Glutamate Receptors In Neurons Research Articles

Obesity alters POMC and kisspeptin neuron crosstalk leading to reduced luteinizing hormone in male mice.

Obesity is associated with hypogonadism in males, characterized by low testosterone and sperm number. Previous studies determined that these stem from dysregulation of hypothalamic circuitry that regulates reproduction, by unknown mechanisms. Herein, we used mice fed chronic high-fat diet, that mimics human obesity, to determine mechanisms of impairment at the level of the hypothalamus, in particular gonadotropin-releasing hormone (GnRH) neurons that regulate luteinizing hormone (LH), which then regulates testosterone. Consistent with obese humans, we demonstrated lower LH, and lower pulse frequency of LH secretion, but unchanged pituitary responsiveness to GnRH. LH pulse frequency is regulated by pulsatile GnRH secretion, which is controlled by kisspeptin. Peripheral and central kisspeptin injections, and DREADD-mediated activation of kisspeptin neurons, demonstrated that kisspeptin neurons were suppressed in obese mice. Thus, we investigated regulators of kisspeptin secretion. We determined that the LH response to NMDA was lower in obese mice, corresponding to fewer glutamate receptors in kisspeptin neurons, which may be critical for kisspeptin synchronization. Given that kisspeptin neurons also interact with POMC neurons, which regulate satiety and are affected by obesity, we examined their crosstalk, and determined that the LH response to either DREADD-mediated activation of POMC neurons or central injection of αMSH, a product of POMC, is abolished in obese mice. This was accompanied by diminished levels of αMSH receptor, MC4R, in kisspeptin neurons. Together, our studies determined that obesity leads to the downregulation of receptors that regulate kisspeptin neurons, which is associated with lower LH pulse frequency, leading to lower LH and hypogonadism.Significance Statement Obesity presents a significant health concern, with multiple comorbidities, including impaired reproduction. However, mechanisms are not clear, and studies are confounded by the chronic nature of this condition that leads to synaptic changes and alterations in neuron responsiveness to stimuli. Here, we demonstrate that the interaction between feeding circuitry and reproductive circuitry is altered by chronic obesity. The reason may be that chronically higher activity of POMC neurons in response to higher leptin in obesity, downregulates αMSH receptors on target neurons, including kisspeptin. This may lead to the suppression of kisspeptin neurons, and their inability to regulate pulsatile secretion of GnRH, which then lowers LH pulse frequency, leading to lower LH in the circulation, lower testosterone, and lower sperm count.

Silica Nanoparticles Decrease Glutamate Uptake in Blood–Brain Barrier Components

Glutamate is the major excitatory amino acid in the vertebrate brain, playing an important role in most brain functions. It exerts its activity through plasma membrane receptors and transporters, expressed both in neurons and glia cells. Overstimulation of neuronal glutamate receptors is linked to cell death in a process known as excitotoxicity, that is prevented by the efficient removal of the neurotransmitter through glutamate transporters enriched in the glia plasma membrane and in the components of the blood–brain barrier (BBB). Silica nanoparticles (SiO2-NPs) have been widely used in biomedical applications and directed to enter the circulatory system; however, little is known about the potential adverse effects of SiO2-NPs exposure on the BBB transport systems that support the critical isolation function between the central nervous system (CNS) and the peripheral circulation. In this contribution, we investigated the plausible SiO2-NPs-mediated disruption of the glutamate transport system expressed by BBB cell components. First, we evaluated the cytotoxic effect of SiO2-NPs on human brain endothelial (HBEC) and Uppsala 87 Malignant glioma (U-87MG) cell lines. Transport kinetics were evaluated, and the exposure effect of SiO2-NPs on glutamate transport activity was determined in both cell lines. Exposure of the cells to different SiO2-NP concentrations (0.4, 4.8, 10, and 20 µg/ml) and time periods (3 and 6 h) did not affect cell viability. We found that the radio-labeled D-aspartate ([3H]-D-Asp) uptake is mostly sodium-dependent, and downregulated by its own substrate (glutamate). Furthermore, SiO2-NPs exposure on endothelial and astrocytes decreases [3H]-D-Asp uptake in a dose-dependent manner. Interestingly, a decrease in the transporter catalytic efficiency, probably linked to a diminution in the affinity of the transporter, was detected upon SiO2-NPs. These results favor the notion that exposure to SiO2-NPs could disrupt BBB function and by these means shed some light into our understanding of the deleterious effects of air pollution on the CNS.

