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Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

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Abstract

The subunit composition of synaptic NMDA receptors (NMDAR), such as the relative content of GluN2A- and GluN2B-containing receptors, greatly influences the glutamate synaptic transmission. Receptor co-agonists, glycine and D-serine, have intriguingly emerged as potential regulators of the receptor trafficking in addition to their requirement for its activation. Using a combination of single-molecule imaging, biochemistry and electrophysiology, we show that glycine and D-serine relative availability at rat hippocampal glutamatergic synapses regulate the trafficking and synaptic content of NMDAR subtypes. Acute manipulations of co-agonist levels, both ex vivo and in vitro, unveil that D-serine alter the membrane dynamics and content of GluN2B-NMDAR, but not GluN2A-NMDAR, at synapses through a process requiring PDZ binding scaffold partners. In addition, using FRET-based FLIM approach, we demonstrate that D-serine rapidly induces a conformational change of the GluN1 subunit intracellular C-terminus domain. Together our data fuels the view that the extracellular microenvironment regulates synaptic NMDAR signaling.

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... In contrast, synaptic insertion of GluN2B occurred even when agonist or co-agonist binding was inhibited. In another study tracking NMDAR subunits in cultured hippocampal neurons using antibody-conjugated quantum dots, D-serine was shown to decrease the lateral mobility of GluN2B-, but not GluN2A-containing NMDARs even when ion flow through the NMDAR was blocked by AP5 (Ferreira et al., 2017;Papouin et al., 2012). Furthermore, glycine was shown to decrease the lateral mobility of GluN2A-, but not GluN2B-, containing NMDARs independent of NMDAR currents (Ferreira et al., 2017;Papouin et al., 2012). ...
... In another study tracking NMDAR subunits in cultured hippocampal neurons using antibody-conjugated quantum dots, D-serine was shown to decrease the lateral mobility of GluN2B-, but not GluN2A-containing NMDARs even when ion flow through the NMDAR was blocked by AP5 (Ferreira et al., 2017;Papouin et al., 2012). Furthermore, glycine was shown to decrease the lateral mobility of GluN2A-, but not GluN2B-, containing NMDARs independent of NMDAR currents (Ferreira et al., 2017;Papouin et al., 2012). Thus, the relative local abundance of glutamate, glycine and D-serine can act to tune NMDAR trafficking and synaptic insertion, and thus NMDAR signaling, independent of ion flux. ...
... To assess conformational movements of the NMDAR during non-ionotropic NMDAR signaling, several studies have used FRET to monitor movements of the C terminus of the NMDAR. These studies reported that NMDA or glutamate binding to the NMDAR drives GluN1 C-tails to move away from each other, independent of NMDAR ion flux Dore et al., 2015;Ferreira et al., 2017). Moreover, antibody-mediated restriction of this movement inhibited ion flux-independent NMDAR-LTD . ...
Article
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NMDA receptors play vital roles in a broad array of essential brain functions, from synaptic transmission and plasticity to learning and memory. Historically, the fundamental roles of NMDARs were attributed to their specialized properties of ion flux. More recently, it has become clear that NMDARs also signal in an ion flux-independent manner. Here, we review these non-ionotropic NMDAR signaling mechanisms that have been reported to contribute to a broad array of neuronal functions and dysfunctions including synaptic transmission and plasticity, cell death and survival, and synaptic alterations associated with neurological disorders. This article is part of the Neuropharmacology Special Issue on ‘Glutamate Receptors – NMDA receptors’.
... Cells were acutely exposed to CSF collected from patients with various neurological, inflammatory, and psychiatric conditions (Fig. 1a), and the membrane dynamics of synaptic NMDAR were recorded (see "Methods" section). In control condition (untreated neurons), surface NMDAR diffuse along dendritic segments and were more confined in synapses and spines (Fig. 1b), as previously reported [40,41]. NMDAR surface diffusion was not significantly affected in presence of any of the patients' CSF but the SCZSD CSF. ...
... To address this possibility, we labeled surface NMDAR in live hippocampal neurons exposed to CSF from either psychiatric (i.e., SCZSD) or subarachnoid hemorrhage condition. As previously shown [38,40], most of the surface NMDAR clusters colocalise with postsynaptic markers, such as Homer 1c (Fig. 2b). ...
... Although there is currently no straightforward answer to this question, one can speculate that the receptor agonist, co-agonist, and cytokines are, among others, putative contributors to the cellular phenotype. Indeed, NMDA, Dserine, and glycine have been shown to significantly decrease the surface dynamics of NMDAR onto hippocampal neurons [40,63,64]. The reported low levels of NMDAR agonist and coagonists in the CSF of patients with SCZSD would thus upregulate the receptor surface dynamics. ...
Article
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A dysfunction of the glutamatergic transmission, especially of the NMDA receptor (NMDAR), constitutes one of the main biological substrate of psychotic disorders, such as schizophrenia. The NMDAR signaling hypofunction, through genetic and/or environmental insults, would cause a neurodevelopmental myriad of molecular, cellular, and network alterations that persist throughout life. Yet, the mechanisms underpinning NMDAR dysfunctions remain elusive. Here, we compared the membrane trafficking of NMDAR in three gold-standard models of schizophrenia, i.e., patient’s cerebrospinal fluids, genetic manipulations of susceptibility genes, and prenatal developmental alterations. Using a combination of single nanoparticle tracking, electrophysiological, biochemical, and behavioral approaches in rodents, we identified that the NMDAR trafficking in hippocampal neurons was consistently altered in all these different models. Artificial manipulations of the NMDAR surface dynamics with competing ligands or antibody-induced receptor cross-link in the developing rat brain were sufficient to regulate the adult acoustic startle reflex and compensate for an early pathological challenge. Collectively, we show that the NMDAR trafficking is markedly altered in all clinically relevant models of psychosis, opening new avenues of therapeutical strategies.
... Notably, chronic depletion of SR (>45 days) in the post-synaptic neuron did not change the synaptic content of D-serine but, over time, altered the synaptic content of GluN2B [87]. As D-serine can alter the mobility of GluN2B in the membrane [88], this may indicate that neurons regulate extracellular D-serine at a site apart from the synaptic cleft, potentially controlling the subunit composition of synaptic NMDARs in adult CA1 pyramidal neurons. These findings suggest that neuronal L-serine and SR are critical for LTP but may not involve the co-agonism of synaptic NMDARs. ...
... Although blocking asc-1 only modestly enhanced synaptic NMDAR function, this is consistent with the observation that the sub-saturated co-agonist sites are held close to saturation [9,10]. Extracellular D-serine has several proposed functions, including co-agonism of synaptic NMDARs [203], regulating trafficking of extra-synaptic NMDARs [88], and a pool for shuttling from glutamatergic to GABAergic neurons [79]. Given these distinct roles and the differential regulation of uptake, spatially distinct pools of extracellular D-serine may exist and be served by different pools of transporters ( Figure 3). ...
... The level of NMDA receptor glycine site occupancy under steady-state conditions or low activity is synapse-dependent [8,10,13,103,104], indicating a circuit-dependent role for the glycine site. In addition, the identity of the co-agonist, glycine or D-serine, is synapse-specific [8,12,227,228], developmentally regulated [88,228], and possibly activitydependent [9,12,145,228]. These patterns are tied to the NMDAR subtypes expression pattern at specific synapses [88,228]. ...
Article
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Synaptic plasticity is an extensively studied cellular correlate of learning and memory in which NMDARs play a starring role. One of the most interesting features of NMDARs is their ability to act as a co-incident detector. It is unique amongst neurotransmitter receptors in this respect. Co-incident detection is possible because the opening of NMDARs requires membrane depolarisation and the binding of glutamate. Opening of NMDARs also requires a co-agonist. Although the dynamic regulation of glutamate and membrane depolarization have been well studied in coincident detection, the role of the co-agonist site is unexplored. It turns out that non-neuronal glial cells, astrocytes, regulate co-agonist availability, giving them the ability to influence synaptic plasticity. The unique morphology and spatial arrangement of astrocytes at the synaptic level affords them the capacity to sample and integrate information originating from unrelated synapses, regardless of any pre-synaptic and post-synaptic commonality. As astrocytes are classically considered slow responders, their influence at the synapse is widely recognized as modulatory. The aim herein is to reconsider the potential of astrocytes to participate directly in ongoing synaptic NMDAR activity and co-incident detection.
... Canonical signaling by NMDARs is mediated by its ionotropic function initiated through simultaneous binding of two molecules of each of the coagonists glycine (or D-serine) and glutamate to the ligand-binding domains in extracellular regions of the receptor, which produces conformational changes that open the cationic conductance pathway of the receptor complex (3,4). However, a growing body of evidence is increasingly demonstrating nonionotropic signaling by NMDARs, signaling which is not mediated by opening of the ionic conductance pathway but which is nevertheless caused through conformational changes that are transmitted across the membrane resulting in molecular rearrangements and signaling within the cell (5)(6)(7)(8)(9)(10). Nonionotropic signaling by NMDARs, often referred to as metabotropic signaling of the receptor, is increasingly implicated in development, physiology, and disease, and in novel actions of CNS drugs (11)(12)(13). ...
... A striking example of nonionotropic NMDAR signaling is that glycine binding to its cognate extracellular site on GluN1, without binding of glutamate to its cognate site, drives intracellular signaling through recruiting the AP2 endocytic adaptor complex (10,12,26). Activating the glycine-binding site has been shown to cause transmembrane changes resulting in conformational rearrangement of the receptor C-terminal domain (7). Given the diversity of GluN1 isoforms in the CNS and linkage of splicing to disease (23,27), we focused on effects of alternative splicing of GRIN1 on glycine-induced signaling by NMDARs. ...
... Additional examples of nonionotropic signaling of NMDARs stimulated by glycine or D-serine are increasingly reported (7,32,41). Our findings altogether uncover a previously unanticipated molecular function for the N1 cassette, encoded by exon 5, in controlling nonionotropic glycine site signaling through the GluN1 subunit of NMDARs. ...
Article
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Significance N -methyl-D-aspartate receptors (NMDARs), which are critical in the brain, are increasingly being shown to signal without ion flux (i.e., “metabotropically”). What controls the metabotropic function of NMDARs is unknown. We discovered that a form of metabotropic signaling—glycine priming—is controlled by alternative splicing of the mRNA encoding one NMDAR subunit, GluN1. Our discovery was surprising because the spliced exon encodes a peptide cassette in the extracellular region of GluN1 far from the plasma membrane, and yet, metabotropic function requires signaling across the neuronal membrane. Moreover, we found that this metabotropic function of NMDARs is neuron cell–type specific: excitatory neurons show glycine priming, whereas inhibitory neurons do not. These findings have widespread implications for NMDARs in health and disease.
... Synaptic and extrasynaptic NMDAR are preferentially gated by different co-agonists, D-serine and glycine, respectively. Each co-agonist differentially impacts NMDAR surface trafficking and influences the distribution of NMDAR throughout synaptic and extrasynaptic areas (Papouin et al., 2012;Ferreira et al., 2017). The activation of extrasynaptic NMDAR is implicated in several brain diseases, as extrasynaptic / GluN2B-containing NMDAR can trigger cell-death associated signalling pathways (Giles E Parsons and Raymond, 2014). ...
... Interestingly, using the same FRET approach as Dore, Aow and Malinow, Ferreira and colleagues showed that D-serine can lead to a conformational change in NMDAR, which is counteracted by agonist binding to the receptor. High levels of D-serine or an increase in D-serine/glycine ratio decreases GluN1/2B-NMDAR surface mobility and synaptic GluN1/2B-NMDAR content (Ferreira et al., 2017). Of note, this may correspond to NMDAR ionotropic functions. ...
