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NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy. [ABSTRACT FROM AUTHOR]

N -methyl-D-aspartate receptors (NMDARs) belong to a family of ionotropic glutamate receptors that play essential roles in excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). Functional NMDARs consist of heterotetramers comprised of GluN1, GluN2A-D, and/or GluN3A-B subunits, each of which contains four membrane domains (M1 through M4), an intracellular C-terminal domain, a large extracellular N-terminal domain composed of the amino-terminal domain and the S1 segment of the ligand-binding domain (LBD), and an extracellular loop between M3 and M4, which contains the S2 segment of the LBD. Both the number and type of NMDARs expressed at the cell surface are regulated at several levels, including their translation and posttranslational maturation in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, lateral diffusion in the plasma membrane, and internalization and degradation. This review focuses on the roles played by the extracellular regions of GluN subunits in ER processing. Specifically, we discuss the presence of ER retention signals, the integrity of the LBD, and critical N -glycosylated sites and disulfide bridges within the NMDAR subunits, each of these steps must pass quality control in the ER in order to ensure that only correctly assembled NMDARs are released from the ER for subsequent processing and trafficking to the surface. Finally, we discuss the effect of pathogenic missense mutations within the extracellular domains of GluN subunits with respect to ER processing of NMDARs. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptors (NMDARs) play critical roles in both excitatory neurotransmission and synaptic plasticity. NMDARs containing the nonconventional GluN3A subunit have different functional properties compared to receptors comprised of GluN1/GluN2 subunits. Previous studies showed that GluN1/GluN2 receptors are regulated by N-glycosylation; however, limited information is available regarding the role of N-glycosylation in GluN3A-containing NMDARs. Using a combination of microscopy, biochemistry, and electrophysiology in mammalian cell lines and rat hippocampal neurons, we found that two asparagine residues (N203 and N368) in the GluN1 subunit and three asparagine residues (N145, N264 and N275) in the GluN3A subunit are required for surface delivery of GluN3A-containing NMDARs. Furthermore, deglycosylation and lectin-based analysis revealed that GluN3A subunits contain extensively modified N-glycan structures, including hybrid/complex forms of N-glycans. We also found (either using a panel of inhibitors or by studying human fibroblasts derived from patients with a congenital disorder of glycosylation) that N-glycan remodeling is not required for the surface delivery of GluN3A-containing NMDARs. Finally, we found that the surface mobility of GluN3A-containing NMDARs in hippocampal neurons is increased following incubation with 1-deoxymannojirimycin (DMM, an inhibitor of the formation of the hybrid/complex forms of N-glycans) and decreased in the presence of specific lectins. These findings provide new insight regarding the mechanisms by which neurons can regulate NMDAR trafficking and function. [ABSTRACT FROM AUTHOR]

In mammals, excitatory synapses contain two major types of ionotropic glutamate receptors: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl- d-aspartate receptors ( NMDARs). Both receptor types are comprised of several subunits that are post-translationally modified by N-glycosylation. However, the precise N-glycans that are attached to these receptor types are largely unknown. Here, we used biochemistry to confirm that native NMDARs are extensively N-glycosylated; moreover, we found that the NMDAR GluN2B subunit differs from GluN1 subunits with respect to endoglycosidase H sensitivity. Next, we used a complete panel of lectins to determine the glycan composition of NMDARs in both cerebellar tissue and cultured cerebellar granule cells. Our experiments identified 23 lectins that pulled down both the GluN1 and GluN2B NMDAR subunits. We then performed an electrophysiological analysis using representative lectins and found that pre-incubating cerebellar granule cells with the AAL, WGA, or ConA alters the receptor's biophysical properties; this lectin-mediated effect was eliminated when the cells were deglycosylated with peptide-N-glycosidase F. Similar lectin-mediated effects were observed using HEK293 cells that express recombinant GluN1/GluN2B receptors. Finally, using mutant recombinant GluN subunits expressed in HEK293 cells, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data provide new insight into the role that N-glycosylation plays in regulating the function of NMDA receptors in the central nervous system. All animal experiments were performed in accordance with relevant institutional ethics guidelines and regulations with respect to protecting animal welfare. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate (NMDA) receptors mediate fast excitatory synaptic transmission in the mammalian central nervous system. The activation of NMDA receptors plays a key role in brain development, synaptic plasticity, and memory formation, and is a major contributor to many neuropsychiatric disorders. Here, we investigated the mechanisms that underlie the trafficking of GluN1/GluN2C receptors. Using an approach combining molecular biology, microscopy, and electrophysiology in mammalian cell lines and cultured cerebellar granule cells, we found that the surface delivery of GluN2C-containing receptors is reduced compared to GluN2A- and GluN2B-containing receptors. Furthermore, we identified three distinct regions within the N-terminus, M3 transmembrane domain, and C-terminus of GluN2C subunits that are required for proper intracellular processing and surface delivery of NMDA receptors. These results shed new light on the regulation of NMDA receptor trafficking, and these findings can be exploited to develop new strategies for treating some forms of neuropsychiatric disorders. [ABSTRACT FROM AUTHOR]

