PACAP: a master regulator of neuroendocrine stress circuits and the cellular stress response

Abstract

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is released from stress-transducing neurons. It exerts postsynaptic effects required to complete the hypothalamo-pituitary-adrenocortical (HPA) and hypothalamo-sympatho-adrenal (HSA) circuits activated by psychogenic and metabolic stressors. Upon activation of these circuits, PACAP-responsive (in cell culture models) and PACAP-dependent (in vivo) transcriptomic responses in the adrenal gland, hypothalamus, and pituitary have been identified. Gene products produced in response circuits during stress include additional neuropeptides, neurotransmitter biosynthetic enzymes, and neuroprotective factors. Major portions of HPA and HSA stress responses are abolished in PACAP-deficient mice. This deficit occurs at the level of both the hypothalamus (HPA axis) and the adrenal medulla (HSA axis). PACAP-dependent transcriptional stress responses are conveyed through noncanonical cyclic AMP- and calcium-initiated signaling pathways within the HSA circuit. PACAP transcriptional regulation of the HPA axis, in the hypothalamus, is likely to be mediated via canonical cyclic AMP signaling through protein kinase A.

Figure 1. PACAP a neurotransmitter at the mouse adrenomedullary synapse in vivo

A. PACAP -38 and a marker for cholinergic neurons, VAChT, are co-localized in neuronal cell bodies the intermediolateral column (IMLC) of the spinal cord, from whence splanchnic sympathetic innervation of the adrenal gland originates, and the splanchnicoadrenomedullary nerve terminals innervating chromaffin cells of the adrenal medulla. B. PACAP-deficient mice treated with insulin (2 units/kg) die within hours (black) unless rescued with i.p. PACAP (blue) or glucose (red). Wild-type mice invariably survive insulin challenge. C. Following insulin administration, wild-type mice show decrease blood levels of glucose (filled squares) compared to saline injection (open squares) while PACAP-deficient mice show a more profound hypoglycemia (filled circles) than wild-type after insulin compared to saline (open circles). D. Lack of compensatory glucose elevation in PACAP-deficient mice can be explained by a less pronounced elevation in plasma epinephrine after insulin administration in PACAP-deficient compared to wild-type mice (symbols mean the same as in C.). E. Decreased epinephrine levels in blood may be explained in part by lack of epinephrine release which is stimulated by PACAP, but also a failure of repletion of released epinephrine due to a failure of PACAP knock-out mice to up-regulate tyrosine hydroxlyase (not shown) resulting in adrenomedullary depletion of epinephrine in the stress PACAP-deficient, but not in the stressed wild-type, adrenal gland. Adapted from Hamelink et al., Proc. Natl. Acad. Sci. USA 99: 461-466, 2002.

Figure 2. PACAP-dependent induction of mRNAs encoding TH and PNMT in adrenal gland during restraint stress

Restraint stress, as well as insulin shock (Stroth et al., in preparation), results in up-regulation of both tyrosine hydroxylase (TH) and phenylethanolamine methyltransferase (PNMT) mRNA in adrenal gland after 6 hours, and this up-regulation is absent in PACAP-deficient mice. Note that 1 hour of restraint stress is not sufficient for PACAP-dependent induction of TH and PNMT mRNA, either when measured immediately following the stressor (1h) or after 5 hours of non-restraint following on the 1 hours restraint stress (1h + 5h). As indicated in the key, data obtained from wild-type C57Bl/6 mice are shown in grey, while those obtained from PACAP-deficient mice of the same strain are shown in black. Adapted from Stroth and Eiden, Neuroscience 165: 1025-1030, 2010.

Figure 3. Analysis of scheme used to identify signaling pathways to activation of key genes controlling various facets of cellular function in PC12 cells

PACAP signaling through the PAC1 receptor activates Gq- and Gs-dependent signaling that results in gene activation. Protein products of induced genes are required for various cellular functions including increased cell size, growth arrest, neuritogenesis, activation of specialized genes for chemical coding of neurotransmission (TH, NPY, sodium and calcium channels, etc), and protection from cell death. By correlating the effects of specific inhibitors on the total PC12 cel transcriptome using microarray, with their effects on cellular phenotype (neuritogenesis, growth arrest, etc), linkages between signaling pathways, gene induction, and cellular function can be established, and then tested with siRNA treatment directed at candidate genes underlying function. Examples of this approach are discussed in text, and see Ravni et al., Mol. Pharmacol. 73: 1688-1708, 2008.

Figure 4. PACAP an endogenous neuroprotectant in stroke in the mouse

A. The effects of middle cerebral artery occlusion (MCAO) on ischemic lesion size and neurological deficits were compared in wild-type and PACAP-deficient mice in which PACAP or saline was administered one hour after MCAO, and lesion size and neurological function assessed 24 hours later. B. Effects of PACAP administered either intravenously (i.v., red bars) or intracerebroventricularly (i.c.v., red hatched bars) one hour after MCAO on neurological outcome (change in number of walking faults-higher number equals greater recovery/less damage) assessed at 24 hours post-MCAO. C. Effects of PACAP administered either intravenously or intracerebroventricularly (symbols as in B.) one hour after MCAO on neurological outcome (larger infarct volume size reflects greater extent of brain damage) assessed at 24 hours post-MCAO Adapted from Chen et al., Regul Pept. 137: 4-19, 2006 and see this reference for additional details.

Figure 5. Induction of immediate early genes in adrenal gland, CRH mRNA in hypothalamus, and corticosterone in blood after restraint stress is PACAP-dependent

A. PACAP-dependent regulation of immediate early genes in hypothalamus following restraint stress. B. Regulation of CRH mRNA in hypothalamus during restraint is abolished in PACAP-deficient mice. C. Sustained corticosterone secretion induced by restraint stress requires PACAP. Adapted from Stroth and Eiden, Neuroscience 165: 1025-1030, 2010.

Figure 6. PACAP is a master regulator of stress circuits in brain and periphery

Depiction of the levels of the central and peripheral nervous system at which PACAP acts to modulate the efferent limb of systemic and processive stress responses at the levels of the HPA and HAS axes, and afferent (or processing nodes) within the limbic system that integrate processive stress responses, such as anxiogenesis. The involvement of PACAP in the output of the sympathetic nervous system (independently of the HSA axis) is not yet clear (see text).

Figure 7. Gene expression analysis reveals mechanism, biomarkers, and therapeutic targets in stress