Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation.

Alterations in pituitary adenylate cyclase-activating polypeptide (PACAP), a multifunctional neuropeptide, and its receptors have been identified as risk factors for certain psychiatric disorders, including schizophrenia. Increasing evidence from human genetic and animal model studies suggest an association between various psychiatric disorders and altered dendritic spine morphology. In the present study, we investigated the role of exogenous and endogenous PACAP in spine formation and maturation. PACAP modified the density and morphology of PSD-95-positive spines in primary cultured hippocampal neurons. Notably, PACAP increased the levels of microRNA (miR)-132 and decreased expression of corresponding miR-132 target genes and protein expression of p250GAP, a miR-132 effector known to be involved in spine morphology regulation. In corroboration, PSD-95-positive spines were reduced in PACAP-deficient (PACAP-/-) mice versus WT mice. Golgi staining of hippocampal CA1 neurons revealed a reduced spine densities and atypical morphologies in the male PACAP-/- mice. Furthermore, viral miR-132 overexpression reversed the reduction in hippocampal spinal density in the male PACAP-/- mice. These results indicate that PACAP signaling plays a critical role in spine morphogenesis possibly via miR-132. We suggest that dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through its effects on spine formation.SIGNIFICANCE STATEMENT Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling dysfunction and dendritic spine morphology alterations have recently been suggested as important pathophysiological mechanisms underlying several psychiatric and neurological disorders. In this study, we investigated whether PACAP regulates dendritic spine morphogenesis. In a combination of pharmacological and viral gain- and loss-of-function approaches in vitro and in vivo experiments, we found PACAP to increase the size and density of dendritic spines via miR-132 upregulation. Together, our data suggest that a dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through abnormal spine formation.

Knockdown of the mitochondria-localized protein p13 protects against experimental parkinsonism.

Mitochondrial dysfunction in the nigrostriatal dopaminergic system is a critical hallmark of Parkinson's disease (PD). Mitochondrial toxins produce cellular and behavioural dysfunctions resembling those in patients with PD Causative gene products for familial PD play important roles in mitochondrial function. Therefore, targeting proteins that regulate mitochondrial integrity could provide convincing strategies for PD therapeutics. We have recently identified a novel 13-kDa protein (p13) that may be involved in mitochondrial oxidative phosphorylation. In the current study, we examine the mitochondrial function of p13 and its involvement in PD pathogenesis using mitochondrial toxin-induced PD models. We show that p13 overexpression induces mitochondrial dysfunction and apoptosis. p13 knockdown attenuates toxin-induced mitochondrial dysfunction and apoptosis in dopaminergic SH-SY5Y cells via the regulation of complex I. Importantly, we generate p13-deficient mice using the CRISPR/Cas9 system and observe that heterozygous p13 knockout prevents toxin-induced motor deficits and the loss of dopaminergic neurons in the substantia nigra. Taken together, our results suggest that manipulating p13 expression may be a promising avenue for therapeutic intervention in PD.

Autism-associated protein kinase D2 regulates embryonic cortical neuron development.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder, characterized by impaired social interaction, repetitive behavior and restricted interests. Although the molecular etiology of ASD remains largely unknown, recent studies have suggested that de novo mutations are significantly involved in the risk of ASD. We and others recently identified spontaneous de novo mutations in PKD2, a protein kinase D family member, in sporadic ASD cases. However, the biological significance of the de novo PKD2 mutations and the role of PKD2 in brain development remain unclear. Here, we performed functional analysis of PKD2 in cortical neuron development using in utero electroporation. PKD2 is highly expressed in cortical neural stem cells in the developing cortex and regulates cortical neuron development, including the neuronal differentiation of neural stem cells and migration of newborn neurons. Importantly, we determined that the ASD-associated de novo mutations impair the kinase activity of PKD2, suggesting that the de novo PKD2 mutations can be a risk factor for the disease by loss of function of PKD2. Our current findings provide novel insight into the molecular and cellular pathogenesis of ASD.

Unbiased compound screening with a reporter gene assay highlights the role of p13 in the cardiac cellular stress response.

We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart.

