N-Methyl-D-aspartate (NMDA) receptor subunit NR1 forms the substrate for oligomeric assembly of the NMDA receptor

The time course of the assembly of the N-methyl-D-aspartate receptor was examined in a cell line expressing it under the control of the dexamethasone promoter. These studies suggested a delay between the appearance of the NR1 and NR2A subunits and their stable association as examined by co-immunoprecipitation of NR1 and NR2A. This prompted us to examine the stability and folding of the individual subunits using nonreduced polyacrylamide gels and the sulfhydryl cross-linker BMH. Both studies showed that the NR1 subunit was expressed in a monomer and dimer form, whereas both NR2 and NR3 showed substantial aggregation on both nonreduced gels and after cross-linking. Protein degradation experiments showed that NR1 was relatively stable, whereas NR2 and NR3 were more rapidly degraded. When co-expressed with NR1, NR2 was more stable. Fluorescence recovery after photobleaching experiments showed that, under conditions of reduced ATP, the diffusion rate of NR2 and NR3 in the endoplasmic reticulum was reduced, whereas that of NR1 was unaffected. Together these data show that NR1 folds stably when expressed alone, unlike NR2 and NR3, and provides the substrate for assembly of the N-methyl-D-aspartate receptor.     

NMDA and glycine regulate the affinity of the Mg2+-block site in NR1-1a/NR2A NMDA receptor channels expressed in Xenopus oocytes

NMDA receptors are glutamate-regulated ion channels of critical importance for many neurophysiological and neuropathological processes. Mg2+ blocks the NMDA receptor by binding to the channel pore with an apparent affinity that depends on the membrane potential. We have investigated the effect of NMDA and the required co-agonist glycine on the affinity of the Mg2+ block site in NR1-1a/NR2A NMDA receptors expressed in Xenopus oocytes. We found that NMDA and glycine increase the IC50 value of the Mg2+-block site at pH 7.4 and in the presence of physiological concentration of Ca2+. The increase the IC50 value may correspond to a decrease in Mg2+-block affinity. This effect may result in an increased influx of Ca2+, and this influx may constitute up to a third of the total Ca2+ influx induced by NMDA. At high pH, or at low concentrations of Ca2+, NMDA and glycine have an opposite effect and instead decreased the IC50 value of the Mg2+-block. These results indicate that glutamate and glycine can regulate the affinity of the Mg2+-block site. This effect may have implications for the understanding the role of NMDA receptors both under physiological and pathophysiological conditions.     

Increased phosphorylation of the NR1 subunit of the NMDA receptor following cerebral ischemia

The effects of transient cerebral ischemia on phosphorylation of the NR1 subunit of the NMDA receptor by protein kinase C (PKC) and protein kinase A (PKA) were investigated. Adult rats received 15 min of cerebral ischemia followed by various times of recovery. Phosphorylation was examined by immunoblotting hippocampal homogenates with antibodies that recognized NR1 phosphorylated on the PKC phosphorylation sites Ser890 and Ser896, the PKA phosphorylation site Ser897, or dually phosphorylated on Ser896 and Ser897. The phosphorylation of all sites examined increased following ischemia. The increase in phosphorylation by PKC was greater than by PKA. The ischemia-induced increase in phosphorylation was predominantly associated with the population of NR1 that was insoluble in 1% deoxycholate. Enhanced phosphorylation of NR1 by PKC and PKA may contribute to alterations in NMDA receptor function in the postischemic brain.     

Reactive derivatives for affinity labeling in the ifenprodil site of NMDA receptors

To prepare thiol-reactive ifenprodil derivatives designed as potential probes for cysteine-substituted NR2B containing NMDA receptors, electrophilic centers were introduced in different areas of the ifenprodil structure. Intermediates and final compounds were evaluated by binding studies and by electrophysiology to determine the structural requirements for their selectivity. The reactive compounds were further tested for their stability and for their reactivity in model reactions; some were found suitable as structural probes to investigate the binding site and the docking mode of ifenprodil in the NR2B subunit.     

