Transient global ischemia in rat brain promotes different NMDA receptor regulation depending on the brain structure studied

The mRNA expression of the major subunits of N-methyl-d-aspartate receptors (NR1, NR2A and NR2B) following ischemia–reperfusion was studied in structures with different vulnerabilities to ischemic insult in the rat brain. The study was performed using quantitative real-time PCR on samples from 3-month-old male Sprague–Dawley rats after global transient forebrain ischemia followed by 48 h of reperfusion. Expression of NMDA receptor subunits mRNAs decreased significantly in all structures studied in the injured animals as compared to the sham-operated ones. The hippocampal subfields (CA1, CA3 and dentate gyrus) as well as the caudate-putamen, both reported to be highly ischemic-vulnerable structures, showed outstandingly lower mRNA levels of NMDA receptor subunits than the cerebral cortex, which is considered a more ischemic-resistant structure. The ratios of the mRNA levels of the different subunits were analyzed as a measure of the NMDA receptor expression pattern for each structure studied. Hippocampal areas showed changes in NMDA receptor expression after the insult, with significant decreases in the NR2A with respect to the NR1 and NR2B subunits. Thus, the NR1:NR2A:NR2B (1:1:2) ratios observed in the sham-operated animals became (2:1:4) in insulted animals. This modified expression pattern was similar in CA1, CA3 and the dentate gyrus, in spite of the different vulnerabilities reported for these hippocampal areas. In contrast, no significant differences in the expression pattern were observed in the caudate-putamen or cerebral cortex on comparing the sham-operated animals with the ischemia-reperfused rats. Our results support the notion that the regulation of NMDA receptor gene expression is dependent on the brain structure rather than on the higher or lower vulnerability of the area studied.     

Transient Ischemia Differentially Increases Tyrosine Phosphorylation of NMDA Receptor Subunits 2A and 2B

Abstract: Activation of the N -methyl- d -aspartate (NMDA) receptor has been implicated in the events leading to ischemia-induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four-vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti-tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit-specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23–29-fold relative to sham-operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia-induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.     

The N-Methyl-d-Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C-γ

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-γ (PLC-γ). A glutathione S -transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-γ was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.     

脑缺血/再灌对蒙古沙土鼠海马突触体酪氨酸磷酸化的影响

用蒙古沙土鼠双侧颈总动脉结扎（ＢＣＡＯ）前脑缺血模型,研究缺血／再灌对海马突触体蛋白酪氨酸磷酸休的影响及ＮＭＤＡ受体（ＮＲ）非竞争性拮抗剂氯胺酮（Ｋｅｔａｍｉｎｅ,ＫＴ）、Ｌ－型电压门控钙离子通道（Ｌ－ｔｙｐｅ ｖｏｌｔａｇｅ ｇａｔｅｄｃａｌｃｉｕｍ ｃｈａｎｎｅｌ,Ｌ－型ＶＧＣＣ）拮抗剂硝苯吡啶（ｎｉｆｅｄｉｐｉｎｅ,ＮＤ）及非ＮＲ拮抗６,７－二硝基喹恶啉上卫四（６,７－ｄｉ－ｎｉｔｒｏｐｕ     

Altered interaction between PSD-95 and the NMDA receptor following transient global ischemia

The postsynaptic density (PSD) is a cytoskeletal specialization involved in the anchoring of neurotransmitter receptors and in regulating the response of postsynaptic neurons to synaptic stimulation. The postsynaptic protein PSD-95 binds to NMDA receptor subunits NR2A and NR2B and to signaling molecules such as neuronal nitric oxide synthase and p135synGAP. We investigated the effects of transient cerebral ischemia on protein interactions involving PSD-95 and the NMDA receptor in the rat hippocampus. Ischemia followed by reperfusion resulted in a decrease in the solubility of the NMDA receptor and PSD-95 in 1% sodium deoxycholate, the decrease being greater in the vulnerable CA1 hippocampal subfield than in the less sensitive CA3/dentate gyrus regions. Solubilization of the kainic acid receptor GluR6/7 and the PSD-95 binding proteins, neuronal nitric oxide synthase and p135synGAP, also decreased following ischemia. The association between PSD-95 and NR2A and NR2B, as indicated by coimmunoprecipitation, was less in postischemic samples than in sham-operated controls. Ischemia also resulted in a decrease in the size of protein complexes containing PSD-95, but had only a small effect on the size distribution of complexes containing the NMDA receptor. The results indicate that molecular interactions involving PSD-95 and the NMDA receptor are modified by an ischemic challenge.     

