Comparison of expression and proliferative effect of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors on human astrocytoma cell lines.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide considered to be a potent regulator of astrocytes. It has been reported that PACAP also affects astrocytoma cell properties, but the proliferative effects of this peptide in previous reports were inconsistent. The purpose of this study was to search for correlations between malignant potential, PACAP/PACAP receptor expression, and the proliferative potential of four astrocytoma cell lines (KNS-81, KINGS-1, SF-126, and YH-13). Immunohistochemical observations were performed using astrocyte lineage markers with a view to establishing malignant potential, which is inversely correlated to differentiation status in astrocytoma cells. YH-13 showed the most undifferentiated astrocyte-like status, and was immunopositive to a cancer stem cell marker, CD44. These observations suggest that YH-13 is the most malignant of the astrocytoma cell lines tested. Moreover, the strongest PAC1-R immunoreactivity was observed in YH-13 cells. Using real-time PCR analysis, no significant differences among cell lines were detected with respect to PACAP mRNA, but PAC1-R and VPAC1-R mRNA levels were significantly increased in YH-13 cells compared with the other cell lines. Furthermore, when cell lines were treated with PACAP (10(-11) M) for 3 days, the YH-13 cell line, but not of the other cell lines, exhibited a significantly increased cell number. These results suggest that PACAP receptor expression is correlated with the malignant and proliferative potential of astrocytoma cell lines.

PACAP stimulates functional recovery after spinal cord injury through axonal regeneration.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuroprotective peptide expressed in the central nervous system. Although many studies have shown a neuroprotective effect of PACAP, the mechanism of PACAP in the treatment of spinal cord injury (SCI) is yet to be elucidated. The purpose of this study was to examine the efficacy and underlying mechanism of PACAP in a mouse SCI model where PACAP was delivered via a biodegradable hydrogel. When PACAP or saline was delivered immediately after SCI, the functional motor recovery 14 days after SCI was significantly improved in the PACAP group compared with that in the saline group. Expression levels of messenger RNA (mRNA) for collapsin response mediator protein 2 (CRMP2), a factor related to axonal regeneration, were increased in the PACAP group 14 days after SCI compared with those in the saline group. A significantly increased number of CRMP2-positive cells were observed around the injury lesion in the PACAP group, while CRMP2 co-labeling with neuronal and oligodendrocyte markers was detected in intact spinal cord. Fourteen days after SCI, anterograde tracing revealed that a significantly increased number of neuronal fibers extended caudally from the lesion epicenter in the PACAP group. These results suggest that PACAP stimulates functional motor recovery after SCI through axonal regeneration mediated by CRMP2.

PACAP attenuates NMDA-induced retinal damage in association with modulation of the microglia/macrophage status into an acquired deactivation subtype.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been known as a neuroprotectant agent in several retinal injury models. However, a detailed mechanism of this effect is still not well understood. In this study, we examined the retinoprotective effects and associated underlying mechanisms of action of PACAP in the mouse N-methyl-D-aspartic acid (NMDA)-induced retinal injury model, focusing on the relationship between PACAP and retinal microglia/macrophage (MG/MΦ) status. Adult male C57BL/6 mice received an intravitreal injection of NMDA to induce retinal injury. Three days after NMDA injection, the number of MG/MΦ increased significantly in the retinas. The concomitant intravitreal injection of PACAP suppressed NMDA-induced cell loss in the ganglion cell layer (GCL) and significantly increased the number of MG/MΦ. These outcomes associated with PACAP were attenuated by cotreatment with PACAP6-38, while the beneficial effects of PACAP were not seen in interleukin-10 (IL-10) knockout mice. PACAP significantly elevated the messenger RNA levels of anti-inflammatory cytokines such as transforming growth factor beta 1 and IL-10 in the injured retina, with the immunoreactivities seen to overlap with markers of MG/MΦ. These results suggest that PACAP enhances the proliferation and/or infiltration of retinal MG/MΦ and modulates their status into an acquired deactivation subtype to favor conditions for neuroprotection.

