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Background Fragile X syndrome (FXS) is a genetic condition that causes a range of developmental problems, including intellectual disability, aggressive behavior, anxiety, abnormal sensory processing, and cognitive impairment. Despite intensive preclinical research in Fmr1-targeted transgenic mice, an effective treatment for FXS has yet to be developed. We previously demonstrated that ASP5736, a 5-Hydroxytryptamine (serotonin) receptor 5A receptor antagonist, ameliorated scopolamine-induced working memory deficits in mice, reference memory impairment in aged rats, and methamphetamine-induced positive symptoms and phencyclidine-induced cognitive impairment in animal models of schizophrenia. We hypothesized that ASP5736 may be effective for ameliorating similar behavior deficits in male Fmr1-targeted transgenic rats as a preclinical model of FXS. Methods We evaluated the effect of acute oral administration of ASP5736 on the abnormal behavior of hyperactivity (0.01, 0.1 mg/kg), prepulse inhibition (0.01, 0.03, 0.1 mg/kg), and the novel object recognition task (0.1 mg/kg) in Frmr1-knockout (KO) rats. Results Fmr1-KO rats showed body weight gain, hyperactivity, abnormal sensory motor gating, and cognitive impairment. ASP5736 (0.1 mg/kg) reversed the hyperactivity and ameliorated the sensory motor gating deficits (0.03–0.1 mg/kg). ASP5736 (0.01 mg/kg) also improved cognitive impairment. Conclusions ASP5736 is a potential drug candidate for FXS. Further studies are needed to confirm its clinical efficacy.

The transcription factor nuclear factor kappa B (NF-κB) regulates the expression of many inflammatory genes that are overexpressed in a subset of people with schizophrenia. Transcriptional reduction in one NF-κB inhibitor, Human Immunodeficiency Virus Enhancer Binding Protein 2 (HIVEP2), is found in the brain of patients, aligning with evidence of NF-κB over-activity. Cellular co-expression of HIVEP2 and cytokine transcripts is a prerequisite for a direct effect of HIVEP2 on pro-inflammatory transcription, and we do not know if changes in HIVEP2 and markers of neuroinflammation are occurring in the same brain cell type. We performed in situ hybridisation on postmortem dorsolateral prefrontal cortex tissue to map and compare the expression of HIVEP2 and Serpin Family A Member 3 (SERPINA3), one of the most consistently increased inflammatory genes in schizophrenia, between schizophrenia patients and controls. We find that HIVEP2 expression is neuronal and is decreased in almost all grey matter cortical layers in schizophrenia patients with neuroinflammation, and that SERPINA3 is increased in the dorsolateral prefrontal cortex grey matter and white matter in the same group of patients. We are the first to map the upregulation of SERPINA3 to astrocytes and to some neurons, and find evidence to suggest that blood vessel-associated astrocytes are the main cellular source of SERPINA3 in the schizophrenia cortex. We show that a lack of HIVEP2 in mice does not cause astrocytic upregulation of Serpina3n but does induce its transcription in neurons. We speculate that HIVEP2 downregulation is not a direct cause of astrocytic pro-inflammatory cytokine synthesis in schizophrenia but may contribute to neuronally-mediated neuroinflammation.

Cognitive deficit remains an intractable symptom of schizophrenia, accounting for substantial disability. Despite this, little is known about the cause of cognitive dysfunction in schizophrenia. Recent studies suggest that schizophrenia patients show several changes in dentate gyrus structure and functional characteristic of immaturity. The immature dentate gyrus (iDG) has been replicated in several mouse models, most notably the CaMKIIα heterozygous mouse (CaMKIIα-hKO). The current study characterizes behavioral phenotypes of CaMKIIα-hKO mice and determines their neurophysiological profile using electroencephalogram (EEG) recording from hippocampus. CaMKIIα-hKO mice were hypoactive in home-cage environment; however, they displayed less anxiety-like phenotype, suggestive of impulsivity-like behavior. In addition, severe cognitive dysfunction was evident in CaMKIIα-hKO mice as examined by novel object recognition and contextual fear conditioning. Several EEG phenomena established in both patients and relevant animal models indicate key pathological changes associated with the disease, include auditory event-related potentials and time-frequency EEG oscillations. CaMKIIα-hKO mice showed altered event-related potentials characterized by an increase in amplitude of the N40 and P80, as well as increased P80 latency. These mice also showed increased power in theta range time-frequency measures. Additionally, CaMKIIα-hKO mice showed spontaneous bursts of spike wave activity, possibly indicating absence seizures. The GABAB agonist baclofen increased, while the GABAB antagonist CGP35348 and the T-Type Ca2

Autism spectrum disorder (ASD) is often signaled by atypical cries during infancy. Copy number variants (CNVs) provide genetically identifiable cases of ASD, but how early atypical cries predict a later onset of ASD among CNV carriers is not understood in humans. Genetic mouse models of CNVs have provided a reliable tool to experimentally isolate the impact of CNVs and identify early predictors for later abnormalities in behaviors relevant to ASD. However, many technical issues have confounded the phenotypic characterization of such mouse models, including systematically biased genetic backgrounds and weak or absent behavioral phenotypes. To address these issues, we developed a coisogenic mouse model of human proximal 16p11.2 hemizygous deletion and applied computational approaches to identify hidden variables within neonatal vocalizations that have predictive power for postpubertal dimensions relevant to ASD. After variables of neonatal vocalizations were selected by least absolute shrinkage and selection operator (Lasso), random forest, and Markov model, regression models were constructed to predict postpubertal dimensions relevant to ASD. While the average scores of many standard behavioral assays designed to model dimensions did not differentiate a model of 16p11.2 hemizygous deletion and wild-type littermates, specific call types and call sequences of neonatal vocalizations predicted individual variability of postpubertal reciprocal social interaction and olfactory responses to a social cue in a genotype-specific manner. Deep-phenotyping and computational analyses identified hidden variables within neonatal social communication that are predictive of postpubertal behaviors.