Whole-exome sequencing and neurite outgrowth analysis in autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex group of clinically heterogeneous neurodevelopmental disorders with unclear etiology and pathogenesis. Genetic studies have identified numerous candidate genetic variants, including de novo mutated ASD-associated genes; however, the function of these de novo mutated genes remains unclear despite extensive bioinformatics resources. Accordingly, it is not easy to assign priorities to numerous candidate ASD-associated genes for further biological analysis. Here we developed a convenient system for identifying an experimental evidence-based annotation of candidate ASD-associated genes. We performed trio-based whole-exome sequencing in 30 sporadic cases of ASD and identified 37 genes with de novo single-nucleotide variations (SNVs). Among them, 5 of those 37 genes, POGZ, PLEKHA4, PCNX, PRKD2 and HERC1, have been previously reported as genes with de novo SNVs in ASD; and consultation with in silico databases showed that only HERC1 might be involved in neural function. To examine whether the identified gene products are involved in neural functions, we performed small hairpin RNA-based assays using neuroblastoma cell lines to assess neurite development. Knockdown of 8 out of the 14 examined genes significantly decreased neurite development (P<0.05, one-way analysis of variance), which was significantly higher than the number expected from gene ontology databases (P=0.010, Fisher's exact test). Our screening system may be valuable for identifying the neural functions of candidate ASD-associated genes for further analysis and a substantial portion of these genes with de novo SNVs might have roles in neuronal systems, although further detailed analysis might eliminate false positive genes from identified candidate ASD genes.

Central CRTH2, a second prostaglandin D2 receptor, mediates emotional impairment in the lipopolysaccharide and tumor-induced sickness behavior model.

Chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) is a second prostaglandin D2 receptor involved in mediating the allergic response; however, its central function is not yet known. Here, we demonstrate that central CRTH2 mediates emotional impairment. Lipopolysaccharide (LPS)-induced decreases in social interaction and novel exploratory behavior were observed in wild-type (CRTH2(+/+)) mice but not CRTH2-deficient (CRTH2(-/-)) mice, but both genotypes showed hypolocomotion and anorexia following LPS injection. Tumor (colon 26) inoculation, a more pathologically relevant model, induced decreases in social interaction and novel exploratory behavior in CRTH2(+/+), but not CRTH2(-/-) mice. In addition, the CRTH2 antagonists including clinically available ramatroban reversed impaired social interaction and novel exploratory behavior after either LPS or tumor inoculation in CRTH2(+/+) mice. Finally, LPS-induced c-Fos expression in the hypothalamic paraventricular nucleus (PVN) and central amygdala (CeA) was selectively abolished in CRTH2(-/-) mice. These results show that CRTH2 participates in LPS-induced emotional changes and activation in the PVN and CeA. Our study provides the first evidence that central CRTH2 regulates specific emotional behaviors, and that CRTH2 antagonism has potential as a therapeutic target for behavioral symptoms associated with tumors and infectious diseases.

Identification of the role of bone morphogenetic protein (BMP) and transforming growth factor-ß (TGF-ß) signaling in the trajectory of serotonergic differentiation in a rapid assay in mouse embryonic stem cells in vitro.

The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock-in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet-1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate-based screening and identified BMP type I receptor kinase inhibitors LDN-193189 and DMH1 as activators of luciferase. LDN-193189 induced ES cells to express the genes encoding Pet-1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF-ß receptor inhibitor SB-431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN-193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF-ß target gene Lefty, whereas the opposite effect was observed with SB-431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF-ß receptor signaling are implicated in serotonergic differentiation. Candidate-based screening for serotonergic induction using a rapid assay in mouse embryonic stem cells revealed that the bone morphogenetic protein (BMP) type I receptor kinase inhibitors selectively induce serotonergic differentiation, whereas the TGF-ß receptor inhibitor SB-431542 inhibits the differentiation. These results suggest that inhibition of BMP type I receptors and concomitant activation of transforming growth factor-ß (TGF-ß) receptor signaling are involved in the early trajectory of serotonergic differentiation.

Simultaneous neuron- and astrocyte-specific fluorescent marking.

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains.

p13 overexpression in pancreatic ß-cells ameliorates type 2 diabetes in high-fat-fed mice.

