GluN2B Subunit Of N-methyl-d-aspartate Receptor Research Articles

Dynamic regulation of phosphorylation of NMDA receptor GluN2B subunit tyrosine residues mediates ketamine rapid antidepressant effects

The rapid antidepressant effects of ketamine depend on the N-methyl-D-aspartate (NMDA) receptor containing 2B subunit (NR2B), whose function is influenced by its phosphorylated regulation and distribution within and outside synapses. It remains unclear if ketamine’s rapid onset of antidepressant effects relies on the dynamic phosphorylated regulation of NR2B within and outside synapses. Here, we show that ketamine rapidlyalleviated depression-like behaviors and normalized abnormal expression of pTyr1472NR2B and striatal-enriched protein tyrosine phosphatase (STEP) 61 within and outside synapses in the medial prefrontal cortex (mPFC) induced by chronic unpredictable stress (CUS) and conditional knockdown of STEP 61, a key phosphatase of NR2B, within 1 hour after administration Together, our results delineate the rapid initiation of ketamine’s antidepressant effects results from the restoration of NR2B phosphorylation homeostasis within and outside synapses. The dynamic regulation of phosphorylation of NR2B provides a new perspective for developing new antidepressant strategies.

Activation of the N-methyl-D-aspartate receptor and calcium/calmodulin-dependent protein kinase IIα signal in the rostral anterior cingulate cortex is involved in pain-related aversion in rats with peripheral nerve injury

The rostral anterior cingulate cortex (rACC) of rat brain is associated with pain-related emotions. However, the underlying molecular mechanism remains unclear. Here, we investigated the effects of the N-methyl-D-aspartate (NMDA) receptor and Ca2+/Calmodulin-dependent protein kinase type II (CaMKII)α signal on pain-related aversion in the rACC of a rat model of neuropathic pain (NP). Mechanical and thermal hyperalgesia were examined using von Frey and hot plate tests in a rat model of NP induced by spared nerve injury (SNI) of the unilateral sciatic nerve. Bilateral rACC pretreatment with the CaMKII inhibitor tat-CN21 (derived from the cell-penetrating tat sequence and CaM-KIIN amino acids 43–63) or tat-Ctrl (the tat sequence and the scrambled sequence of CN21) was performed on postoperative days 29–35 in Sham rats or rats with SNI. Spatial memory performance was tested using an eight-arm radial maze on postoperative days 34–35. Pain-related negative emotions (aversions) were evaluated using the place escape/avoidance paradigm on postoperative day 35 following the spatial memory performance test. The percentage of time spent in the light area was used to assess pain-related negative emotions (i.e., aversion). The expression levels of the NMDA receptor GluN2B subunit, CaMKIIα, and CaMKII-Threonine at position 286 (Thr286) phosphorylation in contralateral rACC specimens were detected by Western blot or real time PCR following the aversion test. Our data showed that pretreatment of the rACC with tat-CN21 increased determinate behavior but did not alter hyperalgesia or spatial memory performance in rats with SNI. In addition, tat-CN21 reversed the enhanced CaMKII-Thr286 phosphorylation and had no effect on the upregulated expression of GluN2B, CaMKIIα protein, and mRNA. Our data suggested that activation of the NMDA receptor-CaMKIIα signal in rACC is associated with pain-related aversion in rats with NP. These data may provide a new approach for the development of drugs that modulate cognitive and emotional pain aspects.

GluN2B subunit selective N-methyl-D-aspartate receptor ligands: Democratizing recent progress to assist the development of novel neurotherapeutics.

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.

The Apelin/APJ system modulates seizure activity and endocytosis of the NMDA receptor GluN2B subunit