Tyrosine phosphorylation of the N-methyl-D-aspartate receptor by exogenous and postsynaptic density-associated Src-family kinases

Phosphorylation of the NMDA receptor by Src-family tyrosine kinases has been implicated in the regulation of receptor function. We have investigated the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exogenous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the receptor by exogenous Src and Fyn was dependent upon initial binding of the kinases to PSDs via their SH2-domains. Src and Fyn phosphorylated similar sites in NR2A and NR2B, tryptic peptide mapping identifying seven and five major tyrosine-phosphorylated peptides derived from NR2A and NR2B, respectively. All five tyrosine phosphorylation sites on NR2B were localized to the C-terminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyrosine kinases yielded only three tyrosine-phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Phosphorylation-site specific antibodies identified NR2B Tyr1472 as a phosphorylation site for intrinsic PSD tyrosine kinases. Phosphorylation of this site was inhibited by the Src-family-specific inhibitor PP2. The results identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosphorylation by kinases located within the structural framework of the PSD.     

Insulin causes a transient tyrosine phosphorylation of NR2A and NR2B NMDA receptor subunits in rat hippocampus

NMDA receptors play a critical role in various aspects of CNS function. Hence, it is important to identify mechanisms that regulate NMDA receptor activity. We have shown previously that insulin rapidly potentiates NMDA receptor activity in both native and recombinant expression systems. Here we report that insulin causes a transient phosphorylation of NR2A and NR2B NMDA receptor subunits on tyrosine residues. Rat hippocampal slices were exposed to 1 microM insulin for 20 and 60 min and then solubilized. NR2A and NR2B subunits were immunoprecipitated and probed for tyrosine phosphorylation. Insulin incubation of hippocampal slices for 20 min elicited an increase in tyrosine phosphorylation to 176 +/- 16% (NR2A) and 203 +/- 15% (NR2B) of control levels. In contrast, 60 min of insulin incubation did not alter NR2 tyrosine phosphorylation levels (NR2A: 85 +/- 13% of control; NR2B: 93 +/- 10% of control). Although the consequence of insulin-stimulated tyrosine phosphorylation is unknown, it is possible that this site(s) is responsible for insulin potentiation of NMDA receptor activity. This possibility is consistent with our earlier finding that insulin potentiates hippocampal NMDA receptor activity after 20 min, but not after 60 min, of insulin exposure.     

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.     

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.     

mGluR1 antagonist decreases tyrosine phosphorylation of NMDA receptor and attenuates infarct size after transient focal cerebral ischemia

The contribution of metabotropic glutamate receptors to brain injury after in vivo cerebral ischemia remains to be determined. We investigated the effects of the metabotropic glutamate receptor 1 (mGluR1) antagonist LY367385 on brain injury after transient (90 min) middle cerebral artery occlusion in the rat and sought to explore their mechanisms. The intravenous administration of LY367385 (10 mg/kg) reduced the infarct volume at 24 h after the start of reperfusion. As the Gq-coupled mGluR1 receptor is known to activate the PKC/Src family kinase cascade, we focused on changes in the activation and amount of these kinases. Transient focal ischemia increased the amount of activated tyrosine kinase Src and PKC in the post-synaptic density (PSD) at 4 h of reperfusion. The administration of LY367385 attenuated the increases in the amounts of PSD-associated PKCγ and Src after transient focal ischemia. We further investigated phosphorylation of the NMDA receptor, which is a major target of Src family kinases to modulate the function of the receptor. Transient focal ischemia increased the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B. Tyrosine phosphorylation of NR2A, but not that of NR2B, in the PSD at 4 h of reperfusion was inhibited by LY367385. These results suggest that the mGluR1 after transient focal ischemia is involved in the activation of Src, which may be linked to the modification of properties of the NMDA receptor and the development of cerebral infarction.     

