Norihito Shintani

Pituitary adenylate cyclase-activating polypeptide is associated with schizophrenia

Pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1: adenylate cyclase-activating polypeptide 1), a neuropeptide with neurotransmission modulating activity, is a promising schizophrenia candidate gene. Here, we provide evidence that genetic variants of the genes encoding PACAP and its receptor, PAC1, are associated with schizophrenia. We studied the effects of the associated polymorphism in the PACAP gene on neurobiological traits related to risk for schizophrenia. This allele of the PACAP gene, which is overrepresented in schizophrenia patients, was associated with reduced hippocampal volume and poorer memory performance. Abnormal behaviors in PACAP knockout mice, including elevated locomotor activity and deficits in prepulse inhibition of the startle response, were reversed by treatment with an atypical antipsychotic, risperidone. These convergent data suggest that alterations in PACAP signaling might contribute to the pathogenesis of schizophrenia.

Phenotypic Characterization of Transgenic Mice Overexpressing Neuregulin-1

Background Neuregulin-1 (NRG1) is one of the susceptibility genes for schizophrenia and implicated in the neurotrophic regulation of GABAergic and dopaminergic neurons, myelination, and NMDA receptor function. Postmortem studies often indicate a pathologic association of increased NRG1 expression or signaling with this illness. However, the psychobehavioral implication of NRG1 signaling has mainly been investigated using hypomorphic mutant mice for individual NRG1 splice variants. Methodology/Principal Findings To assess the behavioral impact of hyper NRG1 signaling, we generated and analyzed two independent mouse transgenic (Tg) lines carrying the transgene of green fluorescent protein (GFP)-tagged type-1 NRG1 cDNA. The promoter of elongation-factor 1α gene drove ubiquitous expression of GFP-tagged NRG1 in the whole brain. As compared to control littermates, both heterozygous NRG1-Tg lines showed increased locomotor activity, a nonsignificant trend toward decreasing prepulse inhibition, and decreased context-dependent fear learning but exhibited normal levels of tone-dependent learning. In addition, social interaction scores in both Tg lines were reduced in an isolation-induced resident-intruder test. There were also phenotypic increases in a GABAergic marker (parvalbumin) as well as in myelination markers (myelin basic protein and 2′,3′-cyclic nucleotide 3′-phosphodiesterase) in their frontal cortex, indicating the authenticity of NRG1 hyper-signaling, although there were marked decreases in tyrosine hydroxylase levels and dopamine content in the hippocampus. Conclusions These findings suggest that aberrant hyper-signals of NRG1 also disrupt various cognitive and behavioral processes. Thus, neuropathological implication of hyper NRG1 signaling in psychiatric diseases should be evaluated with further experimentation.

Retardation of Retinal Vascular Development in Apelin-Deficient Mice

Objective—Apelin is an endogenous ligand for the G protein–coupled receptor, APJ, and participates in multiple physiological processes. To identify the roles of endogenous apelin, we investigated the phenotype of apelin-deficient (apelin-KO) mice. Methods and Results—Apelin-KO mice showed impaired retinal vascularization and ocular development, which were analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and the mouse corneal micropocket assay. Apelin-KO mice showed significantly impaired retinal vascularization in the early postnatal period. Retinal apelin/APJ mRNAs were transiently upregulated during the first 2 postnatal weeks but were undetectable in adults. There were no differences in VEGF or FGF2 mRNA expression, or in the morphology and localization of GFAP-positive astrocytes, in the apelin-KO retinas at P5. The corneal pocket assay showed that angiogenic responses to VEGF and FGF2 were remarkably decreased in apelin-KO mice. The reduced responses to VEGF and FGF2 in apelin-KO mice were partially restored by apelin, but apelin alone did not induce angiogenesis. Conclusions—Our results suggest that spatiotemporally regulated apelin/APJ signaling participates in retinal vascularization in a cooperative manner with VEGF or FGF2, and contributes to normal ocular development.

Pituitary Adenylate Cyclase-Activating Polypeptide Is Required for the Development of Spinal Sensitization and Induction of Neuropathic Pain

The prolonged sensitization of pain transmission after nerve injury by increasing excitability of spinal neurons and thereby promoting repair is an adaptive response of the body. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is widely distributed in the nervous system and implicated in neurotransmission, neural plasticity, and neurotrophic actions. Although PACAP is distributed in the spinal cord and dorsal root ganglia, a role of PACAP in pain responses remains essentially unknown. Here we show that mice lacking the PACAP gene (PACAP-/-) did not exhibit inflammatory pain induced by intraplantar injection of carrageenan or neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses. Intrathecal administration of NMDA induced mechanical allodynia in wild-type mice, but not in PACAP-/- mice. The NMDA-induced allodynia in PACAP-/- mice was reproduced by simultaneous intrathecal injection of PACAP with NMDA. Concomitant with the increase in PACAP immunoreactivity after nerve injury, NADPH-dependent nitric oxide synthase (NOS) activity visualized by NADPH diaphorase histochemistry markedly increased in the superficial layer of the spinal cord of wild-type mice, which was not observed in PACAP-/- mice. Simultaneous addition of PACAP and NMDA caused translocation of neuronal NOS from the cytosol to the membrane and stimulated NO production in vitro. These results demonstrate that PACAP might promote the functional coupling of neuronal NOS to NMDA receptors for both inflammatory and neuropathic pain to occur.