Vagus nerve stimulation inhibits cortical spreading depression via glutamate-dependent TrkB activation mechanism in the nucleus tractus solitarius.

Vagus nerve stimulation (VNS) was recently found to inhibit cortical spreading depression (CSD), the underlying mechanism of migraine aura, through activation of the nucleus tractus solitarius (NTS), locus coeruleus (LC) and dorsal raphe nucleus (DRN). The molecular mechanisms underlying the effect of VNS on CSD in these nuclei remain to be explored. We hypothesized that VNS may activate glutamate receptor-mediated tropomyosin kinase B (TrkB) signaling in the NTS, thereby facilitating the noradrenergic and serotonergic neurotransmission to inhibit CSD. To investigate the role of TrkB and glutamate receptors in non-invasive VNS efficacy on CSD, a validated KCl-evoked CSD rat model coupled with intra-NTS microinjection of selective antagonists, immunoblot and immunohistochemistry was employed. VNS increased TrkB phosphorylation in the NTS. Inhibition of intra-NTS TrkB abrogated the suppressive effect of VNS on CSD and CSD-induced cortical neuroinflammation. TrkB was found colocalized with glutamate receptors in NTS neurons. Inhibition of glutamate receptors in the NTS abrogated VNS-induced TrkB activation. Moreover, the blockade of TrkB in the NTS attenuated VNS-induced activation of the LC and DRN. VNS induces the activation of glutamate receptor-mediated TrkB signaling in the NTS, which might modulate serotonergic and norepinephrinergic innervation to the cerebral cortex to inhibit CSD and cortical inflammation.

Bioenergetic and excitotoxic determinants of cofilactin rod formation.

Cofilactin rods (CARs), which are 1:1 aggregates of cofilin-1 and actin, lead to neurite loss in ischemic stroke and other disorders. The biochemical pathways driving CAR formation are well-established, but how these pathways are engaged under ischemic conditions is less clear. Brain ischemia produces both ATP depletion and glutamate excitotoxicity, both of which have been shown to drive CAR formation in other settings. Here, we show that CARs are formed in cultured neurons exposed to ischemia-like conditions: oxygen-glucose deprivation (OGD), glutamate, or oxidative stress. Of these conditions, only OGD produced significant ATP depletion, showing that ATP depletion is not required for CAR formation. Moreover, the OGD-induced CAR formation was blocked by the glutamate receptor antagonists MK-801 and kynurenic acid; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors GSK2795039 and apocynin; as well as an ROS scavenger. The findings identify a biochemical pathway leading from OGD to CAR formation in which the glutamate release induced by energy failure leads to activation of neuronal glutamate receptors, which in turn activates NADPH oxidase to generate oxidative stress and CARs.

Reduction of p11 in dorsal raphe nucleus serotonergic neurons mediates depression-like behaviors

The pathology of depression is related to the imbalance of various neurotransmitters. The dorsal raphe nucleus (DRN), the main brain region producing 5-HT, is crucially involved in the pathophysiology of depression. It contains several neuron types, in which GABAergic neurons are activated by stimuli associated with negative experiences and 5-HT neurons are activated by reward signals. However, little is known about its underlying molecular mechanisms. Here, we found that p11, a multifunctional protein associated with depression, was down-regulated by chronic social defeat stress in 5-HTDRN neurons. Knockdown of p11 in DRN induced depression-like behaviors, while its overexpression in 5-HTDRN neurons alleviated depression-like behavior caused by chronic social defeat stress. Further, p11 regulates membrane trafficking of glutamate receptors in 5-HTDRN neurons, suggesting a possible molecular mechanism underlying the participation of p11 in the pathological process of depression. This may facilitate the understanding of the molecular and cellular basis of depression.

