Distinct NMDA receptors provide differential modes of transmission at mossy fiber-interneuron synapses

Dentate gyrus granule cells innervate inhibitory interneurons via a continuum of synapses comprised of either Ca(2+)-impermeable (CI) or Ca(2+)-permeable (CP) AMPA receptors. Synapses at the extreme ends of this continuum engage distinct postsynaptic responses, with activity at CI synapses being strongly influenced by NMDA receptor activation. NMDARs at CI synapses have a lower NR2B subunit composition and a higher open probability, which generate larger amplitude and more rapid EPSCs than their CP counterparts. A novel form of NMDAR-dependent long-term depression (iLTD) is associated with CI-mossy fiber synapses, whereas iLTD at CP synapses is dependent on Ca(2+)-permeable AMPA receptor activation. Induction of both forms of iLTD required elevation of postsynaptic calcium. Thus mossy fibers engage CA3 interneurons via multiple synapse types that will act to expand the computational repertoire of the mossy fiber-CA3 network.     

Post-translational modification of NMDA receptor GluN2B subunit and its roles in chronic pain and memory

N-methyl-d-aspartate receptors (NMDA receptors) play critical roles in brain functions and diseases. The expression, trafficking, synaptic location and function of different NMDA receptor subtypes are not static, but regulated dynamically in a cell-specific and synapse-specific manner during physiological and pathological conditions. In this review, we will examine recent evidence on the post-translational modulation of NMDA receptors subunit, in particular GluN2B subunit, such as phosphorylation, palmitoylation, and ubiquitination. In parallel, we will overview the roles of these modifications of GluN2B-NMDA receptor subtype in physiological functions, such as learning and memory, and pathophysiological conditions, such as chronic pain, ischemia and neurodegenerative diseases.     

Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening

The defining functional feature of N-methyl-d-aspartate (NMDA) receptors is activation gating, the energetic coupling of ligand binding into opening of the associated ion channel pore. NMDA receptors are obligate heterotetramers typically composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits that gate in a concerted fashion, requiring all four ligands to bind for subsequent opening of the channel pore. In an individual subunit, the extracellular ligand-binding domain, composed of discontinuous polypeptide segments S1 and S2, and the transmembrane channel-forming domain, composed of M1-M4 segments, are connected by three linkers: S1-M1, M3-S2, and S2-M4. To study subunit-specific events during pore opening in NMDA receptors, we impaired activation gating via intrasubunit disulfide bonds connecting the M3-S2 and S2-M4 in either the GluN1 or GluN2A subunit, thereby interfering with the movement of the M3 segment, the major pore-lining and channel-gating element. NMDA receptors with gating impairments in either the GluN1 or GluN2A subunit were dramatically resistant to channel opening, but when they did open, they showed only a single-conductance level indistinguishable from wild type. Importantly, the late gating steps comprising pore opening to its main long-duration open state were equivalently affected regardless of which subunit was constrained. Thus, the NMDA receptor ion channel undergoes a pore-opening mechanism in which the intrasubunit conformational dynamics at the level of the ligand-binding/transmembrane domain (TMD) linkers are tightly coupled across the four subunits. Our results further indicate that conformational freedom of the linkers between the ligand-binding and TMDs is critical to the activation gating process.     

Transient expression of NMDA receptor subunit NR2B in the developing rat heart

NMDA receptors represent a subtype of the ionotropic glutamate receptor family, comprising three classes of subunits (NR1, NR2A-D, NR3), which exhibit distinct patterns of regional and developmental expression in the CNS. Recently, some NMDA receptor subunits have also been described in adult extraneuronal tissues and keratinocytes. However, their developmental expression patterns are currently unknown. With use of RT-PCR and western blot analysis, the expression of NMDA receptor subunit NR2B was investigated in the developing rat heart. NR2B mRNA and protein were detected in heart tissue of rats from embryonic day 14 until postnatal day 21 but disappeared 10 weeks after birth. In contrast, no NMDA receptor subunit NR1, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluR2, or anchoring postsynaptic density protein-95 could be detected in rat heart at any developmental stage. Confocal microscopy of cultured cardiac myocytes (CMs) from neonatal rats revealed distinct NR2B staining mainly of intracellular structures. However, no functional NMDA receptor could be detected on CMs by whole-cell recordings. In conclusion, high concentrations of NR2B protein can be detected in early rat heart development, but its function still remains elusive.     

