Ionotropic Glutamate Receptors Research Articles

Divergent Presentation of GRIN2B Neurodevelopmental Disorder in Monozygotic Twins: Case Report with Unique Imaging Phenotypes.

We describe a set of monozygotic twins with Glutamate Ionotropic Receptor N-methyl-D-aspartate Type Subunit 2B-related neurodevelopmental disorder (GRIN2B-ND) who exhibited distinct clinical and imaging characteristics due to a de novo heterozygous pathogenic variant in the GRIN2B gene (c.2453T > C, p.Met818Thr). Twin A displayed extensive symmetric malformation of cortical development (MCD) resembling polymicrogyria, accompanied by shallow sulci, dilated lateral ventricles, and dysplastic appearances of the basal ganglia, corpus callosum, and hippocampi. In twin B, malformative features, such as reduced brain volume, MCD, shallow sulci, and dilated lateral ventricle, were confined to the left hemisphere. In combination with previously published data, our report highlights variable phenotypes associated with the p.(Met818Thr) pathogenic variant, specifically with a potential for asymmetric or even unilateral presentation. We discuss the potential interplay between genetic and environmental factors underlying this phenomenon within the context of monozygotic twins. In addition, we also highlight the importance of recognizing potential genetic underpinnings in the assessment of apparently unilateral brain malformations.

Exome sequencing reveals a rare damaging variant in GRIN2C in familial late-onset Alzheimer's disease

BackgroundAlzheimer's disease (AD) is a progressive neurodegenerative disorder with both genetic and environmental factors contributing to its pathogenesis. While early-onset AD has well-established genetic determinants, the genetic basis for late-onset AD remains less clear. This study investigates a large Italian family with late-onset autosomal dominant AD, identifying a novel rare missense variant in GRIN2C gene associated with the disease, and evaluates the functional impact of this variant.MethodsAffected and unaffected members from a Northern Italian family were included. Genomic DNA from family members was extracted and initially screened for pathogenic mutations in APP, PSEN1, and PSEN2, and screened for 77 genes associated with neurodegenerative conditions using NeuroX array assay. Exome sequencing was performed on three affected individuals and two healthy relatives. Bioinformatics analyses were conducted. Functional analysis was performed using primary neuronal cultures, and the impact of the variant was assessed through immunocytochemistry and electrophysiology.ResultsPathogenic variants were not identified in APP, PSEN1, or PSEN2, nor in the 77 genes in NeuroX array assay. Exome Sequencing revealed the c.3215C > T p.(A1072V) variant in GRIN2C gene (NM 000835.6), encoding for the glutamate ionotropic receptor N-methyl-D-aspartate receptor (NMDA) type subunit 2C (GluN2C). This variant segregated in 6 available AD patients in the family and was absent in 9 healthy relatives. Primary rat hippocampal neurons overexpressing GluN2CA1072V showed an increase in NMDAR-induced currents, suggesting altered glutamatergic transmission. Surface expression assays demonstrated an elevated surface/total ratio of the mutant GluN2C, correlating with the increased NMDAR current. Additionally, immunocytochemistry revealed in neurons expressing the mutant variant a reduced colocalization between the GluN2C subunit and 14-3-3 proteins, which are known to facilitate membrane trafficking of NMDARs.DiscussionWe identified a rare missense variant in GRIN2C associated with late-onset autosomal dominant Alzheimer's disease. These findings highlight the role of GluN2C-containing NMDARs in glutamatergic signaling and their potential contribution to AD pathogenesis.

Discovery of Brain-Penetrative Negative Allosteric Modulators of NMDA Receptors Using FEP-Guided Structure Optimization and Membrane Permeability Prediction.

