Contribution of NMDA and Non-NMDA Receptors to In vivo Glutamate-Induced Calpain Activation in the Rat Striatum. Relation to Neuronal Damage

Glutamate, the major excitatory neurotransmitter, can cause the death of neurons by a mechanism known as excitotoxicity. This is a calcium-dependent process and activation of the NMDA receptor subtype contributes mainly to neuronal damage, due to its high permeability to calcium. Activation of calpain, a calcium-dependent cysteine protease, has been implicated in necrotic excitotoxic neuronal death. We have investigated the contribution of NMDA and non-NMDA ionotropic receptors to calpain activation and neuronal death induced by the acute administration of glutamate into the rat striatum. Calpain activity was assessed by the cleavage of the cytoskeletal protein, α-spectrin. Caspase-3 activity was also studied because glutamate can also lead to apoptosis. Results show no caspase-3 activity, but a strong calpain activation involving both NMDA and non-NMDA receptors. Although neuronal damage is mediated mainly by the NMDA receptor subtype, it can not be attributed solely to calpain activity. Special issue article in honor of Dr. Ricardo Tapia.     

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.     

Glucagon-like peptide 1 modulates calcium responses to glutamate and membrane depolarization in hippocampal neurons

Glucagon-like peptide 1 (GLP-1) activates receptors coupled to cAMP production and calcium influx in pancreatic cells, resulting in enhanced glucose sensitivity and insulin secretion. Despite evidence that the GLP-1 receptor is present and active in neurons, little is known of the roles of GLP-1 in neuronal physiology. As GLP-1 modulates calcium homeostasis in pancreatic beta cells, and because calcium plays important roles in neuronal plasticity and neurodegenerative processes, we examined the effects of GLP-1 on calcium regulation in cultured rat hippocampal neurons. When neurons were pre-treated with GLP-1, calcium responses to glutamate and membrane depolarization were attenuated. Whole-cell patch clamp analyses showed that glutamate-induced currents and currents through voltage-dependent calcium channels were significantly decreased in neurons pre-treated with GLP-1. Pre-treatment of neurons with GLP-1 significantly decreased their vulnerability to death induced by glutamate. Acute application of GLP-1 resulted in a transient elevation of intracellular calcium levels, consistent with the established effects of GLP-1 on cAMP production and activation of cAMP response element-binding protein. Collectively, our findings suggest that, by modulating calcium responses to glutamate and membrane depolarization, GLP-1 may play important roles in regulating neuronal plasticity and cell survival.     

Bidirectional regulation of neuronal nitric-oxide synthase phosphorylation at serine 847 by the N-methyl-D-aspartate receptor

At glutamatergic synapses, the scaffolding protein PSD95 links the neuronal isoform of nitric-oxide synthase (nNOS) to the N-methyl-d-aspartate (NMDA) receptor. Phosphorylation of nNOS at serine 847 (Ser(847)) by the calcium-calmodulin protein kinase II (CaMKII) inhibits nNOS activity, possibly by blocking the binding of Ca(2+)-CaM. Here we show that the NMDA mediates a novel bidirectional regulation of Ser(847) phosphorylation. nNOS phosphorylated at Ser(847) colocalizes with the NMDA receptor at spines of cultured hippocampal neurons. Treatment of neurons with 5 microm glutamate stimulated CaMKII phosphorylation of nNOS at Ser(847), whereas excitotoxic concentrations of glutamate, 100 and 500 microm, induced Ser(847)-PO(4) dephosphorylation by protein phosphatase 1. Strong NMDA receptor stimulation was likely to activate nNOS under these conditions because protein nitration to form nitrotyrosine, a marker of nNOS activity, correlated in individual neurons with Ser(847)-PO(4) dephosphorylation. Of particular note, stimulation with low glutamate that increased phosphorylation of nNOS at Ser(847) could be reversed by subsequent high glutamate treatment which induced dephosphorylation. The reversibility of NMDA receptor-induced phosphorylation at Ser(847) by different doses of glutamate suggests two mechanisms with opposite effects: 1). a time-dependent negative feedback induced by physiological concentrations of glutamate that limits nNOS activation and precludes the overproduction of NO; and 2). a pathological stimulation by high concentrations of glutamate that leads to unregulated nNOS activation and production of toxic levels of NO. These mechanisms may share pathways, respectively, with NMDA receptor-induced forms of synaptic plasticity and excitotoxicity.     