We examined the pancreatic function of p13 encoded by 1110001J03Rik, whose expression is decreased in pancreatic islets in high-fat-fed diabetic mice, by generating transgenic mice overexpressing p13 (p13-Tg) in pancreatic ß-cells. p13-Tg mice showed normal basal glucose metabolism; however, under high-fat feeding, these animals showed augmented glucose-induced first-phase and total insulin secretion, improved glucose disposal, greater islet area and increased mitotic insulin-positive cells. In addition, high-fat diet-induced 4-hydroxynonenal immunoreactivity, a reliable marker and causative agent of lipid peroxidative stress, was significantly decreased in p13-Tg mouse islets. These results indicate that p13 is a novel pancreatic factor exerting multiple beneficial effects against type 2 diabetes.

The selective metabotropic glutamate 2/3 receptor agonist MGS0028 reverses psychomotor abnormalities and recognition memory deficits in mice lacking the pituitary adenylate cyclase-activating polypeptide.

Previous studies suggest that metabotropic glutamate 2/3 receptors are involved in psychiatric disorders. In this study, we examined the effects of the selective metabotropic glutamate 2/3 (mGlu2/3) receptor agonist MGS0028 on behavioral abnormalities in mice lacking the pituitary adenylate cyclase-activating polypeptide (PACAP), an experimental model of psychiatric disorders such as schizophrenia and attention-deficit/hyperactivity disorder. We found that PACAP-deficient mice showed impairments in the novel object recognition test and these impairments were improved by MGS0028 (0.1 mg/kg). Similarly, MGS0028 improved hyperactivity and jumping behaviors, but did not reverse increased immobility times in the forced swim test in PACAP-deficient mice. These results suggest that MGS0028 may be a potential, novel treatment for psychiatric disorders.

The Pivotal Role of Neuropeptide Crosstalk from Ventromedial-PACAP to Dorsomedial-Galanin in the Appetite Regulation in the Mouse Hypothalamus.

We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) in the ventromedial hypothalamus (VMH) enhances feeding during the dark cycle and after fasting, and inhibits feeding during the light cycle. On the other hand, galanin is highly expressed in the hypothalamus and has been reported to be involved in feeding regulation. In this study, we investigated the involvement of the VMH-PACAP to the dorsomedial hypothalamus (DMH)-galanin signaling in the regulation of feeding. Galanin expression in the hypothalamus was significantly increased with fasting, but this increment was canceled in PACAP-knockout (KO) mice. Furthermore, overexpression of PACAP in the VMH increased the expression of galanin, while knockdown (KD) of PACAP in the VMH decreased the expression of galanin, indicating that the expression of galanin in the hypothalamus might be regulated by PACAP in the VMH. Therefore, we expressed the synaptophysin-EGFP fusion protein (SypEGFP) in PACAP neurons in the VMH and visualized the neural projection to the hypothalamic region where galanin was highly expressed. A strong synaptophysin-EGFP signal was observed in the DMH, indicating that PACAP-expressing cells of the VMH projected to the DMH. Furthermore, galanin immunostaining in the DMH showed that galanin expression was weak in PACAP-KO mice. When galanin in the DMH was knocked down, food intake during the dark cycle and after fasting was decreased, and food intake during the light cycle was increased, as in PACAP-KO mice. These results indicated that galanin in the DMH may regulate the feeding downstream of PACAP in the VMH.

Inhibition of the mitochondria-shaping protein Opa1 restores sensitivity to Gefitinib in a lung adenocarcinomaresistant cell line.

Drug resistance limits the efficacy of chemotherapy and targeted cancer treatments, calling for the identification of druggable targets to overcome it. Here we show that the mitochondria-shaping protein Opa1 participates in resistance against the tyrosine kinase inhibitor gefitinib in a lung adenocarcinoma cell line. Respiratory profiling revealed that oxidative metabolism was increased in this gefitinib-resistant lung cancer cell line. Accordingly, resistant cells depended on mitochondrial ATP generation, and their mitochondria were elongated with narrower cristae. In the resistant cells, levels of Opa1 were increased and its genetic or pharmacological inhibition reverted the mitochondrial morphology changes and sensitized them to gefitinib-induced cytochrome c release and apoptosis. In vivo, the size of gefitinib-resistant lung orthotopic tumors was reduced when gefitinib was combined with the specific Opa1 inhibitor MYLS22. The combo gefitinib-MYLS22 treatment increased tumor apoptosis and reduced its proliferation. Thus, the mitochondrial protein Opa1 participates in gefitinib resistance and can be targeted to overcome it.