CNSC-06. REDIRECTING IMMUNOSUPPRESSIVE GLIOMA MICROENVIRONMENT THROUGH PHARMACOLOGICAL INTERVENTION TARGETING NEURONAL ACTIVITY

Abstract Recent single-cell RNA-sequencing analyses have revealed a strong association between glioma-neuronal circuit remodeling and regional immunosuppression. Subsequent preclinical investigations using syngeneic tumor models demonstrated that genetic disruption of circuit assembly genes, including Thrombospondin-1 (TSP-1/Thbs1) in GBM cells suppressed synaptogenesis, excitatory neurotransmitter ligand and receptor density in addition to neuronal activities while simultaneously restoring inflammatory responses, indicating glioma-neuron-immune cross talk. However, the underlying mechanisms and directions of causality remain unclear. Here we test the hypothesis that tumor-derived synaptogenic factors directly reprogram the tumor immune microenvironment (“model 1”). Alternatively, tumor intrinsic neuronal activity may indirectly regulate adaptive immunity (“model 2”). In this study, we performed in-vitro bone-marrow-derived macrophage (BMDM) reprogramming assays evaluating various stimulation conditions and in-vivo experiments evaluating the effect of neuronal activity-oriented therapy on the tumor-associated macrophages (TAMs). Our in-vitro assays demonstrated that, secreted factors from mouse neonatal cortical neurons (not tumor-derived synaptogenic factors) induced an anti-inflammatory, M2-like phenotype of BMDMs, corroborating the proposed model 2. This M2-inducing effect was inhibited when the neuronal activities were suppressed pharmacologically, using perampanel (PER), a noncompetitive inhibitor of neuronal AMPA-type glutamate receptors. Direct treatment of BMDMs with PER did not affect the polarization patterns, further supporting the M2-inducing effect of glutamatergic excitatory neuronal signals. In in-vivo syngeneic orthotopic tumor models, treatment with PER significantly prolonged survival compared to the non-treated group (27.5 [PER] vs. 23 days [control], p = 0.01). Flow cytometric analysis of brain-infiltrating leukocytes showed that TAMs isolated from PER-treated mice exhibited a more proinflammatory M1-like polarization compared to the control group (median M1/M2 ratio, 1.2 [PER] vs. 0.7 [control], p = 0.004), which explained the improved prognosis. Collectively, these findings unveil novel immunosuppression mechanisms induced by glioma-neuronal circuit remodeling and the resulting excitatory neuronal signals, and potential therapeutic vulnerabilities that could be targeted in combination with cancer immunotherapy.

Glutamate and GABA receptors in non-neural animals (Placozoa): Preadaptation to neural transmission

Origins of neural system is one of the major transitions in planetary evolution. Many details of these transitions are still unknown. In particular, high diversity of neurotransmitters lacks convincing explanation so far. We analyze homologues of neuronal glutamate and gamma-aminobutyric acid (GABA) receptors of Placozoa – animal phyla lacking neurons but displaying motility and complex behaviour. Phylogenetic analysis and comparison of amino acids in ligand-binding pockets show that glutamate and GABA-like receptors of Placozoa are surprisingly numerous, diverse and fast-evolving. All these traits are characteristic of odorant rather than neurotransmitter receptors of higher animals. We argue that chemoreception system was an important source of diverse receptors for emerging nervous system to recruit, and that amino acid neurotransmitters (glutamate, GABA, glycine) were relevant external stimuli for early animals before the emergence of nervous system.

PLPP/CIN inhibits dopamine D1 receptor-mediated seizure activity via DARPP-32 serine 97 dephosphorylation in the mouse hippocampus

Dopamine plays a central role in the regulation of psychomotor functions in the brain. Furthermore, the dopaminergic system is involved in the ictogenesis in human patients and animal models of epilepsy. Dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32) plays an important role in the regulation of interactions between dopamine and glutamate receptors in neurons. Indeed, SKF 83822 (a specific D1 receptor agonist) facilitates DARPP-32-mediated protein phosphatase 1 (PP1) inhibition leading to the increase in phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR), which potentiates channel activities and currents and thereby generates seizure activity. In the present study, we found that pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN), a selective phosphatase for serine (S) residues, attenuated seizure susceptibility in response to SKF 83822 by dephosphorylating DARPP-32 S97 site. Similarly, inhibition of DARPP-32 S97 phosphorylation by 2-[4,5,6,7-Tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazole-1-yl]acetic acid (TMCB; a selective casein kinase 2 inhibitor) attenuated SKF 83822-induced seizure activity. These inhibitory effects of PLPP/CIN and TMCB were relevant to the regulations of DARPP-32-PP1-AMPAR signaling pathway. Therefore, our findings suggest that PLPP/CIN may be a modulator in dopaminergic neurotransmission as well as glutamatergic systems, and that the PLPP/CIN-mediated DARPP-32 regulation may be one of the potential therapeutic targets for medication of seizure or epilepsy induced by D1 receptor hyperactivation.