Basic Fibroblast Growth Factor Selectively Increases AMPA-Receptor Subunit GluR1 Protein Level and Differentially Modulates Ca2+ Responses to AMPA and NMDA in Hippocampal Neurons

Abstract: The excitatory neurotransmitter glutamate is believed to play important roles in development, synaptic plasticity, and neurodegenerative conditions. Recent studies have shown that neurotrophic factors can modulate neuronal excitability and survival and neurite outgrowth responses to glutamate, but the mechanisms are unknown. The present study tested the hypothesis that neurotrophic factors modulate responses to glutamate by affecting the expression of specific glutamate-receptor proteins. Exposure of cultured embryonic rat hippocampal cells to basic fibroblast growth factor (bFGF) resulted in a concentration-dependent increase in levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor subunit GluR1 protein as determined by western blot, dot-blot, and immunocytochemical analyses. In contrast, bFGF did not alter levels of GluP2/3, GluR4, or the NMDA-receptor subunit NR1. Nerve growth factor did not affect GluR1 levels. Calcium-imaging studies revealed that elevation of [Ca²⁺]_i, resulting from selective AMPA-receptor activation, was enhanced in bFGF-pretreated neurons. On the other hand, [Ca²⁺]_i responses to NMDA-receptor activation were suppressed in bFGF-treated neurons, consistent with previous studies showing that bFGF can protect neurons against NMDA toxicity. Moreover, neurons pretreated with bFGF were relatively resistant to the toxicities of glutamate and AMPA, both of which were shown to be mediated by NMDA receptors. These data suggest that differential regulation of the expression of specific glutamate-receptor subunits may be an important mechanism whereby neurotrophic factors modulate activity-dependent neuronal plasticity and vulnerability to excitotoxicity.     

Involvement of the GluN2A and GluN2B Subunits in Synaptic and Extrasynaptic N-methyl-d-aspartate Receptor Function and Neuronal Excitotoxicity

GluN2A and GluN2B are the major subunits of functional NMDA receptors (NMDAR). Previous studies have suggested that GluN2A and GluN2B may differentially mediate NMDAR function at synaptic and extrasynaptic locations and play opposing roles in excitotoxicity, such as neurodegeneration triggered by ischemic stroke and brain injury. By using pharmacological and molecular approaches to suppress or enhance the function of GluN2A and GluN2B in cultured cortical neurons, we examined NMDAR-mediated, bidirectional regulation of prosurvival signaling (i.e. the cAMP response element-binding protein (CREB)-Bdnf cascade) and cell death. Inhibition of GluN2A or GluN2B attenuated the up-regulation of prosurvival signaling triggered by the activation of either synaptic or extrasynaptic NMDAR. Inhibition of GluN2A or GluN2B also attenuated the down-regulation of prosurvival signaling triggered by the coactivation of synaptic and extrasynaptic receptors. The effects of GluN2B on CREB-Bdnf signaling were larger than those of GluN2A. Consistently, compared with suppression of GluN2A, suppression of GluN2B resulted in more reduction of NMDA- and oxygen glucose deprivation-induced excitotoxicity as well as NMDAR-mediated elevation of intracellular calcium. Moreover, excitotoxicity and down-regulation of CREB were exaggerated in neurons overexpressing GluN2A or GluN2B. Together, we found that GluN2A and GluN2B are involved in the function of both synaptic and extrasynaptic NMDAR, demonstrating that they play similar rather than opposing roles in NMDAR-mediated bidirectional regulation of prosurvival signaling and neuronal death.     

Excitotoxicity induced by enhanced excitatory neurotransmission in cultured hippocampal pyramidal neurons.

Cultures of rat hippocampal pyramidal neurons were used to examine the roles of excitatory synaptic transmission, NMDA receptors, and elevated [Ca2+]i in the production of excitotoxicity. In integral of 70% of the cells observed, perfusion with Mg2(+)-free, glycine-supplemented medium induced large spontaneous fluctuations or maintained plateaus of [Ca2+]i. [Ca2+]i fluctuations could be blocked by tetrodotoxin, NMDA receptor antagonists, dihydropyridines, or compounds that inhibit synaptic transmission in the hippocampus, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. When cells were treated with Mg2(+)-free, glycine-supplemented medium and examined 24 hr later, integral of 30% of the neurons were found to have died. Cell death could be inhibited by the same agents that reduced [Ca2+]i fluctuations. These results support a role for direct excitatory synaptic transmission, as opposed to the general release of glutamate, in excitotoxicity. A major role for synaptically activated NMDA receptors, rather than kainate/quisqualate receptors, is also indicated. Neuronal death may be produced by abnormal changes in neuronal [Ca2+]i.     

