Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.

Deciphering the mechanisms of regulation of an excitatory synapse via cyclooxygenase-2. A review.

Cyclooxygenase (COX) is a heme-containing enzyme that produces prostaglandins (PGs) via a pathway known as the arachidonic acid (AA) cascade. Two isoforms of COX enzyme (COX-1 and COX-2) and splice variant (COX-3) have been described so far. COX-2 is a neuronal enzyme that is intensively produced during activation of the synapse and glutamate (Glu) release. The end product of COX-2 action, prostaglandin E2 (PGE2), regulates Glu level in a retrograde manner. At the same time, the level of Glu, the primary excitatory neurotransmitter, is regulated in the excitatory synapse via Glu receptors, both ionotropic and metabotropic ones. Glu receptors are known modulators of behavior, engaged in cognition and mood. So far, the interaction between ionotropic N-methyl-D-aspartate (NMDA) receptors or metabotropic glutamate (mGluRs) receptors and COX-2 was found. Here, based on literature data and own research, a new mechanism of action of COX-2 in an excitatory synapse will be presented.

Differential glutamatergic and GABAergic contributions to the tetrad effects of Δ9-tetrahydrocannabinol revealed by cell-type-specific reconstitution of the CB1 receptor.

Δ9-tetrahydrocannabinol (THC), the major psychoactive ingredient of Cannabis sativa, exerts its actions through the endocannabinoid system by stimulation of the cannabinoid type 1 (CB1) receptor. The widespread distribution of this receptor in different neuronal cell types and the plethora of functions that is modulated by the endocannabinoid system explain the versatility of the effects of THC. However, the cell types involved in the different THC effects are still not fully known. Conditional CB1 receptor knock-out mice were previously used to identify CB1 receptor subpopulations that are "necessary" for the tetrad effects of a high dose of THC: hypothermia, hypolocomotion, catalepsy and analgesia. Here, we used mouse models for conditional CB1 receptor "rescue" in dorsal telencephalic glutamatergic and forebrain GABAergic neurons to determine which CB1 receptor subpopulations are "sufficient" for these tetrad effects. Glutamatergic CB1 receptor was not only necessary but also sufficient for THC-induced hypothermia and hypolocomotion. Analgesic and cataleptic effects of THC are largely independent of glutamatergic and GABAergic CB1 receptors, since no sufficiency was found, in agreement with the previously reported lack of necessity. We also revealed a novel aspect of GABAergic CB1 receptor signaling. In animals with CB1 receptors exclusively in forebrain GABAergic neurons, THC stimulated rather than reduced locomotion. This cell-type selective and hitherto unsuspected hyperlocomotive effect may be occluded in wild-types and conditional knockouts and only be exposed when CB1 signaling is absent in all other cell types, thus underlining the importance of investigating both necessary and sufficient functions to unequivocally unravel cell-type specific actions.

Anxiolytic Effects of 8-O-Acetyl Shanzhiside Methylester on Acute and Chronic Anxiety via Inflammatory Response Inhibition and Excitatory/Inhibitory Transmission Imbalance.

Anxiety leads to a global decline in quality of life and increase in social burden. However, treatments are limited, because the molecular mechanisms underlying complex emotional disorders are poorly understood. We explored the anxiolytic effects of 8-O-acetyl shanzhiside methylester (8-OaS), an active component in Lamiophlomis rotata (L. rotata; Benth.) or Kudo, a traditional herb that has been shown to be effective in the clinical treatment of chronic pain syndromes in China. Two mouse anxiety models were used: forced swimming stress (FSS)-induced anxiety and complete Freund's adjuvant (CFA)-induced chronic inflammatory pain. All animal behaviors were analyzed on the elevated plus maze and in the open-field test. 8-OaS significantly ameliorated anxiety-like behaviors in both anxiety models and inhibited the translation enhancement of GluN2A, GluN2B, and PSD95. Moreover, a reduction in GABA receptors disrupted the excitatory/inhibitory (E/I) balance in the basolateral amygdala (BLA), indicated by increased excitatory and decreased inhibitory presynaptic release. 8-OaS also blocked microglia activation and reduced the phosphorylation of p38, c-Jun N-terminal kinase (JNK), NF-κB p65, and tumor necrosis factor alpha (TNF-α) in the BLA of anxiety mice. 8-OaS exhibits obvious anxiolytic effects by regulating the excitatory/inhibitory (E/I) synaptic transmission and attenuating inflammatory responses in the BLA.