N-Methyl-d-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptors in the central nervous system (CNS), have garnered attention for their role in brain disorders. Specifically, GluN2A-containing NMDA receptors have emerged as a potential therapeutic target for the treatment of depressive disorders and epilepsy. However, the development of GluN2A-containing NMDA receptor-selective antagonists, represented by N-(4-(2-benzoylhydrazine-1-carbonyl)benzyl)-3-chloro-4-fluorobenzenesulfonamide (TCN-201) and its derivatives, faces a significant challenge due to their limited ability to penetrate the blood-brain barrier (BBB), hampering their in vivo characterization and further advancement. In this study, we reported a series of 2-((5-(phemylamino)-1,3,4-thiadiazol-2-yl)thio)-N-(cyclohexylmethyl)acetamide derivatives, achieved through a structure-guided optimization strategy using free energy perturbation (FEP) and BBB permeability estimation. Through systematic exploration of various phenyl substitutions, compound 1f emerged as a standout compound, demonstrating substantially enhanced inhibitory activity compared with the lead compound TCN-213. Compound 1f not only displayed satisfactory BBB permeability but also showed antidepressant-like potency in the hydrocortisone-induced zebrafish depression-like model. All results position it as a promising candidate for developing innovative therapeutics for NMDA receptor-related disorders.

Identification of a Subpopulation of Astrocyte Progenitor Cells in the Neonatal Subventricular Zone: Evidence that Migration is Regulated by Glutamate Signaling

In mice engineered to express enhanced green fluorescent protein (eGFP) under the control of the entire glutamate transporter 1 (GLT1) gene, eGFP is found in all ‘adult’ cortical astrocytes. However, when 8.3 kilobases of the human GLT1/EAAT2 promoter is used to control expression of tdTomato (tdT), tdT is only found in a subpopulation of these eGFP-expressing astrocytes. The eGFP mice have been used to define mechanisms of transcriptional regulation using astrocytes cultured from cortex of 1–3 day old mice. Using the same cultures, we were never able to induce tdT+ expression. We hypothesized that these cells might not have migrated into the cortex by this age. In this study, we characterized the ontogeny of tdT+ cells, performed single-cell RNA sequencing (scRNA-seq), and tracked their migration in organotypic slice cultures. At postnatal day (PND) 1, tdT+ cells were observed in the subventricular zone and striatum but not in the cortex, and they did not express eGFP. At PND7, tdT+ cells begin to appear in the cortex with their numbers increasing with age. At PND1, scRNA-seq demonstrates that the tdT+ cells are molecularly heterogeneous, with a subpopulation expressing astrocytic markers, subsequently validated with immunofluorescence. In organotypic slices, tdT+ cells migrate into the cortex, and after 7 days they express GLT1, NF1A, and eGFP. An ionotropic glutamate receptor (iGluR) antagonist reduced by 50% the distance tdT+ cells migrate from the subventricular zone into the cortex. The pan-glutamate transport inhibitor, TFB-TBOA, increased, by sixfold, the number of tdT+ cells in the cortex. In conclusion, although tdT is expressed by non-glial cells at PND1, it is also expressed by glial progenitors that migrate into the cortex postnatally. Using this fluorescent labeling, we provide novel evidence that glutamate signaling contributes to the control of glial precursor migration.

Molecular Insights Into the Sensory Adaption of the Cave‐Dwelling Leech Sinospelaeobdella wulingensis to the Karst Cave Environment

ABSTRACTKarst caves are a unique environment significantly different from the external environment; adaptation of cave‐dwelling animals to the cave environment is often accompanied by shifts in the sensory systems. Aquatic and terrestrial leeches have been found in the karst caves. In this study, we conducted a transcriptome analysis on the cave‐dwelling leech Sinospelaeobdella wulingensis. A total of 29,286 unigenes were obtained by assembling the clean reads, and only 395 genes are differentially expressed in winter and summer samples. Two piezo‐type mechanosensitive ion channels (Piezos), eight transient receptor potential channels (TRPs), and six ionotropic glutamate receptors (iGluRs) were identified in the transcriptome. These channels/receptors are transmembrane proteins sharing conserved structural features in the respective protein families. SwPiezo1 shares high identity with Piezos in non‐caving leeches. SwiGluRs are conserved in protein sequence and share high identities with homologous proteins in other leeches. In contrast, SwTRPs belong to different subfamilies and share diverse identities with TRPs in other species. Gene expression analysis showed that two SwPiezos, five SwTRPs, and one SwiGluR are abundantly expressed in both winter and summer samples. These results suggest that SwPiezos, SwTRPs, and SwiGluRs are candidate sensory channels/receptors that may have roles in mechanosensory and chemosensory systems. High expression levels of Piezo and TRP genes imply a mechanosensory adaptation of S. wulingensis to the hanging living style in caves. Furthermore, enrichment of sensory genes in the oral sucker indicates the important role of this tissue in response to environmental stimuli. Similar gene expression profiles in winter and summer samples imply a stable physiological status of S. wulingensis in the cave environment.

