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

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

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

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

A role for polyamines in retinal ganglion cell excitotoxic death

Neuronal death due to excessive activation of N -methyl- d -aspartate (NMDA) receptors is a hallmark of neurodegenerative diseases. The polyamines: putrescine, spermine, and spermidine, bind to specific sites on the NMDA receptor and promote its activation, but their role in NMDA-induced neuronal death is ill defined. In this study, we characterized the role of polyamines in excitotoxic death of retinal ganglion cells (RGCs), a population of central neurons susceptible to NMDA-induced damage. Our data show that endogenous arginase I, the rate limiting enzyme for polyamine biosynthesis, is expressed in the intact, adult retina. Intraocular injection of NMDA visibly increased arginase I expression in Müller cells, the predominant glial cell-type in the mammalian retina. Inhibition of polyamine synthesis using di-fluoro-methyl-ornithine (DFMO) was markedly neuroprotective, while injection of exogenous polyamines in conjunction with NMDA exacerbated RGC death. Blockade of the polyamine binding sites on NMDA receptors using the non-competitive antagonist ifenprodil was neuroprotective, suggesting that polyamines contribute to excitotoxic death, at least partly, by binding to NMDA receptors. Importantly, we also demonstrate that NMDA leads to activation of both the Erk1/2 and PI3 K/Akt pathways, but only the PI3 K/Akt kinase was required for di-fluoro-methyl-ornithine-induced RGC survival. In summary, our study reveals that polyamines modulate neuronal death in the retina via different mechanisms that potentiate NMDA-triggered excitotoxicity.     

NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury

Excitotoxic neuronal damage via over-activation of the NMDA receptor has been implicated in many neurodegenerative diseases. In vitro modeling of excitotoxic injury has shown that activation of G-protein coupled receptors (GPCRs) counteracts such injury through modulation of neuronal pro-survival pathways and/or NMDA receptor signaling. We have previously demonstrated that the GPCR APJ and its endogenous neuropeptide ligand apelin can protect neurons against excitotoxicity, but the mechanism(s) of this neuroprotection remain incompletely understood. We hypothesized that apelin can promote neuronal survival by activating pro-survival signaling as well as inhibiting NMDA receptor-mediated excitotoxic signaling cascades. Our results demonstrate that (i) apelin activates pro-survival signaling via inositol trisphosphate (IP(3) ), protein kinase C (PKC), mitogen-activated protein kinase kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase-1/2 (ERK1/2) to protect against excitotoxicity, and (ii) apelin inhibits excitotoxic signaling by attenuating NMDA receptor and calpain activity, and by modulating NMDA receptor subunit NR2B phosphorylation at serine 1480. These studies delineate a novel apelinergic signaling pathway that concurrently promotes survival and limits NMDA receptor-mediated injury to protect neurons against excitotoxicity. Defining apelin-mediated neuroprotection advances our understanding of neuroprotective pathways and will potentially improve our ability to develop therapeutics for excitotoxicity-associated neurodegenerative disorders.     

Calpain-mediated mGluR1alpha truncation: a key step in excitotoxicity

Excitotoxicity mediated by glutamate receptors plays crucial roles in ischemia and other neurodegenerative diseases. Whereas overactivation of ionotropic glutamate receptors is neurotoxic, the role of metabotropic glutamate receptors (mGluRs), and especially mGluR1, remains equivocal. Here we report that activation of NMDA receptors results in calpain-mediated truncation of the C-terminal domain of mGluR1alpha at Ser(936). The truncated mGluR1alpha maintains its ability to increase cytosolic calcium while it no longer activates the neuroprotective PI(3)K-Akt signaling pathways. Full-length and truncated forms of mGluR1alpha play distinct roles in excitotoxic neuronal degeneration in cultured neurons. A fusion peptide derived from the calpain cleavage site of mGluR1alpha efficiently blocks NMDA-induced truncation of mGluR1alpha in primary neuronal cultures and exhibits neuroprotection against excitotoxicity both in vitro and in vivo. These findings shed light on the relationship between NMDA and mGluR1alpha and indicate the existence of a positive feedback regulation in excitotoxicity involving calpain and mGluR1alpha.     

