PARP cleavage, DNA fragmentation, and pyknosis during excitotoxin-induced neuronal death

Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear enzyme activated by DNA breaks and serves a role in DNA repair through the formation of polymers (poly(ADP)ribosylation) at sites of DNA damage. PARP-1 is activated by DNA damage in neurons of the hippocampus and cerebral cortex following excessive exposure to glutamate receptor agonists such as NMDA or kainic acid. In addition, recent studies suggest that degradation of PARP-1 occurs in cells that undergo apoptotic versus nonapoptotic forms of cell death. To investigate this process further, we examined the spatiotemporal aspects of excitotoxic injury in the rodent visual cortex by making focal intracerebral injections of kainic acid. These injections resulted in DNA damage, PARP-1 activation, and neuronal cell death over a 5-day period. Rapid neuronal cell injury assessed by Fluoro-Jade staining appeared within hours, but increased TUNEL staining occurred only after 24 h. A dramatic increase in caspase-3 activity, as well as an increase in the number of neurons containing active caspase-3, peaked 2 days after injury. Last, increased PARP-1 immunoreactivity and PARP-1 cleavage reached peak levels 2 to 3 days after delivering the excitotoxin. These findings suggest that increased caspase-3 activity may regulate the degradation of PARP-1 in subsets of cortical neurons during excitotoxic cell death.     

N-Methyl-D-aspartate receptor blockade induces neuronal apoptosis in cortical culture.

Whereas excessive activation of the NMDA receptor may contribute to ischemic neuronal injury, physiologic activation may promote neuronal survival under certain conditions. Consistently, it has recently been shown that NMDA antagonists induce apoptosis of central neurons in immature rats. In the present study, we have examined whether NMDA antagonists induce neuronal apoptosis also in a culture condition. Exposure of cortical cultures (DIV 10-13) to MK-801 (1-10 microM) for 48 h resulted in death of about 30-40% of neurons. Similar neuronal death was induced by exposure to other NMDA antagonists, D-AP5 and dextromethorphan. The neuronal death was dependent on the culture age; MK-801 induced much less neuronal death in younger (DIV 7) and older (DIV 16-19) cultures. The NMDA antagonist-induced neuronal death was accompanied by cell body shrinkage, nuclear fragmentation, and cleavage/activation of caspase-3. Furthermore, it was attenuated by cycloheximide and zVAD-fmk, indicating that the death occurred mainly by the apoptosis mechanism. As in several other apoptosis models, high-potassium medium blocked the NMDA antagonist-induced apoptosis, which was reversed by voltage-gated calcium channel blockers. The present results demonstrate that NMDA antagonists induce neuronal apoptosis in cortical culture, consistent with the findings obtained in immature rats. Since the activation of the voltage-gated calcium channels attenuated the NMDA antagonist-induced apoptosis, it may be another example of the "calcium set point hypothesis." Copyright 1999 Academic Press.     

Neuronal trauma model: in search of Thanatos

Trauma to the nervous system triggers responses that include oxidative stress due to the generation of reactive oxygen species (ROS). DNA is a major macromolecular target of ROS, and ROS-induced DNA strand breaks activate poly(ADP-ribose)polymerase-1 (PARP-1). Upon activation PARP-1 uses NAD(+) as a substrate to catalyze the transfer of ADP-ribose subunits to a host of nuclear proteins. In the face of extensive DNA strand breaks, PARP-1 activation can lead to depletion of intracellular NAD(P)(H) pools, large decreases in ATP, that threaten cell survival. Accordingly, inhibition of PARP-1 activity after acute oxidative injury has been shown to increase cell survival. When NGF-differentiated PC12 cells, an in vitro neuronal model, are exposed to H(2)O(2) there is increased synthesis of poly ADP-ribose and decreases in intracellular NAD(P)(H) and ATP. Addition of the chemical PARP inhibitor 3-aminobenzamide (AB) prior to H(2)O(2) exposure blocks the synthesis of poly ADP-ribose and maintains intracellular NAD(P)(H) and ATP levels. H(2)O(2) injury is characterized by an immediate, necrotic cell death 2h after injury and a delayed apoptotic-like death 12-24h after injury. This apoptotic-like death is characterized by apoptotic membrane changes and apoptotic DNA fragmentation but is not associated with measurable caspase-3 activity. AB delays cell death beyond 24h and increases cell survival by approximately 25%. This protective effect is accompanied by significantly decreased necrosis and the apoptotic-like death associated with H(2)O(2) exposure. AB also restores caspase-3 which can be attributed to the activation of the upstream activator of caspase-3, caspase-9. Thus, the maintenance of intracellular ATP levels associated with PARP-1 inhibition shifts cell death from necrosis to apoptosis and from apoptosis to cell survival. Furthermore, the shift from necrosis to apoptosis may be explained, in part, by an energy-dependent activation of caspase-9.     

