Mitochondria transfer-based therapies reduce the morbidity and mortality of Leigh syndrome.

Mitochondria transfer is a recently described phenomenon in which donor cells deliver mitochondria to acceptor cells1-3. One possible consequence of mitochondria transfer is energetic support of neighbouring cells; for example, exogenous healthy mitochondria can rescue cell-intrinsic defects in mitochondrial metabolism in cultured ρ0 cells or Ndufs4-/- peritoneal macrophages4-7. Exposing haematopoietic stem cells to purified mitochondria before autologous haematopoietic stem cell transplantation allowed for treatment of anaemia in patients with large-scale mitochondrial DNA mutations8,9, and mitochondria transplantation was shown to minimize ischaemic damage to the heart10-12, brain13-15 and limbs16. However, the therapeutic potential of using mitochondria transfer-based therapies to treat inherited mitochondrial diseases is unclear. Here we demonstrate improved morbidity and mortality of the Ndufs4-/- mouse model of Leigh syndrome (LS) in multiple treatment paradigms associated with mitochondria transfer. Transplantation of bone marrow from wild-type mice, which is associated with release of haematopoietic cell-derived extracellular mitochondria into circulation and transfer of mitochondria to host cells in multiple organs, ameliorates LS in mice. Furthermore, administering isolated mitochondria from wild-type mice extends lifespan, improves neurological function and increases energy expenditure of Ndufs4-/- mice, whereas mitochondria from Ndufs4-/- mice did not improve neurological function. Finally, we demonstrate that cross-species administration of human mitochondria to Ndufs4-/- mice also improves LS. These data suggest that mitochondria transfer-related approaches can be harnessed to treat mitochondrial diseases, such as LS.

Polymorphisms in Cha o 1 and Cha o 2, major allergens of Japanese cypress (Chamaecyparis obtusa) pollen from a restricted region in Japan.

Japanese cedar pollinosis is a major seasonal allergy in Japan, and Japanese cypress pollinosis is a growing concern because the cypress pollen season follows the cedar pollen season and cross-reactivity among allergens occurs between these closely related species. Allergens purified from pollen under unspecified collecting conditions can potentially heterogenous allergens profiles and batch to batch variability, and amino acid sequence variants in allergens possibly exist among trees. Polymorphisms have not been investigated for the cypress pollen major allergens, Cha o 1 and Cha o 2. Our aim was to examine the homogeneity of allergen amino acid sequences. DNA sequences of Cha o 1 and Cha o 2 from pollen collected from Chiba and Ibaraki prefectures and from needles of 47 plus trees located at seed orchards in Chiba Prefecture were examined by amplicon sequencing and amino acid substitutions were deduced. Sequence analysis of the pollen samples revealed that eight and seven residues of Cha o 2 were polymorphic, respectively. Thirteen residues in Cha o 2, including those residues identified in pollen, were deduced to be polymorphic for the plus trees. Cha o 2 expressed by the 47 plus trees included amino acid differences when compared with that of isoallergen Cha o 2.0101. No substitution was deduced in Cha o 1 for pollen taken from the two prefectures. One conservative amino acid substitution was deduced in Cha o 1 for the plus trees. Of the 47 plus trees examined, 38 were deduced to express only the isoallergen Cha o 1.0101 isoform, whereas eight trees were heterozygous and a single tree was homozygous for the non-synonymous mutation, which indicates relative uniformity of Cha o 1. Cha o 2 was found to be a heterogeneous allergen which suggests that studies using pollen from different trees may not give the same results.

