Overactivation Of NMDA Receptors Research Articles

Mitochondrial Calcium Uniporter (MCU) is Involved in an Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mice

The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-KATP) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca2+ present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca2+ concentration, mitochondrial membrane potential, and N-methyl-d-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca2+ concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca2+ concentrations against I/R injury.Graphical

The Expression and Functionality of CB1R-NMDAR Complexes Are Decreased in A Parkinson's Disease Model.

One of the hallmarks of Parkinson's disease (PD) is the alteration in the expression and function of NMDA receptor (NMDAR) and cannabinoid receptor 1 (CB1R). The presence of CB1R-NMDAR complexes has been described in neuronal primary cultures. The activation of CB1R in CB1R-NMDAR complexes was suggested to counteract the detrimental NMDAR overactivation in an AD mice model. Thus, we aimed to explore the role of this receptor complex in PD. By using Bioluminescence Resonance Energy Transfer (BRET) assay, it was demonstrated that α-synuclein induces a reorganization of the CB1R-NMDAR complex in transfected HEK-293T cells. Moreover, α-synuclein treatment induced a decrease in the cAMP and MAP kinase (MAPK) signaling of both CB1R and NMDAR not only in transfected cells but also in neuronal primary cultures. Finally, the interaction between CB1R and NMDAR was studied by Proximity Ligation Assay (PLA) in neuronal primary cultures, where it was observed that the expression of CB1R-NMDAR complexes was decreased upon α-synuclein treatment. These results point to a role of CB1R-NMDAR complexes as a new therapeutic target in Parkinson's disease.

Glycine transporter-1 inhibition by NFPS promotes neuroprotection against striatal damage models

The striatum, an essential component of the brain's motor and reward systems, plays a pivotal role in a wide array of cognitive processes. Its dysfunction is a hallmark of neurodegenerative diseases like Parkinson's disease (PD) and Huntington's disease (HD), leading to profound motor and cognitive deficits. These conditions are often related to excitotoxicity, primarily due to overactivation of NMDA receptors (NMDAR). In the synaptic cleft, glycine transporter type 1 (GlyT1) controls the glycine levels, a NMDAR co-agonist, which modulates NMDAR function. This research explored the neuroprotective potential of NFPS, a GlyT1 inhibitor, in murine models of striatal injury. Employing models of neurotoxicity induced by 6-hydroxydopamine (PD model) and quinolinic acid (HD model), we assessed the effectiveness of NFPS pre-treatment in maintaining the integrity of striatal neurons and averting neuronal degeneration. The results indicated that NFPS pre-treatment conferred significant neuroprotection, reducing neuronal degeneration, protecting dopaminergic neurons, and preserving dendritic spines within the striatum. Additionally, this pre-treatment notably mitigated motor impairments resulting from striatal damage. The study revealed that GlyT1 inhibition led to substantial changes in the ratios of NMDAR subunits GluN2A/GluN1 and GluN2B/GluN1, 24 h after NFPS treatment. These findings underscore the neuroprotective efficacy of GlyT1 inhibition, proposing it as a viable therapeutic strategy for striatum-related damage.

STIM2 regulates NMDA receptor endocytosis that is induced by short-term NMDA receptor overactivation in cortical neurons

Recent findings suggest an important role for the dysregulation of stromal interaction molecule (STIM) proteins, activators of store-operated Ca2+ channels, and the prolonged activation of N-methyl-D-aspartate receptors (NMDARs) in the development of neurodegenerative diseases. We previously demonstrated that STIM silencing increases Ca2+ influx through NMDAR and STIM–NMDAR2 complexes are present in neurons. However, the interplay between NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM1/STIM2 with regard to intracellular trafficking remains unknown. Here, we found that the activation of NMDAR endocytosis led to an increase in STIM2–GluN2A and STIM2–GluN2B interactions in primary cortical neurons. STIM1 appeared to migrate from synaptic to extrasynaptic sites. STIM2 silencing inhibited post-activation NMDAR translocation from the plasma membrane and synaptic spines and increased NMDAR currents. Our findings reveal a novel molecular mechanism by which STIM2 regulates NMDAR synaptic trafficking by promoting NMDAR endocytosis after receptor overactivation, which may suggest protection against excessive uncontrolled Ca2+ influx through NMDARs.