Transient Global Ischemia Alters NMDA Receptor Expression in Rat Hippocampus: Correlation with Decreased Immunoreactive Protein Levels of the NR2A/2B Subunits, and an Altered NMDA Receptor Functionality

Abstract: We investigated the gene expression levels, the immunoreactive protein prevalence, and the functional activity of N -methyl- d -aspartate (NMDA) receptor complexes at early times after severe global ischemia challenge in rats. The mRNA expression levels for the NR2A and NR2B subunits of NMDA receptors changed to different degrees within different subregions of the hippocampus after reperfusion with respect to sham-operated control. No significant change in expression was observed in the vulnerable CA1 subfield at or before 6 h after challenge for either receptor subunit, although changes in expression in other hippocampal subfields were observed. At 12 and 24 h after challenge, significant decreases in expression for both subunits were found in the vulnerable CA1 subfield, as well as in other hippocampal regions. At the protein level, a significant decrease in the amount of NR2A/NR2B immunoreactivity in the total hippocampus was observed at both 6 and 24 h after reperfusion compared with sham control. Electrophysiological assessment of single-channel NMDA receptor activity in the CA1 subfield indicates that the main conductance state of NMDA receptor channels is maintained 6 h after challenge, although by 18–24 h after challenge, this main conductance state is rarely observed. The NMDA receptor component of the excitatory postsynaptic field potential was found to be significantly diminished from sham control 24 h after challenge, such that only ∼10% of the sham response remained, but was not significantly altered from sham control at 6 h after challenge. These results indicate that decreases in the expression levels, the immunoreactive protein prevalence, and that alterations in the functionality of NMDA receptors occur in the hippocampus at early times after severe transient global ischemia.     

Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.     

Insulin-induced oxidative neuronal injury in cortical culture: mediation by induced N-methyl-D-aspartate receptors

While effectively attenuating neuronal apoptosis in mouse cortical culture, insulin paradoxically induced neuronal necrosis with 48 h of exposure. The insulin neurotoxicity was blocked by an antioxidant but not by caspase inhibitors. Exposure to insulin led to tyrosine phosphorylation of the insulin receptor and the insulin-like growth factor-1 (IGF-1) receptor and activation of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3-kinase). Inhibitors of tyrosine kinase and PKC, but not PI3-kinase, attenuated the insulin neurotoxicity. Conversely, the inhibitor of PI3-kinase but not PKC reversed the antiapoptotic effect of insulin. Suggesting that the gene activity-dependent emergence of excitotoxicity contributed to insulin neurotoxicity, macromolecule synthesis inhibitors and N-methyl-D-aspartate (NMDA) antagonists blocked it. Consistently, exposure to insulin increased the level of the NR2A subunit of the NMDA receptor without much altering NR1 or NR2B levels. The present study suggests that insulin can be both neuroprotective and neurotoxic in the same cell system but by way of different signaling cascades.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95

Both tyrosine phosphorylation and calpain-mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src significantly reduced calpain-mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn significantly protected calpain-mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain-mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C-terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C-terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD-95. Thus, tyrosine phosphorylation impairs or facilitates calpain-mediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.     

Relative extent of tyrosine phosphorylation of the NR2A and NR2B subunits in the rat forebrain postsynaptic density fraction

The activity of the N-methyl-D-aspartate (NMDA) receptor, a subclass of ionotropic glutamate receptor, is modulated by a complex network of phosphorylation and dephosphorylation. I investigated the relative extent of tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) and 2B (NR2B) subunits in the rat forebrain postsynaptic density (PSD) fraction. Immunoblot analysis of immunoprecipitates with antiphosphotyrosine antibodies indicated that tyrosine phosphorylation of NR2A was only 28.6% of that of NR2B. When phosphotyrosine-containing peptides were isolated by affinity-purification or immunoprecipitation, and probed for the two subunits, NR2B was detected but not NR2A. Furthermore, depletion of NR2B removed the phosphotyrosine-containing 180 kDa peptide from the solution while the converse was not true. The small extent of tyrosine phosphorylation of NR2A in the unstimulated condition may explain the dramatic increase in tyrosine phosphorylation in various physiological and pathological conditions.     

Spatial learning and memory deficits after whole-brain irradiation are associated with changes in NMDA receptor subunits in the hippocampus

Whole-brain irradiation is used for the treatment of brain tumors, but can it also induce neural changes, with progressive dementia occurring in 20-50% of long-term survivors. The present study investigated whether 45 Gy of whole-brain irradiation delivered to 12-month-old Fischer 344 x Brown Norway rats as nine fractions over 4.5 weeks leads to impaired Morris water maze (MWM) performance 12 months later. Compared to sham-irradiated rats, the irradiated rats demonstrated impaired MWM performance. The relative levels of the NR1 and NR2A but not the NR2B subunits of the NMDA receptor were significantly higher in hippocampal CA1 of irradiated rats compared to control rats. No significant differences were detected for these NMDA subunits in CA3 or dentate gyrus. Further analysis of CA1 revealed that the relative levels of the GluR1 and GluR2 subunits of the AMPA receptor and synaptophysin were not altered by whole-brain irradiation. In summary, a clinically relevant regimen of fractionated whole-brain irradiation led to significant impairments in spatial learning and reference memory and alterations in the relative levels of subunits of the NMDA, but not the AMPA, receptors in hippocampal CA1. These findings suggest for the first time that radiation-induced cognitive impairments may be associated with alterations in glutamate receptor composition.     

