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N-methyl-D-aspartate receptors (NMDARs) are glutamate receptors with key roles in synaptic communication and plasticity. The activation of synaptic NMDARs initiates plasticity and stimulates cell survival. In contrast, the activation of extrasynaptic NMDARs can promote cell death underlying a potential mechanism of neurodegeneration occurring in Alzheimer's disease (AD). The distribution of synaptic versus extrasynaptic NMDARs has emerged as an important parameter contributing to neuronal dysfunction in neurodegenerative diseases including AD. Here, we review the concept of extrasynaptic NMDARs, as this population is present in numerous neuronal cell membranes but also in the membranes of various non-neuronal cells. Previous evidence regarding the membranal distribution of synaptic versus extrasynaptic NMDRs in relation to AD mice models and in the brains of AD patients will also be reviewed. [ABSTRACT FROM AUTHOR]

Monocular deprivation (MD) causes an initial decrease in synaptic responses to the deprived eye in juvenile mouse primary visual cortex (V1) through Hebbian long-term depression (LTD). This is followed by a homeostatic increase, which has been attributed either to synaptic scaling or to a slide threshold for Hebbian long-term potentiation (LTP) rather than scaling. We therefore asked in mice of all sexes whether the homeostatic increase during MD requires GluN2B-containing NMDA receptor activity, which is required to slide the plasticity threshold but not for synaptic scaling. Selective GluN2B blockade from 2–6 d after monocular lid suture prevented the homeostatic increase in miniature excitatory postsynaptic current (mEPSC) amplitude in monocular V1 of acute slices and prevented the increase in visually evoked responses in binocular V1 in vivo. The decrease in mEPSC amplitude and visually evoked responses during the first 2 d of MD also required GluN2B activity. Together, these results support the idea that GluN2B-containing NMDA receptors first play a role in LTD immediately following eye closure and then promote homeostasis during prolonged MD by sliding the plasticity threshold in favor of LTP. [ABSTRACT FROM AUTHOR]

Here, we describe the characterization of a radioligand selective for GluN2B‐containing NMDA receptors, 3‐[3H] 1‐(azetidin‐1‐yl)‐2‐(6‐(4‐fluoro‐3‐methyl‐phenyl)pyrrolo[3,2‐b]pyridin‐1‐yl)ethanone ([3H]‐JNJ‐ GluN2B‐5). In rat cortical membranes, the compound bound to a single site, and the following kinetic parameters were measured; association rate constant Kon = 0.0066 ± 0.0006 min−1 nM−1, dissociation rate constant Koff = 0.0210 ± 0.0001 min−1 indicating calculated KD = Koff/Kon = 3.3 ± 0.4 nM, (mean ± SEM, n = 3). The equilibrium dissociation constant determined from saturation binding experiments in rat cortex was KD of 2.6 ± 0.3 nM (mean ± SEM, n = 3). In contrast to the widely used GluN2B radioligand [3H]‐Ro 25‐6981, whose affinity Ki for sigma 1 and sigma 2 receptors are 2 and 189 nM, respectively, [3H]‐JNJ‐GluN2B‐5 exhibits no measurable affinity for sigma 1 and sigma 2 receptors (Ki > 10 μM for both) providing distinct selectivity advantages. Anatomical distribution of [3H]‐JNJ‐GluN2B‐5 binding sites in rat, mouse, dog, monkey, and human brain tissue was studied using in vitro autoradiography, which showed high specific binding in the hippocampus and cortex and negligible binding in the cerebellum. Enhanced selectivity for GluN2B‐containing receptors translated to a good signal‐to‐noise ratio in both in vitro radioligand binding and in vitro autoradiography assays. In conclusion, [3H]‐JNJ‐GluN2B‐5 is a high‐affinity GluN2B radioligand with excellent signal‐to‐noise ratio and unprecedented selectivity. [ABSTRACT FROM AUTHOR]

N-Methyl-D-aspartate receptors (NMDARs), encoded by GRIN genes, are ionotropic glutamate receptors playing a critical role in synaptic transmission, plasticity, and synapse development. Genome sequence analyses have identified variants in GRIN genes in patients with neurodevelopmental disorders, but the underlying disease mechanisms are not well understood. Here, we have created and evaluated a transgenic mouse line carrying a missense variant Grin2bL825V, corresponding to a de novo GRIN2B variant encoding GluN2B(L825V) found in a patient with intellectual disability (ID) and autismspectrum disorder (ASD). We used HEK293T cells expressing recombinant receptors and primary hippocampal neurons prepared from heterozygous Grin2bL825V/+ (L825V/+) and wild-type (WT) Grin2b+/+ (+/+) male and female mice to assess the functional impact of the variant. Whole-cell NMDAR currents were reduced in neurons from L825V/+ compared with +/+ mice. The peak amplitude of NMDAR-mediated evoked excitatory postsynaptic currents (NMDAR-eEPSCs) was unchanged, but NMDAR-eEPSCs in L825V/+ neurons had faster deactivation compared with +/+ neurons and were less sensitive to a GluN2B-selective antagonist ifenprodil. Together, these results suggest a decreased functional contribution of GluN2B subunits to synaptic NMDAR currents in hippocampal neurons from L825V/+ mice. The analysis of the GluN2B(L825V) subunit surface expression and synaptic localization revealed no differences compared with WT GluN2B. Behavioral testing of mice of both sexes demonstrated hypoactivity, anxiety, and impaired sensorimotor gating in the L825V/+ strain, particularly affecting males, as well as cognitive symptoms. The heterozygous L825V/+ mouse offers a clinically relevant model of GRIN2B-related ID/ASD, and our results suggest synaptic-level functional changes that may contribute to neurodevelopmental pathology. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptors (NMDARs) play essential roles in vital aspects of brain functions. NMDARs mediate clinical features of neurological diseases and thus, represent a potential therapeutic target for their treatments. Many findings implicated the GluN2B subunit of NMDARs in various neurological disorders including epilepsy, ischemic brain damage, and neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, Huntington's chorea, and amyotrophic lateral sclerosis. Although a large amount of information is growing consistently on the importance of GluN2B subunit, however, limited recent data is available on how subunit-selective ligands impact NMDAR functions, which blunts the ability to render the diagnosis or craft novel treatments tailored to patients. To bridge this gap, we have focused on and summarized recently reported GluN2B selective ligands as emerging subunit-selective antagonists and modulators of NMDAR. Herein, we have also presented an overview of the structure–function relationship for potential GluN2B/NMDAR ligands with their binding sites and connection to CNS functionalities. Understanding of design rules and roles of GluN2B selective compounds will provide the link to medicinal chemists and neuroscientists to explore novel neurotherapeutic strategies against dysfunctions of glutamatergic neurotransmission. [ABSTRACT FROM AUTHOR]

Introduction: Neonatal hypoxic-ischemic brain damage (HIBD) refers to brain damage in newborns caused by hypoxia and reduced or even stopped cerebral blood flow during the perinatal period. Currently, there are no targeted treatments for neonatal ischemic hypoxic brain damage, primarily due to the incomplete understanding of its pathophysiological mechanisms. Especially, the role of NMDA receptors is less studied in HIBD. Therefore, this study explored the molecular mechanism of endogenous protection mediated by GluN2B-NMDAR in HIBD. Method: Hypoxic ischemia was induced in mice aged 9-11 days. The brain damage was examined by Nissl staining and HE staining, while neuronal apoptosis was examined by Hoechst staining and TTC staining. And cognitive deficiency of mice was examined by various behavior tests including Barnes Maze, Three Chamber Social Interaction Test and Elevated Plus Maze. The activation of ER stress signaling pathways were evaluated by Western blot. Results: We found that after HIBD induction, the activation of GluN2B-NMDAR attenuated neuronal apoptosis and brain damage. Meanwhile, the ER stress PERK/eIF2α signaling pathway was activated in a time-dependent manner after HIBE. Furthermore, after selective inhibiting GluN2B-NMDAR in HIBD mice with ifenprodil, the PERK/eIF2α signaling pathway remains continuously activated, leading to neuronal apoptosis, morphological brain damage. and aggravating deficits in spatial memory, cognition, and social abilities in adult mice. Discussion: The results of this study indicate that, unlike its role in adult brain damage, GluN2B in early development plays a neuroprotective role in HIBD by inhibiting excessive activation of the PERK/eIF2α signaling pathway. This study provides theoretical support for the clinical development of targeted drugs or treatment methods for HIBD. [ABSTRACT FROM AUTHOR]