SAD-B modulates epileptic seizure by regulating AMPA receptors in patients with temporal lobe epilepsy and in the PTZ-induced epileptic model.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the predominant mediators of glutamate-induced excitatory neurotransmission. It is widely accepted that AMPA receptors are critical for the generation and spread of epileptic seizure activity. Dysfunction of AMPA receptors as a causal factor in patients with intractable epilepsy results in neurotransmission failure. Brain-specific serine/threonine-protein kinase 1 (SAD-B), a serine-threonine kinase specifically expressed in the brain, has been shown to regulate AMPA receptor-mediated neurotransmission through a presynaptic mechanism. In cultured rat hippocampal neurons, the overexpression of SAD-B significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Here, we showed that SAD-B downregulation exerted antiepileptic activity by regulating AMPA receptors in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We first used immunoblotting and immunohistochemistry analysis to demonstrate that SAD-B expression was increased in the epileptic rat brain. Subsequently, to explore the function of SAD-B in epilepsy, we used siRNA to knock down SAD-B protein and observed behavior after PTZ-induced seizures. We found that SAD-B downregulation attenuated seizure severity and susceptibility in the PTZ-induced epileptic model. Furthermore, we showed that the antiepileptic effect of SAD-B downregulation on PTZ-induced seizure was abolished by CNQX (an AMPA receptor inhibitor), suggesting that SAD-B modulated epileptic seizure by regulating AMPA receptors in the brain. Taken together, these findings suggest that SAD-B may be a potential and novel therapeutic target to limit epileptic seizures.

D-Serine, the Shape-Shifting NMDA Receptor Co-agonist.

Shape-shifting, a phenomenon wide-spread in folklore, refers to the ability to physically change from one identity to another, typically from an innocuous entity to a destructive one. The amino acid D-serine over the last 25 years has "shape-shifted" into several identities: a purported glial transmitter activating N-methyl-D-aspartate receptors (NMDARs), a co-transmitter concentrated in excitatory glutamatergic neurons, an autocrine that is released at dendritic spines to prime their post-synaptic NMDARs for an instantaneous response to glutamate and an excitotoxic moiety released from inflammatory (A1) astrocytes. This article will review evidence in support of these scenarios and the artifacts that misled investigators of the true identity of D-serine.

Desensitization of NMDA channels requires ligand binding to both GluN1 and GluN2 subunits to constrict the pore beside the activation gate.

N-methyl-D-aspartate (NMDA) receptor channels are activated by glutamate (or NMDA) and glycine. The channels also undergo desensitization, which denotes decreased channel availability, after prolonged exposure to the activating ligands. Glycine apparently has a paradoxical negative effect on desensitization, as the increase in ambient glycine in concentrations required for channel activation would increase sustained NMDA receptor currents. We hypothesized that this classical "glycine-dependent desensitization" could be glycine-dependent activation in essence. By performing electrophysiological recordings and biophysical analyses with rat brain NMDA receptors heterogeneously expressed in Xenopus laevis oocytes, we characterized that the channel opened by "only" NMDA (in nominally glycine-free condition probably with the inevitable nanomolar glycine) would undergo a novel form of deactivation rather than desensitization, and is thus fully available for subsequent activation. Moreover, external tetrapentylammonium ions (TPentA), tetrabutylammonium ions, and tetrapropylammonium ions (TPA, in higher concentrations) block the pore and prohibit channel desensitization with a simple "foot-in-the-door" hindrance effect. TpentA and TPA have the same voltage dependence but show different flow dependence in binding affinity, revealing a common binding site at an electrical distance of ~0.7 from the outside yet differential involvement of the flux-coupling region in the external pore mouth. The smaller tetraethylammonium ion and the larger tetrahexylammonium and tetraheptylammonium ions may block the channel but could not affect desensitization. We conclude that NMDA receptor desensitization requires concomitant binding of both glycine and glutamate, and thus movement of both GluN1 and GluN2 subunits. Desensitization gate itself embodies a highly restricted pore reduction with a physical distance of ~4 Å from the charged nitrogen atom of bound tetraalkylammonium ions, and is located very close to the activation gate in the bundle-crossing region in the external pore vestibule.

