F. Gasparini

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

An antagonist for the metabotropic glutamate receptor may improve symptoms in patients with fragile X syndrome whose FMR1 promoters are fully methylated.

In this Review, a group of experts in fragile X syndrome analyses why the considerable drug development effort based on robust preclinical findings describing the mechanisms underlying this neurodevelopmental disorder has failed to translate into effective treatment and offers possible solutions to improve clinical trial design and therapeutic approaches. Neurodevelopmental disorders such as fragile X syndrome (FXS) result in lifelong cognitive and behavioural deficits and represent a major public health burden. FXS is the most frequent monogenic form of intellectual disability and autism, and the underlying pathophysiology linked to its causal gene, FMR1, has been the focus of intense research. Key alterations in synaptic function thought to underlie this neurodevelopmental disorder have been characterized and rescued in animal models of FXS using genetic and pharmacological approaches. These robust preclinical findings have led to the implementation of the most comprehensive drug development programme undertaken thus far for a genetically defined neurodevelopmental disorder, including phase IIb trials of metabotropic glutamate receptor 5 (mGluR5) antagonists and a phase III trial of a GABAB receptor agonist. However, none of the trials has been able to unambiguously demonstrate efficacy, and they have also highlighted the extent of the knowledge gaps in drug development for FXS and other neurodevelopmental disorders. In this Review, we examine potential issues in the previous studies and future directions for preclinical and clinical trials. FXS is at the forefront of efforts to develop drugs for neurodevelopmental disorders, and lessons learned in the process will also be important for such disorders.

Several lines of evidence suggest a crucial involvement of glutamate in the mechanism of action of anxiolytic and/or antidepressant drugs. The involvement of group I mGlu receptors in anxiety and depression has also been proposed. Given the recent discovery of a selective and brain penetrable mGlu5 receptor antagonists, the effect of 2‐methyl‐6‐(phenylethynyl)‐pyridine (MPEP), i.e. the most potent compound described, was evaluated in established models of anxiety and depression. Experiments were performed on male Wistar rats or male Albino Swiss or C57BL/6J mice. The anxiolytic‐like effects of MPEP was tested in the conflict drinking test and the elevated plus‐maze test in rats as well as in the four‐plate test in mice. The antidepressant‐like effect was estimated using the tail suspension test in mice and the behavioural despair test in rats. MPEP (1 – 30 mg kg −1 ) induced anxiolytic‐like effects in the conflict drinking test and the elevated plus‐maze test in rats as well as in the four‐plate test in mice. MPEP had no effect on locomotor activity or motor coordination. MPEP (1 – 20 mg kg −1 ) did shorten the immobility time in a tail suspension test in mice, however it was inactive in the behavioural despair test in rats. These data suggest that selective mGlu5 receptor antagonists may play a role in the therapy of anxiety and/or depression, further studies are required to identify the sites and the mechanism of action of MPEP. British Journal of Pharmacology (2001) 132 , 1423–1430; doi: 10.1038/sj.bjp.0703923

We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [3H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [3H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues. We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [3H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [3H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues. metabotropic glutamate receptor human metabotropic glutamate receptor 2-methyl-6-(phenylethynyl)pyridine 2-methyl-6-(3-methoxyphenyl)ethynyl pyridine 7-hydroxyiminocyclopropan [b]chromen-1a-carboxylic acid ethyl ester transmembrane seven-transmembrane 6-methyl-2-(phenylazo)-3-pyridinol) (E)-2-methyl-6-(2-phenylethenyl)pyridine inositol phosphate G protein-coupled receptor Metabotropic glutamate receptors are G protein-coupled receptors that play important roles in regulating the activity of many synapses in the central nervous system. At present, eight mGluR1 subtypes (mGluR1 through mGluR8) have been cloned and functionally expressed (1Knoepfel T. Kuhn R. Allgeier H. J. Med. Chem. 1995; 38: 1417-1426Crossref PubMed Scopus (276) Google Scholar, 2Conn P.J. Pin J.-P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2708) Google Scholar). Based on their amino acid sequence homologies, pharmacology, and functional profiles, these subtypes are classified further into three groups. Members of group I (mGluR1 and -5) stimulate the activity of phospholipase C and mobilize intracellular Ca2+. Members of group II (mGluR2 and -3) and group III (mGluR4, -6, -7, -8) inhibit adenylyl cyclase. Despite the differences in primary structures and functional roles, all mGluRs feature a large conserved N-terminal extracellular domain, which is involved in the recognition of agonists and competitive antagonists (3O'Hara P.J. Sheppard P.O. Thogersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (616) Google Scholar, 4Takahashi K. Tsuchida K. Tanabe Y. Masu M. Nakanishi S. J. Biol. Chem. 1993; 268: 19341-19345Abstract Full Text PDF PubMed Google Scholar, 5Tones M.A. Bendali N. Flor P.J. Knoepfel T. Kuhn R. NeuroReport. 1995; 7: 117-120Crossref PubMed Google Scholar, 6Wroblewska B. Wroblewski J.T. Pshenichkin S. Surin A. Sullivan S.E. Neale J.H. J. Neurochem. 1997; 69: 174-181Crossref PubMed Scopus (273) Google Scholar, 7Okamoto T. Sekiyama N. Otsu M. Shimada Y. Sato A. Nakanishi S. Jingami H. J. Biol. Chem. 1998; 273: 13089-13096Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Parmentier M.-L. Joly C. Restituito S. Bockaert J. Grau Y. Pin J.-P. Mol. Pharmacol. 1998; 53: 778-786Crossref PubMed Scopus (70) Google Scholar).Most ligands for mGluRs were derived from amino acids and act at the conserved glutamate binding site (9Schoepp D.D. Jane D.E. Monn J.A. Neuropharmacology. 1999; 38: 1431-1476Crossref PubMed Scopus (1006) Google Scholar). Recently, novel subtype-selective group I mGluR antagonists emerged that are structurally unrelated to amino acids and to each other. The first non-amino acid-like antagonist described was CPCCOEt (Fig. 1), a selective mGluR1 antagonist (10Annoura H. Fukunaga A. Uesugi M. Tatsuoka T. Horikawa Y. Bioorg. Med. Chem. Lett. 1996; 6: 763-766Crossref Scopus (166) Google Scholar, 11Casabona G. Knoepfel T. Kuhn R. Gasparini F. Baumann P. Sortino M.A. Copani A. Nicoletti F. Eur. J. Neurosci. 1997; 9: 12-17Crossref PubMed Scopus (127) Google Scholar). CPCCOEt inhibits receptor activity by a non-competitive mechanism which does not affect the binding affinity of glutamate (12Hermans E. Nahorski S.R. Challiss R.A. Neuropharmacology. 1998; 37: 1645-1647Crossref PubMed Scopus (52) Google Scholar, 13Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.-P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461PubMed Google Scholar). Molecular characterization of the site of inhibition in mGluR1 revealed that CPCCOEt interacts with two non-conserved residues at the top of transmembrane (TM) helix VII (13Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.-P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461PubMed Google Scholar). The first described selective mGluR5 antagonists, SIB-1757 and SIB-1893 (Fig. 1), are also unrelated to amino acids and were shown to act via a non-competitive mechanism (14Varney M.A. Cosford N.D.P. Jachec C. Rao S.P. Sacaan A. Lin F.-F. Bleicher L. Santori E.M. Flor P.J. Allgeier H. Gasparini F. Kuhn R. Hess S.D. Velicelebi G. Johnson E.C.J. Pharmacol. Exp. Ther. 1999; 290: 170-181PubMed Google Scholar).To address the question whether these structurally unrelated mGluR1 and mGluR5 antagonists interact with different sites of the mGluR subtypes or share a common binding site in the 7TM domain, we have studied the binding site and mode of action of the selective mGluR5 antagonist MPEP (15Gasparini F. Lingenhöhl K. Stoehr N. Flor P.J. Heinrich M. Vranesic I. Biollaz M. Allgeier H. Heckendorn R. Urwyler S. Varney M.A. Johnson E.C. Hess S.D. Rao S.P. Sacaan A.I. Santori E.M. Veliçelebi G. Kuhn R. Neuropharmacology. 1999; 38: 1493-1503Crossref PubMed Scopus (700) Google Scholar). MPEP is a novel derivative of SIB-1893 with nanomolar potency (Fig. 1); it is an effective antihyperalgesic in animal models of chronic inflammatory pain (16Walker, K., Bowes, M., Panesar, M., Davis, A., Gentry, C., Kesingland, A., Gasparini, F., Spooren, W., Stoehr, N., Pagano, A., Flor, P. J., Vranesic, I., Lingenhoehl, K., Johnson, E. C., Varney, M., Urban, L., and Kuhn, R. (2000) Neuropharmacology, in press.Google Scholar), a neuroprotectant in excitotoxin-induced striatal lesions (17Bruno V. Ksiazek I. Battaglia G. Lukic S. Leonhardt T. Sauer D. Gasparini F. Kuhn R. Nicoletti F. Flor P.J. Neuropharmacology. 2000; 39: 2223-2230Crossref PubMed Scopus (116) Google Scholar) and an anticonvulsant in several epilepsy models (18Chapman A.G. Nanan K. Williams M. Meldrum B.S. Neuropharmacology. 2000; 39: 1567-1574Crossref PubMed Scopus (140) Google Scholar). We generated a number of chimeric receptors and point mutations in which segments or single residues of hmGluR5 were exchanged with the corresponding residues of hmGluR1 andvice versa. These mutants were tested for inhibition of glutamate-induced calcium signals by MPEP and for binding affinity of [3H]M-MPEP (Fig. 1), a closely related radioligand with a KD of 3.5 nm. We found that the key amino acids for selective and high affinity binding of [3H]M-MPEP to hmGluR5 are Pro-655 and Ser-658 in TMIII and Ala-810 in TMVII. The reciprocal mutations in the hmGluR1 background were sufficient to impart high affinity [3H]M-MPEP binding otherwise absent in the wild-type mGluR1. Furthermore, [3H]M-MPEP binding to these mutants was inhibited by CPCCOEt, suggesting that the subtype-selective antagonists MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs. These experimental findings are plausibly explained by a three-dimensional model of the mGluR5-MPEP complex that we constructed departing from the bovine rhodopsin structure.MATERIALS AND METHODSCompoundsMPEP and CPCCOEt were synthesized as described previously (15Gasparini F. Lingenhöhl K. Stoehr N. Flor P.J. Heinrich M. Vranesic I. Biollaz M. Allgeier H. Heckendorn R. Urwyler S. Varney M.A. Johnson E.C. Hess S.D. Rao S.P. Sacaan A.I. Santori E.M. Veliçelebi G. Kuhn R. Neuropharmacology. 1999; 38: 1493-1503Crossref PubMed Scopus (700) Google Scholar,10Annoura H. Fukunaga A. Uesugi M. Tatsuoka T. Horikawa Y. Bioorg. Med. Chem. Lett. 1996; 6: 763-766Crossref Scopus (166) Google Scholar). [3H]M-MPEP was synthesized using 2-methyl-6-(3-hydroxy-phenylethynyl)pyridine as starting material by methylation with tritiated methyliodide. Further details of the synthesis and characteristics of the radioligand will be published elsewhere. 2I. Vranesic, personal communication. Glutamate was obtained from Tocris (Bristol, United Kingdom). Other chemicals were purchased from Sigma (Buchs, Switzerland).PlasmidsCDNAs encoding wild-type hmGluR1b and hmGluR5a and the chimeric hmGluR1/5a and hmGluR5/1b receptors termed p254, p255, p317, p257, p305, p306, p310, and p322 were described previously (13Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.-P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461PubMed Google Scholar). Additional chimeras were constructed in pCMV-T7–3 (19Daggett L.P. Sacaan A.I. Akong M. Rao S.P. Hess S.D. Liaw C. Urrutia A. Jachec C. Ellis S.B. Dreessen J. Knoepfel T. Landwehrmeyer G.B. Testa C.M. Young A.B. Varney M. Johnson E.C. Velicelebi G. Neuropharmacology. 1995; 34: 871-886Crossref PubMed Scopus (93) Google Scholar) using standard cloning techniques based on unique restriction sites in hmGluR1b and -5a, novel restriction sites introduced by site-directed mutagenesis or the polymerase chain reaction-based overlap extension technique. The authenticity of the chimeric cDNAs (TableI) was confirmed by sequencing of all amplified DNA fragments. Point mutations in TMIII and TMVII of hmGluR1b and hmGluR5a cDNAs were generated using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The authenticity of each point mutation was confirmed by DNA sequencing.Table IChimeric receptor constructsPlasmidsChimeric mGlu receptor constructsp2541–616R5/630–906R1bp2551–679R5/693–906R1bp2571–838R5/852–906R1bp3051–629R1/617–1180R5ap3061–692R1/679–1180R5ap3101–629R1/617–838R5/852–906R1bp3121–692R1/679–838R5/852–906R1bp3161–660R1/648–838R5/852–906R1bp3171–785R5/798–906R1bp3221–785R5/798–851R1/839–1180R5ap3231–629R1/616–679R5/743–798R1/785–838R5/852–906R1bp3241–679R5/693–798R1/786–1180R5p3611–648R5/662–671R1/659–1180R5aNumbering indicates amino acid fragments of wild-type hmGluR1b (R1b) and hmGluR5a (R5a) receptors used to generate chimeric receptors Open table in a new tab The cDNA encoding Gαq (q4WT-pcDNA-I) was described previously by Conklin et al. (20Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (601) Google Scholar), rat mGluR5a (pRKG5a) by Joly et al. (21Joly C. Gomeza J. Brabet I. Curry K. Bockaert J. Pin J.-P. J. Neurosci. 1995; 15: 3970-3981Crossref PubMed Google Scholar), and EAAC1 by Brabet et al. (22Brabet I. Parmentier M.-L. De Colle C. Bockaert J. Acher F. Pin J.-P. Neuropharmacology. 1998; 37: 1043-1051Crossref PubMed Scopus (136) Google Scholar), respectively.Cell Culture and TransfectionsChinese hamster ovary and L cell lines stably expressing human mGluR1b and -5a were grown in Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal calf serum as described previously (13Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.-P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461PubMed Google Scholar). For radioligand binding experiments, wild-type and mutant cDNAs were transfected into COS1 cells using the DEAE-dextran method (23Al-Moslih M.I. Dubes G.R. J. Gen. Virol. 1973; 73: 189-193Crossref Scopus (30) Google Scholar). For measurements of intracellular [Ca2+]i, mutant cDNAs were transiently expressed in HEK293 cells by electroporation using a Gene Pulser apparatus (Bio-Rad). Briefly, 5 μg of plasmid DNA were used to transfect 1.5 × 106 cells in a total volume of 150 μl of electroporation buffer (K2HPO4, 50 mm; CH3COOK, 20 mm; KOH, 20 mm, pH 7.4). After electroporation (250 V, 300 microfarads), cells were resuspended in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum and antibiotics. 5 × 105 cells were plated on glass coverslips (9 × 18 mm) coated with 100 mg/ml poly-d-lysine (Sigma).IP and [Ca2+]i MeasurementsCells were seeded in 24-well tissue culture plates and were labeled to equilibrium with myo-[3H]inositol at 2 × 10−6 Ci/ml in Dulbecco's modified Eagle's medium for 20 h. Preincubation with LiCl, stimulation with agonist and/or antagonist, and extraction of total inositol phosphates (IP) were performed as described previously (13Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.-P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461PubMed Google Scholar). For the experiments addressing constitutive receptor activity, the cells were incubated in the presence of a glutamate-degrading enzyme (1 unit/ml glutamate pyruvate transaminase plus 2 mmpyruvate), 1 h before and during the incubation period with LiCl, except when cells were stimulated by added glutamate. Concentration-response curves were obtained by fitting the four-parametric logistic equation to the data using Prism2.0 (GraphPad Software, San Diego, CA).Microfluorimetric measurements of [Ca2+]i were performed as described previously (13Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.-P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461PubMed Google Scholar).Ligand BindingMembranes from transfected COS1 cells were collected 2 days after transfection. Cells were washed with phosphate-buffered saline and mechanically detached in ice-cold phosphate-buffered saline containing 10 mm EDTA. Cells were centrifuged at 4000 rpm for 20 min at 4 °C and resuspended in binding buffer (30 mm NaHepes, 110 mm NaCl, 1.2 mmMgCl2, 5 mm KCl, 2.5 mmCaCl22H2O, pH 8.0). Cells were then disrupted on ice with a Polytron homogenizer for 20 s, and membranes were collected by centrifugation at 18,000 rpm for 20 min at 4 °C. The pellet was resuspended in binding buffer, homogenized with a Teflon homogenizer, and used immediately for binding. Ligand binding assays were performed using [3H]M-MPEP and cold M-MPEP (10−6m) to determine nonspecific binding.2 Briefly, samples consisted of 200 μl of membrane suspension (50 μg), 25 μl of radioligand (2–30 nm), and 25 μl of binding buffer. The reaction was terminated after a 30-min incubation at 25 °C by dilution and rapid filtration through Whatman GF/B glass fiber filters. The filters were washed three times with cold binding buffer, and the bound radioactivity was counted using a β-counter in 5 ml of Ultima Gold NV Packard (Canberra Packard, Zurich, Switzerland). Specific [3H]M-MPEP binding was defined as total binding minus nonspecific binding in the presence of 1 × 10−6m cold M-MPEP. In one experiment all measurements were performed in duplicate. Saturation analysis and competition curves were analyzed using Prism2.0 (GraphPad Software).Molecular ModelingConstruction of a hmGluR5 Transmembrane Domain ModelTo suggest a plausible binding mode of MPEP to hmGluR5, a seven-transmembrane model was built and optimized using the programs SYBYL (SYBYL 6.4 software; Tripos Inc., St. Louis, MO) and X-PLOR (X-PLOR 3.1 software; Molecular Simulations, Inc., San Diego, CA) based on the α-carbon template of the transmembrane helices of the rhodopsin receptor family (24Moro S. Guo D. Camaioni E. Boyer J.L. Harden T.K. Jacobson K.A. J. Med. Chem. 1998; 41: 1456-1466Crossref PubMed Scopus (151) Google Scholar, 25Baldwin J.M. Schertler G.F.X. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (632) Google Scholar). In brief, seven individual polyalanine standard α helices of lengths 27, 27, 35, 25, 30, 30, and 24 were built, and each helix was superimposed on the corresponding helix of the α-carbon template derived from the rhodopsin family of GPCRs (25Baldwin J.M. Schertler G.F.X. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (632) Google Scholar). The root-mean-square distances of these superimposed C-α atoms of TM helices I–VII were 0.14, 0.79, 0.19, 0.13, 0.84, 1.35, and 0.12, respectively.All 198 alanines were mutated according to the putative assignment of transmembrane segments given in Table IIby making the corresponding side chain changes of alanines. All prolines were fixed using the SYBYL Biopolymer FIX_PROLINE command, hydrogen atoms were added with the ADDH command, and Gasteiger partial charges were computed with the CHARGE GAST_HUC command. Owing to the presence of charged residues (arginines (4Takahashi K. Tsuchida K. Tanabe Y. Masu M. Nakanishi S. J. Biol. Chem. 1993; 268: 19341-19345Abstract Full Text PDF PubMed Google Scholar), lysines (11Casabona G. Knoepfel T. Kuhn R. Gasparini F. Baumann P. Sortino M.A. Copani A. Nicoletti F. Eur. J. Neurosci. 1997; 9: 12-17Crossref PubMed Scopus (127) Google Scholar), aspartic acid (2Conn P.J. Pin J.-P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2708) Google Scholar), and glutamic acids (2Conn P.J. Pin J.-P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2708) Google Scholar)), the net charge was +11e. Groups forming termini of helices where kept neutral as NH2 and COOH.Table IIAssignment of α-helical transmembrane segments of the hmGluR5 receptor based on the rhodopsin receptor familyTMSNAmino acid sequenceENBaldwin numberingI582(PEPI)AAVAFACLGLLATLFVTVVFIIYRDTP608101–127II617LCYIILAGICLGYLCTFCLIAKPKQIY643200–226III649IGIGLSPAMSYSALVTKTNRIARILAGSKKKICTK683298–332IV695VIAFILICIQLGIIVALFIMEPPDI719401–425V738LGVVTPLGYNGLLILSCTFYAFKTRNVPAN767503–532VI770EAKYIAFTMYTTCIIWLAFVPIYFGSNYKI〈IT〉799596–625VII802MCFSVSLSATVALGCMFVPKMYII(LAK)825703–726SN, starting amino acid; EN, ending amino acid. Residues 〈IT〉 have been used for ELIII-loop building. Residues (PEPI) and (LAK) have not been used in the receptor model. Open table in a new tab This raw model was optimized with X-PLOR using the conjugate gradient method and the Tripos force field (X-PLOR/TAFF). Harmonic restraints with a force constant of 1.0 kcal/mol Å were applied to the initial coordinates of all 198 C-α and 188 C-β atoms. Minimization was carried out until the gradient grad(E) and the energyE were 0.4 kcal/mol A and −1814 kcal/mol Å, respectively.The extracellular domain between TMVI and TMVII is, according to our sequence assignment to helical transmembrane segments, formed by a short sequence of about four residues, which may be part of the binding pocket. A plausible third extracellular loop between helix VI and VII was searched with SYBYL/BIOPOLYMER/LOOP assigning residues Asn-796, Tyr-797, and Met-802 as anchor regions and the four residues Lys-798 to Thr-801 as window region. A loop with a sequence homology of 26% and a root-mean-square fit of 0.22 of backbone atoms in the anchor region was selected for the automatic construction of the loop. After calculation of all partial charges the model was again minimized (X-PLOR/TAFF, grad(E) = 0.4 kcal/mol Å, E = −1870 kcal/mol) using the same constraints of the previous minimization.Docking of MPEPTo this initial mGluR5 model we have manually docked MPEP with the pyridine N-atom accepting a H-bond from the hydroxyl group of Ser-658 of helix III and the 2-methyl in hydrophobic contact with the pyrrolidine ring of Pro-655 also in helix III. This model was optimized in 48 independent runs using an X-PLOR molecular dynamics protocol involving heating from 25 to 500 K in 2 ps and cooling again to 25 K in 2 ps, followed by 2000 steps of TAFF/X-PLOR minimization while keeping harmonic restraints to the previous reference coordinates of C-α and C-β atoms.Docking of CPCCOEt to the Mutant hmGluR5-M802T,S805AThe initial receptor model of hmGluR5-M802T,S805A was obtained by changing in the initial wild-type hmGluR5 model the side chains of Met-802 and Ser-805 into respective threonine and alanine side chains, recalculation of partial charges, and X-PLOR/TAFF minimization. To this model CPCCOEt was manually docked such that its oxime group formed a contact with the hydroxyl group of Thr-802 and its benzene moiety was pointing down into the transmembrane region between TMIII and TMVII. Optimization of this complex was performed as described above for the complex of MPEP with wild-type hmGluR5.DISCUSSIONThe major findings of this study concern the involvement of the hmGluR5 7TM domain in the high affinity binding of MPEP, which mediates non-competitive inhibition. This suggests that MPEP inhibits receptor activity without changing the affinity of glutamate to its binding site, and thus MPEP might act at a receptor site different from the glutamate binding domain. In agreement with this hypothesis is the inability of MPEP to displace [ 3V. Mutel, personal communication.H]quisqualate binding to rat mGluR5a3 and the lack of effect of mGluR5 agonists on [3H]M-MPEP binding.2In addition to its non-competitive antagonist action, MPEP also decreased basal IP production in the absence of an agonist in cells transiently overexpressing rat mGluR5a indicating an inverse agonist activity. Indeed, increased basal receptor activity was still detected in the presence of the glutamate transporter EAAC1 and the glutamate degrading enzyme glutamate pyruvate transaminase, conditions expected to drastically decrease the extracellular glutamate concentration to levels below that required for activation of mGluR5. As previously reported for rat mGluR1a (8Parmentier M.-L. Joly C. Restituito S. Bockaert J. Grau Y. Pin J.-P. Mol. Pharmacol. 1998; 53: 778-786Crossref PubMed Scopus (70) Google Scholar), co-expression of rat mGluR5a with the G protein Gαq further increased basal IP production. The competitive antagonist MCPG did not block any basal IP formation at a concentration of 3 mm but almost fully inhibited the action of glutamate. This shows that the basal activity does not result from the activation of the receptor by an endogenous agonist, and therefore likely results from the constitutive activity of the receptor. In contrast, when MPEP was applied alone on cells expressing mGluR5a or mGluR5a plus Gαq, a significant but not complete inhibition of the basal activity was detected. This inverse agonism of MPEP, so far not described for other antagonists of family 3 (mGluR-like) GPCRs, was dose-dependent and in agreement with the potency of MPEP in inhibiting the effect of glutamate.Studies of a large number of antagonists of family 1 (rhodopsin-like) GPCRs led to the hypothesis that these receptors oscillate between (at least) two conformational states, an inactive and an active one. Agonists stabilize the receptor in the active state. Antagonists, divided in two categories, are either neutral antagonists, which have the same affinity to both the inactive and active conformational state, or inverse agonists, which stabilize the inactive state and therefore inhibit the constitutive activity. In contrast to family 1 GPCRs, mGluRs and other family 3 GPCRs consist of two distinct domains, the large extracellular agonist binding domain and the 7TM region, which directly interacts with the G proteins. As none of the known competitive mGluR1 antagonists interacting at the glutamate binding site have been shown to inhibit constitutive activity of mGluRs (27Prezeau L. Gomeza J. Ahern S. Mary S. Galvez T. Bockaert J. Pin J.P. Mol. Pharmacol. 1996; 49: 422-429PubMed Google Scholar), we speculate that the constitutive activity of mGluRs might result from an equilibrium between two conformational states of the 7TM region rather than from an equilibrium between an active and inactive state of the agonist binding domain. According to this hypothesis, one is expecting that non-competitive antagonists acting on the 7TM region of mGluRs are more likely to act as inverse agonist than competitive antagonists acting at the extracellular glutamate binding site.A detailed molecular investigation using chimeric receptors and point mutants of hmGluR5 and hmGluR1 revealed that MPEP binds to and interacts with Ala-810 in TMVII and Ile-651, Pro-655, and Ser-658 in TMIII, respectively. Replacement of Ala-810 with valine, the homologous residue of hmGluR1, completely abolished [3H]M-MPEP binding and MPEP inhibition of glutamate-induced [Ca2+]i responses. Single replacement of Pro-655 and Ser-658 with the corresponding residues of hmGluR1 reduced the binding affinity of [3H]M-MPEP from 3.5 nm to 27.1 and 16.5 nm, respectively. However, a combination of both mutations lacked any significant binding affinity for [3H]M-MPEP, indicating the importance of both residues for radioligand binding. Likewise, introduction of Pro-655, Ser-658, and Ala-810 from hmGluR5 at the corresponding position of hmGluR1 generated a hmGluR1 mutant with a high affinity binding site for [3H]M-MPEP (KD = 20 nm), indicating that these residues are critical molecular determinants for [3H]M-MPEP binding. A further 2-fold increase in binding affinity was achieved by introduction of Ile-651, mutation of which showed no change in binding affinity in wild type hmGluR5a. Because the combination of Pro-655, Ser-658, Ile-651, and Ala-810 is unique to mGluR5 and not found in the homologous position of other mGluR subtypes, it provides a molecular explanation for the high pharmacological selectivity of MPEP.It has not yet been determined whether the structurally unrelated non-competitive antagonists MPEP and CPCCOEt interact with different sites of mGluR subtypes or share a common binding site in the TM domain. We showed in a previous study that two residues unique to hmGluR1, Thr-815 and Ala-818 on the extracellular surface of TMVII, were responsible for the selective action of the non-competitive mGluR1 antagonist CPCCOEt (13Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.-P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461PubMed Google Scholar). The present study shows that binding of the mGluR5 antagonist MPEP requires an interaction with Ala-810 further down in the transmembrane helix VII as well as additional interactions with Pro-655 and Ser-658 in TMIII. Using hmGluR1 and -5 mutants (R1-V664I,S668P, C671S,V823A and R5-M802T,S805A), which show functional inhibition by MPEP and CPCCOEt, we unequivocally demonstrated complete inhibition of [3H]M-MPEP binding by CPCCOEt in a concentration dependent manner. This is further supported by docking studies of MPEP and CPCCOEt to 7TM domain models. These models suggest that the pyridine ring of MPEP precisely occupies the same space between TMVII and TMIII as the benzene ring of CPCCOEt, providing a molecular explanation for the displacement of MPEP by CPCCOEt. However, other parts of these antagonists do not overlap and suggest interactions with different TM helices. Thus, although MPEP and CPCCOEt are structurally unrelated, they recognize overlapping binding pockets in the 7TM region of group I mGluRs that are sufficiently diverse to allow subtype-specific interaction with different classes of compounds.These findings may have important implications for the design of novel mGluR antagonists. Up to now, most compounds acting at the eight subtypes of mGluRs are phenylglycines or rigidified amino acid analogs such as LY354740 (9Schoepp D.D. Jane D.E. Monn J.A. Neuropharmacology. 1999; 38: 1431-1476Crossref PubMed Scopus (1006) Google Scholar). These compounds possess a wide spectrum of agonist, partial agonist, and antagonist a

