NMDA Receptor Signaling Research Articles

Abstract P017: Circulating tumor DNA kinetics identify prodynorphin signaling as a target to radiosensitize non-small cell lung cancer

Abstract Introduction: Definitive chemoradiation therapy (CRT) is essential for controlling locoregionally advanced non-small cell lung cancer (NSCLC); however, up to 30% of patients experience local recurrences within the radiation field. Analyzing favorable responders to treatment could enable identification of genetic alterations associated with radiosensitivity, but standard imaging cannot distinguish residual disease from inflammation and fibrosis during treatment to assess treatment response. We hypothesized that circulating tumor DNA (ctDNA) kinetics during CRT could identify genetic alterations associated with radiosensitivity that could be validated in preclinical models and harnessed to develop new combination therapies with radiotherapy (RT). Methods: We applied cancer personalized profiling by deep sequencing (CAPP-Seq) ctDNA analysis to pre-CRT and mid-treatment (mid-CRT) plasma samples from 61 patients with Stage II-III NSCLC. We identified ctDNA rapid responders as the 10 patients with the largest decrease in ctDNA concentration mid-CRT without local progression and determined the prevalence of genetic alterations in rapid responders versus slow responders using Fisher’s exact tests corrected for multiple hypothesis testing. To investigate the therapeutic potential of targeting PDYN during RT, we performed clonogenic assays on isogeneic PDYN CRISPR-Cas9 knockout and wildtype H1299 and SW1573 human NSCLC cell lines and H1299 cells treated with prodynorphin-neutralizing antibodies. Results: Mid-CRT log-fold change in ctDNA concentration was significantly associated with progression-free survival (P=0.02) in Stage II-III NSCLC. Mutations in PDYN, which encodes the opioid peptide precursor protein prodynorphin, were observed in 30% of ctDNA rapid responders and 0% of ctDNA slow responders (adjusted P=0.04). In NSCLC patients from TCGA treated with RT, the cumulative incidence of local failure was significantly lower in tumors with PDYN mutations (0% vs. 17% at 3 years, P<0.001). PDYN knockout (PDYNNULL) radiosensitized human NSCLC cancer cells by clonogenic assay, and extracellular administration of prodynorphin or its cleavage product dynophin A partially restored radioresistance in PDYNNULL cells. Prodynorphin cleavage products have been reported to signal through opioid receptors and to antagonize N-methyl-D-aspartate receptor (NMDAR) signaling. Although treatment of wildtype H1299 cells with opioid receptor antagonists did not affect radiosensitivity, the NMDAR antagonist MK-801 partially restored RT resistance in PDYNNULL cells. Finally, prodynorphin-neutralizing antibodies significantly reduced clonogenic survival of human NSCLC cells. Conclusions: ctDNA kinetics enable the identification of patients responding favorably to treatment and putative targets to enhance the efficacy of RT. Targeting prodynorphin may enhance the radiosensitivity of NSCLC by increasing NMDAR signaling. Citation Format: Ziwei Wang, Angela B. Hui, Ash A. Alizadeh, Maximilian Diehn, Everett J. Moding.Circulating tumor DNA kinetics identify prodynorphin signaling as a target to radiosensitize non-small cell lung cancer.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P017

EAAT2 Activation Regulates Glutamate Excitotoxicity and Reduces Impulsivity in a Rodent Model of Parkinson's Disease.