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. There are three distinct subtypes of ionotropic glutamate receptors (GluRs) that have been identified including 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptors (AMPARs), N-methyl-D-aspartate receptors (NMDARs) and kainate receptors. The most common GluRs in mature synapses are AMPARs that mediate the fast excitatory neurotransmission and NMDARs that mediate the slow excitatory neurotransmission. There have been large numbers of recent reports studying how a single neuron regulates synaptic numbers and types of AMPARs and NMDARs. Our current research is centered primarily on NMDARs and, therefore, we will focus in this review on recent knowledge of molecular mechanisms occurring (1) early in the biosynthetic pathway of NMDARs, (2) in the transport of NMDARs after their release from the endoplasmic reticulum (ER); and (3) at the plasma membrane including excitatory synapses. Because a growing body of evidence also indicates that abnormalities in NMDAR functioning are associated with a number of human psychiatric and neurological diseases, this review together with other chapters in this issue may help to enhance research and to gain further knowledge of normal synaptic physiology as well as of the etiology of many human brain diseases. [ABSTRACT FROM AUTHOR]

N-methyl- d-aspartate ( NMDA) receptors are glutamate ion channels that are critically involved in excitatory synaptic transmission and plasticity. The functional NMDA receptor is a heterotetramer composed mainly of GluN1 and GluN2 subunits. It is generally thought that only correctly assembled NMDA receptors can pass the quality control checkpoint in the endoplasmic reticulum ( ER) and are transported to the cell surface membranes. The molecular mechanisms underlying these processes remain poorly understood. Using chimeric and mutated GluN1 subunits expressed in heterologous cells, we identified a single amino acid residue within the fourth membrane domain (M4) of GluN1 subunit, L830, that regulates the surface number of NMDA receptors. Our experiments show that this residue is not critical for the interaction between GluN1 and GluN2 subunits or for the formation of functional receptors, but rather that it regulates the forward trafficking of the NMDA receptors. The surface expression of both GluN2A- and GluN2B-containing receptors is regulated by the L830 residue in a similar manner. We also found that the L830 residue is not involved in the trafficking of individually expressed GluN1 subunits. Our data reveal a critical role of the single amino acid residue within the GluN1 M4 domain in the surface delivery of functional NMDA receptors. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the mammalian central nervous system (CNS), but their dysregulation results in the aetiology of many human CNS disorders. Several NMDAR modulators including memantine have been used successfully in clinical trials. Indeed, 1,2,3,4-tetrahydro-9-aminoacridine (tacrine; THA) was the first approved drug for Alzheimer's disease (AD) treatment. 7-methoxyderivative of THA (7-MEOTA) is less toxic and showed promising results in patients with tardive dyskinesia. Here, we employed electrophysiological recordings in HEK293 cells and rat neurones to examine the mechanism of action of THA and 7-MEOTA at the NMDAR. We showed that both THA and 7-MEOTA are "foot-in-the-door" open-channel blockers of GluN1/GluN2 and GluN1/GluN3 NMDARs and that 7-MEOTA is a more potent but slower blocker than THA. Furthermore, the inhibitory potency of 7-MEOTA at synaptic and extrasynaptic hippocampal NMDARs was similar, and 7-MEOTA exhibited better neuroprotective activity in rats exposed NMDA-induced lesions in hippocampus when compared with THA and memantine. Finally, intraperitoneal administration of 7-MEOTA attenuated MK-801-induced hyperlocomotion in rats. We conclude that 7-MEOTA is a promising candidate for the treatment of diseases associated with the dysfunction of NMDARs. [ABSTRACT FROM AUTHOR]

