Glycine Binding Site Research Articles

Pharmacological Study on Alzheimer’s Drugs Targeting Calcium/Calmodulin-Dependent Protein Kinase II

In the brain of Alzheimer's disease patients, down-regulation of both cholinergic and glutamatergic systems have been found and is thought to play an important role in impairment of cognition, learning, and memory. Nefiracetam is a pyrrolidine-related nootropic drug exhibiting various pharmacological actions such as a cognitive-enhancing effect. The present study was undertaken to elucidate mechanisms underlying the action of nefiracetam on glutamatergic receptors and intracellular protein kinases. N-Methyl-D-aspartate (NMDA)-evoked currents were recorded from rat cortical neurons in long-term cultured primary neurons using the whole-cell patch-clamp technique. NMDA-evoked currents were greatly and reversibly potentiated by bath application of nefiracetam, resulting in a bell-shaped dose-response curve. The maximum potentiation of 170% relative to the control was produced at 10 nM. Treatment with an inhibitor of the glycine binding site of the NMDA receptor, 7-chlorokynurenic acid, at 1 µM prevented augmentation of NMDA-evoked currents by nefiracetam. In rat hippocampal CA1 slices, field excitatory postsynaptic potentials were recorded by stimulation of Schaffer collateral/commissural pathways. Nefiracetam treatment significantly enhanced long-term potentiation (LTP) with the same bell-shaped dose-response curve. Furthermore, nefiracetam-induced LTP enhancement was closely associated with calcium/calmodulin-dependent protein kinase II (CaMKII) activation with concomitant increase in phosphorylation of AMPA-type glutamate receptor subunit 1 (GluA1) (Ser-831) as a postsynaptic CaMKII substrate. In conclusion, nefiracetam enhances NMDA-receptor function through stimulation of its glycine binding site and nefiracetam-induced CaMKII activation likely contributes to improvement of cognition, learning, and memory.

Recent Advances in the Discovery of D-Amino Acid Oxidase Inhibitors and Their Therapeutic Utility in Schizophrenia

D-amino acid oxidase (DAAO) catalyzes the oxidative metabolism of D-amino acids including D-serine, a full agonist at the allosteric glycine binding site of the NMDA receptor. D-serine was reported to improve negative and cognitive symptoms of schizophrenia, symptoms poorly addressed by the standard D2 antagonist therapies. Therefore, inhibition of DAAO has gained substantial interest as an effective way to increase D-serine levels in the brain. During the last several years, a growing number of structurally diverse DAAO inhibitors have been identified with significantly higher inhibitory potency compared to the conventional DAAO inhibitors. Some of these new generation of DAAO inhibitors are being evaluated for their ability to enhance D-serine levels in rodents and efficacy in animal models of schizophrenia. This article highlights the progress that has been made toward the discovery of DAAO inhibitors and recent efforts to exploit their therapeutic utility in schizophrenia.

Exposure to Kynurenic Acid During Adolescence Produces Memory Deficits in Adulthood

The glia-derived molecule kynurenic acid (KYNA) is an antagonist of α7 nicotinic acetylcholine receptors and the glycine(B) binding site on n-methyl-d-aspartateglutamate receptors, both of which have critical roles in neural plasticity as well as learning and memory. KYNA levels are increased in the brains and cerebral spinal fluid of persons with schizophrenia, leading to the notion that changes in KYNA concentration might contribute to cognitive dysfunction associated with this disorder. Indeed, recent studies indicate that increasing endogenous KYNA concentration by administering l-kynurenine (L-KYN, the precursor of KYNA) impairs spatial as well as contextual learning and memory in adult rats. In the present study, rats were treated with L-KYN (100 mg/kg) throughout adolescence to increase endogenous KYNA concentration during this critical time in brain development. Rats were then tested drug-free as adults to test the hypothesis that exposure to elevated levels of KYNA during development may contribute to cognitive dysfunction later in life. Consistent with prior studies in which adult rats were treated acutely with L-KYN, juvenile rats exposed to increased KYNA concentration during adolescence exhibited deficits in contextual fear memory, but cue-specific fear memory was not impaired. In addition, rats treated with L-KYN as adolescents were impaired on a novel object recognition memory task when tested as adults. The memory deficits could not be explained by drug-induced changes in locomotor activity or shock sensitivity. Together, these findings add to the growing literature supporting the notion that exposure to increased concentration of KYNA may contribute to cognitive deficits typically observed in schizophrenia.

