Neuroprotective Effects of Hydroxysafflor Yellow A Against Excitotoxic Neuronal Death Partially Through Down-Regulation of NR2B-Containing NMDA Receptors

Hydroxysafflor yellow A (HSYA) is a component of the flower Carthamus tinctorius L. that elicits neuroprotective effects in vivo and in vitro. The purpose of this study was to investigate pharmacological properties of HSYA on neurotoxicity of glutamate in primary cultured rat cortical neurons along with its possible mechanism of action. After challenge with N-methyl-d-aspartate (NMDA, 100 μM) for 30 min, loss of cell viability and excessive apoptotic cell death were observed in cultured cortical neurons. However, the excitotoxic neuronal death was attenuated markedly by HSYA treatment. Western blot analysis revealed that HSYA decreased expression of Bax and rescued the balance of pro-and anti-apoptotic proteins. In addition, HSYA significantly reversed up-regulation of NR2B-containing NMDA receptors by exposure to NMDA, while it did not affect the expression of NR2A-containing NMDA receptors. These finding suggest that HSYA protects cortical neurons, at least partially, from inhibiting the expression NR2B-containing NMDA receptors and by regulating Bcl-2 family.     

Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation

The therapeutic mechanisms of lithium for treating bipolar mood disorder remain poorly understood. Recent studies demonstrate that lithium has neuroprotective actions against a variety of insults. Here, we studied neuroprotective effects of lithium against excitotoxicity in cultured cerebral cortical neurons. Glutamate-induced excitotoxicity in cortical neurons was exclusively mediated by NMDA receptors. Pre-treatment of cortical neurons with LiCl time-dependently suppressed excitotoxicity with maximal protection after 6 days of pre-treatment. Significant protection was observed at the therapeutic and subtherapeutic concentration of 0.2-1.6 mm LiCl with almost complete protection at 1 mM. Neuroprotection was also elicited by valproate, another major mood-stabilizer. The neuroprotective effects of lithium coincided with inhibition of NMDA receptor-mediated calcium influx. Lithium pre-treatment did not alter total protein levels of NR1, NR2A and NR2B subunits of NMDA receptors. However, it did markedly reduce the level of NR2B phosphorylation at Tyr1472 and this was temporally associated with its neuroprotective effect. Because NR2B tyrosine phosphorylation has been positively correlated with NMDA receptor-mediated synaptic activity and excitotoxicity, the suppression of NR2B phosphorylation by lithium is likely to result in the inactivation of NMDA receptors and contributes to neuroprotection against excitotoxicity. This action could also be relevant to its clinical efficacy for bipolar patients.     

芝麻素抑制NMDA诱导的小鼠皮层神经元损伤

目的:研究芝麻素对NMDA所致原代培养小鼠皮层神经元损伤的保护作用及其机制。方法:体外培养原代皮层神经元;免疫荧光染色鉴定细胞纯度;MTT法测定细胞存活率;Hoechst/PI双染色观察细胞凋亡的形态学变化;激光共聚焦显微镜技术观察钙成像,检测细胞内钙离子浓度变化;Western blot检测各组细胞中Bcl-2、Bax、NR2A、NR2B蛋白的表达。结果:NMDA(200μM)60分钟能使神经元细胞存活率显著下降,细胞凋亡百分比明显增加(P0.01),芝麻素(0.1μM)能提高细胞存活率,减少细胞凋亡(P0.01)。与NMDA组相比,芝麻素能抑制钙超载;降低Bax和NR2B蛋白表达;增加和Bcl-2蛋白表达(P0.01)。结论:芝麻素具有神经保护作用,这种作用可能与抑制钙超载、下调NMDA受体亚型NR2B的表达以及调节Bcl-2家族蛋白有关。     

NR2A and NR2B subunits differentially mediate MAP kinase signaling and mitochondrial morphology following excitotoxic insult

NMDA receptors are essential for neurotransmission and key mediators of synaptic signaling, but they can also trigger deleterious degenerative processes that lead to cell death. Growing evidence suggests that selective blockade of the heterogeneous subunits that comprise the NMDA receptor may enable better control of pharmacotherapies for treating neurological diseases and injuries. We investigated the relationship between NMDAR activation, MAPK signaling, and mitochondrial shape following an excitotoxic insult. NR2A- and NR2B-containing NMDARs differentially mediated acute changes in cytosolic calcium, alterations in mitochondrial morphology, and phosphorylation of the MAPKs ERK and JNK. Activation of NR2A-containing NMDARs was associated with JNK phosphorylation that was neuroprotective in neuronal cultures subjected to excitotoxicity. In contrast, activation of NR2B-containing NMDARs triggered calcium accumulation in mitochondria that was strongly associated with mitochondrial swelling and neuronal cell death. Indeed, while blockade of NR2B-containing receptors was neuroprotective, this protection was lost when NR2A-initiated JNK phosphorylation was inhibited. Given the modest selectivity of the NR2A inhibitor, NVP-AAM077, the results highlight the significance of the relative, rather than absolute, activation of these two NMDA subtypes in modulating cell death pathways. Therefore, the balance between concurrent activation of NR2B-containing and NR2A-containing NMDARs dictates neuronal fate following excitotoxicity.     

