MAB5216 Sigma-AldrichAnti-NMDAR2A Antibody

N-methyl-D-aspartate receptors (NMDARs) in cerebellar molecular layer interneurons (MLIs) are expressed and activated in unusual ways: at parallel fibre (PF) synapses they are only recruited by repetitive stimuli, suggesting an extrasynaptic location, whereas their activation by climbing fibre is purely mediated by spillover. NMDARs are thought to play an important role in plasticity at different levels of the cerebellar circuitry. Evaluation of the location, functional properties and physiological roles of NMDARs will be facilitated by knowledge of the NMDAR isoforms recruited. Here we show that MLI-NMDARs activated by both PF and climbing fibre inputs have similar kinetics and contain GluN2B but not GluN2A subunits. On the other hand, no evidence was found of functional NMDARs in the axons of MLIs. At the PF-Purkinje cell (PF-PC) synapse, the activation of GluN2A-containing NMDARs has been shown to be necessary for the induction of long-term depression (LTD). Our results therefore provide a clear distinction between the NMDARs located on MLIs and those involved in plasticity at PF-PC synapses.

N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune neurological disease, diagnosed by a specific autoantibody against NMDAR. Antibody testing using commercially available cell-based assays (CBA) or immunohistochemistry on rat brain tissue has proven high specificity and sensitivity. Here we compare an immunofluorescence live CBA to a flow cytometry (FACS) based assay to detect NMDAR antibodies by their binding to the surface of HEK293A cells functionally expressing NMDAR. Both assays were first established using a discovery group of 76 individuals and then validated in a group of 32 patients in a blinded manner. In the CBA, 23 of 23 patients with NMDAR encephalitis were positive for NMDAR antibodies and 0 of 85 controls (32 healthy controls and 53 patients with other neurological diseases), resulting in a sensitivity and specificity of 100% (95% confidence intervals (CI) 85.1-100.0 and 95.7-100.0, respectively). The FACS based assay detected NMDAR antibodies in 20 of 23 patients and in 0 of 85 controls. Therefore, with an equally high specificity (95% CI 95.7-100.0) the sensitivity of the FACS based assay was 87% (95% CI 66.4-97.2). Comparing antibody titers from CBA with delta median fluorescence intensities from FACS showed a high concordance (kappa = 0.943, pless than 0.0001) and correlation (r = 0.697, pless than 0.0001). In conclusion, evaluation of the FACS based assay revealed a lower sensitivity and high inter-assay variation, making the CBA a more reliable detection method.

Fragile X syndrome is a common inherited form of mental retardation caused by the lack of fragile X mental retardation protein (FMRP) because of Fmr1 gene silencing. Serotonin (5-HT) is significantly increased in the null mutants of Drosophila Fmr1, and elevated 5-HT brain levels result in cognitive and behavioral deficits in human patients. The serotonin type 2A receptor (5-HT2AR) is highly expressed in the cerebral cortex; it acts on pyramidal cells and GABAergic interneurons to modulate cortical functions. 5-HT2AR and FMRP both regulate synaptic plasticity. Therefore, the lack of FMRP may affect serotoninergic activity. In this study, we determined the involvement of FMRP in the 5-HT modulation of synaptic potentiation with the use of primary cortical neuron culture and brain slice recording. Pharmacological inhibition of 5-HT2AR by R-96544 or ketanserin facilitated long-term potentiation (LTP) in the anterior cingulate cortex (ACC) of WT mice. The prefrontal LTP induction was dependent on the activation of NMDARs and elevation of postsynaptic Ca(2+) concentrations. By contrast, inhibition of 5-HT2AR could not restore the induction of LTP in the ACC of Fmr1 knock-out mice. Furthermore, 5-HT2AR inhibition induced AMPA receptor GluR1 subtype surface insertion in the cultured ACC neurons of Fmr1 WT mice, however, GluR1 surface insertion by inhibition of 5-HT2AR was impaired in the neurons of Fmr1KO mice. These findings suggested that FMRP was involved in serotonin receptor signaling and contributed in GluR1 surface expression induced by 5-HT2AR inactivation.

Chronic stress causes various detrimental effects including cognitive and affective dysfunctions. Given the recent findings emphasizing the importance of information processing between the prefrontal cortex (PFC) and limbic structures on cognitive and affective functions, impairments of these functions caused by chronic stress may be associated with stress-induced adaptive and maladaptive responses in limbic structure-PFC interaction. In this study we have shown that chronic stress disrupts limbic structure-PFC interaction by modulating N-methyl-D-aspartate (NMDA) receptor expression in the PFC. We found that chronic stress decreased expression of NR1, NR2A and NR2B subunits of NMDA receptors in the PFC but not in the motor cortex. However, the reduction in NR2B subunits of NMDA receptors was larger in the dorsal part than the ventral part of PFC. In agreement with this observation, administration of the NMDA antagonist that was more selective for NMDA receptors containing NR2B subunits induced alterations of synchronous local field potentials between the PFC and limbic structures, synaptic plasticity induction in the limbic structure-PFC pathway, and spike firing of PFC neurons that were similar to those observed in the dorsal PFC of rats exposed to chronic stress. In contrast, administration of the NMDA antagonist that was not subunit-selective resulted in electrophysiological alterations resembling to those observed in the ventral PFC of rats exposed to chronic stress. These results suggest that chronic stress disrupts NMDA receptor-dependent limbic structure-PFC information processing.© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.