... Importantly, there is a developmental switch on the co-agonist which gates synaptic NMDAR from glycine to D-serine during development, closely paralleling the GluN2B to GluN2A switch on synaptic NMDAR composition (Bellone and Nicoll, 2007;Le Bail et al., 2015;Ferreira et al., 2017). These observations suggest that co-agonists steer the spatial segregation of NMDAR subtypes through modulations of NMDAR surface trafficking. ...
Thesis
N-Methyl-D-Aspartate glutamate receptors (NMDAR) are key actors of excitatory synaptic transmission, synaptic plasticity and higher brain functions such as memory formation and learning. As a consequence, NMDAR dysfunctions are associated to pathological states and high investments have been made to develop modulators of NMDAR activity for clinical applications. While some NMDAR antagonists such as ketamine (anesthetic, antidepressant) or memantine (prescribed as a treatment for Alzheimer’s disease) have proven of great medical value, their clinical use is often limited by severe adverse effects (e.g. psychotic-like states induced by ketamine) and several questions regarding their action mode - including why some antagonists exhibit psychoactive properties when others do not - remain unanswered. Accumulating evidence suggests that beyond their channel function, physiological and pathological NMDAR signaling may involve non-canonical pathways independent from ion flux. Using a combination of epifluorescence, FRET-FLIM, biochemistry and single molecule localization microscopy approaches, we investigated the impact of competitive (D-AP5, CPP) and uncompetitive (MK-801, ketamine, memantine) NMDAR antagonists on the properties, redistribution and subsynaptic organization of surface NMDAR and their cytosolic partners in hippocampal neurons. We found that while all antagonists produce comparable inhibition of NMDAR ionotropic activity, exposure to the psychotomimetic blockers MK-801 and ketamine selectively triggers changes in the conformation of NMDAR. Interestingly, these conformational rearrangements were associated with a decreased surface diffusion and an increased residency time of receptors at synapses, suggesting MK-801 and ketamine binding possibly enhance NMDAR synaptic anchoring. Although drug exposure (1h) did not change the overall receptor abundance at excitatory synapses, super-resolution imaging revealed profound and antagonist-specific nanoscale reorganizations of synaptic NMDAR clusters, with exposure to the competitive antagonist D-AP5 causing a reduction in the size and an increase in the density of receptor nanodomains while inhibition by the uncompetitive psychotomimetic blockers MK-801 and ketamine triggered an enlargement of receptor nanodomains, and exposure to memantine prompted the fragmentation of these nanodomains. Moreover, we found that MK-801 and ketamine selectively enhanced the mobility of Ca2+/calmodulin-dependent protein kinase II (CaMKII) within dendritic spines through an action mode that relies on the direct interaction between both partners, suggesting that drug-induced receptor redistributions may impact the intracellular dynamics and organization of downstream signaling partners of NMDAR. Altogether, our results provide evidence that besides inhibition of ion fluxes through the receptors, competitive and uncompetitive antagonists have a different impact on NMDAR surface dynamics and subsynaptic organization, and suggest that the psychoactive blockers MK-801 and ketamine may act on receptor function through non-canonical rearrangements in the organization of NMDAR signaling complexes.
... Extracellular NMDAR surface dynamics is regulated by components of the extracellular matrix such as matrix metalloproteinase 9, tissue plasminogen activator, stress hormone (corticosterone), and sex hormone (estrogen; Groc et al., 2008;Michaluk et al., 2009;Lesept et al., 2016;Potier et al., 2016;Mikasova et al., 2017). In addition, the NMDAR coagonists, glycine and D-serine, differentially regulate the surface dynamics of both synaptic and extrasynaptic NMDARs (Fig. 1 C;Papouin and Oliet, 2014;Ferreira et al., 2017). For instance, D-serine alters the surface dynamics and synaptic content of GluNB-NMDAR, but not GluN2A-NMDAR, through interaction between its C-terminus and PDZ-binding scaffold proteins (Ferreira et al., 2017). ...
... In addition, the NMDAR coagonists, glycine and D-serine, differentially regulate the surface dynamics of both synaptic and extrasynaptic NMDARs (Fig. 1 C;Papouin and Oliet, 2014;Ferreira et al., 2017). For instance, D-serine alters the surface dynamics and synaptic content of GluNB-NMDAR, but not GluN2A-NMDAR, through interaction between its C-terminus and PDZ-binding scaffold proteins (Ferreira et al., 2017). Other, yet unknown, molecules of the extracellular environment could also be potent regulators of NMDAR dynamics, e.g., polyamines, protons, zinc, or neurosteroids, as they all contribute to NMDAR-mediated synaptic regulation (Paoletti et al., 2013;Hansen et al., 2018). ...
... Although the exact molecular mechanism underpinning the regulation of such protein-protein interaction remain unclear, a change in the conformational structure of the receptors that impacts their interaction is a likely scenario. In support, the activation of NMDARs alters the intracellular C-terminus conformation that impairs direct interaction with PDZ scaffold proteins (Ferreira et al., 2017). Whether such a molecular scenario applies to the other members of the NMDAR cis-interactome remains an open question. ...
Article
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Understanding neurotransmitter system crosstalk in the brain is a major challenge in neurobiology. Several intracellular and genomic cascades have been identified in this crosstalk. However, the discovery that neurotransmitter receptors are highly diffusive in the plasma membrane of neurons, where they form heterocomplexes with other proteins, has profoundly changed our view of neurotransmitter signaling. Here, we review new insights into neurotransmitter crosstalk at the plasma membrane. We focus on the membrane organization and interactome of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) that plays a central role in excitatory synaptic and network physiology and is involved in the etiology of several major neuropsychiatric disorders. The nanoscale organization and dynamics of NMDAR is a key regulatory process for glutamate synapse transmission, plasticity, and crosstalk with other neurotransmitter systems, such as the monoaminergic ones. The plasma membrane appears to be a prime regulatory compartment for spatial and temporal crosstalk between neurotransmitter systems in the healthy and diseased brain. Understanding the molecular mechanisms regulating membrane neurotransmitter receptor crosstalk will likely open research avenues for innovative therapeutical strategies.
... This is consistent with a reduced level of occupancy of the NMDAR co-agonist binding site in 3xTg-AD mice because of a reduced level of D-serine within the cleft. Another possibility would be that the NMDAR subunit composition was switched from GluN2B to GluN2A in 3xTg-AD mice, since GluN2A-containing NMDARs have a lower affinity for D-serine than GluN2B receptors (Ferreira et al., 2017). In that case, the number of NMDARs recruited during synaptic stimulation could be reduced even if endogenous D-serine levels were unchanged. ...
... The majority of l-serine production comes from de novo catabolism of the glycolytic intermediate 3PG. Using [U- 13 C]-glucose, it was found that a substantial fraction of glycolytic carbon is indeed transferred to serine and that the two \uxes (glycolysis and PP) are comparable (Locasale et al., 2011). The fraction of 3PG diverted toward serine synthesis remains unknown in the brain and we are far from a full understanding of the co-ordination of glycolysis and serine biosynthetic pathways. ...
... How can we integrate this information in the context of the brain? Astrocytes share many metabolic similarities with cancer cells (Belanger et al., 2011). A recent study demonstrated that astrocytes express PKM2 instead of the usual expression of PKM1 in differentiated cells . ...
Thesis
Les pertes de mémoire et les changements comportementaux sont les premiers signes de la maladie d’Alzheimer (MA). Les patients présentent souvent un métabolisme du glucose diminué, observable par TEP 18F-fluorodéoxyglucose. L’implication de cette perturbation du métabolisme du glucose dans la pathogenèse de la MA n’est pas connue. Dans le cerveau, le glucose est oxydé pour produire l’ATP nécessaire à l’activité synaptique. Cependant, des observations récentes suggèrent qu’une altération de la glycolyse aérobie sur-vient de façon précoce dans la MA. Le phosphorylated pathway dévie le 3-phosphoglycérate du flux glycolytique pour la production de novo de L-sérine, via trois enzymes (PHGDH, PSAT1, PSPH). Les astrocytes sont la principale source de L-sérine dans le cerveau. Nous émettons l’hypothèse qu’une perturbation de la production astrocytaire de L-sérine, due à l’altération du métabolisme énergétique, puisse participer à l’établissement des déficits observés dans la MA. Nous avons utilisé le modèle murin 3xTg-AD, récapitulant les déficits métaboliques et synaptiques en plus des pathologies tau et amyloïde de la MA. Nous avons mis en évidence une diminution de la concentration en L-sérine dans l’hippocampe de ces souris. En conséquence, nous les avons nourries avec un régime enrichi à 10% en L-sérine pendant deux mois, ayant pour résultat d’améliorer leurs déficits synaptique et cognitif. Dans le but de comprendre les mécanismes sous-jacents à cet effet bénéfique de la L-sérine, nous nous sommes intéressés au métabolisme des lipides. Par analyse 3D de la morphologie cellulaire, nous avons mis en évidence une diminution du territoire couvert par les astrocytes de ces souris, déficit pouvant être amélioré par le régime L-sérine. Pour identifier l’impact de cette diminution sur la neurotransmission, nous avons évalué la couverture astrocytaire des synapses de l’hippocampe par microscopie électronique. Ensemble, ces résultats suggèrent un rôle critique de la L-sérine astrocytaire dans la plasticité synaptique et la mémoire.
... Peptide application TAT-peptides [39,40] [TAT-NS: control, scramble sequence; TAT-GluN2B15: TAT-sequence (YGRKKRRQRRR) -GluN2B C-terminus sequence (NGH VYEKLSSIESDV), CASOL] were administered at 5 µM into culture medium of the electroporated neurons, once every other days from DIV7-12, for four applications in total. ...
... α-Syn proteins (monomer or PFF) were incubated for 20 min before QD labeling and acquisitions. The calculations of single QD tracking followed the formulas and the rules described previously [38,39]. Brie y, the instantaneous diffusion coe cient 'D' was calculated for each trajectory, from linear ts of the rst four points of the mean-square-displacement versus time function using MSD(t) = < r2 > (t) = 4Dt. ...
... Synaptosomes components were puri ed from homogenized hippocampus of 10-month-old WT-and A53T-α-syn transgenic mice, following the methods as previously described [39]. Hippocampal tissues (around 20 mg) were thawed in 300 µL fresh TPS buffer (0.32M sucrose 4 mM HEPES buffer, pH7.4) supplemented with a protease inhibitor cocktail with 15 strokes in glass-Te on homogenizer (500 µL), on the ice. ...
Preprint
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Background α-Synuclein misfolding and aggregation contribute to synaptic dysfunction in synucleinopathies, including Parkinson’s disease. However, the mechanism underlying the effect of α-synuclein on synaptic components remains unclear. Since the N-methyl-D-aspartic acid receptor (NMDAR) plays a key role in glutamate synapse pathophysiology, we here investigated its surface dynamics and functional distribution in neurons exposed to various pathological α-synuclein forms. Methods A combination of single-molecule tracking, immunochemistry, immunoblot and calcium imaging approaches were used to assess the changes in NMDAR membrane dynamics and functions. The NMDAR alterations were evaluated in rat cultured hippocampal networks, in which α-synuclein mutants were overexpressed or exposed to α-synuclein proteins (monomeric/PFF α-synuclein). The surface dynamics of NMDAR subtype was artificially tuned in order to test its instrumental role. Results We observed that mutant α-synuclein (A53T-α-synuclein) restricted NMDAR surface trafficking and impaired synaptic function. In contrast, wild-type α-synuclein did not affect synaptic NMDAR. Further, we found that chronic exposure to α-synuclein preformed fibrils induced molecular dysfunctions that mainly targeted the GluN2B-NMDAR subtype. The deficits of synaptic NMDAR have also been found in A53T transgenic mice α-synuclein. Upon fine-tuning of the surface dynamics of GluN2B-NMDAR, pathological α-synuclein gradually lost its synaptic toxicity. Conclusions Our findings indicate that pathological α-synuclein alters GluN2B-NMDAR synaptic dynamics and organization, which leads to glutamate synapse dysfunction.
