Overstimulation Of N-methyl-D-aspartate Receptors Research Articles

The Computer Simulation of Therapy with the NMDA Antagonist in Excitotoxic Neurodegeneration in an Alzheimer's Disease-like Pathology.

(1) Background: The use of uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists results in neuroprotective benefits in patients with moderate to severe Alzheimer’s disease. In this study, we demonstrated mathematical and computer modelling of the excitotoxicity phenomenon and performed virtual memantine therapy. (2) Methods: A computer simulation environment of the N-methyl-D-aspartate receptor combining biological mechanisms of channel activation by means of excessive extracellular glutamic acid concentration in three models of excitotoxicity severity. The simulation model is based on sliding register tables, where each table is associated with corresponding synaptic inputs. Modelling of the increase in extracellular glutamate concentration, through over-stimulation of NMDA receptors and exacerbation of excitotoxicity, is performed by gradually increasing the parameters of phenomenological events by the power function. Pathological models were virtually treated with 3–30 µM doses of memantine compared to controls. (3) Results: The virtual therapy results of memantine at doses of 3–30 µM in the pathological models of excitotoxicity severity show statistically significant neuroprotective benefits in AD patients with moderate severity, 1.25 (95% CI, 1.18–1.32) vs. 1.76 (95% CI, 1.71–1.80) vs. 1.53 (95% CI, 1.48–1.59), (p < 0.001), to severe, 1.32 (95% CI, 1.12–1.53) vs. 1.77 (95% CI, 1.72–1.82) vs. 1.73 (95% CI, 1.68–1.79), (p < 0.001), in the area of effects on memory. A statistically significant benefit of memantine was demonstrated for all neuronal parameters in pathological models. In the mild severity model, a statistically significant increase in frequency was obtained relative to virtual memantine treatment with a dose of 3 µM, which was 23.5 Hz (95% CI, 15.5–28.4) vs. 38.8 Hz (95% CI, 34.0–43.6), (p < 0.0001). In the intermediate excitotoxicity severity model, a statistically significant increase in frequency was obtained relative to virtual memantine therapy with a 3 µM dose of 26.0 Hz (95% CI, 15.7–36.2) vs. 39.0 Hz (95% CI, 34.2–43.8) and a 10 µM dose of 26.0 Hz (95% CI, 15.7–36.2) vs. 30.9 Hz (95% CI, 26.4–35.4), (p < 0.0001). A statistically significant increase in frequency was obtained in the advanced excitotoxicity severity model as in the medium. (4) Conclusions: The NMDA antagonist memantine causes neuroprotective benefits in patients with moderate to severe AD. One of the most important benefits of memantine is the improvement of cognitive function and beneficial effects on memory. On the other hand, memantine provides only symptomatic and temporary support for AD patients. Memantine is prescribed in the US and Europe if a patient has moderate to severe AD. Memantine has also been approved for mild to moderate AD patients. However, its very modest effect provides motivation for further research into new drugs in AD. We are the first to present a mathematical model of the NMDA receptor that allows the simulation of excitotoxicity and virtual memantine therapy.

ENOS-dependent S-nitrosylation of the NF-\u03baB subunit p65 has neuroprotective effects

Cell death by glutamate excitotoxicity, mediated by N-methyl-d-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

Structural Insights into the Calcium/Calmodulin Dependent Inactivation of N-Methyl-D-Aspartate (NMDA) Receptors