Endoplasmic Reticulum Calcium Signaling in Hippocampal Neurons.

The endoplasmic reticulum (ER) is a key organelle in cellular homeostasis, regulating calcium levels and coordinating protein synthesis and folding. In neurons, the ER forms interconnected sheets and tubules that facilitate the propagation of calcium-based signals. Calcium plays a central role in the modulation and regulation of numerous functions in excitable cells. It is a versatile signaling molecule that influences neurotransmitter release, muscle contraction, gene expression, and cell survival. This review focuses on the intricate dynamics of calcium signaling in hippocampal neurons, with particular emphasis on the activation of voltage-gated and ionotropic glutamate receptors in the plasma membrane and ryanodine and inositol 1,4,5-trisphosphate receptors in the ER. These channels and receptors are involved in the generation and transmission of electrical signals and the modulation of calcium concentrations within the neuronal network. By analyzing calcium fluctuations in neurons and the associated calcium handling mechanisms at the ER, mitochondria, endo-lysosome and cytosol, we can gain a deeper understanding of the mechanistic pathways underlying neuronal interactions and information transfer.

Inhibitory Role of L-theanine, a Structural Analogue of Glutamate, Against GluR5 Kainate Receptor and its Prospective Utility Against Excitotoxicity.

Overactivation of receptors that respond to excitatory neurotransmitters can result in various harmful outcomes, such as the inability to properly modulate calcium levels, generation of free radicals, initiation of the mitochondrial permeability transition, and subsequent secondary damage caused by excitotoxicity. A non-proteinogenic amino acid of tea, L-theanine, is structurally related to glutamate, the major stimulatory neurotransmitter in the brain. Previous reports have emphasised its ability to bind with glutamate receptors. An in-depth understanding of the binding compatibility between ionotropic glutamate receptors and L-theanine is a compelling necessity. In this molecular docking study, the antagonistic effect of L-theanine and its possible therapeutic benefit in GluR5 kainate receptor inhibition has been evaluated and compared to the familiar AMPA and kainite receptor antagonists, cyanoquinoxaline (CNQX) and dinitroquinoxaline (DNQX), using Molegro Virtual Docker 7.0.0. The capacity of L-theanine to cohere with the GluR5 receptor was revealed to be higher than that of glutamate, although it could not surpass the high binding tendency of competitive antagonists CNQX and DNQX. Nonetheless, the drug-likeness score and the blood-brain barrier traversing potential of L-theanine were higher than CNQX and DNQX. The study provides an inference to the advantage of L-theanine, which can be a safe and effective alternative natural therapy for rescuing neuronal death due to excitotoxicity.

The Glutamatergic System in Alzheimer’s Disease: A Systematic Review with Meta‐analysis

AbstractBackgroundGlutamatergic neurotransmission system dysregulation may play an important role in the pathophysiology of Alzheimer’s disease (AD). However, reported results on glutamatergic components across brain regions are contradictory. Here, we conducted a systematic review with meta‐analysis to examine whether there are consistent glutamatergic abnormalities in the human AD brain.MethodWe searched PubMed and Web of Science(database origin‐October 2023) reports evaluating glutamate, glutamine, glutaminase, glutamine synthetase, glutamate reuptake, aspartate, excitatory amino acid transporters, vesicular glutamate transporters, glycine, D‐serine, metabotropic and ionotropic glutamate receptors in the AD human brain. The studies were synthesized by outcome and brain region. We included cortical regions, the whole brain(cortical and subcortical regions combined), the entorhinal cortex and the hippocampus. Pooled effect sizes were determined with standardized mean differences (SMD), random effects adjusted by false discovery rate, and heterogeneity was examined by I2statistics.ResultThe search retrieved 6 936 articles,63 meeting the inclusion criteria(N = 709CN/786AD;mean age 75/79). We showed that the brain of AD individuals presents decreased glutamate (SMD = ‐0.82;P<0.001) and aspartate levels (SMD = ‐0.64; I2 = 89.71%; P = 0.006), and reuptake (SMD = ‐0.75;P<0.001). We also found reduced AMPAR‐GluA2/3 levels(SMD = ‐0.63;P = 0.046), hypofunctional NMDAR (SMD = ‐0.60;P<0.001) and selective reduction of NMDAR‐GluN2B subunit levels(SMD = ‐1.07;P<0.001). Regional differences include lower glutamate levels in cortical areas and aspartate levels in cortical areas and in the hippocampus, reduced glutamate reuptake, reduced AMPAR‐GluA2/3 in the entorhinal cortex, hypofunction of NMDAR in cortical areas, and a decrease in NMDAR‐GluN2B subunit levels in the entorhinal cortex and hippocampus. Other parameters studied were not altered.ConclusionOur findings show depletion of the glutamatergic system and emphasize the importance of understanding glutamate‐mediated neurotoxicity in AD. This study has implications for the development of therapies and biomarkers in AD.