Abstract Background Synaptic degeneration occurs in the early stage of Alzheimer's disease (AD) before devastating symptoms, strongly correlated with cognitive decline. Circular RNAs (circRNAs) are abundantly enriched in neural tissues, and aberrant expression of circRNAs precedes AD symptoms, significantly correlated with clinical dementia severity. However, the direct relationship between circRNA dysregulation and synaptic impairment in the early stage of AD remains poorly understood. Methods Hippocampal whole-transcriptome sequencing was performed to identify dysregulated circRNAs and miRNAs in 4-month-old wild-type and APP/PS1 mice. RNA antisense purification and mass spectrometry were utilized to unveil interactions between circRIMS2 and methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3). The roles of circRIMS2/miR-3968 in synaptic targeting of UBE2K-mediated ubiquitination of GluN2B subunit of NMDA receptor were evaluated via numerous lentiviruses followed by morphological staining, co-immunoprecipitation and behavioral testing. Further, a membrane-permeable peptide was used to block the ubiquitination of K1082 on GluN2B in AD mice. Results circRIMS2 was significantly upregulated in 4-month-old APP/PS1 mice, which was mediated by METTL3-dependent N6-methyladenosine (m6A) modification. Overexpression of circRIMS2 led to synaptic and memory impairments in 4-month-old C57BL/6 mice. MiR-3968/UBE2K was validated as the downstream of circRIMS2. Elevated UBE2K induced synaptic dysfunction of AD through ubiquitinating K1082 on GluN2B. Silencing METTL3 or blocking the ubiquitination of K1082 on GluN2B with a short membrane-permeable peptide remarkably rescued synaptic dysfunction in AD mice. Conclusions In conclusion, our study demonstrated that m6A-modified circRIMS2 mediates the synaptic and memory impairments in AD by activating the UBE2K-dependent ubiquitination and degradation of GluN2B via sponging miR-3968, providing novel therapeutic strategies for AD.

N-methyl-D-aspartate receptors (NMDARs) are glutamate receptors with key roles in synaptic communication and plasticity. The activation of synaptic NMDARs initiates plasticity and stimulates cell survival. In contrast, the activation of extrasynaptic NMDARs can promote cell death underlying a potential mechanism of neurodegeneration occurring in Alzheimer’s disease (AD). The distribution of synaptic versus extrasynaptic NMDARs has emerged as an important parameter contributing to neuronal dysfunction in neurodegenerative diseases including AD. Here, we review the concept of extrasynaptic NMDARs, as this population is present in numerous neuronal cell membranes but also in the membranes of various non-neuronal cells. Previous evidence regarding the membranal distribution of synaptic versus extrasynaptic NMDRs in relation to AD mice models and in the brains of AD patients will also be reviewed.

The N-methyl-D-aspartate(NMDA)receptors are ionotropic glutamate receptors that participate in excitatory postsynaptic signaling in the mammalian central nervous system. In the 1980s, octreotide first became available for the treatment of acromegaly. In acromegaly patients and healthy volunteers, somatostatin and its analogs, such as octreotide and lanreotide, dramatically decrease the release of GH. In this study, we investigated the effect of octreotide, which controls blood lipids, on one of the proteins involved in epilepsy. After examining the genes involved in epilepsy by the DisGeNET server, we selected the ionotropic glutamate receptor NMDA type subunit 2B gene with the symbol GRIN2B. The docking and molecular simulation process was done using the AutoDock Vina 1.2.0 algorithm under the Chimera user interface. The analysis of docking results has been done by the PDBsum and Protein-Ligand Interaction Profiler servers. The VMD program prepared the images. The Pocket Drug server detected eighteen packets. Only the molecular docking results of 5 out of 18 pockets were acceptable. The results of docking for eighteen pockets were found in the target protein after the simulations; 13 have fewer than 14 residues, and 5 have more than 14 residues. We selected the best 18 pockets (P35, P64), (P10, P14, P17). [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptors, are important in regulating sympathetic tone and cardiovascular function in the rostral ventrolateral medulla (RVLM). Amyloid-beta peptide (Aβ) is linked to the pathogenesis of Alzheimer's disease (AD). Cerebro- and cardiovascular diseases might be the risk factors for developing AD. The present study examines the acute effects of soluble Aβ on the function of NMDA receptors in rats RVLM. We used the magnitude of increases in the blood pressure (pressor responses) induced by microinjection of NMDA into the RVLM as an index of NMDA receptor function in the RVLM. Soluble Aβ was applied by intracerebroventricular (ICV) injection. Aβ1-40 at a lower dose (0.2 nmol) caused a slight reduction, and a higher dose (2 nmol) showed a significant decrease in NMDA-induced pressor responses 10 min after administration. ICV injection of Aβ1-42 (2 nmol) did not affect NMDA-induced pressor responses in the RVLM. Co-administration of Aβ1-40 with ifenprodil or memantine blocked the inhibitory effects of Aβ1-40. Immunohistochemistry analysis showed a significant increase in the immunoreactivity of phosphoserine 1480 of GluN2B subunits (pGluN2B-serine1480) in the neuron of the RVLM without significant changes in phosphoserine 896 of GluN1 subunits (pGluN1-serine896), GluN1 and GluN2B, 10 min following Aβ1-40 administration compared with saline. Interestingly, we found a much higher level of Aβ1-40 compared to that of Aβ1-42 in the cerebrospinal fluid (CSF) measured using enzyme-linked immunosorbent assay 10 min following ICV administration of the same dose (2 nmol) of the peptides. In conclusion, the results suggest that ICV Aβ1-40, but not Aβ1-42, produced an inhibitory effect on NMDA receptor function in the RVLM, which might result from changes in pGluN2B-serine1480 (regulated by casein kinase II). The different elimination of the peptides in the CSF might contribute to the differential effects of Aβ1-40 and Aβ1-42 on NMDA receptor function. [ABSTRACT FROM AUTHOR]

Recent findings suggest an important role for the dysregulation of stromal interaction molecule (STIM) proteins, activators of store-operated Ca2+ channels, and the prolonged activation of N-methyl-D-aspartate receptors (NMDARs) in the development of neurodegenerative diseases. We previously demonstrated that STIM silencing increases Ca2+ influx through NMDAR and STIM–NMDAR2 complexes are present in neurons. However, the interplay between NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM1/STIM2 with regard to intracellular trafficking remains unknown. Here, we found that the activation of NMDAR endocytosis led to an increase in STIM2–GluN2A and STIM2–GluN2B interactions in primary cortical neurons. STIM1 appeared to migrate from synaptic to extrasynaptic sites. STIM2 silencing inhibited post-activation NMDAR translocation from the plasma membrane and synaptic spines and increased NMDAR currents. Our findings reveal a novel molecular mechanism by which STIM2 regulates NMDAR synaptic trafficking by promoting NMDAR endocytosis after receptor overactivation, which may suggest protection against excessive uncontrolled Ca2+ influx through NMDARs. [ABSTRACT FROM AUTHOR]

Major depressive disorder (MDD) is a serious public health problem, as it is the most common psychiatric disorder worldwide. Antidepressant drugs increase adult hippocampal neurogenesis, which is required to induce some behavioral effects of antidepressants. Adult‐born granule cells in the dentate gyrus (DG) and the glutamate receptors subunits 2 (GluN2B) subunit of N‐methyl‐D‐aspartate (NMDA) ionotropic receptors play an important role in these effects. However, the precise neurochemical role of the GluN2B subunit of the NMDA receptor on adult‐born GCs for antidepressant‐like effects has yet to be elucidated. The present study aims to explore the contribution of the GluN2B‐containing NMDA receptors in the ventral dentate gyrus (vDG) to the antidepressant drug treatment using a pharmacological approach. Thus, (αR)‐(4‐hydroxyphenyl)‐(βS)‐methyl‐4‐(phenylmethyl)‐1‐piperidinepropanol (Ro25‐6981), a selective antagonist of the GluN2B subunit, was acutely administered locally into the ventral DG (vDG, 1 μg each side) following a chronic fluoxetine (18 mg/kg/day) treatment—known to increase adult hippocampal neurogenesis—in a mouse model of anxiety/depression. Responses in a neurogenesis‐dependent task, the novelty suppressed feeding (NSF), and neurochemical consequences on extracellular glutamate and gamma‐aminobutyric acid (GABA) levels in the vDG were measured. Here, we show a rapid‐acting antidepressant‐like effect of local Ro25‐6981 administration in the NSF independent of fluoxetine treatment. Furthermore, we revealed a fluoxetine‐independent increase in the glutamatergic transmission in the vDG. Our results suggest behavioral and neurochemical effects of GluN2B subunit independent of serotonin reuptake inhibition. [ABSTRACT FROM AUTHOR]