Previous studies have demonstrated that the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) is expressed in the cell bodies of rat primary afferent neurones. We have further investigated the function and expression of mGlu5 receptors in primary afferent neurones, and their role in inflammatory nociception. Freund's complete adjuvant-induced inflammatory hyperalgesia of the rat hind paw was significantly reduced by intraplantar, but not by intracerebroventricular or intrathecal microinjection of the selective mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Pharmacological comparison in vivo of the nociceptive effects of glutamate, and ionotropic and metabotropic glutamate (mGlu) receptor agonists applied to the rat hind paw, indicated that group I mGlu receptor agonists induce a dose-dependent decrease in paw withdrawal threshold (mechanical hyperalgesia). Group I mGlu agonist-induced hyperalgesia was inhibited by co-microinjection of MPEP, but not by the mGlu1 receptor antagonist (S)-4-carboxy-phenylglycine (4-CPG). Carrageenan-induced inflammatory hyperalgesia was inhibited by pre-treatment of the inflamed hind paw with MPEP, but not following MPEP injection into the contralateral hind paw. Dorsal horn neurones receiving peripheral nociceptive and non-nociceptive afferent input were recorded in anaesthetized rats following microinjection of CHPG into their peripheral receptive fields. CHPG significantly increased the frequency and duration of firing of dorsal horn wide dynamic range (WDR) neurones and this activity was prevented by co-administration of CHPG and MPEP into their receptive fields. Immunohistochemical experiments revealed the co-expression of mGlu5 receptor protein and betaIII tubulin in skin from naive rats, indicating the constitutive expression of mGlu5 receptors on peripheral neurones. Double-labelling of adult rat DRG cells with mGlu5 receptor and vanilloid receptor subtype 1 antisera also supports the expression of mGlu5 receptors on peripheral nociceptive afferents. These results suggest that mGlu5 receptors expressed on the peripheral terminals of sensory neurones are involved in nociceptive processes and contribute to the hyperalgesia associated with inflammation.

Although multiple metabotropic glutamate (mglu) receptor subtypes were cloned in the early 1990s, progress in the characterization of these receptors has been slow because of difficulties in obtaining subtype-selective ligands. However, in the past few years exciting progress has been made on the mglu5 receptor subtype following the identification of selective non-amino-acid-like ligands that implicate the mglu5 receptor as a potentially important therapeutic target, particularly for the treatment of pain and anxiety.

To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7 −/− ). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7 −/− , but not in mGluR7 +/− , mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist ( R,S )-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7 −/− mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs.

Metabotropic glutamate receptors (mGluRs) have recently been considered as potential pharmacological targets in the treatment of neurodegenerative disorders and particularly in parkinsonism. Within the basal ganglia, receptors of group I (mGluR1 and mGluR5) are widely expressed; the present study was thus aimed at blocking these receptors in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease in the rat. Considering the prominent expression of mGluR5, we have used the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) to target these receptors. In rats trained to quickly depress a lever after a visual cue, bilateral lesions of the dopaminergic nerve terminals in the striatum produced severe akinetic deficits, which were expressed by increases in delayed responses and reaction times. Acute MPEP injection (1.5, 3, and 6 mg/kg, i.p.) had no effect, whereas chronic administration, ineffective in a control group, significantly reversed the akinetic deficits. Alleviation of these deficits was seen after 1 week of treatment, and the preoperative performance was fully recovered after a 3 week treatment of MPEP at all doses. Chronic MPEP also induced ipsilateral rotation in the unilateral 6-OHDA circling model. However, no effect was seen of MPEP (1.5, 3, or 6 mg/kg, i.p.) on haloperidol-induced catalepsy (1 mg/kg, i.p.). Altogether, these results suggest a specific role of mGluRs in the regulation of extrapyramidal motor functions and a potential therapeutic value for mGluR5 antagonists in the treatment of Parkinson's disease.