Recombinant Fusion Allergens, Cry j 1 and Cry j 2 from Japanese Cedar Pollen, Conjugated with Polyethylene Glycol Potentiate the Attenuation of Cry j 1-Specific IgE Production in Cry j 1-Sensitized Mice and Japanese Cedar Pollen Allergen-Sensitized Monkeys.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is the most prevalent seasonal rhinitis in Japan. A standardized Japanese cedar pollen extract (CPE) containing 1.5-4.2 µg of Cry j 1 is currently the highest-concentration extract available for allergen-specific immunotherapy (SIT) against this pollinosis. Therefore, we developed a PEGylated fusion protein as a more effective SIT vaccine against Japanese cedar pollinosis. METHODS: The fusion protein of major allergens for Japanese cedar pollen, Cry j 1 and Cry j 2, was expressed in Escherichia coli and conjugated with polyethylene glycol (PEG). The purified PEGylated Cry j 1/2 fusion protein (PEG-fusion) was subcutaneously injected four times into Cry j 1- sensitized mice and CPE-sensitized monkeys. The mice were then subcutaneously challenged with Cry j 1 and serum levels of Cry j 1-specific immunoglobulin, and the proliferation and cytokine production of splenocytes were analyzed. The monkeys were intranasally challenged with CPE and analyzed for Cry j 1-specific immunoglobulin levels in plasma. RESULTS: Cry j 1-specific IgE was significantly attenuated in the PEG-fusion-treated group after Cry j 1-challenge and Cry j 1-specific IgG was significantly increased following PEG-fusion treatment in mice and monkeys. Proliferation and Th2-type cytokine production in splenocytes stimulated with Cry j 1 were also reduced in PEG-fusion-treated mice. IL10 and IL2 production were reduced, but not significantly, while IFN-x03B3; was significantly increased in the PEG-fusion-treated group. CONCLUSIONS: A high-dose injection of PEG-fusion appears to be a valid candidate for a safer and more effective vaccine than the conventional SIT extract for Japanese cedar pollinosis.

Transcriptome profiling of white adipose tissue in a mouse model for 15q duplication syndrome.

Obesity is not only associated with unhealthy lifestyles, but also linked to genetic predisposition. Previously, we generated an autism mouse model (patDp/+) that carries a 6.3 Mb paternal duplication homologous to the human 15q11-q13 locus. Chromosomal abnormalities in this region are known to cause autism spectrum disorder, Prader-Willi syndrome, and Angelman syndrome in humans. We found that, in addition to autistic-like behaviors, patDp/+ mice display late-onset obesity and hypersensitivity to a high-fat diet. These phenotypes are likely to be the results of genetic perturbations since the energy expenditures and food intakes of patDp/+ mice do not significantly differ from those of wild-type mice. Intriguingly, we found that an enlargement of adipose cells precedes the onset of obesity in patDp/+ mice. To understand the underlying molecular networks responsible for this pre-obese phenotype, we performed transcriptome profiling of white adipose tissue from patDp/+ and wild-type mice using microarray. We identified 230 genes as differentially expressed genes. Sfrp5 - a gene whose expression is positively correlated with adipocyte size, was found to be up-regulated, and Fndc5, a potent inducer of brown adipogenesis was identified to be the top down-regulated gene. Subsequent pathway analysis highlighted a set of 35 molecules involved in energy production, lipid metabolism, and small molecule biochemistry as the top candidate biological network responsible for the pre-obese phenotype of patDp/+. The microarray data were deposited in NCBI Gene Expression Omnibus database with accession number GSE58191. Ultimately, our dataset provides novel insights into the molecular mechanism of obesity and demonstrated that patDp/+ is a valuable mouse model for obesity research.

Model mice for 15q11-13 duplication syndrome exhibit late-onset obesity and altered lipid metabolism.

Copy number variations on human chromosome 15q11-q13 have been implicated in several neurodevelopmental disorders. A paternal loss or duplication of the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region confers a risk of obesity, although the mechanism remains a mystery due to a lack of an animal model that accurately recreates the obesity phenotype. We performed detailed analyses of mice with duplication of PWS/AS locus (6 Mb) generated by chromosome engineering and found that animals with a paternal duplication of this region (patDp/+) show late-onset obesity, high sensitivity for high-fat diet, high levels of blood leptin and insulin without an increase in food intake. We show that prior to becoming obese, young patDp/+ mice already had enlarged white adipocytes. Transcriptome analysis of adipose tissue revealed an up-regulation of Secreted frizzled-related protein 5 (Sfrp5), known to promote adipogenesis. We additionally generated a new mouse model of paternal duplication focusing on a 3 Mb region (3 Mb patDp/+) within the PWS/AS locus. These mice recapitulate the obese phenotypes including expansion of visceral adipose tissue. Our results suggest paternally expressed genes in PWS/AS locus play a significant role for the obesity and identify new potential targets for future research and treatment of obesity.