In the central nervous system (CNS), the apelin/APJ system is broadly expressed. According to some studies, activation of this system protects against excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors and exerts neuroprotective effects. However, the role of this system in epilepsy remains unclear. In the present study, immunofluorescence staining and western blotting were used to assess APJ localization and expression in the brains of mice with recurrent spontaneous seizures induced by kainic acid (KA). Behavior and local field potentials (LFPs) were assessed in mice with KA-induced seizures. Susceptibility to seizures was assessed in a pentylenetetrazole (PTZ)-induced seizure model. Whole-cell patch-clamp recordings were used to evaluate the role of the apelin/APJ system in regulating synaptic transmission in brain slices from mice in which Mg2+-free medium was used to induce seizures. NMDA receptor GluN2B subunit expression and phosphorylation of GluN2B at Ser1480 were measured in the mouse hippocampus. APJ was primarily localized in neurons, and its expression was upregulated in the epileptic brain. APJ activation after KA-induced status epilepticus (SE) reduced epileptic activity, whereas APJ inhibition aggravated epileptic activity. In the PTZ model, APJ activation reduced and APJ inhibition increased susceptibility to seizures. The apelin/APJ system affected NMDA receptor-mediated postsynaptic currents in patch-clamp recordings. Moreover, APJ regulated the levels of GluN2B phosphorylated at Ser1480 and the abundance of cell-surface GluN2B in neurons. Furthermore, endocytosis of the NMDA receptor GluN2B subunit was regulated by the apelin/APJ system. Together, our findings indicate that the apelin/APJ system modulates seizure activity and may be a novel therapeutic target for epilepsy.

Positive modulation of N-methyl-D-aspartate receptors in the mPFC reduces the spontaneous recovery of fear

N-methyl-D-aspartate receptor (NMDAR) modulators have recently received increased attention as potential therapeutics for posttraumatic stress disorder (PTSD). Here, we tested a novel NMDAR-positive modulator, NYX-783, in the following two rodent models of PTSD: an auditory fear-conditioning model and a single-prolonged stress (SPS) model. We examined the ability of NYX-783 to reduce subsequent fear-based behaviors by measuring enhanced fear extinction and reduced spontaneous recovery (spontaneous return of fear) in male mice. NYX-783 administration significantly reduced spontaneous recovery in both PTSD models and enhanced fear extinction in the SPS model. Furthermore, NYX-783 increased the NMDA-induced inward currents of excitatory and inhibitory neurons in the infralimbic medial prefrontal cortex (IL mPFC) and that the GluN2B subunit of NMDARs on pyramidal neurons in the IL mPFC is required for its effect on spontaneous recovery. The downstream expression of brain-derived neurotrophic factor was required for NYX-783 to achieve its behavioral effect. These results elucidate the cellular targets of NYX-783 and the molecular mechanisms underlying the inhibition of spontaneous recovery. These preclinical findings support the hypothesis that NYX-783 may have therapeutic potential for PTSD treatment and may be particularly useful for inhibiting spontaneous recovery.

Two-Chains Tissue Plasminogen Activator Unifies Met and NMDA Receptor Signalling to Control Neuronal Survival.

Tissue-type plasminogen activator (tPA) plays roles in the development and the plasticity of the nervous system. Here, we demonstrate in neurons, that by opposition to the single chain form (sc-tPA), the two-chains form of tPA (tc-tPA) activates the MET receptor, leading to the recruitment of N-Methyl-d-Aspartate receptors (NMDARs) and to the endocytosis and proteasome-dependent degradation of NMDARs containing the GluN2B subunit. Accordingly, tc-tPA down-regulated GluN2B-NMDAR-driven signalling, a process prevented by blockers of HGFR/MET and mimicked by its agonists, leading to a modulation of neuronal death. Thus, our present study unmasks a new mechanism of action of tPA, with its two-chains form mediating a crosstalk between MET and the GluN2B subunit of NMDARs to control neuronal survival.

Enhancement of the GluN2B subunit of glutamatergic NMDA receptors in rat brain areas after cocaine abstinence.

