Activity-induced synaptic delivery of the GluN2A-containing NMDA receptor is dependent on endoplasmic reticulum chaperone Bip and involved in fear memory

The N-methyl-D-aspartate receptor (NMDAR) in adult forebrain is a heterotetramer mainly composed of two GluN1 subunits and two GluN2A and/or GluN2B subunits. The synaptic expression and relative numbers of GluN2A- and GluN2B-containing NMDARs play critical roles in controlling Ca²⁺-dependent signaling and synaptic plasticity. Previous studies have suggested that the synaptic trafficking of NMDAR subtypes is differentially regulated, but the precise molecular mechanism is not yet clear. In this study, we demonstrated that Bip, an endoplasmic reticulum (ER) chaperone, selectively interacted with GluN2A and mediated the neuronal activity-induced assembly and synaptic incorporation of the GluN2A-containing NMDAR from dendritic ER. Furthermore, the GluN2A-specific synaptic trafficking was effectively disrupted by peptides interrupting the interaction between Bip and GluN2A. Interestingly, fear conditioning in mice was disrupted by intraperitoneal injection of the interfering peptide before training. In summary, we have uncovered a novel mechanism for the activity-dependent supply of synaptic GluN2A-containing NMDARs, and demonstrated its relevance to memory formation.     

A critical role for GluN2B-containing NMDA receptors in cortical development and function

The subunit composition of N-methyl D-aspartate receptors (NMDARs) is tightly regulated during cortical development. NMDARs are initially dominated by GluN2B (NR2B), whereas GluN2A (NR2A) incorporation increases after birth. The function of GluN2B-containing NMDARs during development, however, is incompletely understood. We generated a mouse in which we genetically replaced GluN2B with GluN2A (2B→2A). Although this manipulation restored NMDAR-mediated currents at glutamatergic synapses, it did not rescue GluN2B loss of function. Protein translation-dependent homeostatic synaptic plasticity is occluded in the absence of GluN2B, and AMPA receptor contribution is enriched at excitatory cortical synapses. Our experiments indicate that specificity of GluN2B-mediated signaling is due to its unique interaction with the protein effector alpha calcium-calmodulin kinase II and the regulation of the mTOR pathway. Homozygous 2B→2A mice exhibited high rates of lethality, suppressed feeding, and depressed social exploratory behavior. These experiments indicate that GluN2B-containing NMDARs activate unique cellular processes that cannot be rescued by replacement with GluN2A.     

Differential roles of NR2A- and NR2B-containing NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking

NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). Here we show that NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission.     

Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-d-aspartate (NMDA) Receptors

The number and subunit composition of synaptic N-methyl-d-aspartate receptors (NMDARs) play critical roles in synaptic plasticity, learning, and memory and are implicated in neurological disorders. Tyrosine phosphorylation provides a powerful means of regulating NMDAR function, but the underling mechanism remains elusive. In this study we identified a tyrosine site on the GluN2B subunit, Tyr-1070, which was phosphorylated by a proto-oncogene tyrosine-protein (Fyn) kinase and critical for the surface expression of GluN2B-containing NMDARs. The phosphorylation of GluN2B at Tyr-1070 was required for binding of Fyn kinase to GluN2B, which up-regulated the phosphorylation of GluN2B at Tyr-1472. Moreover, our results revealed that the phosphorylation change of GluN2B at Tyr-1070 accompanied the Tyr-1472 phosphorylation and Fyn associated with GluN2B in synaptic plasticity induced by both chemical and contextual fear learning. Taken together, our findings provide a new mechanism for regulating the surface expression of NMDARs with implications for synaptic plasticity.     