Diphosphorylation and involvement of extracellular signal-regulated kinases (ERK1/2) in glutamate-induced apoptotic-like death in cultured rat cortical neurons

Glutamate-induced excitotoxicity, with certain characteristics of apoptosis, has been implicated in a variety of neuronal degenerative disorders. In some physiological cases, extracellular signal-regulated kinases (ERK1/2) are activated by stimulation of glutamate receptors. In the present study, the activation (diphosphorylation) and role of ERK1/2 in glutamate-induced apoptotic-like death in cultured cortical neurons were investigated. Protein levels and activation (diphosphorylation) levels of ERK1/2 were examined by Western immunoblot, probed with anti-ERK1/2 and anti-active (diphosphorylated) ERK1/2 antibodies, respectively. Apoptotic-like death was determined by DAPI staining. Before a remarkable increase of apoptotic-like cell death was observed at 9-18 h after 15 min exposure to 50 microM glutamate, diphosphorylation levels of ERK1/2 were rapidly increased, peaked at 5-15 min of the exposure, and reverted to sham control level 3 h after the exposure, while the protein levels of ERK1/2 were unaffected. The glutamate concentration effective for inducing apoptotic-like cell death was correlated with that for inducing ERK1/2 diphosphorylation. Both ERK1/2 diphosphorylation and the apoptotic-like cell death were largely prevented by MK-801, a specific NMDA receptor (a subtype receptor of glutamate) antagonist, or the elimination of extracellular Ca(2+) with EGTA. PD98059, a specific inhibitor of ERK1/2 kinase, completely inhibited ERK1/2 diphosphorylation and partially inhibited the apoptotic-like cell death. These results suggest that largely via NMDA receptor-mediated influx of extracellular Ca(2+), ERK1/2 were rapidly and transiently activated and were involved in glutamate-induced apoptotic-like death in cultured rat cortical neurons.     

Calcium dysregulation induces apoptosis-inducing factor release: Cross-talk between PARP-1- and calpain- signaling pathways

Recent discoveries show that caspase-independent cell death pathways are a pervasive mechanism in neurodegenerative diseases, and apoptosis-inducing factor (AIF) is an important effector of this mode of neuronal death. There are currently two known mechanisms underlying AIF release following excitotoxic stress, PARP-1 and calpain. To test whether there is an interaction between PARP-1 and calpain in triggering AIF release, we used the NMDA toxicity model in rat primary cortical neurons. Exposure to NMDA resulted in AIF truncation and nuclear translocation, and shRNA-mediated knockdown of AIF resulted in neuroprotection. Both calpain and PARP-1 are involved with AIF processing as AIF truncation, nuclear translocation and neuronal death were attenuated by calpain inhibition using adeno-associated virus-mediated overexpression of the endogenous calpain inhibitor, calpastatin, or treatment with the PARP-1 inhibitor 3-ABA. Activation of PARP-1 is necessary for calpain activation as PARP-1 inhibition blocked mitochondrial calpain activation. Finally, NMDA toxicity induces mitochondrial Ca²⁺ dysregulation in a PARP-1 dependent manner. Thus, PARP-1 and mitochondrial calpain activation are linked via PARP-1-induced alterations in mitochondrial Ca²⁺ homeostasis. Collectively, these findings link the two seemingly independent mechanisms triggering AIF-induced neuronal death.     

