NMDA Receptor-mediated Neurotoxicity Research Articles

Serine Racemase Deletion Protects Against Cerebral Ischemia and Excitotoxicity

D-Serine, formed from L-serine by serine racemase (SR), is a physiologic coagonist at NMDA receptors. Using mice with targeted deletion of SR, we demonstrate a role for D-serine in NMDA receptor-mediated neurotoxicity and stroke. Brain cultures of SR-deleted mice display markedly diminished nitric oxide (NO) formation and neurotoxicity. In intact SR knock-out mice, NO formation and nitrosylation of NO targets are substantially reduced. Infarct volume following middle cerebral artery occlusion is dramatically diminished in several regions of the brains of SR mutant mice despite evidence of increased NMDA receptor number and sensitivity.

Decoy Peptides that Bind Dynorphin Noncovalently Prevent NMDA Receptor-Mediated Neurotoxicity

Prodynorphin-derived peptides elicit various pathological effects including neurological dysfunction and cell death. These actions are reduced by N-methyl-d-aspartate receptor (NMDAR) but not opioid receptor antagonists suggesting NMDAR-mediation. Here, we show that a conserved epitope (KVNSEEEEEDA) of the NR1 subunit of the NMDAR binds dynorphin peptides (DYNp) noncovalently. Synthetic peptides containing this epitope form stable complexes with DYNp and prevent the potentiation of NMDAR-gated currents produced by DYNp. They attenuate DYNp-evoked cell death in spinal cord and prevent, as well as reverse, DYNp-induced paralysis and allodynia. The data reveal a novel mechanism whereby prodynorphin-derived peptides facilitate NMDAR function and produce neurotoxicity. Furthermore, they suggest that synthetic peptides that bind DYNp, thus preventing their interaction with NMDAR, may be novel therapeutic agents for the treatment of spinal cord injury.

P2 receptor antagonist PPADS confers neuroprotection against glutamate/NMDA toxicity

The present study investigated the role of pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid tetrasodium salt (PPADS), a P2 receptor antagonist, in protecting mouse cerebellar granule neurons (CGNs) against glutamate/NMDA-induced neuronal death. Neurotoxicity caused by 50 μM glutamate or 200 μM NMDA was significantly reduced in CGNs treated with PPADS. Such neuroprotection was in a time- and dose-dependent manner. The possibility that PPADS may block glutamate/NMDA-mediated intracellular Ca 2+ influx to CGNs was investigated using temperature-controlled platereader measurements of fluorescence intensity of CGNs loaded with Ca 2+-sensitive fluorescent dye Fluo-4AM. Interestingly, the rapid increase of calcium influx following glutamate/NMDA treatment was not significantly affected by prior treatment with PPADS. In contrast, MK801, a specific NMDA receptor antagonist, completely blocked intracellular Ca 2+ influx. Taken together, these data suggest that inhibition of the P2 receptor may directly modulate NMDA receptor-mediated neurotoxicity through a Ca 2+-independent mechanism.

Protective effect of minocycline on oxygen/glucose deprivation and NMDA-induced neurotoxicity in rat primary neurons and hippocampal slices

To develop oxygen/glucose deprivation (OGD)-and NMDA-induced neurotoxicity models in rat primary neurons and hippocampal slices, and to determine the protective effect of minocycline. The injuries of primary neurons were induced by OGD or NMDA (50micromol/L). Morphological changes of neurons were observed, and neuron viability was evaluated by MTT assay. The changes of light transmittance (LT) were induced by OGD or NMDA in rat hippocampal slices. The effects of minocycline and MK-801, an NMDA receptor antagonist, were observed in the models of OGD-or NMDA-induced injuries. Minocycline concentration dependently inhibited OGD induced decrease of neuron viability and ameliorated neuron morphological changes at 1 and 10 micromol/L. It also inhibited NMDA insult at 10 and 100 micromol/L. MK-801 inhibited both injuries at 1 micromol/L. However, minocycline at 1 or 10 micromol/L did not inhibit the augment of LT in hippocampal slices induced by OGD or NMDA, while MK-801 inhibited both OGD-and NMDA-induced LT augments. Minocycline protects neurons from OGD insult, which may inhibit NMDA receptor-mediated neurotoxicity through an indirect pathway, but has no effect on OGD-or NMDA-induced immediate injury in hippocampal slices.