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory neurotransmission and play a crucial role in synaptic plasticity and brain development. However, the overstimulation of NMDARs is involved in various neuropathological conditions. NMDARs exist as obligate heterotetramers having two glycine binding (GluN1) and two glutamate binding (GluN2) subunits. The release of voltage-dependent Mg2+ block and the binding of both agonists govern the gating of NMDARs. However, NMDA induced excessive Ca2+ influx downregulates the activation of NMDARs through a process called Ca2+ dependent inactivation (CDI). Extensive functional studies have revealed that the interaction between the C-terminal of GluN1 subunit and calmodulin mediates CDI. However, due to the flexibility of the C-terminal domain of GluN1 subunit, there is a very limited structural insight into the underlying mechanism of calmodulin mediated CDI. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to map various conformational changes that occur near the transmembrane domain of full-length NMDAR upon calmodulin binding. We also performed whole-cell measurements of HEK293 cells expressing NMDAR under different conditions to establish a structure-function relationship. We estimated the distances between the two GluN1 subunits (or GluN2) by smFRET in the presence and absence of calmodulin. To ensure the absence of any calmodulin activity, we used calmodulin inhibitory peptide (CaMIP). We observed that the distribution of FRET states varies under different conditions indicating significant structural changes are involved upon binding of calmodulin. We will perform similar smFRET measurements to map changes in the intracellular and ligand-binding domain. Thus, such investigations will provide structural insights into the calmodulin mediated CDI, which may further provide a direction for designing effective therapeutic strategies against Ca2+ related disorders and calmodulinopathies.

CYP46A1 protects against NMDA-mediated excitotoxicity in Huntington's disease: Analysis of lipid raft content

Huntington's Disease (HD) is an autosomal dominant neurodegenerative disease caused by abnormal polyglutamine expansion in huntingtin (mHtt) protein leading to degeneration of striatal neurons. Excitotoxicity, consecutive to overstimulation of N-methyl d-aspartate receptors (NMDARs) has a pivotal role in many neurological disorders including HD. Mutant Htt causes enhanced NMDA sensitivity, alteration of NMDAR expression and localization in neurons. Excitotoxic events initiate neuronal death in numerous ways, including activation of apoptotic cascades. Among the NMDAR subunits involved in glutamatergic-mediated excitotoxicity, GluN2B has been extensively reported. In addition to excitotoxicity, alteration of cholesterol metabolism has been observed in HD, with a decrease of cholesterol precursor synthesis along with an increase of cholesterol accumulation, which is deleterious for neurons. Expression of Cholesterol Hydroxylase enzyme, CYP46A1, which converts cholesterol into 24 S-hydroxycholesterol is down-regulated in HD. We found that CYP46A1 overexpression is beneficial in HD neurons and mouse model, but the mechanisms involved still remain unclear. In this study we addressed the effect of CYP46A1 on NMDAR-mediated excitotoxicity in HD primary neurons and its role in modulating cholesterol and localization of GLUN2B in lipid rafts. We showed that CYP46A1 is protective against NMDAR-mediated excitotoxicity in two different HD neuronal cell models. Cholesterol as well as GluN2B level in lipid raft, are significantly increased by mHtt. Despite a clear effect of CYP46A1 in reducing cholesterol content in lipid raft extracts from wild type neurons, CYP46A1 overexpression in HD neurons could not normalize the increased cholesterol levels in lipid rafts. This study highlights the beneficial role of CYP46A1 against NMDAR-mediated excitotoxicity and gives further insights into the cellular mechanisms underlying CYP46A1-mediated neuroprotection.

Activation of caspase-6 and cleavage of caspase-6 substrates is an early event in NMDA receptor-mediated excitotoxicity.

Excitotoxicity, due to overstimulation of N-methyl D-aspartate receptors (NMDARs), has a pivotal role in many neurological disorders. However, NMDAR antagonists often cause side effects, and identifying more druggable therapeutic targets for NMDAR excitotoxicity is an important goal. Activation of caspases is a downstream effect of excitotoxicity that may be critically involved in NMDAR-mediated cell death. Caspase-6 (casp6) in particular has been shown to play a key role in the pathogenesis of stroke, Huntington disease, and Alzheimer disease. Using N-methyl D-aspartate (NMDA)-induced excitotoxic injuries of primary rat neurons, we demonstrate that there is an early increase in caspase profiles after an excitotoxic event at the level of mRNA, protein, and activity. Casp6 is elevated and activated first, followed by caspase-8 and caspase-3. Similarly, known casp6 substrates huntingtin, as well as novel casp6 substrates serine/threonine kinase 3 and death domain-associated protein, are cleaved in similar temporal patterns post NMDA. On the basis of these data, we propose that casp6 may be an initiator caspase in apoptotic cascades leading to neuronal death after an excitotoxic event and suggest casp6 as a potential therapeutic target for neurological disorders where NMDAR-mediated excitotoxicity has been shown to play a role.