Ceftriaxone improves impairments in synaptic plasticity and cognitive behavior in APP/PS1 mouse model of Alzheimer's disease by inhibiting extrasynaptic NMDAR-STEP61 signaling.

Abnormal activation of the extrasynaptic N-methyl-d-aspartate receptor (NMDAR) contributes to the pathogenesis of Alzheimer's disease (AD). Ceftriaxone (Cef) can improve cognitive impairment by upregulating glutamate transporter-1 and promoting the glutamate-glutamine cycle in an AD mouse model. This study aimed to investigate the effects of Cef on synaptic plasticity and cognitive-behavioral impairment and to unravel the associated underlying mechanisms. We used an APPswe/PS1dE9 (APP/PS1) mouse model of AD in this study. Extrasynaptic components from hippocampal tissue homogenates were isolated using density gradient centrifugation. Western blot was performed to evaluate the expressions of extrasynaptic NMDAR and its downstream elements. Intracerebroventricular injections of adeno-associated virus (AAV)-striatal enriched tyrosine phosphatase 61 (STEP61 ) and AAV-STEP61 -shRNA were used to modulate the expressions of STEP61 and extrasynaptic NMDAR. Long-term potentiation (LTP) and Morris water maze (MWM) tests were performed to evaluate the synaptic plasticity and cognitive function. The results showed that the expressions of GluN2B and GluN2BTyr1472 in the extrasynaptic fraction were upregulated in AD mice. Cef treatment effectively prevented the upregulation of GluN2B and GluN2BTyr1472 expressions. It also prevented changes in the downstream signals of extrasynaptic NMDAR, including increased expressions of m-calpain and phosphorylated p38 MAPK in AD mice. Furthermore, STEP61 upregulation enhanced, whereas STEP61 downregulation reduced the Cef-induced inhibition of the expressions of GluN2B, GluN2BTyr1472 , and p38 MAPK in the AD mice. Similarly, STEP61 modulation affected Cef-induced improvements in induction of LTP and performance in MWM tests. In conclusion, Cef improved synaptic plasticity and cognitive behavioral impairment in APP/PS1 AD mice by inhibiting the overactivation of extrasynaptic NMDAR and STEP61 cleavage due to extrasynaptic NMDAR activation.

Inhibition of GluN2B pathway is involved in the neuroprotective effect of silibinin on streptozotocin-induced Alzheimer's disease models

BackgroundOver-activation of N-methyl-D-aspartate receptors (NMDARs) is involved in sporadic Alzheimer's disease. Silibinin, a natural flavonoid gained from the seeds of Silybum marianum, exerts neuroprotective effects on sporadic AD models, but its impacts on NMDARs remain unknown. PurposeTo study silibinin's regulatory effects on NMDARs pathway in sporadic AD models. MethodsMTT assay, western blotting, confocal microscopy, flow cytometry, RT-PCR, and siRNA transfection etc. were used for cellular and molecular studies. The direct interactions between silibinin and NMDAR subunits were evaluated by computational molecular docking, drug affinity responsive target stability (DARTS) assay and cellular thermal shift assay (CETSA). Y maze test, novel objects recognition test and Morris water maze test were conducted to examine the learning and memory ability of rats. ResultsAn in vitro AD model was established by treating HT22 murine hippocampal neurons with streptozotocin (STZ), as evidenced by the amyloid β (Aβ) deposition and hyperphosphorylation of tau proteins. Silibinin shows protection of neurons against STZ-induced cell damage. It is noteworthy that STZ-induced cellular calcium influx is inhibited by silibinin-treatment, indicating the possible modulation of calcium channels. Studies on NMDARs, the most widely distributed calcium channel, by using molecular docking, DARTS and CESTA, reveal that the GluN2B subunit, but not GluN2A, is the potential target of silibinin. Further studies using the pharmacological agonist (NMDA) and the GluN2B-specific inhibitor (Ifenprodil) or siRNA, indicate that the protection by silibinin treatment from STZ-induced cytotoxicity is medicated through interference with GluN2B-containing NMDARs, followed by the upregulation of CaMKIIα/ BDNF/ TrkB signaling pathway and improved levels of synaptic proteins (SYP and PSD-95). The results in vivo using rats intracerebroventricularly injected with STZ (ICV-STZ), a well-established sporadic AD model, confirm that silibinin improves learning and memory ability in association with modulation of the GluN2B/CaMKIIα/ BDNF/TrkB signaling pathway. ConclusionInhibiting over-activation of GluN2B-containing NMDARs is involved in the neuroprotective effect of silibinin on STZ-induced sporadic AD models.