... This partitioning suggests that specific NMDAR composition uniquely impacts neuronal integration of synaptic and extrasynaptic inputs (Groc et al. 2009). Previous work has found that the GluN2A and GluN2B subtypes are dynamically trafficked into and out of the synaptic membrane by NMDAR agonists (Barria and Malinow 2002) and co-agonists (Nong et al. 2003;Ferreira et al. 2017). Thus, NMDARs (particularly those containing the GluN2B subtype) are highly mobile and can exchange between synaptic and extrasynaptic sites (Tovar and Westbrook 2002;Ferreira et al. 2017). ...
... Previous work has found that the GluN2A and GluN2B subtypes are dynamically trafficked into and out of the synaptic membrane by NMDAR agonists (Barria and Malinow 2002) and co-agonists (Nong et al. 2003;Ferreira et al. 2017). Thus, NMDARs (particularly those containing the GluN2B subtype) are highly mobile and can exchange between synaptic and extrasynaptic sites (Tovar and Westbrook 2002;Ferreira et al. 2017). ...
... As reviewed by Dore and colleagues (Dore et al. 2017), the existence and biological relevance of metabotropic NMDAR signaling is an active area of research with several investigators finding both supportive (Yang et al. 2004;Kessels et al. 2013;Nabavi et al. 2013;Tamburri et al. 2013;Dore et al. 2015;Kim et al. 2015;Stein et al. 2015) and contradictory (Babiec et al. 2014;Volianskis et al. 2015;Sanderson et al. 2016) evidence. Among the former, several studies have revealed ion flux-independent trafficking of GluN2-containing NMDARs both by NMDAR agonists (Vissel et al. 2001;Barria and Malinow 2002) and by coagonists (Nong et al. 2003;Ferreira et al. 2017). Together, these data add to the rich repertoire by which neuronal communication can be shaped by a growing number of NMDAR modulatory mechanisms. ...
Article
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N‐Methyl‐D‐Aspartate receptors (NMDARs) mediate both physiological and pathophysiological processes, though selective ligands lack broad clinical utility. NMDARs are composed of multiple subunits, but GluN2 is predominately responsible for functional heterogeneity. Specifically, the GluN2A‐ and GluN2B‐containing subtypes are enriched in adult hippocampus and cortex and impact neuronal communication via dynamic trafficking into and out of the synapse. We sought to understand if NYX‐2925, a novel NMDAR modulator, alters synaptic levels of GluN2A‐ or GluN2B‐containing NMDARs. Low‐picomolar NYX‐2925 increased GluN2B colocalization with the excitatory post‐synaptic marker PSD‐95 in rat primary hippocampal neurons within 30 min. Twenty‐four hours following oral administration, 1 mg/kg NYX‐2925 increased GluN2B in PSD‐95‐associated complexes ex vivo, and low‐picomolar NYX‐2925 regulated numerous trafficking pathways in vitro. Because the NYX‐2925 concentration that increases synaptic GluN2B was markedly below that which enhances long‐term potentiation (mid‐nanomolar), we sought to elucidate the basis of this effect. Although NMDAR‐dependent, NYX‐2925‐mediated colocalization of GluN2B with PSD‐95 occurred independent of ion flux, as colocalization increased in the presence of either the NMDAR channel blocker MK‐801 or glycine site antagonist 7CK. Moreover, while mid‐nanomolar NYX‐2925 concentrations, which do not increase synaptic GluN2B, enhanced calcium transients, functional plasticity was only enhanced by picomolar NYX‐2925. Thus, NYX‐2925 concentrations that increase synaptic GluN2B facilitated the chemical long‐term potentiation (chemLTP) induced insertion of synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor GluA1 subunit levels. Basal (unstimulated by chemLTP) levels of synaptic GluA1 were only increased by mid‐nanomolar NYX‐2925. These data suggest that NYX‐2925 facilitates homeostatic plasticity by initially increasing synaptic GluN2B via metabotropic‐like NMDAR signaling. This article is protected by copyright. All rights reserved.
... A decrease in the synaptic residency time of GluN2B-NMDARs is observed during development, accompanied by stronger synaptic stabilization of GluN2A-NMDARs in later stages of development (Groc et al., 2006). Extracellular regulators, such as NMDAR coagonists and extracellular matrix proteins, also affect receptor mobility (Groc et al., 2007;Papouin et al., 2012;Ferreira et al., 2017). D-serine and glycine have been shown to differentially modulate the surface behavior of GluN2 subunits with a preferential negative effect of glycine on GluN2A-NMDARs while D-serine decreases GluN2B-NMDAR surface diffusion (Papouin et al., 2012;Ferreira et al., 2017). ...
... Extracellular regulators, such as NMDAR coagonists and extracellular matrix proteins, also affect receptor mobility (Groc et al., 2007;Papouin et al., 2012;Ferreira et al., 2017). D-serine and glycine have been shown to differentially modulate the surface behavior of GluN2 subunits with a preferential negative effect of glycine on GluN2A-NMDARs while D-serine decreases GluN2B-NMDAR surface diffusion (Papouin et al., 2012;Ferreira et al., 2017). The extracellular matrix protein reelin specifically reduces GluN2B-NMDAR mobility while increasing its time spent within the synaptic area (Groc et al., 2007). ...
... The following primary antibodies were used: anti-flag (Sigma-Aldrich #F1804, 2 µg/ml), anti-GluN2A (Agrobio, custom-made, epitope: GHSHDVTERELRN, 0.1 mg/ml, [Ferreira et al., 2017]), anti-GluN2B (Agrobio, custom-made, epitope: ...
Thesis
NMDA-type glutamate receptors (NMDARs) are a type of ion permeable channels playing critical roles in excitatory neurotransmission in the central nervous system by mediating different forms of synaptic plasticity, a mechanism thought to be the molecular basis of neuronal development, learning and memory formation. NMDARs form tetramers in the postsynaptic membrane, most generally associating two obligatory GluN1 subunits and two modulatory GluN2 (GluN2A-D) or GluN3 (GluN3A-B) subunits. In the hippocampus, the dominant GluN2 subunits are GluN2A and GluN2B, displaying different expression patterns, with GluN2B being highly expressed in early development while GluN2A levels increase gradually during postnatal development. In the forebrain, the plastic processes mediated by NMDARs, such as the adaptation of glutamate synapses and excitatory neuronal networks, mostly rely on the relative implication of GluN2A- and GluN2B-containing NMDARs that have different signaling properties. Although the molecular regulation of synaptic NMDARs has been under intense investigation over the last decades, the exact topology of these two subtypes within the postsynaptic membrane has remained elusive. Here we used a combination of super-resolution microscopy techniques such as direct stochastic optical reconstruction microscopy (dSTORM) and stimulated emission depletion (STED) microscopy to characterize the surface distribution of GluN2A- or GluN2B-containing NMDARs. Both dSTORM and STED microscopy, based on different principles, enable to overcome the resolution barrier due to the diffraction limit of light. Using these techniques, we here unveil a differential nanoscale organization of native GluN2A- and GluN2B-NMDARs in rat hippocampal neurons. Both NMDAR subtypes are organized in nanoscale structures (termed nanodomains) that differ in their number, area, and shape. These observed differences are also maintained in synaptic structures. During development of hippocampal cultures, the membrane organization of both NMDAR subtypes evolves, with marked changes for the topology of GluN2A-NMDARs. Furthermore, GluN2A- and GluN2B-NMDAR nanoscale organizations are differentially affected by alterations of either interactions with PDZ scaffold proteins or CaMKII activity. The regulation of GluN2A-NMDARs mostly implicates changes in the number of receptors in fixed nanodomains, whereas the regulation of GluN2B-NMDARs mostly implicates changes in the nanodomain topography with fixed numbers of receptors. Thus, GluN2A- and GluN2B-NMDARs have distinct organizations in the postsynaptic membrane, likely implicating different regulatory pathways and signaling complexes.
... Our observation of increased synaptic enrichment of NMDARs relative to WT within the hippocampus of young mice is consistent with previous studies on SRKO mice showing increased expression of GluN1 (Balu and Coyle, 2011;Mustafa et al., 2010) and GluN2B (Basu et al., 2009;Wong et al., 2020). These changes in the overall number and composition of NMDARs may be a direct consequence of reduced D-serine levels, as prior studies have demonstrated the role of co-agonist binding in priming of the NMDAR for endocytosis, with D-serine specifically acting on GluN2B subunits (Ferreira et al., 2017;Nong et al., 2003). We found that the increase in synaptic NMDAR levels was associated with an increase in the magnitude of non-ionotropic signaling in SRKO, which would be expected to drive enhanced spine destabilization and shrinkage (Stein et al., 2015;Stein et al., 2021). ...
... Another study implicated mammalian target of rapamycin complex 1 (mTORC1), which is important for protein synthesis, in spine shrinkage induced by non-ionotropic NMDAR signaling (Thomazeau et al., 2020). Furthermore, studies using FRET to monitor movements of the C terminus of the NMDAR, have shown that agonist (glutamate or NMDA) binding drives the GluN1 C-tails to move away from each other, independent of NMDAR ion flux Dore et al., 2015;Ferreira et al., 2017). Intriguingly, over-expression of PSD-95 is sufficient to block both agonist-induced NMDAR conformational changes and non-ionotropic NMDAR-LTD, leading to a proposal that PSD-95 plays an important role linking NMDAR conformational changes to ion flux-independent downstream signaling (Dore and Malinow, 2020). ...
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Schizophrenia is a psychiatric disorder that affects over 20 million people globally. Notably, schizophrenia is associated with decreased density of dendritic spines and decreased levels of d-serine, a co-agonist required for opening of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that lowered d-serine levels associated with schizophrenia would enhance ion flux-independent signaling by the NMDAR, driving destabilization and loss of dendritic spines. We tested our hypothesis using the serine racemase knockout (SRKO) mouse model, which lacks the enzyme for d-serine production. We show that activity-dependent spine growth is impaired in SRKO mice, but can be acutely rescued by exogenous d-serine. Moreover, we find a significant bias of synaptic plasticity toward spine shrinkage in the SRKO mice as compared to wild-type littermates. Notably, we demonstrate that enhanced ion flux-independent signaling through the NMDAR contributes to this bias toward spine destabilization, which is exacerbated by an increase in synaptic NMDARs in hippocampal synapses of SRKO mice. Our results support a model in which lowered d-serine levels associated with schizophrenia enhance ion flux-independent NMDAR signaling and bias toward spine shrinkage and destabilization.
... Combined, the above data strongly support the major contribution of astroglial CACCs into glial release of D-Serine. Our data also show that all three types of GluN2B-mediated neuronal currents (gSICs, gFICs and tonic current) strongly rely on the presence of extracellular D-Serine, this goes in line with recent results (Ferreira et al., 2017). ...
... Thus, astroglial-derived currents occur in the cortical neurons most likely at extra-synaptic and/or peri-synaptic loci, where GluN2B-containing NMDARs are predominately expressed (Paoletti et al., 2013;Papouin and Oliet, 2014). Importantly, our results revealed a dependence of all types of GluN2B-mediated glia-driven neuronal currents on glial release of D-Serine which agrees with the data of (Ferreira et al., 2017) on the preferential action of D-Serine on GluN2B-rather than GluN2A-containing NMDARs. ...