Novel N-methyl-d-aspartate receptor antagonists: a review of compounds patented since 2006

Importance of the field: The NMDA receptor is a complex ligand gated, voltage-dependent ion channel. It has been a drug target for > 25 years for neurological and psychiatric indications. Whereas the initial optimism to turn preclinically active compounds rapidly into drugs for human use was dampened, new insights into cellular receptor localization, role of subunits and receptor operation have kept the interest alive to modulate this receptor for therapeutic intervention.Areas covered in this review: The article describes the NMDA receptor antagonists patented since 2006. Also included are novel NMDA receptor ligands potentially useful for positron emission tomography imaging.What the reader will gain: The first section summarizes the current status of NMDA receptor pharmacology. This serves as a base for the next sections discussing the patented compounds with respect to their mode of action, potency and, in some cases, drugability.Take home message: The most important recent strategies aiming for inhibition of NMDA receptor-mediated neurotransmission avoid for safety reasons full receptor blockade but allow a low degree of normal receptor function. Approaches pursued by the latest patents comprise blocking the channel with compounds of low affinity, antagonizing receptor activity by highly potent NR2B ligands, partial agonism at the glutamate or glycine-binding site and improvement of pharmacokinetic properties of well established, safe antagonists by deuteration.

Mapping Interactions between Germinants and Clostridium difficile Spores

Germination of Clostridium difficile spores is the first required step in establishing C. difficile-associated disease (CDAD). Taurocholate (a bile salt) and glycine (an amino acid) have been shown to be important germinants of C. difficile spores. In the present study, we tested a series of glycine and taurocholate analogs for the ability to induce or inhibit C. difficile spore germination. Testing of glycine analogs revealed that both the carboxy and amino groups are important epitopes for recognition and that the glycine binding site can accommodate compounds with more widely separated termini. The C. difficile germination machinery also recognizes other hydrophobic amino acids. In general, linear alkyl side chains are better activators of spore germination than their branched analogs. However, L-phenylalanine and L-arginine are also good germinants and are probably recognized by distinct binding sites. Testing of taurocholate analogs revealed that the 12-hydroxyl group of taurocholate is necessary, but not sufficient, to activate spore germination. In contrast, the 6- and 7-hydroxyl groups are required for inhibition of C. difficile spore germination. Similarly, C. difficile spores are able to detect taurocholate analogs with shorter, but not longer, alkyl amino sulfonic acid side chains. Furthermore, the sulfonic acid group can be partially substituted with other acidic groups. Finally, a taurocholate analog with an m-aminobenzenesulfonic acid side chain is a strong inhibitor of C. difficile spore germination. In conclusion, C. difficile spores recognize both amino acids and taurocholate through multiple interactions that are required to bind the germinants and/or activate the germination machinery.

D-Cycloserine improves the impaired sociability of the Balb/c mouse

The genetically inbred Balb/c mouse strain shows evidence of impaired sociability in a standard paradigm. For example, relative to 8-week-old male outbred Swiss-Webster mice, 8 week-old male Balb/c mice spend less time sniffing and in the vicinity of an enclosed 4 week-old male ICR stimulus mouse and, when allowed to interact freely with the stimulus mouse for five minutes, make fewer discrete episodes of social approach and show suppression of locomotor activity. We explored the effect of d-cycloserine (320 mg/kg, intraperitoneally), a partial glycine agonist that binds to the obligatory co-agonist glycine binding site on the NMDA receptor, on the sociability of the Balb/c and Swiss-Webster mouse strains in a standard paradigm. The results show that treatment with d-cycloserine increased the locomotor activity of the Balb/c mouse strain in the presence of an enclosed social stimulus mouse and when these mice were allowed to interact freely with each other. Also, d-cycloserine increased the number of discrete episodes of social approach when Balb/c mice were allowed to interact freely with social stimulus mice. However, d-cycloserine had similar effects on measures of sociability in the Swiss-Webster mouse, raising the possibility that the positive effects on the sociability of the Balb/c mouse strain may be mediated by indirect effects on locomotion, arousal, and anxiety.