Evaluation of [¹8F]MK-0911, a positron emission tomography (PET) tracer for opioid receptor-like 1 (ORL1), in rhesus monkey and human.

Antagonism of the central opioid receptor like-1 receptor (ORL1) has been implicated in cognition, and has been a focus of drug discovery efforts to ameliorate the cognitive deficits that remain during the stable treatment of schizophrenia with current antipsychotics. In order to facilitate dose selection for phase II clinical testing an ORL1-specific PET tracer was developed to determine drug plasma concentration versus occupancy relationships in order to ensure that the doses selected and the degree of target engagement were sufficient to ensure adequate proof of concept testing. MK-0911 is a selective, high affinity antagonist for the ORL1 receptor radiolabeled with high specific activity (18)F for positron emission tomography (PET) studies. Evaluation of [(18)F]MK-0911 in rhesus monkey PET studies showed a pattern of brain uptake which was consistent with the known distribution of ORL1. In vitro autoradiography with [(18)F]MK-0911 in rhesus monkey and human brain tissue slices showed a regional distribution that was consistent with in vivo imaging results in monkey. Pre-treatment of rhesus monkeys with high doses of structurally diverse ORL1 antagonists MK-0584, MK-0337, or MK-5757 achieved blockade of [(18)F]MK-0911 in all gray matter regions. Baseline PET studies with [(18)F]MK-0911 in healthy human subjects showed tracer distribution and kinetics similar to that observed in rhesus monkey. Quantification of [(18)F]MK-0911 uptake in repeat human baseline PET studies showed a test-retest variability in volume of distribution (V(T)) averaging 3% across brain regions. Humans dosed orally with MK-5757 showed reduced [(18)F]MK-0911 tracer concentration in brain proportional with MK-5757 dose and plasma level. [(18)F]MK-0911 was useful for determining MK-5757-induced receptor occupancy of ORL1 to guide MK-5757 dose-selection for clinical proof-of-concept studies. Additionally, [(18)F]MK-0911 may be a useful tool for studying the pharmacology of ORL1 in various human populations and disease states.

Characterization of metabolic phenotypes of mice lacking GPR61, an orphan G-protein coupled receptor.

AIMS: GPR61 is an orphan G protein-coupled receptor whose function remains unknown. The purpose of the present study is to elucidate the importance of GPR61 in metabolism by characterization of GPR61-deficient mice. MAIN METHODS: Male GPR61-deficient mice were characterized regarding various metabolic parameters, including food intake, body weight, oxygen consumption, body temperature, locomotor activity, and in a pair feeding study. Hypothalamic gene expression was analyzed using real-time quantitative RT-PCR. KEY FINDINGS: GPR61-deficient mice exhibited marked hyperphagia and heavier body weight than wild-type mice. Hyperphagia of GPR61-deficient mice was observed before the differences in body weight became apparent between the genotypes. When body weight difference did become apparent between genotypes, increases in visceral fat pad weight, liver weight, liver triglyceride (TG) content, plasma leptin, and plasma insulin were observed in GPR61-deficient mice, suggesting that GPR61 deficiency caused obesity associated with hyperphagia. Oxygen consumption, body temperature, and locomotor activity were not significantly different between GPR61-deficient and wild-type mice. Pair-fed GPR61-deficient mice had a greater fat mass than wild-type mice despite comparable body weight in both genotypes. The mRNA levels of proopiomelanocortin (POMC) and brain-derived neurotropic factor (BDNF) in the hypothalamus of GPR61-deficient mice were significantly lower than those of wild-type mice. SIGNIFICANCE: GPR61-deficient mice exhibited obesity associated with hyperphagia. These findings suggest that GPR61 is involved in the regulation of food intake and body weight, and may be of importance when considering GPR61 as a therapeutic target for obesity or eating disorders.