Directional gating of synaptic plasticity by GPCRs and their distinct downstream signalling pathways

EMBO J 31 4, 805–816 (2012); published online December202011Synaptic plasticity, the activity-dependent modification of synaptic strength, plays a fundamental role in learning and memory as well as in developmental maturation of neuronal circuitry. However, how synaptic plasticity is induced and regulated remains poorly understood. In this issue of The EMBO Journal, Yang and colleagues present sets of exciting data, suggesting that G-protein-coupled receptors (GPCRs) selectively execute distinct signalling pathways to differentially regulate induction thresholds of hippocampal long-term potentiation (LTP) and long-term depression (LTD), thereby governing the direction of synaptic plasticity. These results shed significant light on our current understanding of how bidirectional synaptic plasticity is regulated.Synaptic plasticity has been demonstrated at synapses in various brain regions; the most well-characterized forms are LTP and LTD at hippocampal CA1 glutamatergic synapses (Collingridge et al, 2004). In experimental models, LTP and LTD can be, respectively, induced by high-frequency stimulation (HFS) and low-frequency stimulation (LFS) via activation of the N-methyl-D-aspartic acid (NMDA) subtype ionotropic glutamate receptor (NMDAR). However, how HFS and LFS activate NMDARs and thereby lead to synaptic plasticity remains poorly understood and highly controversial. It is even more unclear how the bidirectional synaptic plasticity is produced and regulated in response to physiological or pathological changes.Functional NMDARs consist primarily of two GluN1 subunits and two GluN2 subunits, with GluN2A and GluN2B subunits being the most common NMDAR subunits found in the cortical and hippocampal regions of the adult brain (Cull-Candy et al, 2001). GluN2A and GluN2B subunits may confer distinct gating and pharmacological properties to NMDARs and couple them to distinct intracellular signalling machineries (Cull-Candy et al, 2001). Moreover, the ratio of these two subpopulations of NMDARs at the glutamatergic synapse is dynamically regulated in an activity-dependent manner (Bellone and Nicoll, 2007; Cho et al, 2009; Xu et al, 2009). Although controversial, GluN2A- and GluN2B-containing NMDARs have been suggested to have differential roles in regulating the direction of synaptic plasticity (Collingridge et al, 2004; Morishita et al, 2007). Among the factors shown to regulate NMDAR function, Src family tyrosine kinases may be the best characterized, with both Src and Fyn able to upregulate NMDAR function, and thus LTP induction (Salter and Kalia, 2004). However, if these kinases modulate NMDAR function in a NMDAR subunit-specific manner remains unknown. To explore this concept, Yang et al (2012) investigated the potential subunit-specific regulation of NMDARs by Src and Fyn using whole-cell patch clamp recording of NMDAR-mediated currents from acutely dissociated CA1 hippocampal neurons or from rat hippocampal slices. They found that intracellular perfusion of recombinant Src or Fyn increased the NMDAR-mediated currents. By applying subunit-preferential antagonists of GluN2A- or GluN2B-containing NMDARs, or by using neurons obtained from GluN2A knockout mice, they discovered that Src and Fyn differentially enhanced currents gated through GluN2A- and GluN2B-containing NMDARs, respectively.Can physiological or pathological factors differentially activate Src or Fyn, thereby exerting subunit-specific regulation of NMDAR function? To answer this question, Yang et al focused their investigation on the role of GPCRs, specifically pituitary adenylate cyclase activating peptide receptor (PAC1R) and dopamine D1 receptor (D1R), both of which have recently been shown to potentiate NMDARs through Src family kinases (Macdonald et al, 2005; Hu et al, 2010). Indeed, they found that activation of PAC1R specifically increased GluN2A-NMDAR-mediated currents without affecting currents gated through GluN2B-NMDARs, and this potentiation was prevented by the Src-specific inhibitory peptide Src(40–58) (Salter and Kalia, 2004). To rule out the contribution of Fyn, the authors developed a novel-specific Fyn inhibitory peptide Fyn(39–57), and demonstrated that it had little effect on PAC1R potentiation. In contrast, activation of D1R potentiated GluN2B- (but not GluN2A-) NMDAR-mediated currents, and this potentiation was specifically eliminated by Fyn(39–57), but not by Src(40–58). The authors further demonstrated that stimulation of PAC1Rs resulted in a selective activation of Src kinase and consequent tyrosine phosphorylation of the GluN2A subunit, whereas activation of D1Rs led to a specific increase in Fyn-mediated tyrosine phosphorylation of the GluN2B subunit. To provide convincing evidence that these subunit-differential modulations are indeed the result of tyrosine phosphorylation of the respective NMDAR subunits, the authors then performed electrophysiological experiments using neurons from two knockin mouse lines GluN2A(Y1325F) and GluN2B(Y1472F), in which the tyrosine phosphorylation residues in native GluN2A and GluN2B subunits were, respectively, replaced with non-phosphorylatable phenylalanine residues. As expected, the authors found that PAC1R-mediated potentiation of NMDA currents was lost in neurons from GluN2A(Y1325F) mice (but maintained in neurons from GluN2B(Y1472F) mice), while D1R-mediated enhancement of NMDA currents was only observed in neurons from GluN2A(Y1325F) mice. Together, as illustrated in Figure 1, the authors have made a very convincing case that PAC1R and D1R, respectively, enhance function of GluN2A- and GluN2B-containing NMDARs by differentially activating Src- and Fyn-mediated phosphorylation of respective NMDAR subunits.Open in a separate windowFigure 1GPCRs regulate the direction of synaptic plasticity via activating distinct signalling pathways. Synaptic NMDA receptors, both GluN2A- and