Diarylamidine activation of a brachiopod DEG/ENaC/ASIC channel.

Diarylamidines are a group of widely used small molecule drugs. One common use of diarylamidines is their pharmacological inhibition of ligand-gated cation channels, including tetrameric ionotropic glutamate receptors and trimeric degenerin/epithelial sodium channel channel/acid-sensing ion channels (DEG/ENaC/ASICs). Here, we discover a DEG/ENaC/ASIC channel from the brachiopod (lamp shell) Novocrania anomala, at which diarylamidines act as agonists. The channel is closely related to bile acid-gated, pH-gated, and peptide-gated channels but is not activated by such stimuli. We describe activation of the channel by diminazene, DAPI, and pentamidine, examine several biophysical and pharmacological properties, and briefly explore the molecular determinants of channel activity with site-directed mutagenesis. We term this channel the diarylamidine-activated sodium channel (DiaaNaC).

New insight into the molecular etiopathogenesis of konzo: Cyanate could be a plausible neurotoxin contributing to konzo, contrary to thiocyanate

IntroductionChronic cassava-derived cyanide poisoning is associated with the appearance of konzo, a tropical spastic paraparesis due to selective upper motor neuron damage. Whether the disease is caused by a direct action of cyanide or its metabolites is still an open question. This preliminary study assessed the neurotoxic effects of thiocyanate (SCN) and cyanate (OCN), two cyanide metabolites hypothesized to be plausible toxic agents in konzo. MethodsCultured mouse neuroblastoma (Neuro-2A) and human neuroblastoma (SH-SY5Y) cell lines were incubated (24, 48, and 72hours) in sodium OCN or sodium SCN in a disease-relevant concentration range. Cell viability, caspase (3, 8, and 9) activities, and reactive oxygen species (ROS) generation were evaluated using appropriate assay kits. Additionally, electrophysiological responses induced by OCN and SCN in primary spinal cord neurons (from Sprague Dawley rats) were assessed by whole-cell patch-clamp techniques. ResultsBoth OCN and SCN were toxic in a dose-dependent way, even if SCN toxicity appeared at very high concentrations (30mM, corresponding to more than 100-fold higher than normal plasmatic levels), contrary to OCN (0.3-3mM). OCN was markedly more toxic in a poor culture medium (MEM; IC50 = 3.2mM) compared to a glucose- and amino acid-rich medium (DMEM; IC50=7.6mM). OCN treatment increased the ROS generation by 8.9 folds, as well as the Caspase-3, Caspase-8, and Caspase-9 activities by 3.2, 2.5, and 2.6 folds, respectively. Finally, OCN (and SCN to a lesser extent) induced depolarizing currents in primary spinal cord neurons, through an activation of ionotropic glutamate receptors. ConclusionOur results suggest OCN as the most plausible neurotoxic agent involved in konzo, while SCN toxicity could be questioned at such high concentrations. Also, they support apoptosis, oxidative stress, and excitotoxicity as probable mechanisms of OCN neurotoxicity.

The gating properties of Drosophila NMJ glutamate receptors and their dependence on Neto.