N-Methyl-D-aspartate receptors (NMDARs) composed of different splice variants display distinct pH sensitivities and are crucial for learning and memory, as well as for inflammatory or injury processes. Dysregulation of the NMDAR has been linked to diseases like Parkinson's, Alzheimer's, schizophrenia, and drug addiction. The development of selective receptor modulators, therefore, constitutes a promising approach for numerous therapeutical applications. Here, we identified (R)-OF-NB1 as a promising splice variant selective NMDAR antagonist. We investigated the interaction of (R)-OF-NB1 and NMDAR from a biochemical, bioinformatical, and electrophysiological perspective to characterize the downstream allosteric modulation of NMDAR by 3-benzazepine derivatives. The allosteric modulatory pathway starts at the ifenprodil binding pocket in the amino terminal domain and immobilizes the connecting α5-helix to the ligand binding domain, resulting in inhibition. In contrast, the exon 5 splice variant GluN1-1b elevates the NMDARs flexibility and promotes the open state of its ligand binding domain. [ABSTRACT FROM AUTHOR]

Background: Synaptic degeneration occurs in the early stage of Alzheimer's disease (AD) before devastating symptoms, strongly correlated with cognitive decline. Circular RNAs (circRNAs) are abundantly enriched in neural tissues, and aberrant expression of circRNAs precedes AD symptoms, significantly correlated with clinical dementia severity. However, the direct relationship between circRNA dysregulation and synaptic impairment in the early stage of AD remains poorly understood. Methods: Hippocampal whole-transcriptome sequencing was performed to identify dysregulated circRNAs and miRNAs in 4-month-old wild-type and APP/PS1 mice. RNA antisense purification and mass spectrometry were utilized to unveil interactions between circRIMS2 and methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3). The roles of circRIMS2/miR-3968 in synaptic targeting of UBE2K-mediated ubiquitination of GluN2B subunit of NMDA receptor were evaluated via numerous lentiviruses followed by morphological staining, co-immunoprecipitation and behavioral testing. Further, a membrane-permeable peptide was used to block the ubiquitination of K1082 on GluN2B in AD mice. Results: circRIMS2 was significantly upregulated in 4-month-old APP/PS1 mice, which was mediated by METTL3-dependent N6-methyladenosine (m6A) modification. Overexpression of circRIMS2 led to synaptic and memory impairments in 4-month-old C57BL/6 mice. MiR-3968/UBE2K was validated as the downstream of circRIMS2. Elevated UBE2K induced synaptic dysfunction of AD through ubiquitinating K1082 on GluN2B. Silencing METTL3 or blocking the ubiquitination of K1082 on GluN2B with a short membrane-permeable peptide remarkably rescued synaptic dysfunction in AD mice. Conclusions: In conclusion, our study demonstrated that m6A-modified circRIMS2 mediates the synaptic and memory impairments in AD by activating the UBE2K-dependent ubiquitination and degradation of GluN2B via sponging miR-3968, providing novel therapeutic strategies for AD. [ABSTRACT FROM AUTHOR]

Neonatal hypoxic-ischemic encephalopathy (HIE) is a disease caused by insufficient blood supply in the brain in newborns during the perinatal period. Severe HIE leads to patient death, and patients with mild HIE are at increased risk of cognitive deficits and behavioral abnormalities. The NMDA receptor is an important excitatory receptor in the central nervous system, and in adult hypoxic-ischemic injury both subtypes of the NMDA receptor play important but distinct roles. The GluN2Acontaining NMDA receptor (GluN2A-NMDAR) could activate neuronal protective signaling pathway, while the GluN2B-NMDAR subtype is coupled to the apoptosis-inducing signaling pathway and leads to neuronal death. However, the expression level of GluN2B is higher in newborns than in adults, while the expression of GluN2A is lower. Therefore, it is not clear whether the roles of different NMDA receptor subtypes in HIE are consistent with those in adults. We investigated this issue in this study and found that in HIE, GluN2B plays a protective role by mediating the protective pathway through binding with PSD95, which is quite different to that in adults. The results of this study provided new theoretical support for the clinical treatment of neonatal hypoxic ischemia. [ABSTRACT FROM AUTHOR]

Key points Question: Do GluN2B radioligands 11C-NR2B-Me enantiomers show cross-binding with σ1 receptors in brain in vivo? Pertinent findings: 11C-(+)-NR2B-Me, 11C-(−)-NR2B-Me, 11C-(S)-NR2B-SMe, and 11C-(R)-NR2B-SMe give specific signals in whole rat brain that could be pre-blocked and displaced using ligands that specifically target GluN2B receptors, but not by σ1 ligand FTC-146. Implications for patient care: 11C-(−)-NR2B-Me does not show cross-binding with σ1 receptor in rat brain using PET imaging. This radioligand may be promising for human PET imaging.

Activation of Ca2+/calmodulin kinase II (CaMKII) and the N-Methyl D-aspartate receptor (NMDAR), particularly its GluN2B subunit, contribute to the central sensitization of nociceptive pathways and persistent pain. Using mutant mice wherein the activity-driven binding of CaMKII to S1303 in GluN2B is abrogated (GluN2BKI), this study investigated the importance of this interaction for acute and persistent inflammatory nociception. GluN2BKI, wild type and heterozygote mice did not differ in responses to acute noxious heat stimuli as measured with tail flick, paw flick, or hot plate assays, nor did they differ in their responses to mechanical stimulation with von Frey filaments. Surprisingly, the three genotypes exhibited similar spontaneous pain behaviors and hypersensitivity to heat or mechanical stimuli induced by intraplantar injection of capsaicin; however, GluN2BKI mice did not immediately attend to the paw. WT and GluN2BKI mice also did not differ in the nociceptive behaviors elicited by intraplantar injection of formalin, even though MK801 greatly reduced these behaviors in both genotypes concordant with NMDAR dependence. CaMKII binding to GluN2B at S1303 therefore does not appear to be critical for the development of inflammatory nociception. Finally, intrathecal KN93 reduced formalin-induced nociceptive behaviors in GluN2BKI mice. KN93 does not inhibit CaKMII, but rather binds Ca2+/calmodulin. It has multiple other targets including Ca2+-, Na+- and K+-channels, as well as various kinases. Therefore, the use of GluN2BKI mice provided genetic specificity in assessing the role of CaMKII in inflammatory pain signaling cascades. These results challenge current thinking on the involvement of the CaMKII-NMDAR interaction in inflammatory pain.

Social adversity not only causes severe psychological diseases but also may improve people's ability to learn and grow. However, the beneficial effects of social adversity are often ignored. In this study, we investigated whether and how social adversity affects learning and memory in a mouse social defeat stress (SDS) model. A total of 652 mice were placed in experimental groups of six to 23 mice each. SDS enhanced spatial, novelty, and fear memory with increased synaptosome associated protein 25 (SNAP-25) level and dendritic spine density in hippocampal neurons among young but not middle-aged mice. Chemogenetic inhibition of hippocampal CaMK2A + neurons blocked SDS-induced enhancement of learning or memory. Knockdown of SNAP-25 or blockade of N-methyl-D-aspartate (NMDA) receptor subunit GluN2B in the hippocampus prevented SDS-induced learning memory enhancement in an emotion-independent manner. These findings suggest that social adversity promotes learning and memory ability in youths and provide a neurobiological foundation for biopsychological antifragility. [ABSTRACT FROM AUTHOR]