Fragile X syndrome (FXS) is characteristically displayed intellectual disability, hyperactivity, anxiety, and abnormal sensory processing. Electroencephalography (EEG) abnormalities are also observed in subjects with FXS, with many researchers paying attention to these as biomarkers. Despite intensive preclinical research using Fmr1 knock out (KO) mice, an effective treatment for FXS has yet to be developed. Here, we examined Fmr1-targeted transgenic rats (Fmr1-KO rats) as an alternative preclinical model of FXS. We characterized the EEG phenotypes of Fmr1-KO rats by measuring basal EEG power and auditory steady state response (ASSR) to click trains of stimuli at a frequency of 10–80 Hz. Fmr1-KO rats exhibited reduced basal alpha power and enhanced gamma power, and these rats showed enhanced locomotor activity in novel environment. While ASSR clearly peaked at around 40 Hz, both inter-trial coherence (ITC) and event-related spectral perturbation (ERSP) were significantly reduced at the gamma frequency band in Fmr1-KO rats. Fmr1-KO rats showed gamma power abnormalities and behavioral hyperactivity that were consistent with observations reported in mouse models and subjects with FXS. These results suggest that gamma power abnormalities are a translatable biomarker among species and demonstrate the utility of Fmr1-KO rats for investigating drugs for the treatment of FXS.

Appropriate axonal pathfinding is a necessary step for the function of neuronal circuits. The mossy fibers (MFs) in the hippocampus of CaMKIIα heterozygous knockout (CaMKIIα-hKO) psychiatric model mice project onto not only the stratum lucidum but also the stratum oriens region in the CA3, which is a projection pattern distinct from that in normal mice. Thus, we examined the electrophysiological properties of the MF-CA3 connection in this mutant mouse on field recordings and found a lower synaptic connection. This study suggested that the phenotype of abnormal MF pathfindings could induce aberrant neuronal functions, which may link to cognition and memory.

Neuregulin 1 (NRG1) is a schizophrenia candidate gene whose protein product is involved in neuronal migration, survival, and synaptic plasticity via production of specific isoforms. Importantly, NRG1 type III (NRG1 III) mRNA is increased in humans inheriting a schizophrenia risk haplotype for the NRG1 gene (HapICE), and NRG1 protein levels can be elevated in schizophrenia. The nature by which NRG1 type III overexpression results in schizophrenia-like behavior and brain pathology remains unclear, therefore we constructed a transgenic mouse with Nrg1 III overexpression in forebrain neurons (CamKII kinase+). Here, we demonstrate construct validity for this mouse model, as juvenile and adult Nrg1 III transgenic mice exhibit an overexpression of Nrg1 III mRNA and Nrg1 protein in multiple brain regions. Furthermore, Nrg1 III transgenic mice have face validity as they exhibit schizophrenia-relevant behavioral phenotypes including deficits in social preference, impaired fear-associated memory, and reduced prepulse inhibition. Additionally, microarray assay of hippocampal mRNA uncovered transcriptional alterations downstream of Nrg1 III overexpression, including changes in serotonin receptor 2C and angiotensin-converting enzyme. Transgenic mice did not exhibit other schizophrenia-relevant behaviors including hyperactivity, social withdrawal, or an increased vulnerability to the effects of MK-801 malate. Our results indicate that this novel Nrg1 III mouse is valid for modeling potential pathological mechanisms of some schizophrenia-like behaviors, for determining what other neurobiological changes may be downstream of elevated NRG1 III levels and for preclinically testing therapeutic strategies that may be specifically efficacious in patients with the NRG1 (HapICE) risk genotype.

Failures of investigational new drugs (INDs) for schizophrenia have left huge unmet medical needs for patients. Given the recent lackluster results, it is imperative that new drug discovery approaches (and resultant drug candidates) target pathophysiological alterations that are shared in specific, stratified patient populations that are selected based on pre-identified biological signatures. One path to implementing this paradigm is achievable by leveraging recent advances in genetic information and technologies. Genome-wide exome sequencing and meta-analysis of single nucleotide polymorphism (SNP)-based association studies have already revealed rare deleterious variants and SNPs in patient populations. Areas covered: Herein, the authors review the impact that genetics have on the future of schizophrenia drug discovery. The high polygenicity of schizophrenia strongly indicates that this disease is biologically heterogeneous so the identification of unique subgroups (by patient stratification) is becoming increasingly necessary for future investigational new drugs. Expert opinion: The authors propose a pathophysiology-based stratification of genetically-defined subgroups that share deficits in particular biological pathways. Existing tools, including lower-cost genomic sequencing and advanced gene-editing technology render this strategy ever more feasible. Genetically complex psychiatric disorders such as schizophrenia may also benefit from synergistic research with simpler monogenic disorders that share perturbations in similar biological pathways.