The Drosophila neuromuscular junction (NMJ) is a powerful genetic system that has revealed numerous conserved mechanisms for synapse development and homeostasis. The fly NMJ uses glutamate as the excitatory neurotransmitter and relies on kainate-type glutamate receptors and their auxiliary protein Neto for synapse assembly and function. However, despite decades of study, the reconstitution of NMJ glutamate receptors using heterologous systems has been achieved only recently, and there are no reports on the gating properties for the recombinant receptors. Here, using outside-out, patch clamp recordings and fast ligand application, we examine for the first time the biophysical properties of native type-A and type-B NMJ receptors in complexes with either Neto-α or Neto-β and compare them with recombinant receptors expressed in HEK293T cells. We found that type-A and type-B receptors have strikingly different gating properties that are further modulated by Neto-α and Neto-β. We captured single-channel events and revealed major differences between type-A and type-B receptors and also between Neto splice variants. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of ionotropic glutamate receptor (iGluR) desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals. KEY POINTS: We report the reconstitution of Drosophila neuromuscular junction ionotropic glutamate receptors (iGluRs) with Neto splice forms. Using outside-out patches and fast ligand application, we examine the deactivation and desensitization of the four iGluR/Neto complexes found in vivo. Expression of functional channels is absolutely dependent on Neto. Single-channel recordings revealed different lifetimes for different receptor complexes. The decay of synaptic currents is controlled by desensitization.

Trapping of spermine, Kukoamine A, and polyamine toxin blockers in GluK2 kainate receptor channels

Kainate receptors (KARs) are a subtype of ionotropic glutamate receptor (iGluR) channels, a superfamily of ligand-gated ion channels which mediate the majority of excitatory neurotransmission in the central nervous system. KARs modulate neuronal circuits and plasticity during development and are implicated in neurological disorders, including epilepsy, depression, schizophrenia, anxiety, and autism. Calcium-permeable KARs undergo ion channel block, but the therapeutic potential of channel blockers remains underdeveloped, mainly due to limited structural knowledge. Here, we present closed-state structures of GluK2 KAR homotetramers in complex with ion channel blockers NpTx-8, PhTx-74, Kukoamine A, and spermine. We find that blockers reside inside the GluK2 ion channel pore, intracellular to the closed M3 helix bundle-crossing gate, with their hydrophobic heads filling the central cavity and positively charged polyamine tails spanning the selectivity filter. Molecular dynamics (MD) simulations of our structures illuminate interactions responsible for different affinity and binding poses of the blockers. Our structures elucidate the trapping mechanism of KAR channel block and provide a template for designing new blockers that can selectively target calcium-permeable KARs in neuropathologies.

BHLHE40-mediated transcriptional activation of GRIN2D in gastric cancer is involved in metabolic reprogramming.

Gastric cancer (GC) is the third leading cause of death in developed countries. The reprogramming of energy metabolism represents a hallmark of cancer, particularly amplified dependence on aerobic glycolysis. Here, we aimed to illustrate the functional role of glutamate ionotropic receptor N-methyl-D-aspartate type subunit 2D (GRIN2D) in the regulation of glycolysis in GC and the mechanisms involved. Differentially expressed genes were analyzed using the GEO and GEPIA databases, followed by prognostic value prediction using the Kaplan-Meier Plotter database. The effect of GRIN2D knockdown on the malignant behavior and glycolysis of GC cells was explored. GRIN2D expression was upregulated in GC cells and promoted the malignant behavior of GC cells by activating glycolysis. Class E basic helix-loop-helix protein 40 (BHLHE40) was overexpressed in GC cells and mediated transcriptional activation of GRIN2D. The anti-tumor effects of BHLHE40 knockdown on GC cells in vitro and in vivo were reversed by GRIN2D overexpression. Knockdown of GRIN2D or BHLHE40 downregulated the expression of mRNA of electron transport chain subunits and phosphorylation of p38 MARK and inhibited calcium efflux in GC cells. Overexpression of GRIN2D promoted calcium efflux, phosphorylation of p38 MARK protein, and proliferation of GES1 cells. Altogether, the findings derived from this study suggest that BHLHE40 knockdown suppresses the growth, mobility, and glycolysis of GC cells by inhibiting GRIN2D transcription and disrupting the BHLHE40/GRIN2D axis may be an attractive therapeutic strategy for GC.