A Sustained Activation of Pancreatic NMDARs Is a Novel Factor of β-Cell Apoptosis and Dysfunction.

Type 2 diabetes, which features β-cell failure, is caused by the decrease of β-cell mass and insulin secretory function. Current treatments fail to halt the decrease of functional β-cell mass. Strategies to prevent β-cell apoptosis and dysfunction are highly desirable. Recently, our group and others have reported that blockade of N-methyl-d-aspartate receptors (NMDARs) in the islets has been proposed to prevent the progress of type 2 diabetes through improving β-cell function. It suggests that a sustained activation of the NMDARs may exhibit deleterious effect on β-cells. However, the exact functional impact and mechanism of the sustained NMDAR stimulation on islet β-cells remains unclear. Here, we identify a sustained activation of pancreatic NMDARs as a novel factor of apoptotic β-cell death and function. The sustained treatment with NMDA results in an increase of intracellular [Ca2+] and reactive oxygen species, subsequently induces mitochondrial membrane potential depolarization and a decrease of oxidative phosphorylation expression, and then impairs the mitochondrial function of β-cells. NMDA specifically induces the mitochondrial-dependent pathway of apoptosis in β-cells through upregulation of the proapoptotic Bim and Bax, and downregulation of antiapoptotic Bcl-2. Furthermore, a sustained stimulation of NMDARs impairs β-cell insulin secretion through decrease of pancreatic duodenal homeobox-1 (Pdx-1) and adenosine triphosphate synthesis. The activation of nuclear factor-κB partly contributes to the reduction of Pdx-1 expression induced by overstimulation of NMDARs. In conclusion, we show that the sustained stimulation of NMDARs is a novel mediator of apoptotic signaling and β-cell dysfunction, providing a mechanistic insight into the pathological role of NMDARs activation in diabetes.

Can an FDA-Approved Alzheimer's Drug Be Repurposed for Alleviating Neuronal Symptoms of Zika Virus?

ABSTRACTZika virus caught the world by surprise by its rapid spread and frightening disease outcomes. This major epidemic motivated many scientists to focus their attention on controlling this emerging pathogen. As many as 45 vaccine candidates are being developed, but progress in the antiviral arena has been slower. In a recent article (mBio 8:e00350-17, 2017, https://doi.org/10.1128/mBio.00350-17), Costa and colleagues showed that an FDA-approved drug used to treat Alzheimer’s disease may moderate Zika virus-induced neuronal damage. This work is based on the premise that overstimulation of N-methyl-d-aspartate receptors (NMDARs) may drive neurodegeneration and that this may be responsible for neuronal cell death associated with Zika virus infection of the central nervous system (CNS). Thus, blockage of the NMDAR channel activity with FDA-approved memantine or other antagonists may reduce neurological complications associated with Zika virus infection. Repurposing a preapproved drug and targeting the host represent intriguing strategies and yet require more analysis prior to moving into clinical trials.