Development of a quaternary ammonium photoswitchable antagonist of NMDA receptors

NMDA receptors play critical roles in numerous physiological and pathological processes in CNS that requires development of modulating ligands. In particular, photoswitchable compounds that selectively target NMDA receptors would be particularly useful for analysis of receptor contributions to various processes. Recently, we identified a light-dependent anti-NMDA activity of the azobenzene-containing quaternary ammonium compounds DENAQ (diethylamine-azobenzene-quaternary ammonium) and DMNAQ (dimethylamine-azobenzene-quaternary ammonium). Here, we developed a series of light-sensitive compounds based on the DENAQ structure, and studied their action on glutamate receptors in rat brain neurons using patch-clamp method. We found that the activities of the compounds and the influence of illumination strongly depended on the structural details, as even minor structural modifications greatly altered the activity and sensitivity to illumination. The compound PyrAQ (pyrrolidine-azobenzene-quaternary ammonium) was the most active and produced fast and fully reversible inhibition of NMDA receptors. The IC50 values under ambient and monochromic light conditions were 2 and 14 μM, respectively. The anti-AMPA activity was much weaker. The action of PyrAQ did not depend on NMDA receptor activity, agonist concentration, or membrane voltage, making it a useful tool for photopharmacological studies.

Phosphorylation and regulation of group II metabotropic glutamate receptors (mGlu2/3) in neurons.

Group II metabotropic glutamate (mGlu) receptors (mGlu2/3) are Gαi/o-coupled receptors and are primarily located on presynaptic axonal terminals in the central nervous system. Like ionotropic glutamate receptors, group II mGlu receptors are subject to regulation by posttranslational phosphorylation. Pharmacological evidence suggests that several serine/threonine protein kinases possess the ability to regulate mGlu2/3 receptors. Detailed mapping of phosphorylation residues has revealed that protein kinase A (PKA) phosphorylates mGlu2/3 receptors at a specific serine site on their intracellular C-terminal tails in heterologous cells or neurons, which underlies physiological modulation of mGlu2/3 signaling. Casein kinases promote mGlu2 phosphorylation at a specific site. Tyrosine protein kinases also target group II receptors to induce robust phosphorylation. A protein phosphatase was found to specifically bind to mGlu3 receptors and dephosphorylate the receptor at a PKA-sensitive site. This review summarizes recent progress in research on group II receptor phosphorylation and the phosphorylation-dependent regulation of group II receptor functions. We further explore the potential linkage of mGlu2/3 phosphorylation to various neurological and neuropsychiatric disorders, and discuss future research aimed at analyzing novel biochemical and physiological properties of mGlu2/3 phosphorylation.

Gliotransmission of D-serine promotes thirst-directed behaviors in Drosophila

Thirst emerges from a range of cellular changes that ultimately motivate an animal to consume water. Although thirst-responsive neuronal signals have been reported, the full complement of brain responses is unclear. Here, we identify molecular and cellular adaptations in the brain using single-cell sequencing of water-deprived Drosophila. Water deficiency primarily altered the glial transcriptome. Screening the regulated genes revealed astrocytic expression of the astray-encoded phosphoserine phosphatase to bi-directionally regulate water consumption. Astray synthesizes the gliotransmitter D-serine, and vesicular release from astrocytes is required for drinking. Moreover, dietary D-serine rescues aay-dependent drinking deficits while facilitating water consumption and expression of water-seeking memory. D-serine action requires binding to neuronal NMDA-type glutamate receptors. Fly astrocytes contribute processes to tripartite synapses, and the proportion of astrocytes that are themselves activated by glutamate increases with water deprivation. We propose that thirst elevates astrocytic D-serine release, which awakens quiescent glutamatergic circuits to enhance water procurement.

Precise Detection and Visualization of Nanoscale Temporal Confinement in Single-Molecule Tracking Analysis.