Expression and distribution of pituitary adenylate cyclase-activating polypeptide receptor in reactive astrocytes induced by global brain ischemia in mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neuroprotectant. We previously showed that PACAP receptor (PAC1R) immunoreactivity was elevated in reactive astrocytes after stab wound injury. However, the pattern of PAC1R expression in astrocytes after brain injury is still unknown. In this study, PAC1R expression was evaluated in mouse hippocampal astrocytes after bilateral common carotid artery occlusion. PAC1R mRNA levels in the hippocampus peaked on day 7, and glial fibrillary acidic protein (GFAP) mRNA levels increased from day 3 to day 7 after ischemia. We then observed co-localization of PAC1R and GFAP by double immunostaining. GFAP-immunopositive cells showed signs of hypertrophy 3 days after the ischemia, and by day 7 had fine processes, were hypertrophied, and are known as reactive astrocytes. A low number of PAC1R-immunopositive astrocytes were detectable in the hippocampal area until 3 days after ischemia. PAC1R-positive astrocytes were widely distributed in the hippocampus between day 7 and day 14 after ischemia, and they were converging around the damaged CA1 pyramidal cell layer by day 28. These results suggest that PAC1R might be expressed in the middle to late stage of reactive astrocytes and PACAP plays an important role in the reactive astrocytes after brain injury.

IL-6 and PACAP receptor expression and localization after global brain ischemia in mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts a neuroprotective action against ischemic damage. This action is mediated by the interleukin-6 (IL-6) pathway. However, as the expression patterns of PACAP receptors and IL-6 following ischemia are not understood, we evaluated them in the mouse hippocampus in response to ischemia induced by bilateral common carotid artery occlusion. Real-time PCR determination of PAC1R mRNA expression in the hippocampus was significantly elevated on day 7 after ischemia. VPAC1R mRNA expression was significantly decreased 3 days after the ischemic episode, while VPAC2R mRNA expression showed a nonsignificant tendency to increase on day 7. IL-6 mRNA expression was significantly increased on day 3 and peaked on day 7 after ischemia. The mRNA expression of activity-dependent neuroprotective protein, which is a neuroprotective factor stimulated by PACAP, remained virtually unchanged in response to ischemia. IL-6 immunoreactivity was detected in the CA1 pyramidal cell layer and colocalized with the neuronal marker NeuN on day 1 after ischemia. On day 3, irregularly shaped IL-6-immunopositive cells colocalized with the astrocytic marker glial fibrillary acidic protein but not with the microglial marker Iba1. PAC1R immunoreactivity co-labeled with IL-6 immunoreactivity. These results suggest that PACAP could stimulate IL-6 secretion by neurons during the acute phase after an ischemic episode and thereafter by astrocytes during the subacute phase.

Neuroprotective effect of endogenous pituitary adenylate cyclase-activating polypeptide on spinal cord injury.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuroprotective peptide expressed in the central nervous system. To date, changes in the expression and effect of endogenous PACAP have not been clarified with respect to spinal cord injury (SCI). The aim of this study was to elucidate the expression pattern and function of endogenous PACAP on the contusion model of SCI using heterozygous PACAP knockout (PACAP(+/-)) and wild-type mice. Real-time polymerase chain reaction methods revealed that the level of PACAP mRNA increased gradually for 14 days after SCI and that PAC1R mRNA levels also increased for 7 days compared with intact control mice. PACAP and PAC1R immunoreactivities colabeled with a neuronal marker in the intact spinal cord. Seven days after SCI, PAC1R immunoreactivity was additionally co-expressed with an astrocyte marker. Wild-type mice gradually recovered motor function after 14 days, but PACAP(+/-) mice showed significantly impaired recovery from 3 days compared with wild-type mice. The injury volume at day 7 in PACAP(+/-) mice, and the number of single-stranded DNA-immunopositive cells as a marker of neuronal cell death at day 3 were significantly higher than values measured in wild-type mice. These data suggest that endogenous PACAP is upregulated by SCI and has a neuroprotective effect on the damaged spinal cord.