N -methyl- d -aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory neurotransmission in the mammalian central nervous system (CNS), and their dysregulation results in the aetiology of many CNS syndromes. Several NMDAR modulators have been used successfully in clinical trials (including memantine) and NMDARs remain a promising pharmacological target for the treatment of CNS syndromes. 1,2,3,4-Tetrahydro-9-aminoacridine (tacrine; THA) was the first approved drug for Alzheimer's disease (AD) treatment. 7-methoxyderivative of THA (7-MEOTA) is less toxic and showed promising results in patients with tardive dyskinesia. We employed electrophysiological recordings in HEK293 cells and rat neurones to examine the mechanism of action of THA and 7-MEOTA at the NMDAR. We showed that both THA and 7-MEOTA are “foot-in-the-door” open-channel blockers of GluN1/GluN2 receptors and that 7-MEOTA is a more potent but slower blocker than THA. We found that the IC 50 values for THA and 7-MEOTA exhibited the GluN1/GluN2A < GluN1/GluN2B < GluN1/GluN2C = GluN1/GluN2D relationship and that 7-MEOTA effectively inhibits human GluN1/GluN2A-M817V receptors that carry a pathogenic mutation. We also showed that 7-MEOTA is a “foot-in-the-door” open-channel blocker of GluN1/GluN3 receptors, although these receptors were not inhibited by memantine. In addition, the inhibitory potency of 7-MEOTA at synaptic and extrasynaptic hippocampal NMDARs was similar, and 7-MEOTA exhibited better neuroprotective activity when compared with THA and memantine in rats with NMDA-induced lesions of the hippocampus. Finally, intraperitoneal administration of 7-MEOTA attenuated MK-801-induced hyperlocomotion and pre-pulse inhibition deficit in rats. We conclude that 7-MEOTA may be considered for the treatment of diseases associated with the dysfunction of NMDARs. [ABSTRACT FROM AUTHOR]

N -methyl- D -aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission in the mammalian brain, and their physiological importance is underscored by the large number of pathogenic mutations that have been identified in the receptor's GluN subunits and associated with a wide range of diseases and disorders. Here, we characterized the functional and pharmacological effects of the pathogenic N650K variant in the GluN1 subunit, which is associated with developmental delay and seizures. Our microscopy experiments showed that when expressed in HEK293 cells (from ATCC®), the GluN1-N650K subunit increases the surface expression of both GluN1/GluN2A and GluN1/GluN2B receptors, but not GluN1/GluN3A receptors, consistent with increased surface expression of the GluN1-N650K subunit expressed in hippocampal neurons (from embryonic day 18 of Wistar rats of both sexes). Using electrophysiology, we found that the GluN1-N650K variant increases the potency of GluN1/GluN2A receptors to both glutamate and glycine but decreases the receptor's conductance and open probability. In addition, the GluN1-N650K subunit does not form functional GluN1/GluN2B receptors but does form fully functional GluN1/GluN3A receptors. Moreover, in the presence of extracellular Mg2+, GluN1-N650K/GluN2A receptors have a similar and increased response to ketamine and memantine, respectively, while the effect of both drugs had markedly slower onset and offset compared to wild-type GluN1/GluN2A receptors. Finally, we found that expressing the GluN1-N650K subunit in hippocampal neurons reduces excitotoxicity, and memantine shows promising neuroprotective effects in neurons expressing either wild-type GluN1 or the GluN1-N650K subunit. This study provides the functional and pharmacological characterization of NMDARs containing the GluN1-N650K variant. • We studied the properties of NMDA receptors consisting of GluN1 subunits with the pathogenic N650K variant. • This variant increases the surface expression of GluN1/GluN2 but not GluN1/GluN3A receptors. • This variant decreases open probability of GluN1/GluN2 receptors. • This variant affects NMDA receptor inhibition by memantine and ketamine. [ABSTRACT FROM AUTHOR]

