Sex-specific involvement of the Notch-JAG pathway in social recognition.

Under the hypothesis that olfactory neural epithelium gene expression profiles may be useful to look for disease-relevant neuronal signatures, we examined microarray gene expression in olfactory neuronal cells and underscored Notch-JAG pathway molecules in association with schizophrenia (SZ). The microarray profiling study underscored JAG1 as the most promising candidate. Combined with further validation with real-time PCR, downregulation of NOTCH1 was statistically significant. Accordingly, we reverse-translated the significant finding from a surrogate tissue for neurons, and studied the behavioral profile of Notch1+/- mice. We found a specific impairment in social novelty recognition, whereas other behaviors, such as sociability, novel object recognition and olfaction of social odors, were normal. This social novelty recognition deficit was male-specific and was rescued by rapamycin treatment. Based on the results from the animal model, we next tested whether patients with psychosis might have male-specific alterations in social cognition in association with the expression of NOTCH1 or JAG1. In our first episode psychosis cohort, we observed a specific correlation between the expression of JAG1 and a face processing measure only in male patients. The expression of JAG1 was not correlated with any other cognitive and symptomatic scales in all subjects. Together, although we acknowledge the pioneering and exploratory nature, the present work that combines both human and animal studies in a reciprocal manner suggests a novel role for the Notch-JAG pathway in a behavioral dimension(s) related to social cognition in psychotic disorders in a male-specific manner.

Identification of potent lysophosphatidic acid receptor 5 (LPA5) antagonists as potential analgesic agents.

Lysophosphatidic acid (LPA) plays an important role in a variety of cellular functions. In particular, LPA5 receptor is highly expressed in spinal cord and dorsal root ganglion, which are associated with pain. This fact prompted us to hypothesize that LPA5 antagonists show analgesic effects. To search for potent LPA5 antagonists with blood brain barrier (BBB) permeability, we conducted high throughput screening (HTS). In HTS campaign, we found a 2H-isoquinoline-1-one scaffold showing antagonistic activity against LPA5 and synthesized a series of 2H-isoquinoline-1-one derivatives and evaluated their LPA5 activities. Among these compounds, compound 7e showed potent LPA5 activity with an IC50 value of 0.12 µM, and acceptable BBB permeability. Furthermore, it showed effective analgesic effect in a chronic constriction injury rat model. Therefore, 7e may have a potential as novel pain therapeutic approach.

Analgesic effects of novel lysophosphatidic acid receptor 5 antagonist AS2717638 in rodents.

Lysophosphatidic acid (LPA) is a bioactive lipid that acts via at least six G protein-coupled receptors, LPA receptors 1-6 (LPA1-6), for various physiological functions. We examined (1) whether LPA5 is involved in pain signaling in the spinal cord; and (2) the pharmacological effects of a novel LPA5 antagonist on intrathecal prostaglandin (PG)- and (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced allodynia, and neuropathic and inflammatory pain in rodents. Intrathecal injection of a selective LPA5 agonist, geranylgeranyl diphosphate, and a non-selective agonist, LPA, induced allodynia in wild type, but not in LPA5 knockout mice. These novel results suggest that LPA5 is important for pain signal transmission in the spinal cord. AS2717638 (6,7-dimethoxy-2-(5-methyl-1,2-benzoxazol-3-yl)-4-(piperidin-1-ylcarbonyl)isoquinolin-1(2H)-one) bound to the LPA-binding site on LPA5 and selectively inhibited LPA-induced cyclic adenosine monophosphate accumulation in human LPA5-but not LPA1-, 2-, or 3-expressing cells. Further, oral administration of AS2717638 inhibited LPA5 agonist-induced allodynia in mice. AS2717638 also significantly improved PGE2-, PGF2α-, and AMPA-induced allodynia, while both pregabalin and duloxetine alleviated only PGE2-induced allodynia in mice. Similarly, AS2717638 significantly ameliorated static mechanical allodynia and thermal hyperalgesia in rat models of chronic constriction injury (CCI)-induced neuropathic pain. AS2717638 also showed analgesic effects in a rat model of inflammatory pain. These findings suggest that LPA5 antagonists elicit broad analgesic effects against both neuropathic and inflammatory pain. Accordingly, pharmacological LPA5 antagonists are attractive development candidates for potential novel pain therapies.

Deficits in microRNA-mediated Cxcr4/Cxcl12 signaling in neurodevelopmental deficits in a 22q11 deletion syndrome mouse model.

