilepsie, ein vielversprechender Ansatz. Um den möglichen Einsatz von GluN1/GluN2 Antagonisten in der Behandlung von neurologischen Erkrankungen zu validieren, wurden der Effekt und der Mechanismus verschiedener GluN1 Antagonisten an exzitatorischen GluN1/GluN3A NMDA Rezeptoren untersucht. Im Gegensatz zu den konventionellen NMDA Rezeptoren führt eine Antagonisierung der GluN1-Bindestelle zu einer verstärkten Rezeptoraktivierung. Die elektrophysiologische Analyse verschiedener GluN1 Antagonisten zeigte eine Korrelation zwischen der Potenzierung der GluN1/GluN3A Rezeptor-Ströme und der Affinität des jeweiligen Antagonisten. Diese Ergebnisse zeigten somit einen direkten Zusammenhang zwischen der Antagonistenaffinität und der Potenzierungseffizienz. Zusammenfassend präsentiert diese Arbeit Mutationen in der GluN2A und GluN2B Untereinheit des NMDA Rezeptors als wichtige, genetische Determinanten, in einigen schweren, altersabhängigen Epilepsien. Diese Ergebnisse sind deutliche Hinweise für eine starke Beteiligung von veränderter NMDA-Rezeptor-Signalübertragung an der Epileptogenese. Weiterhin eröffnen diese Daten die Möglichkeit NMDA Rezeptoren als neue Ziele in der Epilepsie-Behandlung zu nutzen und mit Blockern und Antagonisten zu behandeln, aber zeigt zugleich die Komplexibilität des Einflusses von Antagonisten auf NMDA Rezeptoren

The N-terminal domain of the GluN3A subunit determines the efficacy of glycine-activated NMDA receptors.

N-methyl-d-aspartate (NMDA) receptors composed of glycine-binding GluN1 and GluN3 subunits function as excitatory glycine receptors that respond to agonist application only with a very low efficacy. Binding of glycine to the high-affinity GluN3 subunits triggers channel opening, whereas glycine binding to the low-affinity GluN1 subunits causes an auto-inhibition of the maximal glycine-inducible receptor current (Imax). Hence, competitive antagonists of the GluN1 subunit strongly potentiate glycine responses of wild type (wt) GluN1/GluN3 receptors. Here, we show that co-expression of N-terminal domain (NTD) deleted GluN1 (GluN1(ΔNTD)) and GluN3 (GluN3(ΔNTD)) subunits in Xenopus oocytes generates GluN1/GluN3 receptors with a large increase in the glycine-inducible Imax accompanied by a strongly impaired GluN1 antagonist-mediated potentiation. Affinity purification after metabolic or surface labeling revealed no differences in subunit stoichiometry and surface expression between wt GluN1/GluN3A and mutant GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, indicating a specific effect of NTD deletions on the efficacy of receptor opening. Notably, GluN1/GluN3A(ΔNTD) receptors showed a similar increase in Imax and a greatly reduced GluN1 antagonist-mediated current potentiation as GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, whereas the glycine-induced currents of GluN1(ΔNTD)/GluN3A receptors resembled those of wt GluN1/GluN3A receptors. Furthermore, oxidative crosslinking of the homophilic GluN3A NTD intersubunit interface in mutant GluN1/GluN3A(R319C) receptors caused both a decrease in the glycine-induced Imax concomitantly with a marked increase in GluN1 antagonist-mediated current potentiation, whilst mutations within the intrasubunit region linking the GluN3A NTD to the ligand binding domain had opposite effects. Together these results show that the GluN3A NTD constitutes a crucial regulatory determinant of GluN1/GluN3A receptor function

GRIN2B Mutations in West Syndrome and Intellectual Disability with Focal Epilepsy

Lemke JR, Hendrickx R, Geider K, et al. GRIN2B Mutations in West Syndrome and Intellectual Disability with Focal Epilepsy. Annals of Neurology. 2014;75(1):147-154.Objective: To identify novel epilepsy genes using a panel approach and describe the functional consequences of mutations. Methods: Using a panel approach, we screened 357 patients comprising a vast spectrum of epileptic disorders for defects in genes known to contribute to epilepsy and/or intellectual disability (ID). After detection of mutations in a novel epilepsy gene, we investigated functional effects in Xenopus laevis oocytes and screened a follow-up cohort. Results: We revealed de novo mutations in GRIN2B encoding the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in 2 individuals with West syndrome and severe developmental delay as well as 1 individual with ID and focal epilepsy. The patient with ID and focal epilepsy had a missense mutation in the extracellular glutamate-binding domain (p.Arg540His), whereas both West syndrome patients carried missense mutations within the NR2B ion channel-forming re-entrant loop (p.Asn615Ile, p.Val618Gly). Subsequent screening of 47 patients with unexplained infantile spasms did not reveal additional de novo mutations, but detected a carrier of a novel inherited GRIN2B splice site variant in close proximity (c.2011-5_2011-4delTC). Mutations p.Asn615Ile and p.Val618Gly cause a significantly reduced Mg2+ block and higher Ca2+ permeability, leading to a dramatically increased Ca2+ influx, whereas p.Arg540His caused less severe disturbance of channel function, corresponding to the milder patient phenotype. Interpretation: We identified GRIN2B gain-of-function mutations as a cause of West syndrome with severe developmental delay as well as of ID with childhood onset focal epilepsy. Severely disturbed channel function corresponded to severe clinical phenotypes, underlining the important role of facilitated NMDA receptor signaling in epileptogenesis

Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes

N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly Ca²(+)-permeable cation channels which are blocked by extracellular Mg²(+) in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2A(N615K) (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg²(+) block and a decrease in Ca²(+) permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected

Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes.

N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly Ca(2)(+)-permeable cation channels which are blocked by extracellular Mg(2)(+) in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2A(N615K) (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg(2)(+) block and a decrease in Ca(2)(+) permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected

Ocean urea fertilization for carbon credits poses high ecological risks

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed

Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes

Lemke JR, Lal D, Reinthaler EM, et al. Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes. Nature Genetics. 2013;45(9):1067-1072.Idiopathic focal epilepsy (IFE) with rolandic spikes is the most common childhood epilepsy, comprising a phenotypic spectrum from rolandic epilepsy (also benign epilepsy with centrotemporal spikes, BECTS) to atypical benign partial epilepsy (ABPE), Landau-Kleffner syndrome (LKS) and epileptic encephalopathy with continuous spike and waves during slow-wave sleep (CSWS)(1,2). The genetic basis is largely unknown. We detected new heterozygous mutations in GRIN2A in 27 of 359 affected individuals from 2 independent cohorts with IFE (7.5%; P = 4.83 x 10(-18), Fisher's exact test). Mutations occurred significantly more frequently in the more severe phenotypes, with mutation detection rates ranging from 12/245 (4.9%) in individuals with BECTS to 9/51 (17.6%) in individuals with CSWS (P = 0.009, Cochran-Armitage test for trend). In addition, exon-disrupting microdeletions were found in 3 of 286 individuals (1.0%; P = 0.004, Fisher's exact test). These results establish alterations of the gene encoding the NMDA receptor NR2A subunit as a major genetic risk factor for IFE