Laser capture microdissection followed by RNA-seq (LCM-seq) was used to profile the transcriptional landscape of the granule cell layer of the dentate gyrus (DG-GCL) in human hippocampus, and contrasted to homogenate tissue. We identified widespread cell type-specific aging and genetic effects in the DG-GCL that were either missing or directionally discordant in corresponding bulk hippocampus RNA-seq data from largely the same subjects. Of the ∼9 million eQTLs in the DG-GCL, 15% were not in bulk hippocampus, including 15 schizophrenia genome-wide association study (GWAS) risk variants. We then created custom transcriptome-wide association study (TWAS) genetic weights from the DG-GCL which identified many novel schizophrenia-associated genetic signals not found in TWAS from bulk hippocampus, including GRM3 and CACNA1C. These results highlight the biological resolution of cell type-specific expression profiling using targeted sampling strategies like LCM, and complement homogenate and single nuclei approaches in human brain.

Specific cell populations may have unique contributions to schizophrenia but may be missed in studies of homogenate tissue. Here laser capture microdissection followed by RNA sequencing (LCM-seq) was used to transcriptomically profile the granule cell layer of the dentate gyrus (DG-GCL) in human hippocampus and contrast these data to those obtained from bulk hippocampal homogenate. We identified widespread cell-type-enriched aging and genetic effects in the DG-GCL that were either absent or directionally discordant in bulk hippocampus data. Of the ~9 million expression quantitative trait loci identified in the DG-GCL, 15% were not detected in bulk hippocampus, including 15 schizophrenia risk variants. We created transcriptome-wide association study genetic weights from the DG-GCL, which identified many schizophrenia-associated genetic signals not found in transcriptome-wide association studies from bulk hippocampus, including GRM3 and CACNA1C. These results highlight the improved biological resolution provided by targeted sampling strategies like LCM and complement homogenate and single-nucleus approaches in human brain. Jaffe et al. profile the granule cell layer of the human hippocampus and find unique molecular associations for aging and genetic variation, as well as diagnosis with schizophrenia and its genetic risk, that were previously undiscovered in homogenate tissue.

The gamma-aminobutyric acid type B (GABA

The auditory steady-state response (ASSR) has been used to detect auditory processing deficits in patients with psychiatric disorders. However, the methodology of ASSR recording from the brain surface has not been standardized in preclinical studies, limiting its use as a translational biomarker. The sites of maximal ASSR in humans are the vertex and/or middle frontal area, although it has been suggested that the auditory cortex is the source of the ASSR. We constructed and validated novel methods for ASSR recording using a switchable pedestal which allows ASSR recording alternatively from temporal or parietal cortex with a wide range of frequencies in freely moving rats. We further evaluated ASSR as a translational tool by assessing the effect of ketamine. The ASSR measured at parietal cortex did not show clear event-related spectral perturbation (ERSP) or inter-trial coherence (ITC) in any frequency bands or a change with ketamine. In contrast, the ASSR at temporal cortex showed clear ERSP and ITC where 40 Hz was maximal in both gamma-band frequencies. Ketamine exerted a biphasic effect in ERSP at gamma bands. These findings suggest that temporal cortex recording with a wide frequency range is a robust methodology to detect ASSR, potentially enabling application as a translational biomarker in psychiatric and developmental disorders.

Biomarkers are needed to improve the diagnosis of neuropsychiatric disorders, which are often associated to excitatory/inhibitory imbalances in neural transmission and abnormal maturation. Here, we characterized different disease conditions by mapping changes in the expression patterns of maturation-related genes whose expression was altered by experimental neural hyperexcitation in published studies. This analysis revealed two gene expression patterns: decreases in maturity markers and increases in immaturity markers. These two groups of genes were characterized by the over-representation of genes related to synaptic function and chromosomal modification, respectively. Using these two groups in a transdiagnostic analysis of 87 disease datasets for eight neuropsychiatric disorders and 12 datasets from corresponding animal models, we found that transcriptomic pseudoimmaturity inducible by neural hyperexcitation is shared by multiple neuropsychiatric disorders, such as schizophrenia, Alzheimer disorders, and amyotrophic lateral sclerosis. Our results indicate that this endophenotype serves as a basis for the transdiagnostic characterization of these disorders.

Biomarkers are needed to improve the diagnosis of neuropsychiatric disorders. Promising candidates are imbalance of excitation and inhibition in the brain, and maturation abnormalities. Here, we characterized different disease conditions by mapping changes in the expression patterns of maturation-related genes whose expression was altered by experimental neural hyperexcitation in published studies. This revealed two gene expression patterns: decreases in maturity markers and increases in immaturity markers. These two groups of genes were characterized by the overrepresentation of genes related to synaptic function and chromosomal modification, respectively. We used these two groups in a transdiagnostic analysis of 80 disease datasets for eight neuropsychiatric disorders and 12 datasets from corresponding animal models, and found that transcriptomic pseudoimmaturity inducible by neural hyperexcitation is shared by multiple neuropsychiatric disorders, such as schizophrenia, Alzheimer disorders, and ALS. Our results indicate that this endophenotype serve as a basis for transdiagnostic characterization of these disorders.

We outline an ambitious project to characterize the genetic and epigenetic regulation of multiple facets of transcription in distinct brain regions across the human lifespan in samples of major neuropsychiatric disorders and controls. Initially focused on schizophrenia and mood disorders, the goal of this consortium is to elucidate the underlying molecular mechanisms of genetic associations with the goal of identifying novel therapeutic targets. The consortium currently consists of seven pharmaceutical companies and a not-for-profit medical research institution working as a precompetitive team to generate and analyze publicly available archival brain genomic data related to neuropsychiatric illness.