Following the molecular cloning in the early 1990s of the metabotropic glutamate receptors (mGlu1-8), research that focused on the physiology, pharmacology and function of these receptors revealed their potential role in CNS disorders. Numerous psychiatric and neurological disorders are indeed linked to changes in excitatory processes, in which glutamate plays a key role. In contrast to ligand-gated ion channels [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) and kainate], which are responsible for fast excitatory transmission, mGlu receptors have a more modulatory role, by contributing to fine-tuning of synaptic efficacy, and control of the accuracy and sharpness of the transmission. Given the fact that the mGlu receptors are Gprotein coupled, they obviously constitute new 'drugable' targets for the treatment of various CNS disorders. Due to the recent emergence of subtype-specific ligands for Group I and II mGlu receptors, this review will concentrate on the molecular characteristics, brain localization, pharmacology and physiological role of these receptors, in order to provide further insights into their therapeutic potential.Behavioural Pharmacology14:257-277

Abstract Study objectives were to assess the efficacy, safety, and tolerability of AFQ056 in Parkinson's disease patients with levodopa‐induced dyskinesia. Two randomized, double‐blind, placebo‐controlled, parallel‐group, in‐patient studies for Parkinson's disease patients with moderate to severe levodopa‐induced dyskinesia (study 1) and severe levodopa‐induced dyskinesia (study 2) on stable dopaminergic therapy were performed. Patients received 25–150 mg AFQ056 or placebo twice daily for 16 days (both studies). Study 2 included a 4‐day down‐titration. Primary outcomes were the Lang‐Fahn Activities of Daily Living Dyskinesia Scale (study 1), the modified Abnormal Involuntary Movement Scale (study 2), and the Unified Parkinson's Disease Rating Scale–part III (both studies). Secondary outcomes included the Unified Parkinson's Disease Rating Scale–part IV items 32–33. The primary analysis was change from baseline to day 16 on all outcomes. Treatment differences were assessed. Fifteen patients were randomized to AFQ056 and 16 to placebo in study 1; 14 patients were randomized to each group in study 2. AFQ056‐treated patients showed significant improvements in dyskinesias on day 16 versus placebo (eg, Lang‐Fahn Activities of Daily Living Dyskinesia Scale, P = .021 [study 1]; modified Abnormal Involuntary Movement Scale, P = .032 [study 2]). No significant changes were seen from baseline on day 16 on the Unified Parkinson's Disease Rating Scale‐part III in either study. Adverse events were reported in both studies, including dizziness. Serious adverse events (most commonly worsening of dyskinesias, apparently associated with stopping treatment) were reported by 4 AFQ056‐treated patients in study 1, and 3 patients (2 AFQ056‐treated patient and 1 in the placebo group) in study 2. AFQ056 showed a clinically relevant and significant antidyskinetic effect without changing the antiparkinsonian effects of dopaminergic therapy. © 2011 Movement Disorder Society

Zinc iodide catalyzed cycloaddition of furan to 1-cyanovinyl (1′S)-camphanate or 1-cyanovinyl (1′R)-camphanate led to optically pure (1R,2S,4R)-2-exo-cyano-7-oxabicyclo[2.2.1]hept-5-en-2-yl (1′S)-camphanate and (1S,2R,4S)-2-exo-cyano-7-oxabicyclo[2.2.1]hept-5-en-2-yl (1′R)-camphanate, respectively. Saponification afforded (1R,4R)- and (1S,4S)-7-oxabicyclo[2.2.1]hept-5-en-2-one, respectively, allowing concurrent recovery of the chiral auxiliaries, (1S)- and (1R)-camphanic acid, respectively. The 7-oxabicyclo[2.2.1]hept-5-en-2-yl derivatives ("naked sugars") so obtained can be substituted at C(3), C(5) and C(6) by direct techniques with high stereoselectivity. The methods exploit the exo-facial selectivity of the reactions of these bicyclic systems and the regiochemical control exerted by the remote substituents at C(2) in electrophilic attack at C(5) and C(6). The polysubstituted 7-oxabicyclo[2.2.1]heptan-2-ones so obtained are chirons that can be converted into D- or L-hexose derivatives and analogues, into 2,5-anhydrohexonic acid derivatives (precursors for C-nucleosides and C-glycosides), or into polysubstituted 2-cyclohexen-1-ones. Moreover, conditions have been found under which the bicyclic ethers can be rearranged stereoselectively into polysubstituted cyclopentane-carbaldehydes. Compared with natural carbohydrates, the "naked sugars" are chirons with the following advantages: (1) the problem of selective protection and deprotection of polyalcoholic systems does not exist; (2) the functional moieties are grafted sequentially onto C(3), C(5), and C(6) of the 7-oxabicyclo[2.2.1]heptane systems together with the appropriate protective groups; (3) since both enantiomeric forms of the "naked sugars" are available, either enantiomer of a target compound can be reached with equal ease. 1. Introduction 2. The Carbonyl Group as Homoconjugated Electron-Releasing Substituent 3. Total Synthesis of 2,5-Anhydro-3-deoxy- and -4-deoxy-D-hexonic Acids and Related Deoxyadenosines-C 4. Total Syntheses of ribo-C-Nuleosides 5. De Novo Syntheses of D- and L-Hexoses and Analogues 6. Acid-Catalyzed Rearrangements of 5-exo-6-exo-Epoxy-7-oxa-2-norbornyl Derivatives 7. Stereoselective trans-Amino-hydroxylation of 7-Oxa-5-norbornen-2-one 8. The 7-Oxa-2-norbornanone/2-Cyclohexen-1-one Rearrangement: Total Syntheses of Conduritols 9. Conclusion and Perspectives

The s dependence of the electromagnetic proton form factors in the time-like region has been determined from the threshold (s = 4Mp2) up to s = 4.2 GeV2. Data were collected in a dedicated experiment performed at the LEAR antiproton ring at CERN, increasing by one order of magnitude the available statistics. Total and differential cross section of the pp → e−e+ reaction have been measured. The electric and magnetic form factors are found to have comparable value. The observed form factor shows a clear steep s dependence close to the threshold.

Fragile X syndrome, the most common form of inherited intellectual disability, is caused by a lack of FMRP, which is the product of the Fmr1 gene. FMRP is an RNA-binding protein and a component of RNA-granules found in the dendrites of neurons. At the synapse, FMRP is involved in regulation of translation of specific target mRNAs upon stimulation of mGluR5 receptors. In this study, we test the effects of a new mGluR5 antagonist (AFQ056) on the prepulse inhibition of startle response in mice. We show that Fmr1 KO mice have a deficit in inhibition of the startle response after a prepulse and that AFQ056 can rescue this phenotype. We also studied the effect of AFQ056 on cultured Fmr1 KO hippocampal neurons; untreated neurons showed elongated spines and treatment resulted in shortened spines. These results suggest that AFQ056 might be a potent mGluR5 antagonist to rescue various aspects of the fragile X phenotype.

Advances in understanding the underlying mechanisms of conditions such as fragile X syndrome (FXS) and autism spectrum disorders have revealed heterogeneous populations. Recent trials of novel FXS therapies have highlighted several challenges including subpopulations with possibly differential therapeutic responses, the lack of specific outcome measures capturing the full range of improvements of patients with FXS, and a lack of biomarkers that can track whether a specific mechanism is responsive to a new drug and whether the response correlates with clinical improvement.We review the phenotypic heterogeneity of FXS and the implications for clinical research in FXS and other neurodevelopmental disorders.Residual levels of fragile X mental retardation protein (FMRP) expression explain in part the heterogeneity in the FXS phenotype; studies indicate a correlation with both cognitive and behavioral deficits. However, this does not fully explain the extent of phenotypic variance observed or the variability of drug response. Post hoc analyses of studies involving the selective mGluR5 antagonist mavoglurant and the GABAB agonist arbaclofen have uncovered significant therapeutic responses following patient stratification according to FMR1 promoter methylation patterns or baseline severity of social withdrawal, respectively. Future studies designed to quantify disease modification will need to develop new strategies to track changes effectively over time and in multiple symptom domains.Appropriate selection of patients and outcome measures is central to optimizing future clinical investigations of these complex disorders.

Overactivity of glutamatergic transmission has been implicated in Parkinson’s disease (PD) and levodopa (l-Dopa)-induced dyskinesias. Striatal metabotropic glutamate receptors type 5 (mGluR5) are abundant and provide specific targets to modulate glutamatergic activity. This study investigated the acute effects of the novel mGluR5 antagonist AFQ056 on motor behavior in l-Dopa-treated monkeys with a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion to model PD. Six Macaca fascicularis MPTP monkeys were treated repeatedly with l-Dopa; this treatment increased their locomotion and reduced their parkinsonian scores, but also induced dyskinesias. When AFQ056 (doses of 5, 25, 125 or 250 mg/kg) was administered one hour prior to a high dose of l-Dopa, the antiparkinsonian activity of l-Dopa was maintained as measured with locomotion and antiparkinsonian scores, whereas dyskinesias were significantly reduced at 25, 125 and 250 mg/kg AFQ056 for peak dyskinesia score and at 125 and 250 mg/kg for the 1 h peak period of dyskinesia score. Administration of AFQ056 one hour before l-Dopa led to peak or elevated plasma AFQ056 concentrations occurring close to l-Dopa peak-dose dyskinesias. We next investigated AFQ056 25 mg/kg combined with a low dose of l-Dopa. The antiparkinsonian activity of l-Dopa was increased as measured with locomotion, while dyskinesias remained low at these doses. Our results show a beneficial motor effect of AFQ056 with l-Dopa in MPTP monkeys. This supports the therapeutic use of an mGluR5 antagonist to restore normal glutamatergic neurotransmission in PD and decrease dyskinesias.

Background Modulation of metabotropic glutamate receptors may be a novel therapeutic approach to manage l-Dopa-induced dyskinesias in patients with Parkinson's disease. This article reviews the rationale for use of metabotropic glutamate 5-receptor antagonists in experimental and clinical l-Dopa-induced dyskinesias. Methods Systematic literature searches were performed (between May 2012–March 2014) for relevant English language articles using PubMed. Additional articles of interest were identified from reference lists of included publications. Relevant clinical abstracts from Movement Disorder Society meetings were included. Results 16 preclinical studies of metabotropic glutamate 5-receptor antagonists in animal models of l-Dopa-induced dyskinesias and 7 clinical studies in patients with Parkinson's disease and l-Dopa-induced dyskinesias were included. Anti-dyskinetic effects of metabotropic glutamate 5-receptor blockade (MPEP, MTEP, fenobam, or MRZ-8676) were reported in dyskinetic 6-hydroxydopamine-lesioned rats. Studies in MPTP-lesioned non-human primates reported anti-dyskinetic effects of MPEP, MTEP, fenobam and mavoglurant (AFQ056). Three randomized, double-blind clinical trials reported anti-dyskinetic efficacy of mavoglurant, without effects on anti-parkinsonian therapy, with dizziness the most common adverse event. However, two further studies failed to demonstrate significant anti-dyskinetic efficacy. A randomized, double-blind, placebo-controlled safety study of dipraglurant (ADX48621) demonstrated tolerability and positive exploratory secondary outcomes of reduced dyskinesia. Conclusions Animal model studies provide evidence for anti-dyskinetic efficacy of metabotropic glutamate 5-receptor antagonists. Initial proof-of-concept clinical trials of mavoglurant and dipraglurant showed positive results; anti-dyskinetic efficacy was not supported by two recent mavoglurant trials. Further evaluations of optimal dosage and long-term efficacy and safety of metabotropic glutamate 5-receptor antagonists for management of l-Dopa-induced dyskinesias in Parkinson's disease are required.

TREM2 is a transmembrane protein expressed exclusively in microglia in the brain that regulates inflammatory responses to pathological conditions. Proteolytic cleavage of membrane TREM2 affects microglial function and is associated with Alzheimer's disease, but the consequence of reduced TREM2 proteolytic cleavage has not been determined. Here, we generate a transgenic mouse model of reduced Trem2 shedding (Trem2-Ile-Pro-Asp [IPD]) through amino-acid substitution of an ADAM-protease recognition site. We show that Trem2-IPD mice display increased Trem2 cell-surface-receptor load, survival, and function in myeloid cells. Using single-cell transcriptomic profiling of mouse cortex, we show that sustained Trem2 stabilization induces a shift of fate in microglial maturation and accelerates microglial responses to Aβ pathology in a mouse model of Alzheimer's disease. Our data indicate that reduction of Trem2 proteolytic cleavage aggravates neuroinflammation during the course of Alzheimer's disease pathology, suggesting that TREM2 shedding is a critical regulator of microglial activity in pathological states.

Metabotropic glutamate receptors (mGluRs) are a family of G protein-coupled receptors characterized by a large, extracellular N-terminal domain comprising the glutamate-binding site. In the current study, we examined the pharmacological profile and site of action of the non-amino-acid antagonist 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt). CPCCOEt selectively inhibited glutamate-induced increases in intracellular calcium at human mGluR1b (hmGluR1b) with an apparent IC50 of 6.5 microM while having no agonist or antagonist activity at hmGluR2, -4a, -5a, -7b, and -8a up to 100 microM. Schild analysis indicated that CPCCOEt acts in a noncompetitive manner by decreasing the efficacy of glutamate-stimulated phosphoinositide hydrolysis without affecting the EC50 value or Hill coefficient of glutamate. Similarly, CPCCOEt did not displace [3H]glutamate binding to membranes prepared from mGluR1a-expressing cells. To elucidate the site of action, we systematically exchanged segments and single amino acids between hmGluR1b and the related subtype, hmGluR5a. Substitution of Thr815 and Ala818, located at the extracellular surface of transmembrane segment VII, with the homologous amino acids of hmGluR5a eliminated CPCCOEt inhibition of hmGluR1b. In contrast, introduction of Thr815 and Ala818 at the homologous positions of hmGluR5a conferred complete inhibition by CPCCOEt (IC50 = 6.6 microM), i.e., a gain of function. These data suggest that CPCCOEt represents a novel class of G protein-coupled receptor antagonists inhibiting receptor signaling without affecting ligand binding. We propose that the interaction of CPCCOEt with Thr815 and Ala818 of mGluR1 disrupts receptor activation by inhibiting an intramolecular interaction between the agonist-bound extracellular domain and the transmembrane domain.

The excitatory neurotransmitter, glutamate, is particularly important in the transmission of pain information in the nervous system through the activation of ionotropic and metabotropic glutamate receptors. A potent, subtype-selective antagonist of the metabotropic glutamate-5 (mGlu5) receptor, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), has now been discovered that has effective anti-hyperalgesic effects in models of inflammatory pain. MPEP did not affect rotarod locomotor performance, or normal responses to noxious mechanical or thermal stimulation in naı̈ve rats. However, in models of inflammatory pain, systemic administration of MPEP produced effective reversal of mechanical hyperalgesia without affecting inflammatory oedema. In contrast to the non-steroidal anti-inflammatory drugs, indomethacin and diclofenac, the maximal anti-hyperalgesic effects of orally administered MPEP were observed without acute erosion of the gastric mucosa. In contrast to its effects in models of inflammatory pain, MPEP did not produce significant reversal of mechanical hyperalgesia in a rat model of neuropathic pain.

Abstract We investigated the expression and coupling to the phospholipase C signal transduction pathway of metabotropic glutamate receptor (mGluR) subtypes by Western blot analysis and agonist‐stimulated inositol monophosphate formation in several brain regions of postnatal day 9 (P9) and adult rats. In the cerebral cortex, hippocampus, corpus striatum, olfactory bulb, cerebellum and hypothalamus, the expression level of mGluR5 was greater at P9 than in adulthood. The mGluR5 signal was very low or absent in the adult cerebellum and hypothalamus. The expression of mGluR1a was slightly greater at P9 in the hypothalamus, hippocampus and olfactory bulb, whereas it substantially increased with age in the cerebellum, and did not change in the cerebral cortex and corpus striatum. mGluR1b and ‐1c were nearly undetectable by Western blot analysis. The expression level of mGluR5, but not that of mGluR1a, was significantly correlated with the extent of phosphoinositide hydrolysis stimulated by mGluR agonists in slices prepared from these brain regions. The mGluR antagonist cyclopropan[b]chromen‐1a‐carboxylic acid ethylester (CPCCOEt), potently antagonized responses mediated by mGluR1, but much less potently those mediated by mGluR5a in recombinant cells. CPCCOEt, at a concentration which efficently blocks mGluR1 responses, did not substantially affect the polyphosphoinositide response in hippocampal or cerebellar slices from newborn animals, and antagonized only a minor component of the polyphosphoinositide response in adult hippocampal slices. CPCCOEt, however, prevented the small stimulation of polyphosphoinositide hydrolysis by mGluR agonists in adult cerebellar slices. We conclude that (i) the efficient mGluR‐mediated polyphosphoinositide hydrolysis in 9‐day‐old rats is mediated by mGluR5; (ii) the increased expression of mGluR1 in the adult cerebellum does not substitute for the decline of mGluR5 expression in the ability to mediate polyphosphoinositide hydrolysis; and therefore (iii) mGluRla might couple less efficiently than mGluR5 to polyphosphoinositide hydrolysis.

