NMDA and non-NMDA receptor-mediated differential Ca2+ load and greater vulnerability of motor neurons in spinal cord cultures

Glutamate receptor activated neuronal cell death has been implicated in the pathogenesis of motor neuron disease but the molecular mechanism responsible for neuronal dysfunction needs to be elucidated. In the present study, we examined the contribution of NMDA and non-NMDA sub-types of glutamate receptors in selective vulnerability of motor neurons. Glutamate receptor activated Ca2+ signaling, mitochondrial functions and neurotoxicity in motor neurons and other spinal neurons were studied in mixed spinal cord primary cultures. Exposure of cells to glutamate receptor agonists glutamate, NMDA and AMPA elevated the intracellular Ca2+, mitochondrial Ca2+ and caused mitochondrial depolarization and cytotoxicity in both motor neurons and other spinal neurons but a striking difference was observed in the magnitude and temporal patterns of the [Ca2+]i responses between the two neuronal cell types. The motor neurons elicited higher Ca2+ load than the other spinal neurons and the [Ca2+]i levels were elevated for a longer duration in motor neurons. AMPA receptor stimulation was more effective than NMDA. Both the NMDA and non-NMDA receptor antagonists APV and NBQX inhibited the Ca2+ entry and decreased the cell death significantly; however, NBQX was more potent than APV. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors contribute to glutamate-mediated motor neuron damage but AMPA receptors play the major role. AMPA receptor-mediated excessive Ca2+ load and differential handling/regulation of Ca2+ buffering by mitochondria in motor neurons could be central in their selective vulnerability to excitotoxicity.     

Apoptotic Cell Death and Caspase-3 Activation Induced by N-Methyl-d-Aspartate Receptor Antagonists and Their Prevention by Insulin-Like Growth Factor I

The effect of N-methyl-D-aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK-801 and 2-amino-5-phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK-801 (an NMDA antagonist) for 1-2 days induced apoptotic cell death in a dose-dependent manner (1 nM to 10 microM). NMDA (25 microM), however, inhibited the MK-801 (0.1 microM)-induced apoptotic cell death. MK-801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase-3 was accompanied by MK-801-induced cell death in a dose-dependent manner, and an inhibitor of caspase-3 reduced the cell death. Further, cycloheximide (0.2 microg/ml) completely protected the cells from MK-801-induced apoptotic cell death and caspase-3 activation. Insulin-like growth factor I completely attenuated MK-801-induced apoptotic cell death and caspase-3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.     

Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death

Multiple cytokines are secreted in the brain during pro-inflammatory conditions and likely affect neuron survival. Previously, we demonstrated that glutamate and tumor necrosis factor alpha (TNFalpha) kill neurons via activation of the N-methyl-d-aspartate (NMDA) and TNFalpha receptors, respectively. This report continues characterizing the signaling cross-talk pathway initiated during this inflammation-related mechanism of death. Stimulation of mouse cortical neuron cultures with TNFalpha results in a transient increase in NMDA receptor-dependent calcium influx that is additive with NMDA stimulation and inhibited by pre-treatment with the NMDA receptor antagonist, DL-2-amino-5-phosphonovaleric acid, or the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione. Pre-treatment with N-type calcium channel antagonist, omega-conotoxin, or the voltage-gated sodium channel antagonist, tetrodotoxin, also prevents the TNFalpha-stimulated calcium influx. Combined TNFalpha and NMDA stimulation results in a transient increase in activity of extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs). Specific inhibition of ERKs but not JNKs is protective against TNFalpha and NMDA-dependent death. Death is mediated via the low-affinity TNFalpha receptor, TNFRII, as agonist antibodies for TNFRII but not TNFRI stimulate NMDA receptor-dependent calcium influx and death. These data demonstrate how microglial pro-inflammatory secretions including TNFalpha can acutely facilitate glutamate-dependent neuron death.     

AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK,calpain and caspase 3

The molecular mechanisms underlying AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor-mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca(2+) influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N -acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor-mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase-3 and c-Jun N-terminal kinase (JNK). A substrate of calpain and caspase-3, alpha-spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase-3 by DEVD-CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca(2+) influx, depletion of glutathione, and activation of JNK, calpain, and caspase-3.     

