NMDA-induced Excitotoxicity Research Articles

Nicotinamide mononucleotide adenylyl transferase 1 protects against acute neurodegeneration in developing CNS by inhibiting excitotoxic-necrotic cell death

Hypoxic-ischemic (H-I) injury to the developing brain is a significant cause of morbidity and mortality in humans. Other than hypothermia, there is no effective treatment to prevent or lessen the consequences of neonatal H-I. Increased expression of the NAD synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) has been shown to be neuroprotective against axonal injury in the peripheral nervous system. To investigate the neuroprotective role of Nmnat1 against acute neurodegeneration in the developing CNS, we exposed wild-type mice and mice overexpressing Nmnat1 in the cytoplasm (cytNmnat1-Tg mice) to a well-characterized model of neonatal H-I brain injury. As early as 6 h after H-I, cytNmnat1-Tg mice had strikingly less injury detected by MRI. CytNmnat1-Tg mice had markedly less injury in hippocampus, cortex, and striatum than wild-type mice as assessed by loss of tissue volume 7 d days after H-I. The dramatic protection mediated by cytNmnat1 is not mediated through modulating caspase3-dependent cell death in cytNmnat1-Tg brains. CytNmnat1 protected neuronal cell bodies and processes against NMDA-induced excitotoxicity, whereas caspase inhibition or B-cell lymphoma-extra large (Bcl-XL) protein overexpression had no protective effects in cultured cortical neurons. These results suggest that cytNmnat1 protects against neonatal HI-induced CNS injury by inhibiting excitotoxicity-induced, caspase-independent injury to neuronal processes and cell bodies. As such, the Nmnat1 protective pathway could be a useful therapeutic target for acute and chronic neurodegenerative insults mediated by excitotoxicity.

The Role of PSD-95 and Cypin in Morphological Changes in Dendrites Following Sublethal NMDA Exposure

Focal swelling or varicosity formation in dendrites and loss of dendritic spines are the earliest indications of glutamate-induced excitotoxicity. Although it is known that microtubule dynamics play a role in varicosity formation, very little is known about the proteins that directly impact microtubules during focal swelling and dendritic spine loss. Our laboratory has recently reported that the postsynaptic protein PSD-95 and its cytosolic interactor (cypin) regulate the patterning of dendrites in hippocampal neurons. Cypin promotes microtubule assembly, and PSD-95 disrupts microtubule organization. Thus, we hypothesized that cypin and PSD-95 may play a role in altering dendrite morphology and spine number in response to sublethal NMDA-induced excitotoxicity. Using an in vitro model of glutamate-induced toxicity in rat hippocampal cultures, we found that cypin overexpression or PSD-95 knockdown increases the percentage of neurons with varicosities and the number of varicosities along dendrites, decreases the size of varicosities after sublethal NMDA exposure, and protects neurons from NMDA-induced death. In contrast, cypin knockdown or PSD-95 overexpression results in opposite effects. We further show that cypin regulates the density of spines/filopodia: cypin overexpression decreases the number of protrusions per micrometer of dendrite while cypin knockdown results in an opposite effect. Cypin overexpression and PSD-95 knockdown attenuate NMDA-promoted decreases in protrusion density. Thus, we have identified a novel pathway by which the microtubule cytoskeleton is regulated during sublethal changes to dendrites.

Iduna protects the brain from glutamate excitotoxicity and stroke by interfering with poly(ADP-ribose) polymer-induced cell death

Glutamate acting on N-methyl-D-aspartate (NMDA) receptors induces neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 (PARP-1) and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a previously undescribed NMDA receptor-induced survival protein that is neuroprotective against glutamate NMDA receptor-mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer-induced cell death (parthanatos). Iduna's protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer-binding protein, and mutation at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion-induced stroke in mice. To our knowledge, these results define Iduna as the first known endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling could be a new therapeutic strategy for the treatment of neurologic disorders.

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.

