N-methyl-D-aspartate Exposure Research Articles

Magnesium acetyltaurate prevents retinal damage and visual impairment in rats through suppression of NMDA-induced upregulation of NF-κB, p53 and AP-1 (c-Jun/c-Fos).

Magnesium acetyltaurate (MgAT) has been shown to have a protective effect against N-methyl-D-aspartate (NMDA)-induced retinal cell apoptosis. The current study investigated the involvement of nuclear factor kappa-B (NF-κB), p53 and AP-1 family members (c-Jun/c-Fos) in neuroprotection by MgAT against NMDA-induced retinal damage. In this study, Sprague-Dawley rats were randomized to undergo intravitreal injection of vehicle, NMDA or MgAT as pre-treatment to NMDA. Seven days after injections, retinal ganglion cells survival was detected using retrograde labelling with fluorogold and BRN3A immunostaining. Functional outcome of retinal damage was assessed using electroretinography, and the mechanisms underlying antiapoptotic effect of MgAT were investigated through assessment of retinal gene expression of NF-κB, p53 and AP-1 family members (c-Jun/c-Fos) using reverse transcription-polymerase chain reaction. Retinal phospho-NF-κB, phospho-p53 and AP-1 levels were evaluated using western blot assay. Rat visual functions were evaluated using visual object recognition tests. Both retrograde labelling and BRN3A immunostaining revealed a significant increase in the number of retinal ganglion cells in rats receiving intravitreal injection of MgAT compared with the rats receiving intravitreal injection of NMDA. Electroretinography indicated that pre-treatment with MgAT partially preserved the functional activity of NMDA-exposed retinas. MgAT abolished NMDA-induced increase of retinal phospho-NF-κB, phospho-p53 and AP-1 expression and suppressed NMDA-induced transcriptional activity of NF-κB, p53 and AP-1 family members (c-Jun/c-Fos). Visual object recognition tests showed that MgAT reduced difficulties in recognizing the visual cues (i.e. objects with different shapes) after NMDA exposure, suggesting that visual functions of rats were relatively preserved by pre-treatment with MgAT. In conclusion, pre-treatment with MgAT prevents NMDA induced retinal injury by inhibiting NMDA-induced neuronal apoptosis via downregulation of transcriptional activity of NF-κB, p53 and AP-1-mediated c-Jun/c-Fos. The experiments were approved by the Animal Ethics Committee of Universiti Teknologi MARA (UiTM), Malaysia, UiTM CARE No 118/2015 on December 4, 2015 and UiTM CARE No 220/7/2017 on December 8, 2017 and Ethics Committee of Belgorod State National Research University, Russia, No 02/20 on January 10, 2020.

Pemafibrate prevents retinal neuronal cell death in NMDA-induced excitotoxicity via inhibition of p-c-Jun expression

Excitotoxicity is involved in the retinal neuronal cell death in diabetic retinopathy. Although fenofibrate has been shown to ameliorate the progression of diabetic retinopathy, the effect of pemafibrate, which is highly selective for peroxisome proliferator-activated receptor α on retinal neuronal cell death has not been documented. Here, we investigated whether pemafibrate exerts a beneficial effect against retinal ganglion cell (RGC) death induced by N-methyl-D-aspartate (NMDA) in rats. Experiments were performed on adult male Wistar rats that received an intravitreal injection of 20 nmol NMDA. Fluoro-Gold labeled RGC morphometry showed that oral intake of pemafibrate once a day for 7 days resulted in significant protection on RGC death induced by NMDA. Phosphorylated c-Jun protein, which is involved in apoptosis, was upregulated after NMDA exposure, and this increase was significantly lessened by the systemic pemafibrate treatment. Phosphorylated c-Jun immunopositive cells were colocalized with Thy-1 immunopositive cells, and the increased these cells were ameliorated by the pemafibrate treatment. An increase in TUNEL-positive cells was significantly suppressed by the pemafibrate treatment. Phosphorylated c-Jun immunopositive cells were colocalized with TUNEL-positive cells, and they were decreased by pemafibrate treatment. These results suggest that the RGC protection achieved with pemafibrate appears to be associated with inhibition of phosphorylated c-Jun and its anti-apoptotic effect.