GluN2A-Selective NMDA Receptor Antagonists: Mimicking the U-Shaped Bioactive Conformation of TCN-201 by a [2.2]Paracyclophane System.

Under physiological conditions, N-Methyl-D-Aspartate (NMDA) receptors play a crucial role for synaptic plasticity, long-term potentiation and long-term depression. However, overactivation of NMDA receptors can result in excitotoxicity, which is associated with various neurological and neurodegenerative diseases. The physiological properties of NMDA receptors are strongly dependent on the GluN2 subunit incorporated into the heterotetrameric NMDA receptor. Therefore, subtype selective NMDA receptor modulators are of high interest. Since prototypical GluN2A-NMDA receptor antagonists TCN-201 and its MPX-analogs adopt a U-shaped conformation within the binding pocket, paracyclophanes were designed containing the phenyl rings in an already parallel orientation. Docking studies of the designed paracyclophanes show a similar binding pose as TCN-201. [2.2]Paracyclophanes with a benzoate or benzamide side chain were prepared in four-step synthesis, respectively, starting with a radical bromination in benzylic 1-position of [2.2]paracyclophane. In two-electrode voltage clamp experiments using Xenopus laevis oocytes transfected with cRNAs for the GluN1-4a and GluN2A subunits, the esters and amides (conc. 10 μM) did not show considerable inhibition of ion flux. It can be concluded that the GluN2A-NMDA receptor does not accept ligands with a paracyclophane scaffold functionalized in benzylic 1-position, although docking studies had revealed promising binding poses for benzoic acid esters and benzamides.

Recent Investigations on Neurotransmitters' Role in Acute White Matter Injury of Perinatal Glia and Pharmacotherapies-Glia Dynamics in Stem Cell Therapy.

Periventricular leukomalacia (PVL) and cerebral palsy are two neurological disease conditions developed from the premyelinated white matter ischemic injury (WMI). The significant pathophysiology of these diseases is accompanied by the cognitive deficits due to the loss of function of glial cells and axons. White matter makes up 50% of the brain volume consisting of myelinated and non-myelinated axons, glia, blood vessels, optic nerves, and corpus callosum. Studies over the years have delineated the susceptibility of white matter towards ischemic injury especially during pregnancy (prenatal, perinatal) or immediately after child birth (postnatal). Impairment in membrane depolarization of neurons and glial cells by ischemia-invoked excitotoxicity is mediated through the overactivation of NMDA receptors or non-NMDA receptors by excessive glutamate influx, calcium, or ROS overload and has been some of the well-studied molecular mechanisms conducive to the injury of white matter. Expression of glutamate receptors (GluR) and transporters (GLT1, EACC1, and GST) has significant influence in glial and axonal-mediated injury of premyelinated white matter during PVL and cerebral palsy. Predominantly, the central premyelinated axons express extensive levels of functional NMDA GluR receptors to confer ischemic injury to premyelinated white matter which in turn invoke defects in neural plasticity. Several underlying molecular mechanisms are yet to be unraveled to delineate the complete pathophysiology of these prenatal neurological diseases for developing the novel therapeutic modalities to mitigate pathophysiology and premature mortality of newborn babies. In this review, we have substantially discussed the above multiple pathophysiological aspects of white matter injury along with glial dynamics, and the pharmacotherapies including recent insights into the application of MSCs as therapeutic modality in treating white matter injury.