A simplified method to generate serotonergic neurons from mouse embryonic stem and induced pluripotent stem cells.

We have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancer-driven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders.

PACAP centrally mediates emotional stress-induced corticosterone responses in mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide widely distributed in the nervous system. Recently, PACAP was shown to be involved in restraint stress-induced corticosterone release and concomitant expression of the genes involved in hypothalamic-pituitary-adrenal (HPA) axis activation. Therefore, in this study, we have addressed the types of stressors and the levels of the HPA axis in which PACAP signaling is involved using mice lacking PACAP (PACAP⁻/⁻). Among four different types of stressors, open-field exposure, cold exposure, ether inhalation, and restraint, the corticosterone response to open-field exposure and restraint, which are categorized as emotional stressors, but not the other two, was markedly attenuated in PACAP⁻/⁻ mice. Peripheral administration of corticotropin releasing factor (CRF) or adrenocorticotropic hormone induced corticosterone increase similarly in PACAP⁻/⁻ and wild-type mice. In addition, the restraint stress-induced c-Fos expression was significantly decreased in the paraventricular nucleus (PVN) and medial amygdala (MeA), but not the medial prefrontal cortex, in PACAP⁻/⁻ mice. In the PVN of PACAP⁻/⁻ mice, the stress-induced c-Fos expression was blunted in the CRF neurons. These results suggest that PACAP is critically involved in activation of the MeA and PVN CRF neurons to centrally regulate the HPA axis response to emotional stressors.

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. CONCLUSIONS: These results suggest that apelin is a prerequisite factor for hypoxia-induced retinal angiogenesis.

The pivotal role of pituitary adenylate cyclase-activating polypeptide for lactate production and secretion in astrocytes during fear memory.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an essential role in the modulation of astrocyte functions. Although lactate secretion from astrocytes contributes to many forms of neuronal plasticity in the central nervous system, including fear learning and memory, the role of PACAP in lactate secretion from astrocytes is unclear. METHODS: The amygdala and hippocampus of PACAP (+ / +) and PACAP (-/-) mice were acquired 1 h after memory acquisition and recall in the passive avoidance test. The concentration of glycogen and lactate in these regions was measured. The concentration of lactate in the hippocampus's extracellular fluid was also measured by microdialysis during memory acquisition or intracerebroventricular administration of PACAP. RESULTS: We observed that memory acquisition caused a significant decrease in glycogen concentration and increased lactate concentration in the PACAP (+ / +) mice's hippocampus. However, memory acquisition did not increase in the lactate concentration in PACAP (-/-) mice's hippocampus. Further, memory retrieval evoked lactate production in the amygdala and the hippocampus of PACAP (+ / +) mice. Still, there was no significant increase in lactate concentration in the same regions of PACAP (-/-) mice. In vivo microdialysis in rats revealed that the hippocampus's extracellular lactate concentration increased after a single PACAP intracerebroventricular injection. Additionally, the hippocampus's extracellular lactate concentration increased with the memory acquisition in PACAP (+ / +) mice, but not in PACAP (-/-) mice. CONCLUSIONS: PACAP may enhance lactate production and secretion in astrocytes during the acquisition and recall of fear memories.

Increased ethanol preference and serotonin 1A receptor-dependent attenuation of ethanol-induced hypothermia in PACAP-deficient mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice display remarkable behavioral changes including increased novelty-seeking behavior and reduced hypothermia induced by either serotonin (5-HT)(1A) receptor agonists or ethanol. Because 5-HT(1A) receptors have been implicated in the development of alcohol dependence, we have examined ethanol preference in PACAP-deficient mice using a two-bottle choice and a conditioned place preference test, as well as additive effects of ethanol and 5-HT(1A) receptor agents on hypothermia. PACAP-deficient mice showed an increased preference towards ethanol compared with wild-type mice. However, they showed no preference for the ethanol compartment after conditioning and neither preference nor aversion to sucrose or quinine. The 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) restored the attenuated hypothermic response to ethanol in the mutants to similar levels in wild-type mice, with no effect in wild-types. In contrast, the 5-HT(1A) receptor antagonist WAY-100635 attenuated the ethanol-induced hypothermia in wild-type mice, with no effect in the mutants. These results demonstrate increased ethanol preference in PACAP-deficient mice that may be mediated by 5-HT(1A) receptor-dependent attenuation of ethanol-induced central inhibition.