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.     

Developmental expression of NMDA receptors in primary cortical cell culture: relationship to function and excitotoxicity

Over‐activation of the N‐methyl‐d ‐aspartate (NMDA) receptor results in a Ca²⁺‐dependent neurotoxicity termed excitotoxicity. Primary neuronal cell cultures are often used to study the mechanisms of excitotoxicity. While the expression of the NMDA receptor (NR) subunits and their relationship to Ca²⁺ entry/accumulation and excitotoxicity has been studied extensively, all three parameters have not been examined concurrently. To determine unequivocally whether developmental expression of NR protein and function do indeed coincide with the appearance of excitotoxicity, we examined the temporal relationship between NR subunit expression, NMDA‐induced Ca²⁺ accumulation, and NMDA‐mediated excitotoxicity simultaneously using sister plates derived from the same mixed cortical cell culture preparations. Western Blot analysis of total protein isolated from cells cultured for 1, 4, 7, 10 and 14 days revealed a time‐dependent increase in NR1, NR2A and NR2B subunit expression, which surprisingly did not correlate with NMDA receptor function, as assessed by measurement of NMDA‐induced ⁴⁵Ca²⁺ accumulation. However, when only NR subunit surface expression was quantified, a correlation between expression and ⁴⁵Ca²⁺ accumulation did indeed exist. To our surprise, the emergence of excitotoxicity did not show a direct relationship to ⁴⁵Ca²⁺ accumulation as has been reported previously. Thus, it appears that other factors besides total Ca²⁺ accumulation must contribute to the emergence of excitotoxicity in mixed murine cortical cell cultures. Acknowledgements: Supported by a grant from The Patrick and Catherine Weldon Donaghue Medical Foundation.     

Neuroprotection against traumatic brain injury by a peptide derived from the collapsin response mediator protein 2 (CRMP2)

Neurological disabilities following traumatic brain injury (TBI) may be due to excitotoxic neuronal loss. The excitotoxic loss of neurons following TBI occurs largely due to hyperactivation of N-methyl-d-aspartate receptors (NMDARs), leading to toxic levels of intracellular Ca(2+). The axon guidance and outgrowth protein collapsin response mediator protein 2 (CRMP2) has been linked to NMDAR trafficking and may be involved in neuronal survival following excitotoxicity. Lentivirus-mediated CRMP2 knockdown or treatment with a CRMP2 peptide fused to HIV TAT protein (TAT-CBD3) blocked neuronal death following glutamate exposure probably via blunting toxicity from delayed calcium deregulation. Application of TAT-CBD3 attenuated postsynaptic NMDAR-mediated currents in cortical slices. In exploring modulation of NMDARs by TAT-CBD3, we found that TAT-CBD3 induced NR2B internalization in dendritic spines without altering somal NR2B surface expression. Furthermore, TAT-CBD3 reduced NMDA-mediated Ca(2+) influx and currents in cultured neurons. Systemic administration of TAT-CBD3 following a controlled cortical impact model of TBI decreased hippocampal neuronal death. These findings support TAT-CBD3 as a novel neuroprotective agent that may increase neuronal survival following injury by reducing surface expression of dendritic NR2B receptors.     

Interleukin-2 inhibits NMDA receptor-mediated currents directly and may differentially affect subtypes

Using whole-cell patch-clamp recordings, this study investigated the effects of interleukin-2 (IL-2) on N-methyl-d-aspartate (NMDA) receptor-mediated currents (I(NMDA)) in rat cultured hippocampal neurons and human embryonic kidney (HEK) 293 cells expressing recombinant NMDA receptors. We found that IL-2 (0.01-1ng/ml) immediately and significantly decreased peak I(NMDA) in cultured neurons. Interestingly, the peak I(NMDA) induced in HEK 293 cells was also inhibited by IL-2. We also found that IL-2 differentially decreased the peak amplitudes of NR2A- and NR2B-containing NMDA receptor-mediated currents (I(NR2A) and I(NR2B)) by 54+/-5% and 30+/-4%, respectively. These results provide new evidence that IL-2 induces rapid inhibition of peak currents of NMDA receptor-mediated responses with possible NR1/NR2A and NR1/NR2B subtype-differentiation, and suggest that the inhibition is mediated by direct interaction between IL-2 and NMDA receptors.     