The plasma membrane consists of a diverse mixture of molecules that dynamically assemble into a highly non-random organization. The formation of nanoscale domains in the membrane is of particular interest as these domains underlie critical cellular functions. Single-molecule tracking is a powerful method to detect and quantify molecular motion at high temporal and spatial resolution and has therefore been instrumental in understanding mechanisms that underlie membrane organization. In single-molecule trajectories, regions of temporal confinement can be determined that might reveal interesting biophysical interactions important for domain formation. However, analytical methods for the detection of temporal confinement in single-molecule trajectories depend on a variety of parameters that heavily depend on experimental factors and the influence of these factors on the performance of confinement detection are not well understood. Here, we present elaborate confinement analyses on simulated random walks and trajectories that display transient confined behavior to optimize the parameters for different experimental conditions. Furthermore, we demonstrate a heatmap visualization tool that allows spatial mapping of confinement hotspots relative to subcellular markers. Using these optimized tools, we reliably detected subdiffusive behavior of different membrane components and observed differences in the confinement behavior of two types of glutamate receptors in neurons. This study will help in further understanding the dynamic behavior of the complex membrane and its role in cellular functioning.

Thirst increases astrocytic release and synthesis of D-serine to promote water procurement

Thirst increases astrocytic release and synthesis of D-serine to promote water procurement SCCMI uses single-cell sequencing to gain a whole-brain understanding of memory and motivational states. Study of thirst revealed the primary transcriptional response in the brain to occur in glial cells. Astrocytes upregulate the expression of an enzyme required to synthesise D-serine, a coagonist of neuronal NMDA-type glutamate receptors. D-serine, in turn, facilitates neural circuits that promote water procurement behaviours.

Expression of the ionotropic glutamate receptors on neuronostatin neurons in the periventricular nucleus of the hypothalamus.

Neuronostatin, a newly identified peptide, is accepted as an anorexigenic peptide since it suppresses food intake when given intracerebroventricularly. Although the effect mechanisms of neuronostatin have been shown in different studies, there are no reports in the literature describing the mechanisms controlling neuronostatin neurons. In this study, we aimed to determine the presence of the ionotropic glutamate receptor subunits (iGluRs) in neuronostatin neurons in the periventricular nucleus of the hypothalamus. The presence of glutamate receptors in neuronostatin neurons was investigated by dual immunohistochemistry. Immunohistochemistry was performed on 40 μm thick coronal brain sections with antibodies against AMPA (GluA1-4), kainate (GluK1/2/3, and GluK5), and NMDA (GluN1 and GluN2A) receptor subunits. The results showed that the neuronostatin neurons expressed most of the NMDA and non-NMDA receptor subunits. The neuronostatin neurons in the anterior hypothalamic periventricular nucleus were particularly immunopositive for GluA1, GluA4, GluK1/2/3, GluK5 and GluN1 antibodies. No expression was observed for GluA2, GluA3 and GluN2A antibodies. For the first time in the literature, our study demonstrated that the neuronostatin neurons express glutamate receptor subunits which may form homomeric or heteromeric functional receptor complexes. Taken together, these results suggest that multiple subunits of iGluRs are responsible for glutamate transmission on neuronostatin neurons in the anterior hypothalamic periventricular nucleus.

Quantification of AMPA-type glutamate receptors trafficking by ligand-directed two-step labeling

Glutamate receptors mediate excitatory neurotransmission in the central nervous system, which have essential roles in our learning and memory. Recent studies have revealed that the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptors (AMPA receptors) are dynamically regulated during synaptic plasticity, the cellular basis of learning and memory. Conventionally, biochemical methods such as surface-biotin labeling or genetic incorporation of fluorescent proteins have been utilized to analyze the AMPA receptors dynamics. However, conflicting findings have been reported because of serious issues in these conventional methods. As the alternative, we have developed a new method for labeling AMPA receptors endogenously expressed in neurons by chemical approaches. This is based on a covalent chemical labeling strategy driven by selective ligand-protein recognition to tether small fluorophores to the target receptors, termed ligand-directed acyl imidazole chemistry. This method has successfully visualized AMPA receptors endogenously expressed in neurons. However, the original method required several hours for fluorophore labeling, which hampered analyzing the dynamics of AMPA receptors in detail. As the alternative, we have recently developed an improved strategy for rapid and selective labeling of chemical probes to cell-surface AMPA receptors by combining ligand-directed chemistry and bio-orthogonal click chemistry. This method allowed to quantify their trafficking, which revealed unique features of AMPA receptors such as long lifetime and rapid recycling in neurons. Notably, this method can be expanded to other receptors. Thus, the two-step labeling method would be a useful tool for understanding the physiological or pathophysiological roles of glutamate receptors in neurons.