The number of N-Methyl-D-aspartate receptor (NMDAR) linked neurodegenerative diseases such as Alzheimer's disease and dementia is constantly increasing. This is partly due to demographic change and presents new challenges to societies. To date, there are no effective treatment options. Current medications are nonselective and can lead to unwanted side effects in patients. A promising therapeutic approach is the targeted inhibition of NMDARs in the brain. NMDARs containing different subunits and splice variants display different physiological properties and play a crucial role in learning and memory, as well as in inflammatory or injury processes. They become overactivated during the course of the disease, leading to nerve cell death. Until now, there has been a lack of understanding of the general functions of the receptor and the mechanism of inhibition, which need to be understood in order to develop inhibitors. Ideal compounds should be highly targeted and even splice-variant-selective. However, a potent and splice-variant-selective NMDAR-targeting drug has yet to be developed. Recently developed 3-benzazepines are promising inhibitors for further drug development. The NMDAR splice variants GluN1-1b-4b carry a 21-amino-acid-long, flexible exon 5. Exon 5 lowers the NMDAR's sensitivity to allosteric modulators by probably acting as an NMDAR modulator itself. The role of exon 5 in NMDAR modulation is still poorly understood. In this review, we summarize the structure and pharmacological relevance of tetrahydro-3-benzazepines. [ABSTRACT FROM AUTHOR]

Excitotoxicity is classically attributed to Ca2+ influx through NMDA receptors (NMDAr), leading to production of nitric oxide by neuronal nitric oxide synthase and superoxide by mitochondria, which react to form highly cytotoxic peroxynitrite. More recent observations warrant revision of the classic view and help to explain some otherwise puzzling aspects of excitotoxic cell injury. Studies using pharmacological and genetic approaches show that superoxide produced by NMDAr activation originates primarily from NADPH oxidase rather than from mitochondria. As NADPH oxidase is localized to the plasma membrane, this also provides an explanation for the extracellular release of superoxide and cell-to-cell "spread" of excitotoxic injury observed in vitro and in vivo. The signaling pathway linking NMDAr to NADPH oxidase involves Ca2+ influx, phosphoinositol-3-kinase, and protein kinase Cζ, and interventions at any of these steps can prevent superoxide production and excitotoxic injury. Ca2+ influx specifically through NMDAr is normally required to induce excitotoxicity, through a mechanism presumed to involve privileged Ca2+ access to local signaling domains. However, experiments using selective blockade of the NMDAr ion channel and artificial reconstitution of Ca2+ by other routes indicate that the special effects of NMDAr activation are attributable instead to concurrent non-ionotropic NMDAr signaling by agonist binding to NMDAr. The non-ionotropic signaling driving NADPH oxidase activation is mediated in part by phosphoinositol-3-kinase binding to the C-terminal domain of GluN2B receptor subunits. These more recently identified aspects of excitotoxicity expand our appreciation of the complexity of excitotoxic processes and suggest novel approaches for limiting neuronal injury.

N-Methyl-d-aspartate receptor (NMDAR) signaling pathway has been implicated in the pathogenesis and treatment of depression. However, the role of NMDAR subunits in depression is still unclear. In this study, alteration in all seven NMDAR subunits in several brain areas of rats exposed to chronic unpredictable mild stress (CUMS), an animal model of depression, was detected. Our findings demonstrated that: (1) CUMS could induce a reduction in sucrose preference, an indicator of typical depression-like behaviors; (2) CUMS significantly reduced the NMDAR subunits of GluN2B and GluN3 in the medial prefrontal cortex (mPFC), but not altered all seven NMDAR subunits in hippocampus and corpus callosum of rats; (3) subunit composition of NMDARs in corpus callosum was different from that in mPFC, PFC and hippocampus; and (4) the mRNA expressions of GluN2B, GluN3A and GluN3B in mPFC as well as mRNA expression of GluN2C in corpus callosum were correlated to sucrose preference in rats. These findings suggested that GluN2B and GluN3 in mPFC may contribute to the pathophysiology of depression.

Abstract Background Autism spectrum disorder (ASD), like many neurodevelopmental disorders, has complex and varied etiologies. Advances in genome sequencing have identified multiple candidate genes associated with ASD, including dozens of missense and nonsense mutations in the NMDAR subunit GluN2B, encoded by GRIN2B. NMDARs are glutamate-gated ion channels with key synaptic functions in excitatory neurotransmission. How alterations in these proteins impact neurodevelopment is poorly understood, in part because knockouts of GluN2B in rodents are lethal. Methods Here, we use CRISPR-Cas9 to generate zebrafish lacking GluN2B (grin2B −/− ). Using these fish, we run an array of behavioral tests and perform whole-brain larval imaging to assay developmental roles and functions of GluN2B. Results We demonstrate that zebrafish GluN2B displays similar structural and functional properties to human GluN2B. Zebrafish lacking GluN2B (grin2B −/− ) surprisingly survive into adulthood. Given the prevalence of social deficits in ASD, we assayed social preference in the grin2B −/− fish. Wild-type fish develop a strong social preference by 3 weeks post fertilization. In contrast, grin2B −/− fish at this age exhibit significantly reduced social preference. Notably, the lack of GluN2B does not result in a broad disruption of neurodevelopment, as grin2B −/− larvae do not show alterations in spontaneous or photic-evoked movements, are capable of prey capture, and exhibit learning. Whole-brain imaging of grin2B −/− larvae revealed reduction of an inhibitory neuron marker in the subpallium, a region linked to ASD in humans, but showed that overall brain size and E/I balance in grin2B −/− is comparable to wild type. Limitations Zebrafish lacking GluN2B, while useful in studying developmental roles of GluN2B, are unlikely to model nuanced functional alterations of human missense mutations that are not complete loss of function. Additionally, detailed mammalian homologies for larval zebrafish brain subdivisions at the age of whole-brain imaging are not fully resolved. Conclusions We demonstrate that zebrafish completely lacking the GluN2B subunit of the NMDAR, unlike rodent models, are viable into adulthood. Notably, they exhibit a highly specific deficit in social behavior. As such, this zebrafish model affords a unique opportunity to study the roles of GluN2B in ASD etiologies and establish a disease-relevant in vivo model for future studies.

Introduction: We recently reported 11C-NR2B-SMe ([S-methyl-11C](R,S)-7-thiomethoxy-3-(4-(4-methyl-phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol) and its enantiomers as candidate radioligands for imaging the GluN2B subunit within rat N-methyl-D-aspartate receptors. However, these radioligands gave unexpectedly high and displaceable binding in rat cerebellum, possibly due to cross-reactivity with sigma-1 (σ1) receptors. This study investigated 11C-labeled enantiomers of a close analogue (7-methoxy-3-(4-(p-tolyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol; NR2B-Me) of 11C-NR2B-SMe as new candidate GluN2B radioligands. PET was used to evaluate these radioligands in rats and to assess potential cross-reactivity to σ1 receptors. Methods: NR2B-Me was assayed for binding affinity and selectivity to GluN2B in vitro. 11C-NR2B-Me and its enantiomers were prepared by Pd-mediated treatment of boronic ester precursors with 11C-iodomethane. Brain PET scans were conducted after radioligand intravenous injection into rats. Various ligands for GluN2B receptors or σ1 receptors were administered at set doses in pre-blocking or displacement experiments to assess their impact on imaging data. 18F-FTC146 and enantiomers of 11C-NR2B-SMe were used for comparison. Radiometabolites from brain and plasma were measured ex vivo and in vitro. Results: NR2B-Me enantiomers showed high GluN2B affinity and selectivity in vitro. 11C-NR2B-Me enantiomers gave high early whole rat brain uptake of radioactivity, including high uptake in cerebellum, followed by slower decline. Radioactivity in brain at 30 min ex vivo was virtually all unchanged radioligand. Only less lipophilic radiometabolites appeared in plasma. When 11C-(R)-NR2B-Me was used, three high-affinity GluN2B ligands—NR2B-SMe, Ro25-6981, and CO101,244—showed increasing pre-block of whole brain radioactivity retention with increasing dose. Two σ1 receptor antagonists, FTC146 and BD1407, were ineffective pre-blocking agents. Together, these results strongly resemble those obtained with 11C-NR2B-SMe enantiomers, except that 11C-NR2B-Me enantiomers showed faster reversibility of binding. When 18F-FTC146 was used as a radioligand, FTC146 and BD1407 showed strong pre-blocking effects whereas GluN2B ligands showed only weak blocking effects. Conclusion: 11C-NR2B-Me enantiomers showed specific binding to GluN2B receptors in rat brain in vivo. High unexpected specific binding in cerebellum was not due to σ1 receptors. Additional investigation is needed to identify the source of the high specific binding. Key points: Question: Do GluN2B radioligands 11C-NR2B-Me enantiomers show cross-binding with σ1 receptors in brain in vivo? Pertinent findings: 11C-(+)-NR2B-Me, 11C-(−)-NR2B-Me, 11C-(S)-NR2B-SMe, and 11C-(R)-NR2B-SMe give specific signals in whole rat brain that could be pre-blocked and displaced using ligands that specifically target GluN2B receptors, but not by σ1 ligand FTC-146. Implications for patient care: 11C-(−)-NR2B-Me does not show cross-binding with σ1 receptor in rat brain using PET imaging. This radioligand may be promising for human PET imaging. [ABSTRACT FROM AUTHOR]