Barbiturates Induce Mitochondrial Depolarization and Potentiate Excitotoxic Neuronal Death

Barbiturates are widely used as anesthetics, anticonvulsants, and neuroprotective agents. However, barbiturates may also inhibit mitochondrial respiration, and mitochondrial inhibitors are known to potentiate NMDA receptor-mediated neurotoxicity. Here we used rat cortical cultures to examine the effect of barbiturates on neuronal mitochondria and responses to NMDA receptor stimulation. The barbiturates tested, secobarbital, amobarbital, and thiamylal, each potentiated NMDA-induced neuron death at barbiturate concentrations relevant to clinical and experimental use (100-300 microm). By using rhodamine-123 under quenching conditions, barbiturates in this concentration range were shown to depolarize neuronal mitochondria and greatly amplify NMDA-induced mitochondrial depolarization. Barbiturate-induced mitochondrial depolarization was increased by the ATP synthase inhibitor oligomycin, indicating that barbiturates act by inhibiting electron transport sufficiently to cause ATP synthase reversal. Barbiturates similarly amplified the effects of NMDA on cytoplasmic free calcium concentrations. The cell-impermeant barbiturate N-glucoside amobarbital did not influence mitochondrial potential or potentiate NMDA neurotoxicity or calcium responses. However, all of the barbiturates attenuated NMDA-induced calcium elevations and cell death when present at millimolar concentrations. Whole-cell patch-clamp studies showed that these effects may be attributable to actions at the cell membrane, resulting in a block of NMDA-induced current flux at millimolar barbiturate concentrations. Together, these findings reconcile previous reports of opposing effects on barbiturates on NMDA neurotoxicity and show that barbiturate effects on neuronal mitochondria can be functionally significant. Effects of barbiturates on neuronal mitochondria should be considered in experimental and clinical application of these drugs.

Excitotoxicity of quinolinic acid: modulation by endogenous antagonists.

Quinolinic acid (QUIN), a product of tryptophan metabolism by the kynurenine pathway, produces excitotoxicity by activation of NMDA receptors. Focal injections of QUIN can deplete the biochemical markers for dopaminergic, cholinergic, gabaergic, enkephalinergic and NADPH diaphorase neurons, which differ in their sensitivity to its neurotoxic action. This effect of QUIN differs from that of other NMDA receptor agonists in terms of its dependency on the afferent glutamatergic input and its sensitivity to the receptor antagonists. The enzymatic pathway yielding QUIN produces metabolites that inhibit QUIN-induced neurotoxicity. The most active of these metabolites, kynurenic acid (KYNA), blocks NMDA and non-NMDA receptor activity. Treatment with kynurenine hydroxylase and kynureinase inhibitors increases levels of endogenous KYNA in the brain and protects against QUIN-induced neurotoxicity. Other neuroprotective strategies involve reduction in QUIN synthesis from its immediate precursor, or endogenous synthesis of 7-chloro-kynurenic acid, a NMDA antagonist, from its halogenated precursor. Several other tryptophan metabolites--quinaldic acid, hydroxyquinaldic acid and picolinic acid--also inhibit excitotoxic damage but their presence in the brain is uncertain. Picolinic acid is of interest since it inhibits excitotoxic but not neuroexcitatory responses. The mechanism of its anti-excitotoxic action is unclear but might involve zinc chelation. Neurotoxic actions of QUIN are modulated by nitric oxide (NO). Treatment with inhibitors of NO synthase can augment QUIN toxicity in some models of excitotoxicity suggesting a neuroprotective potential of endogenous NO. In recent studies, certain nitroso compounds which could be NO donors, have been reported to reduce the NMDA receptor-mediated neurotoxicity. The existence of endogenous compounds which inhibit excitotoxicity provides a basis for future development of novel and effective neuroprotectants.