Myelin-associated glycoprotein protects neurons from excitotoxicity

In addition to supporting rapid nerve conduction, myelination nurtures and stabilizes axons and protects them from acute toxic insults. One myelin molecule that protects and sustains axons is myelin-associated glycoprotein (MAG). MAG is expressed on the innermost wrap of myelin, apposed to the axon surface, where it interacts with axonal receptors that reside in lateral membrane domains including gangliosides, the glycosylphosphatidylinositol-anchored Nogo receptors, and β1-integrin. We report here that MAG protection extends beyond the axon to the neurons from which those axons emanate, protecting them from excitotoxicity. Compared to wild type mice, Mag-null mice displayed markedly increased seizure activity in response to intraperitoneal injection of kainic acid, an excitotoxic glutamate receptor agonist. Mag-null mice also had larger lesion volumes in response to intrastriatal injection of the excitotoxin NMDA. Prior injection of a soluble form of MAG partially protected Mag-null mice from NMDA-induced lesions. Hippocampal neurons plated on proteins extracted from wild-type rat or mouse myelin were resistant to kainic acid-induced excitotoxicity, whereas neurons plated on proteins from Mag-null myelin were not. Protection was reversed by anti-MAG antibody and replicated by addition of soluble MAG. MAG-mediated protection from excitotoxicity was dependent on Nogo receptors and β1-integrin. We conclude that MAG engages membrane-domain resident neuronal receptors to protect neurons from excitotoxicity, and that soluble MAG mitigates excitotoxic damage in vivo.     

The CB1 cannabinoid receptor mediates excitotoxicity-induced neural progenitor proliferation and neurogenesis

Endocannabinoids are lipid signaling mediators that exert an important neuromodulatory role and confer neuroprotection in several types of brain injury. Excitotoxicity and stroke can induce neural progenitor (NP) proliferation and differentiation as an attempt of neuroregeneration after damage. Here we investigated the mechanism of hippocampal progenitor cell engagement upon excitotoxicity induced by kainic acid administration and the putative involvement of the CB1 cannabinoid receptor in this process. Adult NPs express kainate receptors that mediate proliferation and neurosphere generation in vitro via CB1 cannabinoid receptors. Similarly, in vivo studies showed that excitotoxicity-induced hippocampal NPs proliferation and neurogenesis are abrogated in CB1-deficient mice and in wild-type mice administered with the selective CB1 antagonist rimonabant (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide; SR141716). Kainate stimulation increased basic fibroblast growth factor (bFGF) expression in cultured NPs in a CB1-dependent manner as this response was prevented by rimonabant and mimicked by endocannabinoids. Likewise, in vivo analyses showed that increased hippocampal expression of bFGF, as well as of brain-derived neurotrophic factor and epidermal growth factor, occurs upon excitotoxicity and that CB1 receptor ablation prevents this induction. Moreover, excitotoxicity increased the number of CB1+ bFGF+ cells, and this up-regulation preceded NP proliferation. In summary, our results show the involvement of the CB1 cannabinoid receptor in NP proliferation and neurogenesis induced by excitotoxic injury and support a role for bFGF signaling in this process.     

Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-D-aspartate receptor redistribution