Protective effect of magnesium acetyltaurate against NMDA-induced retinal damage involves restoration of minerals and trace elements homeostasis

Glutamate-mediated excitotoxicity involving N-methyl-d-aspartate (NMDA) receptors has been recognized as a final common outcome in pathological conditions involving death of retinal ganglion cells (RGCs). Overstimulation of NMDA receptors results in influx of calcium (Ca) and sodium (Na) ions and efflux of potassium (K). NMDA receptors are blocked by magnesium (Mg). Such changes due to NMDA overstimulation are also associated with not only the altered levels of minerals but also that of trace elements and redox status. Both the decreased and elevated levels of trace elements such as iron (Fe), zinc (Zn), copper (Cu) affect NMDA receptor excitability and redox status. Manganese (Mn), and selenium (Se) are also part of antioxidant defense mechanisms in retina. Additionally endogenous substances such as taurine also affect NMDA receptor activity and retinal redox status. Therefore, the aim of this study was to evaluate the effect of Mg acetyltaurate (MgAT) on the retinal mineral and trace element concentration, oxidative stress, retinal morphology and retinal cell apoptosis in rats after-NMDA exposure. One group of Sprague Dawley rats received intravitreal injection of vehicle while 4 other groups similarly received NMDA (160nmolL−1). Among the NMDA injected groups, 3 groups also received MgAT (320nmolL−1) as pre-treatment, co-treatment or post-treatment. Seven days after intravitreal injection, rats were sacrificed, eyes were enucleated and retinae were isolated for estimation of mineral (Ca, Na, K, Mg) and trace element (Mn, Cu, Fe, Se, Zn) concentration using Inductively Coupled Plasma (DRC ICP-MS) techniques (NexION 300D), retinal oxidative stress using Elisa, retinal morphology using H&E staining and retinal cell apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Intravitreal NMDA injection resulted in increased concentration of Ca (4.6 times, p<0.0001), Mg (1.5 times, p<0.01), Na (3 times, p<0.0001) and K (2.3 times, p<0.0001) compared to vehicle injected group. This was accompanied with significant increase of Ca/Mg and Na/K ratios, 3 and 1.27 times respectively, compared to control group. The trace elements such as Cu, Fe and Zn also showed a significant increase amounting to 3.3 (p<0.001), 2.3 (p<0.0001) and 3 (p<0.0001) times respectively compared to control group. Se was increased by 60% (p<0.005). Pre-treatment with MgAT abolished effect of NMDA on minerals and trace elements more effectively than co- and post-treatment. Similar observations were made for retinal oxidative stress, retinal morphology and retinal cell apoptosis. In conclusion, current study demonstrated the protective effect of MgAT against NMDA-induced oxidative stress and retinal cell apoptosis. This effect of MgAT was associated with restoration of retinal concentrations of minerals and trace elements. Further studies are warranted to explore the precise molecular targets of MgAT. Nevertheless, MgAT seems a potential candidate in the management of diseases involving NMDA-induced excitotoxicity.

Dinitrobenzene sulphonic acid-induced colitis impairs spatial recognition memory in mice: roles of N-methyl D-aspartate receptors and nitric oxide.

Many peripheral diseases are associated with a decline in cognitive function. In this regard, there have been reports of patients with inflammatory bowel disease and an otherwise unexplained memory impairment. We sought to assess the memory performance of mice with colitis. We also investigated the roles of N-methyl D-aspartate (NMDA) receptors and nitric oxide (NO) as possible mediators of colitis-induced amnesia. To induce colitis, male NMRI mice were intrarectally injected with a solution containing dinitrobenzene sulfonic acid (DNBS; 4 mg in 100 μl) under anesthesia. Three days after intrarectal DNBS instillation, spatial recognition and associative memories were assessed by the Y-maze and passive avoidance tasks, respectively. The NMDA antagonists, MK-801 and memantine, and the inducible NO synthase (iNOS) inhibitor, aminoguanidine, were injected intraperitoneally 45 min before the Y-maze task. Induction of colitis by DNBS impaired spatial recognition memory in the Y-maze task but had no effect on step through latencies in the passive avoidance test. Colitis-induced amnesia was reversed by administering specific doses of MK-801 and memantine (30 μg/kg and 1 mg/kg, respectively) suggesting dysregulated NMDA receptor activation as an underlying mechanism. No effect was seen with lower and higher doses of these drugs, resulting in a bell-shaped dose response curve. Colitis-induced amnesia was also inhibited by aminoguanidine (50 mg/kg), implicating a role for iNOS activation and neuroinflammation in this phenomenon. DNBS-induced colitis impairs memory through NMDA receptor overstimulation and NO overproduction.