Accumulating evidence points to the association of epilepsy, particularly, temporal lobe epilepsy (TLE), with psychiatric disorders, such as schizophrenia. Among these illnesses, the hippocampus is considered the regional focal point of the brain, playing an important role in cognition, psychosis, and seizure activity and potentially suggesting common etiologies and pathophysiology of TLE and schizophrenia. In the present review, we overview abnormal network connectivity between the dentate gyrus (DG) and the Cornus Ammonis area 3 (CA3) subregions of the hippocampus relative to the induction of epilepsy and schizophrenia. In light of our recent finding on the misguidance of hippocampal mossy fiber projection in the rodent model of schizophrenia, we discuss whether ectopic mossy fiber projection is a commonality in order to evoke TLE as well as symptoms related to schizophrenia.

Elevated levels of the type III (III) isoforms of neuregulin 1 (NRG1) have been observed in the brains of schizophrenia patients that carry NRG1 HapICE risk alleles, which is thought to contribute to the aetiology of the disease. We generated transgenic mice with forebrain driven Nrg1 III overexpression (Nrg1 III tg) and previously found that male heterozygous Nrg1 type III tg mice exhibit several schizophrenia-relevant behaviours including social and cognitive deficits as well as impaired sensorimotor gating. A number of mouse models for other Nrg1 isoform types exhibit sex-specific phenotypes yet sex-specific effects of Nrg1 III overexpression had not been evaluated. Thus, in this study we tested female Nrg1 III transgenic mice using a comprehensive behavioural phenotyping battery relevant to positive, negative and cognitive symptoms of schizophrenia. Firstly, forebrain Nrg1 III mRNA overexpression was confirmed in female transgenic mice using by qPCR. In the open field test, female Nrg1 III mice exhibited a blunted response to an acute challenge with the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801. Female Nrg1 III tg mice also exhibited moderately impaired short-term memory. Other behavioural domains including sensory abilities, motor functions, baseline locomotion, anxiety, sociability, social recognition memory, fear conditioning and prepulse inhibition were unperturbed in Nrg1 III tg females. Together these results illustrate that overexpressing forebrain Nrg1 III in female mice modifies the locomotive response to NMDA receptor antagonism without causing severe alterations to a number of other schizophrenia-related behavioural domains. The data suggest that behavioural effects of Nrg1 III overexpression may be sex-dependent.

Fragile X syndrome is a genetic condition resulting fromFMR1gene mutation that leads to intellectual disability, autism-like symptoms, and sensory hypersensitivity. Arbaclofen, a GABA-B agonist, has shown efficacy in some individuals with FXS but has become unavailable after unsuccessful clinical trials, prompting interest in publicly available, racemic baclofen. The present study investigated whether racemic baclofen can remediate abnormalities of neural circuit function, sensory processing, and behavior inFmr1knockout mice, a rodent model of fragile X syndrome.Fmr1knockout mice showed increased baseline and auditory-evoked high-frequency gamma (30–80 Hz) power relative to C57BL/6 controls, as measured by electroencephalography. These deficits were accompanied by decreased T maze spontaneous alternation, decreased social interactions, and increased open field center time, suggestive of diminished working memory, sociability, and anxiety-like behavior, respectively. Abnormal auditory-evoked gamma oscillations, working memory, and anxiety-related behavior were normalized by treatment with baclofen, but impaired sociability was not. Improvements in working memory were evident predominantly in mice whose auditory-evoked gamma oscillations were dampened by baclofen. These findings suggest that racemic baclofen may be useful for targeting sensory and cognitive disturbances in fragile X syndrome.

In the postnatal hippocampus, newly generated neurons contribute to learning and memory. Disruptions in neurogenesis and neuronal development have been linked to cognitive impairment and are implicated in a broad variety of neurological and psychiatric disorders. To identify putative factors involved in this process, we examined hippocampal gene expression alterations in mice possessing a heterozygous knockout of the calcium/calmodulin-dependent protein kinase II alpha heterozygous knockout gene (CaMK2α-hKO), an established model of cognitive impairment that also displays altered neurogenesis and neuronal development. Using this approach, we identified gastrin-releasing peptide (GRP) as the most dysregulated gene. In wild-type mice, GRP labels NeuN-positive neurons, the lone exception being GRP-positive, NeuN-negative cells in the subgranular zone, suggesting GRP expression may be relevant to neurogenesis and/or neuronal development. Using a model of in vitro hippocampal neurogenesis, we determined that GRP signaling is essential for the continued survival and development of newborn neurons, both of which are blocked by transient knockdown of GRP’s cognate receptor (GRPR). Furthermore, GRP appears to negatively regulate neurogenesis-associated proliferation in neural stem cells both in vitro and in vivo. Intracerebroventricular infusion of GRP resulted in a decrease in immature neuronal markers, increased cAMP response element-binding protein (CREB) phosphorylation, and decreased neurogenesis. Despite increased levels of GRP mRNA, CaMK2α-hKO mutant mice expressed reduced levels of GRP peptide. This lack of GRP may contribute to the elevated neurogenesis and impaired neuronal development, which are reversed following exogenous GRP infusion. Based on these findings, we hypothesize that GRP modulates neurogenesis and neuronal development and may contribute to hippocampus-associated cognitive impairment. Stem Cells 2014;32:2454–2466