CNSC-39. INTEGRATED CLINICAL GENETIC ANALYSIS REVEALS NEUROTRANSMITTER RECEPTOR DYSREGULATION IN MENINGIOMAS CAUSING SEIZURE

Abstract Molecular correlates of seizures in meningioma remain unexplored. Previously, we identified MenG molecular grouping, wherein MenG C meningiomas recur more than MenG A or B. We therefore sought to identify molecular and clinical drivers of seizures in meningioma. We identified 144 primary supratentorial meningiomas, excluding those with pre-existing epilepsy. Whole exome sequencing assessed somatic mutation burden. Bulk RNA-sequencing identified differentially expressed genes and gene ontologies (GO). Immunohistochemistry confirmed protein levels. No differences in canonical gene mutation burden (NF2, TRAF7, AKT1, KLF4, SMO, SMARC) was found between seizure and non-seizure groups. MenG C tumors had higher seizure incidence compared to MenG A or B (p=0.03). Seizure causing meningiomas, though extra-axial, exhibited dysregulated neuronal signaling genes. GABAA receptor subunit genes GABRA3 and GABRG1 were downregulated in tumors causing seizure (Log2FC=-1.67, padj=0.014; Log2FC=-2.24, padj=0.009). Immunohistochemistry confirmed expression of GABRB2 subunit, the first documentation of GABA receptor expression in meningiomas. Neuropeptide Y Receptor Y1 (NPY1R) is downregulated in seizure causing meningiomas (Log2FC=-1.64, padj=0.002). NPY1R loss correlates is observed in mesial temporal lobe epilepsy and post-electroconvulsive shock mouse brains. In GO analysis, the terms “GABA signaling pathway,” “neuropeptide signaling,” “excitatory postsynaptic membrane potential,” and “ionotropic glutamate pathway,” were downregulated in meningiomas with seizure (padj<0.05). We assessed which alterations within MenG C tumors caused seizures by identifying genes associated with both seizure presentation and MenG C classification. Glutamate ionotropic receptor AMPA type subunit 1 (GRIA1) is downregulated in MenG C tumors (Log2FC=-2.29, padj=6.02E-07) and seizure causing tumors (Log2FC=-1.75, padj=0.03). Clinically, patients experiencing pre-operative seizures more frequently had cerebral edema, non-homogenous enhancement, intratumoral calcification, higher MIB-1, atypical grade 1, reduced GCS, and ER presentation; they were less likely to have incidental or headache presentation (p<0.05 for all). MenG classification predicts seizure presentation whereas somatic mutation status does not. Meningiomas causing seizures show pathologic neurotransmitter receptor dysregulation.

TMIC-18. ROLE OF NEUROTRANSMITTERS IN GLIOBLASTOMA PATHOPHYSIOLOGY: EMERGING INSIGHTS

Abstract INTRODUCTION Glioblastoma (GBM) are the most common primary brain tumor in adults associated with a dismal patient outcome. While it is well-known that glutamatergic neurons can form functional synapses with glioblastoma cells to enhance tumor growth, lesser is known about the effects of activity of other neuronal types on glioblastoma biology, especially among different glioblastoma molecular subtypes. Here, we examined the effects of various neurotransmitters on the growth of glioblastoma cells characterized as classical, proneural and mesenchymal subtypes. METHODS Patient-derived glioblastoma cell lines were exposed to varying concentrations of different neurotransmitters and assessed for cell viability using CCK-8 assay. Transcriptomic data from TCGA (The Cancer Genome Atlas) database was analyzed for correlation of expression of neurotransmitter receptors (NTRs) with patient survival. Using IVY-GAP database, we compared the expression of NTRs at leading-edge versus tumor core of tumors to assess their possible involvement in cell migration and microenvironmental interactions. RESULTS Consistent with previous findings, we observed an increase in cell viability in all GBM cell lines after being incubated with L-glutamate. The classical subtype has a stronger growth response to L-glutamate compared to the other two subtypes. TCGA data revealed the expression of GRIK4 (glutamate ionotropic receptor kainate type subunit 4) as highest in the classical subtype as well as inversely correlated with worse patient survival in this GBM subtype, as opposed to proneural or mesenchymal patient groups. CCK8 assay also suggested that acetylcholine and dopamine increase glioblastoma cell growth. A subset of cholinergic and glutamatergic receptors (CHRM1, GRM2) was found upregulated in infiltrating/leading-edge regions of glioblastoma tumors compared to the tumor core, suggesting possible involvement in glioblastoma-neuron interactions. CONCLUSION Our findings suggest that GBM subtypes may differentially leverage neuro-modulatory mechanisms to support their growth. Future studies will investigate the differential neuronal responses in GBM subtypes using pharmacological/genetic approaches.

