CGX-1007 prevents excitotoxic cell death via actions at multiple types of NMDA receptors

Glutamate induced excitotoxic injury through over-activation of N-methyl-D-aspartate receptors (NMDARs) plays a critical role in the development of many neurodegenerative diseases. The present study was undertaken to evaluate the role of CGX-1007 (Conantokin G) as a neuroprotective agent against NMDA-induced excitotoxicity. Conantokin G, a cone snail peptide isolated from Conus geographus is reported to selectively inhibit NR2B containing NMDARs with high specificity and is shown to have potent anticonvulsant and antinociceptive effects. CGX-1007 significantly reduced the excitotoxic cell death induced by NMDA in organotypic hippocampal brain slice cultures in a concentration-dependent manner. In contrast, ifenprodil, another NR2B specific antagonist failed to offer neuroprotection against NMDA-induced excitotoxicity. We further determined that the neuroprotection observed is likely due to the action of CGX-1007 at multiple NMDA receptor subtypes. In a series of electrophysiology experiments, CGX-1007 inhibited NMDA-gated currents in human embryonic kidney (HEK) 293 cells expressing NMDA receptors containing either NR1a/NR2B or NR1a/NR2A subunit combinations. CGX-1007 produced a weak inhibition at NR1a/NR2C receptors, whereas it had no effect on NR1a/NR2D receptors. Further, the inhibition of NMDA receptors by CGX-1007 was voltage-dependent with greater inhibition seen at hyperpolarized membrane potentials. The voltage-dependence of CGX-1007 activity was also observed in recordings of NMDA-gated currents evoked in native receptors expressed in cortical neurons in culture. Based on our results, we conclude that CGX-1007 is a potent neuroprotective agent that acts as an antagonist at both NR2A and NR2B containing receptors.     

The effect of CGX-1007 and CI-1041, novel NMDA receptor antagonists, on NMDA receptor-mediated EPSCs

The N-methyl-D-aspartate (NMDA) receptor-gated ion channel is comprised of at least one NR1 subunit and any of four NR2 subunits (NR2A-D). The NR2 subunit confers different pharmacological and kinetic properties to the receptor. CGX-1007 (Conantokin G), a 17-amino acid polypeptide isolated from the venom of Conus geographus, is a novel NMDA receptor antagonist that is thought to be selective for the NR2B subunit. CGX-1007 has been reported to have highly potent, broad-spectrum anticonvulsant activity in animal seizure models. CI-1041 is an investigational compound, which also possesses anticonvulsant activity and has been shown to be highly selective for NR2B containing NMDA receptors. Although both CI-1041 and CGX-1007 are reportedly NR2B specific antagonists, they differ in their ability to block amygdala-kindled seizures, suggesting that the mechanism of action of these compounds differs. The present study was designed to test the hypothesis that CI-1041 and CGX-1007 would differentially modulate the function of NMDA receptors at excitatory synapses. Using the whole cell patch clamp technique, CGX-1007 and CI-1041 were found to block CA1 pyramidal cell, NMDA receptor-mediated excitatory postsynaptic currents (N-EPSCs) in a concentration-dependent manner in hippocampal slices from P4-P6 animals. In contrast, only CGX-1007 decreased NMDA receptor-mediated EPSC peak amplitude in slices from adult animals. The CGX-1007 block of peak amplitude was accompanied by a similar concentration-dependent decrease in decay kinetics of NMDA receptor-mediated EPSCs. These results suggest that while CI-1041 may be selective for NMDA receptors containing the NR2B subunit, CGX-1007 appears to be less selective than previously reported.     

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.     