22q11 deletion syndrome (22q11DS) frequently accompanies psychiatric conditions, some of which are classified as schizophrenia and bipolar disorder in the current diagnostic categorization. However, it remains elusive how the chromosomal microdeletion leads to the mental manifestation at the mechanistic level. Here we show that a 22q11DS mouse model with a deletion of 18 orthologous genes of human 22q11 (Df1/+ mice) has deficits in migration of cortical interneurons and hippocampal dentate precursor cells. Furthermore, Df1/+ mice show functional defects in Chemokine receptor 4/Chemokine ligand 12 (Cxcr4/Cxcl12; Sdf1) signaling, which reportedly underlie interneuron migration. Notably, the defects in interneuron progenitors are rescued by ectopic expression of Dgcr8, one of the genes in 22q11 microdeletion. Furthermore, heterozygous knockout mice for Dgcr8 show similar neurodevelopmental abnormalities as Df1/+ mice. Thus, Dgcr8-mediated regulation of microRNA is likely to underlie Cxcr4/Cxcl12 signaling and associated neurodevelopmental defects. Finally, we observe that expression of CXCL12 is decreased in olfactory neurons from sporadic cases with schizophrenia compared with normal controls. Given the increased risk of 22q11DS in schizophrenia that frequently shows interneuron abnormalities, the overall study suggests that CXCR4/CXCL12 signaling may represent a common downstream mediator in the pathophysiology of schizophrenia and related mental conditions.

Knockdown of DISC1 by in utero gene transfer disturbs postnatal dopaminergic maturation in the frontal cortex and leads to adult behavioral deficits.

Adult brain function and behavior are influenced by neuronal network formation during development. Genetic susceptibility factors for adult psychiatric illnesses, such as Neuregulin-1 and Disrupted-in-Schizophrenia-1 (DISC1), influence adult high brain functions, including cognition and information processing. These factors have roles during neurodevelopment and are likely to cooperate, forming pathways or "signalosomes." Here we report the potential to generate an animal model via in utero gene transfer in order to address an important question of how nonlethal deficits in early development may affect postnatal brain maturation and high brain functions in adulthood, which are impaired in various psychiatric illnesses such as schizophrenia. We show that transient knockdown of DISC1 in the pre- and perinatal stages, specifically in a lineage of pyramidal neurons mainly in the prefrontal cortex, leads to selective abnormalities in postnatal mesocortical dopaminergic maturation and behavioral abnormalities associated with disturbed cortical neurocircuitry after puberty.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia.

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3' UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

Distinct localization of prokineticin 2 and prokineticin receptor 2 mRNAs in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian clock that regulates physiological and behavioral circadian rhythms in mammals. Prokineticin 2 (PK2) is highly expressed in the SCN, and its involvement in the generation of circadian locomotor activity has been reported previously. In the present study, using in situ hybridization methods, we investigated the localization of PK2 and prokineticin receptor 2 (PKR2), a specific receptor for PK2, in the rat SCN. In steady light : dark (L : D = 12 : 12 h) and constant dark conditions, rPK2 mRNA displayed a robust circadian oscillation with a peak occurring during the day. Moreover, during peak expression, the rPK2 mRNA-positive neurons were scattered in both the dorsomedial and ventrolateral SCN, which are two functionally and morphologically distinct subregions. Furthermore, double-labeling in situ hybridization experiments revealed that greater than 50% of the rPK2 mRNA-containing neurons co-expressed either vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) or arginine vasopressin (AVP) in the SCN. In contrast, the rPKR2 mRNA levels did not show significant diurnal alterations. rPKR2 mRNA-containing neurons were also clustered in the dorsolateral part of the SCN, which shows negligible labeling of either rAVP, rVIP, rGRP or rPK2 transcripts. In addition, this region exhibited a delayed cycling of the rPer1 gene. These results suggest an intrinsic PK2 neurotransmission and functionally distinct roles for PKR2-expressing neurons in the SCN.

Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2.

Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-)but Pkr2(-/-)mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.

Molecular cloning of monkey histamine H4 receptor.

Histamine H(4) receptor is considered as a novel therapeutic target for allergic diseases. To enhance the knowledge about species difference, which is essential for drug discovery research, monkey H(4) receptor was identified. Monkey H(4) receptor was characterized to have comparable similarity with its human counterpart. Discovery of monkey H(4) receptor will contribute to a better interpretation of effective drug discovery.

Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides.