Parkinson's disease (PD) is a systemic disease characterized by motor and nonmotor impairments. Loss of dopaminergic neurons in the substantia nigra pars compacta region in PD disrupts dopamine-glutamate homeostasis in the corticostriatal circuit, contributing to cognitive impairment. In addition, excitatory amino acid transporter-2 (EAAT2), localized predominantly to astrocytes and responsible for > 80% of synaptic glutamate clearance, is downregulated in PD, causing glutamate spillover and excitotoxicity. This altered dopamine-glutamate homeostasis and excitotoxicity may affect reward-mediated decision-making behaviors and promote impulsive behaviors in PD. In this study, we hypothesized that GTS467, a small-molecule activator of EAAT2, could effectively reduce excitotoxicity and treat cognitive impairment without promoting impulsive behavior in PD. Rats that were unilaterally lesioned with the 6-OHDA toxin to produce Parkinsonian symptoms were referred to as lesioned rats. Lesioned rats were trained to meet baseline criteria in a 5-choice serial reaction time task, and the chronic effects of GTS467 were assessed after 3weeks of treatment. The results showed that chronic treatment with GTS467 significantly improved correct responses and reduced premature impulsive responses and omissions compared with saline treatment. This improvement in performance correlated with a reduction in glutamate levels, an increase in EAAT2 expression, and normalization of NMDA receptor subunit expression and signaling. Furthermore, transcriptomic studies on the prefrontal cortex tissue have shown the differential expression of genes involved in neuroprotection, neuroinflammation, learning, and memory. These results validate the role of glutamate excitotoxicity in promoting impulsive behaviors and suggest that GTS467 can be developed as a therapeutic agent to reduce cognitive impairment and impulsive behaviors in PD.

NMDA Receptor-Mediated Ca2+ Flux Attenuated by the NMDA Receptor/TRPM4 Interface Inhibitor Brophenexin.

Transient receptor potential melastatin-4 (TRPM4) forms a complex with N-methyl-D-aspartate receptors (NMDARs) that facilitates NMDAR-mediated neurotoxicity. Here we used pharmacological tools to determine how TRPM4 regulates NMDAR signaling. Brophenexin, a compound that binds to TRPM4 at the NMDAR binding interface, protected hippocampal neurons in culture from NMDA-induced death, consistent with published work. Brophenexin (10 μM) reduced NMDA-evoked whole-cell currents recorded at 22°C by 87% ± 14% with intracellular Ca2+ chelated to prevent TRPM4 activation. Brophenexin inhibited NMDA-evoked currents recorded in Na+-free solution by 87% ± 13%, suggesting that brophenexin and TRPM4 modulate NMDAR function. Incubating cultures in Mg2+-free buffer containing tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, and bicuculline for 30 min inhibited NMDA-evoked increases in intracellular Ca2+ concentration ([Ca2+]i) recorded at 22°C by 50% ± 18% and prevented inhibition by brophenexin. In the absence of these inhibitors, brophenexin inhibited the NMDA-evoked response by 51% ± 16%. Treatment with the TRPM4 inhibitor 4-chloro-2-(1-naphthyloxyacetamido)benzoic acid (NBA; 10 μM) increased NMDA-evoked Ca2+ influx by 90% ± 15%. Increasing extracellular NaCl to 237 mM, a treatment that activates TRPM4, inhibited the NMDA-evoked increase in [Ca2+]i by a process that occluded the inhibition produced by brophenexin and was prevented by NBA. In recordings performed at 32°C-34°C, brophenexin inhibited the NMDA-evoked [Ca2+]i response by 42% ± 10% but NBA was without effect. These results are consistent with a model in which TRPM4 interacts with NMDARs to potentiate Ca2+ flux through the NMDAR ion channel and thus provides a potential mechanism for the neuroprotection afforded by NMDAR/TRPM4 interface inhibitors such as brophenexin.

Metabotropic NMDAR Signaling Contributes to Sex Differences in Synaptic Plasticity and Episodic Memory.