Cyclodextrins (CDs) are cyclic oligosaccharides composed of a lipophilic central cavity and a hydrophilic outer surface. Some CDs are capable of extracting cholesterol from cell membranes and can affect function of receptors and proteins localized in cholesterol-rich membrane domains. In this report, we demonstrate the neuroprotective activity of some CD derivatives against oxygen-glucose deprivation (OGD), N-methyl-D-aspartic acid (NMDA) and glutamate in cortical neuronal cultures. Although all CDs complexed with NMDA or glutamate, only beta-, methylated beta- and sulfated beta-CDs displayed neuroprotective activity and lowered cellular cholesterol. Only CDs that lowered cholesterol levels redistributed the NMDA receptor NR2B subunit, PSD-95 (postsynaptic density protein 95 kDa) and neuronal nitric oxide synthase (nNOS) from Triton X-100 insoluble membrane domains to soluble fractions. Cholesterol repletion counteracted the ability of methylated beta-CD to protect against NMDA toxicity, and reversed NR2B, PSD-95 and nNOS localization to Triton X-100 insoluble membrane fraction. Surprisingly, neuroprotective CDs had minimal effect on NMDA receptor-mediated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), but did suppress OGD-induced increases in [Ca(2+)](i). beta-CD, but not Mbeta-CD, also caused a slight block of NMDA-induced currents, suggesting a minor contribution to neuroprotection by direct action on NMDA receptors. Taken together, data suggest that cholesterol extraction from detergent-resistant microdomains affects NMDA receptor subunit distribution and signal propagation, resulting in neuroprotection of cortical neuronal cultures against ischemic and excitotoxic insults. Since cholesterol-rich membrane domains exist in neuronal postsynaptic densities, these results imply that synaptic NMDA receptor subpopulations underlie excitotoxicity, which can be targeted by CDs without affecting overall neuronal Ca(2+) levels.     

Purinergic signaling induces cyclooxygenase-1-dependent prostanoid synthesis in microglia: roles in the outcome of excitotoxic brain injury

Cyclooxygenases (COX) are prostanoid synthesizing enzymes constitutively expressed in the brain that contribute to excitotoxic neuronal cell death. While the neurotoxic role of COX-2 is well established and has been linked to prostaglandin E(2) synthesis, the role of COX-1 is not clearly understood. In a model of N-Methyl-D-aspartic acid (NMDA) induced excitotoxicity in the mouse cerebral cortex we found a distinctive temporal profile of COX-1 and COX-2 activation where COX-1, located in microglia, is responsible for the early phase of prostaglandin E(2) synthesis (10 minutes after NMDA), while both COX-1 and COX-2 contribute to the second phase (3-24 hours after NMDA). Microglial COX-1 is strongly activated by ATP but not excitatory neurotransmitters or the Toll-like receptor 4 ligand bacterial lipopolysaccharide. ATP induced microglial COX-1 dependent prostaglandin E(2) synthesis is dependent on P2X7 receptors, extracellular Ca(2+) and cytoplasmic phospholipase A2. NMDA receptor activation induces ATP release from cultured neurons leading to microglial P2X7 receptor activation and COX-1 dependent prostaglandin E(2) synthesis in mixed microglial-neuronal cultures. Pharmacological inhibition of COX-1 has no effect on the cortical lesion produced by NMDA, but counteracts the neuroprotection exerted by inhibition of COX-2 or observed in mice lacking the prostaglandin E(2) receptor type 1. Similarly, the neuroprotection exerted by the prostaglandin E(2) receptor type 2 agonist butaprost is not observed after COX-1 inhibition. P2X7 receptors contribute to NMDA induced prostaglandin E(2) production in vivo and blockage of P2X7 receptors reverses the neuroprotection offered by COX-2 inhibition. These findings suggest that purinergic signaling in microglia triggered by neuronal ATP modulates excitotoxic cortical lesion by regulating COX-1 dependent prostanoid production and unveil a previously unrecognized protective role of microglial COX-1 in excitotoxic brain injury.     

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

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

Selective and biphasic effect of the membrane lipid peroxidation product 4-hydroxy-2,3-nonenal on N-methyl-D-aspartate channels

Increased oxyradical production and membrane lipid peroxidation occur in neurons under physiological conditions and in neurodegenerative disorders. Lipid peroxidation can alter synaptic plasticity and may increase the vulnerability of neurons to excitotoxicity, but the underlying mechanisms are unknown. We report that 4-hydroxy-2,3-nonenal (4HN), an aldehyde product of lipid peroxidation, exerts a biphasic effect on NMDA-induced current in cultured rat hippocampal neurons with current being increased during the first 2 h and decreased after 6 h. Similarly, 4HN causes an early increase and a delayed decrease in NMDA-induced elevation of intracellular Ca2+ levels. In contrast, 4HN affects neither the ion current nor the Ca2+ response to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). The initial enhancement of NMDA-induced current is associated with increased phosphorylation of the NR1 receptor subunit, whereas the delayed suppression of current is associated with cellular ATP depletion and mitochondrial membrane depolarization. Cell death induced by 4HN is attenuated by an NMDA receptor antagonist, but not by an AMPA receptor antagonist. A secreted form of amyloid precursor protein, previously shown to protect neurons against oxidative and excitotoxic insults, prevented each of the effects of 4HN including the early and late changes in NMDA current, delayed ATP depletion, and cell death. These findings show that the membrane lipid peroxidation product 4HN can modulate NMDA channel activity, suggesting a role for this aldehyde in physiological and pathophysiological responses of neurons to oxidative stress.     