7-Chlorokynurenate Blocks NMDA Receptor-Mediated Neurotoxicity in Murine Cortical Culture

We examined the neuroprotective actions of the glycine site N-methyl-D-aspartate (NMDA) antagonist, 7-chlorokynurenate, in murine neocortical cell cultures. Cultures exposed for 5 min to 100 - 500 microM NMDA in the absence of added glycine developed substantial neuronal degeneration over the next 24 h. The addition of 10 microM glycine did not increase submaximal NMDA-induced neuronal injury, suggesting that endogenous glycine levels were sufficient to saturate its receptor sites on NMDA receptor complexes. Addition of 3 - 300 microM 7-chlorokynurenate produced concentration-dependent reduction in this neuronal damage with an IC50 of approximately 30 microM. Some injury reduction was seen even if the drug was added after completion of the NMDA exposure. The protective effect of 100 microM 7-chlorokynurenate could be overcome by adding 10 - 1000 microM glycine (glycine median effective concentration (EC50) approximately 100 microM) or 1 mM D-serine. As predicted by its ability to block NMDA receptor-mediated injury, 10 - 300 microM 7-chlorokynurenate also produced concentration-dependent reduction in the neuronal loss induced by 50 - 60 min exposure to combined glucose and oxygen deprivation. These data support the suggestion that pharmacologic interference with the binding of glycine to the NMDA receptor complex represents a potentially effective approach to blocking NMDA receptor-induced neurotoxicity in ischemia.     

TrkB mediates BDNF-induced potentiation of neuronal necrosis in cortical culture

In the present study, the signaling mechanisms underlying the effect of brain-derived neurotrophic factor (BDNF) on neuronal necrosis were investigated. Exposure of mature mouse cortical cultures (more than 10 days in vitro (DIV)) to 50-100 ng/ml BDNF for 48 h induced widespread neuronal necrosis that was antioxidant-sensitive. This neuronal necrosis was blocked by the selective NMDA antagonist MK-801, suggesting that prolonged BDNF exposure caused endogenous levels of NMDA receptor activation to become excitotoxic. We examined whether the p75(NTR) played a role in BDNF-induced neuronal death. However, p75(NTR) expression was low in cultured cortical cells, and neutralizing antibodies to p75(NTR) did not attenuate BDNF-triggered neuronal death. In contrast, trkB antisense oligonucleotides and inhibitors of Trk tyrosine kinase blocked BDNF-triggered neuronal death as well as BDNF potentiation of iron-induced oxidative neuronal necrosis, suggesting a critical role for TrkB in this phenomenon. Furthermore, BDNF did not potentiate neuronal necrosis in cortical cultures prepared from embryonic TrkB-null mice. These results suggest that TrkB plays an important role in BDNF-mediated neuronal necrosis.     

The role of NADPH oxidase and neuronal nitric oxide synthase in zinc-induced poly(ADP-ribose) polymerase activation and cell death in cortical culture

In the present study, we examined the role and the mechanism of poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) activation in zinc-induced cell death in cortical culture. After brief exposure to 400 microM zinc, cortical cells exhibited DNA fragmentation, increased poly(ADP-ribosyl)ation, and decreased levels of nicotinamide adenine dinucleotide (NAD) and ATP and subsequently underwent cell death. Inhibitors of PARP/PARG attenuated both zinc-induced NAD/ATP depletion and cell death, thereby implicating the PARP/PARG cascade in these processes. The zinc-inducible enzymes NADPH oxidase and neuronal nitric oxide synthase (nNOS) contributed to PARP activation as their inhibitors attenuated zinc-induced poly(ADP-ribosyl)ation. Levels of nitric oxide and nitrites increased following zinc exposure, consistent with NOS activation. In addition, Western blots and RT-PCR analysis revealed that protein and mRNA levels of nNOS specifically increased following zinc exposure in a manner similar to that of NADPH oxidase. The present study demonstrates that induction of NADPH oxidase and nNOS actively contributes to PARP/PARG-mediated NAD/ATP depletion and cell death induced by zinc in cortical culture.     

Ischemic tolerance in murine cortical cell culture: critical role for NMDA receptors.

Murine cortical cultures containing both neurons and glia (days in vitro 13-15) were exposed to periods of oxygen-glucose deprivation (5-30 min) too brief to induce neuronal death. Cultures "preconditioned" by sublethal oxygen-glucose deprivation exhibited 30-50% less neuronal death than controls when exposed to a 45-55 min period of oxygen-glucose deprivation 24 hr later. This preconditioning-induced neuroprotection was specific in that neuronal death induced by exposure to excitotoxins or to staurosporine was not attenuated. Neuroprotection was lost if the time between the preconditioning and severe insult were decreased to 7 hr or increased to 72 hr and was blocked if the NMDA antagonist 100 microM 3-((D)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid was applied during the preconditioning insult. This was true even if the duration of preconditioning was increased as far as possible (while still remaining sublethal). A similar preconditioning effect was also produced by sublethal exposure to high K+, glutamate, or NMDA but not to kainate or trans-1-aminocyclopentane-1, 3-dicarboxylic acid.     