GluN2B-containing, play key roles in the induction of various forms of synaptic plasticity at the hippocampal CA1 glutamatergic synapse. Under the basal level of GluN2A and GluN2B ratio, stimulation with a train of pulses at frequencies from 1 to 100 Hz produces a frequency and plasticity (LTD–LTP) curve, with maximum LTD and LTP being, respectively, induced at 1 and 100 Hz. Activation of PAC1R with its agonist PACAP38 activates Src and thereby results in tyrosine phosphorylation and consequent functional upregulation of GluN2A-containing NMDARs, resulting in an increase in the ratio of functional GluN2A and GluN2B. The increased ratio in turn causes a left shift of frequency and plasticity curve, favouring LTP induction. In contrast, activation of D1R by the receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF81297","term_id":"1156277425","term_text":"SKF81297"}}SKF81297 triggers Fyn-specific tyrosine phosphorylation and functional upregulation of GluN2B, causing a reduction of GluN2A and GluN2B ratio. This decreased ratio results in a right shift of the curve, favouring LTD induction. The ability of GPCRs to differentially activate distinct downstream signalling pathways involved in synaptic plasticity suggests the potential roles of GPCRs in governing the direction of synaptic plasticity.Given the coupling of NMDARs to the induction of synaptic plasticity, it is then reasonable to ask if activation of the two GPCRs can selectively affect the induction of LTP or LTD at CA1 synapses. Yang and colleagues investigated the effects of pharmacological activation of PAC1R and D1R on the induction of LTP and LTD by recording the field excitatory postsynaptic potentials from hippocampal slices. Consistent with differential roles of NMDAR subunits in governing directions of synaptic plasticity, the authors observed that activation of PAC1Rs reduces the induction threshold of LTP, while stimulation of D1Rs favours LTD induction (Figure 1). Facilitation of LTP by PAC1R and LTD by D1R were, respectively, prevented in the brain slices obtained from GluN2A(Y1325F) and GluN2B(Y1472F) knockin mice, supporting the differential involvements of Src-mediated GluN2A phosphorylation and Fyn-mediated GluN2B phosphorylation.Taken together, the authors'' results have demonstrated that activation of PAC1R and D1R can control the direction of synaptic plasticity at the hippocampal CA1 synapse by differentially regulating NMDAR_EPSCs in a subunit-specific fashion (Figure 1). Specifically, PAC1R enhances the function of GluN2A-containing NMDARs by increasing Src phosphorylation of GluN2A subunit at Y1325, whereas D1R upregulates GluN2B-containing NMDARs through increased Fyn phosphorylation of GluN2B at Y1472. Moreover, by regulating the ratio of functional GluN2A- and GluN2B-containing NMDARs, PAC1R and D1R in turn modulate the direction of synaptic plasticity, favouring the production of LTP and LTD, respectively.While consistent with the recently proposed hypothesis that GluN2A and GluN2B may have preferential roles in the induction of hippocampal CA1 LTP and LTD (Collingridge et al, 2004; but see also Morishita et al, 2007), the current study further emphasizes the importance of GluN2A/GluN2B ratios in regulating LTP and LTD thresholds: increased ratio favours LTP, while reduced ratio promotes LTD. However, this seems to contradict some recent studies where the reduction and increase in the GluN2A/GluN2B ratio appeared to, respectively, favour LTP (Cho et al, 2009; Xu et al, 2009) and LTD (Xu et al, 2009). Therefore, the direction of plasticity change is likely modulated not only by the GluN2A/GluN2B ratio, but also by additional factors such as experimental conditions, developmental stages, and brain regions.Under many experimental conditions, LTP and LTD are usually induced by HFS and LFS stimulating protocols, respectively, but it remains essentially unknown how LTP and LTD are physiologically or pathologically generated in animals. To this end, the identification of different GPCRs as the endogenous upstream regulators of NMDA receptor subpopulations, and hence regulators of synaptic plasticity, is the major novelty of Yang and colleagues'' work. Future studies are needed to investigate if and how PAC1R and/or D1R are critically involved in the production of LTP or LTD in animals under physiological or pathological conditions. Given the fact that Src family kinases may be required for LTP induced by HFS in hippocampal slices (Salter and Kalia, 2004), an equally intriguing question would be whether these GPCRs are actually required for LTP/LTD induced by HFS/LFS experimental paradigms. In line with this conjecture, it would be interesting to determine if ligands for various GPCRs co-exist in the glutamatergic presynaptic terminals and, if so, can be differentially co-released with glutamate in a frequency-dependent manner, thereby contributing to either HFS-induced LTP or LFS-induced LTD.The findings by Yang and colleagues establish an exciting mechanistic model by which GPCRs can govern the direction of synaptic plasticity by determining the contributions of GluN2A- and GluN2B-NMDARs through differential tyrosine phosphorylation of respective NMDA receptor subtypes. Additional studies further validating this model under physiological and pathological conditions will greatly improve our understanding of the molecular mechanisms underlying synaptic plasticity and cognitive brain functions. In addition, NMDARs, depending on their subunit composition and/or subcellular localization, may also have complex roles in mediating neuronal survival and death (Lai et al, 2011). Considering that neurotoxicity produced by over-activation of NMDARs is widely accepted to be a common mechanism for neuronal loss in a number of acute brain injuries and chronic neurodegenerative diseases, Yang and colleagues'' finding of the differential regulation of NMDAR subunits by different GPCRs could have wider implications beyond synaptic plasticity.     