Cocaine use disorder is associated with compulsive drug-seeking and drug-taking, whereas relapse may be induced by several factors, including stress, drug-related places, people, and cues. Recent observations strongly support the involvement of the N-methyl-D-aspartate (NMDA) receptors in cocaine use disorders and abstinence, whereas withdrawal in different environments may affect the intensification of relapse. The aim of this study was to examine the GluN2B subunit expression and its association with the postsynaptic density protein 95 (PSD95) in several brain structures in rats with a history of cocaine self-administration and housed either in an enriched environment or in an isolated condition. Furthermore, a selective antagonist of the GluN2B subunit-CP 101,606 (10 and 20 mg/kg) administered during exposure to cocaine or a drug-associated conditional stimulus (a cue) was used to evaluate seeking behavior in rats. In rats previously self-administering cocaine, we observed an increase in the GluN2B expression in the total homogenate from the dorsal hippocampus under both enriched environment and isolation. Cocaine abstinence under isolation conditions increased the GluN2B and GluN2B/PSD95 complex levels in the PSD fraction of the prelimbic cortex in rats previously self-administering cocaine. Administration of CP 101,606 attenuated cue-induced cocaine-seeking behavior only in isolation-housed rats. In summary, in this study we showed region-specific changes in both the expression of GluN2B subunit and NMDA receptor trafficking during cocaine abstinence under different housing conditions. Furthermore, we showed that the pharmacological blockade of the GluN2B subunit may be useful in attenuating cocaine-seeking behavior.

Perspectives for Treating Alzheimer’s Disease: A Review on Indigeous Plants for Alzheimer Disease

Alzheimer’s disease (AD) is one of the most common forms of dementia. The salient pathological hallmarks of AD are extracellular accumulation of amyloid β peptide (Aβ) forming senile plaques (SP), the aggregates of intracellular neurofibrillary tangles (NFTs) and microglial changes in the brain. N-Methyl-D-aspartate receptor (NMDAR), a member of ionotropic glutamate receptor (iGluR), plays an indispensable mantle in maintaining calcium homeostasis and synaptic plasticity. Activation of NMDAR requires co-binding of glycine and glutamate along with postsynaptic depolarization. Upon activation it facilitates the entry of calcium ion into the neurons. The GluN2B subunit of NMDAR has been involved into the direct interaction with Aβ and tau protein and also in the activation of microglia, thus leading to pathophysiology of AD. N-methyl-d-aspartate receptors (NMDARs) mediated excitotoxicity has been implicated in multi-neurodegenerative diseases. Owing to dearth of efficacy and adverse effects of NMDA receptor antagonists, search for herbal remedies acting like salutary agents is a dynamic expanse of investigation to contest neurodegenerative disease. In this review we have delved deep into different NMDA antagonist and their pivotal role in preventing Aβ-mediated synaptic plasticity and their potential role as therapeutic target to curtail AD.

A Review of Molecular Imaging of Glutamate Receptors.

Molecular imaging with positron emission tomography (PET) and single photon emission computed tomography (SPECT) is a well-established and important in vivo technique to evaluate fundamental biological processes and unravel the role of neurotransmitter receptors in various neuropsychiatric disorders. Specific ligands are available for PET/SPECT studies of dopamine, serotonin, and opiate receptors, but corresponding development of radiotracers for receptors of glutamate, the main excitatory neurotransmitter in mammalian brain, has lagged behind. This state of affairs has persisted despite the central importance of glutamate neurotransmission in brain physiology and in disorders such as stroke, epilepsy, schizophrenia, and neurodegenerative diseases. Recent years have seen extensive efforts to develop useful ligands for molecular imaging of subtypes of the ionotropic (N-methyl-D-aspartate (NMDA), kainate, and AMPA/quisqualate receptors) and metabotropic glutamate receptors (types I, II, and III mGluRs). We now review the state of development of radioligands for glutamate receptor imaging, placing main emphasis on the suitability of available ligands for reliable in vivo applications. We give a brief account of the radiosynthetic approach for selected molecules. In general, with the exception of ligands for the GluN2B subunit of NMDA receptors, there has been little success in developing radiotracers for imaging ionotropic glutamate receptors; failure of ligands for the PCP/MK801 binding site in vivo doubtless relates their dependence on the open, unblocked state of the ion channel. Many AMPA and kainite receptor ligands with good binding properties in vitro have failed to give measurable specific binding in the living brain. This may reflect the challenge of developing brain-penetrating ligands for amino acid receptors, compounded by conformational differences in vivo. The situation is better with respect to mGluR imaging, particularly for the mGluR5 subtype. Several successful PET ligands serve for investigations of mGluRs in conditions such as schizophrenia, depression, substance abuse and aging. Considering the centrality and diversity of glutamatergic signaling in brain function, we have relatively few selective and sensitive tools for molecular imaging of ionotropic and metabotropic glutamate receptors. Further radiopharmaceutical research targeting specific subtypes and subunits of the glutamate receptors may yet open up new investigational vistas with broad applications in basic and clinical research.