N‐methyl‐D‐aspartate receptors (NMDARs) are critical for the maturation and plasticity of glutamatergic synapses. In the hippocampus, NMDARs mainly contain GluN2A and/or GluN2B regulatory subunits. The amyloid precursor protein (APP) has emerged as a putative regulator of NMDARs, but the impact of this interaction to their function is largely unknown. By combining patch‐clamp electrophysiology and molecular approaches, we unravel a dual mechanism by which APP controls GluN2B‐NMDARs, depending on the life stage. We show that APP is highly abundant specifically at the postnatal postsynapse. It interacts with GluN2B‐NMDARs, controlling its synaptic content and mediated currents, both in infant mice and primary neuronal cultures. Upon aging, the APP amyloidogenic‐derived C‐terminal fragments, rather than APP full‐length, contribute to aberrant GluN2B‐NMDAR currents. Accordingly, we found that the APP processing is increased upon aging, both in mice and human brain. Interfering with stability or production of the APP intracellular domain normalized the GluN2B‐NMDARs currents. While the first mechanism might be essential for synaptic maturation during development, the latter could contribute to age‐related synaptic impairments. [ABSTRACT FROM AUTHOR]

N-Methyl-D-aspartate receptors (NMDARs) are central for learning and information processing in the brain. Dysfunction of NMDARs can play a key role in the pathogenesis of neurodegeneration and drug addiction. The development of selective NMDAR modulators represents a promising strategy to target these diseases. Among such modulating compounds are ifenprodil and its 3-benzazepine derivatives. Classically, the effects of these NMDAR modulators have been tested by techniques like two-electrode voltage clamp (TEVC), patch clamp, or fluorescence-based assays. However, testing their functional effects in complex human systems requires more advanced approaches. Here, we established a human induced pluripotent stem cell-derived (hiPSC-derived) neural cell system and proved its eligibility as a test system for investigating NMDAR modulators and pharmaceutical effects on human neurons. [ABSTRACT FROM AUTHOR]

Glutamate receptor ionotropic NMDA 2B (GluN2B) plays an essential role in calcium overload during excitotoxicity. Reverse-phase nano-liquid chromatography-tandem mass spectrometry has revealed an interaction between GluN2B and HECT domain E3 ubiquitin protein ligase 4 (HECTD4), an E3 ubiquitin ligase highly expressed in the brain. As a potential substrate for HECTD4, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) acts as a scaffold with hydrolysis activity. This study explores the relationship between HECTD4, GluN2B, and MALT1, focusing on their role in brain injury in ischemic stroke. Rats were subjected to 2 h-ischemia followed by 24-h reperfusion to establish an ischemic stroke model. We observed the downregulation of HECTD4 and the upregulation of MALT1. Additionally, an increased GluN2B phosphorylation was concomitant with weakened interactions between HECTD4 and GluN2B, followed by decreased striatal-enriched protein phosphatase (STEP61). Knockdown of HECTD4 exacerbated hypoxia- or NMDA-induced injury in nerve cells coincident with a decrease in GluN2B and MALT1 ubiquitination, and an increase in GluN2B phosphorylation as well as an increase in intracellular calcium level, which were counteracted by MALT1 siRNA. Blockage of MALT1 with its inhibitor or siRNA reduced STEP61 degradation, accompanied by a decrease in GluN2B phosphorylation, intracellular calcium concentration, and brain cell injury, which were reversed by overexpression of MALT1. Based on these observations, we conclude that the downregulation of HECTD4 in ischemic stroke rat brain accounts for calcium overload and brain injury due to activating GluN2B directly and indirectly through a mechanism involving the reduced ubiquitination of GluN2B and MALT1, respectively. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is the most common form of neurodegenerative dementia among the elderly. Excitotoxicity has been implicated as playing a dominant role in AD, especially related to the hyperactivation of excitatory neurons. Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin-dependent kinase and involved in the pathogenesis of AD, but the roles and mechanisms of DAPK1 in excitotoxicity in AD are still uncertain. Objective: We mainly explored the underlying mechanisms of DAPK1 involved in the excitotoxicity of AD and its clinical relevance. Methods: Differentiated SH-SY5Y human neuroblastoma cells, PS1 V97 L transgenic mice, and human plasma samples were used. Protein expression was assayed by immunoblotting, and intracellular calcium and neuronal damage were analyzed by flow cytometry. Plasma DAPK1 was measured by ELISA. Results: We found that DAPK1 was activated after amyloid-β oligomers (AβOs) exposure in differentiated SH-SY5Y cells. Besides, we found the phosphorylation of GluN2B subunit at Ser1303 was increased, which contributing to excitotoxicity and Ca2+ overload in SH-SY5Y cells. Inhibiting DAPK1 activity, knockdown of DAPK1 expression, and antagonizing GluN2B subunits could effectively prevent AβOs-induced activation of GluN2B subunit, Ca2+ overload, and neuronal apoptosis. Additionally, we found that DAPK1 was elevated in the brain of AD transgenic mouse and in the plasma of AD patients. Conclusion: Our finding will help to understand the mechanism of DAPK1 in the excitotoxicity in AD and provide a reference for the diagnosis and therapy of AD. [ABSTRACT FROM AUTHOR]

Perioperative neurocognitive disorders (PND) is a common complication that occurs among elderly patients in the perioperative course. Current clinical evidence has shown that isoflurane exposure could cause cognitive decline, but the exact molecular mechanisms remain unclear. As both NMDARs‐dependent synaptic plasticity and histone acetylation play vital roles in processing learning and memory, we postulated that these alternations might occur in the isoflurane‐associated PND. Here, we found that isoflurane impaired fear memory in aged mice, decreased GluN2B‐containing NMDA receptors phosphorylation and trafficking, as well as the expression of EphB2, a key regulator of synaptic localization of NMDA receptors. We also identified that isoflurane could increase the expression of HDAC2, which was significantly enriched at the ephb2 gene promoter and regulated the transcription of ephb2. Furthermore, we showed that suberoylanilide hydroxamic acid (SAHA), a nonselective HDAC inhibitor or knocking‐down HDAC2 rescued the cognitive dysfunction in isoflurane‐treated aged mice via increasing acetylation of H3Ac, expression of EphB2 and promoting NMDA receptor trafficking. Collectively, our study highlighted the crucial role of histone posttranslational modifications for EphB2‐GluN2B signals in isoflurane‐associated PND, and modulating HDAC2 might be a new therapeutic strategy for isoflurane‐associated PND. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptors, are important in regulating sympathetic tone and cardiovascular function in the rostral ventrolateral medulla (RVLM). Amyloid-beta peptide (Aβ) is linked to the pathogenesis of Alzheimer’s disease (AD). Cerebro- and cardiovascular diseases might be the risk factors for developing AD. The present study examines the acute effects of soluble Aβ on the function of NMDA receptors in rats RVLM. We used the magnitude of increases in the blood pressure (pressor responses) induced by microinjection of NMDA into the RVLM as an index of NMDA receptor function in the RVLM. Soluble Aβ was applied by intracerebroventricular (ICV) injection. Aβ1-40 at a lower dose (0.2 nmol) caused a slight reduction, and a higher dose (2 nmol) showed a significant decrease in NMDA-induced pressor responses 10 min after administration. ICV injection of Aβ1-42 (2 nmol) did not affect NMDA-induced pressor responses in the RVLM. Co-administration of Aβ1-40 with ifenprodil or memantine blocked the inhibitory effects of Aβ1-40. Immunohistochemistry analysis showed a significant increase in the immunoreactivity of phosphoserine 1480 of GluN2B subunits (pGluN2B-serine1480) in the neuron of the RVLM without significant changes in phosphoserine 896 of GluN1 subunits (pGluN1-serine896), GluN1 and GluN2B, 10 min following Aβ1-40 administration compared with saline. Interestingly, we found a much higher level of Aβ1-40 compared to that of Aβ1-42 in the cerebrospinal fluid (CSF) measured using enzyme-linked immunosorbent assay 10 min following ICV administration of the same dose (2 nmol) of the peptides. In conclusion, the results suggest that ICV Aβ1-40, but not Aβ1-42, produced an inhibitory effect on NMDA receptor function in the RVLM, which might result from changes in pGluN2B-serine1480 (regulated by casein kinase II). The different elimination of the peptides in the CSF might contribute to the differential effects of Aβ1-40 and Aβ1-42 on NMDA receptor function.