PEGylated d-serine dehydratase as a d-serine reducing agent

d-Serine is an endogenous coagonist for N-methyl-d-aspartate (NMDA) receptors and is involved in excitatory neurotransmission. Excessive receptor activation causes excitotoxicity, leading to various acute and chronic neurological disorders. Decrease in d-serine content may provide a therapeutic strategy for the treatment of the neurological disorders in which overstimulation of NMDA receptors plays a pathological role. Saccharomyces cerevisiaed-serine dehydratase (Dsd1p), which acts dominantly on d-serine, may be a useful d-serine reducing agent. We conjugated a linear 5-kDa polyethylene glycol (PEG) to Dsd1p (PEG-Dsd1p) and examined the effects of PEG-conjugation on its biochemical and pharmacokinetic properties. PEG-Dsd1p retained activity, specificity, and stability of the enzyme. The PEG modification extended the serum half-life of Dsd1p in mice 6-fold, from 3.8h to 22.4h. PEG-Dsd1p was much less immunogenic compared to the unmodified enzyme. Intraperitoneal administration of PEG-Dsd1p was effective in decreasing the d-serine content in the mouse hippocampus. These findings suggest that PEG-Dsd1p may be a novel tool for lowering d-serine levels in vivo.

The effects of glycine, glutamate and their combination on cardiodynamics, coronary flow and oxidative stress in isolated rat hearts

Objectives: To examine the effects of glycine, glutamate and their combination on cardiac function, coronary flow and oxidative stress in isolated rat hearts, and to examine the effects of potential activation of N-methyl-Daspartate (NMDA) receptors in isolated rat hearts. Methods: The hearts of male Wistar albino rats were excised and perfused according to the Langendorff technique, and cardiodynamic parameters (maximum rate of pressure development in the left ventricle [dp/dt max]; minimum rate of pressure development in the left ventricle [dp/dt min], systolic left ventricular pressure, diastolic left ventricular pressure, heart rate) and coronary flow were determined during the subsequent administration of glycine, glutamate and their combination. Oxidative stress biomarkers, including thiobarbituric acid-reactive substances, nitrites (NO2 −), superoxide anion radical (O2 −) and hydrogen peroxide (H2O2), were each determined spectrophotometrically in coronary venous effluent. Results: Treatment with glycine and glutamate alone did not cause a statistically significant change in any of the observed parameters; however, their combined administration induced significant decreases in dp/dt max, dp/dt min, heart rate and coronary flow compared with the control conditions. Treatment with glycine and glutamate together induced significant increases in NO2 −, O2 − and H2O2 levels. After the washout period, all parameters that had changed (cardiodynamic parameters, coronary flow and levels oxidative stress biomarkers) returned to values that were not significantly different from controls. Conclusions: The results of the present study indicate that NMDA receptors likely have important roles in the function of the heart and coronary circulation. In addition, these results are suggestive of a damaging effect of NMDA receptor overstimulation in heart functioning, potentially mediated by oxidative stress.

The effects of the modulation of NMDA receptors by homocysteine thiolactone and dizocilpine on cardiodynamics and oxidative stress in isolated rat heart.