Glutamate receptors play an essential role in fear-related learning and memory. The present study was designed to assess the role of the group I metabotropic glutamate receptor (mGluR) subtype 5 in the acquisition and retrieval of conditioned fear in rats. The selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) was applied systemically (0.0, 0.3, 3.0, 30.0 mg/kg per os) 60 min before the acquisition training and before the expression of conditioned fear, respectively, in the fear-potentiated startle paradigm. MPEP dose-dependently blocked the acquisition of fear. This effect was not due to state-dependent learning. MPEP also prevented the expression of fear at a dose of 30.0 mg/kg. As a positive control for these effects, we showed that the benzodiazepine anxiolytic compound diazepam (1.25 mg/kg intraperitoneally) also blocked acquisition and expression of fear potentiated startle. MPEP did not affect the baseline startle magnitude, short-term habituation of startle, sensitisation of startle by footshocks or prepulse inhibition of startle. These data indicate a crucial role for mGluR5 in the regulation of fear conditioning. In the highest dose MPEP might exert anxiolytic properties.

Recent drug discovery programs aimed at identifying selective metabotropic mGlu receptor ligands by high-throughput functional screening efforts have revealed subtype-selective allosteric modulators of mGlu1 and mGlu5 receptors that are structurally unrelated to glutamate. In contrast to competitive ligands, which bind to the glutamate binding site located in the large N-terminal extracellular domain, these modulators act as non-competitive antagonists, inverse agonists or positive modulators by binding to specific residues in the seven-transmembrane domain. More recent studies to assess the potential of these compounds in in vivo models of nervous system disorders have implicated the mGlu5 receptor subtype as a potentially important therapeutic target for inflammatory pain, anxiety, Parkinson's disease and drug abuse, and mGlu1 and mGlu5 receptors as potential targets for anticonvulsant and neuroprotective therapies. Very recent findings indicate an important regulatory role for intracellular proteins interacting with metabotropic glutamate receptors, which might constitute novel drug targets for modulating metabotropic glutamate receptor activity.

This study investigated the role of ionotropic and metabotropic glutamate receptors in the deficits in brain reward function, as measured by elevations in intracranial self-stimulation (ICSS) reward thresholds, associated with nicotine withdrawal. The group II metabotropic glutamate (mGluII) receptor agonist LY314582 [a racemic mixture of LY354740 ([+]-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid])] (2.5-7.5 mg/kg) precipitated withdrawal-like elevations in ICSS thresholds, a sensitive measure of reward function, in nicotine-dependent but not control rats. LY314582 did not affect response latencies, a measure of performance in the ICSS paradigm. Bilateral microinfusion of LY314582 (10-100 ng/side) into the ventral tegmental area likewise precipitated dose-dependent threshold elevations in nicotine-dependent rats. Furthermore, a single injection of the mGluII receptor antagonist LY341495 (2S-2-amino-2-[1S,2S-2-carboxycyclopropan-1-yl]-3-[xanth-9-yl]propionic acid) (1 mg/kg) attenuated the threshold elevations observed in rats undergoing spontaneous nicotine withdrawal. mGluII receptors are primarily located on glutamatergic terminals throughout the mesocorticolimbic system, where they act as inhibitory autoreceptors. To investigate whether mGluII receptors contributed to nicotine withdrawal by decreasing glutamatergic transmission, we next examined whether direct blockade of postsynaptic glutamate receptors precipitated withdrawal-like reward deficits in nicotine-dependent rats. The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX; 0.01-1 mg/kg) precipitated withdrawal-like threshold elevations in nicotine-dependent but not control rats, whereas 6-methyl-2-[phenylethynyl]-pyridine (MPEP; 0.01-3 mg/kg) and dizocilpine (MK-801; 0.01-0.2 mg/kg), antagonists at metabotropic glutamate 5 and N-methyl-d-aspartate receptors, respectively, did not. Overall, these data demonstrate that mGluII receptors play an important role in the reward deficits associated with nicotine withdrawal. Furthermore, it is likely that mGluII receptors generate this reward deficit, at least in part, by decreasing glutamate transmission at AMPA/kainate receptors.

Phencyclidine (PCP), a non-competitive antagonist of ionotropic N-methyl-D-aspartate (NMDA) receptors, produces psychotomimetic effects, such as a disruption in prepulse inhibition (PPI) of the startle response. NMDA antagonists also induce locomotor hyperactivity in rodents. We hypothesized that, like NMDA receptors, metabotropic glutamate receptors (mGluRs) modulate PPI and locomotor activity either alone or, in the case of mGluR5, via interaction with NMDA receptors. Rats treated with the mGluR5 antagonist MPEP (2-methyl-6-phenylethynylpyridine) or the mGluR2/3 agonist LY314582, either alone or in combination with PCP, were tested in PPI and locomotor activity paradigms. Neither MPEP nor LY314582 altered PPI. MPEP, but not LY314582, potentiated the PPI-disruptive effects of PCP. MPEP alone did not alter locomotor or exploratory behavior, but augmented the complex, time-dependent locomotor-stimulating effects of PCP. LY314582 dose-dependently decreased locomotor activity and exploratory holepokes. LY314582 did not alter the PCP-induced increases in locomotor activity, but further decreased the number of holepokes. The effects of MPEP on the response to PCP may reflect the cooperation and co-localization of NMDA and mGlu5 receptors.

The stress-induced hyperthermia test is a paradigm developed several years ago to model the expression of autonomic hyperactivity in anxiety. Whereas in the classical stress-induced hyperthermia, cohort removal was used, in a recently described modification of the stress-induced hyperthermia model singly housed mice rather than groups of mice were used. The modification of this model can be summarized as follows: rectal temperature is recorded in singly housed animals at two consecutive time-points (T1 and T2) which are interspaced by a defined time-interval (15 min). Since the value at the second temperature-recording exceeds the value of the initial measure it is the difference between these two core-temperatures which reflects stress-induced hyperthermia. In the present study, the stress-induced hyperthermia paradigm, in its modified design, was evaluated in OF1/IC mice. By comparing the effect of various compounds in both the modified as well as the classical (cohort removal) stress-induced hyperthermia paradigm, a very high correlation was found for the pharmacological sensitivity of the two paradigms. Furthermore, it was demonstrated that other anxiolytics, all known to be active in the classical stress-induced hyperthermia paradigm, such as the benzodiazepines chlordiazepoxide (0.3, 1, 3, 10 mg/kg, p.o.), diazepam (0.1, 0.3, 1, 3 mg/kg, p.o.), clobazam (5 or 10 mg/kg, p.o.) and oxazepam (5 or 10 mg/kg, p.o.) as well as the non-benzodiazepines buspirone (7.5 or 15 mg/kg, p.o.) and ethanol (15% or 30%, 10 ml/kg, p.o.), showed a marked reduction in stress-induced hyperthermia in the modified design. New candidate anxiolytics, i.e. the metabotropic glutamate (mGlu) receptor group 2 agonist LY314582 (1 or 10 mg/kg, p.o.; racemic mixture of LY354740 ((2S,4S)-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-dioic acid), the metabotropic glutamate 5 receptor antagonist MPEP (1, 7.5, 15 or 30 mg/kg, p.o.; 2-methyl-6-(phenylethynyl)pyridine) and the neurokinin 1 (NK1) receptor antagonist NKP608 (0.01 or 0.1 mg/kg, p.o.; quinoline-4-carboxylic acid [trans-(2R,4S)-1-(3,5-bis-trifluoromethyl-benzoyl)-2-(4-chloro-benzyl)-piperidin-4-yl]-amide) also reduced stress-induced hyperthermia in the modified paradigm clearly indicating anxiolytic-like activity for these compounds. Finally, the effects of the classical benzodiazepine chlordiazepoxide (10 mg/kg, p.o.), in parallel with its effect on stress-induced hyperthermia, were also investigated for its effect on plasma concentrations of the two stress hormones, adrenocorticotropin (ACTH) and corticosterone. It was shown that all three parameters were significantly increased 15 min after T1 in vehicle-treated mice whereas the increase was significantly attenuated following pre-treatment with chlordiazepoxide. In conclusion, all the data presented here indicate that the modified version of the stress-induced hyperthermia-paradigm is a valid and interesting alternative to the classical stress-induced hyperthermia test.

Recently, selective and systemically active antagonists for the metabotropic glutamate 5 receptor (mGlu(5)) were discovered, and the most potent derivative was found to be MPEP (2-methyl-6-(phenylethynyl)pyridine). Given the high expression of mGlu(5) receptors in limbic forebrain regions, it was decided to evaluate the anxiolytic potential of MPEP. After an acute oral administration, MPEP attenuated the anxiety-dependent variable in a variety of well established anxiety test paradigms. In rats, MPEP (10, 30, and 100 mg/kg) increased punished responses in the Geller-Seifter test, but none of these effects reached statistical significance. MPEP significantly increased the ratio (open/total arm entries; 0.1, 1, and 10 mg/kg), the number of open arm entries (0.1, 1, and 10 mg/kg), as well as time spent on open arm (0.1 and 1 mg/kg) in the elevated plus maze test. Furthermore, MPEP (0.3 and 1 mg/kg) significantly increased the time spent in social contact in the social exploration test. In mice, MPEP attenuated stress-induced hyperthermia (15 and 30 mg/kg) and decreased the number of buried marbles in the marble burying test (7.5 and 30 mg/kg). Finally, MPEP (0.01, 0.1, 1, 10, and 100 mg/kg) was tested on spontaneous locomotor activity in mice, and only a dose of 100 mg/kg significantly reduced vertical activity; no effect was seen on horizontal activity. MPEP (7.5, 15, and 30 mg/kg) was ineffective on d-amphetamine-induced (2.5 mg/kg) locomotor activity in mice and prepulse inhibition in rats (1, 3, or 10 mg/kg). Thus, these findings indicate that MPEP exhibits anxiolytic-like effects and low risks for sedation and psychotomimetic side-effects in rodents.

We have used potent and selective non-competitive antagonists of metabotropic glutamate receptor subtype 5 (mGlu5) —- 2-methyl-6-phenylethynylpyridine (MPEP), [6-methyl-2-(phenylazo)-3-pyridinol] (SIB-1757) and [(E)-2-methyl-6-(2-phenylethenyl)pyridine] (SIB-1893) — to examine whether endogenous activation of this particular metabotropic glutamate receptor subtype contributes to neuronal degeneration. In cortical cultures challenged with N-methyl-d-aspartate (NMDA), all three mGlu5 receptor antagonists were neuroprotective. The effect of MPEP was highly specific because the close analogue, 3-methyl-6-phenylethynylpyridine (iso-MPEP), which did not antagonize heterologously expressed mGlu5 receptors, was devoid of activity on NMDA toxicity. Neuroprotection by mGlu5 receptor antagonists was also observed in cortical cultures challenged with a toxic concentration of β-amyloid peptide. We have also examined the effect of mGlu5 receptor antagonists in in vivo models of excitotoxic degeneration. MPEP and SIB-1893 were neuroprotective against neuronal damage induced by intrastriatal injection of NMDA or quinolinic acid. These results indicate that mGlu5 receptors represent a suitable target for novel neuroprotective agents of potential application in neurodegenerative disorders.

Brain glutamate overactivity is well documented in Parkinson's disease (PD) and antiglutamatergic drugs have been proposed to relieve PD symptoms and decrease dyskinesias. Metabotropic glutamate receptors are topics of recent interest in PD. This study investigated the effects of the metabotropic glutamate receptors type 5 (mGluR5) antagonists MPEP and MTEP on motor behavior in monkeys with a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion to model PD and treated with L-Dopa the gold standard therapy. Six Macaca fascicularis MPTP monkeys were initially treated repeatedly with L-Dopa; this treatment increased their locomotion and reduced their parkinsonian scores but also induced dyskinesias. Then, a dose-response of MPEP and MTEP (1.5-30 mg/kg) administered 15 and 30 min respectively prior to L-Dopa, showed that the antiparkinsonian activity of L-Dopa was generally maintained as measured with locomotion and antiparkinsonian scores as well as the onset and duration of the L-Dopa response. Interestingly the mean dyskinesia score during all the duration of the L-Dopa motor effect, the 1 h peak period dyskinesias scores as well as the maximal dyskinesias scores were dose-dependently reduced with both drugs reaching statistical significance at 10 and 30 mg/kg. Our results showed a beneficial antidyskinetic effect of blocking mGluR5 in L-Dopa-treated MPTP monkeys. This supports the therapeutic use of an mGluR5 antagonist to restore normal brain glutamate neurotransmission in PD and decrease dyskinesias.

Fragile X syndrome is caused by lack of FMR1 protein (FMRP) leading to severe symptoms, including intellectual disability, hyperactivity and autistic-like behaviour. FMRP is an RNA binding protein involved in the regulation of translation of specific target mRNAs upon stimulation of metabotropic glutamate receptor 5 (mGluR5) at the synapse. The absence of FMRP leads to enhanced activity of mGluR5 signal transduction pathways. Many conflicting results have been reported regarding social behaviour deficits in Fmr1 knockout mice, and little is known about the involvement of mGluR5 pathways on social behaviour. In this study, a three-chambered task was used to determine sociability and preference for social novelty in Fmr1 knockout mice. Disruption of Fmr1 functioning resulted in enhanced interaction with stranger mouse during sociability while no significant changes were observed during preference for social novelty assay. Chronic administration of a specific mGluR5 antagonist, AFQ056/Mavoglurant, was able to restore sociability behaviour of Fmr1 knockout mice to levels of wild type littermates. These results support the importance of mGluR5 signalling pathways on social interaction behaviour and that AFQ056/Mavoglurant might be useful as potential therapeutic intervention to rescue various behavioural aspects of the fragile X phenotype.

Metabotropic glutamate receptors type 5 (mGluR5) are implicated in regulation of synaptic plasticity and learning, and were the focus of our investigation in human Parkinson's disease (PD) patients with dyskinesias and wearing-off, and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys with dyskinesias. Using the selective mGluR5 ligand [3H]ABP688 autoradiography, we measured mGluR5 in brain slices from 11 normal and 14 PD patients and from MPTP monkeys, in relation to motor complications (dyskinesias and wearing-off) associated with treatment with l-dopa. In 16 monkeys with a bilateral MPTP lesion and four controls, [3H]ABP688 specific binding was elevated in the striatum of dyskinetic l-dopa-treated MPTP monkeys but not in MPTP monkeys without dyskinesias compared to controls. PD patients with motor complications (either dyskinesias or wearing-off) had higher [3H]ABP688 specific binding compared to those without motor complications and controls in putamen, external and internal globus pallidus. Elevated glutamatergic transmission as measured with increased mGluR5 specific binding was associated with motor complications and its antagonism could be targeted for their treatment.