The Mechanism of the Neuroprotective Effect of Kynurenic Acid in the Experimental Model of Neonatal Hypoxia-Ischemia: The Link to Oxidative Stress.

The over-activation of NMDA receptors and oxidative stress are important components of neonatal hypoxia–ischemia (HI). Kynurenic acid (KYNA) acts as an NMDA receptor antagonist and is known as a reactive oxygen species (ROS) scavenger, which makes it a potential therapeutic compound. This study aimed to establish the neuroprotective and antioxidant potential of KYNA in an experimental model of HI. HI on seven-day-old rats was used as an experimental model. The animals were injected i.p. with different doses of KYNA 1 h or 6 h after HI. The neuroprotective effect of KYNA was determined by the measurement of brain damage and elements of oxidative stress (ROS and glutathione (GSH) level, SOD, GPx, and catalase activity). KYNA applied 1 h after HI significantly reduced weight loss of the ischemic hemisphere, and prevented neuronal loss in the hippocampus and cortex. KYNA significantly reduced HI-increased ROS, GSH level, and antioxidant enzyme activity. Only the highest used concentration of KYNA showed neuroprotection when applied 6 h after HI. The presented results indicate induction of neuroprotection at the ROS formation stage. However, based on the presented data, it is not possible to pinpoint whether NMDA receptor inhibition or the scavenging abilities are the dominant KYNA-mediated neuroprotective mechanisms.

Calcium Permeable-AMPA Receptors and Excitotoxicity in Neurological Disorders.

Excitotoxicity is one of the primary mechanisms of cell loss in a variety of diseases of the central and peripheral nervous systems. Other than the previously established signaling pathways of excitotoxicity, which depend on the excessive release of glutamate from axon terminals or over-activation of NMDA receptors (NMDARs), Ca2+ influx-triggered excitotoxicity through Ca2+-permeable (CP)-AMPA receptors (AMPARs) is detected in multiple disease models. In this review, both acute brain insults (e.g., brain trauma or spinal cord injury, ischemia) and chronic neurological disorders, including Epilepsy/Seizures, Huntington’s disease (HD), Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), chronic pain, and glaucoma, are discussed regarding the CP-AMPAR-mediated excitotoxicity. Considering the low expression or absence of CP-AMPARs in most cells, specific manipulation of the CP-AMPARs might be a more plausible strategy to delay the onset and progression of pathological alterations with fewer side effects than blocking NMDARs.

Protective Effect of Memantine on Bergmann Glia and Purkinje Cells Morphology in Optogenetic Model of Neurodegeneration in Mice.

Spinocerebellar ataxias are a family of fatal inherited diseases affecting the brain. Although specific mutated proteins are different, they may have a common pathogenetic mechanism, such as insufficient glutamate clearance. This function fails in reactive glia, leading to excitotoxicity and overactivation of NMDA receptors. Therefore, NMDA receptor blockers could be considered for the management of excitotoxicity. One such drug, memantine, currently used for the treatment of Alzheimer’s disease, could potentially be used for the treatment of other forms of neurodegeneration, for example, spinocerebellar ataxias (SCA). We previously demonstrated close parallels between optogenetically induced cerebellar degeneration and SCA1. Here we induced reactive transformation of cerebellar Bergmann glia (BG) using this novel optogenetic approach and tested whether memantine could counteract changes in BG and Purkinje cell (PC) morphology and expression of the main glial glutamate transporter—excitatory amino acid transporter 1 (EAAT1). Reactive BG induced by chronic optogenetic stimulation presented increased GFAP immunoreactivity, increased thickness and decreased length of its processes. Oral memantine (~90 mg/kg/day for 4 days) prevented thickening of the processes (1.57 to 1.81 vs. 1.62 μm) and strongly antagonized light-induced reduction in their average length (186.0 to 150.8 vs. 171.9 μm). Memantine also prevented the loss of the key glial glutamate transporter EAAT1 on BG. Finally, memantine reduced the loss of PC (4.2 ± 0.2 to 3.2 ± 0.2 vs. 4.1 ± 0.3 cells per 100 μm of the PC layer). These results identify memantine as potential neuroprotective therapeutics for cerebellar ataxias.