In mature neurons, postsynaptic N-methyl-D-aspartate receptors (NMDARs) are segregated into two populations, synaptic and extrasynaptic, which differ in localization, function, and associated intracellular cascades. These two pools are connected via lateral diffusion, and receptor exchange between them modulates synaptic NMDAR content. Here, we identify the phosphorylation of the PDZ-ligand of the GluN2B subunit of NMDARs (at S1480) as a critical determinant in dynamically controlling NMDAR synaptic content. We find that phosphorylation of GluN2B at S1480 maintains NMDARs at extrasynaptic membranes as part of a protein complex containing protein phosphatase 1 (PP1). Global activation of NMDARs leads to the activation of PP1, which mediates dephosphorylation of GluN2B at S1480 to promote an increase in synaptic NMDAR content. Thus, PP1-mediated dephosphorylation of the GluN2B PDZ-ligand modulates the synaptic expression of NMDARs in mature neurons in an activity-dependent manner, a process with profound consequences for synaptic and structural plasticity, metaplasticity, and synaptic neurotransmission.

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in humans. Cognitive impairment and memory consolidation problems are common among TLE patients. To understand the changes in the cellular process of memory in TLE, we studied the long-term depression (LTD) in Schaffer-collateral (Sc) CA1 synapses in an epilepsy model. Long-term potentiation (LTP) was investigated in patient samples and animal models by several groups, but LTD was not studied with the same interest in epilepsy research. Here we induced epileptiform activity in rat hippocampal slices using magnesium-free high-potassium (7.5 mM K +) artificial cerebrospinal fluid (HK-ACSF) and characterized the LTD in Sc-CA1 synapses. We found that epileptiform activity abolished/impaired LTD and depotentiation in the Sc-CA1 synapses. In control slices, application of NMDA (30 μM for 3 min) induced chemical LTD (c-LTD) in Sc-CA1 synapses, whereas epileptiform activity induced slices showed slow onset potentiation. Induction of LTD using 1 Hz, 900 pulses yielded a similar outcome as c-LTD. Both forms of LTD were NMDA receptor dependent. In addition, we found that the polarity changes in the synaptic plasticity in epileptiform-induced slices were blocked by GluN2B antagonists ifenprodil and Ro 25–6981. Our data suggest that epileptiform-induced metaplasticity inhibits LTD in Sc-CA1 synapses. We provide new insight into the cellular mechanism of memory formation during epilepsy. [ABSTRACT FROM AUTHOR]

Learning and memory are thought to require the induction and maintenance of long-term potentiation (LTP) of synaptic strength. LTP induction requires the Ca2+/calmodulin-dependent protein kinase II (CaMKII) but for structural rather than enzymatic functions. We show that the relevant structural function is regulated by CaMKII binding to the NMDA-type glutamate receptor subunit GluN2B. This binding directly generates Ca2+-independent autonomous CaMKII activity, and we show that this enzymatic activity is dispensable for LTP induction (within 5 min) but required for a subsequent LTP phase (within 15 min). This requirement for CaMKII activity provides an objective temporal definition for the intermediary phase of LTP expression. Later LTP maintenance may still require structural functions of GluN2B-bound CaMKII but not the resulting enzymatic CaMKII activity or their co-condensation. Thus, autonomous CaMKII activity mediates post-induction LTP but (1) via GluN2B binding, not T286 autophosphorylation, and (2) during the intermediary expression phase rather than for long-term maintenance. [Display omitted] • GluN2B binding is the structural CaMKII function required for LTP induction • Autonomous activity of GluN2B-bound CaMKII mediates LTP expression • Enzymatic CaMKII activity is not required for LTP maintenance • CaMKII pT286 is not required for LTP expression or maintenance Rumian et al. demonstrate that enzymatic activity of CaMKII is not required for LTP induction or maintenance but is required for the intermediary phase of LTP expression. CaMKII pT286 was required only during LTP induction, whereas LTP expression was instead mediated by the autonomous activity of GluN2B-bound CaMKII. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancer. Evidence suggests that the ionotropic glutamate receptor N-methyl-D-aspartate (NMDA) receptor, a critical molecule in the central nervous system, is expressed in NSCLC. However, the specific expression patterns, subcellular localization, functional modulation, and pathological implications of NMDA receptor subtypes in NSCLC have not been fully elucidated. In this study, we employed a multi-disciplinary approach, combining biochemical and molecular biology with electrophysiological recordings and behavioral assays, to investigate these aspects. We reveal the expression of GluN2B-containing NMDA receptors in A549 and H460 NSCLC cell lines and the induction of NMDA receptor-mediated currents by glutamate in A549 cells. Furthermore, the GluN2B-specific inhibitors ifenprodil and Ro 25–6981 significantly reduced cell viability and migration, while promoting apoptosis. Importantly, intraperitoneal administration of ifenprodil in nude mice inhibited the growth of subcutaneous tumors derived from A549 and H460 cells and ameliorated depression-like behaviors. These findings underscore the potential antiproliferative effects of ifenprodil and Ro 25–6981 and suggest that GluN2B-containing NMDA receptors may represent novel therapeutic targets for NSCLC, with the added benefit of potential antidepressant action. • GluN2B-containing NMDA receptors are upregulated in non-small cell lung cancer. • GluN2B antagonists reduce the malignant potential of A549 and H460 cancer cells. • Ifenprodil inhibits subcutaneous tumor growth and exerts antidepressant effects. • NMDA receptors represent a promising therapeutic target for cancer treatment strategies. [ABSTRACT FROM AUTHOR]

Background: Autism spectrum disorder (ASD), like many neurodevelopmental disorders, has complex and varied etiologies. Advances in genome sequencing have identified multiple candidate genes associated with ASD, including dozens of missense and nonsense mutations in the NMDAR subunit GluN2B, encoded by GRIN2B. NMDARs are glutamate-gated ion channels with key synaptic functions in excitatory neurotransmission. How alterations in these proteins impact neurodevelopment is poorly understood, in part because knockouts of GluN2B in rodents are lethal. Methods: Here, we use CRISPR-Cas9 to generate zebrafish lacking GluN2B (grin2B−/−). Using these fish, we run an array of behavioral tests and perform whole-brain larval imaging to assay developmental roles and functions of GluN2B. Results: We demonstrate that zebrafish GluN2B displays similar structural and functional properties to human GluN2B. Zebrafish lacking GluN2B (grin2B−/−) surprisingly survive into adulthood. Given the prevalence of social deficits in ASD, we assayed social preference in the grin2B−/− fish. Wild-type fish develop a strong social preference by 3 weeks post fertilization. In contrast, grin2B−/− fish at this age exhibit significantly reduced social preference. Notably, the lack of GluN2B does not result in a broad disruption of neurodevelopment, as grin2B−/− larvae do not show alterations in spontaneous or photic-evoked movements, are capable of prey capture, and exhibit learning. Whole-brain imaging of grin2B−/− larvae revealed reduction of an inhibitory neuron marker in the subpallium, a region linked to ASD in humans, but showed that overall brain size and E/I balance in grin2B−/− is comparable to wild type. Limitations: Zebrafish lacking GluN2B, while useful in studying developmental roles of GluN2B, are unlikely to model nuanced functional alterations of human missense mutations that are not complete loss of function. Additionally, detailed mammalian homologies for larval zebrafish brain subdivisions at the age of whole-brain imaging are not fully resolved. Conclusions: We demonstrate that zebrafish completely lacking the GluN2B subunit of the NMDAR, unlike rodent models, are viable into adulthood. Notably, they exhibit a highly specific deficit in social behavior. As such, this zebrafish model affords a unique opportunity to study the roles of GluN2B in ASD etiologies and establish a disease-relevant in vivo model for future studies. [ABSTRACT FROM AUTHOR]