The effects of glutamate receptor antagonists on cerebellar granule cell survival and development

N-Methyl-d-aspartate (NMDA) receptor stimulation promotes neuronal survival and differentiation under both in vitro and in vivo conditions. We studied the effects of various NMDA receptor antagonists acting at different NMDA receptor binding sites and non-NMDA receptor antagonists on the development and survival of cerebellar granule cell (CGC) culture. Only three of the drugs tested induced neurotoxicity-MK-801 (non-competitive NMDA channel blocking antagonist), ifenprodil (an antagonist of the NR2B site and polyamine site of the NMDA receptor) and L-701.324 (full antagonist at glycine site), while CGP-37849 (a competitive NMDA antagonist), (+)-HA-966 (a partial agonist of the glycine site of the NMDA receptor), and NBQX (a competitively acting AMPA receptor antagonist) were not toxic at any concentration (1-100 microM) used. Among these drugs, only MK-801 was toxic for the immature CGC on second day in vitro (2DIV), and toxicity was diminished parallel to the neuronal maturation. In more mature neurons (7DIV), MK-801 demonstrated some neuroprotection, which diminished spontaneously occurring neuronal death in culture. Neither NMDA nor glutamate were able to prevent the neurotoxic effect of MK-801 at 2DIV. MK-801, ifenprodil and L-701.324 induced DNA fragmentation on 2DIV in CGC culture measured by the TUNEL method. The BOC-D-FMK, the universal caspase inhibitor, completely reversed MK-801-induced DNA fragmentation, suggesting an apoptotic pathway of MK-801-induced cell death. Neurite outgrowth as a characteristic feature of the development of CGC was diminished after treatment with MK-801, ifenprodil and L-701.324. In conclusion, the results of the present study demonstrate that only nonselective channel blocker MK-801 decreases cell viability, induces apoptosis and inhibits neurite outgrowth of CGC in a development-dependent manner.     

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.     

Intrathecal CGX-1007 is neuroprotective in a rat model of focal cerebral ischemia

The NMDA antagonist CGX-1007 (Conantokin-G) has previously been shown to possess potent neuroprotective properties when administered intracranially following experimental ischemic brain injury. Using the same model of middle cerebral artery occlusion (MCAo) in rats we now report the neuroprotective effects of CGX-1007 when delivered intrathecally (i.t.). When given 4 h post-occlusion, a reduction in brain infarction was measured along with significant neurological recovery. Furthermore, we describe an i.t. neuroprotective therapeutic window lasting > or = 8 h from the start of the injury. Critically, this is the first comprehensive report of a neuroprotective agent that can be administered i.t. to ameliorate experimental brain injury and potentially provide an excellent therapeutic window as a neuroprotection treatment.     

Differential role of poly(ADP-ribose) polymerase-1in apoptotic and necrotic neuronal death induced by mild or intense NMDA exposure in vitro

Overactivation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) plays a key role in the mechanisms responsible for neuronal death. In the present study, we examined the effects of the PARP-1 inhibitor 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone (DPQ) in two models of N-methyl-d-aspartate (NMDA)-induced neurotoxicity. The exposure of mixed cultured cortical cells to 300 microM NMDA for 10 min induced a caspase-dependent type of apoptotic neuronal death. Conversely, exposure to 2 mM NMDA for 10 min led to the appearance of morphological features of necrosis, with no increase in caspase-3 activity and depletion in adenosine triphosphate (ATP) levels. DPQ (10 microM) reduced the NMDA-induced PARP activation, restored ATP to near control levels and significantly attenuated neuronal injury only in the severe NMDA exposure model. Similar results were obtained when pure neuronal cortical cultures were used. PARP-1 activation thus appears to play a preferential role in necrotic than in caspase-dependent apoptotic neuronal death.     