Neural cell adhesion molecule-associated polysialic acid inhibits NR2B-containing N-methyl-D-aspartate receptors and prevents glutamate-induced cell death

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.     

Overactivation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate but not kainate receptors inhibits phosphatidylcholine synthesis before excitotoxic neuronal death

Glutamate receptor overactivation induces excitotoxic neuronal death, but the contribution of glutamate receptor subtypes to this excitotoxicity is unclear. We have previously shown that excitotoxicity by NMDA receptor overactivation is associated with choline release and inhibition of phosphatidylcholine synthesis. We have now investigated whether the ability of non-NMDA ionotropic glutamate receptor subtypes to induce excitotoxicity is related to the ability to inhibit phosphatidylcholine synthesis. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced a concentration-dependent increase in extracellular choline and inhibited phosphatidylcholine synthesis when receptor desensitization was prevented. Kainate released choline and inhibited phosphatidylcholine synthesis by an action at AMPA receptors, because these effects of kainate were blocked by the AMPA receptor antagonist LY300164. Selective activation of kainate receptors failed to release choline, even when kainate receptor desensitization was prevented. The inhibition of phosphatidylcholine synthesis evoked by activation of non-desensitizing AMPA receptors was followed by neuronal death. In contrast, specific kainate receptor activation, which did not inhibit phosphatidylcholine synthesis, did not produce neuronal death. Choline release and inhibition of phosphatidylcholine synthesis were induced by AMPA at non-desensitizing AMPA receptors well before excitotoxicity. Furthermore, choline release by AMPA required the entry of Ca(2+) through the receptor channel. Our results show that AMPA, but not kainate, receptor overactivation induces excitotoxic cell death, and that this effect is directly related to the ability to inhibit phosphatidylcholine synthesis. Moreover, these results indicate that inhibition of phosphatidylcholine synthesis is an early event of the excitotoxic process, downstream of glutamate receptor-mediated Ca(2+) overload.     

Nitric oxide-induced blockade of NMDA receptors.

We studied the effects of nitric oxide (NO)-producing agents on N-methyl-D-aspartate (NMDA) receptor activation in cultured neurons. 3-Morpholino-sydnonimine (SIN-1) blocked both NMDA-induced currents and the associated increase in intracellular Ca2+. The actions of SIN-1 were reversible and suppressed by hemoglobin. A degraded SIN-1 solution that did not release NO was unable to block NMDA receptors. This showed that the SIN-1 effects were due to NO and not to another breakdown product. Similar results were obtained with 1-nitrosopyrrolidine (an NO-containing drug) and with NO released from NaNO2. Pretreatment with hemoglobin potentiated NMDA-induced effects, demonstrating that endogenous NO modulates NMDA receptors. Since NMDA receptor activation induces NO synthesis, these results suggest a feedback inhibition of NMDA receptors by NO under physiological condition.     

Ethanol inhibition of NMDA receptors under conditions of altered protein kinase A activity