Adenine nucleotide metabolism and cell fate after oxidant exposure of rat cortical neurons: effects of inhibition of poly(ADP-ribose) polymerase

We exposed cultured neurons prelabeled with 14C-adenine to H2O2 with or without the poly(ADP-ribose) polymerase (PARP) inhibitor 3,4-Dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) to quantify its effects on acute ATP depletion, later ATP synthesis, cellular and nuclear morphology, extent of DNA fragmentation, and PARP cleavage. According to the extent of the acute ATP depletion, the exposures were classified as 'mild' (50 microM H2O2), 'moderate' (100-250 microM H2O2), or 'severe' (500 microM-1 mM H2O2) insults. Mild exposure had no significant effects on the parameters studied. In the 'moderately' exposed neurons, ATP depletion to 59+/-6% of control was associated with a decrease in the cell counts, apoptotic morphology, and cleavage of PARP. In this group, DPQ prevented the acute ATP (to 95+/-15% of control), preserved cell morphology, and improved cell survival. In the 'severe' group, ATP depletion to 18+/-4% was associated with necrosis and intact PARP. DPQ elevated ATP levels (to 44+/-12% of control) and post-insult ATP synthesis, improved cell counts, and altered cell morphology towards apoptosis rather than necrosis. Post-insult application of DPQ was less effective. Our results show that the extent of oxidant-induced ATP depletion and cell fate can be modified by PARP inhibition, to some extent also after the insult.     

Involvement of poly ADP ribosyl polymerase-1 in acute but not chronic zinc toxicity

We have previously suggested that zinc-induced neuronal death may be mediated in part by inhibition of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), secondary to depletion of the essential cosubstrate NAD+. Given convergent evidence implicating the NAD+-catabolizing enzyme, poly ADP ribosyl polymerase (PARP) in mediating ATP depletion and neuronal death after excitotoxic and ischemic insults, we tested the specific hypothesis that the neuronal death induced by exposure to toxic levels of extracellular zinc might be partly mediated by PARP. PARP was activated in cultured mouse cortical astrocytes after a toxic acute Zn2+ exposure (350 microm Zn2+ for 15 min), but not in cortical neurons or glia after exposure to a toxic chronic Zn2+ exposure (40 microm Zn2+ for 1-4 h), an exposure sufficient to deplete NAD+ and ATP levels. Furthermore, the neurotoxicity induced by acute, but not chronic, Zn2+ exposure was reduced in mixed neuronal-glial cultures prepared from mutant mice lacking the PARP gene. These data suggest PARP activation may contribute to more fulminant forms of Zn2+-induced neuronal death.     

Routes of zinc entry in mouse cortical neurons: role in zinc-induced neurotoxicity

Exposure of central neurons to Zn2+ triggers neuronal death. The routes of Zn2+ entry were investigated in living cortical neurons from the mouse using the specific Zn2+ fluorescent dye N-(6-methoxy-8-quinolyl)-p-toluene sulphonamide (TSQ), which preferentially detects membrane-bound Zn2+. Exposure of cortical neurons to increasing concentrations of Zn2+ (1-100 microM) induced a progressive increase in the fluorescence of TSQ. This fluorescence signal was not attenuated by the permeation of plasma membrane with digitonin. Accordingly, the major part of TSQ fluorescence (two-thirds) was associated to the particulate fraction of cortical neurons exposed to Zn2+. These results suggest that Zn2+ detected with TSQ in neurons is mainly bound to membranes. TSQ fluorescence measured in neurons exposed to 3 microM Zn2+ was enhanced by Na+-pyrithione, a Zn2+ ionophore, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate (NMDA) or KCl-induced depolarization. However, in the absence of any treatment, TSQ labelling of neurons exposed to 3 microM Zn2+ was only decreased by NMDA receptor antagonists, whereas it remained unaltered in the presence of antagonists of AMPA receptors or L-type voltage-gated Ca2+ channels. Zn2+ entry through NMDA receptors did not contribute to Zn2+-induced neuronal death, as it was prevented by antagonists of NMDA receptors only when they were added after the Zn2+ exposure. Finally, Zn2+ induced a delayed accumulation of extracellular glutamate which might be responsible for the delayed NMDA receptor activation that leads to neuronal death.     