Optical Control of N-Methyl-d-aspartate Receptors by Azobenzene Quaternary Ammonium Compounds

Azobenzene-based quaternary ammonium compounds provide optical control of ion channels and are considered promising agents for regulation of neuronal excitability and for restoration of the photosensitivity of retinal cells. However, the selectivity of the action of these compounds remains insufficiently known. We studied the action of DENAQ (diethylamine-azobenzene-quaternary ammonium) and DMNAQ (dimethylamine-azobenzene-quaternary ammonium) on ionotropic glutamate receptors in rat brain neurons. In the dark, both compounds applied extracellularly caused fast and reversible inhibition of NMDA (N-methyl-d-aspartate) receptor-mediated currents with IC50 values of 10 and 5 μM, respectively. Light-induced transformation of DENAQ and DMNAQ to their cis forms caused the IC50 values to increase to 30 and 27 μM, respectively. Detailed analysis of this action revealed a complex nature consisting of fast inhibitory and slower potentiating effects. The AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors were only weakly affected independently on illumination. We conclude that, in addition to their long-lasting intracellular action, which persists after washout, azobenzene-based quaternary ammonium compounds should affect glutamatergic transmission and synaptic plasticity during treatment. Our findings also extend the list of soluble photoswitchable inhibitors of NMDA receptors. While the site(s) and mechanisms of action are unclear, the effect of DENAQ demonstrates strong pH dependence. At acidic pH values, DENAQ potentiates both NMDA and AMPA receptors.

Research progress on the effect of transcranial magnetic stimulation on learning, memory and plasticity of brain synaptic

Transcranial magnetic stimulation (TMS) as a noninvasive neuromodulation technique can improve the impairment of learning and memory caused by diseases, and the regulation of learning and memory depends on synaptic plasticity. TMS can affect plasticity of brain synaptic. This paper reviews the effects of TMS on synaptic plasticity from two aspects of structural and functional plasticity, and further reveals the mechanism of TMS from synaptic vesicles, neurotransmitters, synaptic associated proteins, brain derived neurotrophic factor and related pathways. Finally, it is found that TMS could affect neuronal morphology, glutamate receptor and neurotransmitter, and regulate the expression of synaptic associated proteins through the expression of brain derived neurotrophic factor, thus affecting the learning and memory function. This paper reviews the effects of TMS on learning, memory and plasticity of brain synaptic, which provides a reference for the study of the mechanism of TMS.

Function and Breakdown of the Neuron-Glia Dual-Layer Super-Network

Glial cells appear to have the capacity of coding information. Neuronal and glial circuits form a loosely coupled dual-layer super network within the brain. Glial cells react with the excitatory transmitter glutamate released from synapses of neurons and, in turn, release additional glutamate. Thus, they effectively function as excitatory signal amplifiers, with the gain of amplification depending on their cytosolic pH. Activation of metabotropic glutamate receptors in neurons is largely influenced by the state of glial cells. Depending on the state of our mind, an experience can become a lasting memory or fade away. The pH of glial cells at the time of the experience could play a pivotal role in memory formation. Glial function is also susceptible to plasticity. First-time exposure to a brief period of brain hyperactivity results in an acute breakdown of the inter-cellular network of glial cells. Therefore, artificial control of the glial state and glial plasticity may provide a new preventative strategy to manage dementia and epilepsy.

Neuronal metabotropic glutamate receptor 8 protects against neurodegeneration in CNS inflammation.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with continuous neuronal loss. Treatment of clinical progression remains challenging due to lack of insights into inflammation-induced neurodegenerative pathways. Here, we show that an imbalance in the neuronal receptor interactome is driving glutamate excitotoxicity in neurons of MS patients and identify the MS risk-associated metabotropic glutamate receptor 8 (GRM8) as a decisive modulator. Mechanistically, GRM8 activation counteracted neuronal cAMP accumulation, thereby directly desensitizing the inositol 1,4,5-trisphosphate receptor (IP3R). This profoundly limited glutamate-induced calcium release from the endoplasmic reticulum and subsequent cell death. Notably, we found Grm8-deficient neurons to be more prone to glutamate excitotoxicity, whereas pharmacological activation of GRM8 augmented neuroprotection in mouse and human neurons as well as in a preclinical mouse model of MS. Thus, we demonstrate that GRM8 conveys neuronal resilience to CNS inflammation and is a promising neuroprotective target with broad therapeutic implications.

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N-methyl-D-aspartate-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.