NMDA receptor (NMDAR)-mediated calcium influx triggers the induction and initial expression of long-term potentiation (LTP). Here we report that in male rodents, ion flux-independent (metabotropic) NMDAR signaling is critical for a third step in the production of enduring LTP, i.e., cytoskeletal changes that stabilize the activity-induced synaptic modifications. Surprisingly, females rely upon estrogen receptor alpha (ERα) for the metabotropic NMDAR operations used by males. Blocking NMDAR channels with MK-801 eliminated LTP expression in hippocampal field CA1 of both sexes but left intact theta burst stimulation (TBS)-induced actin polymerization within dendritic spines. A selective antagonist (Ro25-6981) of the NMDAR GluN2B subunit had minimal effects on synaptic responses but blocked actin polymerization and LTP consolidation in males only. Conversely, an ERα antagonist thoroughly disrupted TBS-induced actin polymerization and LTP in females while having no evident effect in males. In an episodic memory paradigm, Ro25-6981 prevented acquisition of spatial locations by males but not females, whereas an ERα antagonist blocked acquisition in females but not males. Sex differences in LTP consolidation were accompanied by pronounced differences in episodic memory in tasks involving minimal (for learning) cue sampling. Males did better on acquisition of spatial information whereas females had much higher scores than males on tests for acquisition of the identity of cues (episodic "what") and the order in which the cues were sampled (episodic "when"). We propose that sex differences in synaptic processes used to stabilize LTP result in differential encoding of the basic elements of episodic memory.

Autism spectrum disorder-like behaviors induced by hyper-glutamatergic NMDA receptor signaling through hypo-serotonergic 5-HT1A receptor signaling in the prefrontal cortex in mice exposed to prenatal valproic acid.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behaviors, social deficits, and cognitive impairments. Maternal use of valproic acid (VPA) during pregnancy is associated with an increased risk of ASD in offspring. The prevailing pathophysiological hypothesis for ASD involves excitation/inhibition (E/I) imbalances and serotonergic dysfunction. Here, we investigated the association between glutamatergic-serotonergic neuronal interactions and ASD-like behaviors in mice exposed to prenatal VPA. Prenatal VPA exposure induced excessive repetitive self-grooming behavior and impaired social behavior and object recognition memory in young adult period. Prenatal VPA mice showed hyper-glutamatergic function (increase in basal extracellular glutamate levels and CaMKII phosphorylation) and hypo-serotonergic function (decrease in 5-hydroxyindoleacetic acid and stimulation-induced serotonin [5-HT] release, but an increase in 5-HT transporter expression) in the prefrontal cortex. Treatment with a low-affinity NMDA receptor antagonist (memantine), a selective 5-HT reuptake inhibitor (fluoxetine), and a 5-HT1A receptor agonist (tandospirone) attenuated both the increase in CaMKII phosphorylation and ASD-like behavior of prenatal VPA mice. Opto-genetic activation of the serotonergic neuronal system attenuated impairments in social behavior and object recognition memory in prenatal VPA mice. WAY-100635-a 5-HT1A receptor antagonist-antagonized the effect of fluoxetine on impaired social behavior and object recognition memory. These results suggest that E/I imbalance and ASD-like behavior are associated with hypo-serotonergic receptor signaling through 5-HT1A receptors in prenatal VPA mice.

Kynurenic acid protects against ischemia/reperfusion injury by modulating apoptosis in cardiomyocytes

Acute myocardial infarction, often associated with ischemia/reperfusion injury (I/R), is a leading cause of death worldwide. Although the endogenous tryptophan metabolite kynurenic acid (KYNA) has been shown to exert protection against I/R injury, its mechanism of action at the cellular and molecular level is not well understood yet. Therefore, we examined the potential involvement of antiapoptotic mechanisms, as well as N-methyl-D-aspartate (NMDA) receptor modulation in the protective effect of KYNA in cardiac cells exposed to simulated I/R (SI/R). KYNA was shown to attenuate cell death induced by SI/R dose-dependently in H9c2 cells or primary rat cardiomyocytes. Analysis of morphological and molecular markers of apoptosis (i.e., membrane blebbing, apoptotic nuclear morphology, DNA double-strand breaks, activation of caspases) revealed considerably increased apoptotic activity in cardiac cells undergoing SI/R. The investigated apoptotic markers were substantially improved by treatment with the cytoprotective dose of KYNA. Although cardiac cells were shown to express NMDA receptors, another NMDA antagonist structurally different from KYNA was unable to protect against SI/R-induced cell death. Our findings provide evidence that the protective effect of KYNA against SI/R-induced cardiac cell injury involves antiapoptotic mechanisms, that seem to evoke independently of NMDA receptor signaling.