NMDA receptor 2B-selective antagonist ifenprodil-induced apoptosis was prevented by glycogen synthase kinase-3 inhibitors in cultured rat cortical neurons

The N-methyl-d-aspartate (NMDA) receptor 2B-selective antagonist ifenprodil induced morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation in rat cultured cortical cells. Ifenprodil increased the apoptotic cell death in a dose-dependent manner (0.5-10 microM). In addition, the protein synthesis inhibitor cycloheximide completely blocked ifenprodil-induced apoptotic cell death. The selective inhibitors of glycogen synthase kinase-3 (GSK-3) prevented the ifenprodil-induced apoptosis. Moreover, activation of caspase-3 was accompanied by cell death induced by ifenprodil in a dose-dependent manner. The ifenprodil-induced apoptosis was prevented by a caspase-3 inhibitor. These results suggested that activation of GSK-3 involves in the apoptosis induced by blocking of trophic effect of NMDA receptor consisting of NR2B subunit in rat cortical neurons.     

NMDA receptor activation enhances diazepam binding inhibitor and its mRNA expressions in mouse cerebral cortical neurons

Effects of N-methyl-D-aspartate (NMDA) on diazepam binding inhibitor (DBI) and its mRNA expression in mouse cerebral cortical neurons were examined. A significant increase in DBI mRNA expression was observed 1 day after the exposure to 0.1 microM NMDA and the maximal expression occurred 2 days after the exposure, whereas transient exposure to 0.1 microM NMDA for 15 min, 1 and 3 h produced no changes in the expression. Similarly, no changes in the expression were found by the concomitant exposure to NMDA and MK-801, a NMDA receptor antagonist, for 72 h subsequent to the incubation with NMDA alone for 3 h. Such NMDA-induced increases in DBI mRNA expression were dose-dependently inhibited by MK-801. Moreover, neuronal DBI content significantly increased by treatment with NMDA, which was completely abolished by MK-801. These results indicate that continuous activation of NMDA receptors is an essential factor for increasing DBI expression in the neurons.     

Involvement of independent mechanism upon poly(ADP-ribose) polymerase (PARP) activation in methylmercury cytotoxicity in rat cerebellar granule cell culture

Poly(ADP-ribose) polymerase (PARP) activation plays a role in repairing injured DNA, while its overactivation is involved in various diseases, including neuronal degradation. In the present study, we investigated the use of a PARP inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), whether methylmercury-induced cell death in the primary culture of cerebellar granule cells involved PARP activation. DPQ decreased the methylmercury-induced cell death in a dose-dependent manner. Unexpectedly, this protective effect was DPQ specific; none of the other PARP inhibitors--1,5-dihydroxyisoquinoline, 3-aminobenzamide, or PJ34--affected neuronal cell death. Methylmercury-induced cell death involves the decrease of glutathione (GSH) and production of reactive oxygen species. Therefore, to understand the mechanism by which DPQ inhibits cytotoxicity, we first studied the effect of DPQ on buthionine sulfoximine- or diethyl maleate-induced death of primary cultured cells and human neuroblastoma IMR-32 cells, both of which are mediated by GSH depletion. DPQ inhibited the cell death of both cultured cells, but it did not restore the decrease of cellular GSH by buthionine sulfoximine to the control level. Second, we evaluated the antioxidant activity of PARP inhibitors by methods with ABTS (2-2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) or DPPH (1,1-diphenyl-2-picrylhydrazyl) used as a radical because antioxidants also efficiently suppress methylmercury-induced cell death. The antioxidant activity of DPQ was the lowest among the tested PARP inhibitors. Taken together, our results indicate that DPQ effectively protects cells against methylmercury- and GSH depletion-induced death. Furthermore, they suggest that DPQ exerts its protective effect through a mechanism other than PARP inhibition and direct antioxidation, and that PARP activation is not involved in methylmercury-induced neuronal cell death.     