Role of neuronal NR2B subunit-containing NMDA receptor-mediated Ca2+ influx and astrocytic activation in cultured mouse cortical neurons and astrocytes

The excitatory neurotransmitter glutamate has been shown to mediate such bidirectional communication between neurons and astrocytes. In the present study, we determined the role of N-methyl-D-aspartate (NMDA) receptors on glutamate-evoked Ca(2+) influx into neurons and astrocytes. Either a nonselective NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or selective NR2B subunit-containing NMDA receptor antagonists ifenprodil and (R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol (Ro25-6981) significantly inhibited the glutamate-evoked Ca(2+) influx into neurons, but not into astrocytes. Furthermore, we investigated whether NR2B subunit-containing NMDA receptor antagonists could suppress the astrocytic activation, as detected by glial fibrillary acidic protein (GFAP; as a specific marker of astrocyte)-like immunoreactivities in mouse cortical astrocytes. Here, we demonstrated that the increases in the level of GFAP-like immunoreactivities induced by glutamate were markedly suppressed by cotreatment with ifenprodil in cortical neuron/glia cocultures, but not in purified astrocytes. These results suggest that NR2B subunit-containing NMDA receptor plays a critical role in not only glutamate-evoked Ca(2+) influx into neurons, but also glutamate-induced astrocytic activation. Thus, glutamate-mediated pathway via NR2B subunit-containing NMDA receptor may, at least in part, contribute to neuron-to-astrocyte signaling.     

Tissue kallikrein alleviates glutamate‐induced neurotoxicity by activating ERK1

Glutamate‐induced neurotoxicity consequent to N‐methyl‐D‐aspartic acid (NMDA) and 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl) propionic acid (AMPA) receptor activation underlies the pathogenesis of a wide range of central nervous system disorders, including brain ischemia. Prevention of ischemia/reperfusion (I/R)‐induced neuronal injury has long been regarded as an effective therapeutic strategy for ischemia. Human tissue kallikrein (TK) gene transfer has been shown to protect neurons against cerebral I/R‐induced apoptosis and oxidative stress, via activation of the brandykinin B2 receptor (B2R). However, little is known about the role of TK on glutamate‐induced neurotoxicity. Here we report that pretreatment of cultured cortical neurons with TK largely prevented glutamate‐induced morphological changes and cell death. We found that TK pretreatment alleviated glutamate‐induced oxidative stress by inhibiting neuronal nitric oxide synthase (nNOS) activity, thereby reducing the generation of nitric oxide (NO) and reactive oxygen species (ROS). Blockage of NMDA and AMPA receptors by their specific antagonists MK801 and CNQX had effects similar to those of TK administration. Furthermore, we found that the extracellular signal‐regulated kinase 1/2 cascade (ERK1/2), particularly ERK1, and nuclear factor‐κB (NF‐κB) were involved in TK neuroprotection against glutamate‐induced neurotoxicity. TK pretreatment activated ERK1 and NF‐κB, leading to enhanced expression of brain‐derived neurotrophic factor (BDNF) mRNA and antiapoptotic gene Bcl‐2 protein. Collectively, these findings demonstrate that TK attenuates glutamate‐induced apoptosis through an intracellular signaling pathway including activation of B2R, ERK1/2, and NF‐κB and up‐regulation of BDNF and Bcl‐2 expression. Thus, TK represents a promising therapeutic strategy for ischemic stroke. © 2009 Wiley‐Liss, Inc.     

Protective Effect of Memantine Against Doxorubicin Toxicity in Primary Neuronal Cell Cultures: Influence a Development Stage