Decreased free D-aspartate levels in the blood serum of patients with schizophrenia.

Schizophrenia (SCZ) and autism spectrum disorder (ASD) are neurodevelopmental diseases characterized by different psychopathological manifestations and divergent clinical trajectories. Various alterations at glutamatergic synapses have been reported in both disorders, including abnormal NMDA and metabotropic receptor signaling. We conducted a bicentric study to assess the blood serum levels of NMDA receptors-related glutamatergic amino acids and their precursors, including L-glutamate, L-glutamine, D-aspartate, L-aspartate, L-asparagine, D-serine, L-serine and glycine, in ASD, SCZ patients and their respective control subjects. Specifically, the SCZ patients were subdivided into treatment-resistant and non-treatment-resistant SCZ patients, based on their responsivity to conventional antipsychotics. D-serine and D-aspartate serum reductions were found in SCZ patients compared to controls. Conversely, no significant differences between cases and controls were found in amino acid concentrations in the two ASD cohorts analyzed. This result further encourages future research to evaluate the predictive role of selected D-amino acids as peripheral markers for SCZ pathophysiology and diagnosis.

APP fragment controls both ionotropic and non-ionotropic signaling of NMDA receptors

NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-β precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by invivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.

Probing cognitive flexibility in Shank2-deficient mice: Effects of D-cycloserine and NMDAR signaling hub dynamics

Neurodevelopmental disorders such as autism spectrum disorder (ASD) have a heterogeneous etiology but are largely associated with genetic factors. Robust evidence from recent human genetic studies has linked mutations in the Shank2 gene to idiopathic ASD. Modeling these Shank2 mutations in animal models recapitulates behavioral changes, e.g. impaired social interaction and repetitive behavior of ASD patients. Shank2-deficient mice exhibit NMDA receptor (NMDAR) hypofunction and associated behavioral deficits. Of note, NMDARs are strongly implicated in cognitive flexibility. Their hypofunction, e.g. observed in schizophrenia, or their pharmacological inhibition leads to impaired cognitive flexibility. However, the association between Shank2 mutations and cognitive flexibility is poorly understood. Using Shank2-deficient mice, we explored the role of Shank2 in cognitive flexibility measured by the attentional set shifting task (ASST) and whether ASST performance in Shank2-deficient mice can be modulated by treatment with the partial NMDAR agonist D-cycloserine (DCS). Furthermore, we investigated the effects of Shank2 deficiency, ASST training, and DCS treatment on the expression level of NMDAR signaling hub components in the orbitofrontal cortex (OFC), including NMDAR subunits (GluN2A, GluN2B, GluN2C), phosphoglycerate dehydrogenase and serine racemase. Surprisingly, Shank2 deficiency did not affect ASST performance or alter the expression of the investigated NMDAR signaling hub components. Importantly, however, DCS significantly improved ASST performance, demonstrating that positive NMDAR modulation facilitates cognitive flexibility. Furthermore, DCS increased the expression of GluN2A in the OFC, but not that of other NMDAR signaling hub components. Our findings highlight the potential of DCS as a pharmacological intervention to improve cognitive flexibility impairments downstream of NMDAR modulation and substantiate the key role of NMDAR in cognitive flexibility.

Dual-step pharmacological intervention for traumatic-like memories: implications from D-cycloserine and cannabidiol or clonidine in male and female rats.