Behavioral effects of N-desmethylclozapine on locomotor activity and sensorimotor gating function in mice-Possible involvement of muscarinic receptors.

N-desmethylclozapine (NDMC), a major circulating metabolite of the atypical antipsychotic drug, clozapine, and has M(1) muscarinic receptor partial agonistic property. The purpose of the present study was to examine whether in vivo behavioral effects of NDMC were elicited through the activation of muscarinic receptors. Both a non-selective muscarinic receptor agonist, oxotremorine (0.01-0.1mg/kg), and an M(1) and M(4) muscarinic receptor agonist, xanomeline (0.3-3mg/kg), decreased exploratory locomotor activity in mice. This effect was significantly antagonized by a non-selective muscarinic receptor antagonist, scopolamine, at a dose of 0.3mg/kg without affecting exploratory locomotor activity by itself. NDMC (3-30mg/kg) also decreased exploratory locomotor activity in a dose-dependent manner, and the reduced locomotor activity was significantly antagonized by scopolamine at doses of 0.1 and 0.3mg/kg. These results suggested that NDMC might decrease exploratory locomotor activity at least partly through the activation muscarinic receptors in vivo. NDMC (10-30mg/kg) and clozapine (0.3-1mg/kg) dose-dependently increased prepulse inhibition (PPI) in DBA/2J mice, as did xanomeline (1-3mg/kg). Scopolamine at a dose of 0.3mg/kg without altering PPI by itself significantly antagonized the increase of PPI caused by NDMC (30mg/kg), xanomeline (3mg/kg), and oxotremorine (0.06mg/kg). These findings suggest that the activation of muscarinic receptors may be at least partly responsible for exerting the antipsychotic-like effects of both NDMC and xanomeline in an animal model for schizophrenia.

Ameliorative effect of N-desmethylclozapine in animal models of social deficits and cognitive functions.

One of the major circulating metabolites of clozapine, N-desmethylclozapine (NDMC), has been demonstrated to exhibit partial agonistic activity at M(1) muscarinic receptors. Some of the unique therapeutic effects of clozapine might involve the pharmacological effects of this metabolite. The purpose of the present study was therefore to examine whether NDMC improved behavioral abnormalities in animal models of social deficits and cognitive deficits of schizophrenia. NDMC (3mg/kg) and clozapine (1-3mg/kg) each improved the reduction of social interaction caused by a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, 5R,10S-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) (0.1mg/kg), without affecting locomotor activity in rats. NDMC (3-10mg/kg) and clozapine (1-3mg/kg) each also resulted in better discrimination of a novel from a familiar object 24h after a training trial in a rat object recognition test. These findings suggest that NDMC can improve behavior in animal models of social deficits and cognitive deficits of schizophrenia as well as clozapine.

Antipsychotic effects of N-desmethylclozapine on sensorimotor gating function in rats--possible involvement of activation of M(1) muscarinic receptors.

N-desmethylclozapine (NDMC), one of the major metabolites of clozapine, has been demonstrated to exhibit partial agonistic activity at M(1) muscarinic receptors in vitro. Behavioral effects of NDMC were examined to determine whether NDMC contributed to the antipsychotic effects of clozapine via activation of muscarinic receptors. Both NDMC (10-30 mg/kg) and its parent compound clozapine (3-10 mg/kg) antagonized the disruption of prepulse inhibition (PPI) caused by the indirect dopamine agonist methamphetamine (3 mg/kg) in rats. However, NDMC (30 mg/kg) did not increase plasma levels of prolactin in rats. The same dose ranges of NDMC antagonized the disruption of PPI caused by the N-methyl-D-aspartate receptor antagonist ketamine (5 mg/kg) in rats. Furthermore, NDMC in the same dose ranges antagonized the disruption of PPI caused by the muscarinic receptor antagonist scopolamine (0.3 mg/kg) in rats. These findings suggest that NDMC has potent antipsychotic effects in animal models to examine sensorimotor gating function, and that NDMC may act through the activation of a muscarinic receptor for the treatment of schizophrenia.