Proadrenomedullin N-terminal 20 peptide (PAMP[1-20]/PAMP-20) and its truncated analog, PAMP[9-20]/PAMP-12, are endogenous peptides that elicit hypotension through inhibiting catecholamine secretion from sympathetic nerve endings and adrenal chromaffin cells. Although the binding sites for PAMP are widely distributed, the nature of its receptor has been elusive. In an effort to identify potential PAMP receptor(s), we found that a human G-protein-coupled receptor, MrgX2, was specifically activated by PAMP. Although a previous study revealed that MrgX2 was a receptor for cortistatin, a neuropeptide involved in sleep regulation and locomotor activity, our present data indicated that the rank order of the agonistic effect against MrgX2 was "PAMP-12> or =cortistatin>PAMP-20". These activities were confirmed by the inhibition of the forskolin-elevated cAMP accumulation, Ca(2+) mobilization, and [(35)S]guanosine 5'-(gamma-thio)triphosphate binding assays. These findings suggest that MrgX2 couples with not only G(alpha q) but also G(alpha i), consistent with previous reports on the pharmacological profile of PAMP signaling. Furthermore, by immunostaining, we found that MrgX2 was expressed in the adrenal chromaffin cells as well as the dorsal root ganglia. From these results, we concluded that MrgX2 is a potential human PAMP-12 receptor that regulates catecholamine secretion from adrenal glands. The present discovery will eventually lead to a better understanding of the pathophysiological role of proadrenomedullin peptides.

Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor.

A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.

Molecular identification of nicotinic acid receptor.

Nicotinic acid and its derivative, Acipimox, have been widely used in the treatment of hyperlipidemia. Pharmacological studies have demonstrated that they exert the beneficial effect through the activation of a Gi-protein-coupled receptor on adipocyte, which has remained elusive to date. Here we show that a novel GPCR, designated HM74b because of its high similarity to HM74, is a receptor for nicotinic acid. HM74b mRNA is found in human, murine, and rat adipose tissues. Nicotinic acid and Acipimox inhibit forskolin-stimulated intracellular cAMP accumulation in human HM74b-expressing cells and activate GTP gamma S binding in a dose-dependent manner. [3H]Nicotinic acid specifically binds to HM74b-expressing membrane and its binding is replaced by Acipimox. This finding will open a new phase of research on the physiological role of nicotinic acid and will be a clue to develop novel antihyperlipidemic drugs.

Molecular cloning and characterization of prokineticin receptors.

Recent studies have identified two novel biofunctional proteins, termed prokineticin 1/EG-VEGF and prokineticin 2, which were mammalian homologues of mamba MIT1 and frog Bv8. Prokineticins have been demonstrated to exert their physiological functions through G-protein coupled receptors (GPCRs). In this study, we report the molecular identification of two endogenous prokineticin receptors, designated PK-R1 and PK-R2, through a search of the human genomic DNA database. PK-R1, locating in chromosome 2, and PK-R2, locating in chromosome 20p13, shared 87% homology, which was an extremely high value among known GPCRs. In functional assays, mammalian cells expressing PK-Rs responded to prokineticins in a concentration-dependent manner. Tissue distribution analysis revealed that expression of PK-R1 was observed in the testis, medulla oblongata, skeletal muscle and skin, while that of PK-R2 showed preferential expression in the central nervous system. The tissue distribution of PK-Rs reported in this paper suggests that the prokineticins play multifunctional roles in vivo.

cDNA cloning and characterization of porcine histamine H4 receptor.

The cDNA encoding histamine H4 receptor was cloned from the porcine spleen cDNA library. Porcine H4 receptor, which shares 72% homology with its human counterpart, bound to histamine in receptor-expressing mammalian cells. Isolation of the porcine H4 receptor, which is important for understanding of the pharmacology, will aid in better interpretation of physiological role of this subtype of histamine receptor.

Two budding yeast RAD4 homologs in fission yeast play different roles in the repair of UV-induced DNA damage.

We have identified two fission yeast homologs of budding yeast Rad4 and human xeroderma pigmentosum complementation group C (XP-C) correcting protein, designated Rhp4A and Rhp4B. Here we show that the rhp4 genes encode NER factors that are required for UV-induced DNA damage repair in fission yeast. The rhp4A-deficient cells but not the rhp4B-deficient cells are sensitive to UV irradiation. However, the disruption of both rhp4A and rhp4B resulted in UV sensitivity that was greater than that of the rhp4A-deficient cells, revealing that Rhp4B plays a role in DNA repair on its own. Fission yeast has two pathways to repair photolesions on DNA, namely, nucleotide excision repair (NER) and UV-damaged DNA endonuclease-dependent excision repair (UVER). Studies with the NER-deficient rad13 and the UVER-deficient (Delta)uvde mutants showed the two rhp4 genes are involved in NER and not UVER. Assessment of the ability of the various mutants to remove cyclobutane pyrimidine dimers (CPDs) from the rbp2 gene locus indicated that Rhp4A is involved in the preferential repair of lesions on the transcribed DNA strand and plays the major role in fission yeast NER. Rhp4B in contrast acts as an accessory protein in non-transcribed strand (NTS) repair.