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Astrocytes are an active element of brain signalling, capable of release of small molecule gliotransmitters by vesicular and channel-mediated mechanisms. However, specific physiological roles of astroglial exocytosis of glutamate and D-Serine remain controversial. Our data demonstrate that cortical astrocytes can release glutamate and D-Serine by combination of SNARE-dependent exocytosis and non-vesicular mechanisms dependent on TREK-1 and Best1 channels. Astrocyte-derived glutamate and D-serine elicited complex multicomponent phasic response in neocortical pyramidal neurons, which is mediated by extra-synaptic GluN2B receptors. Impairment of either pathway of gliotransmission (in the TREK1 KO, Best-1 KO or dnSNARE mice) strongly affected the NMDAR-dependent long-term synaptic plasticity in the hippocampus and neocortex. Moreover, impairment of astroglial exocytosis in dnSNARE mice led to the deficit in the spatial working memory which was rescued by environmental enrichment. We conclude that synergism between vesicular and non-vesicular gliotransmission is crucial for astrocyte-neuron communication and astroglia-driven regulation of synaptic plasticity and memory.
... L-Serine can be reversibly converted into glycine by SHMT, in this way affecting the tetrahydrofolate pool. Notably, D-serine is the main coagonist for synaptic NMDARs, and glycine is the dominant coagonist at extracellular NMDARs [20][21][22] An overview on L-serine interplay ...
... While the synaptic NMDARs are preferentially activated by the coagonist D-serine, the extrasynaptic ones better interact with the coagonist glycine [20,21]: the regionalized availability of the coagonist matches the preferential affinity of synaptic receptors for D-serine and of the extrasynaptic NMDARs for glycine. Glycine and D-serine relative availability at rat hippocampal glutamatergic synapses regulate the trafficking and synaptic content of NMDAR subtypes [22]: D-serine alters the membrane dynamics and synaptic content of GluN2B subunit of NMDAR (but not of GluN2A ones) through a process requiring PDZ-binding scaffold partners. Electrophysiological experiments demonstrated that D-serine is required for the induction and expression of Long-Term Potentiation at both excitatory and inhibitory synapses, while glycine does not modulate synaptic plasticity, but controls neuronal gain activity at the dendritic integration level [23]. ...
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L-serine is a nonessential amino acid in eukaryotic cells, used for protein synthesis and in producing phosphoglycerides, glycerides, sphingolipids, phosphatidylserine, and methylenetetrahydrofolate. Moreover, L-serine is the precursor of two relevant coagonists of NMDA receptors: glycine (through the enzyme serine hydroxymethyltransferase), which preferentially acts on extrasynaptic receptors and D-serine (through the enzyme serine racemase), dominant at synaptic receptors. The cytosolic “phosphorylated pathway” regulates de novo biosynthesis of L-serine, employing 3-phosphoglycerate generated by glycolysis and the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase (the latter representing the irreversible step). In the human brain, L-serine is primarily found in glial cells and is supplied to neurons for D-serine synthesis. Serine-deficient patients show severe neurological symptoms, including congenital microcephaly, psychomotor retardation, and intractable seizures, thus highlighting the relevance of de novo production of this amino acid in brain development and morphogenesis. Indeed, the phosphorylated pathway is strictly linked to cancer. Moreover, L-serine has been suggested as a ready-to-use treatment, as also recently proposed for Alzheimer’s disease. Here, we present our current state of knowledge concerning the three mammalian enzymes of the phosphorylated pathway and known mutations related to pathological conditions: although the structure of these enzymes has been solved, how enzyme activity is regulated remains largely unknown. We believe that an in-depth investigation of these enzymes is crucial to identify the molecular mechanisms involved in modulating concentrations of the serine enantiomers and for studying the interplay between glial and neuronal cells and also to determine the most suitable therapeutic approach for various diseases.
... At the SC-CA1 synapses, glycine stimulates synaptic NMDARs in young mice, but the preference to glycine switches to D-serine in adults. However, there is no consensus on the extent of this effect (16,28). We found that inhibiting the glycine synthesis by inactivating the Phgdh pathway depresses the isolated NMDAR potentials and reduces the LTP in SR-KO, indicating that Phgdh-derived glycine also governs synaptic NMDARs in the mature hippocampus. ...
... Conversely, our data suggest that removing extracellular glycine may affect local D-serine metabolism. Previous studies using glycine oxidase treatment to examine NMDAR coagonist identity (16,21,24,28) may have affected local D-serine dynamics and therefore should be interpreted with caution. ...
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Significance Phgdh is a crucial enzyme in the astrocytic synthesis of l -serine from glucose. Our study uncovers the role of glial-neuronal cross-talk in regulating NMDAR synaptic activation through the astrocytic export of Phgdh-derived l -serine to generate the coagonists d -serine and glycine. We clarify the relative roles of d -serine and glycine by showing that like d -serine, Phgdh-derived glycine mediates NMDAR synaptic activation in the mature hippocampus. We show that glycine is a significant regulator of d -serine synthesis and release, revealing a cross-talk between glycine and d -serine metabolism that fine tunes NMDAR activation. Our data reveal a multifaceted mechanism regulating NMDAR activity, which ultimately depends on a Phgdh-dependent serine shuttle.
... This alternative way of action depends on the scaffolding and signaling proteins that interact with NMDAR2 CTD. NMDAR binding by glycine or D-serine only causes allosteric changes and stimulates receptor internalization via AP2 [39,40]. Furthermore, the downregulation of the receptor by tyrosine dephosphorylation occurs in an ion flux-independent manner [41]. ...
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Glutamate is a key player in excitatory neurotransmission in the central nervous system (CNS). The N-methyl-D-aspartate receptor (NMDAR) is a glutamate-gated ion channel which presents several unique features and is involved in various physiological and pathological neuronal processes. Thanks to great efforts in neuroscience, its structure and the molecular mechanisms controlling its localization and functional regulation in neuronal cells are well known. The signaling mediated by NMDAR in neurons is very complex as it depends on its localization, composition, Ca2+ influx, and ion flow-independent conformational changes. Moreover, NMDA receptors are highly diffusive in the plasma membrane of neurons, where they form heterocomplexes with other membrane receptors and scaffold proteins which determine the receptor function and activation of downstream signaling. Interestingly, a recent paper demonstrates that NMDAR signaling is involved in epithelial cell competition, an evolutionary conserved cell fitness process influencing cancer initiation and progress. The idea that NMDAR signaling is limited to CNS has been challenged in the past two decades. A large body of evidence suggests that NMDAR is expressed in cancer cells outside the CNS and can respond to the autocrine/paracrine release of glutamate. In this review, we survey research on NMDAR signaling and regulation in neurons that can help illuminate its role in tumor biology. Finally, we will discuss existing data on the role of the glutamine/glutamate metabolism, the anticancer action of NMDAR antagonists in experimental models, NMDAR synaptic signaling in tumors, and clinical evidence in human cancer.
... GluN2A-NMDAR and GluN2B-NMDAR appear to be differentially located on the neuronal surface. GluN2A-NMDAR is preferentially expressed in the synapse, while the expression of GluN2B-NMDAR tends to be higher in extrasynapse during development phase [112][113][114][115] . The underlying mechanism seems to be that the interaction between CTD of GluN2A with PDZ domain of post-synaptic density protein-95 (PSD-95) is relatively stabler [116,117] . ...
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Proper signal transmission is the fundamental process of the brain activity. Changes and adaption of neuroplasticity based on the strength of synaptic transmission are essential for the information propagation in the central nervous system, which contribute to cognition, learning, and memory. Being the major excitatory neurotransmitter in the central nervous system, glutamate acts primarily through binding to the glutamate receptors, the glutamate-gated ion channels localized on post-synaptic membrane. The ionotropic glutamate receptors, pharmacologically grouped into α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, N-methyl-D-aspartic acid (NMDA) receptors, and kainate receptors, have been shown to play distinct roles in excitatory neurotransmission and synaptic plasticity. Due to their high permeability to Ca 2+ , the NMDA receptors have very unique function in neurotransmission and particular importance in the induction of long-term synaptic plasticity. Dysfunction of NMDA receptors causes impairment in synaptic plasticity and learning and memory. In recent years, with the development of genome-wide association studies and next-generation sequencing technology, mutations of NMDA receptor subunits have been in a variety of neuropsychiatric disorders, such as cognitive impairment, schizophrenia, autism or epilepsy. In clinical practice, NMDA receptors are known as the targets for the treatment of many neuropsychiatric disorders. In current review, we summarize current knowledge of NMDA receptors with different subunit compositions in the context of expression pattern, channel properties, protein trafficking, and synaptic plasticity as well as their roles in neuropsychiatric disorders.
... These findings unveil an unsuspected role of GluN1/GluN3A receptors in setting S1 SST-IN Vm rest and suggest that ambient extracellular glycine concentrations are a contributing factor to SST-INs excitability. To test this hypothesis directly, we incubated the slices with the enzyme glycine oxidase (GO), an approach known to reduce extracellular glycine levels efficiently and specifically (Ferreira et al., 2017;Le Bail et al., 2015;Papouin et al., 2012). Surprisingly, under these conditions, the Vm rest of S1 SST-INs became even more depolarized and increased cell excitability ( Figures 5G and 5H). ...
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GluN3A is an atypical glycine-binding subunit of NMDA receptors (NMDARs) whose actions in the brain are mostly unknown. Here, we show that the expression of GluN3A subunits controls the excitability of mouse adult cortical and amygdalar circuits via an unusual signaling mechanism involving the formation of excitatory glycine GluN1/GluN3A receptors (eGlyRs) and their tonic activation by extracellular glycine. eGlyRs are mostly extrasynaptic and reside in specific neuronal populations, including the principal cells of the basolateral amygdala (BLA) and SST-positive interneurons (SST-INs) of the neocortex. In the BLA, tonic eGlyR currents are sensitive to fear-conditioning protocols, are subject to neuromodulation by the dopaminergic system, and control the stability of fear memories. In the neocortex, eGlyRs control the in vivo spiking of SST-INs and the behavior-dependent modulation of cortical activity. GluN3A-containing eGlyRs thus represent a novel and widespread signaling modality in the adult brain, with attributes that strikingly depart from those of conventional NMDARs.
... Recently, it has emerged that only D-SER gates NMDA receptors at the synaptic level, whereas extra-synaptic receptors are gated by glycine (Papouin et al., 2012;Sullivan & Miller, 2012). Furthermore, D-SER gates NMDA receptors only in the mature brain, whereas glycine gates NMDA receptor activity in the immature brain (Ferreira et al., 2017). In our study, we used adult rats with a weight of about 300-350 g. ...
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Place cells are cells that exhibit location‐dependent responses; they have mostly been studied in the hippocampus. Place cells have also been reported in the rat claustrum, an underexplored paracortical region with extensive corto‐cortical connectivity. It has been hypothesised that claustral neuronal responses are anchored to cortical visual inputs. We show rat claustral place cells remap when visual inputs are eliminated from the environment, and that this remapping is NMDA‐receptor‐dependent. Eliminating visual input decreases claustral delta‐band oscillatory activity, increases theta‐band oscillatory activity, and increases simultaneously‐recorded visual cortical activity. We conclude that, like the hippocampus, claustral place field remapping might be mediated by NMDA receptor activity, and is modulated by visual cortical inputs.