Neuroprotection of sevoflurane against ischemia/reperfusion-induced brain injury through inhibiting GluN2A/GluN2B-PSD-95-MLK3 module.

To investigate the role of GluN2A and GluN2B in neuroprotective effect of sevoflurane preconditioning against cerebral ischemia-reperfusion injury (CIRI). Rats were randomly divided into five groups as follows: control, ischemia-reperfusion (I/R) 6h, sevoflurane preconditioning (SP), SP + amantadine, SP + NMDA. Immunoblot and immunoprecipitation were used to detect the tyrosine phosphorylation of GluN2A/GluN2B, the interaction of GluN2A/GluN2B-PSD-95-MLK3 and the expression of phosphorylation of MLK3, MKK7 and JNK3. Cresyl violet staining was employed to analyse neuronal injury in rat hippocampal CA1 subfields. Sevoflurane preconditioning inhibits the tyrosine phosphorylation of GluN2A/GluN2B, the interaction of GluN2A/GluN2B-PSD-95-MLK3 and the phosphorylation of MLK3, MKK7 and JNK3 in rat hippocampus. An N-methyl-D-aspartate receptor (NMDAR) antagonist amantadine reversed the MLK3-MKK7- JNK3 signal events. Such reversion was also realized by NMDA (60 and 80nmol) and low doses of NMDA (0-40nmol) could not change the inhibitory effect of sevoflurane preconditioning on MLK3-MKK7-JNK3 signal events. Finally, Cresyl violet staining also confirmed that low dose of NMDA reduced neuronal loss in rat hippocampal CA1 subfields. Sevoflurane preconditioning provides neuroprotection against CIRI by inhibiting NMDAR over-activation.

Amyloid β Perturbs Cu(II) Binding to the Prion Protein in a Site-Specific Manner: Insights into Its Potential Neurotoxic Mechanisms.

Amyloid β (Aβ) is a Cu-binding peptide that plays a key role in the pathology of Alzheimer's disease. A recent report demonstrated that Aβ disrupts the Cu-dependent interaction between cellular prion protein (PrPC) and N-methyl-d-aspartate receptor (NMDAR), inducing overactivation of NMDAR and neurotoxicity. In this context, it has been proposed that Aβ competes for Cu with PrPC; however, there is no spectroscopic evidence to support this hypothesis. Prion protein (PrP) can bind up to six Cu(II) ions: from one to four at the octarepeat (OR) region, producing low- and high-occupancy modes, and two at the His96 and His111 sites. Additionally, PrPC is cleaved by α-secretases at Lys110/His111, yielding a new Cu(II)-binding site at the α-cleaved His111. In this study, the competition for Cu(II) between Aβ(1-16) and peptide models for each Cu-binding site of PrP was evaluated using circular dichroism and electron paramagnetic resonance. Our results show that the impact of Aβ(1-16) on Cu(II) coordination to PrP is highly site-specific: Aβ(1-16) cannot effectively compete with the low-occupancy mode at the OR region, whereas it partially removes the metal ion from the high-occupancy modes and forms a ternary OR-Cu(II)-Aβ(1-16) complex. In contrast, Aβ(1-16) removes all Cu(II) ions from the His96 and His111 sites without formation of ternary species. Finally, at the α-cleaved His111 site, Aβ(1-16) yields at least two different ternary complexes depending on the ratio of PrP/Cu(II)/Aβ. Altogether, our spectroscopic results indicate that only the low-occupancy mode at the OR region resists the effect of Aβ, while Cu(II) coordination to the high-occupancy modes and all other tested sites of PrP is perturbed, by either removal of the metal ion or formation of ternary complexes. These results provide important insights into the intricate effect of Aβ on Cu(II) binding to PrP and the potential neurotoxic mechanisms through which Aβ might affect Cu-dependent functions of PrPC, such as NMDAR modulation.