SAD-B modulates epileptic seizure by regulating AMPA receptors in patients with temporal lobe epilepsy and in the PTZ-induced epileptic model

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the predominant mediators of glutamate-induced excitatory neurotransmission. It is widely accepted that AMPA receptors are critical for the generation and spread of epileptic seizure activity. Dysfunction of AMPA receptors as a causal factor in patients with intractable epilepsy results in neurotransmission failure. Brain-specific serine/threonine-protein kinase 1 (SAD-B), a serine-threonine kinase specifically expressed in the brain, has been shown to regulate AMPA receptor-mediated neurotransmission through a presynaptic mechanism. In cultured rat hippocampal neurons, the overexpression of SAD-B significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Here, we showed that SAD-B downregulation exerted antiepileptic activity by regulating AMPA receptors in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We first used immunoblotting and immunohistochemistry analysis to demonstrate that SAD-B expression was increased in the epileptic rat brain. Subsequently, to explore the function of SAD-B in epilepsy, we used siRNA to knock down SAD-B protein and observed behavior after PTZ-induced seizures. We found that SAD-B downregulation attenuated seizure severity and susceptibility in the PTZ-induced epileptic model. Furthermore, we showed that the antiepileptic effect of SAD-B downregulation on PTZ-induced seizure was abolished by CNQX (an AMPA receptor inhibitor), suggesting that SAD-B modulated epileptic seizure by regulating AMPA receptors in the brain. Taken together, these findings suggest that SAD-B may be a potential and novel therapeutic target to limit epileptic seizures.

Structural Mechanisms Underlying Activity Changes in an AMPA-type Glutamate Receptor Induced by Substitutions in Its Ligand-Binding Domain.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors produce postsynaptic current by transmitting an agonist-induced structural change in the ligand-binding domain (LBD) to the transmembrane channel. Receptors carrying T686S/A substitutions in their LBDs produce weaker glutamate-evoked currents than wild-type (WT) receptors. However, the substitutions induce little differences in the crystal structures of their LBDs. To understand the structural mechanism underlying reduced activities of these AMPAR variants, we analyzed the structural dynamics of WT, T686S, and T686A variants of LBD using nuclear magnetic resonance. The HD exchange studies of the LBDs showed that the kinetic step where the ligand-binding cleft closes was changed by the substitutions, and the substitution-induced population shift from cleft-closed to cleft-open structures is responsible for the reduced activities of the variants. The chemical shift analyses revealed another structural equilibrium between cleft-locked and cleft-partially-open conformations. The substitution-induced population shift in this equilibrium may be related to slower desensitization observed for these variants.

Metabolism of the neurotoxic amino acid ß-N-methylamino-L-alanine in human cell culture models.

Human dietary exposure to the environmental neurotoxin ß-N-methylamino-L-alanine (BMAA) has been implicated in an increased risk of developing sporadic neurodegenerative diseases like Alzheimer's and amyotrophic lateral sclerosis. Evidence suggests that humans are exposed to BMAA globally, but very little is known about BMAA metabolism in mammalian systems, let alone in humans. The most plausible, evidence-based mechanisms of BMAA toxicity rely on the metabolic stability of the amino acid and that, following ingestion, it enters the circulatory system unmodified. BMAA crosses from the intestinal lumen into the circulatory system, and the small intestine and liver are the first sites for dietary amino acid metabolism. Both tissues have substantial amino acid metabolic needs, which are largely fulfilled by dietary amino acids. Metabolism of BMAA in these tissues has been largely overlooked, yet is important in gauging the true human exposure risk. Here we investigate the potential for BMAA metabolism by the human liver and small intestine, using in vitro cell systems. Data show that BMAA metabolism via common proteinogenic amino acid metabolic pathways is negligible, and that in the presence of other amino acids cellular uptake of BMAA is substantially reduced. These data suggest that the majority of ingested BMAA remains unmodified following passage through the small intestine and liver. This not only supports oral BMAA exposure as a plausible exposure route to toxic doses of BMAA, but also supports previous notions that protein deficient diets or malnutrition may increase an individual's susceptibility to BMAA absorption and subsequent toxicity.