The role of glutamate receptors and their interactions with dopamine and other neurotransmitters in the development of tardive dyskinesia: preclinical and clinical results.

Tardive dyskinesia is a serious, disabling, movement disorder associated with the ongoing use of antipsychotic medication. Current evidence regarding the pathophysiology of tardive dyskinesia is mainly based on preclinical animal models and is still not completely understood. The leading preclinical hypothesis of tardive dyskinesia development includes dopaminergic imbalance in the direct and indirect pathways of the basal ganglia, cholinergic deficiency, serotonin receptor disturbances, neurotoxicity, oxidative stress, and changes in synaptic plasticity. Although, the role of the glutamatergic system has been confirmed in preclinical tardive dyskinesia models it seems to have been neglected in recent reviews. This review focuses on the role and interactions of glutamate receptors with dopamine, acetylcholine, and serotonin in the neuropathology of tardive dyskinesia development. Moreover, preclinical and clinical results of the differentiated effectiveness of N-methyl-D-aspartate (NMDA) receptor antagonists are discussed with a special focus on antagonists that bind with the GluN2B subunit of NMDA receptors. This review also presents new combinations of drugs that are worth considering in the treatment of tardive dyskinesia.

Glu60 of α-Calcium/calmodulin dependent protein kinase II mediates crosstalk between the regulatory T-site and protein substrate binding region of the active site

Memory formation transpires to be by activation and persistent modification of synapses. A chain of biochemical events accompany synaptic activation and culminate in memory formation. These biochemical events are steered by interplay and modulation of various synaptic proteins, achieved by conformational changes and phosphorylation/dephosphorylation of these proteins. Calcium/calmodulin dependent protein kinase II (CaMKII) and N-methyl-d-aspartate receptors (NMDARs) are synaptic proteins whose interactions play a pivotal role in learning and memory process. Catalytic activity of CaMKII is modulated upon its interaction with the GluN2B subunit of NMDAR. The structural basis of this interaction is not clearly understood. We have investigated the role of Glu60 of α-CaMKII, a conserved residue present in the ATP binding region of kinases, in the regulation of catalysis of CaMKII by GluN2B. Mutation of Glu60 to Gly exerts different effects on the kinetic parameters of phosphorylation of GluN2B and GluN2A, of which only GluN2B binds to the T-site of CaMKII. GluN2B induced modulation of the kinetic parameters of peptide substrate was altered in the E60G mutant. The mutation almost abolished the modulation of the apparent Km value for protein substrate. However, although kinetic parameters for ATP were altered by mutating Glu60, modulation of the apparent Km value for ATP by GluN2B seen in WT was exhibited by the E60G mutant of α-CaMKII. Hence our results posit that the communication of the T-site of CaMKII with protein substrate binding region of active site is mediated through Glu60 while the communication of the T-site with the ATP binding region is not entirely dependent on Glu60.

Potential link between antidepressant-like effects of ketamine and promotion of adult neurogenesis in the ventral hippocampus of mice

Recent studies have shown that ketamine, an open channel blocker of the N-methyl-d-aspartate receptor (NMDAR), is effective for patients with treatment-resistant depression. In this study, we aimed to elucidate the potential link between antidepressant-like effects of a single ketamine administration and dorsoventral differentiation in adult hippocampal neurogenesis. Immunohistochemical analyses revealed that elevation in the densities of neuronal progenitors and newborn granule cells by ketamine was seen in the ventral (related to emotion), but not dorsal (related to spatial memory), hippocampus in adult mice, although the densities of neural stem cells were not affected by ketamine in both the dorsal and ventral regions. Promotion of maturation of newborn granule cells by ketamine was evident in the ventral, but not dorsal, hippocampus. Behavioral analyses showed that ketamine did not affect spatial memory but ameliorated depression-related behavior. Western blot analyses showed that the basal expression of the GluN2B, but not GluN1, subunit of the NMDAR was higher in the ventral hippocampus than in the dorsal hippocampus. The induction of expression of GluN2B subunit of the NMDAR, phosphorylated mammalian target of rapamycin (p-mTOR), GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), and brain derived neurotrophic factor (BDNF), by ketamine was greater in the ventral hippocampus than in the dorsal hippocampus. Our results demonstrate that a single ketamine administration promotes adult neurogenesis in the ventral hippocampus quite selectively. Furthermore, ventral-dominant induction of the GluN2B subunit of NMDAR, p-mTOR, GluA1 subunit of AMPAR, and BDNF, in the hippocampus may underlie the unique antidepressant-like effects of ketamine.