... In support, intracellular and extracellular D-serine levels in PC-12 cell cultures were reduced by (2R,6R; 2S,6S)-HNK with IC 50 values of 0.68 ± 0.09 nM and 0.18 ± 0.04 nM respectively [176]. The potential significance of this finding is that a lack of D-serine might preferentially impact GluN2B containing NMDARs as D-serine restricts their diffusion within membranes and reduces synaptic expression [61]. However, these experiments will need to be repeated using neural cells to confirm the relevance to NMDAR function in the brain. ...
Article
Treating major depression is a medical need that remains unmet by monoaminergic therapeutic strategies that commonly fail to achieve symptom remission. A breakthrough in the treatment of depression was the discovery that the anesthetic (R,S)-ketamine (ketamine), when administered at sub-anesthetic doses, elicits rapid (sometimes within hours) antidepressant effects in humans that are otherwise resistant to monoaminergic-acting therapies. While this finding was revolutionary and led to the FDA approval of (S)-ketamine (esketamine) for use in adults with treatment-resistant depression and suicidal ideation, the mechanisms underlying how ketamine or esketamine elicit their effects are still under active investigation. An emerging view is that metabolism of ketamine may be a crucial step in its mechanism of action, as several metabolites of ketamine have neuroactive effects of their own and may be leveraged as therapeutics. For example, (2R,6R)-hydroxynorketamine (HNK), is readily observed in humans following ketamine treatment and has shown therapeutic potential in preclinical tests of antidepressant efficacy and synaptic potentiation while being devoid of the negative adverse effects of ketamine, including its dissociative properties and abuse potential. We discuss preclinical and clinical studies pertaining to how ketamine and its metabolites produce antidepressant effects. Specifically, we explore effects on glutamate neurotransmission through N-methyl D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), synaptic structural changes via brain derived neurotrophic factor (BDNF) signaling, interactions with opioid receptors, and the enhancement of serotonin, norepinephrine, and dopamine signaling. Strategic targeting of these mechanisms may result in novel rapid-acting antidepressants with fewer undesirable side effects compared to ketamine.
... Glial inhibitor 5-fluoro-2′-deoxyuridine (5 μM) was added at DIV3. At 12DIV, N-methyl-d-aspartate (NMDA, 10 μM) and co-agonist glycine (20 μM) were added to the culture for 15 min followed by 6 h recovery to gate extrasynaptic glutamatergic NMDA receptors 79 . Pridopidine was added for 24 h. ...
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Optic neuropathies such as glaucoma are characterized by retinal ganglion cell (RGC) degeneration and death. The sigma-1 receptor (S1R) is an attractive target for treating optic neuropathies as it is highly expressed in RGCs, and its absence causes retinal degeneration. Activation of the S1R exerts neuroprotective effects in models of retinal degeneration. Pridopidine is a highly selective and potent S1R agonist in clinical development. We show that pridopidine exerts neuroprotection of retinal ganglion cells in two different rat models of glaucoma. Pridopidine strongly binds melanin, which is highly expressed in the retina. This feature of pridopidine has implications to its ocular distribution, bioavailability, and effective dose. Mitochondria dysfunction is a key contributor to retinal ganglion cell degeneration. Pridopidine rescues mitochondrial function via activation of the S1R, providing support for the potential mechanism driving its neuroprotective effect in retinal ganglion cells.
... For NMDA receptors, it is better to add the NMDA receptor blockers when recording neuronal intrinsic excitability. It is commonly known that NMDAR-mediated excitatory postsynaptic currents were recorded at +40 mV (significant depolarization) in the presence of Mg 2+ or in the absence of Mg 2+ (Chen et al., 2016;Ferreira et al., 2017). In our protocol here, the membrane test was recorded at −60 mV with 2 mM Mg 2+ ; therefore, it was unlikely to be affected by NMDA receptors even without NMDA blockers in the recording of isolated DRG neurons. ...
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Inflammatory pain encompasses many clinical symptoms, and there is no satisfactory therapeutic target. Neuronal hyperexcitability and/or sensitization of the primary nociceptive neurons in the dorsal root ganglion (DRG) and spinal dorsal horn are critical to the development and maintenance of inflammatory pain. The sodium leak channel (NALCN), a non-selective cation channel, mediates the background Na ⁺ leak conductance and controls neuronal excitability. It is unknown whether abnormal activity of NALCN mediates the pathological process of inflammatory pain. Complete Freund’s adjuvant (CFA) was injected into the left footpad of rats to induce inflammatory pain. The thresholds of mechanical and thermal sensation and spontaneous pain behaviors were assessed. The expression of NALCN in DRG and spinal dorsal cord was measured. NALCN currents and the contribution of NALCN to neuronal excitability in the DRG and spinal dorsal cord were recorded using whole-cell patch-clamping recording. NALCN was abundantly expressed in neurons of the DRG and spinal dorsal cord. In acutely isolated DRG neurons and spinal cord slices from rats with CFA-induced inflammatory pain, NALCN currents and neuronal excitability were increased. Subsequently, intrathecal and sciatic nerve injection of NALCN-small interfering RNA (siRNA) decreased NALCN mRNA and reverted NALCN currents to normal levels, and then reduced CFA-induced neuronal excitability and alleviated pain symptoms. Furthermore, pain-related symptoms were significantly prevented by the NALCN-shRNA-mediated NALCN knockdown in DRG and spinal cord. Therefore, increased expression and activity of NALCN contributed to neuronal sensitization in CFA-induced inflammatory pain. NALCN may be a novel molecular target for the control of inflammatory pain.
... In the past 20 years, a number of studies focused on D-Ser since, in many areas of the mature brain, it is the preferred coagonist for synaptic N-methyl-D-aspartate (NMDA) receptors, a subtype of the glutamate ionotropic receptor family. D-Ser activates the receptor by binding to the socalled "glycine-binding site" of GluN1 subunits (alternatively to glycine) and potentiates NMDA receptormediated responses (Panatier et al., 2006;Wolosker 2007;Henneberger et al., 2010;LeBail et al., 2015;Ferreira et al., 2017), thus playing an essential role in synaptic plasticity. Accordingly, perturbation of D-Ser levels in brain, cerebrospinal fluid (CSF), and serum has been related to the pathophysiology of various neurological and psychiatric disorders (Pollegioni and Sacchi, 2010), e.g., Alzheimer's disease (Wu et al., 2004;Madeira et al., 2015;Piubelli et al., 2021), schizophrenia (Hashimoto et al., 2003;Bendikov et al., 2006), and amyotrophic lateral sclerosis (Sasabe et al., 2007;Sasabe et al., 2012). ...
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In recent years, the D-enantiomers of amino acids have been recognized as natural molecules present in all kingdoms, playing a variety of biological roles. In humans, d-serine and d-aspartate attracted attention for their presence in the central nervous system. Here, we focus on d-aspartate, which is involved in glutamatergic neurotransmission and the synthesis of various hormones. The biosynthesis of d-aspartate is still obscure, while its degradation is due to the peroxisomal flavin adenine dinucleotide (FAD)-containing enzyme d-aspartate oxidase. d-Aspartate emergence is strictly controlled: levels decrease in brain within the first days of life while increasing in endocrine glands postnatally and through adulthood. The human d-aspartate oxidase (hDASPO) belongs to the d-amino acid oxidase-like family: its tertiary structure closely resembles that of human d-amino acid oxidase (hDAAO), the enzyme that degrades neutral and basic d-amino acids. The structure-function relationships of the physiological isoform of hDASPO (named hDASPO_341) and the regulation of gene expression and distribution and properties of the longer isoform hDASPO_369 have all been recently elucidated. Beyond the substrate preference, hDASPO and hDAAO also differ in kinetic efficiency, FAD-binding affinity, pH profile, and oligomeric state. Such differences suggest that evolution diverged to create two different ways to modulate d-aspartate and d-serine levels in the human brain. Current knowledge about hDASPO is shedding light on the molecular mechanisms underlying the modulation of d-aspartate levels in human tissues and is pushing novel, targeted therapeutic strategies. Now, it has been proposed that dysfunction in NMDA receptor-mediated neurotransmission is caused by disrupted d-aspartate metabolism in the nervous system during the onset of various disorders (such as schizophrenia): the design of suitable hDASPO inhibitors aimed at increasing d-aspartate levels thus represents a novel and useful form of therapy.
... NMDARs containing GluN2A Li et al., 2016;Vissel, Krupp, Heinemann, & Westbrook, 2001) or GluN2B (Bowers et al., 2020;Ferreira et al., 2017;Tamburri, Dudilot, Licea, Bourgeois, & Boehm, 2013) produce nonionotropic signals, but variations in the magnitude, time-course, or molecular components of evoked nonionotropic LTD have not been compared across GluN2 subunits. Third, direct studies of relationships between NMDAR nonionotropic signaling and cognitive abilities are required. ...
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N‐methyl‐D‐aspartate receptors (NMDARs) can be considered to be the de facto “plasticity” receptors in the brain due to their central role in the activity‐dependent modification of neuronal morphology and synaptic transmission. Since the 1980s, research on NMDARs has focused on the second messenger properties of calcium and the downstream signaling pathways that mediate alterations in neural form and function. Recently, NMDARs were shown to drive activity‐dependent synaptic plasticity without calcium influx. How this “nonionotropic” plasticity occurs in vitro is becoming clearer, but research on its involvement in behavior and cognition is in its infancy. There is a partial overlap in the downstream signaling molecules that are involved in ionotropic and nonionotropic NMDAR‐dependent plasticity. Given this, and prior studies of the cognitive impacts of ionotropic NMDAR plasticity, a preliminary model explaining how NMDAR nonionotropic plasticity affects learning and memory can be established. We hypothesize that nonionotropic NMDAR plasticity takes part in latent memory encoding in immature rodents through nonassociative depression of synaptic efficacy, and possibly shrinking of dendritic spines. Further, the late postnatal alteration in NMDAR composition in the hippocampus appears to reduce nonionotropic signaling and remove a restriction on memory retrieval. This framework substantially alters the canonical model of NMDAR involvement in spatial cognition and hippocampal maturation and provides novel and exciting inroads for future studies.
... It is The copyright holder for this preprint this version posted April 22, 2021. ; https://doi.org/10.1101/2021.04.21.440764 doi: bioRxiv preprint NMDA receptor activity in the immature brain (Ferreira et al., 2017). In our study, we used adult rats 342 with a weight of about 300-350 g. ...
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Place cells are cells exhibiting location-dependent responses; they have mostly been studied in the hippocampus. Place cells have also been reported in the rat claustrum, an underexplored paracortical region with extensive corto-cortical connectivity. It has been hypothesised that claustral neuronal responses are anchored to cortical visual inputs. We show rat claustral place cells remap when visual inputs are eliminated from the environment and that this remapping is NMDA-receptor-dependent. Eliminating visual input enhances delta-band oscillatory activity in the claustrum, without affecting simultaneously-recorded visual cortical activity. We conclude that, like the hippocampus, claustral place field remapping might be mediated by NMDA receptor activity, and is modulated by visual cortical inputs. (106 words)
... The copyright holder for this preprint this version posted March 5, 2021. ; https://doi.org/10.1101/2021.03.04.434016 doi: bioRxiv preprint composition of NMDARs are likely to be the consequence of lack of D-serine, as prior studies have demonstrated the role of co-agonist binding in priming of the NMDAR for endocytosis, with D-serine specifically acting on GluN2B subunits (52,53). In addition, we found that this increase in the number of NMDARs led to an increase in the magnitude of non-ionotropic signaling in SRKO, which would be expected to drive enhanced spine loss (11). ...