Enhancement of NMDA-induced functional responses without concomitant NMDA receptor changes following chronic ethanol exposure in cerebellar granule cells.

Primary cultures of rat cerebellar granule cells were used to investigate the effects of chronic ethanol exposure (50-100 mM for 3 days) on NMDA receptor functions (Ca2+ fluxes and neurotoxicity), binding parameters of the non-competitive NMDA receptor antagonist [3H]MK-801, relative abundance of mRNAs coding for NMDA receptor subunits, and expression of NMDA receptor subunit proteins. Ethanol exposure caused a marked increase in NMDA-produced neurotoxicity but produced a differential pattern of effects on NMDA-induced Ca2+ fluxes with a marked enhancement of NMDA-stimulated free cytoplasmic Ca2+ concentrations ([Ca2+]i), but no changes in NMDA-induced 45Ca2+ uptake. As shown by [3H]MK-801 binding experiments, chronic ethanol had no effect on affinity or number of the NMDA receptors. Furthermore, ethanol exposure had no effect on the relative abundance of the mRNAs for any of the NMDA receptor subunits (four splice variants of NR1, or NR2A-C), or on the expression of NMDA receptor subunit proteins. Our data confirm previous observations that chronic ethanol exposure enhances NMDA receptor-mediated neurotoxicity and elevation of [Ca2+]i, but also suggest that the increased responsiveness of NMDA receptors is not necessarily associated with alterations in the subunit composition or the ligand binding properties of NMDA receptors.

Antillatoxin and kalkitoxin, ichthyotoxins from the tropical cyanobacterium Lyngbya majuscula, induce distinct temporal patterns of NMDA receptor-mediated neurotoxicity

Curacin-A, antillatoxin and kalkitoxin, natural products from the marine cyanobacterium Lyngbya majuscula, were tested for neurotoxicity in primary cultures of rat cerebellar granule neurons. Curacin-A was non-toxic, whereas antillatoxin and kalkitoxin produced concentration-dependent cytotoxicity with LC 50 values of 20.1±6.4 and 3.86±1.91 nM, respectively. Antillatoxin neurotoxicity was produced acutely, whereas kalkitoxin caused a delayed neurotoxic response. The cytotoxicity produced by both antillatoxin and kalkitoxin was prevented by the non-competitive NMDA receptor antagonists dextrorphan and MK-801.

Analysis of the neuroprotective effects of various nitric oxide donor compounds in murine mixed cortical cell culture.

Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor-mediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate-type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S-nitrosothiols S-nitroso-L-glutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (NO+ equivalents), decreased NMDA-induced neuronal injury in a concentration-dependent manner. 8-Bromo-cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO-stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H2O2 was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect. NONOates did, however, reduce NMDA-stimulated uptake of 45Ca2+, whereas high potassium-induced 45Ca2+ accumulation, a measurement of entry via voltage-gated calcium channels, was unaffected. The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO-sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5-min time frame. Determination of the formation of NO+ equivalents, as assessed by N-nitrosation of 2,3-diaminonaphthylene, revealed little or no observable N-nitrosation by Sper/NO, GSNO, and SNAP with significant N-nitrosation observed by Papa/NO and Dea/NO. However, addition of ascorbate (400 microM) effectively prevented the nitrosation of 2,3-diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on 45Ca2+ accumulation. Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor-mediated neurotoxicity at the cellular level.

Subtype-specific enhancement of NMDA receptor currents by mutant huntingtin.