One of the serious unwanted effects of the anthracycline anticancer drug doxorubicin (Dox, adriamycin) is its neurotoxicity, which can be evoked by the activation of extracellular (FAS/CD95/Apo-1) pathway of apoptosis in cells. Since memantine, a clinically used N-methyl-d-aspartic acid (NMDA) receptor antagonist, shows antiapoptotic action in several models of neuronal cell damage, in this study we evaluated the effect of memantine on the cell death induced by Dox in primary neuronal cell cultures. First, we investigated the effect of different concentrations of Dox (0.1–5 μM) on mouse neocortical, hippocampal, striatal, and cerebellar neurons on 7- and 12-day in vitro (DIV). The 7 DIV neuronal cell cultures were more prone to Dox-induced cell death than 12 DIV cultures. The cerebellar neurons were the most resistant to Dox-induced apoptosis in comparison to neuronal cell cultures derived from the forebrain. Memantine (0.1–2 μM) attenuated the Dox-evoked lactate dehydrogenase release in 7 DIV neuronal cell cultures with no significant effect on 12 DIV cultures. The ameliorating effect of memantine on Dox-mediated cell death was also confirmed by an increase in cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. There was no effect of memantine on Dox-induced caspase-8 and -3 activity and Dox-evoked decrease in mitochondrial potential, although attenuation in the number of cells with apoptotic DNA fragmentation was observed. We also showed that the antiapoptotic effect of memantine in our model was NMDA receptor-independent, since two other antagonists of this receptor, MK-801 and AP-5, did not attenuate Dox-induced cell death. Furthermore, memantine did not influence the Dox-evoked increase in cytoplasmic Ca²⁺ level. The obtained data suggest developmental regulation of both, the Dox-mediated neurotoxicity and efficacy of memantine in alleviating the Dox-induced cell damage in neuronal cell cultures. Moreover, this neuroprotective effect of memantine seems not to be dependent on caspase-3 activity and on the antagonistic action on NMDA receptor.     

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.     

Kinetic characterization of novel NR2B antagonists using fluorescence detection of calcium flux

To facilitate the discovery of novel N-methyl-d-aspartate (NMDA) receptor antagonists, we have developed a high-throughput functional assay based on fluorescence detection of free intracellular calcium concentrations. Mouse fibroblast L(tk-) cells expressing human NR1a/NR2B NMDA receptors were plated in 96-well plates and loaded with fluorescence calcium indicator fluo-3 AM. NR2B antagonists were added after stimulation of NMDA receptors with 10 microM glutamate and 10 microM glycine. Changes in fluorescence after the addition of the antagonists were fitted by a single exponential equation providing k(obs). The concentration dependence of k(obs) was linear for all NR2B antagonists at concentrations where k(obs) < 0.2 s(-1). The values of k(obs) for six structurally distinct NR2B antagonists were in the range of 1.1 to 7.5 x 10(5) M(-1)s(-1). These values were several orders of magnitude slower than that obtained for diffusion limited Mg(2+) channel block. The rate constants k(off) provided the values of t(1/2) for dissociation of NR2B antagonists in the range of 1.8 min for ifenprodil to 240 min for the slowest novel antagonist. The IC(50) values obtained from the end-point fluorescence measurements agree with K(d) values calculated from kinetic measurements. All kinetic constants, obtained using our fluorescence method, correlate well with data measured by voltage clamp.     

Effect of the N‐methyl‐D‐aspartate NR2B subunit antagonist ifenprodil on precursor cell proliferation in the hippocampus

The N‐methyl‐D‐aspartate (NMDA) receptor, one of the ionotropic glutamate receptor, plays important physiological and pathological roles in learning and memory, neuronal development, acute and chronic neurological diseases, and neurogenesis. This work examines the contribution of the NR2B NMDA receptor subunit to adult neurogenesis/cell proliferation under physiological conditions and following an excitotoxic insult. We have previously shown in vitro that a discrete NMDA‐induced, excitotoxic injury to the hippocampus results in an increase in neurogenesis within the dentate gyrus. Here we have characterized adult neurogenesis or proliferation, using BrdU, in an in vivo model of excitotoxic injury to the CA1 subfield of the hippocampus. We demonstrate a peak in neural stem cell proliferation/neurogenesis between 6 and 9 days after the excitotoxic insult. Treatment with ifenprodil, an NR2B subunit‐specific NMDA receptor antagonist, without prior injury induction, also increased the number of BrdU‐positive cells within the DG and posterior periventricle, indicating that ifenprodil itself could modulate the rate of proliferation. Interestingly, though, the increased level of cell proliferation did not change significantly when ifenprodil was administered following an excitotoxic insult. In conclusion, our results suggest and add to the growing evidence that NR2B subunit‐containing NMDA receptors play a role in neural stem cell proliferation. © 2014 Wiley Periodicals, Inc.     