Selective M3 muscarinic receptor antagonist inhibits small-cell lung carcinoma growth in a mouse orthotopic xenograft model.

In small cell lung carcinoma (SCLC), acetylcholine (ACh) is synthesized and secreted, and it acts as an autocrine growth factor through activation of its receptors, muscarinic receptor (mAChR) and nicotinic receptor (nAChR). Alteration of tumor growth by blockade of M(3) mAChR in a human SCLC cell line, NCI-H82, was investigated in the present study. We used a highly selective M(3) muscarinic antagonist, N-(2-[3-([3R]-1-(cyclohexylmethyl)-3-piperidinyl]methylamino)-3-oxopropyl]amino-2-oxoethyl)-3,3,3-triphenyl-propioamide (J-115311). Our results show that J-115311 inhibited the increased intracellular calcium elicited by carbachol, a muscarinic agonist, in SCLC cells. J-115311 also inhibited SCLC cell growth in vitro. In a mouse orthotopic xenograft model, J-115311 dose-dependently reduced tumor growth when NCI-H82 cells were inoculated into the upper left lobe of the lung. These findings indicate that blockade of M(3) mAChR can suppress tumor growth in SCLC, suggesting the potential therapeutic utility of M(3) muscarinic antagonists as anti-cancer agents.

Synthesis, characterization, and monkey positron emission tomography (PET) studies of [18F]Y1-973, a PET tracer for the neuropeptide Y Y1 receptor.

Neuropeptide Y receptor subtype 1 (NPY Y1) has been implicated in appetite regulation, and antagonists of NPY Y1 are being explored as potential therapeutics for obesity. An NPY Y1 PET tracer is useful for determining the level of target engagement by NPY Y1 antagonists in preclinical and clinical studies. Here we report the synthesis and evaluation of [(18)F]Y1-973, a novel PET tracer for NPY Y1. [(18)F]Y1-973 was radiolabeled by reaction of a primary chloride with [(18)F]KF/K2.2.2 followed by deprotection with HCl. [(18)F]Y1-973 was produced with high radiochemical purity (>98%) and high specific activity (>1000 Ci/mmol). PET studies in rhesus monkey brain showed that the distribution of [(18)F]Y1-973 was consistent with the known NPY Y1 distribution; uptake was highest in the striatum and cortical regions and lowest in the pons, cerebellum nuclei, and brain stem. Blockade of [(18)F]Y1-973 uptake with NPY Y1 antagonist Y1-718 revealed a specific signal that was dose-dependently reduced in all regions of grey matter to a similarly low level of tracer uptake, indicative of an NPY Y1 specific signal. In vitro autoradiographic studies with [(18)F]Y1-973 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the in vivo PET studies. Highest binding density was observed in the dentate gyrus, caudate-putamen, and cortical regions; moderate binding density in the hypothalamus and thalamus; and lowest binding density in the globus pallidus and cerebellum. In vitro saturation binding studies in rhesus monkey and human caudate-putamen homogenates confirmed a similarly high B(max)/K(d) ratio for [(18)F]Y1-973, suggesting the tracer may provide a specific signal in human brain of similar magnitude to that observed in rhesus monkey. [(18)F]Y1-973 is a suitable PET tracer for imaging NPY Y1 in rhesus monkey with potential for translation to human PET studies.

Synthesis, characterization, and monkey PET studies of [¹8F]MK-1312, a PET tracer for quantification of mGluR1 receptor occupancy by MK-5435.