The gamma-aminobutyric acid type B (GABAB) receptor agonist, the sodium salt of gamma-hydroxybutyrate (GHB), significantly improved pain, sleep disturbance and fatigue in fibromyalgia (FM) patients. However, the use of GABAB receptor agonists is limited by their undesirable side-effects. To clarify whether GABAB receptor positive allosteric modulator (PAM) approach would achieve analgesia with less side-effects than GABAB receptor agonist in FM, we investigated the potential of a novel GABAB receptor PAM, ASP8062, for FM treatment. We examined the in vitro profiles of ASP8062, the effects of a GABAB receptor PAM and an agonist on pain in a rat model of FM, and the sleep/wake cycle, EEG during sleep stages and motor coordination in rats. ASP8062 showed PAM activity on human and rat GABAB receptors. Oral administration of ASP8062 significantly reversed the decrease in muscle pressure threshold in reserpine-induced myalgia rats. The analgesic effects of ASP8062 were significantly blocked by a GABAB receptor antagonist. ASP8062 had a significant effect on motor coordination at a 1000-fold higher dose than the analgesic dose in rats. ASP8062 significantly decreased total REM sleep time and frequency of sleep interruptions, and increased the power in delta waves frequency during non-REM sleep in rats. ASP8062, a novel GABAB receptor PAM, has therapeutic potential to exert analgesic effects with less side-effects compared to GABAB receptor agonists in patients with FM.

Antipsychotic drugs are widely prescribed to elderly patients for the treatment of a variety of psychopathological conditions, including psychosis and the behavioral disturbances associated with dementia. However, clinical experience suggests that these drugs may be less efficacious in the elderly individuals than in the young. Recent studies suggest that aging may be associated with epigenetic changes and that valproic acid (VPA), a histone deacetylase inhibitor, may reverse such changes. However, it is not yet known whether HDAC inhibitors can modulate age-related epigenetic changes that may impact antipsychotic drug action. In this study, we analyzed conditioned avoidance response (CAR) and c-Fos expression patterns to elucidate the effect of HDAC inhibitors VPA and entinostat (MS-275) on behavioral and molecular markers of the effects of haloperidol (HAL) in aged mice. Our results showed that HAL administration failed to suppress the avoidance response during the CAR test, suggesting an age-related decrease in drug efficacy. In addition, HAL-induced c-Fos expression in the nucleus accumbens shell and prefrontal cortex was significantly lower in aged mice as compared with young mice. Pretreatment with VPA and MS-275 significantly improved HAL effects on the CAR test in aged mice. Also, VPA and MS-275 pretreatment restored HAL-induced increases in c-Fos expression in the nucleus accumbens shell and prefrontal cortex of aged mice to levels comparable with those observed in young mice. Lastly, but most importantly, increases in c-Fos expression and HAL efficacy in the CAR test of the HAL+VPA and HAL+MS-275 groups were correlated with elevated histone acetylation at the c-fos promoter region in aged mice. These findings suggest that pretreatment with VPA or MS-275 increases the behavioral and molecular effects of HAL in aged mice and that these effects occur via modulation of age-related histone hypoacetylation in the nucleus accumbens shell and prefrontal cortex.

Shin R, Kobayashi K, Hagihara H, Kogan JH, Miyake S, Tajinda K,Walton NM, Gross AK, Heusner CL, Chen Q, Tamura K, Miyakawa T,Matsumoto M. The immature dentate gyrus represents a sharedphenotype of mouse models of epilepsy and psychiatric disease.Bipolar Disord 2013: 15: 405–421. © 2013 John Wiley & Sons A/S.Published by John Wiley & Sons Ltd.Objectives: There is accumulating evidence to suggest psychiatricdisorders, such as bipolar disorder and schizophrenia, share commonetiologies, pathophysiologies, genetics, and drug responses with many ofthe epilepsies. Here, we explored overlaps in cellular/molecular,electrophysiological, and behavioral phenotypes between putative mousemodels of bipolar disorder/schizophrenia and epilepsy. We tested thehypothesis that an immature dentate gyrus (iDG), whose associationwith psychosis in patients has recently been reported, represents acommon phenotype of both diseases.Methods: Behaviors of calcium/calmodulin-dependent protein kinase IIalpha (a-CaMKII) heterozygous knock-out (KO) mice, which are arepresentative bipolar disorder/schizophrenia model displaying iDG, andpilocarpine-treated mice, which are a representative epilepsy model, weretested followed by quantitative polymerase chain reaction (qPCR)/immunohistochemistry for mRNA/protein expression associated with aniDG phenotype. In vitro electrophysiology of dentate gyrus granule cells(DG GCs) was examined in pilocarpine-treated epileptic mice.Results: The two disease models demonstrated similar behavioraldeficits, such as hyperactivity, poor working memory performance, andsocial withdrawal. Significant reductions in mRNA expression andimmunoreactivity of the mature neuronal marker calbindin andconcomitant increases in mRNA expression and immunoreactivity of theimmature neuronal marker calretinin represent iDG signatures that arepresent in both mice models. Electrophysiologically, we have confirmedthat DG GCs from pilocarpine-treated mice represent an immature state.A significant decrease in hippocampal a-CaMKII protein levels was alsofound in both models.Conclusions: Our data have shown iDG signatures from mouse modelsof both bipolar disorder/schizophrenia and epilepsy. The evidencesuggests that the iDG may, in part, be responsible for the abnormalbehavioral phenotype, and that the underlying pathophysiologies inepilepsy and bipolar disorder/schizophrenia are strikingly similar.