Abstract: Glutamate is the main neurotransmitter released at synapses in the central nervous system of vertebrates. Its excitatory role is mediated through activation of specific glutamatergic ionotropic receptors, among which the N-methyl-d-aspartate (NMDA) receptor subtype has attracted considerable attention in recent years. Substantial progress has been made in elucidating the roles these receptors play under physiological and pathological conditions and in our understanding of the functional, structural, and pharmacological properties of NMDA receptors. Many pharmacological compounds have been identified that affect the activity of NMDA receptors, including neurosteroids. This review summarizes our knowledge about molecular mechanisms underlying the neurosteroid action at NMDA receptors as well as about the action of neurosteroids in animal models of human diseases. [Copyright &y& Elsevier]

Tacrine is a classic drug whose efficacy against neurodegenerative diseases is still shrouded in mystery. It seems that besides its inhibitory effect on cholinesterases, the clinical benefit is co-determined by NMDAR-antagonizing activity. Our previous data showed that the direct inhibitory effect of tacrine, as well as its 7-methoxy derivative (7-MEOTA), is ensured via a "foot-in-the-door" open-channel blockage, and that interestingly both tacrine and 7-MEOTA are slightly more potent at the GluN1/GluN2A receptors when compared with the GluN1/GluN2B receptors. Here, we report that in a series of 30 novel tacrine derivatives, designed for assessment of structure-activity relationship, blocking efficacy differs among different compounds and receptors using electrophysiology with HEK293 cells expressing the defined types of NMDARs. Selected compounds (4 and 5) potently inhibited both GluN1/GluN2A and GluN1/GluN2B receptors; other compounds (7 and 23) more effectively inhibited the GluN1/GluN2B receptors; or the GluN1/GluN2A receptors (21 and 28). QSAR study revealed statistically significant model for the data obtained for inhibition of GluN1/Glu2B at −60 mV expressed as IC 50 values, and for relative inhibition of GluN1/Glu2A at +40 mV caused by a concentration of 100 μM. The models can be utilized for a ligand-based virtual screening to detect potential candidates for inhibition of GluN1/Glu2A and/or GluN1/Glu2B subtypes. Using in vivo experiments in rats we observed that unlike MK-801, the tested novel compounds did not induce hyperlocomotion in open field, and also did not impair prepulse inhibition of startle response, suggesting minimal induction of psychotomimetic side effects. We conclude that tacrine derivatives are promising compounds since they are centrally available subtype-specific inhibitors of the NMDARs without detrimental behavioral side-effects. [Display omitted] • A series of 30 tacrine derivatives were designed and synthesized. • SAR analysis on anticholinesterase and QSAR analysis NMDA receptors activity is reported. • NMDA receptors inhibition significantly differs among the compounds. • Highlighted compounds with promising anti-ChE and NMDA activity are centrally active. • Top-ranked compounds were devoid of detrimental behavioral side-effects. [ABSTRACT FROM AUTHOR]

N -methyl- d -aspartate receptors (NMDARs) play an essential role in regulating glutamatergic neurotransmission. Recently, pathogenic missense mutations were identified in genes encoding NMDAR subunits; however, their effect on NMDAR activity is often poorly understood. Here, we examined whether three previously identified pathogenic mutations (M641I, A645S, and Y647S) in the M3 domain of the GluN1 subunit affect the receptor's surface delivery, agonist sensitivity, Mg2+ block, and/or inhibition by the FDA-approved NMDAR blocker memantine. When expressed in HEK293 cells, we found reduced surface expression of GluN1-M641I/GluN2A, GluN1-Y647S/GluN2A, and GluN1-Y647S/GluN2B receptors; other mutation-bearing NMDAR combinations, including GluN1/GluN3A receptors, were expressed at normal surface levels. When expressed in rat hippocampal neurons, we consistently found reduced surface expression of the GluN1-M641I and GluN1-Y647S subunits when compared with wild-type GluN1 subunit. At the functional level, we found that GluN1-M641I/GluN2 and GluN1-A645S/GluN2 receptors expressed in HEK293 cells have wild-type EC 50 values for both glutamate and glycine; in contrast, GluN1-Y647S/GluN2 receptors do not produce glutamate-induced currents. In the presence of a physiological concentration of Mg2+, we found that GluN1-M641I/GluN2 receptors have a lower memantine IC 50 and slower offset kinetics, whereas GluN1-A645S/GluN2 receptors have a higher memantine IC 50 and faster offset kinetics when compared to wild-type receptors. Finally, we found that memantine was the most neuroprotective in hippocampal neurons expressing GluN1-M641I subunits, followed by neurons expressing wild-type GluN1 and then GluN1-A645S subunits in an NMDA-induced excitotoxicity assay. These results indicate that specific pathogenic mutations in the M3 domain of the GluN1 subunit differentially affect the trafficking and functional properties of NMDARs. • We studied NMDA receptors containing three pathogenic mutations in the GluN1 subunit. • These pathogenic mutations alter surface numbers of specific GluN1/GluN2 receptors. • Two of the mutations alter the sensitivity of the NMDA receptor to memantine. • Altered sensitivity to memantine correlates with its neuroprotective effect. [ABSTRACT FROM AUTHOR]