Data are presented on the details of the interaction γ+p→p+ρ0 observed in a 12-in. hydrogen bubble chamber exposed to a bremsstrahlung photon beam of 6-BeV maximum energy at the Cambridge Electron Accelerator. The energy dependence of the cross sections, the production angular distributions, and the decay angular distributions of the ρ0's are compared with the predictions of the one-pion-exchange (OPE) mechanism and with a diffraction or multiperipheral model proposed by Berman and Drell. These data, as well as a comparison with ω0 and with ρ0+N*(1238) production, reject the OPE model and favor a diffraction mechanism.Received 22 December 1965DOI:https://doi.org/10.1103/PhysRev.146.994©1966 American Physical Society

This study investigated the hypothesis that AFQ056 (mavoglurant), a selective metabotropic glutamate receptor 5 antagonist, reduces chorea in Huntington's disease (HD).This 32-day randomized, double-blind, parallel-group, proof-of-concept study investigated AFQ056 (25-150 mg [incremental doses], twice-daily) versus placebo in patients with HD. Primary efficacy assessments were the chorea-sum score and orientation index (nondominant hand) from the quantitative motor (Q-Motor) grasping task at day 28. Key secondary efficacy assessments included finger-tapping in the Unified Huntington's Disease Rating Scale-Total Motor Score and Q-Motor measures. Safety and tolerability were assessed.Overall, 42 patients were randomized. At day 28, no improvement was observed on the primary efficacy assessments (P > 0.10) with AFQ056 versus placebo. The Q-Motor speeded-tapping interonset interval variability was reduced with AFQ056 versus placebo for the nondominant hand (P = 0.01). The incidence of adverse events was 66.7% with AFQ056 and 57.1% with placebo.AFQ056 did not reduce choreatic movements in HD, but was well tolerated. The clinical relevance of the Q-Motor findings (speeded-tapping) are unknown and may warrant further investigation.

Preclinical and postmortem studies have implicated the metabotropic glutamate receptor 5 (mGluR5) in the pathophysiology of major depressive disorder (MDD). The goal of the present study was to determine the role of mGluR5 in a large group of individuals with MDD compared to healthy controls (HC) in vivo with [18F]FPEB and positron emission tomography (PET). Furthermore, we sought to determine the role glutamate plays on mGluR5 availability in MDD.Sixty-five participants (30 MDD and 35 HC) completed [18F]FPEB PET to estimate the primary outcome measure - mGluR5 volume of distribution (VT), and the secondary outcome measure - mGluR5 distribution volume ratio (DVR). A subgroup of 39 participants (16 MDD and 23 HC) completed proton magnetic resonance spectroscopy (1H MRS) to estimate anterior cingulate (ACC) glutamate, glutamine, and Glx (glutamate + glutamine) levels relative to creatine (Cr).No significant between-group differences were observed in mGluR5 VT or DVR. Compared to HC, individuals with MDD had higher ACC glutamate, glutamine, and Glx levels. Importantly, the ACC mGluR5 DVR negatively correlated with glutamate/Cr and Glx/Cr levels.In this novel in vivo examination, we show an inverse relationship between mGluR5 availability and glutamate levels. These data highlight the need to further investigate the role of glutamatergic system in depression.

Fragile X syndrome (FXS) is a monogenic form of intellectual disability and autism spectrum disorder caused by the absence of the fragile X mental retardation protein (FMRP). In biological models for the disease, this leads to upregulated mRNA translation and as a consequence, deficits in synaptic architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here, we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here, we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those in neurons we suggest the validity of this peripheral biomarker. Our study offers a potential explanation for the comprehensive drug development program undertaken thus far yielding negative results and suggests that a significant proportion, but not all individuals with FXS, may benefit from the reduction of excessive levels of protein synthesis.

In the present study, we evaluated the effect of the prototypical metabotropic glutamate receptor 5 (mGlu5) antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) on motor behaviour in rats using the accelerating rotarod, spontaneous locomotor activity and the 6-hydroxy-dopamine (6-OHDA) lesion model to assess its treatment potential for Parkinson's disease. The data indicate that MPEP at doses between 7.5 and 300 mg/kg, p.o. did not disrupt endurance performance on the accelerating rotarod (4–40 rpm in 300 s) which indicates that MPEP has a relatively high safety margin. However, while ineffective at doses of 3.75, 7.5 and 15 mg/kg (p.o.) MPEP inhibited spontaneous locomotor activity at doses of 30 and 100 mg/kg (p.o.). In the 6-OHDA rat rotation model, at doses of 7.5, 15 and 30 mg/kg (p.o.), MPEP induced a dose-dependent ipsilateral rotational response that reached statistical significance at the highest dose tested. This effect was relatively small but consistent. In combination with direct or indirect dopamine agonists, i.e. apomorphine (0.25 mg/kg, s.c.) and d-amphetamine (2.5 mg/kg, i.p.), MPEP (7.5, 15 or 30 mg/kg, p.o.) was found to significantly inhibit these dopamine receptor mediated rotational responses. MPEP injected at a dose of 30 mg/kg also inhibited the rotational response induced by l-DOPA (25 mg/kg, i.p.). (+)MK-801 was used in these rotation experiments as the reference compound. In view of these findings, it could be concluded that MPEP and potentially other mGlu5 receptor antagonists are probably not appropriate drug candidates for the symptomatic treatment of Parkinson's disease.

Activation of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, and mGluR8) has been established to be neuroprotective in vitro and in vivo. To disclose the identity of the receptor subtype(s) that exert(s) the protective effect, we have used group III agonists in combination with mGluR4 subtype-deficient mice (-/-). In cortical cultures prepared from wild-type (+/+) mice and exposed to a toxic pulse of NMDA, the selective group III agonist (+)-4-phosphonophenylglycine [(+)-PPG] reversed excitotoxicity with an EC(50) value of 4.9 microm, whereas its enantiomer (-)-PPG was inactive. This correlated closely with the potency of (+)-PPG in activating recombinant mGluR4a. In cortical neurons from -/- mice, (+)-PPG showed no protection against the NMDA insult up to 300 microm, whereas group I/II mGluR ligands still retained their protective activity. Classical group III agonists (l-2-amino-4-phosphonobutyrate and l-serine-O-phosphate) were also substantially neuroprotective against NMDA toxicity in +/+ and heterozygous (+/-) cultures but were inactive in -/- cultures. Interestingly, -/- cultures were more vulnerable to low concentrations of NMDA and showed higher extracellular glutamate levels compared with +/+ cultures. We have also examined neurodegeneration induced by intrastriatal infusion of NMDA in wild-type or mGluR4-deficient mice. Low doses of (R,S)-PPG (10 nmol/0.5 microl) substantially reduced NMDA toxicity in +/+ mice but were ineffective in -/- mice. Higher doses of (R,S)-PPG were neuroprotective in both strains of animals. Finally, microdialysis studies showed that intrastriatal infusion of NMDA increased extracellular glutamate levels to a greater extent in -/- than in +/+ mice, supporting the hypothesis that the mGluR4 subtype is necessary for the maintenance of the homeostasis of extracellular glutamate levels.

Annals of the New York Academy of SciencesVolume 1003, Issue 1 p. 415-418 Metabotropic Glutamate 5 Receptor Antagonist MPEP Decreased Nicotine and Cocaine Self-Administration but Not Nicotine and Cocaine-Induced Facilitation of Brain Reward Function in Rats P. J. KENNY, Corresponding Author P. J. KENNY The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USAAddress for correspondence: P.J. Kenny, The Scripps Research Institute, Department of Neuropharmacology, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Voice: 858-784-7305; fax: 858-784-7405. pjkenny@scripps.eduSearch for more papers by this authorN. E. PATERSON, N. E. PATERSON The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorB. BOUTREL, B. BOUTREL The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorS. SEMENOVA, S. SEMENOVA The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorA. A. HARRISON, A. A. HARRISON The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorF. GASPARINI, F. GASPARINI Nervous System Research, Novartis-Pharma AG, 4002 Basel, SwitzerlandSearch for more papers by this authorG. F. KOOB, G. F. KOOB The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorP. D. SKOUBIS, P. D. SKOUBIS The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorA. MARKOU, A. MARKOU The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this author P. J. KENNY, Corresponding Author P. J. KENNY The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USAAddress for correspondence: P.J. Kenny, The Scripps Research Institute, Department of Neuropharmacology, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Voice: 858-784-7305; fax: 858-784-7405. pjkenny@scripps.eduSearch for more papers by this authorN. E. PATERSON, N. E. PATERSON The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorB. BOUTREL, B. BOUTREL The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorS. SEMENOVA, S. SEMENOVA The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorA. A. HARRISON, A. A. HARRISON The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorF. GASPARINI, F. GASPARINI Nervous System Research, Novartis-Pharma AG, 4002 Basel, SwitzerlandSearch for more papers by this authorG. F. KOOB, G. F. KOOB The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorP. D. SKOUBIS, P. D. SKOUBIS The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this authorA. MARKOU, A. MARKOU The Scripps Research Institute, Department of Neuropharmacology, 10550 North Torrey Pines Road, La Jolla, California 92037, USASearch for more papers by this author First published: 24 January 2006 https://doi.org/10.1196/annals.1300.040Citations: 80Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume1003, Issue1GLUTAMATE AND DISORDERS OF COGNITION AND MOTIVATIONNovember 2003Pages 415-418 RelatedInformation

In metabotropic glutamate receptor 2 (mGlu(2)) knockout mice, the group 2 metabotropic glutamate receptor agonist LY314582 (20 mg/kg, i.p.), a racemate of LY354740, inhibits neither spontaneous nor phencyclidine (PCP)-induced (2.5 mg/kg, s.c.) locomotor activity. Since LY314582 attenuated spontaneous and PCP-induced locomotor activity in wild-type control mice, these data indicate that the effects of LY314582 are mediated via the mGlu(2) receptor and not via the mGlu(3) receptor.

(11)C-ABP688 (3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-(11)C-methyl-oxime), a noncompetitive and highly selective antagonist for the metabotropic glutamate receptor subtype 5 (mGluR5), was evaluated for its potential as a PET agent.ABP688 was radiolabeled with (11)C by reacting (11)C-methyl iodide with the sodium salt of desmethyl-ABP688 (3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone oxime). The affinity of (11)C-ABP688 for mGluR5 was determined by Scatchard analysis using rat whole-brain membranes (without cerebellum). Ex vivo autoradiography, biodistribution, and PET studies with (11)C-ABP688 were performed on rats, wild-type mice, and mGluR5-knock-out mice.The overall synthesis time was 45-50 min from the end of radionuclide production. (11)C-ABP688 was obtained in good radiochemical yield (35% +/- 8%, n = 17, decay corrected), and the specific radioactivity was 150 +/- 50 GBq/mumol (n = 17) at the end of the synthesis. Scatchard analysis revealed a single high-affinity binding site with a dissociation constant of 1.7 +/- 0.2 nmol/L and a maximum number of binding sites of 231 +/- 18 fmol/mg of protein. Ex vivo autoradiography in wild-type mice and rats showed a heterogeneous distribution pattern consistent with the known distribution of mGluR5 in the brain, with the highest uptake in hippocampus, striatum, and cortex. Blocking studies by coinjection of (11)C-ABP688 and unlabeled 2-methyl-6-(3-methoxyphenyl)ethynyl-pyridine (1 mg/kg), an antagonist for mGluR5, revealed up to 80% specific binding in rat brain. In mGluR5-knock-out mouse brain, a homogeneous and markedly reduced accumulation of (11)C-ABP688 was observed. PET studies on rats and mice using a small-animal PET scanner also demonstrated radioactivity uptake in the brain regions known to be rich in mGluR5. In contrast, radioactivity uptake in mGluR5-knock-out mice was fairly uniform, substantiating the specificity of (11)C-ABP688 binding to mGluR5.(11)C-ABP688 is a selective tracer for imaging mGluR5 in vivo in rodents and may offer a future tool for imaging mGluR5 in humans using PET.

In the early 1990s, a new family of receptors were cloned that were found to mediate the intracellular metabolic effects of glutamate via coupling to secondary messenger systems, that is, the metabotropic glutamate (mGlu) receptors. Eight such receptors (mGlu1 to mGlu8) have been cloned to date, and according to their amino acid sequence, pharmacology and second-messenger coupling, these receptors have been clustered into three groups (I-III). In contrast to the glutamate-gated ion channels (NMDA, AMPA and kainate receptors), which are responsible for fast excitatory transmission, mGlu receptors have been shown to play a modulatory role in the glutamatergic synaptic transmission either by modulating the ion channel activity or by influencing neurotransmitter release. Given the fact that the mGlu receptors are G-protein- coupled, they obviously constitute a new attractive group of "drugable" targets for the treatment of various CNS disorders. The recent discovery of small molecules that selectively bind to receptors of group I (mGlu1 and mGlu5) and group II (mGlu2 and mGlu3) allowed significant advances in our understanding of the roles of these receptors in brain physiology and pathophysiology. The identification of MPEP (2-methyl-6-(phenylethynyl)-pyridine), a highly selective and brain-penetrant mGlu5 receptor antagonist, allowed the exploration of the therapeutic potential of this class of compounds. Subsequent behavior studies revealed that--with the exception of benzodiazepines--mGlu5 receptor antagonists exhibit the widest and most robust anxiolytic activity in preclinical models seen to date. Upcoming clinical studies will soon indicate if the preclinical anxiolytic-like efficacy translates into anxiolytic activity in humans.

3-(6-Methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-11C-methyl-oxime (11C-ABP688), a noncompetitive and highly selective antagonist for the metabotropic glutamate receptor subtype 5 (mGluR5), was evaluated for its potential as a PET agent.Six healthy male volunteers (mean age, 25 y; range, 21-33 y) were studied. Brain perfusion (15O-H2O) was measured immediately before each 11C-ABP688 PET scan. For anatomic coregistration, T1-weighted MRI was performed on each subject. Arterial blood samples for the determination of the arterial input curve were obtained at predefined time points, and 11C-ABP688 uptake was assessed quantitatively using a 2-tissue-compartment model.An initial rapid uptake of radioactivity followed by a gradual clearance from all examined brain regions was observed. Relatively high radioactivity concentrations were observed in mGluR5-rich brain regions such as the anterior cingulate, medial temporal lobe, amygdala, caudate, and putamen, whereas radioactivity uptake in the cerebellum and white matter, regions known to contain low densities of mGluR5, was low. Specific distribution volume as an outcome measure of mGluR5 density in the various brain regions ranged from 5.45 +/- 1.47 (anterior cingulate) to 1.91 +/- 0.32 (cerebellum), and the rank order of the corresponding specific distribution volumes of 11C-ABP688 in cortical regions was temporal > frontal > occipital > parietal. The metabolism of 11C-ABP688 in plasma was rapid; at 60 min after injection, 25% +/- 0.03% of radioactivity measured in the plasma of healthy volunteers was intact parent compound.The results of these studies indicate that 11C-ABP688 has suitable characteristics and is a promising PET ligand for imaging mGluR5 distribution in humans. Furthermore, it could be of great value for the selection of appropriate doses of clinically relevant candidate drugs that bind to mGluR5 and for PET studies of patients with psychiatric and neurologic disorders.

An experiment designed to study the π−p total neutral cross section and its breakdown into several channels has been performed at eleven incident pion momenta ranging from 654 to 1247 MeV/c. Angular distributions for the charge exchange π0 and for η0 production are given in terms of Legendre-polynomial expansion coefficients. Forward and backward differential cross sections are presented for the charge-exchange channel and comparisons with recent dispersion-relation predictions for the forward cross section are made.Received 2 June 1969DOI:https://doi.org/10.1103/PhysRev.187.1827©1969 American Physical Society

l-3,4-Dihydroxyphenylalanine (l-DOPA), the gold standard therapy for Parkinson disease (PD), is associated with motor fluctuations and dyskinesias. This study sought to prevent the development of l-DOPA-induced dyskinesias (LID) with the metabotropic glutamate receptor type 5 (mGlu5 receptor) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) in the de novo treatment of monkeys lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a PD model. MPTP-lesioned monkeys were treated once daily for one month with either l-DOPA or l-DOPA + MPEP (10 mg/kg). MPEP (administered 15 min before l-DOPA) plasma concentrations were elevated during all the l-DOPA motor activation and did not accumulate during a month. The antiparkinsonian effect was maintained throughout the treatment period in MPTP-lesioned monkeys treated with l-DOPA + MPEP, while the duration of this effect decreased over time in MPTP-lesioned monkeys treated with l-DOPA alone, suggesting wearing-off. Over the month-long treatment, the mean dyskinesia score increased in l-DOPA-treated monkeys; interestingly, this increase was reduced by overall 72% in the l-DOPA + MPEP group. Mean dyskinesia scores of monkeys correlated inversely with plasma MPEP concentrations. Normal control and saline-treated MPTP-lesioned monkeys were also included for biochemical analyses. All MPTP-lesioned monkeys were extensively and similarly denervated. [3H]ABP688 specific binding to mGlu5 receptors increased in the putamen of l-DOPA-treated monkeys compared to control, saline or l-DOPA + MPEP-treated monkeys. Mean dyskinesia scores of MPTP-lesioned monkeys correlated positively with [3H]ABP688 specific binding in the putamen. This study showed a beneficial chronic antidyskinetic effect of MPEP in de novo l-DOPA-treated MPTP-lesioned monkeys, supporting the therapeutic use of mGlu5 receptor antagonists in PD to prevent LID. This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’.