Failure of glycine site NMDA receptor antagonists to protect againstl-2-chloropropionic acid-induced neurotoxicity highlights the uniqueness of cerebellar NMDA receptors

Cultured cerebellar granule cells and cerebellar slices from neonatal rats have been widely used to examine the biochemistry of excitatory amino acid-induced cell death mediated in part by the activation of NMDA receptors. However, the NMDA subunit stoichiometry, producing functional NMDA receptors is different in cultured granule cells, neonatal and adult rat cerebellum as compared to the NMDA receptors in forebrain regions. We have used thel-2-chloropropionic acid (l-CPA) (750 mg/kg) model of NMDA-medialed selective cerebellar granule cell necrosis in vivo to examine the role of the glycine binding site and possible effect of the NR2C subunit (which is largely expressed only in the cerebellum) on granule cell necrosis. The abilities of various NMDA receptor antagonists were examined in vivo to determine the relative contribution of both glutamate and glycine sites involved in thel-CPA-induced neurotoxicity. The potent neuroprotective, non-competitive NMDA receptor antagonist dizocilpine (MK-801) was compared with glutamate and glycine site NMDA antagonists. We have examined a number of markers for thel-CPA-induced granule cell necrosis. Thel-CPA-induced reduction in cerebellar aspartate and glutamate concentrations were used as markers of granule cell necrosis. We also measured the cerebellar water content and sodium concentrations as measures of thel-CPA-induced cerebellar edema that accompanies the granule cell necrosis. Finally the ability of the NMDA antagonists to attenuate thel-CPA-induced reductions in body weight gain and the prevention of the loss in hindlimb function using a behavioral measure of hindlimb retraction were examined. The potent glutamate antagonists, CPP and CGP40116 and dizocilpine prevented thel-CPA-induced locomotor dysfunction and granule cell necrosis as measured by their ability to preventl-CPA-induced reductions in aspartate and glutamate concentrations. CPR CGP40116 and dizocilpine also prevented the appearance of cerebellar edema followingl-CPA administration. In addition, dizocilpine, CPP and CGP40116 were able to partially prevent thel-CPA-induced loss in body weight over the 48 h experimental period. In contrast, none of the glycine partial agonists or antagonists, namely (±)HA-966,d-cycloserine, MDL-29951, DPCQ, MNQX or L-701 252 were able to prevent thel-CPA-induced loss in body weight,l-CPA-induced granule cell necrosis and behavioral disturbances when administered to rats. None of the NMDA antagonists had any effect on the cerebellar neurochemistry when injected alone or had any effect on animal behavior except for dizocilpine, CPP, CGP401 l6 and (±)HA-966 which resulted in a transient sedation for between three and five hours immediately following their administration. In conclusion, we demonstrate that NMDA open channel blockade and glutamate antagonists can provide full neuroprotection against thel-CPA-induced granule cell necrosis. The failure of the glycine partial agonists and antagonists to provide any neuroprotection againstl-CPA-induced neurotoxicity in the cerebellum contrasts with their neuroprotective efficacy in other animal models of excitatory amino acid-induced cell death in forebrain regions in vivo. We therefore suggest that the glycine site plays a lesser role in modulating NMDA receptor function in the cerebellum and may explain why cells expressing NMDA receptors composed of NR1/NR2C subunits are particularly resistant to excitatory amino acid-induced neurotoxicity.     

Modulation of calcium/calmodulin kinase-II provides partial neuroprotection against beta-amyloid peptide toxicity