SREB2/GPR85, a member of the super-conserved receptor expressed in brain (SREB) family, is the most conserved G-protein-coupled receptor in vertebrate evolution. Previous human and mouse genetic studies have indicated a possible link between SREB2 and schizophrenia. SREB2 is robustly expressed in the hippocampal formation, especially in the dentate gyrus, a structure with an established involvement in psychiatric disorders and cognition. However, the function of SREB2 in the hippocampus remains elusive. Here we show that SREB2 regulates hippocampal adult neurogenesis, which impacts on cognitive function. Bromodeoxyuridine incorporation and immunohistochemistry were conducted in SREB2 transgenic (Tg, over-expression) and knockout (KO, null-mutant) mice to quantitatively assay adult neurogenesis and newborn neuron dendritic morphology. Cognitive responses associated with adult neurogenesis alteration were evaluated in SREB2 mutant mice. In SREB2 Tg mice, both new cell proliferation and new neuron survival were decreased in the dentate gyrus, whereas an enhancement of new neuron survival occurred in SREB2 KO mouse dentate gyrus. Doublecortin staining revealed dendritic morphology deficits of newly generated neurons in SREB2 Tg mice. In a spatial pattern separation task, SREB2 Tg mice displayed a decreased ability to discriminate spatial relationships, whereas SREB2 KO mice had enhanced abilities in this task. Additionally, SREB2 Tg and KO mice had reciprocal phenotypes in a Y-maze working memory task. Our results indicate that SREB2 is a negative regulator of adult neurogenesis and consequential cognitive functions. Inhibition of SREB2 function may be a novel approach to enhance hippocampal adult neurogenesis and cognitive abilities to ameliorate core symptoms of psychiatric patients.

SREB2 (GPR85) is an orphan G-protein coupled receptor (GPCR) whose function is unknown. We previously prepared a SREB2-overexpressing transgenic mouse for functional analysis but were unable to confirm SREB2 protein expression level by immunochemical or biochemical methods. In this article, we report mass spectrometric identification and relative quantitative analysis of SREB2 in the forebrains of transgenic and wild type mice using nanoliquid chromatography coupled with a linear ion-trap mass spectrometer. By analyzing Chinese hamster ovary (CHO) cells overexpressing the SREB2 gene, we identified a proteotypic SREB2 peptide, GPTPPTLLGIR. Using a stable isotope-labeled analog as an authentic peptide for protein identification and as an internal control for relative quantitation, SREB2 was directly identified from the membrane fraction of forebrains from wild type and SREB2 transgenic mice. SREB2 protein expression level in the transgenic mouse was estimated to be 3-fold higher than that in the wild type littermate.

Dopaminergic stabilizers are recognized as compounds that can either enhance or antagonize dopamine (DA)-dependent behaviors depending on the prevailing dopaminergic tone. The dopaminergic stabilizer ASP2314 is being tested clinically and has been reported to have antipsychotic effects in a clinical trial as an add on medication. To elucidate the mechanisms of action of this dopaminergic stabilizer, its potency on the functional dopamine D2High receptors was examined. In competition with D2 receptors selectively labeled by [3H]domperidone, ASP2314 had a dissociation constant, KiHigh, of 1.62 μM for D2High in human cloned D2Long receptors and 0.83 μM for rat homogenized striata. Using the D2 agonist ligand [3H](+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ((+)PHNO), ASP2314 had a high-affinity Ki of 32 nM for D2High for rat homogenized striata. ASP2314 stimulated the incorporation of [35S]GTP-γ-S into rat striata by 50% at 43 nM, and into the cloned D2Long membranes by 50% at 3.2 μM (compared to 100% stimulation by 10 μM dopamine). With similar concentrations of ASP2314 inhibiting the binding of ligands at D2High and stimulating [35S]GTP-γ-S incorporation, the data indicate that the dopaminergic stabilizing action of ASP2314 may be related to the selectivity for the D2high state of the D2 receptor. Synapse 63:930–934, 2009. © 2009 Wiley-Liss, Inc.

The chromosome 15q13.3 microdeletion is a pathogenic copy number variation conferring epilepsy, intellectual disability, schizophrenia, and autism spectrum disorder (ASD). We generated mice carrying a deletion of 1.2 Mb homologous to the 15q13.3 microdeletion in human patients. Here, we report that mice with a heterozygous deletion on a C57BL/6 background (D/+ mice) demonstrated phenotypes including enlarged/heavier brains (macrocephaly) with enlarged lateral ventricles, decreased social interactions, increased repetitive grooming behavior, reduced ultrasonic vocalizations, decreased auditory-evoked gamma band EEG, and reduced event-related potentials. D/+ mice had normal body weight, activity levels, sensory gating, and cognitive abilities and no signs of epilepsy/seizures. Our results demonstrate that D/+ mice represent ASD-related phenotypes associated with 15q13.3 microdeletion syndrome. Further investigations using this chromosome-engineered mouse model may uncover the common mechanism(s) underlying ASD and other neurodevelopmental/psychiatric disorders representing the 15q13.3 microdeletion syndrome, including epilepsy, intellectual disability, and schizophrenia.SIGNIFICANCE STATEMENTRecently discovered pathologic copy number variations (CNVs) from patients with neurodevelopmental/psychiatric disorders show very strong penetrance and thus are excellent candidates for mouse models of disease that can mirror the human genetic conditions with high fidelity. A 15q13.3 microdeletion in humans results in a range of neurodevelopmental/psychiatric disorders, including epilepsy, intellectual disability, schizophrenia, and autism spectrum disorder (ASD). The disorders conferred by a 15q13.3 microdeletion also have overlapping genetic architectures and comorbidity in other patient populations such as those with epilepsy and schizophrenia/psychosis, as well as schizophrenia and ASD. We generated mice carrying a deletion of 1.2 Mb homologous to the 15q13.3 microdeletion in human patients, which allowed us to investigate the potential causes of neurodevelopmental/psychiatric disorders associated with the CNV.