Reduced excitatory neurotransmitter levels in anterior insulae are associated with abdominal pain in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a visceral pain condition with psychological comorbidity. Brain imaging studies in IBS demonstrate altered function in anterior insula (aINS), a key hub for integration of interoceptive, affective, and cognitive processes. However, alterations in aINS excitatory and inhibitory neurotransmission as putative biochemical underpinnings of these functional changes remain elusive. Using quantitative magnetic resonance spectroscopy, we compared women with IBS and healthy women (healthy controls [HC]) with respect to aINS glutamate + glutamine (Glx) and γ-aminobutyric acid (GABA+) concentrations and addressed possible associations with symptoms. Thirty-nine women with IBS and 21 HC underwent quantitative magnetic resonance spectroscopy of bilateral aINS to assess Glx and GABA+ concentrations. Questionnaire data from all participants and prospective symptom-diary data from patients were obtained for regression analyses of neurotransmitter concentrations with IBS-related and psychological parameters. Concentrations of Glx were lower in IBS compared with HC (left aINS P < 0.05, right aINS P < 0.001), whereas no group differences were detected for GABA+ concentrations. Lower right-lateralized Glx concentrations in patients were substantially predicted by longer pain duration, while less frequent use of adaptive pain-coping predicted lower Glx in left aINS. Our findings provide first evidence for reduced excitatory but unaltered inhibitory neurotransmitter levels in aINS in IBS. The results also indicate a functional lateralization of aINS with a stronger involvement of the right hemisphere in perception of abdominal pain and of the left aINS in cognitive pain regulation. Our findings suggest that glutaminergic deficiency may play a role in pain processing in IBS.

Use of the 4-Hydroxytriazole Moiety as a Bioisosteric Tool in the Development of Ionotropic Glutamate Receptor Ligands.

We report a series of glutamate and aspartate analogues designed using the hydroxy-1,2,3-triazole moiety as a bioisostere for the distal carboxylic acid. Compound 6b showed unprecedented selectivity among ( S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtypes, confirmed also by an unusual binding mode observed for the crystal structures in complex with the AMPA receptor GluA2 agonist-binding domain. Here, a methionine (Met729) was highly disordered compared to previous agonist-bound structures. This observation provides a possible explanation for the pharmacological profile. In the structure with 7a, an unusual organization of water molecules around the bioisostere arises compared to previous structures of ligands with other bioisosteres. Aspartate analogue 8 with the hydroxy-1,2,3-triazole moiety directly attached to glycine was unexpectedly able to activate both the glutamate and glycine agonist-binding sites of the N-methyl-d-aspartic acid receptor. These observations demonstrate novel features that arise when employing a hydroxytriazole moiety as a bioisostere for the distal carboxylic acid in glutamate receptor agonists.

Population Shift Mechanism for Partial Agonism of AMPA Receptor.

α-amino-3-hydroxy-5-methyl-4-isoaxazolepropionic acid (AMPA) ionotropic glutamate receptors mediate fast excitatory neurotransmission in the central nervous system, and their dysfunction is associated with neurological diseases. Glutamate binding to ligand-binding domains (LBDs) of AMPA receptors induces channel opening in the transmembrane domains of the receptors. The T686A mutation reduces glutamate efficacy so that the glutamate behaves as a partial agonist. The crystal structures of wild-type and mutant LBDs are very similar and cannot account for the observed behavior. To elucidate the molecular mechanism inducing partial agonism of the T686A mutant, we computed the free-energy landscapes governing GluA2 LBD closure using replica-exchange umbrella sampling simulations. A semiclosed state, not observed in crystal structures, appears in the mutant during simulation. In this state, the LBD cleft opens slightly because of breaking of interlobe hydrogen bonds, reducing the efficiency of channel opening. The energy difference between the LBD closed and semiclosed states is small, and transitions between the two states would occur by thermal fluctuations. Evidently, glutamate binding to the T686A mutant induces a population shift from a closed to a semiclosed state, explaining the partial agonism in the AMPA receptor.

Inhibition of alpha7 nicotinic receptors in the ventral hippocampus selectively attenuates reinstatement of morphine-conditioned place preference and associated changes in AMPA receptor binding.

Recurrent relapse is a major problem in treating opiate addiction. Pavlovian conditioning plays a role in recurrent relapse whereby exposure to cues learned during drug intake can precipitate relapse to drug taking. α7 nicotinic acetylcholine receptors (nAChRs) have been implicated in attentional aspects of cognition and mechanisms of learning and memory. In this study we have investigated the role of α7 nAChRs in morphine-conditioned place preference (morphine-CPP). CPP provides a model of associative learning that is pertinent to associative aspects of drug dependence. The α7 nAChR antagonist methyllycaconitine (MLA; 4 mg/kg s.c.) had no effect on the acquisition, maintenance, reconsolidation or extinction of morphine-CPP but selectively attenuated morphine-primed reinstatement of CPP, in both mice and rats. Reinstatement of morphine-CPP in mice was accompanied by a selective increase in [3 H]-AMPA binding (but not in [3 H]-MK801 binding) in the ventral hippocampus that was prevented by prior treatment with MLA. Administration of MLA (6.7 µg) directly into the ventral hippocampus of rats prior to a systemic priming dose of morphine abolished reinstatement of morphine-CPP, whereas MLA delivered into the dorsal hippocampus or prefrontal cortex was without effect. These results suggest that α7 nAChRs in the ventral hippocampus play a specific role in the retrieval of associative drug memories following a period of extinction, making them potential targets for the prevention of relapse.