N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia.

Although stroke is very often the cause of death worldwide, the burden of ischemic and hemorrhagic stroke varies between regions and over time regarding differences in prognosis, prevalence of risk factors, and treatment strategies. Excitotoxicity, oxidative stress, dysfunction of the blood-brain barrier, neuroinflammation, and lysosomal membrane permeabilization, sequentially lead to the progressive death of neurons. In this process, protein kinases-related checkpoints tightly regulate N-methyl-D-aspartate (NMDA) receptor signaling pathways. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca2+ overload and ultimately neuronal death. Thus, reduced expression of postsynaptic density-95 protein and increased protein S-nitrosylation in neurons is responsible for neuronal vulnerability in cerebral ischemia. In this chapter death-associated protein kinases, cyclin-dependent kinase 5, endoplasmic reticulum stress-induced protein kinases, hyperhomocysteinemia-related NMDA receptor overactivation, ephrin-B-dependent amplification of NMDA-evoked neuronal excitotoxicity and lysosomocentric hypothesis have been discussed.Consequently, ample evidences have demonstrated that enhancing extrasynaptic NMDA receptor activity triggers cell death after stroke. In this context, considering the dual roles of NMDA receptors in both promoting neuronal survival and mediating neuronal damage, selective augmentation of NR2A-containing NMDA receptor activation in the presence of NR2B antagonist may constitute a promising therapy for stroke.

A Bioactive Olive Pomace Extract Prevents the Death of Murine Cortical Neurons Triggered by NMDAR Over-Activation.

We have recently demonstrated that bioactive molecules, extracted by high pressure and temperature from olive pomace, counteract calcium-induced cell damage to different cell lines. Here, our aim was to study the effect of the same extract on murine cortical neurons, since the preservation of the intracellular Ca2+-homeostasis is essential for neuronal function and survival. Accordingly, we treated neurons with different stimuli in order to evoke cytotoxic glutamatergic activation. In these conditions, the high-pressure and temperature extract from olive pomace (HPTOPE) only abolished the effects of N-methyl-d-aspartate (NMDA). Particularly, we observed that HPTOPE was able to promote the neuron rescue from NMDA-induced cell death. Moreover, we demonstrated that HPTOPE is endowed with the ability to maintain the intracellular Ca2+-homeostasis following NMDA receptor overactivation, protecting neurons from Ca2+-induced adverse effects, including aberrant calpain proteolytic activity. Moreover, we highlight the importance of the extraction conditions used that, without producing toxic molecules, allow us to obtain protecting molecules belonging to proanthocyanidin derivatives like procyanidin B2. In conclusion, we can hypothesize that HPTOPE, due to its functional and nontoxic properties on neuronal primary culture, can be utilized for future therapeutic interventions for neurodegeneration.