Humanin Attenuates NMDA-Induced Excitotoxicity by Inhibiting ROS-dependent JNK/p38 MAPK Pathway.

Humanin (HN) is a novel 24-amino acid peptide that protects neurons against N-methyl-d-aspartate (NMDA)-induced toxicity. However, the contribution of the different mitogen-activated protein kinases (MAPKs) signals to HN neuroprotection against NMDA neurotoxicity remains unclear. The present study was therefore aimed to investigate neuroprotective mechanisms of HN. We analyzed intracellular Ca2+ levels, reactive oxygen species (ROS) production, and the MAPKs signal transduction cascade using an in vitro NMDA-mediated excitotoxicity of cortical neurons model. Results showed that: (1) HN attenuated NMDA-induced neuronal insults by increasing cell viability, decreasing lactate dehydrogenase (LDH) release, and increasing cell survival; (2) HN reversed NMDA-induced increase in intracellular calcium; (3) pretreatment by HN or 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, decreased ROS generation after NMDA exposure; (4) administration of HN or N-Acetyl-l-cysteine (NAC), a ROS scavenger, inhibited NMDA-induced JNK and p38 MAPK activation. These results indicated that HN reduced intracellular elevation of Ca2+ levels, which, in turn, inhibited ROS generation and subsequent JNK and p38 MAPK activation that are involved in promoting cell survival in NMDA-induced excitotoxicity. Therefore, the present study suggests that inhibition of ROS-dependent JNK/p38 MAPK signaling pathway serves an effective strategy for HN neuroprotection against certain neurological diseases.

The excitotoxity of NMDA receptor NR2D subtype mediates human fetal lung fibroblasts proliferation and collagen production

Studies have suggested that endogenous glutamate and N-methyl-d-aspartate (NMDA) receptor have an excitotoxity role during acute lung injury. Fibroblasts play a critical role in lung development and chronic lung disease after acute lung injury. This study aims to explore the immediate role of NMDAR activation in human lung fibroblasts. The expression of NMDAR 1 subtype (NR1) and four individual NMDAR 2 (NR2) subtypes (NR 2 A to D) was measured in human fetal lung fibroblasts (HFL-1 and MRC-5). Five NMDARs expression were all detectable in two cell lines. Although the expressions of NMDARs were different between MRC-5 and HFL-1, 1mM NMDA elicited the same trend in the downregulation of NR2A expression, the upregulation of NR2D, and the increase of cells proliferation and collagen production. Glutamate stimulation after 24-h of NMDA exposure resulted in weaker and more delayed but more prolonged iCa2+ elevation in HFL-1 than no NMDA exposed cells. NMDA increased the level of pERK1/2, cells proliferation and collagen production, whereas nonspecific NMDAR antagonist MK-801, NR2D-preferring receptor antagonist UBP141 and ERK1/2 phosphorylation inhibitor U0126 suppressed it, respectively. In conclusion, we found that NMDAR activation, NR2D in particular, is involved in human fetal lung fibroblast proliferation and collagen production through a potential ERK1/2-mediated mechanism.

Glutamate-Mediated Down-Regulation of the Multidrug-Resistance Protein BCRP/ABCG2 in Porcine and Human Brain Capillaries

Breast cancer resistance protein (BCRP) functions as a major molecular gatekeeper at the blood-brain barrier. Considering its impact on access to the brain by therapeutic drugs and harmful xenobiotics, it is of particular interest to elucidate the mechanisms of its regulation. Excessive glutamate concentrations have been reported during epileptic seizures or as a consequence of different brain insults including brain ischemia. Previously, we have demonstrated that glutamate can trigger an induction of the transporter P-glycoprotein. These findings raised the question whether other efflux transporters are affected in a comparable manner. Glutamate exposure proved to down-regulate BCRP transport function and expression in isolated porcine capillaries. The reduction was efficaciously prevented by coincubation with N-methyl-d-aspartate (NMDA) receptor antagonist MK-801. The involvement of the NMDA receptor in the down-regulation of BCRP was further confirmed by experiments showing an effect of NMDA exposure on brain capillary BCRP transport function and expression. Pharmacological targeting of cyclooxygenase-1 and -2 (COX-1 and -2) using the nonselective inhibitor indomethacin, COX-1 inhibitor SC-560, and COX-2 inhibitor celecoxib revealed a contribution of COX-2 activity to the NMDA receptor's downstream signaling events affecting BCRP. Translational studies were performed using human capillaries isolated from surgical specimens of epilepsy patients. The findings confirmed a glutamate-induced down-regulation of BCRP transport activity in human capillaries, which argued against major species differences. In conclusion, our data reveal a novel mechanism of BCRP down-regulation in porcine and human brain capillaries. Moreover, together with previous data sets for P-glycoprotein, the findings point to a contrasting impact of the signaling pathway on the regulation of BCRP and P-glycoprotein. The effect of glutamate and arachidonic acid signaling on BCRP function might have implications for brain drug delivery and for radiotracer brain access in epilepsy patients and patients with other brain insults.

