AB1557 Sigma-AldrichAnti-NMDAR2B Antibody, pain

Glaucoma is an optic neuropathy and the second major cause of blindness worldwide next to cataracts. The protection from retinal ganglion cell (RGC) loss, one of the main characteristics of glaucoma, would be a straightforward treatment for this disorder. However, the clinical application of neuroprotection has not, so far, been successful. Here, we report that apolipoprotein E-containing lipoproteins (E-LPs) protect primary cultured RGCs from Ca(2+)-dependent, and mitochondrion-mediated, apoptosis induced by glutamate. Binding of E-LPs to the low density lipoprotein receptor-related protein 1 recruited the N-methyl-d-aspartate receptor, blocked intracellular Ca(2+) elevation, and inactivated glycogen synthase kinase 3β, thereby inhibiting apoptosis. When compared with contralateral eyes treated with phosphate-buffered saline, intravitreal administration of E-LPs protected against RGC loss in glutamate aspartate transporter-deficient mice, a model of normal tension glaucoma that causes glaucomatous optic neuropathy without elevation of intraocular pressure. Although the presence of α2-macroglobulin, another ligand of the low density lipoprotein receptor-related protein 1, interfered with the neuroprotective effect of E-LPs against glutamate-induced neurotoxicity, the addition of E-LPs overcame the inhibitory effect of α2-macroglobulin. These findings may provide a potential therapeutic strategy for normal tension glaucoma by an LRP1-mediated pathway.

The cell adhesion molecule pair neuroligin1 (Nlg1) and beta-neurexin (beta-NRX) is a powerful inducer of postsynaptic differentiation of glutamatergic synapses in vitro. Because Nlg1 induces accumulation of two essential components of the postsynaptic density (PSD) - PSD-95 and NMDA receptors (NMDARs) - and can physically bind PSD-95 and NMDARs at mature synapses, it has been proposed that Nlg1 recruits NMDARs to synapses through its interaction with PSD-95. However, PSD-95 and NMDARs are recruited to nascent synapses independently and it is not known if Nlg1 accumulates at synapses before these PSD proteins. Here, we investigate how a single type of cell adhesion molecule can recruit multiple types of synaptic proteins to new synapses with distinct mechanisms and time courses.Nlg1 was present in young cortical neurons in two distinct pools before synaptogenesis, diffuse and clustered. Time-lapse imaging revealed that the diffuse Nlg1 aggregated at, and the clustered Nlg1 moved to, sites of axodendritic contact with a rapid time course. Using a patching assay that artificially induced clusters of Nlg, the time course and mechanisms of recruitment of PSD-95 and NMDARs to those Nlg clusters were characterized. Patching Nlg induced clustering of PSD-95 via a slow palmitoylation-dependent step. In contrast, NMDARs directly associated with clusters of Nlg1 during trafficking. Nlg1 and NMDARs were highly colocalized in dendrites before synaptogenesis and they became enriched with a similar time course at synapses with age. Patching of Nlg1 dramatically decreased the mobility of NMDAR transport packets. Finally, Nlg1 was biochemically associated with NMDAR transport packets, presumably through binding of NMDARs to MAGUK proteins that, in turn, bind Nlg1. This interaction was essential for colocalization and co-transport of Nlg1 with NMDARs.Our results suggest that axodendritic contact leads to rapid accumulation of Nlg1, recruitment of NMDARs co-transported with Nlg1 soon thereafter, followed by a slower, independent recruitment of PSD-95 to those nascent synapses.

Integrin class adhesion proteins are concentrated at adult brain synapses. Whether synaptic integrins engage kinase signaling cascades has not been determined, but is a question of importance to ideas about integrin involvement in functional synaptic plasticity. Accordingly, synaptoneurosomes from adult rat brain were used to test if matrix ligands activate integrin-associated tyrosine kinases, and if integrin signaling targets include NMDA-class glutamate neurotransmitter receptors. The integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) induced rapid (within 5 min) and robust increases in tyrosine phosphorylation of focal adhesion kinase, proline-rich tyrosine kinase 2 and Src family kinases. Increases were similarly induced by the native ligand fibronectin, blocked with neutralizing antibodies to beta1 integrin, and not obtained with control peptides, indicating that kinase activation was integrin-mediated. Both GRGDSP and fibronectin caused rapid Src kinase-dependent increases in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Tests of the physiological significance of the latter result showed that ligand treatment caused a rapid and beta1 integrin-dependent increase in NMDA receptor-mediated synaptic responses. These results provide the first evidence that, in adult brain, synaptic integrins activate local kinase cascades with potent effects on the operation of nearby neurotransmitter receptors implicated in synaptic plasticity.