Neuronal Sirt3 Protects against Excitotoxic Injury in Mouse Cortical Neuron Culture

BackgroundSirtuins (Sirt), a family of nicotinamide adenine nucleotide (NAD) dependent deacetylases, are implicated in energy metabolism and life span. Among the known Sirt isoforms (Sirt1-7), Sirt3 was identified as a stress responsive deacetylase recently shown to play a role in protecting cells under stress conditions. Here, we demonstrated the presence of Sirt3 in neurons, and characterized the role of Sirt3 in neuron survival under NMDA-induced excitotoxicity.Methodology/Principal FindingsTo induce excitotoxic injury, we exposed primary cultured mouse cortical neurons to NMDA (30 µM). NMDA induced a rapid decrease of cytoplasmic NAD (but not mitochondrial NAD) in neurons through poly (ADP-ribose) polymerase-1 (PARP-1) activation. Mitochondrial Sirt3 was increased following PARP-1 mediated NAD depletion, which was reversed by either inhibition of PARP-1 or exogenous NAD. We found that massive reactive oxygen species (ROS) produced under this NAD depleted condition mediated the increase in mitochondrial Sirt3. By transfecting primary neurons with a Sirt3 overexpressing plasmid or Sirt3 siRNA, we showed that Sirt3 is required for neuroprotection against excitotoxicity.ConclusionsThis study demonstrated for the first time that mitochondrial Sirt3 acts as a prosurvival factor playing an essential role to protect neurons under excitotoxic injury.

Curcumin Protects against NMDA-Induced Toxicity: A Possible Role for NR2A Subunit

Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDA-induced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca(2+)](i). Curcumin dose- and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDA-induced [Ca(2+)](i) rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca(2+)](i). The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs.

The optic nerve head is the site of axonal transport disruption, axonal cytoskeleton damage and putative axonal regeneration failure in a rat model of glaucoma

The neurodegenerative disease glaucoma is characterised by the progressive death of retinal ganglion cells (RGCs) and structural damage to the optic nerve (ON). New insights have been gained into the pathogenesis of glaucoma through the use of rodent models; however, a coherent picture of the early pathology remains elusive. Here, we use a validated, experimentally induced rat glaucoma model to address fundamental issues relating to the spatio-temporal pattern of RGC injury. The earliest indication of RGC damage was accumulation of proteins, transported by orthograde fast axonal transport within axons in the optic nerve head (ONH), which occurred as soon as 8 h after induction of glaucoma and was maximal by 24 h. Axonal cytoskeletal abnormalities were first observed in the ONH at 24 h. In contrast to the ONH, no axonal cytoskeletal damage was detected in the entire myelinated ON and tract until 3 days, with progressively greater damage at later time points. Likewise, down-regulation of RGC-specific mRNAs, which are sensitive indicators of RGC viability, occurred subsequent to axonal changes at the ONH and later than in retinas subjected to NMDA-induced somatic excitotoxicity. After 1 week, surviving, but injured, RGCs had initiated a regenerative-like response, as delineated by Gap43 immunolabelling, in a response similar to that seen after ON crush. The data presented here provide robust support for the hypothesis that the ONH is the pivotal site of RGC injury following moderate elevation of IOP, with the resulting anterograde degeneration of axons and retrograde injury and death of somas.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-011-0807-1) contains supplementary material, which is available to authorized users.

The Mitochondrial Inner Membrane GTPase, Optic Atrophy 1 (Opa1), Restores Mitochondrial Morphology and Promotes Neuronal Survival following Excitotoxicity

Mitochondrial dynamics have been extensively studied in the context of classical cell death models involving Bax-mediated cytochrome c release. Excitotoxic neuronal loss is a non-classical death signaling pathway that occurs following overactivation of glutamate receptors independent of Bax activation. Presently, the role of mitochondrial dynamics in the regulation of excitotoxicity remains largely unknown. Here, we report that NMDA-induced excitotoxicity results in defects in mitochondrial morphology as evident by the presence of excessive fragmented mitochondria, cessation of mitochondrial fusion, and cristae dilation. Up-regulation of the mitochondrial inner membrane GTPase, Opa1, is able to restore mitochondrial morphology and protect neurons against excitotoxic injury. Opa1 functions downstream of the calcium-dependent protease, calpain. Inhibition of calpain activity by calpastatin, an endogenous calpain inhibitor, significantly rescued mitochondrial defects and maintained neuronal survival. Opa1 was required for calpastatin-mediated neuroprotection because the enhanced survival found following NMDA-induced toxicity was significantly reduced upon loss of Opa1. Our results define a mechanism whereby breakdown of the mitochondrial network mediated through loss of Opa1 function contributes to neuronal death following excitotoxic neuronal injury. These studies suggest Opa1 as a potential therapeutic target to promote neuronal survival following acute brain damage and neurodegenerative diseases.