Neuroprotective effects of daphnetin against NMDA receptor-mediated excitotoxicity.

The accumulation of glutamate can excessively activate the N-methyl-d-aspartate (NMDA) receptors and cause excitotoxicity. Daphnetin (Dap), a coumarin derivative, is a protein kinase inhibitor that exhibits antioxidant and neuroprotective properties. However, little is known about the neuroprotective effects of Dap on glutamate-induced excitotoxicity. We evaluated the neuroprotective activities in the primary cultured cortical neurons against NMDA-induced excitotoxicity. Pretreatment with Dap significantly prevented NMDA-induced neuronal cell loss. Dap significantly inhibited the neuronal apoptosis by regulating balance of Bcl-2 and Bax expression. Furthermore, pretreatment of Dap reversed the up-regulation of NR2B-containing NMDA receptors and inhibited the intracellular Ca2+ overload induced by NMDA exposure. In addition, Dap prevented cerebral ischemic injury in mice induced via a 2 h middle cerebral artery occlusion and a 24 h reperfusion in vivo. The findings suggest that Dap prevents the excitotoxicity through inhibiting the NR2B-containing NMDA receptors and the subsequent calcium overload in cultured cortical neurons.

Neuroprotective effects of vitexin by inhibition of NMDA receptors in primary cultures of mouse cerebral cortical neurons

The accumulation of glutamate can excessively activate the N-methyl-D-aspartate (NMDA) receptors and cause excitotoxicity. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside, Vit) is a c-glycosylated flavone which was found in the several herbs, exhibiting potent hypotensive, anti-inflammatory, and neuroprotective properties. However, little is known about the neuroprotective effects of Vit on glutamate-induced excitotoxicity. In present study, primary cultured cortical neurons were treated with NMDA to induce the excitotoxicity. Pretreatment with Vit significantly prevented NMDA-induced neuronal cell loss and reduced the number of apoptotic neurons. Vit significantly inhibited the neuronal apoptosis induced by NMDA exposure by regulating balance of Bcl-2 and Bax expression and the cleavages of poly (ADP-ribose) polymerase and pro-caspase 3. Furthermore, pretreatment of Vit reversed the up-regulation of NR2B-containing NMDA receptors and the intracellular Ca(2+) overload induced by NMDA exposure. The neuroprotective effects of Vit are related to inhibiting the activities of NR2B-containing NMDA receptors and reducing the calcium influx in cultured cortical neurons.

Erythropoietin Protects Adult Retinal Ganglion Cells against NMDA-, Trophic Factor Withdrawal-, and TNF-α-Induced Damage