GluN2B-containing NMDA receptors as possible targets for the neuroprotective and antidepressant effects of fluoxetine

Accumulating evidence has indicated the involvement of glutamatergic neurotransmission in the pathophysiology of excitotoxicity and in the mechanism of action of antidepressants. We have previously shown that tricyclic desipramine and the selective serotonin reuptake inhibitor fluoxetine inhibit NMDA receptors (NMDARs) in the clinically relevant, low micromolar concentration range. As the different subtypes of NMDARs are markedly different in their physiological and pathological functions, our aim was to investigate whether the effect of antidepressants is subtype-specific. Using whole-cell patch-clamp recordings in rat cortical cell cultures, we studied the age-dependence of inhibition of NMDA-induced currents after treatment with desipramine and fluoxetine, as the expression profile of the NMDAR subtypes changes as a function of days in vitro. We also investigated the inhibitory effect of these antidepressants on NMDA-induced currents in HEK 293 cell lines that stably expressed rat recombinant NMDARs with GluN1a/GluN2A or GluN1a/GluN2B subunit compositions. The inhibitory effect of desipramine was not age-dependent, whereas fluoxetine displayed a continuously decreasing inhibitory profile, which was similar to the GluN1/GluN2B subtype-selective antagonist ifenprodil. In HEK 293 cells, desipramine equally inhibited NMDA currents in both cell lines, whereas fluoxetine showed an inhibitory effect only in cells that expressed the GluN1/GluN2B subtype. Our data show that fluoxetine is a selective inhibitor of GluN2B-containing NMDARs, whereas desipramine inhibits both GluN1/GluN2A and GluN1/GluN2B subtypes. As the clinical efficacy of these drugs is very similar, the putative NMDAR-associated therapeutic effect of antidepressants may be mediated only via inhibition of the GluN2B-containing subtype. The manifestation of the GluN1/GluN2B-selectivity of fluoxetine suggests the neuroprotective potential for this drug in both acute and chronic neurodegenerative disorders.     