Assessment of NMDA receptor NR1 subunit hypofunction in mice as a model for schizophrenia

N-methyl- D -aspartate receptors (NMDARs) play a pivotal role in excitatory neurotransmission, synaptic plasticity and brain development. Clinical and experimental evidence suggests a dysregulation of NMDAR function and glutamatergic pathways in the pathophysiology of schizophrenia. We evaluated electrophysiological and behavioral properties of NMDAR deficiency utilizing mice that express only 5–10% of the normal level of NMDAR NR1 subunit. Auditory and visual event related potentials yielded significantly increased amplitudes for the P20 and N40 components in NMDAR deficient (NR1^neo−/−) mice suggesting decreased inhibitory tone. Compared to wild types, NR1^neo−/− mice spent less time in social interactions and showed reduced nest building. NR1^neo−/− mice displayed a preference for open arms of a zero maze and central zone of an open field, possibly reflecting decreased anxiety-related behavioral inhibition. However, locomotor activity did not differ between groups in either home cage environment or during behavioral testing. NR1^neo−/− mice displayed hyperactivity only when placed in a large unfamiliar environment, suggesting that neither increased anxiety nor non-specific motor activation accounts for differential behavioral patterns. Data suggest that NMDAR NR1 deficiency causes disinhibition in sensory processing as well as reduced behavioral inhibition and impaired social interactions. The behavioral signature in NR1^neo−/− mice supports the impact of impaired NMDAR function in a mouse model with possible relevance to negative symptoms in schizophrenia.     

Translationally distinct populations of NMDA receptor subunit NR1 mRNA in the developing rat brain

The translational activity of the NMDA subunit 1 (NR1) mRNA was examined in the developing rat brain by sucrose gradient fractionation. One translationally-active pool of NR1 mRNA was associated with large polyribosomes (polysomes) over the entire developmental period examined. A second NR1 mRNA pool, approximately half of the NR1 mRNA at post-natal day 4, sedimented only within the two to three ribosome range, indicating that it was translationally blocked during early brain development despite active translation of mRNAs coding for the NR2 subunits of the receptor. At post-natal day 4, both NR1 mRNA pools were distributed throughout the brain and contained similar profiles of NR1 mRNA splice variants, except that NR1-3 appeared to be present only in the translationally-blocked NR1 pool. After post-natal day 8, the translationally-blocked NR1 mRNA pool became progressively active within a background of globally-decreasing brain translational activity.     

Serotonin 5-HT1A receptor activation prevents phosphorylation of NMDA receptor NR1 subunit in cerebral ischemia

The mechanisms involved in the neuroprotective effect of serotonin 5-HT1A receptor agonists on brain damage induced by ischemia remain to be fully elucidated. Given that serotonergic drugs may regulate N-methyl-D-aspartate (NMDA) receptor function, which is implicated in events leading to ischemia-induced neuronal cell death, this study sought to determine the effects of the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on the levels of NMDA receptor NR1 subunit in gerbil hippocampus after transient global cerebral ischemia. Pretreatment with 8-OH-DPAT (1 mg/kg) prevented the neuronal loss in CA1 subfield 72 h after ischemia. NMDA receptor NR1 levels in whole hippocampus were not affected 24 h after ischemia, but the levels of the subunit phosphorylated at the protein kinase A (PKA) site, pNR1(Ser897), were significantly increased, and this increase was prevented by the same 8-OH-DPAT dose, a probable consequence of the increased phosphatase 1 (PP1) enzyme activity found in ischemic gerbils pretreated with the 5-HT1A receptor agonist. The results suggest that NR1 subunit phosphorylation plays a role in the neuroprotective effect of 8-OH-DPAT on cell damage induced by global cerebral ischemia in the gerbil hippocampus and support the potential interest of 5-HT1A receptor activation in the search for neuroprotective strategies.     