In light of the limited data concerning the role of N-methyl-D-aspartate (NMDA) receptors in cardiac function, the aim of the present study was to determine the role of NMDA receptors in cardiac function, as well as the possible role played by the oxidative stress induced by the overstimulation of NMDA receptors in isolated rat heart. The hearts of male, Wistar albino rats (n = 24, 12 in each experimental group, BM 180-200 g) were retrogradely perfused at a constant perfusion pressure (70 cm H₂O₂), using the Langendorff technique, and cardiodynamic parameters were determined during the subsequent administration of DL-homocysteine thiolactone (DL-Hcy TLHC) alone, the combination of DL-Hcy TLHC and dizocilpine (MK-801), and MK-801 alone. In the second experimental group, the order of the administration of each of the substances was reversed. The oxidative stress biomarkers, including thiobarbituric acid reactive substances (TBARS), NO(2)(-), O(2)(-) and H₂O₂, were each determined spectrophotometrically. DL-Hcy TLHC and MK-801 depressed cardiac function. DL-Hcy TLHC decreased oxidative stress, a finding that contrasted with the results of the experiments in which MK-801 was administered first. The findings of this study were suggestive of the likely role played by NMDA receptors in the regulation of cardiac function and coronary circulation in isolated rat heart.

XENON in medical area: emphasis on neuroprotection in hypoxia and anesthesia

Xenon is a medical gas capable of establishing neuroprotection, inducing anesthesia as well as serving in modern laser technology and nuclear medicine as a contrast agent. In spite of its high cost, its lack of side effects, safe cardiovascular and organoprotective profile and effective neuroprotective role after hypoxic-ischemic injury (HI) favor its applications in clinics. Xenon performs its anesthetic and neuroprotective functions through binding to glycine site of glutamatergic N-methyl-D-aspartate (NMDA) receptor competitively and blocking it. This blockage inhibits the overstimulation of NMDA receptors, thus preventing their following downstream calcium accumulating cascades. Xenon is also used in combination therapies together with hypothermia or sevoflurane. The neuroprotective effects of xenon and hypothermia cooperate synergistically whether they are applied synchronously or asynchronously. Distinguishing properties of Xenon promise for innovations in medical gas field once further studies are fulfilled and Xenon’s high cost is overcome.

Nothing Is Written in Stone

a s c p “N othing is written in stone,” the late Seymour Levine once said about the amazing plasticity of the developing brain in adaptation to an ever-changing environment (1). These adaptations, in response to environmental input, may cause lasting changes in the function of the evolutionary older brain circuitry, underlying emotion and cognition. As a result, adverse experiences in early life are thought to enhance disease vulnerability. However, recent evidence suggests that early adversity does not inevitably lead to a negative outcome (2). Rather, depending on genes and environmental context, early experience may program the brain for life to come. In this issue, Bagot et al. (3) describe the crucial role of the N-methyl-D-aspartate (NMDA) receptor in this programming effect. Bagot et al. recorded synaptic transmission in the dentate gyrus of adult rats that were raised by mothers spending either extremely high or low amounts of time licking and grooming (LG) their offspring during the first postnatal week (High vs. Low LG mothers, respectively). Earlier studies, particularly from the group of Michael Meaney, have shown that offspring from High compared with Low LG mothers show more complex hippocampal cells, more efficient long-term potentiation (LTP), better spatial learning, and more efficient release patterns of corticosterone after stress (2,4). Crossfostering could reverse some of these effects, emphasizing the relevance of early life environment rather than genetic background in the development of these phenotypes. Using this model, the present study shows that the ratio between NMDA and alphaamino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor–mediated currents in dentate granule cells is enhanced in Low compared with High LG offspring. Field potential recordings indicate that this is caused by enhanced transmission via NMDA receptors, whereas AMPA receptor function seems unchanged. In agreement, protein levels of NMDA receptor subunits are higher in Low than High LG offspring. In other words, an adverse early postnatal environment appears to upregulate NMDA transmission in the dentate gyrus over the long term. This finding comes as a bit of a surprise, because earlier studies have shown that NMDA receptor expression is decreased and LTP impaired in the adult offspring from Low compared with High LG mothers (5). The incongruity in NMDA receptor expression drives home the message that the proof of the pudding is in the recording: the only way to determine the functionality of receptors is by doing the actual electrophysiologic recordings. The second point—reduced LTP in light of enhanced NMDA receptor function—is experimentally addressed by the authors. They argue that the location of the NMDA receptors, that is, intraversus extrasynaptic, is important and moreover that steady overstimulation of NMDA receptors may impair the ability to induce LTP. They confirm this view by showing that partial blockade of NMDA receptors in Low LG off-