Modulation of synaptic plasticity by stress hormone associates with plastic alteration of synaptic NMDA receptor in the adult hippocampus

Stress exerts a profound impact on learning and memory, in part, through the actions of adrenal corticosterone (CORT) on synaptic plasticity, a cellular model of learning and memory. Increasing findings suggest that CORT exerts its impact on synaptic plasticity by altering the functional properties of glutamate receptors, which include changes in the motility and function of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype of glutamate receptor (AMPAR) that are responsible for the expression of synaptic plasticity. Here we provide evidence that CORT could also regulate synaptic plasticity by modulating the function of synaptic N-methyl-D-aspartate receptors (NMDARs), which mediate the induction of synaptic plasticity. We found that stress level CORT applied to adult rat hippocampal slices potentiated evoked NMDAR-mediated synaptic responses within 30 min. Surprisingly, following this fast-onset change, we observed a slow-onset (>1 hour after termination of CORT exposure) increase in synaptic expression of GluN2A-containing NMDARs. To investigate the consequences of the distinct fast- and slow-onset modulation of NMDARs for synaptic plasticity, we examined the formation of long-term potentiation (LTP) and long-term depression (LTD) within relevant time windows. Paralleling the increased NMDAR function, both LTP and LTD were facilitated during CORT treatment. However, 1-2 hours after CORT treatment when synaptic expression of GluN2A-containing NMDARs is increased, bidirectional plasticity was no longer facilitated. Our findings reveal the remarkable plasticity of NMDARs in the adult hippocampus in response to CORT. CORT-mediated slow-onset increase in GluN2A in hippocampal synapses could be a homeostatic mechanism to normalize synaptic plasticity following fast-onset stress-induced facilitation.     

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.     

Palmitoylation at Two Cysteine Clusters on the C-Terminus of GluN2A and GluN2B Differentially Control Synaptic Targeting of NMDA Receptors

Palmitoylation of NMDARs occurs at two distinct cysteine clusters in the carboxyl-terminus of GluN2A and GluN2B subunits that differentially regulates retention in the Golgi apparatus and surface expression of NMDARs. Mutations of palmitoylatable cysteine residues in the membrane-proximal cluster to non-palmitoylatable serines leads to a reduction in the surface expression of recombinant NMDARs via enhanced internalization of the receptors. Mutations in a cluster of cysteines in the middle of the carboxyl-terminus of GluN2A and GluN2B, leads to an increase in the surface expression of NMDARs via an increase in post-Golgi trafficking. Using a quantitative electrophysiological assay, we investigated whether palmitoylation of GluN2 subunits and the differential regulation of surface expression affect functional synaptic incorporation of NMDARs. We show that a reduction in surface expression due to mutations in the membrane-proximal cluster translates to a reduction in synaptic expression of NMDARs. However, increased surface expression induced by mutations in the cluster of cysteines that regulates post-Golgi trafficking of NMDARs does not increase the synaptic pool of NMDA receptors, indicating that the number of synaptic receptors is tightly regulated.     

Computational investigation of the changing patterns of subtype specific NMDA receptor activation during physiological glutamatergic neurotransmission

NMDA receptors (NMDARs) are the major mediator of the postsynaptic response during synaptic neurotransmission. The diversity of roles for NMDARs in influencing synaptic plasticity and neuronal survival is often linked to selective activation of multiple NMDAR subtypes (NR1/NR2A-NMDARs, NR1/NR2B-NMDARs, and triheteromeric NR1/NR2A/NR2B-NMDARs). However, the lack of available pharmacological tools to block specific NMDAR populations leads to debates on the potential role for each NMDAR subtype in physiological signaling, including different models of synaptic plasticity. Here, we developed a computational model of glutamatergic signaling at a prototypical dendritic spine to examine the patterns of NMDAR subtype activation at temporal and spatial resolutions that are difficult to obtain experimentally. We demonstrate that NMDAR subtypes have different dynamic ranges of activation, with NR1/NR2A-NMDAR activation sensitive at univesicular glutamate release conditions, and NR2B containing NMDARs contributing at conditions of multivesicular release. We further show that NR1/NR2A-NMDAR signaling dominates in conditions simulating long-term depression (LTD), while the contribution of NR2B containing NMDAR significantly increases for stimulation frequencies that approximate long-term potentiation (LTP). Finally, we show that NR1/NR2A-NMDAR content significantly enhances response magnitude and fidelity at single synapses during chemical LTP and spike timed dependent plasticity induction, pointing out an important developmental switch in synaptic maturation. Together, our model suggests that NMDAR subtypes are differentially activated during different types of physiological glutamatergic signaling, enhancing the ability for individual spines to produce unique responses to these different inputs.     