Beta-amyloid (Abeta) peptide-induced neurotoxicity has been implicated in the pathogenesis of Alzheimer's disease (AD). The exact mechanism by which Abeta peptides trigger neuronal death is not well defined and may be related to an abrupt increase in intracellular calcium, leading to the activation of many pro-apoptotic pathways. While modulation of intracellular calcium increase receives much attention for pharmaceutical intervention, Ca2+-mediated pro-apoptotic signalling pathways have not been systematically studied. We have reported our study on the roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in Abeta peptide neurotoxicity. By treating the primary cortical neurons exposed to Abeta peptides (Abeta(25-35) and Abeta(1-42)) with two selective CaMKII inhibitors, autocamtide-related inhibitory peptide (AIP) and KN93, Abeta peptide neurotoxicity was significantly reduced. Release of LDH and DNA fragmentation/condensation (by DAPI staining) in neurons exposed to Abeta peptides were significantly decreased in the presence of AIP and KN93. While these inhibitors significantly attenuated Abeta peptide-triggered activation of caspase-2 and caspase-3, and AIP significantly decreased the degree of tau phosphorylation of the Abeta peptide-treated neurons at early time, they could elicit partial neuroprotection only. Pharmacological inhibitor targeting calmodulin, W7, did not provide neuroprotection. Morphine, which activates CaMKII via micro receptors, augments Abeta-induced LDH release, caspase-2 and caspase-3 activities and neuronal apoptosis. Taken together, although CaMKII plays a role in Abeta peptide neurotoxicity, pharmacological inhibition cannot afford complete neuroprotection.     

Neuroprotective Effects of NMDA and Group I Metabotropic Glutamate Receptor Antagonists Against Neurodegeneration Induced by Homocysteine in Rat Hippocampus: In Vivo Study

Homocysteine (Hcy), a neurotoxic amino acid, is a risk factor for neurodegenerative diseases. Previous in vitro studies have demonstrated that group I metabotropic glutamate receptors along with N-methyl-d-aspartic acid (NMDA) receptors participate in acute and chronic aspects of Hcy-induced neuronal damage. In the present study, we examined whether the same mechanism may be involved in homocysteine neurotoxicity in vivo. Memantine, MPEP, and LY367385 were used as NMDA, mGlu5, and mGlu1 antagonists, respectively. Repeated i.c.v injection of Hcy was performed for three consecutive days. Neuronal loss in different zones of the hippocampus was assessed by Nissl, Fluoro-Jade B, and TUNEL staining. Neuronal degeneration was observed in both types of apoptosis and necrosis. All glutamate receptor antagonists, even when given alone, provided some degree of neuroprotection. The degree of protection was dependent on the area of the hippocampus. While memantine was more potent against Hcy-induced apoptosis, the potency of mGluR antagonists in neuronal protection against apoptosis and necrosis was almost equal. No more protection was observed when all three antagonists were used simultaneously. It seems that Fluoro-Jade could be a useful marker of apoptotic cell death. Taken together, results demonstrate that, in vivo, Hcy neurotoxicity is mediated mainly by the NMDA receptors and group I mGluRs.     

Characterization of Excitatory Amino Acid Neurotoxicity in N-methyl-D-aspartate Receptor-deficient Mouse Cortical Neuronal Cells

Roles and mechanisms of N -methyl-D-aspartate (NMDA) receptors in glutamate neurotoxicity were investigated in cultures of NMDA receptor-deficient cortical neuronal cells. Mutant mice lacking a functional NMDA receptor were generated by gene targeting of the NR1 NMDA receptor subunit. Cortical neuronal cells prepared from wild-type NR1 ^+/+, heterozygous NR1 ^+/- and homozygous mutant NR1^-/- mice at 15–17 days of gestation grew indistinguishably from each other. Brief exposures (5 min) of both NR1 ^+/+ and NR1 ^+/- neuronal cells to glutamate or NMDA, but not kainate or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), resulted in widespread neuronal degeneration by the following day. In contrast, neither glutamate nor NMDA treatment caused neuronal degeneration in NR1 ^-/- cells, indicating that NMDA receptors are responsible for rapidly triggered glutamate neurotoxicity. The above four compounds were all effectivein inducing the death of NR1 ^+/+ and NR1 ^+/- neuronal cells after prolonged exposure (20–24 h). However, NMDA had no neurotoxic effects on NR1 -/- cells, although the other three compounds were neurotoxic with potencies comparable to those for NR1 ^+/+ and NR1 ^+/- cells. The AMPA and kainate receptors are thus sufficient for inducing slowly triggered glutamate neurotoxicity. Brief exposure of a mixed population of NR1 ^+/+ and NR1 ^-/- neuronal cells to NMDA selectively killed the NMDA receptor-expressing cells without any appreciable effects on neighbouring NMDA receptor-deficient cells. This finding further supports a direct and indispensable role for NMDA receptors in NMDA-evoked neuronal cell death.     