Roles of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling pathway in activity-dependent dendritic protein synthesis in hippocampal neurons

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by alphaCaMKII 5' and 3' untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous alphaCaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.     

Excitotoxic stimulation downregulates the ubiquitin–proteasome system through activation of NMDA receptors in cultured hippocampal neurons

Overactivation of glutamate receptors contributes to neuronal damage (excitotoxicity) in ischemic stroke but the detailed mechanisms are not fully elucidated. Brain ischemia is also characterized by an impairment of the activity of the proteasome, one of the major proteolytic systems in neurons. We found that excitotoxic stimulation with glutamate rapidly decreases ATP levels and the proteasome activity, and induces the disassembly of the 26S proteasome in cultured rat hippocampal neurons. Downregulation of the proteasome activity, leading to an accumulation of ubiquitinated proteins, was mediated by calcium entry through NMDA receptors and was only observed in the nuclear fraction. Furthermore, excitotoxicity-induced proteasome inhibition was partially sensitive to cathepsin-L inhibition and was specifically induced by activation of extrasynaptic NMDA receptors. Oxygen and glucose deprivation induced neuronal death and downregulated the activity of the proteasome by a mechanism dependent on the activation of NMDA receptors. Since deubiquitinating enzymes may regulate proteins half-life by counteracting ubiquitination, we also analyzed how their activity is regulated under excitotoxic conditions. Glutamate stimulation decreased the total deubiquitinase activity in hippocampal neurons, but was without effect on the activity of Uch-L1, showing that not all deubiquitinases are affected. These results indicate that excitotoxic stimulation with glutamate has multiple effects on the ubiquitin–proteasome system which may contribute to the demise process in brain ischemia and in other neurological disorders.     

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.     

Neuroprotective effects of vitexin by inhibition of NMDA receptors in primary cultures of mouse cerebral cortical neurons

The accumulation of glutamate can excessively activate the N-methyl-d-aspartate (NMDA) receptors and cause excitotoxicity. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside, Vit) is a c-glycosylated flavone which was found in the several herbs, exhibiting potent hypotensive, anti-inflammatory, and neuroprotective properties. However, little is known about the neuroprotective effects of Vit on glutamate-induced excitotoxicity. In present study, primary cultured cortical neurons were treated with NMDA to induce the excitotoxicity. Pretreatment with Vit significantly prevented NMDA-induced neuronal cell loss and reduced the number of apoptotic neurons. Vit significantly inhibited the neuronal apoptosis induced by NMDA exposure by regulating balance of Bcl-2 and Bax expression and the cleavages of poly (ADP-ribose) polymerase and pro-caspase 3. Furthermore, pretreatment of Vit reversed the up-regulation of NR2B-containing NMDA receptors and the intracellular Ca²⁺ overload induced by NMDA exposure. The neuroprotective effects of Vit are related to inhibiting the activities of NR2B-containing NMDA receptors and reducing the calcium influx in cultured cortical neurons.     

Psoralidin Stimulates Expression of Immediate-Early Genes and Synapse Development in Primary Cortical Neurons

Upon synaptic stimulation and glutamate release, glutamate receptors are activated to regulate several downstream effectors and signaling pathways resulting in synaptic modification. One downstream intracellular effect, in particular, is the expression of immediate-early genes (IEGs), which have been proposed to be important in synaptic plasticity because of their rapid expression following synaptic activation and key role in memory formation. In this study, we screened a natural compound library in order to find a compound that could induce the expression of IEGs in primary cortical neurons and discovered that psoralidin, a natural compound isolated from the seeds of Psoralea corylifolia, stimulated synaptic modulation. Psoralidin activated mitogen-activated protein kinase (MAPK) signaling, which in turn induced the expression of neuronal IEGs, particularly Arc, Egr-1, and c-fos. N-methyl-d-aspartate (NMDA) receptors activation and extracellular calcium influx were implicated in the psoralidin-induced intracellular changes. In glutamate dose–response curve, psoralidin shifted glutamate EC₅₀ to lower values without enhancing maximum activity. Interestingly, psoralidin increased the density, area, and intensity of excitatory synapses in primary hippocampal neurons, which were mediated by NMDA receptor activation and MAPK signaling. These results suggest that psoralidin triggers synaptic remodeling through activating NMDA receptor and subsequent MAPK signaling cascades and therefore could possibly serve as an NMDA receptor modulator.