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl-d-aspartate receptors in cultured rat hippocampal neurons

We have previously shown that mitochondrial membrane potential disruption is involved in mechanisms underlying differential vulnerabilities to the excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors between primary cultured neurons prepared from rat cortex and hippocampus. To further elucidate the role of mitochondria in the excitotoxicity after activation of NMDA receptors, neurons were loaded with the fluorescent dye calcein diffusible in the cytoplasm and organelles for determination of the activity of mitochondrial permeability transition pore (mPTP) responsible for the leakage of different mitochondrial molecules. The addition of CoCl₂ similarly quenched the intracellular fluorescence except mitochondria in both cultured neurons, while further addition of NMDA led to a leakage of the dye into the cytoplasm in hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of viability in hippocampal neurons, while an activator of mPTP induced a similarly potent loss of viability in cortical and hippocampal neurons. Although NMDA was more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium indicator in hippocampal than cortical neurons, a mitochondrial calcium uniporter inhibitor significantly prevented the NMDA-induced loss of viability in hippocampal neurons. Expression of mRNA was significantly higher for the putative uniporter uncoupling protein-2 in hippocampal than cortical neurons. These results suggest that mitochondrial calcium uniporter would be at least in part responsible for the NMDA neurotoxicity through a mechanism relevant to promotion of mPTP orchestration in hippocampal neurons.     

Genetic evidence for adenylyl cyclase 1 as a target for preventing neuronal excitotoxicity mediated by N-methyl-D-aspartate receptors

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.     

CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival

Homeostatic chemokines, such as CXCL12, can affect neuronal activity by the regulation of inhibitory and excitatory neurotransmission, but the mechanisms involved are still undefined. Our previous studies have shown that CXCL12 protects cortical neurons from excitotoxicity by promoting the function of the gene-repressor protein Rb, which is involved in the recruitment of chromatin modifiers (such as histone deacetylases (HDACs)) to gene promoters. In neurons, Rb controls activity-dependent genes essential to neuronal plasticity and survival, such as the N-methyl--aspartic acid (NMDA) receptor''s subunit NR2B, the expression of which in the tetrameric ion channel largely affects calcium signaling by glutamate. In this study, we report that CXCL12 differentially modulates intracellular responses after stimulation of synaptic and extrasynaptic NMDA receptors, by a specific regulation of the NR2B gene that involves HDACs. Our results show that CXCL12 selectively inhibits NR2B expression in vitro and in vivo altering NMDA-induced calcium responses associated with neuronal death, while promoting prosurvival pathways that depend on stimulation of synaptic receptors. Along with previous studies, these findings underline the role of CXCL12/CXCR4 in the regulation of crucial components of glutamatergic transmission. These novel effects of CXCL12 may be involved in the physiological function of the chemokine in both developing and mature brains.     

Neuroprotection of the leaf and stem of Vitis amurensis and their active compounds against ischemic brain damage in rats and excitotoxicity in cultured neurons

Vitis amurensis (Vitaceae) has been reported to have anti-oxidant and anti-inflammatory activities. The present study investigated a methanol extract from the leaf and stem of V. amurensis for neuroprotective effects on cerebral ischemic damage in rats and on excitotoxicity induced by glutamate in cultured rat cortical neurons. Transient focal cerebral ischemia was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion (MCAO/reperfusion) in rats. Orally administered V. amurensis (25-100 mg/kg) reduced MCAO/reperfusion-induced infarct and edema formation, neurological deficits, and neuronal death. Depletion of glutathione (GSH) level and lipid peroxidation induced by MCAO/reperfusion was inhibited by administration of V. amurensis. The increase of phosphorylated mitogen-activated protein kinases (MAPKs), cyclooxygenase-2 (COX-2), and pro-apoptotic proteins and the decrease of anti-apoptotic protein in MCAO/reperfusion rats were significantly inhibited by treatment with V. amurensis. Exposure of cultured cortical neurons to 500 μM glutamate for 12 h induced neuronal cell death. V. amurensis (1-50 μg/ml) and (+)-ampelopsin A, γ-2-viniferin, and trans-?-viniferin isolated from the leaf and stem of V. amurensis inhibited glutamate-induced neuronal death, the elevation of intracellular calcium ([Ca²⁺]_i), the generation of reactive oxygen species (ROS), and changes of apoptosis-related proteins in cultured cortical neurons, suggesting that the neuroprotective effect of V. amurensis may be partially attributed to these compounds. These results suggest that the neuroprotective effect of V. amurensis against focal cerebral ischemic injury might be due to its anti-apoptotic effect, resulting from anti-excitotoxic, anti-oxidative, and anti-inflammatory effects and that the leaf and stem of V. amurensis have possible therapeutic roles for preventing neurodegeneration in stroke.     