Cytoskeletal Breakdown and Apoptosis Elicited by NO Donors in Cerebellar Granule Cells Require NMDA Receptor Activation

Abstract: We have recently demonstrated that nitric oxide (NO) donors can trigger either apoptosis or necrosis of neurons as a function of the intensity of the exposure. Here, we show that the apoptosis induced by the NO donors S -nitrosocysteine (SNOC) or S -nitroso- N -acetylpenicillamine (SNAP) in cultured cerebellar granule cells (CGCs) depends on NMDA receptor (NMDA-R) activation leading to intracellular Ca²⁺ overload. Early dissolution of actin filaments followed by breakdown of microtubules and nuclear lamins preceded the appearance of typical apoptotic features. NO donors induced tyrosine nitration in neurons, in a small population of contaminating astrocytes, and in cultures of cerebellar astroglial cells. However, astrocytes neither displayed cytoskeletal alterations nor underwent apoptosis. Competitive and uncompetitive NMDA receptor antagonists, such as d -aminophosphonovaleric acid and MK-801, did not influence tyrosine nitration but prevented the accumulation of intracellular Ca²⁺, cytoskeletal breakdown, and apoptosis induced by either SNOC or SNAP in CGCs. Taken together, these data strongly suggest that Ca²⁺ influx through NMDA-R-gated ion channels is a critical event in CGC apoptosis induced by NO donors.     

A comparison between acute exposures to ethanol and acetaldehyde on neurotoxicity, nitric oxide production and NMDA-induced excitotoxicity in primary cultures of cortical neurons

Chronic exposure of primary neuronal cultures to ethanol has been shown to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated processes, such as nitric oxide (NO) formation and excitotoxicity. In the present study, we compared the effects of acute ethanol and acetaldehyde on NMDA receptor-mediated excitotoxicity and NO production in primary cultures of rat cortical neurons. The delayed cell death induced by NMDA (300 mM, 25 min) was evaluated by morphological examination and by measuring the release of the cytotoxic indicator, lactate dehydrogenase, in the culture media 24 hours after the NMDA exposure. The accumulation of nitrite, as an index of NO production, was also measured 24 hours after NMDA treatment. NMDA caused a dose-dependent cell death and nitrite accumulation, both effects were blocked by pretreatment of MK-801 (100 microM). Acute exposure to ethanol (1-1000 mM) or acetaldehyde (0.1-1 mM) for 35 minutes did not affect neuronal viability in the following 24-hr period. However, acute exposure to acetaldehyde (> or =10 mM) was neurotoxic. Neither ethanol nor acetaldehyde changed basal nitrite levels in the culture media. Acute ethanol (50-400 mM, 10 min) given before the NMDA treatment (25 min) resulted in a concentration-dependent suppression of the delayed cell death. The NMDA-induced NO production was, however, not affected by ethanol. Neither the NMDA excitotoxicity nor NO production was affected by acute ethanol given after NMDA treatment. Acute acetaldehyde (0.01-0.5 mM, 10 min) given before or after NMDA treatment had no effect on delayed NMDA neurotoxicity and NO production. Our data suggest that acute exposure to ethanol is not neurotoxic and is even protective against delayed NMDA-excitotoxicity when given before but not after NMDA treatment. Neither NO nor metabolism of ethanol to acetaldehyde is required for ethanol-mediated suppression of NMDA excititoxicity. Acetaldehyde, on the other hand, is toxic by itself at low concentrations (> or =10 mM). Furthermore, acute exposure to non-toxic concentrations of acetaldehyde could not protect cortical neurons against NMDA-induced excitotoxicity.     

Low‐level laser therapy (810 nm) protects primary cortical neurons against excitotoxicity in vitro

Excitotoxicity describes a pathogenic process whereby death of neurons releases large amounts of the excitatory neurotransmitter glutamate, which then proceeds to activate a set of glutamatergic receptors on neighboring neurons (glutamate, N‐methyl‐D‐aspartate (NMDA), and kainate), opening ion channels leading to an influx of calcium ions producing mitochondrial dysfunction and cell death. Excitotoxicity contributes to brain damage after stroke, traumatic brain injury, and neurodegenerative diseases, and is also involved in spinal cord injury. We tested whether low level laser (light) therapy (LLLT) at 810 nm could protect primary murine cultured cortical neurons against excitotoxicity in vitro produced by addition of glutamate, NMDA or kainate. Although the prevention of cell death was modest but significant, LLLT (3 J/cm² delivered at 25 mW/cm² over 2 min) gave highly significant benefits in increasing ATP, raising mitochondrial membrane potential, reducing intracellular calcium concentrations, reducing oxidative stress and reducing nitric oxide. The action of LLLT in abrogating excitotoxicity may play a role in explaining its beneficial effects in diverse central nervous system pathologies. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Knockdown of the aryl hydrocarbon receptor attenuates excitotoxicity and enhances NMDA-induced BDNF expression in cortical neurons