P53 induction contributes to excitotoxic neuronal death in rat striatum through apoptotic and autophagic mechanisms

The present study sought to investigate mechanisms by which p53 induction contributes to excitotoxic neuronal injury. Rats were intrastriatally administered the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA), the changes in the expression of p53 and its target genes involved in apoptosis and autophagy, including p53-upregulated modulator of apoptosis (PUMA), Bax, Bcl-2, damage-regulated autophagy modulator (DRAM) and other autophagic proteins including microtubule-associated protein 1 light chain 3 (LC3) and beclin 1 were assessed. The contribution of p53-mediated autophagy activation to apoptotic death of striatal neurons was assessed with co-administration of the nuclear factor-kappaB (NF-kappaB) inhibitor SN50, the p53 inhibitor Pifithrin-alpha (PFT-alpha) or the autophagy inhibitor 3-methyladenine (3-MA). The increased formation of autophagosomes and secondary lysosomes were observed with transmission electron microscope after excitotoxin exposure. QA induced increases in the expression of p53, PUMA, Bax and a decrease in Bcl-2. These changes were significantly attenuated by pre-treatment with SN50, PFT-alpha or 3-MA. SN50, PFT-alpha or 3-MA also reversed QA-induced upregulation of DRAM, the ratio of LC3-II/LC3-I and beclin 1 protein levels in the striatum. QA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by SN50, PFT-alpha or 3-MA. These results suggest that overstimulation of NMDA receptors can induce NF-kappaB-dependent expression of p53. p53 participates in excitotoxic neuronal death probably through both apoptotic and autophagic mechanisms.

P53 induction contributes to excitotoxic neuronal death in rat striatum through apoptotic and autophagic mechanisms

The present study sought to investigate mechanisms by which p53 induction contributes to excitotoxic neuronal injury. Rats were intrastriatally administered the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA), the changes in the expression of p53 and its target genes involved in apoptosis and autophagy, including p53-upregulated modulator of apoptosis (PUMA), Bax, Bcl-2, damage-regulated autophagy modulator (DRAM) and other autophagic proteins including microtubule-associated protein 1 light chain 3 (LC3) and beclin 1 were assessed. The contribution of p53-mediated autophagy activation to apoptotic death of striatal neurons was assessed with co-administration of the nuclear factor-kappaB (NF-kappaB) inhibitor SN50, the p53 inhibitor Pifithrin-alpha (PFT-alpha) or the autophagy inhibitor 3-methyladenine (3-MA). The increased formation of autophagosomes and secondary lysosomes were observed with transmission electron microscope after excitotoxin exposure. QA induced increases in the expression of p53, PUMA, Bax and a decrease in Bcl-2. These changes were significantly attenuated by pre-treatment with SN50, PFT-alpha or 3-MA. SN50, PFT-alpha or 3-MA also reversed QA-induced upregulation of DRAM, the ratio of LC3-II/LC3-I and beclin 1 protein levels in the striatum. QA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by SN50, PFT-alpha or 3-MA. These results suggest that overstimulation of NMDA receptors can induce NF-kappaB-dependent expression of p53. p53 participates in excitotoxic neuronal death probably through both apoptotic and autophagic mechanisms.

Achyranthes bidentata polypeptides confer neuroprotection through inhibition of reactive oxygen species production, Bax expression, and mitochondrial dysfunction induced by overstimulation of N‐methyl‐D‐aspartate receptors