NMDAR-Activated PP1 Dephosphorylates GluN2B to Modulate NMDAR Synaptic Content

SUMMARYIn 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.

Src activation in the hypothalamic arcuate nucleus may play an important role in pain hypersensitivity

Src family of kinases (SFKs) has been found to play an important role in the regulation of nociception. However, how each member of this family acts in the central nervous system (CNS) structures involved in the relay and/or modulation of nociceptive signals, and thereby contributes to the formation and maintenance of pain hypersensitivity, is still a challenge. In this work, a combined study using biochemical, genetic and behavioral approaches was conducted. We found that the expression of activated SFKs in the hypothalamic arcuate nucleus (ARC) area was significantly increased following the development of inflammation induced by injection of complete freund’s adjuvant (CFA) into the hind paw of rats. Furthermore, we identified that Src, but not Fyn or Lyn in the Src family, was activated, and that Src knockdown in the ARC area blocked the inflammation-induced increases in the expression of activated SFKs, the N-Methyl-D-aspartate receptor (NMDAR) GluN2B subunit and phosphorylated GluN2B at Y1472 in this region. Moreover, the CFA injection-induced allodynia and hyperalgesia, and the analgesic effect produced by systemic application of the SFK inhibitor, SU6656, were significantly diminished. However, the Src knockdown did not induce any change in the expression of activated SFKs and the NMDAR GluN2B subunit in normal rats which were not injected with CFA. Neither the Src knockdown nor the systemic application of SU6656 affected the mechanical and thermal sensitivity of the normal rats. Thus, Src activation in the ARC may be a key event for formation and maintenance of pain hypersensitivity associated with peripheral inflammation.

Identification and Preclinical Evaluation of a Radiofluorinated Benzazepine Derivative for Imaging the GluN2B Subunit of the Ionotropic NMDA Receptor.

The previously reported carbon-11 labeled GluN2B PET radioligand 11C-Me-NB1 served as a starting point for derivatization and led to the successful development of a radiofluorinated analogue designated (R)-18F-OF-Me-NB1. Given the short physical half-life of 20.3 min for carbon-11, (R)-18F-OF-Me-NB1 with a physical half-life of 109.8 min would allow satellite distribution to nuclear medicine facilities without an on-site cyclotron. Methods: Two fluorinated Me-NB1 derivatives, OF-Me-NB1 and PF-Me-NB1, were synthesized. Upon chiral resolution, the respective enantiomers were radiolabeled with carbon-11 and assessed in a proof-of-concept study by applying in vitro autoradiography on rodent brain sections. Based on the autoradiograms, (R)-OF-Me-NB1 was selected for radiofluorination and preclinical evaluation by ex vivo autoradiography, PET imaging, biodistribution and metabolite studies in Wistar rats. To rule out off-target binding to the σ1 receptor, the brain uptake of (R)-18F-OF-Me-NB1 in wild-type mice was compared with σ1 receptor knock-out mice. Results: Autoradiographic assessment revealed that both enantiomers of 11C-PF-Me-NB1 distributed homogenously across all brain regions on rodent brain sections. In contrast, the two enantiomers of 11C-OF-Me-NB1 exhibited an entirely different behaviour. While (S)-11C-OF-Me-NB1 bound virtually to all brain regions with considerable σ1 receptor binding, (R)-11C-OF-Me-NB1 exhibited high selectivity and specificity for the GluN2B-rich rat forebrain. These findings were confirmed for the radiofluorinated analogue (R)-11C-OF-Me-NB1, which was obtained via copper-mediated radiofluorination in radiochemical yields of 13-25% and molar activities ranging from 61-168 GBq/µmol. PET imaging and biodistribution studies in Wistar rats indicated appropriate pharmacokinetic profile and high in vivo specific binding of (R)-18F-OF-Me-NB1 as revealed by blocking studies with GluN2B-antagonist CP101,606. Off-target binding to the σ1 receptor was excluded by PET imaging with σ1 receptor knock-out mice. Receptor occupancy experiments with CP101,606 revealed a D50-value of 8.3 µmol/kg (intravenous). Conclusion: (R)-18F-OF-Me-NB1 is a promising radiofluorinated probe that exhibits specificity and selectivity for the GluN2B-containing N-methyl-D-aspartate (NMDA) complex and enables in vivo target occupancy studies in rodents.