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

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

Association of the small GTPase Rheb with the NMDA receptor subunit NR3A

The NMDAR subunit NR3A is most highly expressed during the second postnatal week, when synaptogenesis reaches peak levels. Genetic ablation or overexpression of the NR3A subunit negatively interferes with the maturation of cortical synapses and leads to changes in the shape and number of dendritic spines, the density of which is increased in NR3A knock-out mice and decreased in NR3A-overexpressing transgenic mice. Alterations in spine density have been linked to dysregulation of mTOR signaling and synaptic protein translation. Using a yeast two-hybrid system, we identified the mTOR-activating GTPase Rheb as an interacting protein of the NMDAR subunit NR3A. We confirmed the interaction in mammalian cells by expressing recombinant Rheb and NR3A and showed that Rheb and NR3A could be co-immunoprecipitated from synaptic plasma membranes from the developing rat brain. These data suggest that NR3A sequesters synaptic Rheb and might thus function as a break of the mTOR-dependent synaptic translation of protein.     

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

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

Sociability development in mice with cell‐specific deletion of the NMDA receptor NR1 subunit gene

Social affiliative behavior is an important component of everyday life in many species and is likely to be disrupted in disabling ways in various neurodevelopmental and neuropsychiatric disorders. Therefore, determining the mechanisms involved in these processes is crucial. A link between N‐methyl‐d ‐aspartate (NMDA) receptor function and social behaviors has been clearly established. The cell types in which NMDA receptors are critical for social affiliative behavior, however, remain unclear. Here, we use mice carrying a conditional allele of the NMDA R1 subunit to address this question. Mice bearing a floxed NMDAR1 (NR1) allele were crossed with transgenic calcium/calmodulin‐dependent kinase IIα (CaMKIIα)‐Cre mice or parvalbumin (PV)‐Cre mice targeting postnatal excitatory forebrain or PV‐expressing interneurons, respectively, and assessed using the three‐chambered Social Approach Test. We found that deletion of NR1 in PV‐positive interneurons had no effect on social sniffing, but deletion of NR1 in glutamatergic pyramidal cells resulted in a significant increase in social approach behavior, regardless of age or sex. Therefore, forebrain excitatory neurons expressing NR1 play an important role in regulating social affiliative behavior.     

Mechanism of partial agonist action at the NR1 subunit of NMDA receptors

Partial agonists produce submaximal activation of ligand-gated ion channels. To address the question of partial agonist action at the NR1 subunit of the NMDA receptor, we performed crystallographic and electrophysiological studies with 1-aminocyclopropane-1-carboxylic acid (ACPC), 1-aminocyclobutane-1-carboxylic acid (ACBC), and 1-aminocyclopentane-1-carboxylic acid (cycloleucine), three compounds with incrementally larger carbocyclic rings. Whereas ACPC and ACBC partially activate the NMDA receptor by 80% and 42%, respectively, their cocrystal structures of the NR1 ligand binding core show the same degree of domain closure as found in the complex with glycine, a full agonist, illustrating that the NR1 subunit provides a new paradigm for partial agonist action that is distinct from that of the evolutionarily related GluR2, AMPA-sensitive receptor. Cycloleucine behaves as an antagonist and stabilizes an open-cleft conformation. The NR1-cycloleucine complex forms a dimer that is similar to the GluR2 dimer, thereby suggesting a conserved mode of subunit-subunit interaction in AMPA and NMDA receptors.     

GFAP and antibodies against NMDA receptor subunit NR2 as biomarkers for acute cerebrovascular diseases

We studied whether the serum levels of glial fibrillary acidic protein (GFAP) and of antibodies against the N‐methyl‐d ‐aspartate receptor subunit NR2 (NR2 R_NMDA) can discriminate between intracerebral haemorrhage (ICH) and ischaemic stroke (IS) in stroke patients. We prospectively recruited patients with suspected stroke (72 confirmed) and 52 healthy controls. The type of brain lesion (ICH or IS) was established using brain imaging. The levels of GFAP and of antibodies against NR2 R_NMDA were measured in blood samples obtained within 12 hrs after stroke onset and 24, 48 and 72 hrs and 1 and 2 weeks later using ELISA immunoassay. Improvement in diagnostic performance was assessed in logistic regression models designed to predict the diagnosis and the type of stroke. GFAP peaks early during haemorrhagic brain lesions (at significantly higher levels), and late in ischaemic events, whereas antibodies against NR2 R_NMDA have significantly higher levels during IS at all time‐points. Neither of the two biomarkers used on its own could sufficiently discriminate patients, but when they are used in combination they can differentiate at 12 hrs after stroke, between ischaemic and haemorrhagic stroke with a sensitivity and specificity of 94% and 91%, respectively.     