Metabotropic glutamate 5 (mGlu5) receptor antagonists reduce L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias (LID) in Parkinson's disease (PD). The aim of this study was to investigate the long-term effect of the prototypal mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) on glutamate receptors known to be involved in the development of LID in the de novo chronic treatment of monkeys lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP monkeys were treated for one month with L-DOPA and developed dyskinesias while those treated with L-DOPA and MPEP (10 mg/kg) developed significantly less. Normal control and saline-treated MPTP monkeys were also included. All MPTP monkeys were extensively and similarly denervated. The basal ganglia [(3)H]ABP688 specific binding (mGlu5 receptors) was elevated in L-DOPA-treated MPTP monkeys compared to controls but not in those treated with L-DOPA and MPEP; dyskinesia scores of these monkeys correlated positively with their [(3)H]ABP688 specific binding. Striatal density (B(max)) of [(3)H]ABP688 specific binding increased in L-DOPA-treated MPTP monkeys compared to other groups and affinity (Kd) remained unchanged. Striatal mGlu5 receptor mRNA remained unchanged following treatments. Elevated basal ganglia specific binding of [(3)H]Ro 25-6981 (NMDA NR1/NR2B receptors), [(3)H]Ro 48-8587 (AMPA receptors) but not [(3)H]CGP-39653 (NMDA NR1/NR2A receptors) was observed only in L-DOPA-treated MPTP monkeys; dyskinesias scores correlated with binding. By contrast, basal ganglia [(3)H]LY341495 specific binding (mGlu2/3 receptors) decreased in L-DOPA-treated MPTP monkeys compared to controls, saline and L-DOPA + MPEP treated MPTP monkeys; dyskinesias scores correlated negatively with this binding. Hence, chronic MPEP treatment reduces the development of LID and is associated with a normalization of glutamate neurotransmission.

Fragile X syndrome is an inherited disease with cognitive, behavioral, and neurologic manifestations, resulting from a single genetic mutation. A variety of treatments that target individual symptoms of fragile X syndrome are currently utilized with limited efficacy. Research in animal models has resulted in the development of potential novel pharmacologic treatments that target the underlying molecular defect in fragile X syndrome, rather than the resultant symptoms. This review describes recent advances in our understanding of the molecular basis of fragile X syndrome and summarizes the ongoing clinical research programs.

Here we describe the identification, structure–activity relationship and the initial pharmacological characterization of AFQ056/mavoglurant, a structurally novel, non-competitive mGlu5 receptor antagonist. AFQ056/mavoglurant was identified by chemical derivatization of a lead compound discovered in a HTS campaign. In vitro, AFQ056/mavoglurant had an IC50 of 30 nM in a functional assay with human mGluR5 and was selective over the other mGluR subtypes, iGluRs and a panel of 238 CNS relevant receptors, transporter or enzymes. In vivo, AFQ056/mavoglurant showed an improved pharmacokinetic profile in rat and efficacy in the stress-induced hyperthermia test in mice as compared to the prototypic mGluR5 antagonist MPEP. The efficacy of AFQ056/mavoglurant in humans has been assessed in L-dopa induced dyskinesia in Parkinson’s disease and Fragile X syndrome in proof of principle clinical studies.

Positron emission tomography tracers [ 11 C]ABP688 and [ 18 F]FPEB target the metabotropic glutamate receptor subtype 5 providing quantification of the brain glutamatergic system in vivo. Previous [ 11 C]ABP688 positron emission tomography human test–retest studies indicate that, when performed on the same day, significant binding increases are observed; however, little deviation is reported when scans are >7 days apart. Due to the small cohorts examined previously (eight and five males, respectively), we aimed to replicate the same-day test–retest studies in a larger cohort including both males and females. Results confirmed large within-subject binding differences (ranging from −23% to 108%), suggesting that measurements are greatly affected by study design. We further investigated whether this phenomenon was specific to [ 11 C]ABP688. Using [ 18 F]FPEB and methodology that accounts for residual radioactivity from the test scan, four subjects were scanned twice on the same day. In these subjects, binding estimates increased between 5% and 39% between scans. Consistent with [ 11 C]ABP688, mean absolute test–retest variability was previously reported as <12% when scans were >21 days apart. This replication study and pilot extension to [ 18 F]FPEB suggest that observed within-day binding variation may be due to characteristics of mGluR5; for example, diurnal variation in mGluR5 may affect measurement of this receptor.

Prion infections cause inexorable, progressive neurological dysfunction and neurodegeneration. Expression of the cellular prion protein PrPC is required for toxicity, suggesting the existence of deleterious PrPC-dependent signaling cascades. Because group-I metabotropic glutamate receptors (mGluR1 and mGluR5) can form complexes with the cellular prion protein (PrPC), we investigated the impact of mGluR1 and mGluR5 inhibition on prion toxicity ex vivo and in vivo. We found that pharmacological inhibition of mGluR1 and mGluR5 antagonized dose-dependently the neurotoxicity triggered by prion infection and by prion-mimetic anti-PrPC antibodies in organotypic brain slices. Prion-mimetic antibodies increased mGluR5 clustering around dendritic spines, mimicking the toxicity of Aβ oligomers. Oral treatment with the mGluR5 inhibitor, MPEP, delayed the onset of motor deficits and moderately prolonged survival of prion-infected mice. Although group-I mGluR inhibition was not curative, these results suggest that it may alleviate the neurological dysfunctions induced by prion diseases.

Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of excitatory amino acids, via mechanisms of presynaptic inhibition, such as regulation of neurotransmitter release. Here, we describe (R,S)-4-phosphonophenylglycine (PPG) as a novel, potent, and selective agonist for group III mGluRs. In recombinant cell lines expressing the human receptors hmGluR4a, hmGluR6, hmGluR7b, or hmGluR8a, EC50 values for (R,S)-PPG of 5.2 +/- 0.7 microM, 4.7 +/- 0.9 microM, 185 +/- 42 microM, and 0.2 +/- 0.1 microM, respectively, were measured. The compound showed EC50 and IC50 values of >/=200 microM at group I and II hmGluRs and was inactive at cloned human N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, and kainate receptors (>300 microM). On the other hand, it showed micromolar affinity for a Ca2+/Cl--dependent L-glutamate binding site in rat brain, similar to other phosphono-substituted amino acids like L-2-amino-4-phosphonobutyrate. In cultured cortical neurons, (R, S)-PPG provided protection against a toxic pulse of N-methyl-D-aspartate (EC50 = 12 microM), which was reversed by the group III mGluR antagonist (R,S)-alpha-methylserine-O-phosphate but not by the group II antagonist (2S)-alpha-ethylglutamate. Moreover, (R,S)-PPG protected against N-methyl-D-aspartate- and quinolinic acid-induced striatal lesions in rats and was anticonvulsive in the maximal electroshock model in mice. In contrast to the group III mGluR agonists L-2-amino-4-phosphonobutyrate and L-serine-O-phosphate, (R,S)-PPG showed no proconvulsive effects (2200 nmol i.c.v.). These data provide novel in vivo evidence for group III mGluRs as attractive targets for neuroprotective and anticonvulsive therapy. Also, (R,S)-PPG represents an attractive tool to analyze the roles of group III mGluRs in nervous system physiology and pathology.

Metabotropic glutamate receptors form a family of currently eight subtypes (mGluR1-8), subdivided into three groups (I-III). Activation of group-II (mGluR2 and -3) or group-III metabotropic glutamate receptors (mGluR4, -6, -7 and -8) has been established to be neuroprotective in vitro and in vivo. In contrast, group-I mGluRs (mGluR1 and -5) need to be antagonized in order to evoke protection. Initially, all neuroprotective mGluR ligands were analogues of L-glutamate. Those compounds were valuable to demonstrate protection in vitro, but showed limited applicability in animal models, particularly in chronic tests, due to low blood-brain-barrier penetration. Recently, systemically active and more potent and selective ligands became available, e.g., the group-II mGluR agonists LY354740 and LY379268 or group-I antagonists like MPEP (mGluR5-selective) and BAY36-7620 (mGluR1-selective). This new generation of pharmacological agents allows a more stringent assessment of the role of individual mGluR-subtypes or groups of receptors in various nervous system disorders, including ischaemia-induced brain damage, traumatic brain injury, Huntington's and Parkinson's-like pathology or epilepsy. Moreover, the use of genetically modified animals (e.g., knock-out mice) is starting to shed light on specific functions of mGluR-subtypes in experimental neuropathologies.

We have constructed and operated a 3 ton liquid argon time projection chamber for the R&D programme of the ICARUS project. The chamber has been in op

The synthesis of a new potent, subtype-selective radioligand [(3)H]-M-MPEP (2-methyl-6-((3-methoxyphenyl)ethynyl)-pyridine) and its in vitro pharmacological characteristics are described. Science Ltd.

Smoking is a major health problem and is propelled, at least in part, by the addictive properties of nicotine. Two types of pharmacological therapies have been approved for smoking cessation by the US Food and Drug Administration. The first therapy consists of nicotine replacement, substituting the nicotine from cigarettes with safer nicotine formulations. The second therapy is bupropion (Zyban), an atypical antidepressant, whose use has raised much debate as to how a non-nicotine-based agent can aid in smoking cessation. This review focuses on recent advances that could lead to the development of improved novel pharmacological treatments. These strategies focus on altering reward processes in the brain by modulating various neurotransmitter systems: the most promising include dopamine D(3) receptor antagonists, noradrenaline reuptake inhibitors, GABA(B) receptor agonists, metabotropic glutamate 5 (mGluR5) receptor antagonists, cannabinoid CB1 receptor antagonists, and corticotropin releasing factor (CRF) 1 receptor antagonists.

[11C]ABP688 (2) has recently been demonstrated to be a useful PET tracer for in vivo imaging of the metabotropic glutamate receptors type 5 (mGluR5) in rodents. We describe here the identification and preclinical profiling of ABP688 and its tritiated version [3H]ABP688, and show that its high affinity (Kd = 2 nM), selectivity, and pharmacokinetic properties fulfill all requirements for development as a PET tracer for clinical imaging of the mGlu5 receptor.

(11)C-ABP688 is a new PET ligand to assess the subtype 5 metabotropic glutamate receptor (mGlu(5)). The purpose of this study was to evaluate different methods for the analysis of human (11)C-ABP688 data acquired from 6 healthy, young volunteers.The methods were a 1-tissue-compartment model (K(1), k(2)''), a 2-tissue-compartment model (K1-k4), and the noncompartmental method developed by Logan. Parameters related to receptor density were the total distribution volume (DV), DV'' (= K(1)/k(2)'', 1 tissue compartment); specific DV, DV(C2) (= K(1)/k(2)' x k(3)'/k(4), 2 tissue compartments); and DV(tot) for the noncompartmental method.The 1-tissue-compartment model was too simple to adequately fit the data. DV(C2) calculated with the 2-tissue-compartment model ranged from 5.45 +/- 1.47 (anterior cingulate) to 1.91 +/- 0.32 (cerebellum). The corresponding values for DV(tot), calculated with the 2-tissue-compartment model and the Logan method (in parentheses), were 6.57 +/- 1.45 (6.35 +/- 1.32) and 2.93 +/- 0.53 (2.48 +/- 0.40). There was no clear evidence of a region devoid of mGlu(5) receptors. The first-pass extraction fraction exceeded 95%. The minimal scan duration to obtain stable results was estimated to be 45 min.(11)C-ABP688 displays favorable kinetics for assessing mGlu(5) receptors. For tracer kinetic modeling, 2-tissue-compartment models are clearly superior to models with only 1 tissue compartment. In comparison to the compartmental models, the Logan method is equally useful if only DV(tot) values are required and fast pixelwise parametric maps are desired. The lack of regions devoid of receptors limits the use of reference region methods that do not require arterial blood sampling. Another advantage of the tracer is the fast kinetics that allow for relatively short acquisitions.

Growing evidence supports a role for the central nervous system (CNS) neurotransmitter L-glutamate and its metabotropic receptors (mGluRs) in drug addiction in general and alcohol-use disorders in particular. Alcohol dependence, for instance, has a genetic component, and the recent discovery that variations in the gene coding for mGluR7 modulate alcohol consumption further validates involvement of the L-glutamate system. Consequently, increasing interest emerges in developing L-glutamatergic therapies for the treatment of alcohol abuse and dependence. To this end, we performed a detailed behavioral pharmacology study to investigate the regulation of alcohol consumption and preference following administration of the mGluR7-selective drugs N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082) and 6-(4-Methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-c]pyridin-4(5H)-one hydrochloride (MMPIP). Upon administration of the allosteric agonist AMN082 (10 mg/kg, i.p.) in rats, there was a significant decrease in ethanol consumption and preference, without affecting ethanol blood metabolism. In contrast, mGluR7 blockade with MMPIP (10 mg/kg, i.p.) showed an increase in alcohol intake and reversed AMN082's effect on ethanol consumption and preference. Both mGluR7-directed pharmacological tools had no effect on total fluid intake, taste preference, or on spontaneous locomotor activity. In conclusion, these findings support a specific regulatory role for mGluR7 on alcohol drinking and preference and provide evidence for the use of AMN082-type drugs as potential new treatments for alcohol-use disorders in man.

Excessive glutamatergic signalling within the basal ganglia is implicated in the progression of Parkinson's disease (PD) and inthe emergence of dyskinesia associated with long-term treatment with L-DOPA. There is considerable research focus on the discovery and development of compounds that modulate glutamatergic signalling via glutamate receptors, as treatments for PD and L-DOPA-induced dyskinesia (LID). Although initial preclinical studies with ionotropic glutamate receptor antagonists showed antiparkinsonian and antidyskinetic activity, their clinical use was limited due to psychiatric adverse effects, with the exception of amantadine, a weak N-methyl-d-aspartate (NMDA) antagonist, currently used to reduce dyskinesia in PD patients. Metabotropic receptor (mGlu receptor) modulators were considered to have a more favourable side-effect profile, and several agents have been studied in preclinical models of PD. The most promising results have been seen clinically with selective antagonists of mGlu5 receptor and preclinically with selective positive allosteric modulators of mGlu4 receptor. The growing understanding of glutamate receptor crosstalk also raises the possibility of more precise modulation of glutamatergic transmission, which may lead to the development of more effective agents for PD.

Fragile X syndrome is the most common monogenetic form of intellectual disability and autism. Although the Fmr1 knockout mouse model recapitulates many aspects of the human FXS condition, the establishment of robust social behavioural phenotypes suitable for drug screening has been difficult. Here, we describe a novel social behavioural paradigm, the Automated Tube Test (ATT), for which Fmr1 knockout mice demonstrate a highly reliable and robust phenotype. Fmr1 KO mice show highly dominant behaviour over wild-type littermates in the ATT. Consistent with previous findings, we observed a highly significant, albeit partial, rescue of the altered social behaviour of Fmr1 knockout mice in the ATT, using genetic (mGluR5 deletion) or pharmacological inhibition (mGluR5 antagonist) of mGluR5 signalling independently. Together, our results validate the Automated Tube Test as a robust outcome measure for social behaviour in preclinical research for FXS, and confirm the pathophysiological relevance of mGluR5 signalling. Moreover, our findings highlight the strategy of initiating pharmacological intervention in adulthood as holding significant clinical potential.

The fractalkine (CX3CR1) ligand is expressed in astrocytes and reported to be neuroprotective. When cleaved from the membrane, soluble fractalkine (sCX3CL1) activates the receptor CX3CR1. Although somewhat controversial, CX3CR1 is reported to be expressed in neurons and microglia. The membrane-bound form of CX3CL1 additionally acts as an adhesion molecule for microglia and infiltrating white blood cells. Much research has been done on the role of fractalkine in neuronal cells; however, little is known about the regulation of the CX3CL1 ligand in astrocytes.The mechanisms involved in the up-regulation and cleavage of CX3CL1 from human astrocytes were investigated using immunocytochemistry, Q-PCR and ELISA. All statistical analysis was performed using GraphPad Prism 5.A combination of ADAM17 (TACE) and ADAM10 protease inhibitors was found to attenuate IL-1β-, TNF-α- and IFN-γ-induced sCX3CL1 levels in astrocytes. A specific ADAM10 (but not ADAM17) inhibitor also attenuated these effects, suggesting ADAM10 proteases induce release of sCX3CL1 from stimulated human astrocytes. A p38 MAPK inhibitor also attenuated the levels of sCX3CL1 upon treatment with IL-1β, TNF-α or IFN-γ. In addition, an IKKβ inhibitor significantly reduced the levels of sCX3CL1 induced by IL-1β or TNF-α in a concentration-dependent manner, suggesting a role for the NF-kB pathway.In conclusion, this study shows that the release of soluble astrocytic fractalkine is regulated by ADAM10 proteases with p38 MAPK also playing a role in the fractalkine shedding event. These findings are important for understanding the role of CX3CL1 in healthy and stimulated astrocytes and may benefit our understanding of this pathway in neuro-inflammatory and neurodegenerative diseases.