Over-activation of NMDA receptors promotes ABCA1 degradation and foam cell formation

ATP-binding cassette transporter A1 (ABCA1) is an essential regulator of intracellular cholesterol efflux. Secreted cholesterol binds to lipid-free apolipoprotein A-I (apoA-I) in peripheral blood to constitute high-density lipoprotein cholesterol (HDL) complexes. ABCA1 protein on the surface of macrophages acts as a crucial controller in preventing cholesterol accumulation. Importantly, ABCA1 is unstable and easily degraded via a series of biochemical activities, including but not limited to calpain-mediated and ubiquitin-proteasome system-mediated processes. How accelerated ABCA1 degradation impacts disordered lipid metabolism in macrophages and foam cell formation is unclear. N-methyl d-aspartate receptors (NMDARs) are ionotropic glutamate receptors with high calcium permeability. Calcium influx via NMDARs activates downstream signaling pathways. Over-activation of NMDARs stimulated by NMDA contributes to dysfunctional lipid metabolism in macrophages and foam cell formation via promotion of calpain-mediated ABCA1 proteolysis. However, increased NMDAR activity does not affect liver X receptor expression or ABCA1 mRNA levels. Following NMDA receptor silencing or calpain inhibition, NMDA treatment did not reduce ABCA1 protein levels, nor caused lipid accumulation in macrophages. In addition, NMDAR over-activation activates NF-κB signaling to promote IL-1β and IL-6 macrophage marker expression. However, NMDAR silencing and calpain inhibition reduce inflammatory macrophage responses. In summary, our study suggests that NMDAR activation reduces surface ABCA1 protein, promotes lipid accumulation, and induces the production and secretion of many inflammatory mediators in macrophages, possibly through enhanced calpain-mediated ABCA1 protein degradation. Thus, the NMDAR receptor may be a novel pharmacologic target for atherosclerosis therapy.

Protopanaxadiol ginsenoside Rd protects against NMDA receptor-mediated excitotoxicity by attenuating calcineurin-regulated DAPK1 activity

Neuroprotective strategies in the treatment of stroke have been attracting a great deal of attentions. Our previous clinical and basic studies have demonstrated that protopanaxadiol ginsenoside-Rd (Rd), a monomer compound extracted from Panax ginseng or Panax notoginseng, has neuroprotective effects against ischemic stroke, probably due to its ability to block Ca2+ overload, an usual consequence of the overactivation of NMDA receptor (NMDAR). As an extending study, we explored here whether Rd exerted its neuroprotection as a novel NMDAR blocker. Our whole-cell patch-clamp results showed that Rd reduced NMDAR currents of cultured rat cortical neurons (EC50 = 7.7 μM) dose-dependently by acting on extrasynaptic NMDAR NR2b subunit. However, unexpectedly, cell transfection and radioligand binding assays revealed that Rd did not bind to the NMDAR channel directly. Alternatively, it inhibited the phosphorylation of NR2b at Ser-1303, a target of death associated protein kinase 1 (DAPK1). Moreover, cell-based and cell-free enzymatic assays showed that Rd did not inhibit the activity of DAPK1 directly, but blocked the activity of calcineurin, a key phosphatase for activating DAPK1. Importantly, other protopanaxadiol ginsenosides were also found to have potential inhibitory effects on calcineurin activity. Furthermore, as expected, calcineurin inhibition by cyclosporin A could mimic Rd’s effects and protect against NMDA-, oxygen glucose deprivation- or transient ischemic stroke-induced neuronal injury. Therefore, our present study provided the first evidence that Rd could exert an inhibitive effect on NMDAR-triggered currents and sequential excitotoxicity through mitigation of DAPK1-mediated NR2b phosphorylation by attenuating calcineurin activity.

Pananx notoginseng saponins attenuate CCL2-induced cognitive deficits in rats via anti-inflammation and anti-apoptosis effects that involve suppressing over-activation of NMDA receptors