Two moderately lipophilic, high affinity ligands for metabotropic glutamate receptor subtype 1 (mGluR1) were radiolabeled with a positron-emitting radioisotope and evaluated in rhesus monkey as potential PET tracers. Both ligands were radiolabeled with fluorine-18 via nucleophilic displacement of the corresponding 2-chloropyridine precursor with [¹8F]potassium fluoride. [¹8F]MK-1312 was found to have a suitable signal for quantification of mGluR1 receptors in nonhuman primates and was more thoroughly characterized. In vitro autoradiographic studies with [¹8F]MK-1312 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the known distribution of mGluR1, with the highest uptake in the cerebellum, moderate uptake in the hippocampus, thalamus, and cortical regions, and lowest uptake in the caudate and putamen. In vitro saturation binding studies in rhesus monkey and human cerebellum homogenates confirmed that [¹8F]MK-1312 binds to a single site with a B(max) /K(d) ratio of 132 and 98, respectively. PET studies in rhesus monkey with [¹8F]MK-1312 showed high brain uptake and a regional distribution consistent with in vitro autoradiography results. Blockade of [¹8F]MK-1312 uptake with mGluR1 allosteric antagonist MK-5435 dose-dependently reduced tracer uptake in all regions of gray matter to a similarly low level of tracer uptake. This revealed a large specific signal useful for determination of mGluR1 receptor occupancy in rhesus monkey. Taken together, these results are promising for clinical PET studies with [¹8F]MK-1312 to determine mGluR1 occupancy of MK-5435.

Effects of a novel metabotropic glutamate receptor 7 negative allosteric modulator, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one (MMPIP), on the central nervous system in rodents.

We recently identified 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), the first allosteric metabotropic glutamate (mGlu) 7 receptor-selective negative allosteric modulator. In this study, we examined the in vivo pharmacological effects of MMPIP on the central nervous system. MMPIP was distributed into the brain after systemic administration in both mice and rats. Pharmacokinetic study revealed that the half-life of MMPIP in circulation was about 1h in rats. Results of various behavioral studies revealed that MMPIP impaired non-spatial and spatial cognitive performances in the object recognition test and the object location test in mice, respectively. In rats, MMPIP increased time to complete the task in the 8-arm radial maze test without increasing error. In addition to impairing cognition, MMPIP decreased social interaction with reduction of line crossing in rats, while MMPIP had no effects on locomotor activity in rats and mice, rota-rod performance in mice, prepulse inhibition in rats, maternal separation-induced ultrasonic vocalization in rat pups, stress-induced hyperthermia in mice, or the tail suspension test in mice. No analgesic effects of MMPIP were detected in either the tail immersion test or formalin test in mice. MMPIP did not alter the threshold for induction of seizures by electrical shock or pentylenetetrazole in mice. These findings suggest that blockade of mGlu(7) receptors by MMPIP may modulate both non-spatial and spatial cognitive functions without non-selective inhibitory effects on the central nervous system.

Pharmacological effects of metabotropic glutamate receptor ligands on prepulse inhibition in DBA/2J mice.

Schizophrenic patients typically exhibit impairment of sensorimotor gating, which can be modeled in animals using acoustic prepulse inhibition of the startle. Both classical and atypical antipsychotics have been shown to improve prepulse inhibition in DBA/2J mice, a non-pharmacological model for impaired sensorimotor gating. The purpose of the present study was to clarify whether metabotropic glutamate receptors participate in control of sensorimotor gating. We evaluated various metabotropic glutamate receptor ligands on prepulse inhibition in DBA/2J mice. This basal level of prepulse inhibition in DBA/2J mice was increased by only the mGlu(1) receptor antagonists [2-cyclopropyl-5-[1-(2-fluoro-3-pyridinyl)-5-methyl-1H-1,2,3-triazol-4-yl]-2,3-dihydro-1H-isoindol-1-one] (CFMTI), 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-alpha]benzimidazole-2-carboxamide hydrochloride (YM-298198), and (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone (JNJ16259685). There was no effect after treatments with the mGlu(5) receptor antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), the mGlu(2/3) receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), the mGlu(2/3) receptor antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), the mGlu(7) receptor agonist N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), the mGlu(7) receptor antagonist 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one (MMPIP), or the mGlu(8) receptor agonist (S)-3,4-dicarboxyphenylglycine (DCPG). These findings indicate that inhibition of mGlu(1) receptor selectively increases prepulse inhibition in DBA/2J mice and suggest that mGlu(1) receptor antagonists could be a novel treatment for some aspects of schizophrenia.