Cell lines expressing the human metabotropic glutamate receptor subtype 5a (hmGluR5a) and hmGluR1b were used as targets in an automated high-throughput screening (HTS) system that measures changes in intracellular Ca2+ ([Ca2+]i) using fluorescence detection. This functional screen was used to identify the mGluR5-selective antagonist, SIB-1757 [6-methyl-2-(phenylazo)-3-pyridinol], which inhibited the glutamate-induced [Ca2+]i responses at hmGluR5 with an IC50 of 0.37 microM compared with an IC50 of >100 microM at hmGluR1. Schild analysis demonstrated a noncompetitive mechanism of inhibition. Pharmacophore mapping was used to identify an additional compound, SIB-1893 [(E)-2-methyl-6-(2-phenylethenyl)pyridine], which was also shown to block glutamate-induced increases in [Ca2+]i at hmGluR5 with an IC50 of 0.29 microM compared with an IC50 of >100 microM at hmGluR1. SIB-1757 and SIB-1893 showed little or no activity when tested for agonist and antagonist activity at the other recombinant human mGluR subtypes, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and N-methyl-D-aspartate receptors. In rat neonatal brain slices, SIB-1757 and SIB-1893 inhibited (S)-3,5-dihydroxyphenylglycine (DHPG)-evoked inositol phosphate accumulation in hippocampus and striatum by 60% to 80%, with a potency similar to that observed on recombinant mGluR5. However, in the cerebellum, a brain region with low mGluR5 expression, SIB-1757 failed to inhibit DHPG-evoked inositol phosphate accumulation. In cultured rat cortical neurons, SIB-1757 and SIB-1893 largely inhibited DHPG-evoked [Ca2+]i signals, revealing a population of neurons that were less sensitive to SIB-1757 and SIB-1893. This is the first description of highly selective, noncompetitive mGluR5 antagonists. These compounds will be useful tools in evaluating the role of mGluR5 in normal physiology and in animal models of disease.

Activation of group I metabotropic glutamate receptors (mGlu1 or -5 receptors) is known to either enhance or attenuate excitotoxic neuronal death depending on the experimental conditions. We have examined the possibility that these receptors may switch between two different functional modes in regulating excitotoxicity. In mixed cultures of cortical cells, the selective mGlu1/5 agonist, 3,5-dihydroxyphenylglycine (DHPG), amplified neurodegeneration induced by a toxic pulse of NMDA. This effect was observed when DHPG was either combined with NMDA or transiently applied to the cultures prior to the NMDA pulse. However, two consecutive applications of DHPG consistently produced neuroprotection. Similar effects were observed with DHPG or quisqualate (a potent agonist of mGlu1/5 receptors) in pure cultures of cortical neurons virtually devoid of astrocytes. In cultures of hippocampal pyramidal neurons, however, only protective effects of DHPG were seen suggesting that, in these particular cultures, group I mGlu receptors were endogenously switched into a "neuroprotective mode". The characteristics of the activity-dependent switch from facilitation to inhibition were examined in mixed cultures of cortical cells. The switch in the response to DHPG was observed when the two applications of the drug were separated by an interval ranging from 1-45 min, but was lost when the interval was extended to 90 min. In addition, this phenomenon required the initial activation of mGlu5 receptors (as indicated by the use of subtype-selective antagonists) and was mediated by the activation of protein kinase C. We conclude that group I mGlu receptors are subjected to an activity-dependent switch in regulating excitotoxic neuronal death and, therefore, the recent "history" of these receptors is critical for the response to agonists or antagonists.

Overactivity of glutamate neurotransmission is suspected to be implicated in Parkinson's disease and levodopa-induced dyskinesia. The fast glutamatergic transmission in the striatum from the cortex is mediated mainly by non-n-methyl-d-aspartate (non-NMDA) receptors. Animal models of Parkinson's disease reveal conflicting data concerning striatal glutamate AMPA receptors. The present study thus sought to shed light on the relationship of striatal AMPA receptors to the development of levodopa-induced dyskinesia. [(3)H]Ro 48-8587, a highly potent and selective-specific antagonist ligand for AMPA receptors, was used to investigate, by autoradiography, striatal AMPA receptors in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys treated for 1 month with levodopa alone, levodopa+CI-1041 (NMDA receptor antagonist) or levodopa+cabergoline (D2 receptor agonist). Levodopa-treated MPTP monkeys developed dyskinesias while those that received levodopa+CI-1041 or levodopa+cabergoline did not. In the anterior caudate nucleus and putamen, specific binding of [(3)H]Ro 48-8587 was reduced in all MPTP-treated monkeys compared to control monkeys, but no significant effect of MPTP was measured in the posterior striatum. In dyskinetic monkeys, specific binding of [(3)H]Ro 48-8587 was elevated in subregions of the posterior caudate nucleus and putamen as compared to saline-treated MPTP monkeys. Levodopa+CI-1041 treatment left unchanged specific binding of [(3)H]Ro 48-8587 whereas levodopa+cabergoline treatment reduced it in subregions of the posterior caudate nucleus and putamen compared to control and levodopa-treated MPTP monkeys. Specific binding of [(3)H]Ro 48-8587 was low in the globus pallidus and remained unchanged following both lesion and treatments. In conclusion, the elevated values of AMPA receptors in dyskinetic monkeys (and their prevention through treatments) were only observed in subregions of the striatum.

Activation of astrocytic metabotropic glutamate receptor 5 (mGluR5) is postulated to elicit calcium transients, triggering a chain of events that ultimately regulates cerebral blood flow by changing the tone of smooth muscle cells of nearby arterioles. Using concurrent in vivo optical imaging and determination of receptor occupancy with (11)C-ABP688, we report here that blocking ∼80% of mGluR5 in vivo does not affect transient hemodynamic responses on brief whisker stimulations while transiently reducing neuronal activity as measured by voltage-sensitive dye imaging. Our results show that mechanisms other than activation of mGluR5 are required to trigger the initial hemodynamic response in normal physiological conditions.

J. Neurochem. (2010) 113 , 715–724. Abstract Behavioral investigations of selective and potent metabotropic glutamate receptor type 5 (mGluR5) antagonists in animal models suggest involvement of mGluR5 in compensatory mechanisms of the basal ganglia circuitry in Parkinson’s disease and levodopa (l‐Dopa) induced motor complications. This study investigated mGluR5 changes in MPTP lesioned monkeys. The effect of a chronic 1 month treatment with l‐Dopa on mGluR5‐specific binding and mRNA levels was investigated in MPTP monkeys killed 4 or 24 h after their last l‐Dopa administration. [ 3 H]ABP688 specific binding in the putamen was elevated in l‐Dopa‐treated MPTP monkeys killed 24 h but not 4 h after their last l‐Dopa dose compared with vehicle‐treated MPTP monkeys. Caudate nucleus [ 3 H]ABP688‐specific binding was elevated in both groups of l‐Dopa treated compared with vehicle–treated MPTP monkeys. In contrast, caudate nucleus and putamen mGluR5 mRNA levels were elevated only in l‐Dopa‐treated MPTP monkeys killed 4 h after their last l‐Dopa administration. MPTP monkeys killed 4 h after their last l‐Dopa treatment showed higher caudate nucleus and putamen l‐Dopa concentrations compared with those killed after 24 h. Hence, mGluR5 in the putamen are sensitive to presence of l‐Dopa leading to a rapid decrease of [ 3 H]ABP688‐specific binding possibly involving a direct mGluR5/dopamine receptors interaction.

Psychiatric and neurological disorders are linked to changes in synaptic excitatory processes with a key role for glutamate, that is, the most abundant excitatory amino-acid. Molecular cloning of the metabotropic glutamate (mGlu) receptors has led to the identification of eight mGlu receptors, which, in contrast to ligand-gated ion channels (responsible for fast excitatory transmission), modulate and fine-tune the efficacy of synaptic transmission. mGlu receptors are G protein-coupled and constitute a new group of "drugable" targets for the treatment of various CNS disorders. The recent discovery of small molecules that selectively bind to receptors of Groups I (mGlu1 and mGlu5) and II (mGlu2 and mGlu3) allowed significant advances in our understanding of the roles of these receptors in brain function and dysfunction including anxiety. Although investigation of the role of the Group III (mGlu4, 6, 7, and 8) receptors is less advanced, the generation of genetically manipulated animals and recent advances in the identification of subtype-selective compounds have revealed some first insights into the therapeutic potential of this group of receptors.

Previous work has demonstrated that neuroimaging biomarkers which capture functional connectivity of the brain can be used to define a specific and robust endophenotype in Fmr1-/y mice, a well-established animal model of human Fragile-X Syndrome (FXS). However, it is currently unknown whether this macroscopic measure of brain connectivity is sufficiently sensitive to reliably detect changes caused by pharmacological interventions. Here we inhibited the activity of the metabotropic glutamate receptor-5 (mGluR5) using AFQ056/Mavoglurant, a drug that is assumed to normalize excitatory/inhibitory neural signaling imbalances in FXS. We employed resting-state-fMRI (rs-fMRI) and diffusion-weighted imaging (DWI) to test whether Mavoglurant re-established brain connectivity - at least partly - within some of the affected circuits in Fmr1-/y mice that are related to social behavior deficits. In line with previous findings, we observed that Fmr1-/y mice exhibited impaired social interaction, reduced connectivity in three main functional networks and altered network topology. At the group level, Mavoglurant did neither rescue abnormal social behavioral nor white matter abnormalities; however, for some, but not all of these circuits Mavoglurant had a genotype-specific effect of restoring functional connectivity. These results show that rs-fMRI connectivity is sufficiently sensitive to pick up system-level changes after the pharmacological inhibition of mGluR5 activity. However, our results also show that the effects of Mavoglurant are confined to specific networks suggesting that behavioral benefits might be restricted to narrow functional domains.

The mGluR-mediated EPSP at parallel fibre-Purkinje cell synapses in the cerebellum was blocked concentration-dependently and reversibly by antagonists acting selectively on group-I mGluRs but not by an inhibitor of group-III receptors. The results provide pharmacological evidence that the receptor type responsible for the mGluR-EPSP is mGluR1.

The two reported metabotropic glutamate receptor (mGluR) antagonists, α-methyl-cyclopropyl glycine (MCCG) and α-methyl-aminophosphonobutyrate (MAP4) were tested on the mGluR1b, mGluR2 and mGluR4a subtypes of human mGluRs. Neither MCCG (500 μM) nor MAP4 (500 μM) antagonized the activation of mGluR1b by 10 μM quisqualate. MCCG was found to potently antagonize the action of 30 μM (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] at mGluR2 (IC50 = 87.5 μM; apparent KD = 25 μM) but did not block the action of 1 μM S-2-amino-4-phosphonobutyric acid at mGluR4a (IC50 > 1 mM). MAP4 was found to be a weak antagonist or partial agonist at mGluR4a (IC50 > 500 μM) and, less potently, also antagonized the action of 30 μM (1S,3R)-ACPD) at mGluR2 (IC50 ~2 mM).

Several lines of evidence indicate that inhibition of the metabotropic glutamate (mGlu) receptor 5 produces anxiolytic-like effects in rodents. Peptide neurotransmitter neuropeptide Y (NPY) produces an anxiolytic effect in rats after intraventricular or intra-amygdalar administration. Many classes of anxiolytic drugs exert their effect through the GABA–benzodiazepine (BZD) receptor complex. Therefore, in the present study we have investigated whether the anxiolytic action of MPEP (2-methyl-6-(phenylethynyl)pyridyne), an mGlu5 receptor antagonist, is mediated by a mechanism involving either the GABA–BZD receptor complex or NPY receptor. In the behavioral studies, the anxiolytic activity of MPEP (10 mg/kg, i.p.) was examined using plus-maze test. The BZD antagonist flumazenil (10 mg/kg, i.p.) was given to one group of rats and Y1 receptor antagonist BIBO 3304 (((R)-N-[[4-(aminocarbonylaminomethyl) phenyl] methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate)3304) (200 pmol/site, intra-amygdala) to the other. It was found that anxiolytic effects of MPEP were not changed by flumazenil, but were abolished by BIBO 3304. Immunohistochemical studies showed a high density of mGlu5 receptor immunoreactivity (IR) in the amygdala. The effect of MPEP on NPY expression in the amygdala was studied using immunohistochemistry (IH) and radioimmunoassay (RIA). Both methods showed a diminution of NPY IR expression, to about 43% (IH) or 81% (RIA) of the control level after multiple administrations, but we observed an increase up to 148% of the control after single MPEP administration. These effects may suggest a release of NPY from nerve terminals after MPEP administration. Our results indicate that the anxiolytic action of MPEP is conveyed through NPY neurons with the involvement of Y1 receptors in the amygdala and that BZD receptors do not significantly contribute to these effects.

As part of the development program for the ICARUS experiment, which consists of a very large time projection chamber (TPC) filled with ultra-pure liquid Ar (LAr), we have carried out tests with different purifier systems, in order to evaluate the performance of the various parts and to improve the purification techniques developed so far. Electron lifetime τ in LAr has been determined with an improved method based on the measurement of the attenuation of a current due to an electron cloud, photoproduced by a laser pulse impinging on a metallic cathode and moving in a small drift chamber filled with the purified LAr. Results of the above-mentioned tests are reported. During these tests, we observed repeatedly and reproducibly an increase of τ that took place over a period of 10 to 20 h after liquefaction. Several tests performed in an attempt to elucidate this effect, suggest that the increase in τ is due to adsorption of electron-attaching impurities on the walls of the stainless steel container, a process governed by thermal diffusion. The electron lifetime monitoring system reported here was used to measure the electric field dependence of τ in purified LAr doped with O2 and CO2, for fields 100 V/cm < E < 800 V/cm: The results for O2 are consistent with published data.

Glutamatergic mechanisms regulate neuronal circuits implicated in mood and anxiety. Emotional disorders as anxiety and depression are particularly difficult to treat during aging and mechanisms underlying emotional disturbances in the brain of the elderly are poorly understood. This may result from the small number of studies investigating these disorders in aged animals. Among glutamate receptors, metabotropic mGlu5 receptors are thought to play an important role, since their pharmacological blockade induces strong anxiolytic effects. However, the implication of mGlu5 in regulating anxiety is not yet completely understood. Here we analyzed both young adult and aged mice lacking mGlu5 receptors, to clarify, if genetic deletion of the receptor induces similar to pharmacological blockade anxiolytic effects. Unexpectedly, mGlu5 receptor knockout (KO) mice showed increased anxiety accentuating with aging. In contrast, young adult mice displayed an anti-depressive-like phenotype that was no longer detectable in aged animals. Our data support important distinct roles of mGlu5 receptors in modulating anxiety and depression during aging.