HIV-associated neurocognitive disorders (HAND) are characterized by synaptic damage and neuronal loss in the brain, ultimately leading to progressive decline of cognitive abilities and memory. Chemokine CC motif ligand 2 (CCL2) is elevated in cerebrospinal fluid (CSF), and has been believed to contribute to HAND. Previous studies by our research team have shown that CCL2 enhances N-Methyl-D-aspartate receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs) and causes nerve cell damage. However, there are few drugs currently available to treat nerve damage that is caused by CCL2. Panax notoginseng saponins (PNS) are isolated from Panax notoginseng and benefit the human body in various ways, including the neuroprotective effect. However, the protective effect of PNS on CCL2-induced neurotoxicity remains unknown. In this study, we found that PNS improved CCL2-induced learning and memory impairment, and inhibited CCL2-induced cell death. These effects may be due to inhibiting over-activation of NMDA receptors by alleviating the dysfunction of glutamate metabolism. Furthermore, PNS-modulated CCL2-inducd intracellular oxidative stress was found to attenuate cell inflammation. Additionally, PNS pretreatment evidently inhibited apoptotic pathways by reducing the Bax/BCL-2 ratio and caspase-3, 8, 9 expressions. In conclusion, this study demonstrates that PNS provides substantial neuroprotection against CCL2-induced neurotoxicity, and may be a novel therapeutic agent in CCL2-induced HAND or other neurodegenerative diseases.

Merging memantine and ferulic acid to probe connections between NMDA receptors, oxidative stress and amyloid-β peptide in Alzheimer's disease

N-methyl-d-aspartate receptors (NMDAR) are critically involved in the pathogenesis of Alzheimer's disease (AD). Acting as an open-channel blocker, the anti-AD drug memantine preferentially targets NMDAR overactivation, which has been proposed to trigger neurotoxic events mediated by amyloid β peptide (Aβ) and oxidative stress. In this study, we applied a multifunctional approach by conjugating memantine to ferulic acid, which is known to protect the brain from Aβ neurotoxicity and neuronal death caused by ROS. The most interesting compound (7) behaved, like memantine, as a voltage-dependent antagonist of NMDAR (IC50 = 6.9 μM). In addition, at 10 μM concentration, 7 exerted antioxidant properties both directly and indirectly through the activation of the Nrf-2 pathway in SH-SY5Y cells. At the same concentration, differently from the parent compounds memantine and ferulic acid alone, it was able to modulate Aβ production, as revealed by the observed increase of the non-amyloidogenic sAPPα in H4-SW cells. These findings suggest that compound 7 may represent a promising tool for investigating NMDAR-mediated neurotoxic events involving Aβ burden and oxidative damage.

Modification of the 4-phenylbutyl side chain of potent 3-benzazepine-based GluN2B receptor antagonists

Excitotoxicity driven by overactivation of NMDA receptors represents a major mechanism of acute and chronic neurological and neurodegenerative disorders. Negative allosteric modulators interacting with the ifenprodil binding site of the NMDA receptor are able to interrupt this ongoing neurodamaging process. Starting from the potent 3-benzazepine-1,7-diol 4a novel NMDA receptor antagonists were designed by modification of the N-(4-phenylbutyl) side chain. With respect to developing novel fluorinated PET tracers, regioisomeric fluoroethoxy derivatives 11, 12, 14, and 15 were synthesized. Analogs 19 and 20 with various heteroaryl moieties at the end of the N-side chain were prepared by Sonogashira reaction and nucleophilic substitution. The fluoroethyl triazole 37 was obtained by 1,3-dipolar cycloaddition. In several new ligands, the flexibility of the (hetero)arylbutyl side chain was restricted by incorporation of a triple bond. The affinity towards the ifenprodil binding site was tested in an established competition assay using [3H]ifenprodil as radioligand. Introduction of a fluoroethoxy moiety at the terminal phenyl ring, replacement of the terminal phenyl ring by a heteroaryl ring and incorporation of a triple bond into the butyl spacer led to considerable reduction of GluN2B affinity. The phenol 15 (Ki = 193 nM) bearing a p-fluoroethoxy moiety at the terminal phenyl ring represents the most promising GluN2B ligand of this series of compounds. With exception of 15 showing moderate σ2 affinity (Ki = 79 nM), the interaction of synthesized 3-benzazepines towards the PCP binding site of the NMDA receptor, σ1 and σ2 receptors was rather low (Ki > 100 nM).