Exploring the Landscape of Pre- and Post-Synaptic Pediatric Disorders with Epilepsy: A Narrative Review on Molecular Mechanisms Involved.

Neurodevelopmental disorders (NDDs) are a group of conditions affecting brain development, with variable degrees of severity and heterogeneous clinical features. They include intellectual disability (ID), autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), often coexisting with epilepsy, extra-neurological comorbidities, and multisystemic involvement. In recent years, next-generation sequencing (NGS) technologies allowed the identification of several gene pathogenic variants etiologically related to these disorders in a large cohort of affected children. These genes encode proteins involved in synaptic homeostasis, such as SNARE proteins, implicated in calcium-triggered pre-synaptic release of neurotransmitters, or channel subunit proteins, such as post-synaptic ionotropic glutamate receptors involved in the brain's fast excitatory neurotransmission. In this narrative review, we dissected emerged molecular mechanisms related to NDDs and epilepsy due to defects in pre- and post-synaptic transmission. We focused on the most recently discovered SNAREopathies and AMPA-related synaptopathies.

Evaluation of allosteric NMDA receptor modulation by GluN2A-selective antagonists using pharmacological equilibrium modeling.

NMDA-type ionotropic glutamate receptors are critically involved in excitatory neurotransmission and their dysfunction is implicated in many brain disorders. Allosteric modulators with selectivity for specific NMDA receptor subtypes are therefore attractive as therapeutic agents, and sustained drug discovery efforts have resulted in a wide range of new allosteric modulators. However, evaluation of allosteric NMDA receptor modulators is limited by the lack of operational ligand-receptor models to describe modulator binding dissociation constants (KB) and effects on agonist binding affinity (α) and efficacy (β). Here, we describe a pharmacological equilibrium model that encapsulates activation and modulation of NMDA receptors, and we apply this model to afford deeper understanding of GluN2A-selective negative allosteric modulators (NAMs), TCN-201, MPX-004, and MPX-007. We exploit slow NAM unbinding to examine receptors at hemi-equilibrium when fully occupied by agonists and modulators to demonstrate that TCN-201 display weaker binding and negative modulation of glycine binding affinity (KB = 42 nM, α = 0.0032) compared to MPX-004 (KB = 9.3 nM, α = 0.0018) and MPX-007 (KB = 1.1 nM, α = 0.00053). MPX-004 increases agonist efficacy (β = 1.19), whereas TCN-201 (β = 0.76) and MPX-007 (β = 0.82) reduce agonist efficacy. These values describing allosteric modulation of diheteromeric GluN1/2A receptors with two modulator binding sites are unchanged in triheteromeric GluN1/2A/2B receptors with a single binding site. This evaluation of NMDA receptor modulation reveals differences between ligand analogs that shape their utility as pharmacological tool compounds and facilitates the design of new modulators with therapeutic potential. Significance Statement Detailed understanding of allosteric NMDA receptor modulation requires pharmacological methods to quantify modulator binding affinity and the strengths of modulation of agonist binding and efficacy. We describe a generic ligand-receptor model for allosteric NMDA receptor modulation and use this model for the characterization of GluN2A-selective NAMs. The model enables quantitative evaluation of a broad range of NMDA receptor modulators and provides opportunities to optimize these modulators by embellishing the interpretation of their structure-activity relationships.

Structural studies of ion channels: achievements, problems and perspectives

The superfamily of membrane proteins known as P-loop channels encompasses potassium, sodium, and calcium channels, as well as TRP channels and ionotropic glutamate receptors. An increasing number of crystal and cryo-EM structures are uncovering both general and specific features of these channels. Fundamental folding principles, the arrangement of structural segments, key residues that influence ionic selectivity, gating, and binding sites for toxins and medically relevant ligands have now been firmly established. The advent of AlphaFold2 (AF2) models represents another significant step in computationally predicting protein structures. Comparison of experimental P-loop channel structures with their corresponding AF2 models shows consistent folding patterns in experimentally resolved regions. Despite this remarkable progress, many crucial structural details, particularly important for predicting the outcomes of mutations and designing new medically relevant ligands, remain unresolved. Certain methodological challenges currently hinder the direct assessment of such details. Until the next methodological breakthrough occurs, a promising approach to analyzing ion channel structures in greater depth involves integrating various experimental and theoretical methods.