N-methyl-D-aspartate receptors (NMDA) are glutamate-activated ligand-gated ion channels that participate in diverse forms of synaptic plasticity as well as glutamate-dependent excitotoxicity. Inhibition of the NMDA receptor function may underlie some of the behavioral actions associated with acute exposure to ethanol. The sensitivity of NMDA receptors to ethanol is influenced by the subunit composition of the receptor and, by association, with certain cytoskeletal proteins. Previous studies have also suggested that phosphorylation may regulate the sensitivity of NMDA receptors to ethanol. In this study, the ethanol inhibition of recombinant NMDA receptor currents was determined under conditions designed to enhance or inhibit the activity of protein kinase A (PKA). Human embryonic kidney 293 (HEK293) cells were transfected with cDNAs encoding NMDA subunits and channel activity was monitored with whole-cell patch-clamp electrophysiology. Under control recording conditions, ethanol (100 mM) inhibited NR1/2A and NR1/2B receptor currents by approximately 25-30%. The degree of ethanol inhibition was not affected or was slightly enhanced under conditions designed to enhance PKA activity. This included treatment of cells with cAMP analogs, inclusion of phosphatase inhibitors or purified PKA in the pipette filling solution, co-expression of catalytically active PKA, expression of the NR1 PKA-site phosphorylation site mimic (S897D) or by co-expression of the PKA scaffolding protein yotiao or the dopamine D(1) receptor. Ethanol inhibition of NR1/2A and NR1/2B receptors was not altered when PKA effects were suppressed, either by co-expression of a PKI inhibitory peptide or the phosphorylation-deficient NR1 mutants (S897A, S896A, S896A/S897A). In addition, ethanol inhibition of NMDA-induced currents in cultured cortical or hippocampal neurons was not affected by modulators of PKA. These results suggest that PKA does not appear to play a major role in determining the acute ethanol sensitivity of NMDA receptors.     

Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death

Excitotoxicity is one of the most extensively studied processes of neuronal cell death, and plays an important role in many central nervous system (CNS) diseases, including CNS ischemia, trauma, and neurodegenerative disorders. First described by Olney, excitotoxicity was later characterized as an excessive synaptic release of glutamate, which in turn activates postsynaptic glutamate receptors. While almost every glutamate receptor subtype has been implicated in mediating excitotoxic cell death, it is generally accepted that the N-methyl-D-aspartate (NMDA) subtypes play a major role, mainly owing to their high calcium (Ca²⁺) permeability. However, other glutamate receptor subtypes such as 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate (AMPA) or kainate receptors have also been attributed a critical role in mediating excitotoxic neuronal cell death. Although the molecular basis of glutamate toxicity is uncertain, there is general agreement that it is in large part Ca²⁺-dependent. The present review is aimed at summarizing the molecular mechanisms of NMDA receptor and AMPA/kainate receptor-mediated excitotoxic neuronal cell death.     

S-Nitrosylation and uncompetitive/fast off-rate (UFO) drug therapy in neurodegenerative disorders of protein misfolding

Although activation of glutamate receptors is essential for normal brain function, excessive activity leads to a form of neurotoxicity known as excitotoxicity. Key mediators of excitotoxic damage include overactivation of N-methyl-D-aspartate (NMDA) receptors, resulting in excessive Ca(2+) influx with production of free radicals and other injurious pathways. Overproduction of free radical nitric oxide (NO) contributes to acute and chronic neurodegenerative disorders. NO can react with cysteine thiol groups to form S-nitrosothiols and thus change protein function. S-nitrosylation can result in neuroprotective or neurodestructive consequences depending on the protein involved. Many neurodegenerative diseases manifest conformational changes in proteins that result in misfolding and aggregation. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. Molecular chaperones - such as protein-disulfide isomerase, glucose-regulated protein 78, and heat-shock proteins - can provide neuroprotection by facilitating proper protein folding. Here, we review the effect of S-nitrosylation on protein function under excitotoxic conditions, and present evidence that NO contributes to degenerative conditions by S-nitrosylating-specific chaperones that would otherwise prevent accumulation of misfolded proteins and neuronal cell death. In contrast, we also review therapeutics that can abrogate excitotoxic damage by preventing excessive NMDA receptor activity, in part via S-nitrosylation of this receptor to curtail excessive activity.     