Activation of N-Methyl-d-aspartate (NMDA) Receptors in the Dorsal Vagal Complex Lowers Glucose Production

Diabetes is characterized by hyperglycemia due partly to increased hepatic glucose production. The hypothalamus regulates hepatic glucose production in rodents. However, it is currently unknown whether other regions of the brain are sufficient in glucose production regulation. The N-methyl-D-aspartate (NMDA) receptor is composed of NR1 and NR2 subunits, which are activated by co-agonist glycine and glutamate or aspartate, respectively. Here we report that direct administration of either co-agonist glycine or NMDA into the dorsal vagal complex (DVC), targeting the nucleus of the solitary tract, lowered glucose production in vivo. Direct infusion of the NMDA receptor blocker MK-801 into the DVC negated the metabolic effect of glycine. To evaluate whether NR1 subunit of the NMDA receptor mediates the effect of glycine, NR1 in the DVC was inhibited by DVC NR1 antagonist 7-chlorokynurenic acid or DVC shRNA-NR1. Pharmacological and molecular inhibition of DVC NR1 negated the metabolic effect of glycine. To evaluate whether the NMDA receptors mediate the effects of NR2 agonist NMDA, DVC NMDA receptors were inhibited by antagonist D-2-amino-5-phosphonovaleric acid (D-APV). DVC D-APV fully negated the ability of DVC NMDA to lower glucose production. Finally, hepatic vagotomy negated the DVC glycine ability to lower glucose production. These findings demonstrate that activation of NR1 and NR2 subunits of the NMDA receptors in the DVC is sufficient to trigger a brain-liver axis to lower glucose production, and suggest that DVC NMDA receptors serve as a therapeutic target for diabetes and obesity.

Identification of Novel Specific Allosteric Modulators of the Glycine Receptor Using Phage Display

The glycine receptor (GlyR) is a member of the Cys-loop superfamily of ligand-gated ion channels and the major mediator of inhibitory neurotransmission in the spinal cord and brainstem. Many allosteric modulators affect the functioning of members of this superfamily, with some such as benzodiazepines showing great specificity and others such as zinc, alcohols, and volatile anesthetics acting on multiple members. To date, no potent and efficacious allosteric modulator acting specifically at the GlyR has been identified, hindering both experimental characterization of the receptor and development of GlyR-related therapeutics. We used phage display to identify novel peptides that specifically modulate GlyR function. Peptide D12-116 markedly enhanced GlyR currents at low micromolar concentrations but had no effects on the closely related gamma-aminobutyric acid type A receptors. This approach can readily be adapted for use with other channels that currently lack specific allosteric modulators.

Effects of Chronic Introduction of Antidepressants on NMDA-Induced Damage to Neurons of the Rat Hippocampus and Cerebral Cortex

In in vitro studies on superfused slices obtained from the rat hippocampus and cortex, we found that 50 μM N-methyl-D-aspartate (NMDA) applied to the slices in the presence of 10 μM glycine for 15 min exerts a significant damaging action to neurons of these structures. One hour after termination of the action of NMDA, this was manifested in more than a twofold decrease in the synaptic reactivity of pyramidal neurons of the hippocampal СА1 area and layers II/III of the cerebral cortex. The excitotoxic effect of NMDA was prevented by application of competitive (D-2-amino-5-phosphonovaleric acid, 50 μM) and noncompetitive (ketamine, 100 μM) blockers of NMDA receptors. A blocker of glycine-binding sites of NMDA receptors (compound ТСВ 24.15, 10 μM) weakened NMDA-induced damage to the neurons. A competitive blocker of glutamate АМРА receptors, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μM), and a local anesthetic, lidocaine hydrochloride (50 μM), did not modify the excitotoxic effect of NMDA. A blocker of voltagedependent L-type calcium channels, verapamil (20 μM), demonstrated some trend to intensification of NMDA excitotoxic action. An inhibitor of tyrosine-protein phosphatases, sodium vanadate, when i.p. injected into rats in a dose of 15 mg/kg 6 h prior to the electrophysiological experiment, decreased the damaging action of NMDA. Two-hour-long treatment of cerebral slices with 1 μM genistein, an inhibitor of tyrosine kinases, weakened the neuroprotective effect of sodium vanadate. Chronic injections (14 days in daily doses of 20 mg/kg) of antidepressants belonging to different functional classes (imipramine, fluoxetine, and pyrazidol) into rats decreased (similarly to blockers of NMDA receptors) the excitotoxic action of NMDA receptors. Neuroprotective effects of antidepressants were weakened upon the action of genistein. We conclude that the neuroprotective activity of antidepressants under conditions of excitotoxic action of NMDA is mainly determined by an increase in the activity of tyrosine kinases in the cytoplasm and/or neuronal nucleus.