NMDA receptors play dual and opposing roles in neuronal survival by mediating the activity-dependent neurotrophic signaling and excitotoxic cell death via synaptic and extrasynaptic receptors, respectively. In this study, we demonstrate that the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, is involved in the expression and the opposing activities of NMDA receptors. In primary cultured cortical neurons, we found that NMDA excitotoxicity is significantly enhanced by an AhR agonist 2,3,7,8-tetrachlorodibenzo- p -dioxin, and AhR knockdown with small interfering RNA significantly reduces NMDA excitotoxicity. AhR knockdown also significantly reduces NMDA-increases intracellular calcium concentration, NMDA receptor expression and surface presentation, and moderately decreases the NMDA receptor-mediated spontaneous as well as miniature excitatory post-synaptic currents. However, AhR knockdown significantly enhances the bath NMDA application– but not synaptic NMDA receptor-induced brain-derived neurotrophic factor (BDNF) gene expression, and activating AhR reduces the bath NMDA-induced BDNF expression. Furthermore, AhR knockdown reveals the calcium dependency of NMDA-induced BDNF expression and the binding activity of cAMP-responsive element binding protein (CREB) and its calcium-dependent coactivator CREB binding protein (CBP) to the BDNF promoter upon NMDA treatment. Together, our results suggest that AhR opposingly regulates NMDA receptor-mediated excitotoxicity and neurotrophism possibly by differentially regulating the expression of synaptic and extrasynaptic NMDA receptors.     

Excitotoxic Death of a Subset of Embryonic Rat Motor Neurons In Vitro

Abstract : We have used cultures of purified embryonic rat spinal cord motor neurons to study the neurotoxic effects of prolonged ionotropic glutamate receptor activation. NMDA and non-NMDA glutamate receptor agonists kill a maximum of 40% of the motor neurons in a concentration- and time-dependent manner, which can be blocked by receptor subtype-specific antagonists. subunit-specific antibodies stain all of the motor neurons with approximately the same intensity and for the same repertoire of subunits, suggesting that the survival of the nonvulnerable population is unlikely to be due to the lack of glutamate receptor expression. Extracellular Ca²⁺ is required for excitotoxicity, and the route of entry initiated by activation of non-NMDA, but not NMDA, receptors is L-type Ca²⁺ channels. Ca²⁺ imaging of motor neurons after application of specific glutamate receptor agonists reveals a sustained rise in intracellular Ca²⁺ that is present to a similar degree in most motor neurons, and can be blocked by appropriate receptor/channel antagonists. Although the lethal effects of glutamate receptor agonists are seen in only a subset of cultured motor neurons, the basis of this selectivity is unlikely to be simply the glutamate receptor phenotype or the level/pattern of rise in agonist-evoked intracellular Ca²⁺.     

Pharmacological activation/inhibition of the cannabinoid system affects alcohol withdrawal-induced neuronal hypersensitivity to excitotoxic insults

Cessation of chronic ethanol consumption can increase the sensitivity of the brain to excitotoxic damages. Cannabinoids have been proposed as neuroprotectants in different models of neuronal injury, but their effect have never been investigated in a context of excitotoxicity after alcohol cessation. Here we examined the effects of the pharmacological activation/inhibition of the endocannabinoid system in an in vitro model of chronic ethanol exposure and withdrawal followed by an excitotoxic challenge. Ethanol withdrawal increased N-methyl-D-aspartate (NMDA)-evoked neuronal death, probably by altering the ratio between GluN2A and GluN2B NMDA receptor subunits. The stimulation of the endocannabinoid system with the cannabinoid agonist HU-210 decreased NMDA-induced neuronal death exclusively in ethanol-withdrawn neurons. This neuroprotection could be explained by a decrease in NMDA-stimulated calcium influx after the administration of HU-210, found exclusively in ethanol-withdrawn neurons. By contrast, the inhibition of the cannabinoid system with the CB1 receptor antagonist rimonabant (SR141716) during ethanol withdrawal increased death of ethanol-withdrawn neurons without any modification of NMDA-stimulated calcium influx. Moreover, chronic administration of rimonabant increased NMDA-stimulated toxicity not only in withdrawn neurons, but also in control neurons. In summary, we show for the first time that the stimulation of the endocannabinoid system is protective against the hyperexcitability developed during alcohol withdrawal. By contrast, the blockade of the endocannabinoid system is highly counterproductive during alcohol withdrawal.     

Distinct mechanisms underlie distinct polyphenol-induced neuroprotection

Glutamate excitotoxicity is mediated by intracellular Ca(2+) overload, caspase-3 activation, and ROS generation. Here, we show that curcumin, tannic acid (TA) and (+)-catechin hydrate (CA) all inhibited glutamate-induced excitotoxicity. Curcumin inhibited PKC activity, and subsequent phosphorylation of NR1 of the NMDA receptor. As a result, glutamate-mediated Ca(2+) influx was reduced. TA attenuated glutamate-mediated Ca(2+) influx only when simultaneously administered, directly interfering with Ca(2+). Both curcumin and TA inhibited glutamate-induced caspase-3 activation. Although Ca(2+) influx was not attenuated by CA, caspase-3 was reduced by direct inhibition of the enzyme. All polyphenols reduced glutamate-induced generation of ROS.     