Enhanced poly(ADP-ribose) polymerase-1 activation contributes to recombinant tissue plasminogen activator-induced aggravation of ischemic brain injury in vivo

Recombinant tissue plasminogen activator (rt-PA) treatment improves functional outcome after acute ischemic stroke, inducing reperfusion by its thrombolytic activity. Conversely, there is evidence that rt-PA can mediate neuronal damage after ischemic brain injury in vivo. In addition to other mechanisms, enhancement of N-methyl-D-aspartate (NMDA) receptor signalling has been proposed to underlie rt-PA-mediated neurotoxicity. However, the role of poly(ADP-ribose) polymerase-1 (PARP-1) activation, which mediates postischemic excitotoxic cell death, in rt-PA-mediated aggravation of ischemic brain injury has not been established and was therefore addressed in this study. After permanent focal cerebral ischemia, intravenous rt-PA application significantly increased early postischemic PARP-1 activation within ischemic hemispheres and infarct volumes compared with control mice without affecting cerebral blood flow. Rt-PA induced increase in PARP-1 activation, and infarct volumes could be blocked by the PARP inhibitor 3-aminobenzamide. Moreover, the rt-PA-induced increase in PARP-1 activation was also prevented by the NMDA antagonist MK-801. In summary, we demonstrate that rt-PA treatment enhances postischemic PARP-1 activation, which contributes to rt-PA induced aggravation of ischemic brain injury in vivo. Furthermore, we provide evidence that NMDA receptor activation is required for rt-PA-mediated effects on postischemic PARP-1 activation.     

Neuroprotection against NMDA excitotoxicity by group I metabotropic glutamate receptors is associated with reduction of NMDA stimulated currents

The neurotransmitter glutamate can have both excitotoxic and protective effects on neurons. The excitotoxic effects have been intensively studied, whereas the protective effects, including the involvement of metabotropic glutamate receptors (mGluRs), remain unclear. In the present study, we tested the protective effects of the group-I-mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) on organotypic hippocampal slice cultures exposed to excitotoxic concentrations of N-methyl-D-aspartate (NMDA). Effects of DHPG on electrophysiological responses induced by NMDA receptor activation were also recorded. Experiments were performed on organotypic hippocampal slice cultures derived from 7-day-old rats, with cellular uptake of propidium iodide as a marker for neuronal cell death. Slice cultures pretreated with DHPG (10 or 100 microM) for 2 h prior to exposure to 50 microM NMDA for 30 min displayed reduced propidium iodide uptake, compared to cultures exposed to NMDA only. The neuroprotective effect was confirmed by Hoechst 33342 staining, where the appearance of pycnotic nuclei after NMDA treatment was prevented by the DHPG pretreatment. Using caspase-3 activity to monitor the presence of apoptosis, failed to demonstrate this type of cell death in CA1 after NMDA application. The protective effect of DHPG was abolished by the mGluR1 selective antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385; 5 or 10 microM), whereas the mGluR5-selective antagonist 2-methyl-6-phenylethynylpyridine (MPEP; 1 microM) had no effect. Voltage-clamping of CA1 pyramidal cells in cultures treated with 10 microM DHPG for 2 h showed a significant depression of NMDA-induced inward currents compared to untreated controls. We conclude that neuroprotection induced by activation of group-I-mGluRs involve mGluR1 and is associated with decreased NMDA-stimulated currents.     

Ischemic brain injury in vitro: protective effects of NMDA receptor antagonists and calmidazolium

Excessive Ca2+ influx through NMDA receptor-coupled channels has been linked to neuronal cell death. Using an in vitro model of transient brain ischemia, we investigated possible protective effects of NMDA receptor antagonists ketamine or MK-801 and of calmidazolium, an inhibitor of intracellular Ca2(+)-activated proteins. Brain ischemia/recovery was simulated in isolated hippocampal slices and injury monitored by measurement of ATP levels. Omission of both glucose and oxygen (but not oxygen alone) for 20 min led to persistent ATP deficits after 4 h recovery. Addition of ketamine or MK-801 at 1 microM permitted ATP to recover within 1 h, as did addition of calmidazolium at 10 microM. Our findings are consistent with other reports that NMDA receptor antagonists can protect neuronal tissue from ischemic damage. The role of inappropriately activated Ca2(+)-mediated signaling processes in the mechanism(s) of such injury is suggested by the protection also seen with calmidazolium, an inhibitor of calmodulin and other structurally related proteins such as calpain(s) and protein kinase C. The inhibition of intracellular Ca2+ target proteins may be an alternative for protection of the brain against injury due to insults that activate NMDA receptors.