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Schizophrenia is a psychiatric disorder that affects over 20 million people globally. Notably, schizophrenia is associated with decreased density of dendritic spines and decreased levels of D-serine, a co-agonist of the N-methyl-D-aspartate receptor (NMDAR), and hypofunction of NMDARs is thought to play a role in the pathophysiology of schizophrenia. We hypothesized that lowered D-serine levels associated with schizophrenia would bias toward ion flux-independent signaling by the NMDAR, which drives spine shrinkage and loss. Using a schizophrenia mouse model lacking the enzyme for D-serine production (serine racemase knock out; SRKO), we show that activity-dependent spine growth is inhibited in SRKO mice but can be acutely rescued by exogenous D-serine. When examining a wider range of stimulus strengths, we observed activity-dependent spine growth at higher stimulus strengths, but overall found a strong bias toward spine shrinkage in the SRKO mice as compared to wild-type littermates. Furthermore, we demonstrate that enhanced ion flux-independent signaling through the NMDAR contributes to this bias toward spine shrinkage, which is likely exacerbated by an increase in synaptic NMDARs in hippocampal synapses of SRKO mice. Our results support a model in which the lowered D-serine levels associated with schizophrenia lead to increased ion flux-independent NMDAR signaling and a bias toward spine shrinkage that could play an important role in the loss of dendritic spines associated with schizophrenia.
... These findings have supported a model in which glutamate binding leads to conformational changes in the NMDAR that drive spine shrinkage and synaptic weakening. Indeed, imaging studies using fluorescence resonance energy transfer (FRET) reporters have shown that NMDA or glutamate binding triggers conformational changes in the NMDAR intracellular domains and changes its interaction with calcium/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 1 (PP1) Dore et al., 2015;Ferreira et al., 2017). p38 mitogen-activated protein kinase (MAPK) has been identified as a key component of the molecular pathway downstream of the NMDAR conformational signaling (Birnbaum et al., 2015;Nabavi et al., 2013;Stein et al., 2015), and a recent study further identified neuronal nitric oxide synthase (nNOS), nNOS-NOS1AP interactions, MAPKactivated protein kinase 2 (MK2), and cofilin as part of this signaling pathway (Stein et al., 2020). ...
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Experience-dependent refinement of neuronal connections is critically important for brain development and learning. Here, we show that ion-flow-independent NMDA receptor (NMDAR) signaling is required for the long-term dendritic spine growth that is a vital component of brain circuit plasticity. We find that inhibition of p38 mitogen-activated protein kinase (p38 MAPK), which is downstream of non-ionotropic NMDAR signaling in long-term depression (LTD) and spine shrinkage, blocks long-term potentiation (LTP)-induced spine growth but not LTP. We hypothesize that non-ionotropic NMDAR signaling drives the cytoskeletal changes that support bidirectional spine structural plasticity. Indeed, we find that key signaling components downstream of non-ionotropic NMDAR function in LTD-induced spine shrinkage are also necessary for LTP-induced spine growth. Furthermore, NMDAR conformational signaling with coincident Ca²⁺ influx is sufficient to drive CaMKII-dependent long-term spine growth, even when Ca²⁺ is artificially driven through voltage-gated Ca²⁺ channels. Our results support a model in which non-ionotropic NMDAR signaling gates the bidirectional spine structural changes vital for brain plasticity.
... Data represent mean 6 SEM, pp , 0.05. rate of GluN2B surface diffusion and decreases residence at postsynaptic sites (Papouin et al., 2012;Ferreira et al., 2017), which may be required for LTP (Dupuis et al., 2014). Thus, loss of Dserine at synaptic sites by the removal of SR may alter the balance of synaptic GluN2 subunits through changes in trafficking mechanisms and have complex effects on synaptic plasticity. ...
Article
D-serine is the primary NMDAR coagonist at mature forebrain synapses and is synthesized by the enzyme serine racemase (SR). However, our understanding of the mechanisms regulating the availability of synaptic D-serine remains limited. Though early studies suggested D-serine is synthesized and released from astrocytes, more recent studies have demonstrated a predominantly neuronal localization of SR. More specifically, recent work intriguingly suggests that SR may be found at the postsynaptic density, yet the functional implications of postsynaptic SR on synaptic transmission are not yet known. Here, we show an age-dependent dendritic and postsynaptic localization of SR and D-serine by immunohistochemistry and electron microscopy in mouse CA1 pyramidal neurons. In addition, using a single-neuron genetic approach in SR conditional KO mice from both sexes, we demonstrate a cell-autonomous role for SR in regulating synaptic NMDAR function at Schaffer collateral (CA3)-CA1 synapses. Importantly, single-neuron genetic deletion of SR resulted in the elimination of LTP at 1 month of age, which could be rescued by exogenous D-serine. Interestingly, there was a restoration of LTP by 2 months of age that was associated with an upregulation of synaptic GluN2B. Our findings support a cell-autonomous role for postsynaptic neuronal SR in regulating synaptic NMDAR function and suggests a possible autocrine mode of D-serine action.
... These data, together with the observation that the interaction between GluK2 and b-catenin is increased in the presence of glutamate, allowed us to propose a model whereby glutamate binding to GluK2 induces the rearrangement of the GluK2 CTD, thus increasing its affinity for the N-cadherin/b-catenin complex and consequently promoting the immobilization of LiGluK2 receptors at synapses. While agonist-induced changes of the CTD conformation has been previously demonstrated at NMDARs and AMPARs (Dore et al., 2015;Zachariassen et al., 2016;Ferreira et al., 2017), at KARs, several studies have only focused on the interaction of the GluK2 CTD with intracellular proteins without considering the role of possible agonist-induced alterations of the CTD structure and interactions (Coussen et al., 2002;Maraschi et al., 2014). Other X-ray crystallography and cryo-EM studies have shown major GluK2 structural rearrangements following desensitization at the level of the ligand binding domain (LBD), leaving the potential changes of the GluK2 CTD during receptor opening/desensitization unaddressed (Meyerson et al., 2016;Møllerud et al., 2017). ...
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Kainate receptors (KARs) mediate postsynaptic currents with a key impact on neuronal excitability. However, the molecular determinants controlling KAR postsynaptic localization and stabilization are poorly understood. Here, we exploit optogenetic and single-particle tracking approaches to study the role of KAR conformational states induced by glutamate binding on KAR lateral mobility at synapses. We report that following glutamate binding, KARs are readily and reversibly trapped at glutamatergic synapses through increased interaction with the β-catenin/N-cadherin complex. We demonstrate that such activation-dependent synaptic immobilization of KARs is crucial for the modulation of short-term plasticity of glutamatergic synapses. Thus, the present study unveils the crosstalk between conformational states and lateral mobility of KARs, a mechanism regulating glutamatergic signaling, particularly in conditions of sustained synaptic activity.
... Our data confirm [67] that the NMDA receptor can mediate some Zn 2+ uptake (Fig. 2b), but the NMDA receptor is not known to be a significant route for Zn 2+ entry. However, Zn 2+ binds to and inhibits the activity of the NMDA receptor [68][69][70], and so we hypothesize that the Zn 2+ intake that was blocked by memantine and MK-801 (Fig. 2b) might reflect internalization through recycling of the NMDA receptor [71]. This could be consistent with the report of Zn 2+ promoting attachment of Aβ to the NMDA receptor [49]. ...
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Aging and female sex are the major risk factors for Alzheimer’s disease and its associated brain amyloid-β (Aβ) neuropathology, but the mechanisms mediating these risk factors remain uncertain. Evidence indicates that Aβ aggregation by Zn²⁺ released from glutamatergic neurons contributes to amyloid neuropathology, so we tested whether aging and sex adversely influences this neurophysiology. Using acute hippocampal slices, we found that extracellular Zn²⁺-elevation induced by high K⁺ stimulation was significantly greater with older (65 weeks vs 10 weeks old) rats, and was exaggerated in females. This was driven by slower reuptake of extracellular Zn²⁺, which could be recapitulated by mitochondrial intoxication. Zn²⁺:Aβ aggregates were toxic to the slices, but Aβ alone was not. Accordingly, high K⁺ caused synthetic human Aβ added to the slices to form soluble oligomers as detected by bis-ANS, attaching to neurons and inducing toxicity, with older slices being more vulnerable. Age-dependent energy failure impairing Zn²⁺ reuptake, and a higher maximal capacity for Zn²⁺ release by females, could contribute to age and sex being major risk factors for Alzheimer’s disease.
... This is consistent with a reduced level of occupancy of the NMDAR co-agonist-binding site in 3xTg-AD mice because of a reduced level of D-serine within the cleft. Another possibility would be that the NMDAR subunit composition was switched from GluN2B to GluN2A in 3xTg-AD mice, since GluN2A-containing NMDARs have a lower affinity for D-serine than GluN2B receptors (Ferreira et al., 2017). In that case, the amount of NMDARs recruited during synaptic stimulation could be reduced even if endogenous D-serine levels were unchanged. ...
Article
Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer’s disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD.
... Moreover, in parallel with the classic view of ion influx as the only NMDAR signaling, several studies pointed out a NMDAR metabotropic cascade independently of ion-flow. In particular, the NMDAR metabotropic activity results from a complex intracellular "signalosome", which is mostly involved in synaptic receptor endocytosis and trafficking [11][12][13][14], and depression of neurotransmission such as NMDAR-dependent Long-Term Depression (LTD) [15][16][17][18][19]. ...
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N-Methyl-d-Aspartate Receptors (NMDARs) are ionotropic glutamate-gated receptors. NMDARs are tetramers composed by several homologous subunits of GluN1-, GluN2-, or GluN3-type, leading to the existence in the central nervous system of a high variety of receptor subtypes with different pharmacological and signaling properties. NMDAR subunit composition is strictly regulated during development and by activity-dependent synaptic plasticity. Given the differences between GluN2 regulatory subunits of NMDAR in several functions, here we will focus on the synaptic pool of NMDARs containing the GluN2A subunit, addressing its role in both physiology and pathological synaptic plasticity as well as the contribution in these events of different types of GluN2A-interacting proteins.
... This is consistent with a reduced level of occupancy of the NMDAR co-agonist binding site in 3xTg-AD mice because of a reduced level of D-serine within the cleft. Another possibility would be that the NMDAR subunit composition was switched from GluN2B to GluN2A in 3xTg-AD mice, since GluN2A-containing NMDARs have a lower affinity for D-serine than GluN2B receptors (Ferreira et al., 2017). In that case, the number of NMDARs recruited during synaptic stimulation could be reduced even if endogenous D-serine levels were unchanged. ...
Article
In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD, we recently developed viral vectors targeting astrocytes that either activate or inhibit the JAK2-STAT3 pathway, a central cascade controlling astrocyte reaction. We aimed to evaluate whether reactive astrocytes contribute to Tau as well as amyloid pathologies in the hippocampus of 3xTg-AD mice, an AD model that develops Tau hyper-phosphorylation and aggregation in addition to amyloid deposition. JAK2-STAT3 pathway-mediated modulation of reactive astrocytes in 25% of the hippocampus of 3xTg-AD mice, did not significantly influence Tau phosphorylation or amyloid processing and deposition at early, advanced and terminal stage of the disease. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did not improve short-term spatial memory in the Y maze but it did reduce anxiety in the elevated plus maze. Our unique approach to specifically manipulate reactive astrocytes in situ show these cells may impact behavioral outcomes without influencing Tau or amyloid pathology.