[Display omitted] N -methyl-D-aspartaterecepro receptor (NMDARs) are a subclass of glutamate receptors, which play an essential role in excitatory neurotransmission, but their excessive overactivation by glutamate leads to excitotoxicity. NMDARs are hence a valid pharmacological target for the treatment of neurodegenerative disorders; however, novel drugs targeting NMDARs are often associated with specific psychotic side effects and abuse potential. Motivated by currently available treatment against neurodegenerative diseases involving the inhibitors of acetylcholinesterase (AChE) and NMDARs, administered also in combination, we developed a dually-acting compound 7-phenoxytacrine (7-PhO-THA) and evaluated its neuropsychopharmacological and drug-like properties for potential therapeutic use. Indeed, we have confirmed the dual potency of 7-PhO-THA, i.e. potent and balanced inhibition of both AChE and NMDARs. We discovered that it selectively inhibits the GluN1/GluN2B subtype of NMDARs via an ifenprodil-binding site, in addition to its voltage-dependent inhibitory effect at both GluN1/GluN2A and GluN1/GluN2B subtypes of NMDARs. Furthermore, whereas NMDA-induced lesion of the dorsal hippocampus confirmed potent anti-excitotoxic and neuroprotective efficacy, behavioral observations showed also a cholinergic component manifesting mainly in decreased hyperlocomotion. From the point of view of behavioral side effects, 7-PhO-THA managed to avoid these, notably those analogous to symptoms of schizophrenia. Thus, CNS availability and the overall behavioral profile are promising for subsequent investigation of therapeutic use. [ABSTRACT FROM AUTHOR]

N -methyl- d -aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission within the mammalian central nervous system (CNS). NMDARs are heteromultimers containing GluN1, GluN2, and/or GluN3 subunits, thus giving rise to a wide variety of subunit combinations, each with unique functional and pharmacological properties. Importantly, GluN1/GluN3A and GluN1/GluN3B receptors form glycine-gated receptors. Here, we combined electrophysiology with rapid solution exchange in order to determine whether the presence of specific N -glycans and/or interactions with specific lectins regulates the functional properties of GluN1/GluN3A and GluN1/GluN3B receptors expressed in human embryonic kidney 293 (HEK293) cells. We found that removing putative N -glycosylation sites alters the functional properties of GluN1/GluN3B receptors, but has no effect on GluN1/GluN3A receptors. Moreover, we found that the functional properties of both GluN1/GluN3A and GluN1/GluN3B receptors are modulated by a variety of lectins, including Concanavalin A (ConA), Wheat Germ Agglutinin (WGA), and Aleuria Aurantia Lectin (AAL), and this effect is likely mediated by a reduction in GluN1 subunit-mediated desensitization. We also found that AAL has the most profound effect on GluN1/GluN3 receptors, and this effect is mediated partly by a single N -glycosylation site on the GluN3 subunit (specifically, N565 on GluN3A and N465 on GluN3B). Finally, we found that lectins mediate their effect only when applied to non-activated receptors and have no effect when applied in the continuous presence of glycine. These findings provide further evidence to distinguish GluN1/GluN3 receptors from the canonical GluN1/GluN2 receptors and offer insight into how GluN1/GluN3 receptors may be regulated in the mammalian CNS. • NMDA receptors play a key role in mediating mammalian excitatory neurotransmission. • GluN1/GluN3 NMDA receptors exhibit unique functional and pharmacological properties. • We show that lectins reduce desensitization of GluN1/GluN3 NMDA receptors. • Our data further distinguish GluN1/GluN3 receptors from GluN1/GluN2 NMDA receptors. [ABSTRACT FROM AUTHOR]