Memantine attenuates staurosporine-induced activation of caspase-3 and LDH release in mouse primary neuronal cultures

Developmental aspects of pro- and antiapoptotic action of some NMDA receptor antagonists in the central nervous system have been postulated. In order to further elucidate this problem, we investigated effect of memantine, an uncompetitive NMDA receptor antagonist and staurosporine alone and in combination on caspase-3 activity and lactate dehydrogenase (LDH) release in primary hippocampal, neocortical and striatal cell cultures on 7 and 12 days in vitro. The data showed that the vulnerability of neuronal cells to induction of caspase-3 activity by staurosporine was higher on 7 DIV than on 12 DIV, whereas staurosporine-mediated LDH release increased with days in vitro in striatal culture only. A specific inhibitor of caspase-3, AcDEVDCHO (60 microM), completely abolished the effect of staurosporine on this enzyme's activity, but only partially attenuated staurosporine-induced LDH release in hippocampal cells. Memantine alone (0.05-2.0 microM) did not induce any cytotoxic effect but attenuated the staurosporine-induced caspase-3 activity and LDH release in hippocampal cultured neurons on each investigated day in vitro. In striatal culture, memantine had a moderate inhibitory effect on staurosporine-evoked LDH release only on 7 DIV with no significant influence on caspase-3 activity. As for neocortical cultures, memantine partially inhibited staurosporine-induced neuronal injury only on 7 DIV. These data showed that the induction of caspase-3 activity by staurosporine was more profound in immature cells, however, the staurosporine neurotoxicity, as reflected by LDH release, only partially depended on caspase-3 activation and stage of cell development. Furthermore, memantine attenuated staurosporine-induced apoptosis more efficiently in hippocampal cultures than in neocortical and striatal ones, which points to tissue specificity of effects of this neuroprotectant.     

Pharmacology of N-methyl-D-aspartate-induced brain injury in an in vivo perinatal rat model

Intrastriatal injection of the glutamate analogue N-methyl-D-aspartate (NMDA, 25 nmol) in postnatal day (PND) 7 rats provides a rapid, sensitive, and reproducible assay in which potential neuroprotective strategies against excitotoxic neuronal injury can be examined in vivo. Brain injury is quantified 5 days postinjection by comparison of the weights of the injected and contralateral cerebral hemispheres. Intraperitoneal injections (15 minutes post-NMDA) of competitive and noncompetitive NMDA receptor antagonists attenuated the severity of NMDA-induced brain injury. The rank order of neuroprotective potency of these antagonists was CGS-19755 greater than DOIPG greater than dextromethorphan greater than HA-966. Of these compounds only the competitive antagonist CGS-19755 provided complete neuroprotection. NMDA-mediated brain injury was also reduced by the specific sigma receptor ligands +PPP and haloperidol (35% reduction). In contrast, drugs that reduce presynaptic neurotransmitter release (adenosine) or enhance neuronal inhibition (baclofen) were not effective against NMDA toxicity. Although all five of the anticonvulsants tested limited NMDA-induced seizure activity, only carbamazepine reduced NMDA-mediated brain injury (36% reduction). These findings extend earlier observations that NMDA receptor antagonists can limit NMDA-induced toxicity in vivo and suggest that sigma receptors contribute to the pathophysiology of NMDA-mediated brain injury in vivo. Furthermore, NMDA-induced seizures and brain injury appear dissociable in this in vivo model. The results illustrate important practical limitations of neuroprotection in vivo vs. in vitro.