Abstracts Biological Sciences

Pituitary Adenylate Cyclase Activating Polypeptide Signaling Alters Gene Expression In Chick Ciliary Ganglion Neurons

by Adriane Dee Sumner

Pituitary adenylate cyclase activating polypeptide (PACAP) modulates a spectrum of cellular functions, with many effects persisting for hours to days. PACAP and its high affinity receptors (PAC<sub>1</sub>) are known to be present in the chick ciliary ganglion (CG). Here it is demonstrated that PACAP is localized to presynaptic terminals and can be released by membrane depolarization. This study also establishes the presence of the PACAP and VIP equal affinity receptors, VPAC<sub>1</sub> and VPAC<sub>2</sub>, as well as several splice variants of the PAC<sub>1</sub> receptors in the CG. PAC<sub>1</sub> receptors have previously been shown to couple through adenylate cyclase (AC) and phospholipase-C dependent (PLC) signaling cascades to increase levels of intracellular cAMP and Ca2+ respectively. Since both messengers influence gene transcription, the ability of PACAP to activate the transcription factor CREB (cAMP/Ca<sup>2+</sup> response element binding protein) and to alter subsequent neuronal gene expression was examined. PACAP persistently activated CREB, as indicated by dose- and time-dependent increases in the incidence and intensity of Ser133 phosphorylated CREB (pCREB) labeling in CG neuron nuclei that remained elevated =90 min after PACAP exposures (15 min). Despite the presence of VPAC receptors in the CG, pCREB induction required PAC<sub>1</sub> receptors since it was mimicked by a PAC<sub>1</sub> agonist (maxadilan) and inhibited by a PAC<sub>1</sub> antagonist (P6-38). The cAMP-dependent protein kinase (PKA) may be involved in signal transduction cascades since pCREB was reduced by PKA inhibition (with H89 or KT5720). Since PACAP is abundant in the CG and released from depolarized cholinergic terminals, it could alter gene expression relevant to synapses and other activity-dependent processes. Affymetrix 28K chicken genome arrays were therefore screened using RNA extracted from 4-day CG neuron cultures grown with or without 100 nM PACAP for 15min, 24h, or 96h. Diverse categories of genes were altered by PACAP treatment, including those implicated in synaptic function, neuronal growth, survival, and development. Regulation of particular genes of interest; WAS3, WAS1, NESH3, Synapsin IIa, and Urocortin 3 were further explored with real-time RT-PCR. These results underscore the importance of PACAP in exerting modulatory and trophic influences on neurons by differentially altering gene expression in a time dependent fashion.