     

Neuron-derived D-serine release provides a novel means to activate N-methyl-D-aspartate receptors

D-serine is a coagonist of N-methyl-D-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of D-serine is carried out by serine racemase, which converts L- to D-serine. D-serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of D-serine. We now report significant amounts of serine racemase and D-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and D-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and D-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived D-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous D-serine by addition of the recombinant enzyme D-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal D-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked D-serine release. Release of D-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. D-serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of D-serine. Glutamate-induced neuronal D-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.     

A highly sulfated chondroitin sulfate preparation, CS-E, prevents excitatory amino acid-induced neuronal cell death

Chondroitin sulfate (CS) is a major microenvironmental molecule in the CNS, and there have been few reports about its neuroprotective activity. As neuronal cell death by excitotoxicity is a crucial phase in many neuronal diseases, we examined the effect of various CS preparations on neuronal cell death induced by the excitotoxicity of glutamate analogs. CS preparations were added to cultured neurons before and after the administration of glutamate analogs. Then, the extents of both neuronal cell death and survival were estimated. Pre-administration of a highly sulfated CS preparation, CS-E, significantly reduced neuronal cell death induced by not only NMDA but also ( S )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate. Neither CS preparations other than CS-E nor other highly sulfated polysaccharides such as heparin and dextran sulfate exerted any neuroprotective effects. NMDA-induced current in neurons was not changed by pre-administration of CS-E, but the pattern of protein-tyrosine phosphorylation was changed. In addition, the elevation of caspase 3 activity was significantly suppressed in CS-E-treated neurons. These results indicate that CS-E prevents neuronal cell death mediated by various glutamate receptors, and suggest that phosphorylation-related intracellular signals and the suppression of caspase 3 activation are implicated in neuroprotection by CS-E.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

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.     

Enhanced expression of RNase L as a novel intracellular signal generated by NMDA receptors in mouse cortical neurons

Recently we showed that the level of mitochondrial mRNA was decreased prior to neuronal death induced by glutamate. As the level of mRNA is regulated by ribonuclease (RNase), we examined RNase activity and its expression in the primary cultures of cortical neurons after glutamate treatment in order to evaluate the involvement of RNase in glutamate-induced neuronal death. A 15-min exposure of the cultures to glutamate at the concentration of 100muM produced marked neuronal damage (more than 70% of total cells) at 24-h post-exposure. Under the experimental conditions used, RNA degradation was definitely observed at a period of 4-12-h post-exposure, a time when no damage was seen in the neurons. Glutamate-induced RNA degradation was completely prevented by the N-methyl-d-aspartic acid (NMDA) receptor channel blocker MK-801 or the NR2B-containing NMDA receptor antagonist ifenprodil. Glutamate exposure produced enhanced expression of RNase L at least 2-12h later, which was absolutely abolished by MK-801. However, no significant change was seen in the level of RNase H1 mRNA at any time point post-glutamate treatment. Immunocytochemical studies revealed that RNase L expressed in response to glutamate was localized within the nucleus, mitochondria, and cytoplasm in the neurons. Taken together, our data suggest that expression of RNase L is a signal generated by NMDA receptor in cortical neurons. RNase L expression and RNA degradation may be events that cause neuronal damage induced by NMDA receptor activation.     

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.     

NMDA受体信号复合体中蛋白质的相互作用

谷氨酸能兴奋性突触的突触后密集区(postsynaptic density,PSD)包含多种受体蛋白、骨架蛋白和信号蛋白,它们通过分子中特定的结构域相互识别并动态地结合,形成多个信号复合体,参与突触后受体功能的调节及其下游特异性信号转导通路的激活。其中,NMDA受体信号复合体中蛋白质-蛋白质的相互作用及其调控机制的阐明,对于深入了解神经发育、突触可塑性、兴奋性毒性等生理病理的分子机制有重要意义。