New insights into the central PACAPergic system from the phenotypes in PACAP- and PACAP receptor-knockout mice.

Abstract:  Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a structurally highly conserved neuropeptide and displays pleiotropic activity, including functioning as a neurotransmitter, neuromodulator, and neurotrophic factor. A series of recent experiments, including genetic manipulation of PACAP and its receptors, has led to better understanding of both normal and pathological processes in which PACAP has been proposed to play a role, and sheds light on previously uncharacterized functions of endogenous PACAP. The aim of this article is to briefly review the recent advances in understanding the role of PACAP in the central nervous system from PACAP‐ and PACAP receptor‐deficient mice, particularly with respect to behavioral and neurological features, including psychomotor behavior, feeding, stress responses, memory performance, ethanol sensitivity, chronic pain, and circadian rhythms. This article also discusses their potential involvement in human diseases.

A novel DISC1-interacting partner DISC1-Binding Zinc-finger protein: implication in the modulation of DISC1-dependent neurite outgrowth

Disrupted-in-schizophrenia 1 (DISC1) is a gene disrupted by a (1;11) (q42.1;q14.3) translocation that segregates with major psychiatric disorders in a Scottish family. To investigate how DISC1 confers susceptibility to psychiatric disorders, we previously identified fasciculation and elongation protein zeta-1 and Kendrin as DISC1-interacting molecules in a yeast two-hybrid screen of a human brain complementary DNA library. Here, we have further identified a novel DISC1-interacting protein, termed DISC1-Binding Zinc-finger protein (DBZ), which has a predicted C2H2-type zinc-finger motif and coiled-coil domains. DBZ was co-immunoprecipitated with DISC1 in lysates of PC12 cells and rat brain tissue. The domain of DISC1 interacting with DBZ was close to the translocation breakpoint in the DISC1 gene. DBZ messenger RNA (mRNA) was expressed in human brains, but not in peripheral tissues. In situ hybridization revealed high expression of DBZ mRNA in the hippocampus, olfactory tubercle, cerebral cortex and striatum in rats. Because this pattern of localization was similar to that of the pituitary adenylate cyclase (PAC1) receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), which has recently been implicated in neuropsychological functions, we examined whether DISC1/DBZ interaction was involved in the PACAP signaling pathway. PACAP upregulated DISC1 expression and markedly reduced the association between DISC1 and DBZ in PC12 cells. A DISC1-binding domain of DBZ reduced the neurite length in PC12 cells after PACAP stimulation and in primary cultured hippocampal neurons. The present results provide some new molecular insights into the mechanisms of neuronal development and neuropsychiatric disorders.

Depression-like behavior in the forced swimming test in PACAP-deficient mice: amelioration by the atypical antipsychotic risperidone

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a neuropeptide with pleiotropic functions. We report here that PACAP‐deficient (PACAP−/−) mice showed increased immobility in a forced swimming test, which was reduced by the antidepressant desipramine, to a similar extent as in wild‐type mice. The atypical antipsychotic risperidone and the selective serotonin (5‐HT)2 antagonist ritanserin normalized the depression‐like behavior in PACAP−/− mice. The 5‐HT2 agonist (±)‐2,5‐dimethoxy‐4‐iodoamphetamine‐induced 5‐HT syndrome was exaggerated in PACAP−/− mice, which suggests a 5‐HT2‐receptor‐dependent mechanism in the depression‐like behavior. The circadian rhythm of plasma corticosterone and body core temperature was significantly flattened in the mutants. mRNA expression of glucocorticoid receptor was reduced in the mutant hippocampus. The present results suggest that alterations in PACAP signaling might contribute to the pathogenesis of certain depressive conditions amenable to atypical antipsychotic drugs.