Regulation of spontaneous Ca(2+) spikes by metabotropic glutamate receptors in primary cultures of rat cortical neurons.

Periodic and spontaneous Ca(2+) spikes are observed in neurons during development of the central nervous system, and spontaneous changes in intracellular Ca(2+) concentration in neurons play important roles in the development of neural circuits. To clarify the roles of metabotropic glutamate receptors (mGluRs) in the regulation of spontaneous Ca(2+) spikes, we investigated the effects of selective and nonselective mGluRs ligands on primary cultures of rat cortical neurons. Cultured cortical neurons expressed all eight mGluR subtypes on reverse transcription-PCR. The mGluR2 and mGluR3 agonists LY379268, LY354740, and (2R,4R)-APDC increased the amplitude but decreased the frequency of spontaneous Ca(2+) spikes in cultured cortical neurons. The effects of these mGluR2 and mGluR3 agonists were completely inhibited by the presence of a potent mGluR2 and mGluR3 antagonist, LY341495, and by pretreatment with pertussis toxin. No significant effect was observed with either activation or inhibition of mGluR1, mGluR4, mGluR5, mGluR6, mGluR7, and mGluR8 on the spontaneous Ca(2+) spikes in cultured cortical neurons. These findings indicate that, among mGluRs, the group II mGluR subtypes mGluR2 and mGluR3 play principal roles in modulation of spontaneous Ca(2+) spikes.

Development of a beta-lactamase reporter gene assay for metabotropic glutamate receptor 1 by using coexpression of glutamate transporter.

mGluR1 antagonists have been postulated to be novel CNS drugs, including antipsychotics. Toward this end, the authors developed a beta-lactamase reporter assay to identify mGluR1 antagonists. beta-Lactamase has several interesting features for high-throughput screening, including very high sensitivity and less well-to-well variation than other reporter enzymes. mGluR1-expressing Chinese hamster ovary (CHO) cells with the beta-lactamase gene under control of the nuclear factor of activated T cells (NFAT) promoter (CHO-NFAT-bla-hmGluR1b) exhibited very high basal activity, resulting in an inadequate signal-to-basal (S/B) ratio. Coexpression of glutamate/aspartate transporter (GLAST) with mGluR1 in the cell line (CHO-NFAT-bla-hmGluR1b-GLAST) dramatically decreased basal activity and improved the S/B ratio (from 2- to 20-fold). The contribution of GLAST to lowering basal activity and increasing the S/B ratio was validated by the expression level of GLAST mRNA and by a GLAST inhibitor. Antagonistic activities of known mGluR1 antagonists in the beta-lactamase reporter assay were comparable with those in the conventional Ca(2+) mobilization assay. The Z' factor of the beta-lactamase reporter assay was 0.89 under optimized conditions. Taken together, the beta-lactamase reporter assay with CHO-NFAT-bla-hmGluR1b-GLAST could be a novel high-throughput assay for mGluR1 antagonist screening. This is the first description of a successful beta-lactamase reporter assay among all mGluR subtypes.

Isoxazolopyridone derivatives as allosteric metabotropic glutamate receptor 7 antagonists.

This Letter describes the synthesis and evaluation of mGluR7 antagonists in the isoxazolopyridone series. In the course of modification in this class, novel solid support synthesis of the isoxazolopyridone scaffold was developed. Subsequent chemical modification led to the identification of several potent derivatives with improved physicochemical properties compared to a hit compound 1. Among these, 2 showed good oral bioavailability and brain penetrability, suggesting that 2 may be useful for in vivo study to elucidate the role of mGluR7.

Discovery and in vitro and in vivo profiles of 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide as novel class of an orally active metabotropic glutamate receptor 1 (mGluR1) antagonist.

We identified 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide 27 as a potent mGluR1 antagonist. The compound possessed excellent subtype selectivity and good PK profile in rats. It also demonstrated relatively potent antipsychotic-like effects in several animal models. Suitable for development as a PET tracer, compound 27 would have great potential for elucidation of mGluR1 functions in human.