N-methyl-d-aspartate receptor (NMDAR) stimulation activates many downstream mechanisms involved in both cell survival and cell death. The manner in which the NMDAR regulates one of these pathways, the p38 mitogen-activated protein kinase (p38) pathway, is currently unknown. In the present study, we have defined a developmental-, concentration-, and time-dependent phosphorylation and subsequent dephosphorylation of p38. In cultured hippocampal neurons 7-8 days in vitro (DIV7-8), NMDAR stimulation leads to a concentration-dependent increase in p38 phosphorylation (phospho-p38). However, in more mature neurons (greater than DIV17) application of NMDA produces concentration-dependent effects, such that low concentrations result in sustained increases in phospho-p38 levels, and high concentrations dephosphorylate p38 within 5 min. Conantokin G, an antagonist of NR1/2A/2B and NR1/2B receptors, inhibits p38 phosphorylation, while NR1/2B-specific antagonists prevent the rapid dephosphorylation of p38 without affecting p38 activation. Furthermore, inhibition of calcineurin prevents the activation of p38, whereas inhibition of phosphoinositide 3-kinase (PI3K) prevents the rapid dephosphorylation of p38. Our results support the presence of subtype-dependent pathways regulating p38 activation and deactivation: one involves NR1/2A/2B receptors activating calcineurin and resulting in p38 phosphorylation, and the other utilizes NR1/2B receptors binding to and activating PI3K and leading to the dephosphorylation of p38 in a manner involving both NR1/2A/2B receptor activation and tyrosine phosphorylation of NR2B. The ability of NMDAR subtype-specific mechanisms to regulate p38 has implications for NMDAR-mediated synaptic plasticity, gene regulation, and excitotoxicity.

N-methyl-D-aspartate receptor dysfunction has been strongly suggested to link with the abnormalities seen in fetal alcohol syndrome. Thus, the effects of prenatal ethanol exposure on the total expression of NR1 splice variants and the cell surface expression of both NR1 and NR2 subunits in brain were investigated in rats. Western blot studies of membrane homogenates from cerebral cortices at postnatal days 1 through 21 indicate that prenatal ethanol treatment does not alter total NR1 expression or differential expression of NR1 splice variants during development. However, immunoprecipitation studies using PSD95 suggest that both C2'-terminal variants and NR2A subunits at the cortical postsynaptic membrane of postnatal day 21 were significantly reduced after prenatal ethanol treatment. Moreover, C1-terminal variants were decreased in both pair-fed and ethanol-treated groups, while no significant differences in the levels of total NR1 subunits, NR1 splice variants containing the N- or C2-terminal cassettes, or NR2B subunits were observed. Thus, these results suggest that prenatal exposure to ethanol may influence neuronal function by selective regulation of expression of C2'-terminal variants and NR2A subunits at the cell surface.

Eighty ICR mice were randomly assigned to one of four groups given daily intraperitoneal injections of 0, 0.1, 1 or 3 mg/kg MeHg chloride respectively from postnatal days (PD) 15-17, and then tested with the Morris water maze on PD45. After that the mice were sacrificed by cervical dislocation, and the protein levels of NMDA receptor subtypes in the hippocampus were measured by Western blot analysis. A significant increase in the latency (F=2.88, P<0.05) before finding the platform was observed in the 1 and 3 mg/kg MeHg exposure groups. Further, the 3 mg/kg MeHg exposure group also had a longer swim distance (F=2.97, P<0.05) for finding the platform. In the probe test, the MeHg exposure groups displayed a smaller number of platform crossings when the hidden platform was moved, but this did not reach statistical significance. Western blot analysis results showed significant increases in the levels of NR1, NR2A and NR2B proteins of the hippocampus in the 1 and 3 mg/kg MeHg exposure groups. Overall, the current study found that MeHg exposure at 1 and 3 mg/kg doses during the postnatal brain growth spurt induces subtle and persistent learning deficits, and the neurobehavioral abnormalities of MeHg-exposed mice might be ascribed to alteration of the gene expression of specific NMDA receptor subunits in the hippocampus.

Activation of the NMDA subtype of glutamate receptor is required for activity-dependent structural plasticity in many areas of the young brain. Previous work has shown that NMDA receptor currents decline approximately at the time that developmental synaptic plasticity ends, and in situ hybridization studies have suggested that receptor subunit changes may be occurring during the same developmental interval. To establish a system in which the relationship between these properties of developing synapses can be explored, we have combined patch-clamp recordings with mRNA- and protein-level biochemical analyses to study the developmental regulation of NMDA receptors in the superficial layers of the rat superior colliculus. These experiments document an abrupt decrease in the NMDA receptor contribution to synaptic currents that occurs before eye opening and is closely associated with changes in NR1 protein, rapidly rising levels of the NMDA receptor subunit NR2A, and decreasing levels of NR2B. The functional and molecular changes also are correlated with the developmental decline in structural plasticity in these layers. In addition, both physiological and biochemical methods show evidence of GABA-mediated inhibition in the superficial collicular layers beginning after eye opening. This may provide an additional heterosynaptic mechanism for controlling excitation and plasticity in this neuropil by pattern vision. Thus our findings lend support to the idea that high levels of NMDA receptor function are associated with the potential for structural rearrangement in CNS neuropil and that the functional downregulation of this molecule results, at least partially, from changes in its subunit composition.