Psychostimulant-Induced Attenuation of Hyperactivity and Prepulse Inhibition Deficits in Adcyap1-Deficient Mice

Psychostimulants, including amphetamine, act as antihyperkinetic agents in humans with hyperkinetic disorder such as attention-deficit hyperactivity disorder and are known to be effective in enhancing attention-related processes; however, the underlying mechanisms have not been adequately addressed. Mice lacking the Adcyap1 gene encoding the neuropeptide pituitary adenylate cyclase-activating polypeptide (Adcyap1−/−) display psychomotor abnormalities, including increased novelty-seeking behavior and hyperactivity. In this study, Adcyap1−/− mice showed sensory-motor gating deficits, measured as deficits in prepulse inhibition (PPI), and showed normal PPI in response to amphetamine. Amphetamine also significantly decreased hyperlocomotion in Adcyap1−/− mice, and this paradoxical antihyperkinetic effect depended on serotonin 1A (5-HT1A) receptor signaling. c-Fos-positive neurons were increased in the prefrontal cortex in amphetamine-treated Adcyap1−/− mice, suggesting increased inhibitory control by prefrontal neurons. Additionally, amphetamine produced an antihyperkinetic effect in wild-type mice that received the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin. These results indicate that Adcyap1−/− mice act as a model of hyperlocomotion and PPI deficits and suggest that 5-HT1A-mediated pathways are important determinants of the psychostimulant-elicited, rate-dependent effects that are in a negative function of the baseline rate of activity.

Synergistic Induction of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Gene Expression by Nerve Growth Factor and PACAP in PC12 Cells

Abstract: Pituitary adenylate cyclase‐activating polypeptide (PACAP) gene expression was analyzed in PC12 cells. PC12 cells transfected with a PACAP promoter‐luciferase reporter construct were utilized to investigate the effects of PACAP, either alone or in combination with nerve growth factor (NGF), on PACAP transcriptional response. PACAP induced transcription from the PACAP promoter through PACAP type I receptor (PAC1 receptor). PACAP gene transcription was also induced by NGF. Simultaneous treatment with PACAP and NGF resulted in a synergistic transcriptional response that was more than three times the predicted response, based on a simple additive effect of both agents. This synergism in transcriptional response paralleled the PACAP mRNA levels, as determined by RT‐PCR and northern blotting. The level of PACAP mRNA peaked 3 h after stimulation and gradually returned to basal levels by 48 h. PC12 cells are known to express predominantly the hop isoform of the PAC1 receptor, which positively couples to both adenylate cyclase and phospholipase C. To determine the role of the cyclic AMP and protein kinase C pathways in PACAP gene expression, the effects of forskolin and phorbol 12‐myristate 13‐acetate (PMA) were then examined. PMA did not alter PACAP mRNA levels but enhanced forskolin‐induced PACAP mRNA expression. Down‐regulation of protein kinase C blocked the ability of PACAP to stimulate PACAP mRNA expression. The mitogen‐activated protein kinase extracellular signal‐regulated kinase (ERK) kinase 1/2 (MEK1/2) inhibitor PD98059 also blocked the PACAP mRNA expression induced by either PACAP or NGF but not that induced by a combination of PACAP and NGF. These results suggest that PACAP stimulates the PACAP gene expression in PC12 cells at least in part through activation of adenylate cyclase and protein kinase C signaling pathways and that the ERK1/2 cascade is involved in PACAP and NGF‐induced PACAP gene expression, although redundant signaling pathways may also be involved. The present finding showing that PACAP in combination with NGF causes a synergistic increase in PACAP gene expression in PC12 cells supports the idea that PACAP acts as an autocrine regulatory factor.

Apelin Is a Crucial Factor for Hypoxia-Induced Retinal Angiogenesis

Objective—To investigate the role of endogenous apelin in pathological retinal angiogenesis. Methods and Results—The progression of ischemic retinal diseases, such as diabetic retinopathy, is closely associated with pathological retinal angiogenesis, mainly induced by vascular endothelial growth factor (VEGF) and erythropoietin. Although antiangiogenic therapies using anti-VEGF drugs are effective in treating retinal neovascularization, they show a transient efficacy and cause general adverse effects. New therapeutic target molecules are needed to resolve these issues. It was recently demonstrated that the apelin/APJ system, a newly deorphanized G protein–coupled receptor system, is involved in physiological retinal vascularization. Retinal angiography and mRNA expression were examined during hypoxia-induced retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Compared with age-matched control mice, retinal apelin expression was dramatically increased during the hypoxic phase in oxygen-induced retinopathy model mice. APJ was colocalized in proliferative cells, which were probably endothelial cells of the ectopic vessels in the vitreous body. Apelin deficiency hardly induced hypoxia-induced retinal angiogenesis despite the upregulation of VEGF and erythropoietin mRNA in oxygen-induced retinopathy model mice. Apelin small and interfering RNA suppressed the proliferation of endothelial cells independent of the VEGF/VEGF receptor 2 signaling pathway. Conclusion—These results suggest that apelin is a prerequisite factor for hypoxia-induced retinal angiogenesis.