Evidence for low GluR2 AMPA receptor subunit expression at synapses in the rat basolateral amygdala

Fast excitatory synaptic responses in basolateral amygdala (BLA) neurons are mainly mediated by ionotropic glutamate receptors of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype. AMPA receptors containing an edited GluR2 subunit are calcium impermeable, whereas those that lack this subunit are calcium permeable and also inwardly rectifying. Here, we sought to determine the extent to which synapses in the rat BLA have AMPA receptors with GluR2 subunits. We assessed GluR2 protein expression in the BLA by immunocytochemistry with a GluR2 subunit-specific antiserum at the light and electron microscopic level; for comparison, a parallel examination was carried out in the hippocampus. We also recorded from amygdala brain slices to examine the voltage-dependent properties of AMPA receptor- mediated evoked synaptic currents in BLA principal neurons. At the light microscopic level, GluR2 immunoreactivity was localized to the perikarya and proximal dendrites of BLA neurons; dense labeling was also present over the pyramidal cell layer of hippocampal subfields CA1 and CA3. In electron micrographs from the BLA, most of the synapses were asymmetrical with pronounced postsynaptic densities (PSD). They contained clear, spherical vesicles apposed to the PSD and were predominantly onto spines (86%), indicating that they are mainly with BLA principal neurons. Only 11% of morphological synapses in the BLA were onto postsynaptic elements that showed GluR2 immunoreactivity, in contrast to hippocampal subfields CA1 and CA3 in which 76% and 71% of postsynaptic elements were labeled (p < 0.001). Synaptic staining in the BLA and hippocampus, when it occurred, was exclusively postsynaptic, and particularly heavy over the PSD. In whole-cell voltage clamp recordings, 72% of BLA principal neurons exhibited AMPA receptor-mediated synaptic currents evoked by external capsule stimulation that were inwardly rectifying. Although BLA principal neurons express perikaryal and proximal dendritic GluR2 immunoreactivity, few synapses onto these neurons express GluR2, and a preponderance of principal neurons have inwardly rectifying AMPA-mediated synaptic currents, suggesting that targeting of GluR2 to synapses is restricted. Many BLA synaptic AMPA receptors are likely to be calcium permeable and could play roles in synaptic plasticity, epileptogenesis and excitoxicity.     

Effect of Src kinase phosphorylation on disordered C-terminal domain of N-methyl-D-aspartic acid (NMDA) receptor subunit GluN2B protein

NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.     

A model of cooperative effect of AMPA and NMDA receptors in glutamatergic synapses

Glutamatergic synapses play a pivotal role in brain excitation. The synaptic response is mediated by the activity of two receptor types (AMPA and NMDA). In the present paper we propose a model of glutamatergic synaptic activity where the fast current generated by the AMPA conductance produces a local depolarization which activates the voltage- and [Mg²⁺]-dependent NMDA conductance. This cooperative effect is dependent on the biophysical properties of the synaptic spine which can be considered a high input resistance specialized compartment. Herein we present results of simulations where different values of the spine resistance and of the Mg²⁺ concentrations determine different levels of cooperativeness between AMPA and NMDA receptors in shaping the post-synaptic response.     

Bipolar disorder: involvement of signaling cascades and AMPA receptor trafficking at synapses

There is increasing evidence that severe mood disorders are associated with impairment of structural plasticity and cellular resilience. Cumulative data demonstrate that mood stabilizers regulate intracellular signaling cascades, including protein kinase C (PKC), PKA, mitogen-activated protein (MAP) kinase, glycogen synthase kinase 3-beta (GSK3-beta) and intracellular calcium, which are signaling pathways that regulate synaptic plasticity. In this context, it is noteworthy that a growing body of data indicates that the glutamatergic system, has a major role in neuronal plasticity and cellular resilience, might be involved in the pathophysiology and treatment of mood disorders. AMPA glutamate-receptor trafficking is important in synaptic plasticity and might play crucial roles in maintaining critical neuronal circuits associated with mood. Two clinically effective, structurally dissimilar, antimanic agents, lithium and valproate (VPA), down-regulate synaptic expression of AMPA receptor subunit GluR1 in hippocampus in chronically treated rats. This reduction in synaptic GluR1 by lithium and VPA is due to attenuated phosphorylation of GluR1 at a specific PKA site (residue 845 of GluR1), which is crucial for AMPA receptor insertion. By contrast,imipramine, which can provoke mania, increases synaptic expression of GluR1 in the hippocampus in vivo. Furthermore, there is ample evidence from preclinical and clinical research that the glutamatergic system is involved in the pathophysiology of mood disorders and that many of the somatic treatments used for mood disorders including antidepressants, mood stabilizers, atypical antipsychotic drugs and electroconvulsive therapy have both direct and indirect effects on the glutamatergic system. Given these findings, further research with medications that specifically affect the glutamatergic system is warranted. Recent studies in our lab have shown that riluzole, a FDA approved medicine that regulates the glutamatergic system, shows antidepressant efficacy in unipolar and bipolar depression. These studies indicate that regulation of glutamate-mediated synaptic plasticity might play a role in the treatment of mood disorders, and raise new avenues for novel therapies for this devastating illness.     