... Glycine priming may also be significant for controlling synaptic signalling in the presence of allosteric modulators that change the potency of glycine/D-serine binding to GluN1 [32]. In addition, glycine priming may be functionally important for changes to glycine and D-serine signalling, which may mediate the migration of receptors between synaptic and extrasynaptic compartments [33], or during developmental changes in subunit composition [34]. Glycine levels also increase in many different pathological conditions such as brain trauma, ischemia, or epilepsy [32], where glycine priming could act as a homeostatic mechanism to remove functional NMDARs and prevent excitotoxic or neurotoxic signalling cascades. ...
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Abstract N-methyl-d-aspartate receptors (NMDARs) are excitatory glutamatergic receptors that are fundamental for many neuronal processes, including synaptic plasticity. NMDARs are comprised of four subunits derived from heterogeneous subunit families, yielding a complex diversity in NMDAR form and function. The quadruply-liganded state of binding of two glutamate and two glycine molecules to the receptor drives channel gating, allowing for monovalent cation flux, Ca2+ entry and the initiation of Ca2+-dependent signalling. In addition to this ionotropic function, non-ionotropic signalling can be initiated through the exclusive binding of glycine or of glutamate to the NMDAR. This binding may trigger a transmembrane conformational change of the receptor, inducing intracellular protein-protein signalling between the cytoplasmic domain and secondary messengers. In this review, we outline signalling cascades that can be activated by NMDARs and propose that the receptor transduces signalling through three parallel streams: (i) signalling via both glycine and glutamate binding, (ii) signalling via glycine binding, and (iii) signalling via glutamate binding. This variety in signal transduction mechanisms and downstream signalling cascades complements the widespread prevalence and rich diversity of NMDAR activity throughout the central nervous system and in disease pathology.
... In contrast, cLTP does not alter the surface diffusion of GluN2A-NMDARs and does not exert any effects on GluN2B-NMDARs in immature neurons. Interestingly, the binding of co-agonists, glycine and D-serine, also differentially affects the surface mobility of NMDARs in a subunit-specific manner (Ferreira et al., 2017;Papouin, Ladepeche, & Ruel, 2012). ...
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The N‐methyl‐D‐aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate the flux of calcium (Ca2+) into the postsynaptic compartment. Calcium influx subsequently triggers the activation of various intracellular signaling cascades that underpin multiple forms of synaptic plasticity. Functional NMDARs are assembled as heterotetramers composed of two obligatory GluN1 subunits and two GluN2 or GluN3 subunits. Four different GluN2 subunits (GluN2A‐D) are present throughout the central nervous system; however, they are differentially expressed, both developmentally and spatially, in a cell‐ and synapse‐specific manner. Each GluN2 subunit confers NMDARs with distinct ion channel properties and intracellular trafficking pathways. Regulated membrane trafficking of NMDARs is a dynamic process that ultimately determines the number of NMDARs at synapses, and is controlled by subunit‐specific interactions with various intracellular regulatory proteins. Here we review recent progress made towards understanding the molecular mechanisms that regulate the trafficking of GluN2‐containing NMDARs, focusing on the roles of several key synaptic proteins that interact with NMDARs via their carboxyl termini.
... This is consistent with a reduced level of occupancy of the NMDAR co-agonist binding site in 3xTg-AD mice because of a reduced level of D-serine within the cleft. Another possibility would be that the NMDAR subunit composition was switched from GluN2B to GluN2A in 3xTg-AD mice, since GluN2A-containing NMDARs have a lower affinity for D-serine than GluN2B receptors (Ferreira et al., 2017). In that case, the number of NMDARs recruited during synaptic stimulation could be reduced even if endogenous D-serine levels were unchanged. ...
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N‐methyl‐D‐aspartate receptors (NMDARs) are critical for the maturation and plasticity of glutamatergic synapses. In the hippocampus, NMDARs mainly contain GluN2A and/or GluN2B regulatory subunits. The amyloid precursor protein (APP) has emerged as a putative regulator of NMDARs, but the impact of this interaction to their function is largely unknown. By combining patch‐clamp electrophysiology and molecular approaches, we unravel a dual mechanism by which APP controls GluN2B‐NMDARs, depending on the life stage. We show that APP is highly abundant specifically at the postnatal postsynapse. It interacts with GluN2B‐NMDARs, controlling its synaptic content and mediated currents, both in infant mice and primary neuronal cultures. Upon aging, the APP amyloidogenic‐derived C‐terminal fragments, rather than APP full‐length, contribute to aberrant GluN2B‐NMDAR currents. Accordingly, we found that the APP processing is increased upon aging, both in mice and human brain. Interfering with stability or production of the APP intracellular domain normalized the GluN2B‐NMDARs currents. While the first mechanism might be essential for synaptic maturation during development, the latter could contribute to age‐related synaptic impairments. Rajão‐Saraiva et al. identified the amyloid precursor protein (APP) as an important regulator of NMDA receptors, acting through a dual age‐dependent mechanism. During postnatal development, APP controls GluN2B‐NMDAR synaptic content and currents, potentially contributing to synaptic maturation. Upon aging, the APP amyloidogenic intracellular fragments contribute to aberrant GluN2B‐NMDAR currents. This work highlights the importance of keeping APP processing under tight control, to ensure the normal functioning of glutamatergic synapses, being particularly relevant to understand age‐related synaptic impairments and Alzheimer's disease.
Article
N-Methyl-d-aspartate (NMDA) receptor hyperfunction plays a key role in the pathological processes of depression and neurodegenerative diseases, whereas NMDA receptor hypofunction is implicated in schizophrenia. Considerable efforts have been made to target NMDA receptor function for the therapeutic intervention in those brain disorders. In this mini-review, we first discuss ion flux-dependent NMDA receptor signaling and ion flux-independent NMDA receptor signaling that result from structural rearrangement upon binding of endogenous agonists. Then, we review current strategies for exploring druggable targets of the NMDA receptor signaling and promising future directions, which are poised to result in new therapeutic agents for several brain disorders.
Thesis
Les échecs cliniques de ces dernières années concernant le traitement de la maladie d’Alzheimer (MA) poussent aujourd’hui les chercheurs à mieux en comprendre la physiopathologie. L’accumulation toxique du peptide amyloïde-β (Aβ) est l’une des caractéristiques cardinales de la MA. L’amyloïdogénèse est impliquée dans la perturbation de l’homéostasie des réseaux neuronaux et contribue à terme aux déficits cognitifs retrouvés chez les patients. L’acide aminé D-sérine est important pour cette homéostasie en étant le co-agoniste préférentiel du récepteur N-methyl-D-aspartate (NMDA-R) du glutamate, un acteur indispensable au contrôle de la plasticité fonctionnelle des réseaux neuronaux de l’hippocampe. Plusieurs études ont directement lié la D-sérine à la MA en rapportant une modification de ses taux ou ceux de son enzyme de conversion – la sérine racémase (SR) – dans le sang, le LCR ou bien le tissu cérébral de patients. Cependant, la direction des modifications et l’implication réelle de la D-sérine dans la MA restent encore débattues. Ce travail de thèse à préciser le rôle de cet acide aminé à l’aide d’un modèle animal innovant associant une amyloïdogénèse à une délétion de la SR (souris 5xFAD/SR-KO). Des études in vivo, ex vivo et in vitro ont été menées de manière à avoir une vision la plus intégrée possible. Dans un premier temps, les travaux ont montré l’implication de la D-sérine dans de nombreux déficits cognitifs retrouvés chez les souris 5xFAD âgées présentant une amyloïdogénèse accentuée. Ces déficits étaient associés à une altération de la plasticité fonctionnelle au niveau des synapses CA3-CA1 de l’hippocampe. Ces atteintes comportementales et fonctionnelles n’étaient pas retrouvées chez la souris 5xFAD/SR-KO indiquant une contribution majeure de la D-sérine dans la physiopathologie amyloïde. Un des résultats majeurs de ce travail est la mise en évidence d’une augmentation précoce et transitoire des taux hippocampiques de D-sérine apparaissant conjointement au début de l’accumulation amyloïde chez les souris 5xFAD et qui se corrèle à une diminution progressive du recrutement des NMDA-R synaptiques. L’ensemble de ces résultats permet une meilleure compréhension de la physiopathologie associée à l'augmentation précoce des taux d’Aβ, en apportant la preuve in vivo d'une implication précoce de la D-sérine dans les désordres fonctionnels des réseaux hippocampiques induits dans un contexte d’amyloïdogénèse accentuée et dans les déficiences cognitives qui en résultent.
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ATB 0 , + ( SLC6A14 ) is a member of the amino acid transporter branch of the SLC6 family along with GlyT1 ( SLC6A9 ) and GlyT2 ( SLC6A5 ), two glycine-specific transporters coupled to 2:1 and 3:1 Na + :Cl − , respectively. In contrast, ATB 0 , + exhibits broad substrate specificity for all neutral and cationic amino acids, and its ionic coupling remains unsettled. Using the reversal potential slope method, we demonstrate a 3:1:1 Na + :Cl − :Gly stoichiometry for ATB 0 , + that is consistent with its 2.1 e /Gly charge coupling. Like GlyT2, ATB 0 , + behaves as a unidirectional transporter with virtually no glycine efflux at negative potentials after uptake, except by heteroexchange as remarkably shown by leucine activation of NMDARs in Xenopus oocytes coexpressing both membrane proteins. Analysis and computational modeling of the charge movement of ATB 0 , + reveal a higher affinity for sodium in the absence of substrate than GlyT2 and a gating mechanism that locks Na + into the apo-transporter at depolarized potentials. A 3:1 Na + :Cl − stoichiometry justifies the concentrative transport properties of ATB 0 , + and explains its trophic role in tumor growth, while rationalizing its phylogenetic proximity to GlyT2 despite their extreme divergence in specificity.
Article
The spinal N-methyl-D-aspartate receptor (NMDAR), particularly their subtypes NR2A and NR2B, plays pivotal roles in neuropathic and inflammatory pain. However, the roles of NR2A and NR2B in orofacial pain and the exact molecular and cellular mechanisms mediating nervous system sensitization are still poorly understood. Here, we exhaustively assessed the regulatory effect of NMDAR in mediating peripheral and central sensitization in orofacial neuropathic pain. Von-Frey filament tests showed that the inferior alveolar nerve (IANX) induced ectopic allodynia behavior in the whisker pad of mice. Interestingly, mechanical allodynia was reversed in mice lacking NR2A and NR2B. IANX also promoted the production of peripheral sensitization-related molecules, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, brain-derived neurotrophic factor (BDNF), and chemokine upregulation (C-C motif) ligand 2 (CCL2), and decreased the inward potassium channel (Kir) 4.1 on glial cells in the trigeminal ganglion, but NR2A conditional knockout (CKO) mice prevented these alterations. In contrast, NR2B CKO only blocked the changes in Kir4.1, IL-1β, and TNF-α and further promoted the production of CCL2. Central sensitization-related c-fos, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) were promoted and Kir4.1 was reduced in the spinal trigeminal caudate nucleus by IANX. Differential actions of NR2A and NR2B in mediating central sensitization were also observed. Silencing of NR2B was effective in reducing c-fos, GFAP, and Iba-1 but did not affect Kir4.1. In contrast, NR2A CKO only altered Iba-1 and Kir4.1 and further increased c-fos and GFAP. Gain-of-function and loss-of-function approaches provided insight into the differential roles of NR2A and NR2B in mediating peripheral and central nociceptive sensitization induced by IANX, which may be a fundamental basis for advancing knowledge of the neural mechanisms’ reaction to nerve injury.