PurposeThis study aimed to evaluate the neuroprotective effect of EPO in the presence of N-methyl-d-aspartate (NMDA)-, trophic factor withdrawal (TFW)-, and tumor necrosis factor-alpha (TNF-α)-induced toxicity on total, small, and large retinal ganglion cells (RGCs).MethodsRetinal cells from adult rats were cultured in a medium containing brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), and forskolin. Expression of RGC markers and EPOR was examined using immunocytochemistry. RGCs were classified according to their morphological properties. Cytotoxicity was induced by NMDA, TFW, or TNF-α. RGC survival was assessed by counting thy-1 and neurofilament-l double-positive cells.ResultsEPO offered dose-dependent (EC50 = 5.7 ng/mL) protection against NMDA toxicity for small RGCs; protection was not significant for large RGCs. Time-course analysis showed that the presence of EPO either before or after NMDA exposure gave effective protection. For both small and large RGCs undergoing trophic factor withdrawal, EPO at concentrations of 1, 10, or 100 ng/mL improved survival. However, EPO had to be administered soon after the onset of injury to provide effective protection. For TNF-α-induced toxicity, survival of small RGCs was seen only for the highest examined concentration (100 ng/mL) of EPO, whereas large RGCs were protected at concentrations of 1, 10, or 100 ng/mL of EPO. Time-course analysis showed that pretreatment with EPO provided protection only for large RGCs; early post-treatment with EPO protected both small and large RGCs. Inhibitors of signal transduction and activators of transcription such as (STAT)-5, mitogen-activated protein kinases (MAPK)/extracellular-regulated kinase (ERK), and phosphatidyl inositol-3 kinase (PI3K)/Akt impaired the protective effect of EPO on RGCs exposed to different insults.ConclusionEPO provided neuroprotection to cultured adult rat RGCs; however, the degree of protection varied with the type of toxic insult, RGC subtype, and timing of EPO treatment.

The neuroprotective effect of praeruptorin C against NMDA-induced apoptosis through down-regulating of GluN2B-containing NMDA receptors

Praeruptorin C (Pra-C), one of the principal bioactive components derived from the root of Peucedanum praeruptorum Dunn, has been widely used as an antioxidant and a calcium antagonist to treat diseases. The present study investigated the protective effect of Pra-C on cultured cortical neuron injury induced by glutamate. After challenge with 200μM N-methyl-d-aspartate (NMDA) for 30min, loss of cell viability and excessive apoptotic cell death were observed in cultured cortical neurons. Pra-C conferred protective effects against loss of cellular viability in a concentration-dependent manner. Pra-C also significantly inhibited neuronal apoptosis induced by NMDA exposure by reversing intracellular Ca2+ overload and balancing Bcl-2 and Bax expression. Furthermore, Pra-C significantly reversed the upregulation of GluN2B-containing NMDA receptors by exposure to NMDA but did not affect the expression of GluN2A-containing NMDA receptors. These findings suggest that Pra-C partially protects cortical neurons by inhibiting the expression of GluN2B-containing NMDA receptors and regulating the Bcl-2 family.

Cytisine confers neuronal protection against excitotoxic injury by down-regulating GluN2B-containing NMDA receptors

Cytisine (CYT), one of the principal bioactive components derived from the seeds of Cytisus laborinum L, has been widely used for central nervous system (CNS) diseases treatment. The present study investigated the protective effect of CYT on cultured cortical neural injury induced by N-methyl-d-aspartate (NMDA). Our data showed that CYT conferred protective effect against loss of cellular viability induced by brief exposure to 200μM NMDA in a concentration-dependent manner. CYT significantly inhibited the neuronal apoptosis induced by NMDA exposure by reversing intracellular Ca2+ overload and balancing Bcl-2 and Bax expression levels. Furthermore, CYT significantly reversed the up-regulation of GluN2B-containing NMDA receptors by exposure to NMDA, but it did not affect the level of GluN2A-containing NMDA receptors. These findings suggest that CYT protects cortical neurons, at least partially, by inhibiting the level of GluN2B-containing NMDA receptors and regulating Bcl-2 family.

Neuroprotective Effects of Flax Lignan Against NMDA‐Induced Neurotoxicity In Vitro

Flax Lignan (FLL), a chemical widespread within the plant and animal kingdoms, has antioxidant, antiinfectious, and antitumor activities. However, little is known about the effects of FLL on the central nervous system (CNS). The neuroprotective actions of FLL against N-methyl-d-aspartate (NMDA) are investigated in primary cultured cortical neurons by MTT assay. The expression levels of proteins related to apoptosis and GluN2-containing receptor were detected by Western blot analysis. Intracellular Ca(2+) was measured under a confocal laser scanning microscope. After challenged with 100μM NMDA for 30min, loss of cell viability and excessive apoptotic cell death were observed in cultured cortical neurons. FLL protected the neurons against the NMDA-induced cell loss in a concentration-dependent manner. FLL also significantly inhibited the neuronal apoptosis induced by NMDA exposure through reversing intracellular concentration of Ca(2+) overload and balancing of Bcl-2 and Bax expression. Furthermore, FLL significantly reversed the upregulation of GluN2B-containing NMDA receptors by exposure to NMDA, but did not affect the expression of GluN2A-containing NMDA receptor. These findings suggest that FLL protects cortical neurons by inhibiting the expression of GluN2B-containing NMDA receptor and regulating the Bcl-2 family.