mGluR5 and NMDA receptors drive the experience- and activity-dependent NMDA receptor NR2B to NR2A subunit switch

In cerebral cortex there is a developmental switch from NR2B- to NR2A-containing NMDA receptors (NMDARs) driven by activity and sensory experience. This subunit switch alters NMDAR function, influences synaptic plasticity, and its dysregulation is associated with neurological disorders. However, the mechanisms driving the subunit switch are not known. Here, we show in hippocampal CA1 pyramidal neurons that the NR2B to NR2A switch driven acutely by activity requires activation of NMDARs and mGluR5, involves PLC, Ca(2+) release from IP(3)R-dependent stores, and PKC activity. In mGluR5 knockout mice the developmental NR2B-NR2A switch in CA1 is deficient. Moreover, in visual cortex of mGluR5 knockout mice, the NR2B-NR2A switch evoked in?vivo by visual experience is absent. Thus, we establish that mGluR5 and NMDARs are required for the activity-dependent NR2B-NR2A switch and play a critical role in experience-dependent regulation of NMDAR subunit composition in?vivo.     

Molecular basis of positive allosteric modulation of GluN2B NMDA receptors by polyamines

NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions.     

Identifying potential GluN2B subunit containing N-Methyl-D-aspartate receptor inhibitors: an integrative in silico and molecular modeling approach

Abstract

N-methyl-D-aspartate receptors (NMDARs), a class of ligand-gated ion channels, are involved in non-selective cation transport across the membrane. These are contained in glutamatergic synapse and produce excitatory effects leading to synaptic plasticity and memory function. GluN1-GluN2B, a subtype of NMDAR(s), has significant role in neurodegeneration, amyloid β (Aβ) induced synaptic dysfunction and loss. Thus, targeting and inhibiting GluN1-GluN2B may be effective in the management of neurodegenerative diseases including Alzheimer’s disease. In the present study, ligand and structure-based approaches were tried to identify the inhibitors. The pharmacophore, developed from co-crystallised ifenprodil, afforded virtual hits, which were further subjected through drug likeliness and PAINS filters to remove interfering compounds. Further comprehensive docking studies, free energy calculations and ADMET studies resulted in two virtual leads. The leads, ZINC257261614 and ZINC95977857 displayed good docking scores of ?12.90 and ?12.20?Kcal/mol and free binding energies of ?60.83 and ?61.83?Kcal/mol, respectively. The compounds were having acceptable predicted ADMET profiles and were subjected to molecular dynamic (MD) studies. The MD simulation produced stable complexes of these ligands with GluN1-GluN2B subunit having protein and ligand RMSD in acceptable limit. Abbreviations AD Alzheimer's disease

ADME Absorption distribution metabolism and excretion

ATD Amino terminal domain

BBB Blood-brain barrier

CNS Central nervous system

CREB cAMP response element binding protein

CTD Carboxy-terminal domain

Glu Glutamate

GMQE Global model quality estimation

HTVS High throughput virtual screening

HIA Human intestinal absorption

LGA Lamarckian genetic algorithm

MD Molecular dynamics

MM-GBSA Molecular mechanics, the Generalised Born model for Solvent Accessibility

NMDAR N-methyl-D-aspartate receptors

PAINS Pan assay interference compounds

RMSD Root-mean square deviation

RMSF Root-mean-square fluctuation

SMARTS SMILES arbitrary target specification

SP standard precision

XP extra precision

Communicated by Ramaswamy H. Sarma     

Regulation of phosphorylation at Ser of GluN2B receptor in the postsynaptic density

Neuronal N-methyl-D-aspartate subtype of ionotropic glutamate receptor (NMDAR) that plays essential roles in excitatory synaptic transmission is regulated by phosphorylation. However, the kinases and phosphatases involved in this regulation are not completely known. We show that the GluN2B subunit of NMDAR is phosphorylated at Ser¹³⁰³ by protein kinase C (PKC) and is dephosphorylated by protein phosphatase 1 (PP1), but not protein phosphatase 2A (PP2A) in isolated postsynaptic density (PSD). Although PSD is known to harbor PKC, PP1 and PP2A, their ability to regulate phosphorylation of GluN2B-Ser¹³⁰³ would depend on the accessibility of GluN2B-Ser¹³⁰³ to these proteins. Since PSD preparation is likely to maintain the organization of its component proteins as inside neurons, accessibility of kinases and phosphatases to GluN2B-Ser¹³⁰³in vivo would be addressed by experiments using this system. Using an antibody specific for the phosphorylated state of GluN2B-Ser¹³⁰³ we demonstrate that PP1 is the major phosphatase in rat brain PSD that can dephosphorylate the GluN2B-Ser¹³⁰³ endogenous to PSD. We also show that PKC present in PSD can phosphorylate GluN2B-Ser¹³⁰³. The events reported here might be important in regulating GluN2B-Ser¹³⁰³ phosphorylation in vivo.     