Co-Induction of LTP and LTD and Its Regulation by Protein Kinases and Phosphatases

The cellular properties of long-term potentiation (LTP) following pairing of pre- and postsynaptic activity were examined at a known glutamatergic synapse in the leech, specifically between the pressure (P) mechanosensory and anterior pagoda (AP) neurons. Stimulation of the presynaptic P cell (25 Hz) concurrent with a 2 nA depolarization of the postsynaptic AP cell significantly potentiated the P-to-AP excitatory postsynaptic potential (EPSP) in an N-methyl-d-aspartate receptor (NMDAR)-dependent manner based on inhibitory effects of the NMDAR antagonist MK801 and inhibition of the NMDAR glycine binding site by 7-chlorokynurenic acid. LTP was blocked by injection of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the postsynaptic (AP) cell, indicating a requirement for postsynaptic elevation of intracellular Ca(2+). Autocamtide-2-related inhibitory peptide (AIP), a specific inhibitor of Ca(2+)/calmodulin-dependent kinase II (CaMKII), and Rp-cAMP, an inhibitor of protein kinase A (PKA), also blocked pairing-induced potentiation, indicating a requirement for activation of CaMKII and PKA. Interestingly, application of AIP during pairing resulted in significantly depressed synaptic transmission. Co-application of AIP with the protein phosphatase inhibitor okadaic acid restored synaptic transmission to baseline levels, suggesting an interaction between CaMKII and protein phosphatases during induction of activity-dependent synaptic plasticity. When postsynaptic activity preceded presynaptic activity, NMDAR-dependent long-term depression (LTD) was observed that was blocked by okadaic acid. Postsynaptic injection of botulinum toxin blocked P-to-AP potentiation while postsynaptic injection of pep2-SVKI, an inhibitor of AMPA receptor endocytosis, inhibited LTD, supporting the hypothesis that glutamate receptor trafficking contributes to both LTP and LTD at the P-to-AP synapse in the leech.

Long-Term Depression in the Hippocampal CA1 Area of Aged Rats, Revisited: Contribution of Temporal Constraints Related to Slice Preparation

BackgroundThe effects of low-frequency conditioning stimulation (LFS, 900 pulses at 1 Hz) of glutamatergic afferents in CA1 hippocampal area using slices from two different strains of adult (3–5 month-old) and aged (23–27 month-old) rats were reinvestigated regarding the discrepancies in the literature concerning the expression of long-term depression (LTD) in the aging brain.Methodology/Principal FindingsN-methyl-D-aspartate receptor (NMDA-R) dependent LTD was examined in both adult (n = 21) and aged (n = 22) Sprague-Dawley rats. While equivalent amounts of LTD could be obtained in both ages, there was significant variability depending upon the time between the slices were made and when they were tested. LTD was not apparent if slices were tested within 3 hours of dissection. The amount of LTD increased over the next three hours but more in adult than in aged rats. This age-related impairment was abolished by exogenous d-serine, thus reflecting the reduced activation of the NMDA-R glycine-binding site by the endogenous agonist in aged rats. Then, the amount of LTD reached asymptote at 5–7 hours following dissection. Similar temporal profiles of LTD expression were seen in young and aged Wistar rats.Conclusions/SignificanceTaken together, these results sound a cautionary note regarding the existence of an experimental “window of opportunity” for studying the effects of aging on LTD expression in hippocampal slice preparation.