Synaptotagmin-7–mediated activation of spontaneous NMDAR currents is disrupted in bipolar disorder susceptibility variants

Synaptotagmin-7 (Syt7) plays direct or redundant Ca²⁺ sensor roles in multiple forms of vesicle exocytosis in synapses. Here, we show that Syt7 is a redundant Ca²⁺ sensor with Syt1/Doc2 to drive spontaneous glutamate release, which functions uniquely to activate the postsynaptic GluN2B-containing NMDARs that significantly contribute to mental illness. In mouse hippocampal neurons lacking Syt1/Doc2, Syt7 inactivation largely diminishes spontaneous release. Using 2 approaches, including measuring Ca²⁺ dose response and substituting extracellular Ca²⁺ with Sr²⁺, we detect that Syt7 directly triggers spontaneous release via its Ca²⁺ binding motif to activate GluN2B-NMDARs. Furthermore, modifying the localization of Syt7 in the active zone still allows Syt7 to drive spontaneous release, but the GluN2B-NMDAR activity is abolished. Finally, Syt7 SNPs identified in bipolar disorder patients destroy the function of Syt7 in spontaneous release in patient iPSC-derived and mouse hippocampal neurons. Therefore, Syt7 could contribute to neuropsychiatric disorders through driving spontaneous glutamate release.

Synaptotagmin-7 (Syt7) variants are associated with susceptibility to bipolar disorder; this study shows that Syt7 acts as a calcium sensor to drive spontaneous glutamate release which uniquely activates postsynaptic GluN2B-containing glutamate receptors. Interestingly, this function of Syt7 is disrupted in bipolar disorder susceptibility variants.     

Amyloid beta peptide 1-42 disturbs intracellular calcium homeostasis through activation of GluN2B-containing N-methyl-d-aspartate receptors in cortical cultures

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to debilitating cognitive deficits. Recent evidence demonstrates that glutamate receptors are dysregulated by amyloid beta peptide (Aβ) oligomers, resulting in disruption of glutamatergic synaptic transmission which parallels early cognitive deficits. Although it is well accepted that neuronal death in AD is related to disturbed intracellular Ca(2+) (Ca(2+)(i)) homeostasis, little is known about the contribution of NMDARs containing GluN2A or GluN2B subunits on Aβ-induced Ca(2+)(i) rise and neuronal dysfunction. Thus, the main goal of this work was to evaluate the role of NMDAR subunits in dysregulation of Ca(2+)(i) homeostasis induced by Aβ 1-42 preparation containing both oligomers (in higher percentage) and monomers in rat cerebral cortical neurons. The involvement of NMDARs was evaluated by pharmacological inhibition with MK-801 or the selective GluN2A and GLUN2B subunit antagonists NVP-AAM077 and ifenprodil, respectively. We show that Aβ, like NMDA, increase Ca(2+)(i) levels mainly through activation of NMDARs containing GluN2B subunits. Conversely, GluN2A-NMDARs antagonism potentiates Ca(2+)(i) rise induced by a high concentration of Aβ (1μM), suggesting that GluN2A and GluN2B subunits have opposite roles in regulating Ca(2+)(i) homeostasis. Moreover, Aβ modulate NMDA-induced responses and vice versa. Indeed, pre-exposure to Aβ (1μM) decrease NMDA-evoked Ca(2+)(I) rise and pre-exposure to NMDA decrease Aβ response. Interestingly, simultaneous addition of Aβ and NMDA potentiate Ca(2+)(I) levels, this effect being regulated by GluN2A and GluN2B subunits in opposite manners. This study contributes to the understanding of the molecular basis of early AD pathogenesis, by exploring the role of GluN2A and GluN2B subunits in the mechanism of Aβ toxicity in AD.     