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Through the decades, 2-photon fluorescence microscopy has allowed visualization of microstructures, such as synapses, with high spatial resolution in deep brain tissue. However, signal transduction, such as protein activity and protein-protein interaction in neurons in tissues and in vivo, has remained elusive because of the technical difficulty of observing biochemical reactions at the level of subcellular resolution in light-scattering tissues. Recently, 2-photon fluorescence microscopy combined with fluorescence lifetime imaging microscopy (2pFLIM) has enabled visualization of various protein activities and protein-protein interactions at submicrometer resolution in tissue with a reasonable temporal resolution. Thus far, 2pFLIM has been extensively applied for imaging kinase and small GTPase activation in dendritic spines of hippocampal neurons in slice cultures. However, it has been recently applied to various subcellular structures, such as axon terminals and nuclei, and has increased our understanding of spatially organized molecular dynamics. One of the future directions of 2pFLIM utilization is to combine various optogenetic tools for manipulating protein activity. This combination allows the activation of specific proteins with light and visualization of its readout as the activation of downstream molecules. Here, we have introduced the recent application of 2pFLIM for neurons and present the utilization of a new optogenetic tool in combination with 2pFLIM.
Article
Proteasome activity at the excitatory synapse plays an important role in neuronal communication. The proteasome translocation to synapses is mediated by neuronal activity, in particular the activation of N-methyl-d-aspartate receptors (NMDARs). These receptors are composed of different subunits with distinct trafficking properties that provide various signaling and plasticity features to the synapse. Yet, whether the interplay between the proteasome and NMDAR relies on specific subunit properties remain unclear. Using a combination of single molecule and immunocytochemistry imaging approaches in rat hippocampal neurons, we unveil a specific interplay between GluN2B-containing NMDARs (GluN2B-NMDARs) and the synaptic proteasome. Sustained proteasome activation specifically increases GluN2B-NMDAR (not GluN2A-NMDAR) lateral diffusion. In addition, when GluN2B-NMDAR expression is downregulated the proteasome localization decreases at glutamatergic synapses. Collectively, our data fuel a model in which the cellular dynamics and location of GluN2B-NMDARs and proteasome are intermingled, shedding new lights on the NMDAR-dependent regulation of synaptic adaptation.
Article
Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the vertebrate CNS. Classified as AMPA, kainate, delta and NMDA receptors, iGluRs are central drivers of synaptic plasticity widely considered as a major cellular substrate of learning and memory. Surprisingly however, five out of the eighteen vertebrate iGluR subunits do not bind glutamate but glycine, a neurotransmitter known to mediate inhibitory neurotransmission through its action on pentameric glycine receptors (GlyRs). This is the case of GluN1, GluN3A, GluN3B, GluD1 and GluD2 subunits, all also binding the D amino acid D-serine endogenously present in many brain regions. Glycine and D-serine action and affinities broadly differ between glycinergic iGluR subtypes. On ‘conventional’ GluN1/GluN2 NMDA receptors, glycine (or D-serine) acts in concert with glutamate as a mandatory co-agonist to set the level of receptor activity. It also regulates the receptor’s trafficking and expression independently of glutamate. On ‘unconventional’ GluN1/GluN3 NMDARs, glycine acts as the sole agonist directly triggering opening of excitatory glycinergic channels recently shown to be physiologically relevant. On GluD receptors, D-serine on its own mediates non-ionotropic signaling involved in excitatory and inhibitory synaptogenesis, further reinforcing the concept of glutamate-insensitive iGluRs. Here we present an overview of our current knowledge on glycine and D-serine agonism in iGluRs emphasizing aspects related to molecular mechanisms, cellular function and pharmacological profile. The growing appreciation of the critical influence of glycine and D-serine on iGluR biology reshapes our understanding of iGluR signaling diversity and complexity, with important implications in neuropharmacology.
Article
Key points: NMDA receptors expressed by dopamine neurons of the ventral tegmental area (VTA) play a central role in glutamate synapse plasticity, neuronal firing and adaptative behaviours. The NMDAR surface dynamics shape synaptic adaptation in hippocampal networks, as well as associative memory. We investigated the basic properties and role of the NMDAR surface dynamics onto cultured mesencephalic and ventral tegmental area dopamine neurons in rodents. Using a combination of single molecule imaging and electrophysiological recordings our finding demonstrate that NMDAR are highly diffusive at the surface of mesencephalic dopamine neurons. Unexpectidly, the NMDAR membrane dynamics per se regulate the firing pattern of VTA DA neurons, likely through a functional interplay between NMDA receptors and small conductance calcium-dependent potassium (SK) channels. Abstract: Midbrain dopaminergic (DA) neurons play a central role in major physiological brain functions, and their dysfunctions have been associated to neuropsychiatric diseases. The activity of midbrain DA neurons is controlled by ion channels and neurotransmitter receptors, such as the glutamate NMDA receptor (NMDAR) and small conductance calcium-dependent potassium channels (SK). The cellular mechanisms through which these channels tune the firing pattern of midbrain DA neurons remain however still unclear. Here, we investigated whether the surface dynamics and distribution of NMDAR tunes the firing pattern of midbrain DA neurons. Using a combination of single molecule imaging and electrophysiological recordings, we report that NMDAR are highly diffusive at the surface of cultured midbrain DA neurons from rodents and humans. Reducing acutely the NMDAR membrane dynamics, which leaves intact the ionotropic function of the receptor, robustly altered the firing pattern of midbrain DA neurons without altering the synaptic glutamatergic transmission. The reduction of NMDAR surface dynamics reduced apamin (SK channel blocker)-induced firing change and the distribution of SK3 channels in DA neurons. Altogether, these data unveil that the surface dynamics of NMDAR, and not solely its ionotropic function, tune the firing pattern of midbrain DA neurons partly through a functional interplay with SK channel function. This article is protected by copyright. All rights reserved.
Article
Fluorescence lifetime microscopy (FLIM) and Förster's resonance energy transfer (FRET) are advanced optical tools that neuroscientists can employ to interrogate the structure and function of complex biological systems in vitro and in vivo using light. In neurobiology they are primarily used to study protein‐protein interactions, to study conformational changes in protein complexes, and to monitor genetically encoded FRET‐based biosensors. These methods are ideally suited to optically monitor changes in neurons that are triggered optogenetically. Utilization of this technique by neuroscientists has been limited, since a broad understanding of FLIM and FRET requires familiarity with the interactions of light and matter on a quantum mechanical level, and because the ultra‐fast instrumentation used to measure fluorescent lifetimes and resonance energy transfer are more at home in a physics lab than in a biology lab. In this overview, we aim to help neuroscientists overcome these obstacles and thus feel more comfortable with the FLIM‐FRET method. Our goal is to aid researchers in the neuroscience community to achieve a better understanding of the fundamentals of FLIM‐FRET and encourage them to fully leverage its powerful ability as a research tool. Published 2020. U.S. Government.
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Although numerous pathogenic mutations have been identified in various subunits of N -methyl-D-aspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Here, we combined in silico modelling with microscopy, biochemistry, and electrophysiology in cultured HEK293 cells and hippocampal neurons to examine how the pathogenic missense mutation S688Y in the GluN1 NMDAR subunit affects receptor function and trafficking. We found that the S688Y mutation significantly increases the EC 50 of both glycine and d -serine in GluN1/GluN2A and GluN1/GluN2B receptors, and significantly slows desensitisation of GluN1/GluN3A receptors. Moreover, the S688Y mutation reduces the surface expression of GluN3A-containing NMDARs in cultured hippocampal neurons, but does not affect the trafficking of GluN2-containing receptors. Finally, we found that the S688Y mutation reduces Ca ²⁺ influx through NMDARs and reduces NMDA-induced excitotoxicity in cultured hippocampal neurons. These findings provide key insights into the molecular mechanisms that underlie the regulation of NMDAR subtypes containing pathogenic mutations.
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Structural plasticity of dendritic spines is a key component of the refinement of synaptic connections during learning. Recent studies highlight a novel role for the NMDA receptor (NMDAR), independent of ion flow, in driving spine shrinkage and LTD. Yet little is known about the molecular mechanisms that link conformational changes in the NMDAR to changes in spine size and synaptic strength. Here, using two-photon glutamate uncaging to induce plasticity at individual dendritic spines on hippocampal CA1 neurons from mice and rats of both sexes, we demonstrate that p38 MAPK is generally required downstream of conformational NMDAR signaling to drive both spine shrinkage and LTD. In a series of pharmacological and molecular genetic experiments, we identify key components of the non-ionotropic NMDAR signaling pathway driving dendritic spine shrinkage, including the interaction between NOS1AP and nNOS, nNOS enzymatic activity, activation of MK2 and cofilin, and signaling through CaMKII. Our results represent a large step forward in delineating the molecular mechanisms of non-ionotropic NMDAR signaling that can drive shrinkage and elimination of dendritic spines during synaptic plasticity.Significance statementSignaling through the NMDA receptor (NMDAR) is vitally important for the synaptic plasticity that underlies learning. Recent studies highlight a novel role for the NMDAR, independent of ion flow, in driving synaptic weakening and dendritic spine shrinkage during synaptic plasticity. Here, we delineate several key components of the molecular pathway that links conformational signaling through the NMDAR to dendritic spine shrinkage during synaptic plasticity.
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Key points: We present a novel protocol to quantify extrasynaptic NMDA receptor function utilizing the semi-selective activation of extrasynaptic receptors by ambient extracellular glutamate in acute brain slices from adult rats. We use whole cell patch clamp to measure the effect of the NMDA receptor antagonist MK-801 on both synaptic and brief, local agonist application-evoked responses. The level of ambient glutamate was estimated from tonic NMDA receptor activity to be ∼77 nm and an equivalent concentration of NMDA was used to estimate the degree of extrasynaptic blockade (>82%) by our MK-801 protocol. The extrasynaptic component of the total NMDA receptor pool can be mathematically derived from these data and was estimated to be 29-39% in the stratum radiatum of the CA1 region of the rat hippocampus. This technique could be used to quantify extrasynaptic NMDA receptor function in rodent models of diseases where extrasynaptic NMDA receptors are implicated in neuron death. Abstract: Synaptic NMDA receptors (NMDARs) play a central role in pro-survival signalling and synaptic plasticity in the majority of excitatory synapses in the central nervous system whereas extrasynaptic NMDARs (ES-NMDARs) activate pro-death pathways and have been implicated in many neurodegenerative diseases. ES-NMDARs have been characterized in acute brain slice preparations using the largely irreversible, activity-dependent NMDAR antagonist, MK-801 to block synaptic NMDARs. This approach is limited by the concomitant MK-801 blockade of ES-NMDARs activated by ambient extracellular glutamate, which is largely absent from the synaptic cleft due to the high density of nearby glutamate transporters. In acute hippocampal slices from P35-42 rats we estimated ambient glutamate to be 72 nm to 83 nm resulting in a block of more than 82% of ES-NMDARs during a 5 min MK-801 application. This paper describes a novel electrophysiological and mathematical method to quantify the proportion of NMDARs located at extrasynaptic locations in a confined region of an acute brain slice preparation using MK-801 to preferentially block ES-NMDARs. The protocol uses whole cell patch clamp measurement of NMDAR responses to synaptic stimulation and brief local pressure application of NMDA before and after MK-801 application. After mathematically correcting for the relative block of both synaptic and extrasynaptic receptors, ES-NMDARs were estimated to comprise 29-39% of the total NMDAR pool in the apical dendrites of hippocampal CA1 pyramidal neurons. This new method may prove useful for accurate quantification NMDAR distributions in neurodegenerative diseases that are associated with increased toxic ES-NMDAR signalling. This article is protected by copyright. All rights reserved.
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The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDAR-dependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A- and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NMDAR subtypes depends on the regulation of the receptor surface trafficking. • development • glutamate receptor • lateral mobility
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