Here, we describe the characterization of a radioligand selective for GluN2B-containing NMDA receptors, 3-[ 3 H] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-1-yl)ethanone ([ 3 H]-JNJ- GluN2B-5). In rat cortical membranes, the compound bound to a single site, and the following kinetic parameters were measured; association rate constant K on  = 0.0066 ± 0.0006 min -1  nM -1 , dissociation rate constant K off  = 0.0210 ± 0.0001 min -1 indicating calculated K D  = K off /K on  = 3.3 ± 0.4 nM, (mean ± SEM, n = 3). The equilibrium dissociation constant determined from saturation binding experiments in rat cortex was K D of 2.6 ± 0.3 nM (mean ± SEM, n = 3). In contrast to the widely used GluN2B radioligand [ 3 H]-Ro 25-6981, whose affinity K i for sigma 1 and sigma 2 receptors are 2 and 189 nM, respectively, [ 3 H]-JNJ-GluN2B-5 exhibits no measurable affinity for sigma 1 and sigma 2 receptors (K i  > 10 μM for both) providing distinct selectivity advantages. Anatomical distribution of [ 3 H]-JNJ-GluN2B-5 binding sites in rat, mouse, dog, monkey, and human brain tissue was studied using in vitro autoradiography, which showed high specific binding in the hippocampus and cortex and negligible binding in the cerebellum. Enhanced selectivity for GluN2B-containing receptors translated to a good signal-to-noise ratio in both in vitro radioligand binding and in vitro autoradiography assays. In conclusion, [ 3 H]-JNJ-GluN2B-5 is a high-affinity GluN2B radioligand with excellent signal-to-noise ratio and unprecedented selectivity., (© 2024 International Society for Neurochemistry.)

The number of N-Methyl-D-aspartate receptor (NMDAR) linked neurodegenerative diseases such as Alzheimer’s disease and dementia is constantly increasing. This is partly due to demographic change and presents new challenges to societies. To date, there are no effective treatment options. Current medications are nonselective and can lead to unwanted side effects in patients. A promising therapeutic approach is the targeted inhibition of NMDARs in the brain. NMDARs containing different subunits and splice variants display different physiological properties and play a crucial role in learning and memory, as well as in inflammatory or injury processes. They become overactivated during the course of the disease, leading to nerve cell death. Until now, there has been a lack of understanding of the general functions of the receptor and the mechanism of inhibition, which need to be understood in order to develop inhibitors. Ideal compounds should be highly targeted and even splice-variant-selective. However, a potent and splice-variant-selective NMDAR-targeting drug has yet to be developed. Recently developed 3-benzazepines are promising inhibitors for further drug development. The NMDAR splice variants GluN1-1b-4b carry a 21-amino-acid-long, flexible exon 5. Exon 5 lowers the NMDAR’s sensitivity to allosteric modulators by probably acting as an NMDAR modulator itself. The role of exon 5 in NMDAR modulation is still poorly understood. In this review, we summarize the structure and pharmacological relevance of tetrahydro-3-benzazepines.

Rationale: Binge drinking during adolescence impairs learning and memory on the long term, and many studies suggest a role of neuroinflammation. However, whether neuroinflammation occurs after the very first exposures to alcohol remains unclear, while initial alcohol exposure impairs learning for several days in male rats. Objectives: To investigate the role of neuroinflammation in the effects of only two binge-like episodes on learning and on neuronal plasticity in adolescent male rat hippocampus. Methods: Animals received two ethanol i.p. injections (3 g/kg) 9 h apart. Forty-eight hours later, we recorded long-term depression (LTD) and potentiation (LTP) in CA1 area of hippocampus slices. In isolated CA1, we measured immunolabelings for microglial activation and Toll-like receptor 4 (TLR4) and mRNA levels for several cytokines. Results: Forty-eight hours after the two binges, rats performed worse than control rats in novel object recognition, LTD was reduced, LTP was increased, and excitatory neurotransmission was more sensitive to an antagonist of the GluN2B subunit of the NMDA receptor. Exposure to ethanol with minocycline or indomethacin, two anti-inflammatory drugs, or with a TLR4 antagonist, prevented all effects of ethanol. Immunolabelings at 48 h showed a reduction of neuronal TLR4 that was prevented by minocycline pretreatment, while microglial reactivity was undetected and inflammatory cytokines mRNA levels were unchanged. Conclusion: Two binge-like ethanol exposures during adolescence in rat involved neuroinflammation leading to changes in TLR4 expression and in GluN2B functioning inducing disturbances in synaptic plasticity and cognitive deficits. Anti-inflammatory drugs are good candidates to prevent brain function and memory deficits induced by few binge-drinking episodes. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a neurodevelopmental condition with core clinical features of abnormal communication, social interactions, atypical intelligence, and a higher risk of epilepsy. Prior work has suggested that de novo heterozygous mutations in the GRIN2B gene that encodes the GluN2B subunit of N-methyl-d-aspartic acid receptors are likely linked to ASD. However, whether GLuN2B-Trp373 mutation derived from autistic individuals causes ASD-like behavioral aberrations in rats remains to be determined. Here, through in utero electroporation and in vivo studies, we conducted a battery of tests to examine ASD-associated behaviors, cognitive impairments, and susceptibility to pentylenetetrazol-induced seizures. Whole-cell patch recording was utilized to determine whether the GluN2B-Trp373 mutation influences GluN2B-containing NMDA receptor currents in rats. Results show that, behaviorally, GLuN2B-Trp373 mutant rats exhibited core behavioral manifestations of ASD, such as social interaction deficits, increases in stereotyped behaviors and anxiety stereotyped/repetitive, impaired spatial memory, and enhanced risk of pentylenetetrazol-induced seizures, consistent with many of the hallmarks of low-functioning ASD in humans. Functionally, the GluN2B-Trp373 mutation results in reduced GluN2B surface protein expression together with decreased hippocampal NMDA receptor currents. Collectively, our findings highlight that GluN2B-Trp373 mutations can drive the manifestation of ASD-associated symptoms via the suppression of NMDA receptor currents. [ABSTRACT FROM AUTHOR]

The death-associated protein kinase 1 (DAPK1) is a potent mediator of neuronal cell death. Here, we find that DAPK1 also functions in synaptic plasticity by regulating the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII). CaMKII and T286 autophosphorylation are required for both long-term potentiation (LTP) and depression (LTD), two opposing forms of synaptic plasticity underlying learning, memory, and cognition. T286-autophosphorylation induces CaMKII binding to the NMDA receptor (NMDAR) subunit GluN2B, which mediates CaMKII synaptic accumulation during LTP. We find that the LTP specificity of CaMKII synaptic accumulation is due to its LTD-specific suppression by calcineurin (CaN)-dependent DAPK1 activation, which in turn blocks CaMKII binding to GluN2B. This suppression is enabled by competitive DAPK1 versus CaMKII binding to GluN2B. Negative regulation of DAPK1/GluN2B binding by Ca2+/CaM results in synaptic DAPK1 removal during LTP but retention during LTD. A pharmacogenetic approach showed that suppression of CaMKII/GluN2B binding is a DAPK1 function required for LTD.

A high rate of heavy tobacco smoking among people with schizophrenia has been suggested to reflect self-medication and amelioration of cognitive dysfunction, a core feature of schizophrenia. NMDAR hypofunction is hypothesized to be a mechanism of cognitive dysfunction, and excessive schizophrenia-linked neuregulin 1 (NRG1) signaling through its receptor ErbB4 can suppress NMDAR function by preventing Src-mediated enhancement of NMDAR responses. Here we investigated whether chronic nicotine exposure in rats by subcutaneous injection of nicotine (0.5-1 mg/kg, twice daily for 10-15 days) counteracts the suppressive effect of NRG1β on NMDAR-mediated responses recorded from CA1 pyramidal cells in acute hippocampal slices. We found that NRG1β, which prevents the enhancement of NMDAR responses by the Src-family-kinase-activating peptide pYEEI in naive rats, failed to block the effect of pYEEI in nicotine-exposed rats. In naive rats, NRG1β acts only on GluN2B-NMDARs by blocking their Src-mediated upregulation. Chronic nicotine exposure causes enhanced GluN2B-NMDAR responses via Src upregulation and recruits Fyn for the enhancement of GluN2A-NMDAR responses. NRG1β has no effect on both enhanced basal GluN2B-NMDAR responses and Fyn-mediated enhancement of GluN2A-NMDAR responses. Src-mediated enhancement of GluN2B-NMDAR responses and Fyn-mediated enhancement of GluN2A-NMDAR responses initiate long-term potentiation (LTP) of AMPAR synaptic responses in naive and nicotine-exposed CA1 pyramidal cells, respectively. These results suggest that NRG1β suppresses LTP by blocking Src-mediated enhancement of GluN2B-NMDAR responses, but has no effect on LTP in nicotine-exposed rats. These effects of chronic nicotine exposure may counteract the negative effect of increased NRG1-ErbB4 signaling on the cellular mechanisms of learning and memory in individuals with schizophrenia, and therefore may motivate heavy smoking.