Sustained Activation of N-Methyl-d-Aspartate Receptors in Podoctyes Leads to Oxidative Stress, Mobilization of Transient Receptor Potential Canonical 6 Channels, Nuclear Factor of Activated T Cells Activation, and Apoptotic Cell Death

Atypical N-methyl-D-aspartate (NMDA) receptors are expressed in podocytes. Sustained (≥24 h) application of 50 to100 μM NMDA to immortalized mouse podocytes evoked a marked increase in the production of reactive oxygen species(ROS) such as H₂O₂. This effect of NMDA was associated with increased cell-surface expression of p47(phox), a cytosolic regulatory subunit of the NADPH oxidase NOX2. NMDA-evoked generation of ROS drove an increase in steady-state surface expression of transient receptor potential canonical (TRPC) 6 channels, which was blocked by the NMDA antagonist dizocilpine(MK-801) and by a membrane-permeable scavenger of ROS. The effect of NMDA on TRPC6 was observed using cell surface biotinylation assays and also with whole-cell recordings made under conditions designed to facilitate detection of current through TRPC6. NMDA mobilization of TRPC6 channels was blocked by concurrent treatment with the NMDA antagonist MK-801 and by a membrane-permeable scavenger ofROS. Mobilization of TRPC6 was also evoked by L-homocysteic acid. NMDA treatment also increased nuclear localization of endogenous nuclear factor of activated T cells, which could be blocked by MK-801, by scavenging ROS, by the calcineurin inhibitor cyclosporine, and by the TRPC channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole (SKF-96365). NMDA treatment also evoked robust activation of Rho but not Rac,consistent with previous studies of downstream effectors of TRPC6 activation. Exposing cells to NMDA for 24 h reduced total and cell surface expression of the podocyte markers nephrin and podocin, but there was no loss of cells. With longer NMDA exposure (72 h), we observed loss of cells associated with nuclear fragmentation and increased expression of caspase-3, caspase-6, and Bax, suggesting an apoptotic process.

Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype

BackgroundAutophagy, an intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes. Prolonged autophagy is known to result in autophagic (Type II) cell death. This study examined the potential role of an autophagic response in cultured cerebellar granule neurons challenged with excitotoxin N-methyl-D-aspartate (NMDA).ResultsNMDA exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in both cell bodies and neurites as early as 3 hours post-treatment. Elevated levels of Beclin-1 and the autophagosome-targeting LC3-II were also observed following NMDA exposure. Prolonged exposure of the cultures to NMDA (8-24 h) generated MDC-, LC3-positive autophagosomal bodies, concomitant with the neurodegenerative phase of NMDA challenge. Lysosomal inhibition studies also suggest that NMDA-treatment diverted the autophagosome-associated LC3-II from the normal lysosomal degradation pathway. Autophagy inhibitor 3-methyladenine significantly reduced NMDA-induced LC3-II/LC3-I ratio increase, accumulation of autophagosomes, and suppressed NMDA-mediated neuronal death. ATG7 siRNA studies also showed neuroprotective effects following NMDA treatment.ConclusionsCollectively, this study shows that autophagy machinery is robustly induced in cultured neurons subjected to prolonged exposure to excitotoxin, while autophagosome clearance by lysosomal pathway might be impaired. Our data further show that prolonged autophagy contributes to cell death in NMDA-mediated excitotoxicity.

