No abstract

The neurotrophic peptide PACAP (pituitary adenylate cyclaseactivating polypeptide) elevates cAMP in PC12 cells. Forskolin and dibutyryl cAMP mimic PACAP's neuritogenic and cell morphological effects, suggesting that they are driven by cAMP. Comparison of microarray expression profiles after exposure of PC12 cells to either forskolin, dibutyryl cAMP, or PACAP revealed a small group of cAMP-dependent target genes. Neuritogenesis induced by all three agents is protein kinase A (PKA)-independent [not blocked by N- [2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89)] and extracellular signal-regulated kinase (ERK)-dependent [blocked by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene (U0126)], and therefore cAMP-dependent target genes potentially mediating neuritogenesis were selected for further analysis based on the pharmacological profile of their induction by PACAP (i.e., mimicking that of neuritogenesis). Small interfering RNA (siRNA) targeting one of these genes, Egr1, blocked PACAPinduced neuritogenesis, and siRNA targeting another, Vil2, blocked a component of the cell size increase elicited by PACAP. Neither siRNA blocked PACAP's PKA-dependent antiproliferative effects. PACAP signaling to neuritogenesis was also impaired by dominant-negative Rap1 expression but was not affected by inhibition of protein kinase C (PKC), indicating a G-protein-coupled receptor-mediated differentiation pathway distinct from the one activated by receptor tyrosine kinase ligands such as nerve growth factor (NGF), that involves both Rap1 and PKC. We have thus identified a cAMP-dependent, PKA-independent pathway proceeding through ERK that functions to up-regulate the transcription of two genes, Egr1 and Vil2, required for PACAP-dependent neuritogenesis and increased cell size, respectively. Dominant-negative Rap1 expression impairs both PACAP-induced neuritogenesis and Egr1 activation by PACAP, suggesting that cAMP elevation and ERK activation by PACAP are linked through Rap1.Neurotrophic factors activating receptor tyrosine kinases, such as nerve growth factor (NGF), promote neurite extension through a cAMP-independent signaling pathway involving Ras, PKC, and ERK (Ginty et al., 1991;Vaudry et al., 2002b), although other effects of NGF, such as induction of sodium channel expression, do require cAMP (Ginty et al., 1992;Yao et al., 1998). A significant literature also implicates cAMP in a broad range of neuronal differentiation responses, including neuritogenesis, survival, regeneration, repair, and expression of genes encoding neuron-specific proteins, such as neurotransmitter biosynthetic enzymes, neuropeptides, receptors, and ion channels (Qiu et al., 2002),

Oxidative stress, resulting from accumulation of reactive oxygen species, plays a critical role in neuronal cell death associated with neurodegenerative diseases and stroke. In the present study, we have investigated the potential neuroprotective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on oxidative stress-induced apoptosis. Incubation of cerebellar granule cells with PACAP inhibited hydrogen peroxide-evoked cell death in a concentration-dependent manner. The effect of PACAP on granule cell survival was not mimicked by vasoactive intestinal polypeptide and was blocked by the antagonist PACAP6-38. The protective action of PACAP upon hydrogen peroxide-induced neuronal cell death was abolished by the MAP-kinase kinase (MEK) inhibitor U0126 and mimicked by the caspase-3 inhibitor Z-DEVD-FMK. PACAP markedly inhibited hydrogen peroxide-evoked caspase-3 activation and DNA fragmentation. Taken together, these data indicate that PACAP, acting through PACAP receptor type 1, exerts a potent protective effect against neuronal degeneration induced by hydrogen peroxide. The anti-apoptotic effect of PACAP is mediated through the MAP-kinase pathway and can be accounted for by inhibition of caspase-3 activation resulting from oxidative stress.