Biased agonism and allosteric modulation of metabotropic glutamate receptor 5.

Metabotropic glutamate receptors belong to class C G-protein-coupled receptors and consist of eight subtypes that are ubiquitously expressed throughout the central nervous system. In recent years, the metabotropic glutamate receptor subtype 5 (mGlu5) has emerged as a promising target for a broad range of psychiatric and neurological disorders. Drug discovery programs targetting mGlu5 are primarily focused on development of allosteric modulators that interact with sites distinct from the endogenous agonist glutamate. Significant efforts have seen mGlu5 allosteric modulators progress into clinical trials; however, recent failures due to lack of efficacy or adverse effects indicate a need for a better understanding of the functional consequences of mGlu5 allosteric modulation. Biased agonism is an interrelated phenomenon to allosterism, describing how different ligands acting through the same receptor can differentially influence signaling to distinct transducers and pathways. Emerging evidence demonstrates that allosteric modulators can induce biased pharmacology at the level of intrinsic agonism as well as through differential modulation of orthosteric agonist-signaling pathways. Here, we present key considerations in the discovery and development of mGlu5 allosteric modulators and the opportunities and pitfalls offered by biased agonism and modulation.

Impact of allosteric modulation: Exploring the binding kinetics of glutamate and other orthosteric ligands of the metabotropic glutamate receptor 2.

While many orthosteric ligands have been developed for the mGlu2 receptor, little is known about their target binding kinetics and how these relate to those of the endogenous agonist glutamate. Here, the kinetic rate constants, i.e. kon and koff, of glutamate were determined for the first time followed by those of the synthetic agonist LY354740 and antagonist LY341495. To increase the understanding of the binding mechanism and impact of allosteric modulation thereon, kinetic experiments were repeated in the presence of allosteric modulators. Functional assays were performed to further study the interplay between the orthosteric and allosteric binding sites, including an impedance-based morphology assay. We found that dissociation rate constants of orthosteric mGlu2 ligands were all within a small 6-fold range, whereas association rate constants were ranging over more than three orders of magnitude and correlated to both affinity and potency. The latter showed that target engagement of orthosteric mGlu2 ligands is kon-driven in vitro. Moreover, only the off-rates of the two agonists were decreased by a positive allosteric modulator (PAM), thereby increasing their affinity. Interestingly, a PAM increased the duration of a glutamate-induced cellular response. A negative allosteric modulator (NAM) increased both on- and off-rate of glutamate without changing its affinity, while it did not affect these parameters for LY354740, indicating probe-dependency. In conclusion, we found that affinity- or potency-based orthosteric ligand optimization primarily results in ligands with high kon values. Moreover, positive allosteric modulators alter the binding kinetics of orthosteric agonists mainly by decreasing koff, which we were able to correlate to a lengthened cellular response. Together, this study shows the importance of studying binding kinetics in early drug discovery, as this may provide important insights towards improved efficacy in vivo.

Slow moving neural source in the epileptic hippocampus can mimic progression of human seizures.

Fast and slow neural waves have been observed to propagate in the human brain during seizures. Yet the nature of these waves is difficult to study in a surgical setting. Here, we report an observation of two different traveling waves propagating in the in-vitro epileptic hippocampus at speeds similar to those in the human brain. A fast traveling spike and a slow moving wave were recorded simultaneously with a genetically encoded voltage sensitive fluorescent protein (VSFP Butterfly 1.2) and a high speed camera. The results of this study indicate that the fast traveling spike is NMDA-sensitive but the slow moving wave is not. Image analysis and model simulation demonstrate that the slow moving wave is moving slowly, generating the fast traveling spike and is, therefore, a moving source of the epileptiform activity. This slow moving wave is associated with a propagating neural calcium wave detected with calcium dye (OGB-1) but is independent of NMDA receptors, not related to ATP release, and much faster than those previously recorded potassium waves. Computer modeling suggests that the slow moving wave can propagate by the ephaptic effect like epileptiform activity. These findings provide an alternative explanation for slow propagation seizure wavefronts associated with fast propagating spikes.