Bicarbonate-Independent Sodium Conductance of Na/HCO3 Cotransporter NBCn1 Decreases NMDA Receptor Function

The sodium bicarbonate cotransporter NBCn1 is an electroneutral transporter with a channel activity that conducts Na⁺ in a HCO₃^–-independent manner. This channel activity was suggested to functionally affect other membrane proteins which permeate Na⁺ influx. We previously reported that NBCn1 is associated with the NMDA receptors (NMDARs) at the molecular and physiological levels. In this study, we examined whether NBCn1 channel activity affects NMDAR currents and whether this effect involves the interaction between the two proteins. NBCn1 and the NMDAR subunits GluN1A/GluN2A were expressed in Xenopus oocytes, and glutamate currents produced by the receptors were measured using two-electrode voltage clamp. In the absence of CO₂/HCO₃^–, NBCn1 channel activity decreased glutamate currents mediated by GluN1A/GluN2A. NBCn1 also decreased the slope of the current–voltage relationships for the glutamate current. Similar effects on the glutamate current were observed with and without PSD95, which can cluster NBCn1 and NMDARs. The channel activity was also observed in the presence of CO₂/HCO₃^–. We conclude that NBCn1 channel activity decreases NMDAR function. Given that NBCn1 knockout mice develop a downregulation of NMDARs, our results are unexpected and suggest that NBCn1 has dual effects on NMDARs. It stabilizes NMDAR expression but decreases receptor function by its Na⁺ channel activity. The dual effects may play an important role in fine-tuning the regulation of NMDARs in the brain.     

GluN2D‐containing NMDA receptors inhibit neurotransmission in the mouse striatum through a cholinergic mechanism: implication for Parkinson's disease

The GluN2 subunits that compose NMDA receptors (NMDARs) determine functional and pharmacological properties of the receptor. In the striatum, functions and potential dysfunctions of NMDARs attributed to specific GluN2 subunits have not been clearly elucidated, although NMDARs play critical roles in the interactions between glutamate and dopamine. Through the use of amperometry and field potential recordings in mouse brain slices, we found that NMDARs that contain the GluN2D subunit contribute to NMDA‐induced inhibition of evoked dopamine release and of glutamatergic neurotransmission in the striatum of control mice. Inhibition is likely mediated through increased firing in cholinergic interneurons, which were shown to express GluN2D. Indeed, NMDA‐induced inhibition of both dopamine release and glutamatergic neurotransmission is reduced in the presence of muscarinic receptor antagonists and is mimicked by a muscarinic receptor agonist. We have also examined whether this function of GluN2D‐containing NMDARs is altered in a mouse model of Parkinson's disease. We found that the inhibitory role of GluN2D‐containing NMDARs on glutamatergic neurotransmission is impaired in the 6‐hydroxydopamine lesioned striatum. These results identify a role for GluN2D‐containing NMDARs and adaptive changes in experimental Parkinsonism. GluN2D might constitute an attractive target for the development of novel pharmacological tools for therapeutic intervention in Parkinson's disease.

     