Evidence suggests that NMDA receptor-mediated neurotoxicity plays a role in the selective neurodegeneration underlying Huntington's disease (HD). The gene mutation that causes HD encodes an expanded polyglutamine tract of >35 in huntingtin, a protein of unknown function. Both huntingtin and NMDA receptors interact with cytoskeletal proteins, and, for NMDA receptors, such interactions regulate surface expression and channel activity. To determine whether mutant huntingtin alters NMDA receptor expression or function, we coexpressed mutant or normal huntingtin, containing 138 or 15 glutamine repeats, respectively, with NMDA receptors in a cell line and then assessed receptor channel function by patch-clamp recording and surface expression by western blot analysis. It is interesting that receptors composed of NR1 and NR2B subunits exhibited significantly larger currents when coexpressed with mutant compared with normal huntingtin. Moreover, this effect was selective for NR1/NR2B, as NR1/NR2A showed similar currents when coexpressed with mutant versus normal huntingtin. However, ion channel properties and total surface expression of the NR1 subunit were unchanged in cells cotransfected with NR1/NR2B and mutant huntingtin. Our results suggest that mutant huntingtin may increase numbers of functional NR1/NR2B-type receptors at the cell surface. Because NR1/NR2B is the predominant NMDA receptor subtype expressed in medium spiny neostriatal neurons, our findings may help explain the selective vulnerability of these neurons in HD.

Extracellular Acidity Potentiates AMPA Receptor-Mediated Cortical Neuronal Death

The extracellular acidity that accompanies brain hypoxia-ischemia is known to reduce both NMDA and AMPA-kainate receptor-mediated currents and NMDA receptor-mediated neurotoxicity. Although a protective effect of acidic pH on AMPA-kainate receptor-mediated excitotoxicity has been assumed, such has not been demonstrated. Paradoxically, we found that lowering extracellular pH selectively increased AMPA-kainate receptor-mediated neurotoxicity in neocortical cell cultures, despite reducing peak elevations in intracellular free Ca2+. This injury potentiation may, at least in part, be related to a slowed recovery of intracellular Ca2+ homeostasis, observed after AMPA-kainate receptor activation, but not after NMDA receptor activation or exposure to high K+. The ability of acidic pH to selectively augment AMPA-kainate receptor-mediated excitotoxicity may contribute to the prominent role that these receptors play in selective neuronal death after transient global ischemia.

Exposure to gp120 of HIV-1 induces an increased release of arachidonic acid in rat primary neuronal cell culture followed by NMDA receptor-mediated neurotoxicity.

After incubation of highly enriched neurons from rat cerebral cortex with the HIV-1 coat protein gp120 for 18 h, cells showed fragmentation of DNA at internucleosomal linkers followed by NMDA receptor-mediated neurotoxicity. We report that in response to exposure to gp120 cells react with an increased release of arachidonic acid (AA) via activation of phospholipase A2. This process was not inhibited by NMDA receptor antagonists. To investigate the role of AA on the sensitivity of the NMDA receptor towards its agonist, low concentrations of NMDA were co-administered with AA. This condition enhanced the NMDA-mediated cytotoxicity. Administration of mepacrine reduced cytotoxicity caused by gp120. We conclude that gp120 causes an activation of phospholipase A2, resulting in the increased release of AA, which may in turn sensitize the NMDA receptor.

Comparison of the Potency of Competitive NMDA Antagonists Against the Neurotoxicity of Glutamate and NMDA

The object of this investigation was to determine whether glutamate uptake affects the apparent potency of the competitive antagonists DL-2-amino-5-phosphonovalerate and CGS-19755 in blocking NMDA receptor-mediated neurotoxicity. In astrocyte-rich rat cortical cultures we observed that DL-2-amino-5-phosphonovalerate and CGS-19755 were 24 and 16 times more potent against NMDA than against glutamate-induced toxicity. In contrast, DL-2-amino-5-phosphonovalerate was equipotent against the two agonists in astrocyte-poor cultures, in which dendrites are directly exposed to the extracellular medium. With the noncompetitive NMDA antagonist MK-801, similar potencies were observed against glutamate (212 +/- 16 nM) and against NMDA (155 +/- 9 nM) neurotoxicity. These results may be explained if we assume that the neuronal cell body is less susceptible than the dendrites to NMDA receptor-mediated toxicity, and that the action of glutamate in astrocyte-rich cultures is confined to the cell body. In this case, one would expect that higher concentrations of glutamate would be needed to produce toxicity in astrocyte-rich cultures, and that higher concentrations of competitive antagonists would be needed to overcome this toxicity. Our observations help explain the pharmacology of the competitive NMDA antagonists against NMDA receptor-mediated neurotoxicity but also suggest the possibility that, because the cell body and dendrites may be distinct sites for neurotoxicity, they might also involve different mechanisms of toxicity.