Therapeutic approaches to mitigating traumatic memories have often faced resistance. Exploring safe reconsolidation blockers, drugs capable of reducing the emotional valence of the memory upon brief retrieval and reactivation, emerges as a promising pharmacological strategy. Towards this objective, preclinical investigations should focus on aversive memories resulting in maladaptive outcomes and consider sex-related differences to enhance their translatability. After selecting a relatively high training magnitude leading to the formation of a more intense and generalized fear memory in adult female and male rats, we investigated whether two clinically approved drugs disrupting its reconsolidation remain effective. We found resistant reconsolidation impairment by the α2-adrenergic receptor agonist clonidine or cannabidiol, a major non-psychotomimetic Cannabis sativa component. However, pre-retrieval administration of D-cycloserine, a partial agonist at the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor complex, facilitated their impairing effects on reconsolidation. A similar reconsolidation blockade by clonidine or cannabidiol was achieved following exposure to a non-conditioned but generalized context after D-cycloserine administration. This suggests that sufficient memory destabilization can accompany generalized fear expression. Combining clonidine with cannabidiol without potentiating memory destabilization by D-cycloserine was ineffective. These findings highlight the importance of NMDA receptor signaling in memory destabilization and underscore the efficacy of a dual-step pharmacological intervention in attenuating traumatic-like memories, even in a context different from the original learning environment.

Edaravone dexborneol attenuates cognitive impairment in a rat model of vascular dementia by inhibiting hippocampal oxidative stress and inflammatory responses and modulating the NMDA receptor signaling pathway

Exploring the intricate pathogenesis of Vascular Dementia (VD), there is a noted absence of potent treatments available in the current medical landscape. A new brain-protective medication developed in China, Edaravone dexboeol (EDB), has shown promise due to its antioxidant and anti-inflammatory properties, albeit with a need for additional research to elucidate its role and mechanisms in VD contexts. In a research setup, a VD model was established utilizing Sprague-Dawley (SD) rats, subjected to permanent bilateral typical carotid artery occlusion (2VO). Behavioral assessment of the rats was conducted using the Bederson test and pole climbing test, while cognitive abilities, particularly learning and memory, were evaluated via the novel object recognition test and the Morris water maze test. Ensuing, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), IL-1β, IL-6, IL-4, and tumor necrosis factor-α (TNF-α) were determined through Enzyme-Linked Immunosorbent Assay (ELISA). Synaptic plasticity-related proteins, synaptophysin (SYP), post-synaptic density protein 95 (PSD-95), and N-methyl-D-aspartate (NMDA) receptor proteins (NR1, NR2A, NR2B) were investigated via Western blotting technique. The findings imply that EDB has the potential to ameliorate cognitive deficiencies, attributed to VD, by mitigating oxidative stress, dampening inflammatory responses, and modulating the NMDA receptor signaling pathway, furnishing new perspectives into EDB’s mechanism and proposing potential avenues for therapeutic strategies in managing VD.

Quercetin attenuated ischemic stroke induced neurodegeneration by modulating glutamatergic and synaptic signaling pathways

Ischemic strokes originate whenever the circulation to the brain is interrupted, either temporarily or permanently, resulting in a lack of oxygen and other nutrients. This deprivation primarily impacts the cerebral cortex and striatum, resulting in neurodegeneration. Several experimental stroke models have demonstrated that the potent antioxidant quercetin offers protection against stroke-related damage. Multiple pathways have been associated with quercetin's ability to safeguard the brain from ischemic injury. This study examines whether the administration of quercetin alters glutamate NMDA and GluR1 receptor signaling in the cortex and striatum 72 h after transient middle cerebral artery occlusion. The administration of 10 mg/kg of quercetin shielded cortical and striatal neurons from cell death induced by ischemia in adult SD rats. Quercetin reversed the ischemia-induced reduction of NR2a/PSD95, consequently promoting the pro-survival AKT pathway and reducing CRMP2 phosphorylation. Additionally, quercetin decreased the levels of reactive oxygen species and inflammatory pathways while increasing the expression of the postsynaptic protein PSD95. Our results suggest that quercetin may be a promising neuroprotective drug for ischemic stroke therapy as it recovers neuronal damage via multiple pathways.

Enhanced NMDA receptor pathway and glutamate transmission in the hippocampal dentate gyrus mediate the spatial learning and memory impairment of obese rats.