H-Ras modulates N-methyl-D-aspartate receptor function via inhibition of Src tyrosine kinase activity

Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hippocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction.     

SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin

Leptin regulates energy homeostasis primarily by binding and activating its long form receptor (LRb). Deficiency of either leptin or LRb causes morbid obesity. Leptin stimulates LRb-associated JAK2, thus initiating multiple pathways including the Stat3 and phosphatidylinositol (PI) 3-kinase pathways that mediate leptin biological actions. Here we report that SH2-B, a JAK2-interacting protein, promotes activation of the PI 3-kinase pathway by recruiting insulin receptor substrate 1 (IRS1) and IRS2 in response to leptin. SH2-B directly bound, via its PH and SH2 domain, to both IRS1 and IRS2 both in vitro and in intact cells and mediated formation of a JAK2/SH2-B/IRS1 or IRS2 tertiary complex. Consequently, SH2-B dramatically enhanced leptin-stimulated tyrosine phosphorylation of IRS1 and IRS2 in HEK293 cells stably expressing LRb, thus promoting association of IRS1 and IRS2 with the p85 regulatory subunit of PI 3-kinase and phosphorylation and activation of Akt. SH2-B mutants with lower affinity for IRS1 and IRS2 exhibited reduced ability to promote association of JAK2 with IRS1, tyrosine phosphorylation of IRS1, and association of IRS1 with p85 in response to leptin. Moreover, deletion of the SH2-B gene impaired leptin-stimulated tyrosine phosphorylation of endogenous IRS1 in mouse embryonic fibroblasts (MEF), which was reversed by reintroduction of SH2-B. Similarly, SH2-B promoted growth hormone-stimulated tyrosine phosphorylation of IRS1 in both HEK293 and MEF cells. Our data suggest that SH2-B is a novel mediator of the PI 3-kinase pathway in response to leptin or other hormones and cytokines that activate JAK2.     

NADPH oxidase mediates the oxygen-glucose deprivation/reperfusion-induced increase in the tyrosine phosphorylation of the N-methyl-D-aspartate receptor NR2A subunit in retinoic acid differentiated SH-SY5Y Cells

ABSTRACT: BACKGROUND: Evidence exists that oxidative stress promotes the tyrosine phosphorylation of N-methyl-D-aspartate receptor (NMDAR) subunits during post-ischemic reperfusion of brain tissue. Increased tyrosine phosphorylation of NMDAR NR2A subunits has been reported to potentiate receptor function and exacerbate NMDAR-induced excitotoxicity. Though the effect of ischemia on tyrosine phosphorylation of NMDAR subunits has been well documented, the oxidative stress signaling cascades mediating the enhanced tyrosine phosphorylation of NR2A subunits remain unclear. RESULTS: We report that the reactive oxygen species (ROS) generator NADPH oxidase mediates an oxidative stress-signaling cascade involved in the increased tyrosine phosphorylation of the NR2A subunit in post-ischemic differentiated SH-SY5Y neuroblastoma cells. Inhibition of NADPH oxidase attenuated the increased tyrosine phosphorylation of the NMDAR NR2A subunit, while inhibition of ROS production from mitochondrial or xanthine oxidase sources failed to dampen the post-ischemic increase in tyrosine phosphorylation of the NR2A subunit. Additionally, inhibition of NADPH oxidase blunted the interaction of activated Src Family Kinases (SFKs) with PSD-95 induced by ischemia/reperfusion. Lastly, inhibition of NADPH oxidase also markedly reduced cell death in post-ischemic SH-SY5Y cells stimulated by NMDA. CONCLUSIONS: These data indicate that NADPH oxidase has a key role in facilitating NMDAR NR2A tyrosine phosphorylation via SFK activation during post-ischemic reperfusion.