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.

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-12or =cortistatinPAMP-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.

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.

Catechol-O-methyltransferase (COMT) is a key enzyme in the elimination of dopamine in the prefrontal cortex of the human brain. Genetic variation in the COMT gene (MIM 116790) has been associated with altered prefrontal cortex function and higher risk for schizophrenia, but the specific alleles and their functional implications have been controversial. We analyzed the effects of several single-nucleotide polymorphisms (SNPs) within COMT on mRNA expression levels (using reverse-transcriptase polymerase chain reaction analysis), protein levels (using Western blot analysis), and enzyme activity (using catechol methylation) in a large sample (n = 108) of postmortem human prefrontal cortex tissue, which predominantly expresses the -membrane-bound isoform. A common coding SNP, Val158Met (rs4680), significantly affected protein abundance and enzyme activity but not mRNA expression levels, suggesting that differences in protein integrity account for the difference in enzyme activity between alleles. A SNP in intron 1 (rs737865) and a SNP in the 3' flanking region (rs165599)--both of which have been reported to contribute to allelic expression differences and to be associated with schizophrenia as part of a haplotype with Val--had no effect on mRNA expression levels, protein immunoreactivity, or enzyme activity. In lymphocytes from 47 subjects, we confirmed a similar effect on enzyme activity in samples with the Val/Met genotype but no effect in samples with the intron 1 or 3' SNPs. Separate analyses revealed that the subject's sex, as well as the presence of a SNP in the P2 promoter region (rs2097603), had small effects on COMT enzyme activity. Using site-directed mutagenesis of mouse COMT cDNA, followed by in vitro translation, we found that the conversion of Leu at the homologous position into Met or Val progressively and significantly diminished enzyme activity. Thus, although we cannot exclude a more complex genetic basis for functional effects of COMT, Val is a predominant factor that determines higher COMT activity in the prefrontal cortex, which presumably leads to lower synaptic dopamine levels and relatively deleterious prefrontal function.

Microarray expression studies have reported decreased mRNA expression of histidine triad nucleotide-binding protein (HINT1) and cytosolic malate dehydrogenase (MDH1) in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. Microarray results for neuroserpin (SERPINI1) mRNA in the DLPFC have reported increased and decreased expression in individuals with schizophrenia. The relative abundances of HINT1, MDH1, and SERPINI1 mRNA in the DLPFC in individuals with schizophrenia and controls were measured by real-time quantitative polymerase chain reaction (Q-PCR) and for HINT1 expression by in situ hybridization. The Q-PCR results were compared by analysis of covariance between individuals with schizophrenia and controls. Gene expression levels for HINT1, MDH1, and SERPINI1 were significantly different between the groups. The male individuals with schizophrenia compared to male controls showed reductions by 2.8- to 3.7-fold of HINT1, neuroserpin, and MDH1 by Q-PCR. The decreases in mRNA abundance for MDH1 (P = 0.006), HINT1 (P = 0.050), and neuroserpin (P = 0.005) in DLPFC of male individuals with schizophrenia is consistent with prior reports. HINT1 mRNA was reduced significantly by 34% in layer VI. Though there were no significant interactions with gender, gene expression between female patients and the female control group did not differ. These results confirm earlier reports and suggest abnormalities of specific genes related to metabolic and protease activities in the DLPFC might be considered as part of a molecular pathway in male patients with schizophrenia.

Catechol O-methyltransferase (COMT) is involved in the inactivation of catecholamines, including the neurotransmitter dopamine. A Val108/158 Met functional polymorphism of the COMT gene has been shown to affect working memory-associated frontal lobe function in humans. In the present study, in situ hybridization histochemistry was employed to determine the mRNA expression profile of COMT in the human prefrontal cortex, striatum and midbrain and in the rat forebrain. In both species, COMT mRNA signals were observed in large pyramidal and smaller neurons in all cortical layers of the prefrontal cortex as well as in medium and large neurons in the striatum. Levels of COMT mRNA were obviously higher in neurons than in glia. The striatum, which receives a dense dopaminergic input, expressed lower levels of COMT mRNA as compared with the prefrontal cortex. Consistent with previous protein expression data, COMT mRNA was abundant in ependymal cells lining the cerebral ventricles. In the midbrain, COMT mRNA was detected in dopaminergic neurons in both species, albeit at low levels. In the rat forebrain, dense labeling was also detected in choroid plexus and hippocampal dentate gyrus and Ammon’s horn neurons. Contrary to expectations that COMT would be expressed predominantly in non-neuronal cells, the present study shows that neurons are the main cell populations expressing COMT mRNA in the prefrontal cortex and striatum. Combined with previous data about protein localization, the present results suggest that the membrane-bound isoform of COMT having a high affinity for dopamine is expressed at neuronal dendritic processes in human cortex, consistent with functional evidence that it plays an important role in dopaminergic neurotransmission. Published by Elsevier Science Ltd on behalf of IBRO.

Background: Fragile X syndrome (FXS) is a genetic condition which results from FMR1 gene mutation and leads to intellectual disability, autism-like symptoms and sensory hypersensitivity. Arbaclofen, a GABA-B agonist, showed efficacy for some individuals with FXS but was withdrawn from clinical trials, prompting interest in publicly available, racemic baclofen.