Selective vulnerability of hippocampal cornu ammonis 1 pyramidal cells to excitotoxic insult is associated with the expression of polyamine-sensitive N-methyl- d-asparate-type glutamate receptors

Excess glutamate release and stimulation of post-synaptic glutamatergic receptors have been implicated in the pathophysiology of many neurological diseases. The hippocampus, and the pyramidal cell layer of the cornu ammonus 1 (CA1) region in particular, has been noted for its selective sensitivity to excitotoxic insults. The current studies examined the role of N-methyl- d-aspartate (NMDA) receptor subunit composition and sensitivity to stimulatory effects of the polyamine spermidine, an allosteric modulator of NMDA NR2 subunit activity, in hippocampal CA1 region sensitivity to excitotoxic insult. Organotypic hippocampal slice cultures of 8 day-old neonatal rat were obtained and maintained in vitro for 5 days. At this time, immunohistochemical analysis of mature neuron density (NeuN); microtubule associated protein-2(a,b) density (MAP-2); and NMDA receptor NR1 and NR2B subunit density in the primary cell layers of the dentate gyrus (DG), CA3, and CA1 regions, was conducted. Further, autoradiographic analysis of NMDA receptor distribution and density (i.e. [ 125I]MK-801 binding) and spermidine (100 μM)-potentiated [ 125I]MK-801 binding in the primary cell layers of these regions was examined. A final series of studies examined effects of prolonged exposure to NMDA (0.1–10 μM) on neurodegeneration in the primary cell layers of the DG, CA3, and CA1 regions, in the absence and presence of spermidine (100 μM) or ifenprodil (100 μM), an allosteric inhibitor of NR2B polypeptide subunit activity. The pyramidal cell layer of the CA1 region demonstrated significantly greater density of mature neurons, MAP-2, NR1 and NR2B subunits, and [ 125I]MK-801 binding than the CA3 region or DG. Twenty-four hour NMDA (10 μM) exposure produced marked neurodegeneration (∼350% of control cultures) in the CA1 pyramidal cell region that was significantly reduced by co-exposure to ifenprodil or dl-2-Amino-5-phosphonopentanoic acid (APV). The addition of spermidine significantly potentiated [ 125I]MK-801 binding and neurodegeneration induced by exposure to a non-toxic concentration of NMDA, exclusively in the CA1 region. This neurodegeneration was markedly reduced with co-exposure to ifenprodil. These data suggest that selective sensitivity of the CA1 region to excitotoxic stimuli may be attributable to the density of mature neurons expressing polyamine-sensitive NR2B polypeptide subunits.

GDNF Selectively Induces Microglial Activation and Neuronal Survival in CA1/CA3 Hippocampal Regions Exposed to NMDA Insult through Ret/ERK Signalling

The glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuronal populations in different brain regions, including the hippocampus. However, no information is available on the: (1) hippocampal subregions involved in the GDNF-neuroprotective actions upon excitotoxicity, (2) identity of GDNF-responsive hippocampal cells, (3) transduction pathways involved in the GDNF-mediated neuroprotection in the hippocampus. We addressed these questions in organotypic hippocampal slices exposed to GDNF in presence of N-methyl-D-aspartate (NMDA) by immunoblotting, immunohistochemistry, and confocal analysis. In hippocampal slices GDNF acts through the activation of the tyrosine kinase receptor, Ret, without involving the NCAM-mediated pathway. Both Ret and ERK phosphorylation mainly occurred in the CA3 region where the two activated proteins co-localized. GDNF protected in a greater extent CA3 rather than CA1 following NMDA exposure. This neuroprotective effect targeted preferentially neurons, as assessed by NeuN staining. GDNF neuroprotection was associated with a significant increase of Ret phosphorylation in both CA3 and CA1. Interestingly, confocal images revealed that upon NMDA exposure, Ret activation occurred in microglial cells in the CA3 and CA1 following GDNF exposure. Collectively, this study shows that CA3 and CA1 hippocampal regions are highly responsive to GDNF-induced Ret activation and neuroprotection, and suggest that, upon excitotoxicity, such neuroprotection involves a GDNF modulation of microglial cell activity.

Transcriptional regulation of PSA-NCAM expression by NMDA receptor activation in RA-differentiated C6 glioma cultures