Genetic evidence for adenylyl cyclase 1 as a target for preventing neuronal excitotoxicity mediated by N-methyl-D-aspartate receptors

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.     

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 Ca²⁺ 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.     

Induction of Ornithine Decarboxylase by N-Methyl-D-Aspartate Receptor Activation Is Unrelated to Potentiation of Glutamate Excitotoxicity by Polyamines in Cerebellar Granule Neurons

Abstract: Polyamines positively modulate the activity of the N -methyl- d -aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)—the key regulatory enzyme in polyamine synthesis—and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.     

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.     

HIV-1 Tat through phosphorylation of NMDA receptors potentiates glutamate excitotoxicity

Toxic effects of HIV-1 proteins contribute to altered function and decreased survival of select populations of neurons in HIV-1-infected brain. One such HIV-1 protein, Tat, can activate calcium release from IP3-sensitive intracellular pools, induce calcium influx in neural cells, and, as a result, can increase neuronal cell death. Here, we provide evidence that Tat potentiates excitatory amino acid (glutamate and NMDA) triggered calcium flux, as well as glutamate- and staurosporine-mediated neurotoxicity. Calcium flux in cultured rat hippocampal neurons triggered by the transient application of glutamate or NMDA was facilitated by pre-exposure to Tat. Facilitation of glutamate-triggered calcium flux by Tat was prevented by inhibitors of ADP-ribosylation of G(i)/G(o) proteins (pertussis toxin), protein kinase C (H7 and bisindolymide), and IP3-mediated calcium release (xestospongin C), but was not prevented by an activator of G(s) (cholera toxin) or an inhibitor of protein kinase A (H89). Facilitation of NMDA-triggered calcium flux by Tat was reversed by inhibitors of tyrosine kinase (genestein and herbimycin A) and by an inhibitor of NMDA receptor function (zinc). Tat increased 32P incorporation into NMDA receptor subunits NR2A and NR2B and this effect was blocked by genestein. Subtoxic concentrations of Tat combined with subtoxic concentrations of glutamate or staurosporine increased neuronal cell death significantly. Together, these findings suggest that NMDA receptors play an important role in Tat neurotoxicity and the mechanisms identified may provide additional therapeutic targets for the treatment of HIV-1 associated dementia.     

Tyrosine phosphatase SHP-2 is a mediator of activity-dependent neuronal excitotoxicity

Calcium influx can promote neuronal differentiation and survival, at least in part by activating Ras and its downstream targets, including the Erk pathway. However, excessive calcium influx can initiate molecular signals leading to neuronal death during excitotoxicity or in neurodegenerative diseases. Here we describe a new signaling pathway associated with calcium influx that contributes to neuronal cell death in cerebellar neurons. Influx of calcium, mediated either by L-type voltage-sensitive calcium channels or glutamate receptors, is associated with the suppression of brain-derived neurotrophic factor (BDNF) activation of Ras and its effectors Erk and Akt. This is the result of enhanced association of the tyrosine phosphatase Shp-2 with TrkB receptors, which inhibits BDNF-induced TrkB autophosphorylation and activation. Deletion of the Shp2 gene in neuronal cultures reverses inhibition of TrkB function and increases neuronal survival after extended depolarization or glutamate treatment. These findings implicate Shp-2 in a feedback system initiated by calcium that negatively regulates neurotrophin signaling and sensitizes neurons to excitotoxicity.     

Tex261 modulates the excitotoxic cell death induced by N-methyl-D-aspartate (NMDA) receptor activation

N-methyl-D-aspartate (NMDA) receptor is a calcium-permeable ionotropic glutamate receptor and plays a role in many neurologic disorders such as brain ischemia through its involvement in excitotoxicity. We have performed differential display PCR to identify changes in gene expression that occur in the hippocampus of the mouse brain after intraperitoneal injection of NMDA and identified a gene, Tex261 as an inducible gene by NMDA stimulation in vivo. Tex261 mRNA was gradually induced in response to NMDA and reached about 4.5-fold at 24 h. When HEK 293 cells are transfected with NMDA receptors, the cells die in a manner that mimics excitotoxicity in neurons. HEK 293 cells transfected with the combination of Tex261 and the NMDA receptors NR1/NR2A produced the greater cell death compared with the cells transfected with the NMDA receptors alone. These findings suggest that Tex261 modulates the excitotoxic cell death induced by NMDA receptor activation.