Insensitivity to glutamate neurotoxicity mediated by NMDA receptors in association with delayed mitochondrial membrane potential disruption in cultured rat cortical neurons

We have attempted to elucidate mechanisms underlying differential vulnerability to glutamate (Glu) using cultured neurons prepared from discrete structures of embryonic rat brains. Brief exposure to Glu led to a significant decrease in the mitochondrial activity in hippocampal neurons cultured for 9 or 12 days at 10 μM to 1 mM with an apoptosis-like profile, without markedly affecting that in cortical neurons. Brief exposure to Glu also increased lactate dehydrogenase release along with a marked decrease in the number of cells immunoreactive for a neuronal marker protein in hippocampal, but not cortical, neurons. Similar insensitivity was seen to the cytotoxicity by NMDA, but not to that by tunicamycin, 2,4-dinitrophenol, hydrogen peroxide or A23187, in cortical neurons. However, NMDA was more efficient in increasing intracellular free Ca²⁺ levels in cortical neurons than in hippocampal neurons. Antagonists for neuroprotective metabotropic Glu receptors failed to significantly affect the insensitivity to Glu, while NMDA was more effective in disrupting mitochondrial membrane potentials in hippocampal than cortical neurons. These results suggest that cortical neurons would be insensitive to the apoptotic neurotoxicity mediated by NMDA receptors through a mechanism related to mitochondrial membrane potentials, rather than intracellular free Ca²⁺ levels, in the rat brain.     

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.     

Pore dilation occurs in TRPA1 but not in TRPM8 channels

Pain-related sensitization and synaptic plasticity in the central nucleus of the amygdala (CeA) depend on the endogenous activation of NMDA receptors and phosphorylation of the NR1 subunit through a PKA-dependent mechanism. Functional NMDA receptors are heteromeric assemblies of NR1 with NR2A-D or NR3A, B subunits. NMDA receptors composed of NR1 and NR2B subunits have been implicated in neuroplasticity and are present in the CeA. Here we used a selective NR2B antagonist (Ro-256981) to determine the contribution of NR2B-containing NMDA receptors to pain-related sensitization of CeA neurons. Extracellular single-unit recordings were made from CeA neurons in anesthetized adult male rats before and during the development of an acute arthritis. Arthritis was induced in one knee joint by intraarticular injections of kaolin and carrageenan. Brief (15 s) mechanical stimuli of innocuous (100–500 g/30 mm²) and noxious (1000–2000 g/30 mm²) intensity were applied to the knee and other parts of the body. In agreement with our previous studies, all CeA neurons developed increased background and evoked activity after arthritis induction. Ro-256981 (1, 10 and 100 μM; 15 min each) was administered into the CeA by microdialysis 5–6 h postinduction of arthritis. Ro-256981 concentration-dependently decreased evoked responses, but not background activity. This pattern of effect is different from that of an NMDA receptor antagonist (AP5) in our previous studies. AP5 (100 μM – 5 mM) inhibited background activity and evoked responses. The differential effects of AP5 and Ro-256981 may suggest that NMDA receptors containing the NR2B subunit are important but not sole contributors to pain-related changes of CeA neurons.     

Overstimulation of Glutamate Signals Leads to Hippocampal Transcriptional Plasticity in Hamsters

It’s known that neurons in mammalian hibernators are more tolerant to hypoxia than those in non-hibernating species and as a consequence animals are capable of awakening from the arousal state without exhibiting cerebral damages. In addition, evidences have suggested that euthermic hamster neurons display protective adaptations against hypoxia, while those of rats are not capable, even though molecular mechanisms involved in similar neuroprotective strategies have not been yet fully studied. In the present work, overstimulation of glutamatergic receptors NMDA recognized as one of the major death-promoting element in hypoxia, accounted for altered network complexity consistent with a moderate reduction of hippocampal neuronal survival (p < 0.05) in hamsters. These alterations appeared to be featured concomitantly with altered glutamatergic signaling as indicated by significant down-regulation (p < 0.01) of NMDAergic (NR2A) and AMPAergic (GluR1, R2) receptor subtypes together with the metabotropic mGluR5 subtype. Diminished mRNA levels were also reported for NMDA receptor binding factors and namely PSD95 plus DREAM, which exert positive and negative regulatory properties, respectively, on receptor trafficking events. Conversely, involvement of glutamatergic signaling systems on neuronal excitotoxicity was strengthened by the co-activation of GABA_AR-mediated effects as indicated by toxic morphological effects being notably reduced along with up-regulated GluR1, GluR2, mGluR5, DREAM, and Homer1c scaffold proteins when muscimol was added. Overall, these results point to a neuroprotective role of the GABAergic system against excitotoxicity episodes via DREAM-dependent inhibition of NMDA receptor and activation of AMPA receptor plus mGluR5, respectively, thus proposing them as novel therapeutic targets against cerebral ischemic damages in humans.     