Obesity has been linked with the impairment of spatial memory and synaptic plasticity but the molecular mechanisms remained unidentified. Since glutamatergic transmission and NMDA receptor neural pathways in hippocampal dentate gyrus (DG) are essential in the learning and memory, we aimed to investigate glutamate (Glu) and NMDA receptor signaling of DG in spatial learning and memory in diet-induced obesity (DIO) rats. Spatial learning and memory were assessed via Morris water maze (MWM) test on control (Ctr) and DIO rats. Extracellular concentration of Glu in the DG was determined using in vivo microdialysis and HPLC. The protein expressions of NMDA receptor subunit 2B (NR2B), brain-derived neurotrophic factor (BDNF), the activation of calcium/calmodulin-dependent kinase II (CaMKII) and cAMP-response-element-binding protein (CREB) in the DG were observed by western blot. Spatial learning and memory were impaired in DIO rats compared to those of Ctr. NR2B expression was increased, while BDNF expression and CaMKII and CREB activation were decreased in DG of DIO rats. Extracellular concentration of Glu was increased in Ctr on the 3rd and 4th days of the MWM test, but significant further increment was observed in DIO rats. Microinjection of an NMDA antagonist (MK-801) into the DG reversed spatial learning and memory impairment. Such effects were accompanied by greater BDNF expression and CaMKII/CREB activation in the DG of DIO rats. In conclusion, the enhancement of Glu-NMDA receptor transmission in the hippocampal DG contributes to the impairment of spatial learning and memory in DIO rats, maybe via the modulation of CaMKII-CREB-BDNF signaling pathway.

Radiation-induced Bystander Effects on Glioblastoma Tumor Cells via NMDA Receptor Signaling.

Proton therapy has been widely applied on treating inaccessible and inoperable tumors, such as tumors deep within the brain or close to the critical brain stem. Nevertheless, the damaging effect of radiation for central nervous system (CNS) tumors is difficult to be confined within the irradiated region and has led to decline of neurological function in especially children with congenital CNS tumors. Currently, the involvement of n-methyl-d-aspartate (NMDA) receptors or secretary cytokines and chemokines in proton-induced bystander effects remains unclear. To understand the modulatory effects of NMDA receptor inhibition on the survival and proliferation of glioblastoma-derived cells, mesenchymal-like U373 cells were applied along with U87 neural glioblastoma cells for single doses of proton radiation at different LET in the presence or absence of pretreatment with memantine and/or collimation. Under collimation, neuronal tumor cells that are not directly irradiated (i.e., bystander cells) encounter similar biological effects potentially through cell coupling and synaptic transmission. Furthermore, whether proton LET plays a role in the mediation of bystander effect awaits to be elucidated. From this study, synaptic transmission was found to play differential roles in the proliferation of U373 and U87 cells after exposure to collimated radiation. Also, radiation-induced cell proliferation at the late stage was more correlated with bystander cell survival than early manifested γH2AX foci, suggesting that proton-induced glutamatergic synapse may act as a more important contributor than proton-induced direct effect on DNA double-stranded breaks to the late-stage responses of glioblastoma cells.

Locally Synthetized 17-β-Estradiol Reverses Amyloid-β-42-Induced Hippocampal Long-Term Potentiation Deficits.

Amyloid beta 1-42 (Aβ42) aggregates acutely impair hippocampal long-term potentiation (LTP) of synaptic transmission, and 17β-estradiol is crucial for hippocampal LTP. We tested whether boosting the synthesis of neural-derived 17β-estradiol (nE2) saves hippocampal LTP by the neurotoxic action of Aβ42. Electrophysiological recordings were performed to measure dentate gyrus (DG) LTP in rat hippocampal slices. Using a pharmacological approach, we tested the ability of nE2 to counteract the LTP impairment caused by acute exposure to soluble Aβ42 aggregates. nE2 was found to be required for LTP in DG under physiological conditions. Blockade of steroid 5α-reductase with finasteride, by increasing nE2 synthesis from testosterone (T), completely recovered LTP in slices treated with soluble Aβ42 aggregates. Modulation of the glutamate N-methyl-D aspartate receptor (NMDAR) by memantine effectively rescued the LTP deficit observed in slices exposed to Aβ42, and memantine prevented LTP reduction observed under the blocking of nE2 synthesis. nE2 is able to counteract Aβ42-induced synaptic dysfunction. This effect depends on a rapid, non-genomic mechanism of action of nE2, which may share a common pathway with glutamate NMDAR signaling.

