Shuhei Tomimoto

Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP)

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been conserved remarkably during evolution and is widely expressed in the mammalian brain. In Drosophila, mutation of the PACAP homologue results in behavioral defects, including impaired olfaction-associated learning and changes in ethanol sensitivity. Here, we report the generation of mice lacking the PACAP gene (PACAP−/−). PACAP−/− mice were born in the expected Mendelian ratios but had a high early-mortality rate. The surviving adult PACAP−/− mice displayed remarkable behavioral changes; they exhibited hyperactive and explosive jumping behaviors in an open field, increased exploratory behavior, and less anxiety in the elevated plus maze, emergence, and novel-object tests. Analysis of PACAP−/− mice brains revealed that the serotonin metabolite 5-hydroxyindoleacetic acid was slightly decreased in the cortex and striatum compared with wild-type mice. The present study provides evidence that PACAP plays a previously uncharacterized role in the regulation of psychomotor behaviors.

Defects in reproductive functions in PACAP-deficient female mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved neuropeptide and widely expressed in both brain and peripheral tissues, including several reproductive organs (e.g., testis and ovary). PACAP stimulates syntheses of several sexual hormones and steroids, suggesting it has possible roles in reproductive function. In this study, the role of PACAP in female reproductive functions such as fertility, mating behavior and maternal behaviors were investigated by using mice lacking PACAP (PACAP(-/-)). PACAP(-/-) females showed reduced fertility (the number of parturitions relative to the number of pairings). Mating experiments using vasectomized males revealed that mating frequency and its intervals in some PACAP(-/-) females were quite different (zero to eight times/4 weeks), whereas the frequency was relatively constant (two to three times/4 weeks) in wild-type females. In PACAP(-/-) females, maternal crouching behavior tended to decrease compared to wild-type females, although the influence of litter size on maternal behavior needs to be considered. These data suggest a role for endogenous PACAP in female reproductive processes.

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.

Neuroprotective action of endogenous PACAP in cultured rat cortical neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic effects both in vitro and in vivo. Here we demonstrate the upregulation of PACAP mRNA expression in cultured rat cortical neurons after excitotoxic glutamate exposure, and the exacerbating effect of the PACAP receptor antagonist, PACAP(6-38), on neuronal viability. PACAP mRNA levels were increased up to 3.5-fold 8 h after glutamate exposure. PACAP(6-38) decreased the viability of cortical neurons, irrespective of whether the cells were exposed to glutamate or not. PACAP(6-38) also inhibited glutamate-induced expression of PACAP mRNA, suggesting that PACAP acts via an autocrine or paracrine mechanism to enhance PACAP expression itself. Glutamate exposure is known to increase brain-derived neurotrophic factor (BDNF) mRNA expression. This increased expression was markedly suppressed by PACAP(6-38). Our previous study has shown that PACAP stimulates the PACAP gene transcription in PC12 cells. Taken together, these data may suggest that endogenous PACAP regulates the expression of PACAP itself and BDNF. Although it may also be possible that PACAP(6-38)-induced death of PACAP and BDNF mRNA-expressing cells, per se, results in reduced levels of these mRNAs, the present results support the idea that endogenous PACAP has a neuroprotective action.

Mice with markedly reduced PACAP (PAC1) receptor expression by targeted deletion of the signal peptide

Abstract: In an attempt to study the pituitary adenylate cyclase‐activating polypeptide (PACAP) type 1 (PAC1) receptor (PAC1R) function in vivo and to produce a mouse model with altered expression of PAC1R, we have used gene targeting in embryonic stem cells to disrupt exon 2 of the PAC1R gene, which contains the ATG translation start site and the signal peptide. Un‐expectedly, active transcription of PAC1R mRNA was detected in the mutant mice; however, exon 1 was spliced to exon 3 (skipping exon 2), and 125I‐PACAP27 binding in brain was greatly reduced. PAC1R exon 2‐/‐ mice were viable, fertile, and morphologically and histologically indistinguishable from their wild‐type counterparts. We next examined the ligand binding and cell surface expression of the mutant receptor lacking the signal peptide in transfected COS‐7 cells. 125I‐PACAP27 binding of the mutant receptor was approximately one‐tenth of that in the wild‐type receptor. Although the wild‐type receptor was expressed abundantly in both the plasma membrane and the cytoplasm around the nucleus, the mutant receptor was expressed in the plasma membrane with a markedly reduced level. Digestion of the membranes with endoglycosidase F greatly reduced the size of the wild‐type receptor but only slightly reduced that of the mutant receptor. These results demonstrate that the signal peptide is required for efficient cell surface expression and N‐linked glycosylation of the PAC1R. However, the mutant receptors still functionally coupled to adenylate cyclase in COS‐7 cells, suggesting the presence of sufficient spare receptors such that the mutant receptors are capable of activating the second messenger system. We suggest that the mutant mice with markedly reduced PAC1R expression can serve as a useful animal model or cell culture system for further studies in PAC1R function.

Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells.

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.

Desensitization, surface expression, and glycosylation of a functional, epitope-tagged type I PACAP (PAC1) receptor

To study desensitization and glycosylation of the type I pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PAC(1)R), a hemagglutinin (HA) epitope was inserted within the N-terminal extracellular domain, allowing immunological detection of PAC(1)R both in intact and permeabilized cells. PAC(1)R was tagged without loss of functions in ligand binding and ligand-stimulated cAMP production. In transiently transfected COS-7 cells, PAC(1)R was localized both in the plasma membrane and the cytoplasm around the nucleus. By immunoblot analysis, the immunoreactive bands with relative molecular masses ranging from 45 to 70 kDa were detected in the membrane fractions of PAC(1)R-expressing COS-7 cells. Digestion of the membranes with endoglycosidase F or treatment of the cells with tunicamycin decreased the size of the receptor to major bands of smaller size (approximately 45 and 48 kDa), suggesting that these two forms of PAC(1)R represent core proteins. Flow cytometric analysis indicated that the agonist promoted a disappearance of cell surface receptor. In accordance with this observation, preexposure of cells to PACAP38 induced a desensitization of PAC(1)R to the agonist response, although it did not cause a reduction in PAC(1)R mRNA or protein level and even slightly elevated them. These results suggest that agonist-induced desensitization of PAC(1)R involves the receptor sequestration.

Overexpression of PACAP in the pancreas failed to rescue early postnatal mortality in PACAP-null mice.

PACAP exerts multiple activities as a hormone and neurotransmitter, and has been proposed to play vital roles in a variety of neuronal functions. PACAP is also involved in insulin secretion from pancreatic beta-cells. Recently, we and other groups demonstrated that PACAP-deficient mice (PACAP(-/-)) are viable, but suffer from increased postnatal mortality. To ascertain whether this high mortality is rescued by overexpression of PACAP in peripheral tissue (such as pancreas), we performed a genetic cross between PACAP(-/-) and our recently developed transgenic mice overexpressing PACAP in pancreatic beta-cells; and then examined the survival rate of their F2 progeny. PACAP(-/-) mice were segregated into two groups based on mortality as well as body weight gain: PACAP(-/-) that survived >20 days of age with normal weight gain and PACAP(-/-) that died before 20 days with a marked weight loss. Kaplan-Meier survival analysis demonstrated that PACAP(-/-) mice and those carrying the PACAP transgene have similarly lower survival probability compared with their heterozygous littermates that served as positive controls. Further study using additional tissue-specific transgenic or knockout mouse models will be required to determine the causative defects underlying the high mortality of PACAP(-/-) mice.

p13 overexpression in pancreatic β-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 β-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.

Comparative gene expression profiles in pancreatic islets associated with agouti yellow mutation and PACAP overexpression in mice

In diabetes mellitus, pituitary adenylate cyclase-activating polypeptide (PACAP) has insulinotropic and glucose-lowering properties. We previously demonstrated that transgenic mice overexpressing PACAP in pancreatic β-cells (PACAP-Tg) show attenuated pancreatic islet hyperplasia and hyperinsulinemia in type 2 diabetic models. To explore the underlying mechanisms, here we crossed PACAP-Tg mice with lethal yellow agouti (KKAy) diabetic mice, and performed gene chip analysis of laser capture microdissected pancreatic islets from four F1 offspring genotypes (wild-type, PACAP-Tg, KKAy, and PACAP-Tg:KKAy). We identified 1371 probes with >16-fold differences between at least one pair of genotypes, and classified the probes into five clusters with characteristic expression patterns. Gene ontology enrichment analysis showed that genes involved in the terms ribosome and intracellular organelles such as ribonucleoprotein complex, mitochondrion, and chromosome organization were significantly enriched in clusters characterized by up-regulated genes in PACAP-Tg:KKAy mice compared with KKAy mice. These results may provide insight into the mechanisms of diabetes that accompany islet hyperplasia and amelioration by PACAP.