RETRACTED: Rett-like Severe Encephalopathy Caused by a De Novo GRIN2B Mutation Is Attenuated by D-serine Dietary Supplement

N-Methyl-D-aspartate receptors (NMDARs) play pivotal roles in synaptic development, plasticity, neural survival, and cognition. Despite recent reports describing the genetic association between de novo mutations of NMDAR subunits and severe psychiatric diseases, little is known about their pathogenic mechanisms and potential therapeutic interventions. Here we report a case study of a 4-year-old Rett-like patient with severe encephalopathy carrying a missense de novo mutation in GRIN2B(p.P553T) coding for the GluN2B subunit of NMDAR. We generated a dynamic molecular model of mutant GluN2B-containing NMDARs. We expressed the mutation in cell lines and primary cultures, and we evaluated the putative morphological, electrophysiological, and synaptic plasticity alterations. Finally, we evaluated D-serine administration as a therapeutic strategy and translated it to the clinical practice. Structural molecular modeling predicted a reduced pore size of mutant NMDARs. Electrophysiological recordings confirmed this prediction and also showed gating alterations, a reduced glutamate affinity associated with a strong decrease of NMDA-evoked currents. Moreover, GluN2B(P553T)-expressing neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of GluA1 at stimulated synapses. Notably, the naturally occurring coagonist D-serine was able to attenuate hypofunction of GluN2B(p.P553T)-containing NMDARs. Hence, D-serine dietary supplementation was initiated. Importantly, the patient has shown remarkable motor, cognitive, and communication improvements after 17months of D-serine dietary supplementation. Our data suggest that hypofunctional NMDARs containing GluN2B(p.P553T) can contribute to Rett-like encephalopathy and that their potentiation by D-serine treatment may underlie the associated clinical improvement.

Genetic Mutation of GluN2B Protects Brain Cells Against Stroke Damages.

Immediately following ischemia, glutamate accumulates in the extracellular space and results in extensive stimulation of its receptors including N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. A large amount of Ca2+ influx directly through the receptor-gated ion channels which leads to Ca2+ overload and triggers several downstream lethal reactions. As a result, cell dies via apoptosis or necrosis, or both. Death-associated protein kinase 1 (DAPK1) physically and functionally interacts with the NMDA receptor GluN2B subunit at extra-synaptic sites and this interaction acts as a central mediator for stroke damage. The goal of this study is to explore an effective strategy in the treatment of stroke with a molecular genetic manipulation to interrupt DAPK1-GluN2B interaction. We generated a mutant strain of mice with the conditional deletion of GluN2B C-terminal tail consisting of amino acids 886-1269 in the forebrain excitatory neurons (the GluN2B mutant mice) and tested the protective effects of this mutation in stroke damages. GluN2B mutation effectively disrupted the DAPK1-GluN2B interaction and inhibited extra-synaptic NMDA receptor currents without affecting synaptic NMDA receptor channel activity in the central neurons. GluN2B mutation protected against stroke damages both in vitro and in vivo and hence improved behavioral performance. Disruption of the DAPK1-GluN2B interaction is therapeutically effective against stroke damages.