Abstract Introduction: Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. With no curative treatment available, current therapeutic approaches are aimed at symptom management. FXS is caused by silencing the FMR1 gene, which encodes FMRP; as loss of FMRP leads to the development of symptoms associated with FXS. Areas covered: In this evaluation, the authors examine the role of the metabotropic glutamate receptor 5 (mGluR5) in the pathophysiology of FXS, and its suitability as a target for rescuing the disease state. Furthermore, the authors review the evidence from preclinical studies of pharmacological interventions targeting mGluR5 in FXS. Lastly, the authors assess the findings from clinical studies in FXS, in particular the use of the Aberrant Behavior Checklist-Community Edition (ABC-C) and the recently developed ABC-C for FXS scale, as clinical endpoints to assess disease modification in this patient population. Expert opinion: There is cautious optimism for the successful treatment of the core behavioral and cognitive symptoms of FXS based on preclinical data in animal models and early studies in humans. However, the association between mGluR5-heightened responsiveness and the clinical phenotype in humans remains to be demonstrated. Many questions regarding the optimal treatment and outcome measures of FXS remain unanswered.Keywords:: Aberrant Behavior ChecklistFMRPfragile X syndromeFXSmavoglurantmGluR5 AcknowledgementsThe authors thank K O'Rourke, PhD, of iMed Comms, who provided medical writing assistance with this review.Declaration of interestFinancial support for medical editorial assistance was provided by Novartis Pharma AG. B Gomez-Mancilla is an employee of, holds shares in, and has received cover/reimbursement of travel/accommodation expenses from Novartis Pharma AG. E Berry-Kravis' institution has received consultancy fees from Novartis Pharma AG and Roche, grants from Novartis, Roche and Seaside Therapeutics for running clinical trials as a site for arbaclofen, RO4917523, and mavoglurant trials. E Berry Kravis has also received honoraria from Novartis Pharma AG to do a preceptorship for personnel working on the mavoglurant project at Novartis and cover/reimbursement of travel/accommodation expenses from Novartis Pharma AG. F Gasparini is an employee of, holds shares in, and has received cover/reimbursement of travel/accommodation expenses from Novartis Pharma AG. F von Raison is an employee of, and holds shares in, Novartis Pharma AG. G Apostol is an employee of, and holds shares in, Novartis Pharma AG; G Apostol's spouse is an employee of Novartis International AG. M Ufer is an employee of, and holds shares in, Novartis Pharma AG. R Hagerman's institution has received grants from Curemark, Novartis Pharma AG, Roche and Seaside Therapeutics, and money from NICHD HD 036071; R Hagerman is an employee of the University of California at Davis, and has received consultancy fees and honoraria from Novartis, honoraria from Genentech, book royalties from Oxford University Press and Johns Hopkins University Press, and cover/reimbursement of travel/accommodation expenses from Novartis Pharma AG. No author has received payment in preparation of this manuscript.Notes

Abstract Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts, dendritic cells and macrophages. Heterozygous loss-of-function mutations in TREM2, including mutations enhancing shedding form the cell surface, have been associated with myelin/neuronal loss and neuroinflammation in neurodegenerative diseases, such as Alzheimer`s disease and Frontotemporal Dementia. Using the cuprizone model, we investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes in cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knockout (TREM2-KO) and wild-type (WT) mice. The TREM2-sol mouse is a new model with selective elimination of plasma membrane TREM2 and a reduced expression of soluble TREM2. In the acute cuprizone model demyelination and remyelination events were reflected by a T2-weighted signal intensity change in magnetic resonance imaging (MRI), most prominently in the external capsule (EC). In contrast to WT and TREM2-IPD, TREM2-sol and TREM2-KO showed an additional increase in MRI signal during the recovery phase. Histological analyses of TREM2-IPD animals revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1 and displayed enhanced cytokine/chemokine levels in the brain. TREM2-sol and, to a much lesser extent, TREM2-KO, however, despite presenting reduced levels of some cytokines/chemokines, showed persistent microgliosis and astrocytosis during recovery, with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. This was accompanied, specifically in the EC, by no myelin recovery, with appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic model consisting of 12-week cuprizone administration followed by 3-week recovery TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation led to increased remyelination, whereas microglia without plasma membrane TREM2 and only soluble TREM2 had reduced phagocytic activity despite efficient lysosomal function, as observed in bone marrow-derived macrophages, leading to a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology following cuprizone intoxication.

Mild cognitive impairment (MCI) is characterized by an abnormal decline in mental and cognitive function compared with normal cognitive aging. It is an underlying condition of Alzheimer’s disease (AD), an irreversible neurodegenerative disease. In recent years, neuroinflammation has been investigated as a new leading target that contributes to MCI progression into AD. Understanding the mechanism underlying inflammatory processes involved in the early onset of the disease could help find a safe and effective way to diagnose and treat patients. In this article, we assessed over twenty different blood and cerebrospinal fluid (CSF) inflammatory biomarker concentrations with immunoassay methods in patients with MCI (mild cognitive impairment), non-impaired control (NIC), and serum healthy control (HC). We performed group comparisons and analyzed in-group correlations between the biomarkers. We included 107 participants (mean age: 64.7 ± 7.8, women: 58.9%). CSF osteopontin and YKL-40 were significantly increased in the MCI group, whereas serum C-reactive protein and interleukin-6 were significantly higher (p &lt; 0.001) in the NIC group compared with the MCI and HC groups. Stronger correlations between interleukin-1β and inflammasome markers were observed in the serum of the MCI group. We confirmed specific inflammatory activation in the central nervous system and interleukin-1β pathway upregulation in the serum of the MCI cohort.

We have previously demonstrated that neuronal release of the excitatory amino acid glutamate is facilitated by the selective activation of presynaptic Group I metabotropic autoreceptors. Here we report the release inhibiting actions of the novel mGlu(5) receptor-selective antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), both in vitro and in vivo. These data provide compelling evidence for the presence of functional positive modulatory mGlu(5) subtype autoreceptors in the mammalian central nervous system.

Our previous work has shown that Group I mGlu receptors participate in thalamic sensory processing in vivo . However, unequivocal demonstration of mGlu5 participation has not been possible due to the lack of specific ligands. We have therefore made a preliminary study of the in vivo actions of the agonist (R,S)‐2‐Chloro‐5‐hydroxyphenylglycine [CHPG] and the novel mGlu5 antagonist 6‐methyl‐2‐(phenylethynyl)‐pyridine [MPEP] in order to characterize their suitability for functional studies. Iontophoretically administered MPEP selectively antagonized excitatory responses of single rat thalamic neurones to CHPG compared to the broad‐spectrum mGlu agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylate. In contrast, the established mGlu1 and mGlu5 antagonist (S)‐4‐carboxyphenylglycine reduced responses to both agonists. These findings are the first demonstration of an in vivo action of CHPG and its antagonism by a selective mGlu5 antagonist. Furthermore MPEP appears to be a good tool for functional studies of mGlu5. British Journal of Pharmacology (1999) 127 , 1057–1059; doi: 10.1038/sj.bjp.0702677

The rate of the reaction p̄p→e+e− has been measured in liquid hydrogen for antiprotons at momentum values between 150 and 300 MeV/c. New precise values of the proton electromagnetic form factor in the time-like region have been obtained assuming |Ge|=|Gm| in the investigated region. The q2 dependence of the form factor close to threshold is found to have a steeper slope than indicated by existing calculations based on vector dominance models.

In this study we assessed the new glutamatergic ligand 11C-ABP688 with regard to the following characteristics: (A) brain distribution, (B) first pass extraction fraction, (C) suitable model to describe tracer kinetics and (D) specificity for the mGlu5 receptor. These parameters were assessed using autoradiography and a beta-scintillator positioned in the striatum. The study included 13 male rats. In 2 animals cerebral blood flow was measured using H215O. The 11C-ABP688 data were analyzed using compartmental modeling. A two-tissue compartment model turned out to fit the data more adequately (parameters: K1, k2′, k3′, k4, total distribution volume DVtot = K1/k2′ (1 + k3′/k4) than a one-tissue compartment model. The autoradiographic studies revealed high uptake in hippocampus, striatum and cortex and low accumulation in thalamus and cerebellum. The uptake was markedly reduced following blockade with the mGlu5 antagonist M-MPEP. The first pass extraction fraction exceeded 85%. Baseline DVtot was 15.16 ± 2.67 ml plasma/ml tissue and decreased by 56, 67 and 72% following blockade with 1, 2 and 6 mg/kg M-MPEP, respectively. These results show that 11C-ABP688 is a promising PET ligand for the quantification of mGlu5 receptors in humans and animals. It readily crosses the blood–brain barrier and binds with high specificity to the mGlu5 receptor. The study furthermore demonstrates the usefulness of a beta-scintillator, if necessary in connection with autoradiography, to evaluate new receptor tracers.

Data are given for the polarization parameter and for the differential cross section in pp elastic scattering at 200 GeV/c, in the range 0.5 ⪕−t ⪕ 4.0GeV2. The polarization changes sign in the dip region, as already observed at 150 GeV/c.

Graphical Abstract Putting the brakes on demyelination: Fingolimod (FTY720) was recently shown to significantly decrease relapse rates in patients with multiple sclerosis. This drug attenuates the trafficking of harmful T-cells entering the brain by regulating sphingosine-1-phosphate (S1P) receptors. We designed, synthesized, evaluated 2H-phthalazin-1-one derivatives (e.g., 1 L) as selective S1P5 receptor agonists; these compounds are highly potent and selective, with good PK properties, and significant activity in oligodendrocytes.

Glutamatergic agents have been conceptualized as powerful, fast-acting alternatives to monoaminergic-based antidepressants. NMDA receptor antagonists such as ketamine or MK-801 are therapeutically effective, but their clinical use is hampered by psychotomimetic effects, accompanied by neurotoxicity in the retrosplenial and cingulate cortex. Antagonists of metabotropic mGlu5 receptors like MPEP elicit both robust antidepressant and anxiolytic effects; however, the underlying mechanisms are yet unknown. mGlu5 receptors closely interact with NMDA receptors, but whether MPEP induces neurotoxicity similar to NMDA receptor antagonists has not been elucidated. We show here using c-Fos brain mapping that MPEP administration results in a restricted activation of distinct stress-related brain areas, including the bed nucleus of stria terminalis (BNST), central nucleus of the amygdala, and paraventricular nucleus of the hypothalamus (PVNH), in a pattern similar to that induced by classical antidepressants and anxiolytics. Unlike the NMDA antagonist MK-801, MPEP does not injure the adult retrosplenial cortex, in which it fails to induce heat shock protein 70 (Hsp70). Moreover, MPEP does not elicit to the same extent as MK-801 apoptosis in cortical areas at perinatal stages, as revealed by caspase 3 expression. These data identify new cellular targets for the anxiolytic and antidepressant effect of MPEP, indicating also in addition that in contrast to MK-801, it lacks the cortical neurotoxicity associated with psychotomimetic side-effects.

ABSTRACT Fenobam is a negative allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5) with inverse agonist activity and is expected to contribute to the treatment of neuropsychiatric disorders involving dysfunction of mGluR5 including Fragile X syndrome. This study examined whether [ 11 C]ABP688, an antagonist PET radioligand, competes with fenobam for the same binding site in the nonhuman primate brain and would allow examination of occupancy‐plasma concentration relationships in the evaluation of the drug for target disorders in the human brain. Four paired PET studies with [ 11 C]ABP688 were performed in baboons at a baseline condition and after intravenous treatment with fenobam at different dose levels (0.3–1.33 mg/kg). Total distribution volume ( V T ) and binding potential (BP ND ) using the cerebellum as a reference region were obtained by the plasma reference graphical method. Then it was examined whether occupancy follows a dose‐dependent, saturating pattern that was predicted by a modified first‐order Hill equation in individual regions. Baseline regional V T and BP ND values agreed with previously published data. Occupancy showed dose‐dependent and saturating patterns in individual regions, reaching &gt;90% occupancy at 1.33 mg/kg dose of fenobam in the majority of regions. To our knowledge, this is the first use of PET to characterize the mGluR5 therapeutic drug fenobam. This study demonstrates a proof of principle for determining the in vivo occupancy of fenobam in primates. The results indicate that [ 11 C]ABP688 and PET may be useful for examination of occupancy of mGluR5 by fenobam, which should prove to be useful for designing future studies and treatment of human disease states. Synapse 68:565–573, 2014 . © 2014 Wiley Periodicals, Inc.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status. We have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity. We identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification.

A novel method was developed to obtain precise timing of muon hits in drift tubes at the first trigger level, and hence to associate a detected muon with the bunch crossing in which it originated. A very good time resolution of ∼ 2 ns was obtained. Some other topics related to muon detection were investigated.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTHighly stereoselective total syntheses of 2,5-anhydro-3-deoxy- and -4-deoxy-D-hexonic acids and of the related deoxyadenosines-CFabrizio Gasparini and Pierre VogelCite this: J. Org. Chem. 1990, 55, 8, 2451–2457Publication Date (Print):April 1, 1990Publication History Published online1 May 2002Published inissue 1 April 1990https://doi.org/10.1021/jo00295a039RIGHTS & PERMISSIONSArticle Views821Altmetric-Citations32LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

The metabotropic glutamate receptor family comprises eight subtypes (mGlu1-8) of G-protein coupled receptors. mGlu receptors have a large extracellular domain which acts as recognition domain for the natural agonist glutamate. In contrast to the ionotropic glutamate receptors which mediate the fast excitatory neurotransmission, mGlu receptors have been shown to play a more modulatory role and have been proposed as alternative targets for pharmacological interventions. The potential use of mGluRs as drug targets for various nervous system pathologies such as anxiety, depression, schizophrenia, pain or Parkinson's disease has triggered an intense search for subtype selective modulators and resulted in the identification of numerous novel pharmacological agents capable to modulate the receptor activity through an interaction at an allosteric site located in the transmembrane domain. The present review presents the most recent developments in the identification and the characterization of allosteric modulators for the mGlu receptors.

In this study, we assessed the whole-body biodistribution and radiation dosimetry of the new glutamatergic ligand (11)C-ABP688. This ligand binds specifically to the metabotropic glutamatergic receptor of subtype 5 (mGluR5).The study included five healthy male volunteers aged 20-29 years. After intravenous injection of 240-260 MBq, a series of four to ten whole-body positron emission tomography/computed tomography scans were initiated, yielding 60-80 min of data. Residence times were then calculated in the relevant organs, and the software packages Mirdose and Olinda were used to calculate the absorbed radiation dose and the effective dose equivalent.Of the excreted (11)C activity at 1 hour, approximately 80% were eliminated via the hepato-biliary pathway and 20% through the urinary tract. The absorbed dose (mGy/MBq) was highest in the liver (1.64 E (-2) +/- 5.08 E (-3)), gallbladder (8.13 E (-3) +/- 5.6 E (-3)), and kidneys (7.27 E (-3) +/- 2.79 E (-3)). The effective dose equivalent was 3.68 +/- 0.84 microSv/MBq. Brain uptake in the areas with high mGluR5 density was 2-3 (SUV). The agreement between the values obtained from Mirdose and the Olinda was excellent.(11)C-ABP688 is a very promising ligand for the investigation of mGluR5 receptors in humans. Brain uptake is high and the effective dose equivalent so low that serial examinations in the same subject seem feasible.

Disturbances of glutamate-mediated neurotransmission have been implicated in a broad range of nervous system disorders. Numerous attempts to correct nervous system dysfunction by pharmacological intervention at glutamate receptors have been made, and some of the approaches have achieved a high level of preclinical validation. However, in a number of cases involving agents acting as blockers of the ionotropic glutamate receptors, clinical success could not be achieved, mostly because of the lack of a therapeutic window. The identification of the metabotropic glutamate receptor (mGluR) family and their modulatory role in the control of neurotransmission provided a new means to alter glutamatergic transmission. Furthermore, selective agents acting as allosteric antagonists at the mGluR5 subtype have demonstrated therapeutic potential. The identification and characterization of mGluR5 antagonists and recent progress in clinical development are summarized.

Partial cross sections for π0 and η0 production are measured at 12 energies. Pion multiplicities up to five are considered.Received 24 July 1968DOI:https://doi.org/10.1103/PhysRevLett.21.845©1968 American Physical Society

The Compact Muon Solenoid (CMS) experiment prepares its Phase-2 upgrade for the high-luminosity era of the LHC operation (HL-LHC). Due to the increase of occupancy, trigger latency and rates, the full electronics of the CMS Drift Tube (DT) chambers will need to be replaced. In the new design, the time bin for the digitization of the chamber signals will be of around 1 ns, and the totality of the signals will be forwarded asynchronously to the service cavern at full resolution. The new backend system will be in charge of building the trigger primitives of each chamber. These trigger primitives contain the information at chamber level about the muon candidates position, direction, and collision time, and are used as input in the L1 CMS trigger. The added functionalities will improve the robustness of the system against ageing. An algorithm based on analytical solutions for reconstructing the DT trigger primitives, called Analytical Method, has been implemented both as a software C++ emulator and in firmware. Its performance has been estimated using the software emulator with simulated and real data samples, and through hardware implementation tests. Measured efficiencies are 96 to 98% for all qualities and time and spatial resolutions are close to the ultimate performance of the DT chambers. A prototype chain of the HL-LHC electronics using the Analytical Method for trigger primitive generation has been installed during Long Shutdown 2 of the LHC and operated in CMS cosmic data taking campaigns in 2020 and 2021. Results from this validation step, the so-called Slice Test, are presented.