Schizophrenia and bipolar disorder are severe neuropsychiatric disorders, affecting about 1% of the population. Identifying endophenotypes in the brains of neuropsychiatric patients is now considered the way to understand the underlying mechanisms and to improve therapeutic outcomes. However, the endophenotypes and brain mechanisms of the disorders remain unknown. We have previously reported that alpha-CaMKII heterozygous knockout mice show abnormal behaviors related to neuropsychiatric disorders. In these mutant mice, almost all neurons in the hippocampal dentate gyrus stay at a pseudo-immature state, which we refer to as "immature dentate gyrus (iDG)." So far, the iDG phenotype and similar behavioral abnormalities have been found in Schnurri-2 knockout, SNAP-25 mutant, and forebrain-specific calcineurin knockout mice. In addition, we found that both chronic fluoxetine treatment and pilocarpine-induced seizures can reverse the maturation state of the mature neurons, resulting in the iDG phenotype in wild-type mice. Such an iDG-like phenomenon was observed in the post-mortem brains from patients with schizophrenia/bipolar disorder. Recent studies suggest that cortex and amygdala of schizophrenia patients are also at a pseudo-immature state. Based on the findings, we proposed that immaturity of certain types of cells in the brain is a potential endophenotype of neuropsychiatric disorders.

Cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) are a class of biologically active lipids that exert potent effects on the heart. To assess their roles, we investigated the distribution of their receptors, CysLT(1) and CysLT(2), in the cardiovascular system. CysLT(2) mRNA was detected at high levels in the human atrium and ventricle and at intermediate levels in the coronary artery, whereas CysLT(1) mRNA was barely detected. Further analysis by in situ hybridization revealed that CysLT(2) mRNA was expressed in myocytes, fibroblasts, and vascular smooth muscle cells, but not in endothelial cells. When human coronary smooth muscle cells were stimulated with LTC(4), the intracellular calcium concentration increased in a dose-dependent manner, and this action was partially inhibited by nicardipine. Additionally, these cells showed chemotactic responses to LTC(4). This is the first report on the physiological role of CysLT(2), and the findings suggest that CysLT(2) has biological significance in the cardiovascular system.

Cysteinyl leukotrienes (CysLTs), slow-reacting substances of anaphylaxis, are lipid mediators known to possess potent proinflammatory action. Pharmacological studies using CysLTs indicate that at least two classes of G protein-coupled receptors (GPCRs), named CysLT(1) and CysLT(2), exist; the former is sensitive and the latter is resistant to the CysLT(1) antagonists currently used to treat asthma. Although the CysLT(1) receptor gene has been recently cloned, the molecular identity of the CysLT(2) receptor has remained elusive. Here we show that the pharmacological profile of an orphan GPCR (PSEC0146) is consistent with that of the CysLT(2) receptor. In human embryonic kidney 293 cells that express the PSEC0146 cDNA, leukotriene C(4) (LTC(4)) and leukotriene D(4) (LTD(4)) induce equal increases in intracellular calcium mobilization; these increases are not affected by CysLT(1) antagonists. Additionally, [(3)H]LTC(4) specifically binds to membranes from COS-1 cells transiently transfected with PSEC0146. Large amounts of the PSEC0146 mRNA are found in human heart, placenta, spleen, and peripheral blood leukocytes but not in the lung and the trachea. Pharmacological feature and expression studies will eventually lead to a better understanding of the classification of CysLT receptors, possibly leading to a reconsideration of the pathological and physiological role of CysLTs.

We report here a novel family of G-protein coupled receptor (GPCR) which is extraordinarily conserved among vertebrate species. This family, designated SREB (Super Conserved Receptor Expressed in Brain), consists of at least three members, termed SREB1, SREB2, and SREB3. SREB members share 52-63% amino acid identity with each other and show relatively high similarity to previously known amine amine GPCRs (approximately 25% identity). Amino acid sequence identity between human and rat orthologues is 97% for SREB1 and 99% for SREB3, while the SREB2 sequence is surprisingly completely identical between the species. Furthermore, amino acid sequence of zebrafish SREB2 and SREB3 are 94 and 78% identical to mammal orthologues. Northern blot analysis revealed that SREB members are predominantly expressed in the brain regions and genital organs. Radiation hybrid analysis localized SREB1, SREB2, and SREB3 genes to different human chromosomes, namely 3p21-p14, 7q31 and Xp11, respectively. The high sequence conservation and abundant expression in the central nervous system suggest the existence of undiscovered fundamental neuronal systems consisting of SREB family members and their endogenous ligand(s).

Gastrointestinal (GI) hemorrhaging secondary to stomal ulcers following a pancreaticojejunostomy for chronic pancreatitis is a rare postoperative condition that has not hitherto been reported in the literature. A 25-yr-old Japanese female was referred to Ryukyu University Hospital with GI hemorrhaging of unknown origin. She had undergone a modified Puestow procedure (Partington procedure) for chronic pancreatitis with pancreatolithiasis and an associated dilatation of the main pancreatic duct at 19 yr of age. A technetium-99m blood-flow scan demonstrated the pooling of radionuclides in the area of the jejunal loop, which was highly suggestive of bleeding into the jejunum. Over the next day, she demonstrated persistent melena. At exploratory laparotomy, the anastomotic jejunal loop was filled with clotted blood. Operative endoscopy through an incision of the jejunal loop in close proximity to the anastomosis showed oozing blood from the anastomotic jejunal mucosa. Following a resection of the affected anastomotic segment of the jejunum, a side to side longitudinal pancreaticojejunostomy was again performed on this patient. The resected jejunum showed pathologically pseudopolyp-like edema, congestion, and an ulceration of the stomal mucosa. The patient showed a good postoperative course and has been doing well for the past 8 yr since reoperation.