Calpain-mediated degradation of myocyte enhancer factor 2D contributes to excitotoxicity by activation of extrasynaptic N-methyl-D-aspartate receptors

Synaptic and extrasynaptic NMDA receptors (NMDARs) appear to play opposite roles in neuronal survival and death. Here we report the new findings on the dysregulation of survival factor, myocyte enhancer factor 2D (MEF2D), by extrasynaptic NMDARs. Excitotoxicity led to the NMDAR-dependent degradation of MEF2D protein and inhibition of its transactivation activity in mature cortical neurons. The activation of extrasynaptic NMDARs alone was sufficient for degradation of MEF2D. Calpain directly cleaved MEF2D in vitro and blocking this protease activity greatly attenuated NMDAR signaled degradation of MEF2D in neurons. Consistently, inhibition of calpain protected cortical neurons from NMDA-induced excitotoxicity. Furthermore, knockdown of MEF2D sensitized neurons to NMDA-induced excitotoxicity, which was not protected by calpain inhibition. Collectively, these findings suggest that dysregulation of MEF2D by calpain may mediate excitotoxicity via an extrasynaptic NMDAR-dependent manner.     

Dendrimer-mediated siRNA delivery knocks down Beclin 1 and potentiates NMDA-mediated toxicity in rat cortical neurons

Autophagy is an important process which plays a key role in cellular homeostasis by degrading cytoplasmic components in the lysosomes, which facilitates recycling. Alterations to normal autophagy have been linked to excitotoxicity, but the mechanisms governing its signal transduction remain unclear. The aim of this study was to explore the role of autophagy in neuronal excitotoxic death by delivering small interfering RNA (siRNA) to rat cortical neurons, using a dendrimer to silence the autophagy-related gene 6 (beclin 1) and to determine the role of autophagy in excitotoxicity. We have found that the dendrimer is very efficient to deliver siRNA to rat cortical neurons, leading to almost complete removal of the target protein Beclin 1. In addition, NMDA increases autophagy markers, such as the protein levels of Beclin 1, the microtubule-associated light chain 3 (LC3) B-II/LC3B-I ratio, and monodansylcadaverine (MDC) labeling in rat cortical neurons. Moreover, NMDA also increases the formation of autophagosomes observed under a transmission electron microscope. Silencing beclin 1 expression blocked NMDA-induced autophagy. Moreover, Beclin 1 removal potentiated NMDA-induced neuronal death indicating that autophagy plays a protective role during excitotoxicity and suggesting that targeting autophagy might be a helpful therapeutic strategy in neurodegenerative diseases.     

S-methyl-N,N-diethylthiocarbamate sulfoxide elicits neuroprotective effect against N-methyl-D-aspartate receptor-mediated neurotoxicity

Glutamatergic neurotransmission, particularly of the NMDA receptor type, has been implicated in the excitotoxic response to several external and internal stimuli. In the present investigation, we report that S-methyl-N,N-diethylthiocarbamate sulfoxide (DETC-MeSO) selectively and specifically blocks the NMDA receptor subtype of the glutamate receptors, and attenuates glutamate-induced neurotoxicity in rat-cultured primary neurons. Other major ionotropic glutamate receptor subtypes, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate, were insensitive to DETC-MeSO both in vitro and in vivo. Disulfiram, the parent compound of DETC-MeSO, also inhibits glutamate receptors partially in vivo; however, it fails to inhibit glutamate receptors in mice pretreated with N-butyl imidazole, a cytochrome P450 enzyme inhibitor, implicating the need for bioactivation of disulfiram to be an effective antagonist. We showed that glutamate-induced increase in (45)Ca2+ was attenuated in rat-cultured primary neurons following pretreatment with DETC-MeSO. The Ca2+ influx into primary neurons, studied by confocal microscopy of the fluorescent Ca2+ dye fura-2, demonstrated a complete attenuation of NMDA-induced Ca2+ influx. Similarly, DETC-MeSO attenuated NMDA-induced (45)Ca2+ uptake. Glutamate-induced (45)Ca2+ uptake and Ca2+ influx, however, were partially blocked by DETC-MeSO, and this is consistent with both in vitro and in vivo studies in which DETC-MeSO partially blocked mouse brain glutamate receptors. In addition, DETC-MeSO pretreatment effectively prevented seizures in mice induced either by NMDA, ammonium acetate, or ethanol-induced kindling seizures, all of which are believed to be mediated by NMDA receptors. These data demonstrate that DETC-MeSO produces the neuroprotective effect through antagonism of NMDA receptors in vivo.