A computational approach to the study of AMPA receptor clustering at Purkinje cell synapses

Synapses between parallel fibres and Purkinje cells in the cerebellum exhibit unique forms of synaptic plasticity thought to be associated with the refinement of motor skills. Since the discovery of Long Term Depression (LTD), more than twenty years ago, many molecular signalling pathways potentially underlying LTD have been explored. These have revealed a surprisingly diverse array of cellular and molecular mechanisms. Foremost has been the now well-established discovery that LTD is the electrophysiological manifestation of a reduced density of AMPA receptors at the synapse, following induction. Although LTD is primarily an electrophysiologically defined phenomenon, recent studies have increasingly combined electrophysiological, imaging, proteomic and biochemical approaches to probe its mechanisms. The challenge is now to integrate data from different modalities into a unified formalism that can deal with the complexity of the system, as well as generate experimental predictions. Here, we use particle-based stochastic modelling as a prototype to explore the feasibility of building realistic model of synaptic plasticity, at the molecular level.     

Aspirin increases NMDA receptor subunit 2A concentrations in rat hippocampus

The N-methyl-d-aspartate receptor (NMDAR), a heteromeric protein, is a glutamate receptor that has three classes of subunits: NR1, NR2, and NR3. It has been reported that these receptors are involved in synaptogenesis, synaptic plasticity, and many other processes in the central nervous system. The aim of this study is to investigate the efficacy of aspirin on hippocampal NMDARs. Sixteen rats were studied in two groups, with eight animals in each group. The first group was the control group, and the second one was the aspirin-given group. Aspirin (acetylsalicylic acid) was administered orally to the rats (200 mg/kg). Tissue samples were obtained after 3 h. The brain was removed, and both hippocampi were dissected out for evaluation. It was found that acute doses of aspirin caused increases on the levels of NMDAR 2A (NR2A) receptors and malondialdehyde (MDA), the end product of lipid peroxidation. Production was significantly increased in the aspirin-given group. We know that MDA is a marker for free radical-mediated tissue damage. In conclusion, lipid peroxidation, caused by acute doses of aspirin may lead to excitotoxicity effects by a hippocampal NR2A-mediated mechanism.     

Distinct quantal features of AMPA and NMDA synaptic currents in hippocampal neurons: implication of glutamate spillover and receptor saturation

Excitatory postsynaptic currents (EPSCs) were studied in the CA1 pyramidal cells of rat hippocampal slices. Components mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and by N-methyl-D-aspartate (NMDA) receptors were separated pharmacologically. Quantal parameters of AMPA and NMDA receptor-mediated EPSCs were obtained using both maximal likelihood and autocorrelation techniques. Enhancement of transmitter release with 4-aminopyridine caused a significant increase in quantal size of NMDA EPSC. This was accompanied by a slowing of the EPSC decay. The maximal number of quanta in the NMDA current was unchanged, while the probability of quantal event dramatically enhanced. In contrast, neither the quantal size nor the kinetics of AMPA EPSC was altered by 4-aminopyridine, while the maximal number of quanta increased. These changes in the quantal parameters are consistent with a transition to multivesicular release of the neurotransmitter. Spillover of excessive glutamate on the nonsynaptic areas of dendritic spines causes an increase in the quantal size of NMDA synaptic current. The difference in quantal behavior of AMPA and NMDA EPSCs implies that different mechanisms underlie their quantization: the additive response of nonsaturated AMPA receptors contrasts with the variable involvement of saturated intrasynaptic and nonsaturated extrasynaptic NMDA receptors.     

Metaplasticity gated through differential regulation of GluN2A versus GluN2B receptors by Src family kinases

Metaplasticity is a higher form of synaptic plasticity that is essential for learning and memory, but its molecular mechanisms remain poorly understood. Here, we report that metaplasticity of transmission at CA1 synapses in the hippocampus is mediated by Src family kinase regulation of NMDA receptors (NMDARs). We found that stimulation of G-protein-coupled receptors (GPCRs) regulated the absolute contribution of GluN2A-versus GluN2B-containing NMDARs in CA1 neurons: pituitary adenylate cyclase activating peptide 1 receptors (PAC1Rs) selectively recruited Src kinase, phosphorylated GluN2ARs, and enhanced their functional contribution; dopamine 1 receptors (D1Rs) selectively stimulated Fyn kinase, phosphorylated GluN2BRs, and enhanced these currents. Surprisingly, PAC1R lowered the threshold for long-term potentiation while long-term depression was enhanced by D1R. We conclude that metaplasticity is gated by the activity of GPCRs, which selectively target subtypes of NMDARs via Src kinases.