Improving stroke outcomes in hyperglycemic mice by modulating tPA/NMDAR signaling to reduce inflammation and hemorrhages

AbstractThe pharmacological intervention for ischemic stroke hinges on intravenous administration of the recombinant tissue-type plasminogen activator (rtPA, Alteplase/Actilyse) either as a standalone treatment or in conjunction with thrombectomy. However, despite its clinical significance, broader use of rtPA is constrained because of the risk of hemorrhagic transformations (HTs). Furthermore, the presence of diabetes or chronic hyperglycemia is associated with an elevated risk of HT subsequent to thrombolysis. This detrimental impact of tPA on the neurovascular unit in patients with hyperglycemia has been ascribed to its capacity to induce endothelial N-methyl-D-aspartate receptor (NMDAR) signaling, contributing to compromised blood-brain barrier integrity and neuroinflammatory processes. In a mouse model of thromboembolic stroke with chronic hyperglycemia, we assessed the effectiveness of rtPA and N-acetylcysteine (NAC) as thrombolytic agents. We also tested the effect of blocking tPA/NMDAR signaling using a monoclonal antibody, Glunomab. Magnetic resonance imaging, speckle contrast imaging, flow cytometry, and behavioral tasks were used to evaluate stroke outcomes. In hyperglycemic animals, treatment with rtPA resulted in lower recanalization rates and increased HTs. Conversely, NAC treatment reduced lesion sizes while mitigating HTs. After a single administration, either in standalone or combined with rtPA-induced thrombolysis, Glunomab reduced brain lesion volumes, HTs, and neuroinflammation after stroke, translating into improved neurological outcomes. Additionally, we demonstrated the therapeutic efficacy of Glunomab in combination with NAC or as a standalone strategy in chronic hyperglycemic animals. Counteracting tPA-dependent endothelial NMDAR signaling limits ischemic damages induced by both endogenous and exogenous tPA, including HTs and inflammatory processes after ischemic stroke in hyperglycemic animals.

Leukocyte differential gene expression prognostic value for high versus low seizure frequency in temporal lobe epilepsy

BackgroundThis study was performed to test the hypothesis that systemic leukocyte gene expression has prognostic value differentiating low from high seizure frequency refractory temporal lobe epilepsy (TLE).MethodsA consecutive series of patients with refractory temporal lobe epilepsy was studied. Based on a median baseline seizure frequency of 2.0 seizures per month, low versus high seizure frequency was defined as ≤ 2 seizures/month and > 2 seizures/month, respectively. Systemic leukocyte gene expression was analyzed for prognostic value for TLE seizure frequency. All differentially expressed genes were analyzed, with Ingenuity® Pathway Analysis (IPA®) and Reactome, to identify leukocyte gene expression and biological pathways with prognostic value for seizure frequency.ResultsThere were ten males and six females with a mean age of 39.4 years (range: 16 to 62 years, standard error of mean: 3.6 years). There were five patients in the high and eleven patients in the low seizure frequency cohorts, respectively. Based on a threshold of twofold change (p < 0.001, FC > 2.0, FDR < 0.05) and expression within at least two pathways from both Reactome and Ingenuity® Pathway Analysis (IPA®), 13 differentially expressed leukocyte genes were identified which were all over-expressed in the low when compared to the high seizure frequency groups, including NCF2, HMOX1, RHOB, FCGR2A, PRKCD, RAC2, TLR1, CHP1, TNFRSF1A, IFNGR1, LYN, MYD88, and CASP1. Similar analysis identified four differentially expressed genes which were all over-expressed in the high when compared to the low seizure frequency groups, including AK1, F2R, GNB5, and TYMS.ConclusionsLow and high seizure frequency TLE are predicted by the respective upregulation and downregulation of specific leukocyte genes involved in canonical pathways of neuroinflammation, oxidative stress and lipid peroxidation, GABA (γ-aminobutyric acid) inhibition, and AMPA and NMDA receptor signaling. Furthermore, high seizure frequency-TLE is distinguished prognostically from low seizure frequency-TLE by differentially increased specific leukocyte gene expression involved in GABA inhibition and NMDA receptor signaling. High and low seizure frequency patients appear to represent two mechanistically different forms of temporal lobe epilepsy based on leukocyte gene expression.