Sustained Glutamate Receptor Activation Down-regulates GABAB Receptors by Shifting the Balance from Recycling to Lysosomal Degradation

Metabotropic GABA(B) receptors are abundantly expressed at glutamatergic synapses where they control excitability of the synapse. Here, we tested the hypothesis that glutamatergic neurotransmission may regulate GABA(B) receptors. We found that application of glutamate to cultured cortical neurons led to rapid down-regulation of GABA(B) receptors via lysosomal degradation. This effect was mimicked by selective activation of AMPA receptors and further accelerated by coactivation of group I metabotropic glutamate receptors. Inhibition of NMDA receptors, blockade of L-type Ca(2+) channels, and removal of extracellular Ca(2+) prevented glutamate-induced down-regulation of GABA(B) receptors, indicating that Ca(2+) influx plays a critical role. We further established that glutamate-induced down-regulation depends on the internalization of GABA(B) receptors. Glutamate did not affect the rate of GABA(B) receptor endocytosis but led to reduced recycling of the receptors back to the plasma membrane. Blockade of lysosomal activity rescued receptor recycling, indicating that glutamate redirects GABA(B) receptors from the recycling to the degradation pathway. In conclusion, the data indicate that sustained activation of AMPA receptors down-regulates GABA(B) receptors by sorting endocytosed GABA(B) receptors preferentially to lysosomes for degradation on the expense of recycling. This mechanism may relieve glutamatergic synapses from GABA(B) receptor-mediated inhibition resulting in increased synaptic excitability.

Phosphorylation of Huntingtin at Ser421in YAC128 Neurons Is Associated with Protection of YAC128 Neurons from NMDA-Mediated Excitotoxicity and Is Modulated by PP1 and PP2A

YAC transgenic mice expressing poly(Q)-expanded full-length huntingtin (mhtt) recapitulate many behavioral and neuropathological features of Huntington disease (HD). We have previously observed a reduction in phosphorylation of mhtt at S421 in the presence of the mutation for HD. In addition, phosphorylation of normal S421-htt is reduced after excitotoxic stimulation of NMDA receptors (NMDARs). To test whether NMDAR stimulation contributes to reduced pS421-htt levels in HD, we determined phosphorylation of htt at Ser421 after NMDA-induced excitotoxicity in neurons from YAC128 mice. Here, we report that the total level of pS421-htt is reduced in YAC128 primary neurons after excitotoxic NMDAR stimulation. Similarly, the total level of pS421-htt is reduced in YAC128 transgenic mice after quinolinic acid injection into the striatum. In contrast, loss of phosphorylation of pS421-htt is prevented in YAC mice that never develop clinical or neuropathological features of HD [the caspase 6-resistant YAC128 transgene (C6R)]. To gain insight into the mechanisms underlying these findings, we determined that the Ser/Thr protein phosphatases PP1 and PP2A dephosphorylate pS421-htt in situ and after excitotoxic stimulation of NMDARs in neurons. Furthermore, increasing the phosphorylation of htt at S421 by blocking PP1 and PP2A activity protects YAC128 striatal neurons from NMDA-induced cell death. These results, together with the observed modulation of pS421-htt levels by dopamine, the reduced expression of PP1 inhibitor Darpp-32 in the striatum of YAC128 mice, and the reduced phosphorylation of PP1 substrate CreB, point to altered regulation of phosphatase activity in HD and highlight enhancing phosphorylation of htt at S421 as a therapeutic target.

N‐Methyl‐d‐aspartate receptor subunit‐ and neuronal‐type dependence of excitotoxic signaling through post‐synaptic density 95

NMDA receptors (NMDARs) mediate excitatory synaptic transmission during repetitive or prolonged glutamate release, playing a critical role in synaptic plasticity or cell death, respectively. Evidence indicates that a major pathway of NMDAR signaling to cell death in cortical and hippocampal neurons requires the scaffolding protein post-synaptic density 95 (PSD-95) and activation of neuronal nitric oxide synthase. However, it is not known if this PSD-95-dependent pathway contributes to excitotoxicity in other brain regions. It is also unclear whether the neuroprotective effects of Tat-NR2B9c, a membrane-permeant peptide that disrupts PSD-95/NMDAR binding, correlate with uncoupling NR2B- and/or NR2A-type NMDARs from PSD-95. In this study, we used cultured hippocampal and striatal neurons to test the potency of Tat-NR2B9c on uncoupling NR2 subunits from PSD-95 and protecting against NMDA-induced excitotoxicity. We found that the concentration of Tat-NR2B9c required to dissociate 50% of PSD-95 was fourfold lower for NR2B than NR2A in cultured hippocampal and striatal neurons, and that this concentration correlated tightly with protection against NMDA-induced toxicity in hippocampal neurons without altering NMDAR current. In contrast, NMDAR signaling to cell death in cultured striatal neurons occurred independently of the NR2B/PSD-95 interaction or neuronal nitric oxide synthase activation. These results will facilitate development of neuronal type-specific protective therapies.