Inhibiting pro-death NMDA receptor signaling dependent on the NR2 PDZ ligand may not affect synaptic function or synaptic NMDA receptor signaling to gene expression

NMDA receptors (NMDARs) mediate ischemic brain damage, in part through interactions of the PDZ ligand of NR2 subunits with the PDZ domain proteins PSD-95 and neuronal nitric oxide synthase located within the NMDAR signaling complex. We have recently shown that this PDZ ligand-dependent pathway promotes neuronal death via p38 activation. A peptide mimetic of the NR2B PDZ ligand (TAT-NR2B9c) reduces p38-mediated death in vitro and p38-dependent ischemic damage in vivo. In the absence of the PDZ ligand-p38 pathway, such as in TAT-NR2B9c-treated neurons, or in NMDAR-expressing non-neuronal cells, NMDAR-dependent excitotoxicity is mediated largely by JNK and requires greater Ca2+ influx. A major reason for blocking pro-death signaling events downstream of the NMDAR as an anti-excitotoxic strategy is that it may spare physiological synaptic function and signaling. We find that neuroprotective doses of TAT-NR2B9c do not alter the frequency of spontaneous synaptic events within networks of cultured cortical neurons nor is mini-EPSC frequency altered. Furthermore, TAT-NR2B9c does not inhibit the capacity of synaptic NMDAR activity to promote neuroprotective changes in gene expression, including the up-regulation of PACAP via CREB, and suppression of the pro-oxidative FOXO target gene Txnip. Thus, while the NR2 PDZ ligand does not account for all the excitotoxic effects of excessive NMDAR activity, these findings underline the value of the specific targeting of death pathways downstream of the NMDAR.     

Study of neuroprotection of donepezil, a therapy for Alzheimer's disease

Donepezil is a potent acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease. Although acetylcholinesterase inhibitors are thought to be symptomatic treatment of Alzheimer's disease, it is not clear whether they are effective against progressive degeneration of neuronal cells. In this study, we investigated the neuroprotective effects of donepezil against ischemic damage, N-methyl-d-aspartate (NMDA) excitotoxicity, and amyloid-beta (Abeta) toxicity using rat brain primary cultured neurons. Lactate dehydrogenase (LDH) released into the culture medium was measured as a marker of neuronal cell damage. As an ischemic damage model, we used oxygen-glucose deprivation in rat cerebral cortex primary cultured neurons. Pretreatment with donepezil (0.1, 1 and 10muM) significantly decreased LDH release in a concentration-dependent manner. However, other acetylcholinesterase inhibitors (galantamine, tacrine and rivastigmine) did not significantly decrease LDH release. In a NMDA excitotoxicity model, pretreatment with donepezil (0.1, 1 and 10muM) decreased the LDH release in a concentration-dependent manner. In binding assay for glutamate receptors, donepezil at 100muM only slightly inhibited binding to the glycine and polyamine sites on NMDA receptor complex. We further examined the effect of donepezil on Abeta (1-40)- and Abeta (1-42)-induced toxicity in primary cultures of rat septal neurons. Pretreatment with donepezil (0.1, 1 and 10muM) significantly decreased LDH release induced by Abetas in a concentration-dependent manner. However, other acetylcholinesterase inhibitors (galantamine and tacrine) and NMDA receptor antagonists (memantine and dizocilpine (MK801)) did not significantly decrease LDH release. These results demonstrate that donepezil has protective effects against ischemic damage, glutamate excitotoxicity and Abeta toxicity to rat primary cultured neurons and these effects are not dependent on acetylcholinesterase inhibition and antagonism of NMDA receptors. Thus, donepezil is expected to have a protective effect against progressive degeneration of brain neuronal cells in ischemic cerebrovascular disease and Alzheimer's disease.     

Regulation of N-methyl-D-aspartate receptors by calpain in cortical neurons

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.