Estimating the Ca2+ Block of NMDA Receptors with Single-Channel Electrophysiology.

In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.

Impairments of Spatial Memory and N-methyl-d-aspartate Receptors and Their Postsynaptic Signaling Molecules in the Hippocampus of Developing Rats Induced by As, Pb, and Mn Mixture Exposure.

Exposure to metal mixtures is recognized as a real-life scenario, needing novel studies that can assess their complex effects on brain development. There is still a significant public health concern associated with chronic low levels of metal exposure. In contrast to other metals, these three metals (As, Pb, and Mn) are commonly found in various environmental and industrial contexts. In addition to additive or synergistic interactions, concurrent exposure to this metal mixture may also have neurotoxic effects that differ from those caused by exposure to single components. The NMDA receptor and several important signaling proteins are involved in learning, memory, and synaptic plasticity in the hippocampus, including CaMKII, postsynaptic density protein-95 (PSD-95), synaptic Ras GTPase activating protein (SynGAP), a negative regulator of Ras-MAPK activity, and CREB. We hypothesized that alterations in the above molecular players may contribute to metal mixture developmental neurotoxicity. Thus, the aim of this study was to investigate the effect of these metals and their mixture at low doses (As 4 mg, Pb 4 mg, and Mn 10 mg/kg bw/p.o) on NMDA receptors and their postsynaptic signaling proteins during developing periods (GD6 to PD59) of the rat brain. Rats exposed to As, Pb, and Mn individually or at the same doses in a triple-metal mixture (MM) showed impairments in learning and memory functions in comparison to the control group rats. Declined protein expressions of NR2A, PSD-95, p- CaMKII, and pCREB were observed in the metal mix-exposed rats, while the expression of SynGAP was found to be enhanced in the hippocampus as compared to the controls on PD60. Thereby, our data suggest that alterations in the NMDA receptor complex and postsynaptic signaling proteins could explain the cognitive dysfunctions caused by metal-mixture-induced developmental neurotoxicity in rats. These outcomes indicate that incessant metal mixture exposure may have detrimental consequences on brain development.

The role of glutamate NMDA receptors of the mediodorsal thalamus in scopolamine-induced amnesia in rats

The current study was designed to examine the role of glutamate NMDA receptors of the mediodorsal thalamus (MD) in scopolamine-induced memory impairment. Adult male rats were bilaterally cannulated into the MD. According to the results, intraperitoneal (i.p.) administration of scopolamine (1.5 mg/kg) immediately after the training phase (post-training) impaired memory consolidation. Bilateral microinjection of the glutamate NMDA receptors agonist, N-Methyl-D-aspartic acid (NMDA; 0.05 µg/rat), into the MD significantly improved scopolamine-induced memory consolidation impairment. Co-administration of D-AP5, a glutamate NMDA receptor antagonist (0.001–0.005 µg/rat, intra-MD) potentiated the response of an ineffective dose of scopolamine (0.5 mg/kg, i.p.) to impair memory consolidation, mimicking the response of a higher dose of scopolamine. Noteworthy, post-training intra-MD microinjections of the same doses of NMDA or D-AP5 alone had no effect on memory consolidation. Moreover, the blockade of the glutamate NMDA receptors by 0.003 ng/rat of D-AP5 prevented the improving effect of NMDA on scopolamine-induced amnesia. Thus, it can be concluded that the MD glutamatergic system may be involved in scopolamine-induced memory impairment via the NMDA receptor signaling pathway.