The potential therapeutic targeting of PINK1-PARK2-mediated mitophagy against cerebral ischemia/reperfusion (CI/R) injury involves the pathophysiological processes of neurovascular unit (NVU) and is closely associated with N-methyl-D-aspartate receptors (NMDARs) commonly expressed in NVU. 2,3,5,4′-Tetrahydroxy-stilbene-2- O -β-D-glucoside (THSG), a compound derived from the traditional Chinese medicine Polygonum multiflorum Thunb., has demonstrated notable neuroprotective properties against CI/R injury. However, it remains unclear whether THSG exerts its protective effects through GluN2B related PINK1/ PARK2 pathway. This study aims to explore the pharmacological effects of THSG on alleviating CI/R injury via the GluN2B–CaMKII–ERK1/2 pathway. THSG neuroprotection against CI/R injury was studied in transient middle cerebral artery occlusion/reversion (tMCAO/R) model rats and in oxygen and glucose deprivation/ reoxygenation (OGD/R) induced neurons. PINK1–PARK2-mediated mitophagy involvement in the protective effect of THSG was investigated in tMCAO/R rats and OGD/R-induced neurons via THSG and 3-methyladenine (3-MA) treatment. Furthermore, the beneficial role of GluN2B in reperfusion and its contribution to the THSG effect via CaMKII–ERK1/2 and PINK1–PARK2-mediated mitophagy was explored using the GluN2B-selective antagonist Ro 25–6981 both in vivo and in vitro. Finally, the interaction between THSG and GluN2B was evaluated using molecular docking. THSG significantly reduced infarct volume, neurological deficits, penumbral neuron structure, and functional damage, upregulated the inhibitory apoptotic marker Bcl-2, and suppressed the increase of pro-apoptotic proteins including cleaved caspase-3 and Bax in tMCAO/R rats. THSG (1 μM) markedly improved the neuronal survival under OGD/R conditions. Furthermore, THSG promoted PINK1 and PARK2 expression and increased mitophagosome numbers and LC3-II–LC3-I ratio both in vivo and in vitro. The effects of THSG were considerably abrogated by the mitophagy inhibitor 3-MA in OGD/R-induced neurons. Inhibiting GluN2B profoundly decreased mitophagosome numbers and OGD/R-induced neuronal viability. Specifically, inhibiting GluN2B abolished the protection of THSG against CI/R injury and reversed the upregulation of PINK1–PARK2-mediated mitophagy by THSG. Inhibiting GluN2B eliminated THSG upregulation of ERK1/2 and CaMKII phosphorylation. The molecular docking analysis results demonstrated that THSG bound to GluN2B (binding energy: –5.2 ± 0.11 kcal/mol). This study validates the premise that THSG alleviates CI/R injury by promoting GluN2B expression, activating CaMKII and ERK1/2, and subsequently enhancing PINK1–PARK2-mediated mitophagy. This work enlightens the potential of THSG as a promising candidate for novel therapeutic strategies for treating ischemic stroke. [Display omitted] [ABSTRACT FROM AUTHOR]

Ablation of adult neurogenesis in mice has revealed that young adult-born granule cells (abGCs) are required for some of the behavioral responses to antidepressants (ADs), yet the mechanism by which abGCs contribute to AD action remains unknown. During their maturation process, these immature neurons exhibit unique properties that could underlie their ability to influence behavioral output. In particular, abGCs in the DG exhibit a period of heightened plasticity 4-6 weeks after birth that is mediated by GluN2B-expressing NMDA receptors. The functional contribution of this critical window to AD responsiveness is unclear. Here, we determined the behavioral and neurogenic responses to the AD fluoxetine (FLX) in mice lacking GluN2B-containing NMDA receptors in abGCs. We found that these mice exhibited an attenuated response to FLX in a neurogenesis-dependent behavioral assay of FLX action, while neurogenesis-independent behaviors were unaffected by GluN2B deletion. In addition, deletion of GluN2B attenuated FLX-induced increases in dendritic complexity of abGCs suggesting that the blunted behavioral efficacy of FLX may be caused by impaired differentiation of young abGCs.

• Mechanical allodynia was ameliorated by inhibition of IL-33/ST2 signaling. • IL-33 caused the phosphorylation of synaptosomal GluN2B and potentiated Vc GluN2B-mediated mEPSCs. • IL-33-mediated Fyn, and subsequent GluN2B phosphorylation, induced mechanical allodynia. Orofacial neuropathic pain can cause considerable disruptions in patients' daily lives, especially because of a lack of effective medications as its underlying causative mechanisms are not fully understood. Here, we found neuron-specific expression of the interleukin (IL)-33 receptor in the trigeminal spinal subnucleus caudalis (Vc), distinct from the spinal dorsal horn. Reduction in head withdrawal threshold in response to von Frey filament stimulation of the whisker pad skin was inversely correlated with the upregulation of IL-33 in the Vc after infraorbital nerve injury (IONI). Neutralization of IL-33 in the Vc alleviated mechanical allodynia in the whisker pad skin after IONI; conversely, intracisternal administration of IL-33 elicited mechanical allodynia in the whisker pad skin, which was relieved by GluN2B antagonism. Moreover, IL-33 triggered the potentiation of GluN2B-containing N -methyl-D-aspartate receptor-mediated synaptic currents and phosphorylation of synaptosomal GluN2B in the Vc, whereas IONI-induced GluN2B phosphorylation was inhibited by neutralization of IL-33 in the Vc. IL-33-induced GluN2B phosphorylation was mediated by phosphorylation of Fyn kinase, and inhibition of the Fyn kinase pathway prevented the development of IL-33-induced mechanical allodynia. Our findings provide insights into a new mechanism by which IL-33 directly regulates synaptic transmission and suggest that IL-33 signaling could be a candidate target for therapeutic interventions for orofacial neuropathic pain. [ABSTRACT FROM AUTHOR]

Gamma-L-glutamyl-L-glutamate (γ-Glu-Glu) was synthetized and further characterized for its activity on cultured neurons. We observed that γ-Glu-Glu elicited excitatory effects on neurons likely by activating mainly the N-methyl-D-aspartate (NMDA) receptors. These effects were dependent on the integrity of synaptic transmission as they were blocked by tetrodotoxin (TTX). We next evaluated its activity on NMDA receptors by testing it on cells expressing these receptors. We observed that γ-Glu-Glu partially activated NMDA receptors and exhibited better efficacy for NMDA receptors containing the GluN2B subunit. Moreover, at low concentration, γ-Glu-Glu potentiated the responses of glutamate on NMDA receptors. Finally, the endogenous production of γ-Glu-Glu was measured by LC-MS on the extracellular medium of C6 rat astroglioma cells. We found that extracellular γ-Glu-Glu concentration was, to some extent, directly linked to GSH metabolism as γ-Glu-Glu can be a by-product of glutathione (GSH) breakdown after γ-glutamyl transferase action. Therefore, γ-Glu-Glu could exert excitatory effects by activating neuronal NMDA receptors when GSH production is enhanced. [ABSTRACT FROM AUTHOR]

N-Methyl-D-aspartate receptor (NMDAR) signaling pathway has been implicated in the pathogenesis and treatment of depression. However, the role of NMDAR subunits in depression is still unclear. In this study, alteration in all seven NMDAR subunits in several brain areas of rats exposed to chronic unpredictable mild stress (CUMS), an animal model of depression, was detected. Our findings demonstrated that: (1) CUMS could induce a reduction in sucrose preference, an indicator of typical depression-like behaviors; (2) CUMS significantly reduced the NMDAR subunits of GluN2B and GluN3 in the medial prefrontal cortex (mPFC), but not altered all seven NMDAR subunits in hippocampus and corpus callosum of rats; (3) subunit composition of NMDARs in corpus callosum was different from that in mPFC, PFC and hippocampus; and (4) the mRNA expressions of GluN2B, GluN3A and GluN3B in mPFC as well as mRNA expression of GluN2C in corpus callosum were correlated to sucrose preference in rats. These findings suggested that GluN2B and GluN3 in mPFC may contribute to the pathophysiology of depression. [ABSTRACT FROM AUTHOR]