NMDA receptor NR2B subunit over-expression increases cerebellar granule cell migratory activity

Glutamate acting on NMDA receptors (NMDARs) is known to influence cerebellar granule cell migration. Subunit composition of NMDARs in granule cells changes characteristically during development: NR2B subunit containing receptors are abundant during migration towards the internal granule cell layer but are gradually replaced by NR2A and/or NR2C subunits once the final position is reached. Cerebellar granule cell migration was investigated using mutant mouse lines either with a deletion of the NR2C gene (NR2C^−/− mice) or expressing NR2B instead of the NR2C subunit (NR2C-2B mice). BrdU-labeling revealed that over-expression of NR2B increased granule cell translocation in vivo , while the lack of NR2C subunit did not have any detectable effects on cell migration. Cellular composition of wild-type and mutant dissociated cerebellar granule cell cultures isolated from 10-day-old cerebella were similar, but NR2C-2B cultures had elevated level of NR2B subunits and intracellular Ca²⁺ imaging revealed higher sensitivity towards the addition of NR2B-selective antagonist in vitro . Time-lapse videomicroscopic observations revealed that average migratory velocity and the proportion of translocating cell bodies were significantly higher in NR2C-2B than in wild-type cultures. Our results provide evidence that NR2B-containing NMDARs can have specialized roles during granule cell migration and can increase migratory speed.     

Serines 890 and 896 of the NMDA receptor subunit NR1 are differentially phosphorylated by protein kinase C isoforms

The NR1 subunit of the NMDA receptor has two serines (S890 and S896) whose phosphorylation by protein kinase C (PKC) differentially modulates NMDA receptor trafficking and clustering. It is not known which PKC isoforms phosphorylate these serines. In primary cultures of cerebellar neurons, we examined which PKC isoforms are responsible for the phosphorylation S890 and S896. We used specific inhibitors of PKC isoforms and antibodies recognizing specifically phosphorylated S890 or S896. The results show that PKC alpha phosphorylates preferentially S896 and PKC gamma preferentially S890. Activation of type I metabotropic glutamate receptors (mGluRs) with DHPG (3,5-dihyidroxy-phenylglycine) activates PKC gamma but not PKC alpha or beta. We found that activation of mGluRs by DHPG increases S890 but not S896 phosphorylation, supporting a role for PKC gamma in the physiological modulation of S890 phosphorylation. It is also shown that the pool of NR1 subunits present in the membrane surface contains phosphorylated S890 but not phosphorylated S896. This supports that differential phosphorylation of S890 and S896 by different PKC isoforms modulates cellular distribution of NMDA receptors and may also contribute to the selective modulation of NMDA receptor function and intracellular localization.     

Calpain product of WT-CRMP2 reduces the amount of surface NR2B NMDA receptor subunit

The brain is particularly vulnerable to ischaemia; however, neurons can become tolerant to ischaemic insult. This tolerance has been shown to involve activation of NMDA receptors, but its mechanisms have not yet been fully elucidated. Using a preconditioning protocol, we show that neurons surviving to a transient NMDA exposure become resistant to the glutamatergic agonist. Using a proteomic approach, we found that alterations of the protein pattern of NMDA-resistant neurons are restricted mainly to the five collapsin response mediator proteins (CRMPs). A sustained increase in calpain activity following NMDA treatment is responsible for the production of cleaved CRMPs. Finally, we provide evidence for the involvement of the cleaved form of WT-CRMP2 in the down-regulation of NR2B. Our data suggests that, beside their role in neuronal morphogenesis, CRMPs may contribute to neuronal plasticity.