N-Methyl- d-aspartate (NMDA) receptors exhibit a dichotomy of signaling with both toxic and plastic responses. Recent reports have shown that exposure to subtoxic concentration of NMDA results in a neuroprotective state that was measured when these neurons were subsequently challenged with toxic doses of glutamate or kainate. Control of polysialylated neural cell adhesion molecule (PSA-NCAM) expression by NMDA receptor activation has been described in several systems, suggesting a functional link between these two proteins. The perception of glial role in CNS function has changed dramatically over the past few years from simple trophic functions to that of cells with important roles in development and maintenance of CNS in cooperation with neurons. We report here the transcriptional regulation of PSA-NCAM expression by subtoxic dose of NMDA in retinoic acid differentiated C6 glioma cell cultures. C6 glioma cell cultures differentiated with retinoic acid (10 μM) were exposed to NMDA (100 μM) or to antagonist MK-801 (200 nM) prior to treatment with NMDA and cells were harvested after 24 h of treatment to study the expression of total NCAM, PSA-NCAM, nuclear factor kappaB (NF-κB) and activator protein-1 (AP-1) by Western blotting and dual immunocytofluorescence and expression of PST mRNA by fluorescent in situ hybridization (FISH). Significant increase in the levels of PSA-NCAM, NF-κB, AP-1 and PST mRNA was observed in NMDA treated cultures. Treatment of cultures with MK-801, a non-competitive NMDA receptor antagonist, prior to NMDA exposure prevented the NMDA-mediated changes indicating the involvement of NMDA receptor activation. The results elucidate the possible cellular and molecular mechanisms of regulation of PSA-NCAM expression in astroglial cultures by extracellular signals.

Elevated glutamate and NMDA disrupt production of the second messenger cyclic GMP in the early postnatal mouse cortex

The second messenger cyclic guanosine monophosphate (cGMP) plays many roles during nervous system development. Consequently, cGMP production shows complex patterns of regulation throughout early development. Elevated glutamate levels are known to increase cGMP levels in the mature nervous system. A number of clinical conditions including ischemia and perinatal asphyxia can result in elevated glutamate levels in the developing brain. To investigate the effects of elevated glutamate levels on cGMP in the developing cortex we exposed mouse brain slices to glutamate or N-methyl D-aspartate (NMDA). We find that at early postnatal stages when the endogenous production of cGMP is high, glutamate or NMDA exposure results in a significant lowering of the overall production of cGMP in the cortex, unlike the situation in the mature brain. However, this response pattern is complex with regional and cell-type specific exceptions to the overall lowered cGMP production. These data emphasize that the response of the developing brain to physiological disturbances can be different from that of the mature brain, and must be considered in the context of the developmental events occurring at the time of disturbance.

Protective Effect of Gabapentin on N-methyl-D-aspartate–Induced Excitotoxicity in Rat Hippocampal CA1 Neurons

Gabapentin was developed as an anticonvulsant, but has also been used to alleviate hyperalgesia in neuropathic pain. In this study, the protective effect of gabapentin against N-methyl-D-aspartate (NMDA)-induced excitotoxicity in rat hippocampal CA1 neurons was investigated. Pre-treatment with gabapentin reduced the degree of neuronal damage induced by NMDA exposure in cultured hippocampal slices. Patch-clamp studies revealed that gabapentin significantly inhibited the NMDA receptor–activated ion current in dissociated hippocampal CA1 neurons, resulting in suppression of glutamate-induced neuronal injury. These results show that gabapentin may exert protective effects against glutamate-induced neuronal injury at least in part by inhibiting the NMDA receptor–activated ion current.

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.

Targeting IKK2 by pharmacological inhibitor AS602868 prevents excitotoxic injury to neurons and oligodendrocytes

Among the diverse mechanisms involved in the pathophysiology of post-ischemic and post-traumatic injuries, excitotoxicity and nuclear factor-kappaB (NF-kappaB) activation through induction of IkappaB kinase (IKK) complex have a primary role. We investigated the effects of the selective inhibitor of the IKK2 subunit, the anilinopyrimidine derivative AS602868, on excitotoxic injury produced in rat organotypic hippocampal slices and cerebellar primary neurons. Brief exposure to N-methyl-D-aspartate (NMDA) induces astrocyte reactivity, neuron cell death and oligodendrocyte degeneration in hippocampal slices. Application of AS602868 elicited a long-lasting protection of both neurons and oligodendrocytes. Maximal effect was observed with prolonged application of the compound after NMDA exposure. Neuroprotection was also evident in primary cultures of cerebellar granule cells starting from 20 nM concentration. AS602868-elicited neuroprotection correlated with inhibition of NF-kappaB activity. Our results suggest that AS602868 may prove to be a valuable approach in treating neurodegeneration and demyelination associated with cerebral trauma and ischemia.