Cardioprotective effect of endogenous pituitary adenylate cyclase-activating polypeptide on Doxorubicin-induced cardiomyopathy in mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a cytoprotective polypeptide. PACAP and its receptors are expressed in the heart, but it is unclear whether PACAP exerts its protective effect on the myocardium in vivo. The aim of the present study was to investigate whether endogenous PACAP has a cardioprotective effect on Doxorubicin (Dox)-induced cardiomyopathy. METHODS AND RESULTS: Dox was intraperitoneally injected to induce cardiomyopathy in wild type (WT) and PACAP knockout (ie, PACAP+/- and PACAP-/-) mice. The survival rates up to 15 days of PACAP+/- mice and PACAP-/- mice were significantly less than that of WT mice. Cardiac function, measured by echocardiography, was significantly lower in PACAP+/- mice than in WT mice at day 10. Morphological examination of sections of myocardium showed degenerative change and fibrosis in PACAP+/- mice at day 10. Serum reactive oxygen metabolites (a marker of oxidative stress), the number of 8-hydroxy-deoxyguanosine-positive nuclei and TdT-mediated dUTP nick end-labeling (TUNEL) positive nuclei in the myocardium were higher in PACAP+/- mice than WT mice. However, continuous subcutaneous administration of PACAP38 was able to prevent the myocardial damage typically caused by Dox injection in PACAP+/-. CONCLUSIONS: These results suggest that endogenous PACAP might attenuate Dox-induced myocardial damage and that its mechanism of action is likely to be associated with the reduction of oxidative stress and mediated via anti-apoptotic effects.

15d-prostaglandin J2 enhancement of nerve growth factor-induced neurite outgrowth is blocked by the chemoattractant receptor- homologous molecule expressed on T-helper type 2 cells (CRTH2) antagonist CAY10471 in PC12 cells.

The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is the most recently identified prostaglandin (PG) receptor for both PGD(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). We examined the mechanism by which 15d-PGJ(2) enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. CAY10471 (CRTH2 antagonist) inhibited both the neurite-promotion and p38 mitogen-activated protein (MAP) kinase phosphorylation induced by 15d-PGJ(2). In contrast, 13,14-dihydro-15-keto-PGD(2 )(DK-PGD(2)) (selective CRTH2 agonist) stimulated its phosphorylation but failed to produce neurite-promoting effects. These suggest, for the first time, the action of 15d-PGJ(2) is mediated by CRTH2, although the CRTH2 activation alone is insufficient for the underlying action.

Endogenous pituitary adenylate cyclase activating polypeptide is involved in suppression of edema in the ischemic brain.

Pituitary adenylate cyclase activating polypeptide is a pleiotropic neuropeptide. We previously showed that heterozygous PACAP gene knockout (PACAP(+/-)) mice had larger infarct volumes and worse neurological scores after middle cerebral artery occlusion (MCAO). However, the relationship between endogenous PACAP levels and edema in the ischemic brain has not yet been evaluated. In this study, the formation of edema in the ischemic brain as well as cerebral blood flow was compared between PACAP(+/-) and wild-type (PACAP(+/+)) mice. The amount of brain edema was calculated by subtracting the contralateral volume from the ipsilateral volume 24 h after permanent MCAO. PACAP(+/-) mice showed significantly greater brain edema than PACAP(+/+) mice. To investigate the effects of endogenous PACAP on blood flow during ischemia, cerebral blood flow in the ipsilateral and the contralateral cortices was compared between PACAP(+/-) and PACAP(+/+) mice for 25 min after ischemia. With a two-dimensional laser Doppler perfusion imaging system, the blood flow in the ipsilateral and contralateral cortices was shown to be similar in PACAP(+/-) and PACAP(+/+) mice during ischemia. These results suggest that endogenous PACAP suppresses the formation of edema in the ischemic brain.