An alternating GluN1-2-1-2 subunit arrangement in mature NMDA receptors

NMDA receptors (NMDARs) form glutamate-gated ion channels that play a critical role in CNS physiology and pathology. Together with AMPA and kainate receptors, NMDARs are known to operate as tetrameric complexes with four membrane-embedded subunits associating to form a single central ion-conducting pore. While AMPA and some kainate receptors can function as homomers, NMDARs are obligatory heteromers composed of homologous but distinct subunits, most usually of the GluN1 and GluN2 types. A fundamental structural feature of NMDARs, that of the subunit arrangement around the ion pore, is still controversial. Thus, in a typical NMDAR associating two GluN1 and two GluN2 subunits, there is evidence for both alternating 1/2/1/2 and non-alternating 1/1/2/2 arrangements. Here, using a combination of electrophysiological and cross-linking experiments, we provide evidence that functional GluN1/GluN2A receptors adopt the 1/2/1/2 arrangement in which like subunits are diagonal to one another. Moreover, based on the recent crystal structure of an AMPA receptor, we show that in the agonist-binding and pore regions, the GluN1 subunits occupy a "proximal" position, closer to the central axis of the channel pore than that of GluN2 subunits. Finally, results obtained with reducing agents that differ in their membrane permeability indicate that immature (intracellular) and functional (plasma-membrane inserted) pools of NMDARs can adopt different subunit arrangements, thus stressing the importance of discriminating between the two receptor pools in assembly studies. Elucidating the quaternary arrangement of NMDARs helps to define the interface between the subunits and to understand the mechanism and pharmacology of these key signaling receptors.     

Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.     

Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo

During development there is an activity-dependent switch in synaptic N-Methyl-D-aspartate (NMDA) receptor subunit composition from predominantly GluN2B to GluN2A, though the precise role of this?switch remains unknown. By deleting GluN2 subunits in single neurons during synaptogenesis, we find that both GluN2B and GluN2A suppress AMPA receptor expression, albeit by distinct means. Similar to GluN1, GluN2B deletion increases the number of functional synapses, while GluN2A deletion increases the strength of unitary connections without affecting the number of functional synapses. We propose a model of excitatory synapse maturation in which baseline activation of GluN2B-containing receptors prevents premature synapse maturation until correlated activity allows induction of functional synapses. This activity also triggers the switch to GluN2A, which dampens further potentiation. Furthermore, we analyze the subunit composition of synaptic NMDA receptors in CA1 pyramidal cells, provide electrophysiological evidence for?a large population of synaptic triheteromeric receptors, and estimate the subunit-dependent open probability.     

NMDA receptors in GABAergic synapses during postnatal development

GABA (gamma-aminobutyric-acid), the main inhibitory neurotransmitter in the adult brain, exerts depolarizing (excitatory) actions during development and this GABAergic depolarization cooperates with NMDARs (N-methyl-D-aspartate receptors) to drive spontaneous synchronous activity (SSA) that is fundamentally important for developing neuronal networks. Although GABAergic depolarization is known to assist in the activation of NMDARs during development, the subcellular localization of NMDARs relative to GABAergic synapses is still unknown. Here, we investigated the subcellular distribution of NMDARs in association with GABAergic synapses at the developmental stage when SSA is most prominent in mice. Using multiple immunofluorescent labeling and confocal laser-scanning microscopy in the developing mouse hippocampus, we found that NMDARs were associated with both glutamatergic and GABAergic synapses at postnatal day 6-7 and we observed a direct colocalization of GABA(A)- and NMDA-receptor labeling in GABAergic synapses. Electron microscopy of pre-embedding immunogold-immunoperoxidase reactions confirmed that GluN1, GluN2A and GluN2B NMDAR subunits were all expressed in glutamatergic and GABAergic synapses postsynaptically. Finally, quantitative post-embedding immunogold labeling revealed that the density of NMDARs was 3 times higher in glutamatergic than in GABAergic synapses. Since GABAergic synapses were larger, there was little difference in the total number of NMDA receptors in the two types of synapses. In addition, receptor density in synapses was substantially higher than extrasynaptically. These data can provide the neuroanatomical basis of a new interpretation of previous physiological data regarding the GABA(A)R-NMDAR cooperation during early development. We suggest that during SSA, synaptic GABA(A)R-mediated depolarization assists NMDAR activation right inside GABAergic synapses and this effective spatial cooperation of receptors and local change of membrane potential will reach developing glutamatergic synapses with a higher probability and efficiency even further away on the dendrites. This additional level of cooperation that operates within the depolarizing GABAergic synapse, may also allow its own modification triggered by Ca(2+)-influx through the NMDA receptors.     

Leptin modulates pancreatic β-cell membrane potential through Src kinase–mediated phosphorylation of NMDA receptors

The adipocyte-derived hormone leptin increases trafficking of K_ATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca²⁺ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.