NMDA-induced neuronal survival is mediated through nuclear factor I-A in mice

Identification of the signaling pathways that mediate neuronal survival signaling could lead to new therapeutic targets for neurologic disorders and stroke. Sublethal doses of NMDA can induce robust endogenous protective mechanisms in neurons. Through differential analysis of primary library expression and microarray analyses, here we have shown that nuclear factor I, subtype A (NFI-A), a member of the NFI/CAAT-box transcription factor family, is induced in mouse neurons by NMDA receptor activation in a NOS- and ERK-dependent manner. Knockdown of NFI-A induction using siRNA substantially reduced the neuroprotective effects of sublethal doses of NMDA. Further analysis indicated that NFI-A transcriptional activity was required for the neuroprotective effects of NMDA receptor activation. Additional evidence of the neuroprotective effects of NFI-A was provided by the observations that Nfia(-/-) neurons were highly sensitive to NMDA-induced excitotoxicity and were more susceptible to developmental cell death than wild-type neurons and that Nfia(+/-) mice were more sensitive to NMDA-induced intrastriatal lesions than were wild-type animals. These results identify NFI-A as what we believe to be a novel neuroprotective transcription factor with implications in neuroprotection and neuronal plasticity following NMDA receptor activation.

Inhibition of cytokine-induced connexin43 hemichannel activity in astrocytes is neuroprotective

Astrocytes express high levels of connexin43, a protein that forms two types of channels: gap junction channels for direct intercellular communication, and hemichannels for exchanges with the extracellular space. Inflammation induces connexin43 hemichannel activation, which has been proposed to be involved in neuroglial interactions. Here, we investigated the contribution of connexin43 to NMDA-induced excitotoxicity in neuron/astrocyte co-cultures, after treatment with a pro-inflammatory cytokine mixture, containing TNF-α and IL1-β (Mix), that stimulated astroglial connexin43 hemichannel activity. Interestingly, NMDA treatment induced a higher amount of neurotoxicity in Mix-treated co-cultures than in untreated ones, whereas this extent of neurotoxicity was absent in enriched neuron cultures or in co-cultures with connexin43 knock-out astrocytes. Furthermore, application of connexin43 hemichannel blockers or a synthetic cannabinoid prevented the Mix-induced potentiated NMDA neurotoxicity. Altogether, these data demonstrate that inflammation-induced astroglial hemichannel activation plays a critical role in neuronal death and suggest a neuroprotective role of connexin43 hemichannel blockade.

Chronic in vitro ketosis is neuroprotective but not anti‐convulsant

The ketogenic diet (KD), used successfully to treat a variety of epilepsy syndromes in humans and to attenuate seizures in different animal models, also provides powerful neuroprotection in various CNS injury models. Yet, a direct role for ketone bodies in limiting seizure and neuronal damage remains poorly understood. Using organotypic hippocampal slice cultures, we established an in vitro model of chronic ketosis for parallel studies of its neuroprotective and anti-convulsant effects. Chronic in vitro treatment with a ketone body, D-beta-hydroxybutyrate, protected the cultures against chronic hypoglycemia, oxygen-glucose deprivation, and NMDA-induced excitotoxicity, but failed to suppress intrinsic and induced seizure-like activity, indicating improved neuroprotection is not directly translated into seizure control. However, chronic in vitro ketosis abolished hippocampal network hyperexcitability following a metabolic insult, hypoxia, demonstrating for the first time a direct link between metabolic resistance and better control of excessive, synchronous, abnormal electrical activity. These findings suggest that the KD and, possibly, exogenous ketone administration, can be more beneficial for the treatment of seizures associated with metabolic stress or underlying metabolic abnormalities, and can potentially be used to optimize clinical applications of the traditional KD or its variants.