The N-Methyl-D-Aspartate Receptor Co-agonist D-Cycloserine Facilitates Declarative Learning and Hippocampal Activity in Humans

The N-methyl-D-aspartate receptor (NMDAR) is critical for learning-related synaptic plasticity in amygdala and hippocampus. As a consequence, there is considerable interest in drugs targeting this receptor to help enhance amygdala- and hippocampus-dependent learning. A promising candidate in this respect is the NMDAR glycine-binding site partial agonist D-cycloserine (DCS). Accumulating clinical evidence indicates the efficacy of DCS in the facilitation of amygdala-dependent fear extinction learning in patients with phobic, social anxiety, panic, and obsessive-compulsive disorder. An important unresolved question though is whether the use of DCS can also facilitate hippocampus-dependent declarative learning in healthy people as opposed to being restricted to the fear memory domain. In the present study, we investigated whether or not DCS can facilitate hippocampus-dependent declarative learning. We have therefore combined functional magnetic resonance imaging with two different declarative learning tasks and cytoarchitectonic probabilistic mapping of the hippocampus and its major subdivisions in 40 healthy volunteers administered either a 250 mg single oral dose of DCS or a placebo. We found that DCS facilitates declarative learning as well as blood-oxygen level dependent activity levels in the probabilistically defined cornu ammonis region of the hippocampus. The absence of activity changes in visual control areas underscores the specific action of DCS in the hippocampal cornu ammonis region. Our findings highlight NMDAR glycine-binding site partial agonism as a promising pharmacological mechanism for facilitating declarative learning in healthy people.

Glycine transporter 1 (GlyT1) inhibitors exhibit anticonvulsant properties in the rat maximal electroshock threshold (MEST) test

Glycine can act as either an inhibitory neurotransmitter or as a potentiator of NMDA-dependent excitatory neurotransmission. There is some evidence that glycine can have both pro- and anticonvulsant properties in various rodent models of epilepsy. In the present study we tested several glycine transporter 1 (GlyT1) inhibitors including NFPS, SSR 504734, Lu AA21279, Org 25935, SB-710622, GSK931145, as well as the glycine agonist d-serine, in the maximal electroshock threshold (MEST) test in the rat. In a series of experiments, male Sprague–Dawley rats ( n = 12/group) were pre-treated with a compound of interest and then received an electric shock delivered via corneal electrodes. A cohort of satellite animals ( n = 3/group) was also used to measure blood and brain levels of Org 25935. All GlyT1 inhibitors increased seizure thresholds dose-dependently, indicative of anticonvulsant activity. SB-710622 and GSK931145 had lower minimum effective doses (MEDs) in the MEST test than other GlyT1 inhibitors. At estimated t max, increases in dose administered were paralleled by increases in blood and brain concentrations of Org 25935. Thus, increasing extracellular concentration of glycine via inhibition of its uptake protects from electroshock-induced seizures in the rat. Whether strychnine-sensitive or strychnine-insensitive glycine binding sites are involved in this effect remains to be determined.

Competitive Inhibition at the Glycine Site of the N -Methyl-d-Aspartate Receptor Mediates Xenon Neuroprotection against Hypoxia–Ischemia