Achyranthes bidentata polypeptides (ABPP), the important constituents separated from the aqueous extract of Achyranthes bidentata, have been shown to attenuate N-methyl-D-aspartate (NMDA)-induced cell apoptosis in cultured hippocampal neurons through differential modulation of NR2A- and NR2B-containing NMDA receptors. The present study sought to investigate the possible mechanism underlying the neuroprotective effect of ABPP on NMDA-induced cell death. Western blot analysis and colorimetric enzymatic assay demonstrated that ABPP pretreatment inhibited NMDA-induced increase of Bax protein expression or caspase-3 activity in cultured hippocampal neurons. Fluorescence measurements after staining with 2,7-dichlorofluorescin diacetate and rhodamine 123 showed that ABPP treatment also reversed NMDA-induced intracellular radical oxygen species (ROS) elevation and mitochondrial membrane potential depression in cultured hippocampal neurons. Furthermore, the in vivo effects of ABPP on cerebral neuronal damage during focal ischemia-reperfusion were also investigated. In rat middle cerebral artery occlusion (MCAO) model, ABPP attenuated the increase in the neurological deficit and cerebral infarction induced by focal ischemia-reperfusion, showing in vivo neuroprotective effects. The results collectively suggest that ABPP might exert neuroprotective actions through inhibiting Bax protein expression, caspase-3 activity, ROS production, and mitochondrial dysfunction that are all caused by overstimulation of NMDA receptors.

Shuffling the Deck Anew: How NR3 Tweaks NMDA Receptor Function

The N-methyl-D -aspartate (NMDA) receptors are the most complex members in the family of ionotropic glutamate receptors. They are involved in long-term potentiation and underlie higher cognitive functions like memory formation and learning. On the other hand, overstimulation of NMDA receptors (NMDARs), leading to a massive influx of Ca(2+) ions into the cell, is linked to neurodegenerative disorders such as for example Huntington's disease and epilepsy. NMDARs are generally considered to be heteromeric tetramers and are conventionally thought to assemble from NR1 splice variants and NR2 subunits, which determine crucial channel properties. With the recent discovery of the functionally different NR3 subunits, many of the known features of NMDARs are being reassessed: The presence of NR3 in NMDARs decreases Mg(2+) sensitivity and Ca(2+) permeability and reduces agonist-induced current responses. Between altering those essential key characteristics of conventional NMDARs and forming a new class of excitatory glycine receptors when coassembling with NR1, the NR3 subunits give rise to a functionally entirely new array of "NMDA" receptors. Understanding the multifaceted influence of NR3 is imperative to further the understanding of the complex role of NMDARs in neurotransmission and higher brain functions.

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.

Plasma homocysteine concentrations do not influence craving in alcohol withdrawal

Results of a number of studies indicate that the glutamate system, especially the N-methyl- d-aspartate (NMDA) receptor, has a major function in chronic alcoholism, including craving. Homocysteine and other excitatory amino acids, such as glutamate and aspartate, lead to an overstimulation of NMDA receptors. Because alcoholism is associated with elevated plasma homocysteine concentrations, we designed the current study to determine whether elevated plasma homocysteine concentrations have an influence on craving in alcohol withdrawal. Two groups of patients with an established diagnosis of alcohol dependence were compared. Group A comprised 50 consecutively admitted alcohol-dependent individuals who had been abstinent from alcohol between 24 and 72 h before hospitalization. Group B comprised 146 consecutively recruited alcohol-dependent individuals who were admitted, acutely intoxicated, for withdrawal treatment. All patients were assessed with the Obsessive Compulsive Drinking Scale (OCDS) on the day of admission and after 7 days of treatment. The mean (27.1, S.D. 18.4) plasma homocysteine concentration for group B was significantly higher than the mean (12.5, S.D. 5.3) plasma homocysteine concentration for group A (Mann–Whitney U test: P < .001). No significant influence of homocysteine concentration on the extent of craving was found for either group with the use of the Spearman correlation (day 0: group A, r = −.076, P = .601; group B, r = .120, P = .148) and logistic regression analysis. Although homocysteine is a potent modulator of glutamatergic neurotransmission, results of the current study provide no evidence for a pathophysiologic role of homocysteine in withdrawal craving. Therefore, further research about alcohol craving should focus on neurobiologic factors other than homocysteine.