Secretoneurin and the tachykinins substance P and neurokinin-A/B in NMDA-induced excitotoxicity in the rat retina

In a recent investigation using the NMDA-excitotoxicity model in the rat retina, we found that, whereas, following intravitreal injection of NMDA, a time-dependent decrease of the levels of a neuropeptide, namely vasoactive intestinal polypeptide (VIP), was fully counteracted by topical treatment with flunarizine eye drops, the levels of pituitary adenylate-cyclase activating peptide-38 (PACAP-38), another neuropeptide, remained unchanged. The aim of the present study was to find out if NMDA causes reduction in the levels of other neuropeptides such as secretoneurin (SN), neurokinin-A/B (NKA/NKB) and substance P (SP), and if so, whether flunarizine has the ability to counteract this effect or prevent such reduction. The reduction of the levels of SN and NKA/NKB 14 days after intravitreal injection of 2 μl of 100 nmol NMDA into one eye was more pronounced than after 7 days; topical flunarizine had a slight counteracting effect, but could not prevent the decrease in the levels of these peptides. Reduction in SP levels after 28 and 56 days was fully counteracted by flunarizine. By enabling a pronounced influx of Ca 2+ ions into peptide-expressing cells, NMDA leads to cell death. Since each of these peptides exerts neuroprotective properties in the central nervous system, the drop in their levels caused by acute insult (e.g. NMDA excitotoxicity) or chronic insult (e.g. glaucoma) may cause a breakdown of endogenous neuroprotection in the retina given that these peptides feature neuroprotective properties in the retina as well.

VIP, PACAP-38, BDNF and ADNP in NMDA-induced excitotoxicity in the rat retina

To evaluate the effect of intravitreal injection of N-methyl-D-aspartate (NMDA) on brain-derived neurotrophic factor (BDNF), pituitary adenylate cyclase-activating peptide-38 (PACAP-38), vasoactive intestinal peptide (VIP) and the VIP-associated glial protein activity-dependent neuroprotective protein (ADNP) in the rat retina. These elements have well-documented neuroprotective properties and may thus be integrated in endogenous neuroprotective mechanisms in the retina which break down in NMDA excitotoxicity. A volume of 2 μl of 100 nmol NMDA was intravitreally injected into one eye of rats, the untreated eye served as a control. Time-dependent effects of NMDA on VIP, PACAP-38 and BDNF were detected by radioimmunoassay and ELISA, and the effect on the expression of VIP, PACAP-38 and ADNP was evaluated by quantitative RT-PCR 20 days after NMDA injection. Topical flunarizine served to find out whether the effect of NMDA is counteracted. Compared to PACAP-38, VIP levels significantly decreased on days 1, 7, 14, 28 and 56 after NMDA injection indicating that VIPergic cells are more vulnerable than PACAP-38-expressing cells. The expression of VIP and ADNP but not of PACAP-38 was found to be reduced, and application of topical flunarizine counteracted the decrease of VIP. BDNF levels significantly increased after days 1 and 3. The early upregulation of BDNF seems to act neuroprotectively and leads to a delay of ganglion cell loss. Although there is no direct evidence, the decrease of VIP and ADNP - the consequence of the presence of NMDA receptors on these peptide-expressing cells - might contribute to the breakdown of endogenous neuroprotective mechanisms given that the decrease of the VIP-related ADNP runs in parallel with the decrease of VIP. Activating and maintaining these mechanisms must be the primary aim in the therapy of diseases with retinal neuronal degeneration.

Carnosine protects against permanent cerebral ischemia in histidine decarboxylase knockout mice by reducing glutamate excitotoxicity

Recently, we showed that carnosine protects against NMDA-induced excitotoxicity in differentiated PC12 cells through a histaminergic pathway. However, whether the protective effect of the carnosine metabolic pathway also occurs in ischemic brain is unknown. Utilizing the model of permanent middle cerebral artery occlusion (pMCAO) in mice, we found that carnosine significantly improved neurological function and decreased infarct size in both histidine decarboxylase knockout and the corresponding wild-type mice to the same extent. Carnosine decreased the glutamate levels and preserved the expression of glutamate transporter-1 (GLT-1) but not the glutamate/aspartate transporter in astrocytes exposed to ischemia in vivo and in vitro. It suppressed the dissipation of ΔΨ m and generation of mitochondrial reactive oxygen species (ROS) induced by oxygen–glucose deprivation in astrocytes. Furthermore, carnosine also decreased the mitochondrial ROS and reversed the decrease in GLT-1 induced by rotenone. These findings are the first to demonstrate that the mechanism of carnosine action in pMCAO may not be mediated by the histaminergic pathway, but by reducing glutamate excitotoxicity through the effective regulation of the expression of GLT-1 in astrocytes due to improved mitochondrial function. Thus, our study reveals a novel antiexcitotoxic agent in ischemic injury.