The general anesthetic gas xenon is neuroprotective and is undergoing clinical trials as a treatment for ischemic brain injury. A small number of molecular targets for xenon have been identified, the N-methyl-D-aspartate (NMDA) receptor, the two-pore-domain potassium channel TREK-1, and the adenosine triphosphate-sensitive potassium channel (KATP). However, which of these targets are relevant to acute xenon neuroprotection is not known. Xenon inhibits NMDA receptors by competing with glycine at the glycine-binding site. We test the hypothesis that inhibition of the NMDA receptor at the glycine site underlies xenon neuroprotection against hypoxia-ischemia. We use an in vitro model of hypoxia-ischemia to investigate the mechanism of xenon neuroprotection. Organotypic hippocampal brain slices from mice are subjected to oxygen-glucose deprivation, and injury is quantified by propidium iodide fluorescence. We show that 50% atm xenon is neuroprotective against hypoxia-ischemia when applied immediately after injury or after a delay of 3 h after injury. To validate our method, we show that neuroprotection by gavestinel is abolished when glycine is added, confirming that NMDA receptor glycine site antagonism underlies gavestinel neuroprotection. We then show that adding glycine abolishes the neuroprotective effect of xenon, consistent with competitive inhibition at the NMDA receptor glycine site mediating xenon neuroprotection. We show that xenon neuroprotection against hypoxia- ischemia can be reversed by increasing the glycine concentration. This is consistent with competitive inhibition by xenon at the NMDA receptor glycine site, playing a significant role in xenon neuroprotection. This finding may have important implications for xenon's clinical use as an anesthetic and neuroprotectant.

Genetic loss of D‐amino acid oxidase activity reverses schizophrenia‐like phenotypes in mice

Reduced function of the N-methyl-d-aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine binding site in its NR1 subunit that may be a useful target for the treatment of schizophrenia. In this study, we assessed the therapeutic potential of long-term increases in the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO) in mice. The effects of eliminating DAO function were investigated in mice that display schizophrenia-related behavioral deficits due to a mutation (Grin 1(D481N)) in the NR1 subunit that results in a reduction in NMDAR glycine affinity. Grin 1(D481N) mice show deficits in sociability, prolonged latent inhibition, enhanced startle reactivity and impaired spatial memory. The hypofunctional Dao 1(G181R) mutation elevated brain levels of D-serine, but alone it did not affect performance in the behavioral measures. Compared to animals with only the Grin 1(D481N) mutation, mice with both the Dao1(G181R) and Grin 1(D481N) mutations displayed an improvement in social approach and spatial memory retention, as well as a reversal of abnormally persistent latent inhibition and a partial normalization of startle responses. Thus, an increased level of D-serine resulting from decreased catalysis corrected the performance of mice with deficient NMDAR glycine site activation in behavioral tasks relevant to the negative and cognitive symptoms of schizophrenia. Diminished DAO activity and elevations in D-serine may serve as an effective therapeutic intervention for the treatment of psychiatric symptoms.

Potentiation of glycine-gated NR1/NR3A NMDA receptors relieves Ca2+-dependent outward rectification

Glycine has diverse functions within the mammalian central nervous system. It inhibits postsynaptic neurons via strychnine-sensitive glycine receptors (GlyRs) and enhances neuronal excitation through co-activation of N-methyl-D-aspartate (NMDA) receptors. Classical Ca2+-permeable NMDA receptors are composed of glycine-binding NR1 and glutamate-binding NR2 subunits, and hence require both glutamate and glycine for efficient activation. In contrast, recombinant receptors composed of NR1 and the glycine binding NR3A and/or NR3B subunits lack glutamate binding sites and can be activated by glycine alone. Therefore these receptors are also named “excitatory glycine receptors”. Co-application of antagonists of the NR1 glycine-binding site or of the divalent cation Zn2+ markedly enhances the glycine responses of these receptors. To gain further insight into the properties of these glycine-gated NMDA receptors, we investigated their current-voltage (I–V) dependence. Whole-cell current-voltage relations of glycine currents recorded from NR1/NR3B and NR1/NR3A/NR3B expressing oocytes were found to be linear under our recording conditions. In contrast, NR1/NR3A receptors displayed a strong outwardly rectifying I–V relation. Interestingly, the voltage-dependent inward current block was abolished in the presence of NR1 antagonists, Zn2+ or a combination of both. Further analysis revealed that Ca2+ (1.8 mM) present in our recording solutions was responsible for the voltage-dependent inhibition of ion flux through NR1/NR3A receptors. Since physiological concentrations of the divalent cation Mg2+ did not affect the I–V dependence, our data suggest that relief of the voltage-dependent Ca2+ block of NR1/NR3A receptors by Zn2+ may be important for the regulation of excitatory glycinergic transmission, according to the Mg2+-block of conventional NR1/NR2 NMDA receptors.