Pubertal- and Stress-Dependent Changes in Cellular Activation and Expression of Excitatory Amino Acid Receptor Subunits in the Paraventricular Nucleus of the Hypothalamus in Male and Female Rats

Introduction: Pubertal maturation is marked by significant changes in stress-induced hormonal responses mediated by the hypothalamic-pituitary-adrenal (HPA) axis, with prepubertal male and female rats often exhibiting greater HPA reactivity compared to adult males and females. Though the implications of these changes are unclear, elevated stress responsiveness might contribute to the stress-related vulnerabilities often associated with puberty. Methods: The current experiments sought to determine whether differences in cellular activation, as measured by FOS immunohistochemistry, or excitatory ionotropic glutamate receptor subunit expression, as measured by qRT-PCR, in the paraventricular nucleus (PVN) were associated with these noted pubertal shifts in stress reactivity in male and female rats. As the PVN is the key nucleus responsible for activating the hormonal stress response, we predicted greater cellular activation and higher expression levels of glutamate receptor subunits in the PVN of prepubertal males and females compared to their adult counterparts. Results: Our FOS data revealed that while prepubertal males showed greater stress-induced activation in the PVN than adult males, prepubertal females showed less activation than adult females. Moreover, many of the NMDA, AMPA, and kainate receptor subunits measured, including Grin1, Grin2b, Gria1, Gria2, Grik1, and Grik2, had higher expression levels in adults, particularly in males. Conclusions: Though not supporting our initial predictions, these data do indicate that age and stress influence the activation of the PVN and the expression of glutamate receptor subunits important in its function. These data also suggest that the effects of age and stress are different in males and females. Though still far from a clear understanding of what mechanism(s) mediate pubertal shift in stress reactivity, these data add to our growing understanding of how age, stress, and sex influence HPA function.

Mechanisms of Action Underlying Conductance-Modifying Positive Allosteric Modulators of the NMDA Receptor.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate a slow, Ca2+-permeable component of excitatory neurotransmission. Modulation of NMDAR function has the potential for disease modification as NMDAR dysfunction has been implicated in neurodevelopment, neuropsychiatric, neurologic, and neurodegenerative disorders. We recently described the thieno[2,3-day]pyrimidin-4-one (EU1622) class of positive allosteric modulators, including several potent and efficacious analogs. Here we have used electrophysiological recordings from Xenopus oocytes, human embryonic kidney cells, and cultured cerebellar and cortical neurons to determine the mechanisms of action of a representative member of this class of modulator. EU1622-240 enhances current response to saturating agonist (doubling response amplitude at 0.2-0.5 μM), slows the deactivation time course following rapid removal of glutamate, increases open probability, enhances coagonist potency, and reduces single-channel conductance. We also show that EU1622-240 facilitates NMDAR activation when only glutamate or glycine is bound. EU1622-240-bound NMDARs channels activated by a single agonist (glutamate or glycine) open to a unique conductance level with different pore properties and Mg2+ sensitivity, in contrast to channels arising from activation of NMDARs with both coagonists bound. These data demonstrate that previously hypothesized distinct gating steps can be controlled by glutamate and glycine binding and shows that the 1622-series modulators enable glutamate- or glycine-bound NMDARs to generate open conformations with different pore properties. The properties of this class of allosteric modulators present intriguing therapeutic opportunities for the modulation of circuit function. SIGNIFICANCE STATEMENT: NMDA receptors are expressed throughout the central nervous system and are permeable to calcium. EU1622-240 increases open probability and agonist potency while reducing single-channel conductance and prolonging the deactivation time course. EU1622-240 allows NMDA receptor activation by the binding of one coagonist (glycine or glutamate), which produces channels with distinct properties. Evaluation of this modulator provides insight into gating mechanisms and may lead to the development of new therapeutic strategies.