AMPA receptor activation potentiates zinc neurotoxicity

Extracellular Zn 2+ attenuates NMDA receptor-mediated neurotoxicity and increases AMPA receptor-mediated toxicity. Known electrophysiological effects of Zn 2+ predict only the former. We considered the possibility that the latter rather reflects AMPA potentiation of Zn 2+ toxicity, perhaps mediated by neuronal depolarization and Zn 2+ entry through voltage-gated Ca 2+ channels. High K + or kainate also potentiated Zn 2+ toxicity, and AMPA plus Zn 2+ toxicity was attenuated by raising extracellular Ca 2+, or by Ca 2+ channel blockers. AMPA plus Zn 2+ exposure induced an increase in fluorescence from neurons loaded with the Zn 2+-sensitive dye TS-Q and increased subsequent 45Ca 2+ accumulation. The ability of AMPA receptor activation to potentiate Zn 2+ toxicity may be relevant to neuronal death associated with intense activation of glutamatergic pathways.

Blockade of NMDA receptor-mediated mobilization of intracellular Ca 2+ prevents neurotoxicity

NMDA receptor activation leads to elevated Ca 2+ in cultured rat cortical and retinal ganglion cell neurons. If excessive, this Ca 2+ response in associated with delayed neurotoxicity. We used dantrolene and ionomycin to test if the Ca 2+ response to NMDA was due to mobilization of intracellular Ca 2+ stores rather than merely to Ca 2+ influx. In the presence of EGTA, ionomycin resulted in release and subsequent depletion of intracellular Ca 2+ stores. Henceforth, despite normal extracellular Ca 2+, NMDA elicited only about half of its former Ca 2+ response. Similarly, when dantrolene was used to block Ca 2+ release from intracellular stores, we observed > 50% smaller NMDA-evoked Ca 2+ responses. These results quite surprisingly indicate that at least half the Ca 2+ response to NMDA is due to release of intracellular Ca 2+, a process triggered by influx of extracellular Ca 2+. Dantrolene also protected neurons from NMDA receptor-mediated neurotoxicity. Release of intracellular Ca 2+ may therefore be a necessary step in the cascade leading to neuronal damage induced by excessive NMDA receptor stimulation and may be amenable to pharmacological intervention.

The effect of peripheral loading with kynurenine and probenecid on extracellular striatal kynurenic acid concentrations.

Kynurenic acid (KYA) is the only known endogenous excitatory amino acid antagonist in mammalian brain. In the present study we examined the effects of precursor loading with kynurenine (KYN) and blockade of organic acid transport with probenecid, either alone or in combination, on extracellular striatal KYA concentrations in unanesthetized rats. Baseline KYA concentrations were 1.61 +/- 0.29 pmol/ml. Following administration of KYN 150 mg/kg with increasing doses of probenecid a maximal increase in KYA to 946 +/- 210 pmol/ml was seen with probenecid 200 mg/kg. Probenecid 200 mg/kg alone increased KYA levels to 16.0 +/- 5.2 pmol/ml. The combination of probenecid 200 mg/kg with KYN 450 mg/kg produced a maximal increase of KYA to 2085 +/- 391 pmol/ml, a 1300-fold increase indicating marked potentiation. These results show that pharmacologic manipulation can markedly increase extracellular fluid concentrations of KYA into a range which may be useful in attempts to block NMDA receptor-mediated neurotoxicity.

Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex

Nitric oxide (NO) is an important messenger both systemically and in the CNS. In digital Ca2+ imaging and patch-clamp experiments, clinically available nitroso compounds that generate NO are shown to inhibit responses mediated by the NMDA subtype of the glutamate receptor on rat cortical neurons in vitro. A mechanism of action for this effect was investigated by using the specific NO-generating agent S-nitrosocysteine. We propose that free sulfhydryl groups on the NMDA receptor-channel complex react to form one or more S-nitrosothiols in the presence of NO. If vicinal thiol groups react in this manner, they can form a disulfide bond(s), which is thought to constitute the redox modulatory site of the receptor, resulting in a relatively persistent blockade of NMDA responses. These reactions with NO can afford protection from NMDA receptor-mediated neurotoxicity. Our results demonstrate a new pathway for NO regulation of physiological function that is not via cGMP, but instead involves reactions with membrane-bound thiol groups on the NMDA receptor-channel complex.

Synergistic effects of HIV coat protein and NMDA receptor-mediated neurotoxicity

Exposure of rat retinal cultures to HIV-1 coat protein gp120 for several minutes increases [Ca 2+]i in approximately half of the ganglion cells; this effect is associated with delayed-onset neuronal injury, similar to that previously reported in NMDA receptor-mediated neurotoxicity. Here we show that NMDA antagonists can prevent both the rise in [Ca2+]; and subsequent neuronal damage engendered by 20 pM gp120. However, whole-cell patch-clamp recordings demonstrate that gp 120 does not directly evoke an NMDA-like response or enhance glutamate/NMDA-activated currents. Moreover, complete protection from gp120-induced [Ca 2+]i increases and neurotoxicity is afforded by incubation with glutamate-pyruvate transaminase, which breaks down endogenous glutamate as verified by HPLC. Since, under standard conditions in these cultures, neither glutamate nor a low picomolar concentration of gp120 is deleterious on its own, our results suggest that their neurotoxicity is synergistic.

Enhancement of NMDA receptor-mediated neurotoxicity in the hippocampal slice by depolarization and ischemia

Evidence from animal stroke models suggests that the proximate cause of neuronal degeneration after ischemia is massive release of glutamate and activation of NMDA receptors. However, in the physiologic presence of oxygen and glucose in the rat hippocampal slice preparation, the neurotoxicity of glutamate, as measured by inhibition of protein synthesis, requires high concentrations and is not prevented by glutamate receptor antagonists. Thus, the NMDA receptor-mediated neurotoxic effects of extracellular glutamate accumulation during ischemia might depend on additional factors, such as neuronal depolarization. In the experiments reported here, slices were exposed to glutamate in a medium intended to mimic the ionic conditions found during ischemia, high potassium (128 mM) and low sodium (26 mM). This depolarizing medium itself inhibited protein synthesis in a manner which was partially mediated by NMDA receptor activation, since it was significantly reversed by the noncompetitive NMDA antagonist, MK-801. Furthermore, the effect of glutamate under depolarizing conditions was also significantly decreased by MK-801, suggesting that glutamate was acting at NMDA receptors. Thus, depolarization appears to enhance the sensitivity of neurons to toxic NMDA receptor activation by glutamate. Under conditions that mimic ischemia, hypoxia plus hypoglycemia, a similar protective effect of NMDA receptor antagonists was observed. Depolarization and ischemia both appeared to attenuate the neurotoxicity of non-NMDA receptor agonists. It appears that under conditions of normal glucose and oxygen, high concentrations of bath applied glutamate inhibit protein synthesis at sites other than the NMDA receptor. However, when the Na + gradient is decreased, as occurs during ischemia, glutamate's NMDA effects predominate. These findings suggest that ionic shifts may play a central role in permitting NMDA receptor-mediated ischemic neuronal damage.