Natural neuroprotective substances and their derivatives: kynurenines in neurodegenerative illnesses

Event Abstract Back to Event Natural neuroprotective substances and their derivatives: kynurenines in neurodegenerative illnesses László Vécsei1* 1 University of Szeged, Albert Szent-Györgyi Clinical Center, Hungary It is well known that kynurenic acid (KYNA) has neuroactive properties and synthesized from kynurenine in reactions mediated by mitochondrial aspartate aminotransferase and kynurenine aminotransferases (KATs: KAT-I, KAT-II, KAT-III). Recent data indicate that major amounts of serum KYNA are produced and liberated from the vascular endothelium. KYNA dispalys a broad spectrum of antagonistic properties. In the human cerebrospinal fluid (CSF), the concentration of KYNA is in the nanomolar range. It can act competitively, primarily at the strychnine-insensitive glycine-binding site of the NMDA receptors. At higher concentration, it can competitively inhibit the AMPA and kainate receptors, too. It was recently demonstrated that KYNA in micromolar concentrations still has a neuroinhibitory effect, whereas in nanomolar concentrations it behaves as a facilitator. Moreover, KYNA nono-competitively blocks the alpha-7-nAch receptors activation of which at the presynaptic site takes part in the regulation of glutamate-release, and hence in both glutamatergic and cholinergic neurotransmission. Furthermore, in nanomolar concentrations, acting on alpha-7-nAch receptors, it can reduce the extracellular level of dopamine in the striatum. This fact suggests that even modest increase in the brain KYNA level can contribute to modulation of the dopaminergic transmission. In this presentation the role of the kynurenine system is discussed in neurodegenerative disorders.

Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRalpha1 ECD, we generated a model of the human GlyRalpha1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRalpha1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.

Comparative virtual screening and novelty detection for NMDA-GlycineB antagonists

Identification of novel compound classes for a drug target is a challenging task for cheminformatics and drug design when considerable research has already been undertaken and many potent lead structures have been identified, which leaves limited unclaimed chemical space for innovation. We validated and successfully applied different state-of-the-art techniques for virtual screening (Bayesian machine learning, automated molecular docking, pharmacophore search, pharmacophore QSAR and shape analysis) of 4.6 million unique and readily available chemical structures to identify promising new and competitive antagonists of the strychnine-insensitive Glycine binding site (Glycine(B) site) of the NMDA receptor. The novelty of the identified virtual hits was assessed by scaffold analysis, putting a strong emphasis on novelty detection. The resulting hits were tested in vitro and several novel, active compounds were identified. While the majority of the computational methods tested were able to partially discriminate actives from structurally similar decoy molecules, the methods differed substantially in their prospective applicability in terms of novelty detection. The results demonstrate that although there is no single best computational method, it is most worthwhile to follow this concept of focused compound library design and screening, as there still can new bioactive compounds be found that possess hitherto unexplored scaffolds and interesting variations of known chemotypes.

Alterations of excitatory transmission in the lateral amygdala during expression and extinction of fear memory

Understanding the neurophysiology of fear extinction has important implications for the treatment of post-traumatic stress disorders. Here we report that fear conditioning resulted in an increase in AMPA/NMDA ratio as well as depression of paired-pulse facilitation (PPF) in neurons of the lateral nucleus of amygdala. These conditioning-induced changes in synaptic transmission were not affected by extinction training. D-cycloserine (DCS), a partial agonist at the glycine-binding site of the NMDA receptor, facilitated extinction and reversed the increase in AMPA/NMDA ratio without altering the depression of PPF when administered before extinction training. Extinction training, however, significantly increased the frequency and amplitude of miniature inhibitory post-synaptic currents and these effects were unaffected by the DCS treatment. Disruption of AMPA receptor endocytosis with a synthetic peptide containing a short C-terminal sequence of GluR2 (869YKEGYNVYG877, GluR23Y) specifically blocked DCS-induced reversal of AMPA/NMDA ratio and the facilitation of extinction. These results suggest that extinction training mainly increases inhibitory transmission leaving conditioning-induced excitatory association unaltered. DCS does not affect inhibitory transmission but reverses the conditioning-induced post-synaptic memory trace when administered before extinction training.