Uncoupling DAPK1 from NMDA receptor GluN2B subunit exerts rapid antidepressant-like effects

Several preclinical studies have reported the rapid antidepressant effects of N-methyl-D-aspartate receptor (NMDAR) antagonists, although the underlying mechanisms are still unclear. Death-associated protein kinase 1 (DAPK1) couples GluN2B subunits at extrasynaptic sites to regulate NMDAR channel conductance. In the present study, we found that chronic unpredictable stress (CUS) induced extracellular glutamate accumulation, accompanied by an increase in the DAPK1–NMDAR interaction, the high expression of DAPK1 and phosphorylated GluN2B at Ser1303, a decrease in phosphorylated DAPK1 at Ser308 and synaptic protein deficits in the rat medial prefrontal cortex (mPFC). CUS also enhanced GluN2B-mediated NMDA currents and extrasynaptic responses that were induced by bursts of high-frequency stimulation, which may be associated with the loss of astrocytes and low expression of glutamate transporter-1 (GLT-1). The blockade of GLT-1 in the mPFC was sufficient to induce depressive-like behavior and cause similar molecular changes. Selective GluN2B antagonist, DAPK1 knockdown by adeno-associated virus-mediated short-hairpin RNA or a pharmacological inhibitor, and the uncoupling of DAPK1 from the NMDAR GluN2B subunit produced rapid antidepressant-like effects and reversed CUS-induced alterations in the mPFC. The inhibition of DAPK1 and its interaction with GluN2B subunit in the mPFC also rescued CUS-induced depressive-like behavior 7 days after treatment. A selective GluN2B antagonist did not have rewarding effects in the conditioned place preference paradigm. Altogether, our findings suggest that the DAPK1 interaction with the NMDAR GluN2B subunit acts as a critical component in the pathophysiology of depression and is a potential target for new antidepressant treatments.

Acute stress regulates phosphorylation of N-methyl-d-aspartate receptor GluN2B at S1284 in hippocampus

Exposure to acute stress leads to diverse changes, which include either beneficial or deleterious effects on molecular levels that are implicated in stress-related disorders. N-methyl-d-aspartate receptor (NMDAR)-mediated signalings, are thought to be vital players in stress-related mental disorders as well as attractive therapeutic targets for clinical treatment. In the present study, we utilized acute stress models in mice to explore regulation of phosphorylation level of S1284 in GluN2B subunit of NMDAR. We found out that forced swimming and acute restraint stress increased phosphorylation level of S1284, while phosphorylation level of S1284 was unaltered after brief exposure to open field. Moreover, phosphorylation change of S1284 was negated by treatment of roscovitine which is believed to be a Cyclin-dependent kinase inhibitor. Besides, we showed well correlation of phosphorylation change of S1284 and immobility time during forced swimming. Collectively, our results demonstrated that phosphorylation level of S1284 in GluN2B was regulated by acute stress.

GluN2B N-methyl-D-aspartate receptor and excitatory amino acid transporter 3 are upregulated in primary sensory neurons after 7 days of morphine administration in rats: implication for opiate-induced hyperalgesia.

The contribution of the peripheral nervous system to opiate-induced hyperalgesia (OIH) is not well understood. In this study, we determined the changes in excitability of primary sensory neurons after sustained morphine administration for 7 days. Changes in the expression of glutamate receptors and glutamate transporters after morphine administration were ascertained in dorsal root ganglions. Patch clamp recordings from intact dorsal root ganglions (ex vivo preparation) of morphine-treated rats showed increased excitability of small diameter (≤30 μm) neurons with respect to rheobase and membrane threshold, whereas the excitability of large diameter (>30 μm) neurons remained unchanged. Small diameter neurons also displayed increased responses to glutamate, which were mediated mainly by GluN2B containing N-methyl-D-aspartate (NMDA) receptors, and to a lesser degree by the neuronal excitatory amino acid transporter 3/excitatory amino acid carrier 1. Coadministration in vivo of the GluN2B selective antagonist Ro 25-6981 with morphine for 7 days prevented the appearance of OIH and increased morphine-induced analgesia. Administration of morphine for 7 days led to an increased expression of GluN2B and excitatory amino acid transporter 3/excitatory amino acid carrier 1, but not of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate, kainate, or group I metabotropic glutamate receptors, or of the vesicular glutamate transporter 2. These results suggest that peripheral glutamatergic neurotransmission contributes to OIH and that GluN2B subunit of NMDA receptors in the periphery may be a target for therapy.