Recent neuroimaging and postmortem studies have demonstrated abnormalities in glutamatergic transmission in major depression. Glutamate NMDA (N-methyl-d-aspartate) receptors are one of the major mediators of excitatory neurotransmission in the central nervous system. At synaptic sites, NMDA receptors are linked with postsynaptic density protein-95 (PSD-95) that plays a key role in mediating trafficking, clustering, and downstream signaling events, following receptor activation. In this study, we examined the expression of NMDA receptor subunits NR1, NR2A, and NR2B as well as PSD-95 in the anterior prefrontal cortex (PFC) using Western blot method. Cortical samples were obtained from age, gender and postmortem interval matched depressed and psychiatrically healthy controls. The results revealed that there was a reduced expression of the NMDA receptor subunits NR2A (-54%) and NR2B (-48%), and PSD-95 protein level (-40%) in the PFC of depressed subjects relative to controls, with no change in the NR1 subunit. The alterations in NMDA receptor subunits, especially the NR2A and NR2B, as well as PSD-95 suggest an abnormality in the NMDA receptor signaling in the PFC in major depression. Our findings in conjunction with recent clinical, cellular, and neuroimaging studies further implicate the involvement of glutamate neurotransmission in the pathophysiology of depression. This study provides additional evidence that NMDA receptor complex is a target for discovery of novel antidepressants.

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that play a role in the inflammatory response. These enzymes have been well studied in the context of cancer biology and inflammation. Recent studies, however, suggest that these enzymes also play roles in brain development and neurodegenerative disease. Select MMPs can target proteins critical to synaptic structure and neuronal survival, including integrins and cadherins. Here, we show that one member of the MMP family, MMP-7, which may be released from cells, including microglia, can target a protein critical to synaptic function. Through analysis of extracts from murine cortical slice preparations, we show that MMP-7 cleaves the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor to generate an N-terminal fragment of approximately 65 kDa. Moreover, studies with recombinant protein show that MMP-7-mediated cleavage of NR1 occurs at amino acid 517, which is extracellular and just distal to the first transmembrane domain. Data suggest that NR2A, which shares sequence homology with NR1, is also cleaved following treatment of slices with MMP-7, while select AMPA receptor subunits are not. Consistent with a potential effect of MMP-7 on ligand binding, additional experiments demonstrate that NMDA-mediated calcium flux is significantly diminished by MMP-7 pretreatment of cultures. In addition, the AMPA/NMDA ratio is increased by MMP-7 pretreatment. These data suggest that synaptic function may be altered in neurological conditions associated with increased levels of MMP-7.

Glutamate receptor (GluR) subunit composition of inferior salivatory nucleus (ISN) neurons was studied by immunohistochemical staining of retrogradely labeled neurons. Preganglionic ISN neurons innervating the von Ebner or parotid salivary glands were labeled by application of a fluorescent tracer to the lingual-tonsilar branch of the glossopharyngeal nerve or the otic ganglion respectively. We used polyclonal antibodies to glutamate receptor subunits NR1, NR2A, NR2B, (NMDA receptor subunits) GluR1, GluR2, GluR3, GluR4 (AMPA receptor subunits), and GluR5-7, KA2 (kainate receptor subunits) to determine their expression in ISN neurons. The distribution of the NMDA, AMPA and kainate receptor subunits in retrogradely labeled ISN neurons innervating the von Ebner and parotid glands was qualitatively similar. The percentage of retrogradley labeled ISN neurons innervating the parotid gland expressing the GluR subunits was always greater than those innervating the von Ebner gland. For both von Ebner and parotid ISN neurons, NR2A subunit staining had the highest expression and the lowest expression of GluR subunit staining was NR2B for von Ebner ISN neurons and GluR1 for parotid ISN neurons. The percentage of NR2B and GluR4 expressing ISN neurons was significantly different between the two glands. The percentage of ISN neurons that expressed GluR receptor subunits ranged widely indicating that the distribution of GluR subunit expression differs amongst the ISN neurons. While ISN preganglionic neurons express all the GluR subunits, differences in the percentage of ISN neurons expression between neurons innervating the von Ebner and parotid glands may relate to the different functional roles of these glands.

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.

Members of the N-methyl-d-aspartate (NMDA) class of glutamate receptors (NMDARs) are critical for development, synaptic transmission, learning and memory; they are targets of pathological disorders in the central nervous system. NMDARs are phosphorylated by both serine/threonine and tyrosine kinases. Here, we demonstrate that cyclin dependent kinase-5 (Cdk5) associates with and phosphorylates NR2A subunits at Ser-1232 in vitro and in intact cells. Moreover, we show that roscovitine, a selective Cdk5 inhibitor, blocks both long-term potentiation induction and NMDA-evoked currents in rat CA1 hippocampal neurons. These results suggest that Cdk5 plays a key role in synaptic transmission and plasticity through its up-regulation of NMDARs.