Mapping the high‐affinity binding domain of 5‐substituted benzimidazoles to the proximal N‐terminus of the GluN2B subunit of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors represent an attractive drug target for the treatment of neurological and neurodegenerative disorders associated with glutamate-induced excitotoxicity. The aim of this study was to map the binding domain of high affinity 5-substituted benzimidazole derivatives [N-{2-[(4-benzylpiperidin-1-yl)methyl]benzimidazol-5-yl}methanesulphonamide (XK1) and N-[2-(4-phenoxybenzyl)benzimidazol-5-yl]methanesulphonamide (XK2)] on the GluN2B subunit of the NMDA receptor. The pharmacological antagonistic profiles of XK1 and XK2 were assessed using in vitro rat primary cerebrocortical neurones and two-electrode voltage clamp on Xenopus oocytes expressing heterologous GluN1/GluN2B receptors. Direct ligand binding was determined using the recombinant amino-terminal domain (ATD) of GluN2B. XK1 and XK2 effectively protected against NMDA-induced excitotoxicity in rat primary cortical neurones. Low concentrations of XK1 (10 nM) and XK2 (1 nM) significantly reversed neuronal death. Both compounds failed to inhibit currents measured from oocytes heterologously expressing GluN1-1a subunit co-assembled with the ATD-deleted GluN2B subunit. XK1 and XK2 showed specific binding to recombinant protein of GluN2B ATD with low nanomolar affinities. Several residues in the recombinant ATD of GluN2B were identified to be critical for conferring XK1 and XK2 sensitivity. The inhibitory effects of XK1 and XK2 were pH-sensitive, being increased at acidic pH. These results demonstrate that XK1 and XK2 are effective neuroprotective agents in vitro and indicate that 5-substituted benzimidazole derivatives inhibit GluN1/GluN2B receptors via direct binding to the ATD of the GluN2B subunit. These compounds represent valuable alternatives to the classical antagonist ifenprodil as pharmacological tools for studying GluN2B-containing NMDA receptors.

Ischaemia differentially regulates GABA B receptor subunits in organotypic hippocampal slice cultures

Reduced synaptic inhibition due to dysfunction of ionotropic GABA A receptors has been proposed as one factor in cerebral ischaemia-induced excitotoxic cell death. However, the participation of the inhibitory metabotropic GABA B receptors in these pathological processes has not been extensively investigated. We used oxygen–glucose deprivation (OGD) and NMDA-induced excitotoxicity as models to investigate whether ischaemia-like challenges alter the protein levels of GABA B1 and GABA B2 receptor subunits in rat organotypic hippocampal slice cultures. Twenty-four hours after the insult both OGD and NMDA produced a marked decrease in the total levels of GABA B2 (∼75%), while there was no significant change in the levels of GABA B1 after OGD, but an increase after NMDA treatment (∼100%). The GABA B receptor agonist baclofen (100 μM) was neuroprotective following OGD or NMDA treatment if added before or during the insult. GABA B receptors comprise heterodimers of GABA B1 and GABA B2 subunits and our results suggest that the separate subunits are independently regulated in response to extreme neuronal stress. However, because GABA B2 is required for functional surface expression, down-regulation of this subunit removes an important inhibitory feedback mechanism under pathological conditions.

Neuroprotective Effect of Fucoidan againstN-methyl-D-aspartate-Induced Excitotoxicity in Rat Hippocampus

Fucoidan, a sulfated polyanionic polymer of L-fucose, is obtained from brown marine macroalgae. In the present study, neuroprotective effect of fucoidan against N-methylD-aspartate (NMDA)-induced excitotoxicity in the hippocampus was investigated. The patch clamp study revealed that fucoidan significantly inhibited NMDA receptor-activated ion current in the acutely dissociated hippocampal CA1 neurons. In an organotypic hippocampal slice culture, fucoidan inhibited NMDA-induced neuronal cell death in a dose-dependent manner. The present study showed that fucoidan possesses a neuroprotective effect against NMDA-induced excitotoxicity, and that the suppressive effect of fucoidan on the NMDA-induced ion current can be suggested as being the underlying neuroprotective mechanism of fucoidan.