Disposition of ß-N-methylamino-l-alanine (L-BMAA), a neurotoxin, in rodents following a single or repeated oral exposure.

ß-N-methylamino-l-alanine (L-BMAA) is produced by cyanobacteria (blue-green algae). Human exposure to L-BMAA occurs via consumption of L-BMAA-contaminated water and food. It is speculated that exposure to L-BMAA, and subsequent brain accumulation, may contribute to an increased incidence of neurodegenerative diseases indicating the need to evaluate risk of L-BMAA exposure to humans. As an initial step in this process, we have evaluated disposition following a single or repeated gavage administration of 1, 10 or 100mg/kg [14C]L-BMAA in rats and mice. L-BMAA was well absorbed following a single gavage administration with minimal dose, species, or sex-related effect. In both species, the main excretion route was as exhaled CO2 (46-61%) with 7-13% and 1.4-8% of the administered dose excreted in the urine and feces, respectively. L-BMAA was distributed to all tissues examined; the total radioactivity in tissues increased with the dose and was significant in both species (8-20%). In male rats, L-BMAA was slowly eliminated from blood and tissues (half-lives ≥48h). Following 1, 5 and 10days of dosing in male rats, levels in tissues increased with the number of doses demonstrating potential for accumulation of BMAA-derived equivalents. There was no greater affinity for accumulation in the brain compared to other organs and tissues. Following repeated exposure in rats, amino acid mass shifts associated with L-BMAA were detected in brain peptides. However, the low frequency of occurrence suggests that the substitution of an amino acid with L-BMAA is not significant relative to substitutions and/or modifications by other L-BMAA-derived equivalents.

The gliadin peptide 31-43 exacerbates kainate neurotoxicity in epilepsy models.

Many neurological disorders of gluten-related diseases (GRD), not directly referable to the gastrointestinal tract, have been reported in association with celiac disease (CD), including ataxia, neuropathy and epilepsy. In particular, people with epilepsy diagnosed with CD seems to be characterized by intractable seizure. In these patients, gluten restriction diet has resulted in a reduction of both seizure frequency and antiepileptic medication. Many hypotheses have been suggested, however, molecular mechanisms that associates GRD and epileptogenesis are yet unknown. In this study, we examined the effects of the toxic gliadin peptide 31-43 in in vivo and in vitro models of kainate-induced-epilepsy. We observed that p31-43 exacerbates kainate neurotoxicity in epilepsy models, through the involvement of the enzymatic activity of transglutaminases. Moreover, electrophysiological recordings in CA3 pyramidal neurons of organotypic hippocampal slices show that p31-43 increases the inward current induced by kainate, the average sEPSC amplitude and the total number of evoked action potentials when applicated alone, thus suggesting that p31-43 is able to influence CA3-CA1 neurotransmission and can potentiate postsynaptic kainate receptors. Our results suggest a possible mechanism underlying the relationship between GRD and epilepsy through a potentiation of kainate-induced neurotoxicity and links the toxic effects of gluten to epilepsy.

Plasticity of calcium-permeable AMPA glutamate receptors in Pro-opiomelanocortin neurons.

POMC neurons integrate metabolic signals from the periphery. Here, we show in mice that food deprivation induces a linear current-voltage relationship of AMPAR-mediated excitatory postsynaptic currents (EPSCs) in POMC neurons. Inhibition of EPSCs by IEM-1460, an antagonist of calcium-permeable (Cp) AMPARs, diminished EPSC amplitude in the fed but not in the fasted state, suggesting entry of GluR2 subunits into the AMPA receptor complex during food deprivation. Accordingly, removal of extracellular calcium from ACSF decreased the amplitude of mEPSCs in the fed but not the fasted state. Ten days of high-fat diet exposure, which was accompanied by elevated leptin levels and increased POMC neuronal activity, resulted in increased expression of Cp-AMPARs on POMC neurons. Altogether, our results show that entry of calcium via Cp-AMPARs is inherent to activation of POMC neurons, which may underlie a vulnerability of these neurons to calcium overload while activated in a sustained manner during over-nutrition.