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The neuropeptide PACAP is an informational molecule released from stress-transducing neurons. It exerts post-synaptic effects required to complete hypothalamo-pituitary-adrenocortical (HPA) and hypothalamo-splanchnico-adrenomedullary (HSA) circuits activated by psychogenic and metabolic stressors. PACAP-responsive (in cell culture models) and PACAP-dependent (in vivo) transcriptomic responses in the adrenal gland, hypothalamus, and pituitary upon activation of these circuits have been identified. Gene products produced in response circuits during stress include additional neuropeptides and 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 adrenal medulla (HSA axis) and the hypothalamus (HPA axis). PACAP-dependent transcriptional stress responses are conveyed through non-canonical 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 (cAMP) signaling through protein kinase A.
The stress response may be defined operationally as the response of the neuroendocrine network to systemic or environmental perturbations outside of the normal physiological range. Stress responses in mammals are mediated through specific neural and neuroendocrine circuits. The propagation of signaling through these circuits is itself a cellular stressor. In other words, a ‘stressed neuron’ is one propagating an organismic stress response through a neuronal stress circuit. Thus, the cellular stress response is a point of entry to identifying targets for pharmacological modulation of organismic stress perception and processing.
Neuropeptides play a special role in the stress response. As informational molecules stored in large dense-core vesicles, neuropeptides are released preferentially at higher firing rates, as occur during neurotransmission activated by stress in both the central and peripheral nervous systems. Fast and slow transmission are the two major modes of intercellular communication that underlie nervous system function. Fast transmission by glutamate, GABA, and acetylcholine involves direct gating of plasma membrane ionic channels for point-to-point communication, and slow transmission, by dopamine, neuropeptides, adenosine, glutamate and acetylcholine1 at metabotropic receptors, involves the mobilization of second messengers like calcium and cAMP, producing the long-term changes underlying cellular plasticity that encode experience.
Bloom et al. made the acute observation that neuropeptides represent the language of stress because the enhanced neuronal firing required to message systemic or environmental stressors to the brain, and execute homeostatic signaling to the periphery, is characterized not only by increased release of classical neurotransmitters from small synaptic vesicles, but the selective release of informational molecules, among them neuropeptides, from large dense-core vesicles (LDCVs). Acting mainly through metabotropic, rather than ionotropic post-synaptic receptors, neuropeptides convey information for post-synaptic adaptation through long-term secretory as well as gene-encoded changes, rather than short-term electrical changes, in the post-synaptic cell [1, 2].
Can microarray analysis of neuropeptide signaling help us understand, in an unbiased fashion, the ‘emergency response’ role of neuropeptides such as PACAP during stress? This short review will examine this question and demonstrate how microarray analysis in combination with informed measurement of key proteins involved in transcription and hormone biosynthesis in the neuroendocrine stress axes have helped to uncover the role of PACAP as a key regulators of signaling in major stress transducing regions of the brain and in the periphery (hypothalamus and adrenal gland). The role of PACAP in mediating stress responses may also be connected to its emerging function in neuroprotection during episodes of hypoxia, ischemia, and traumatic injury, and this connection is also briefly explored here.
Pituitary adenylate cyclase-activating polypeptide (PACAP) was discovered in 1989 by Miyata, Arimura, Coy and co-workers in a screen for hypothalamic hypophysiotropic hormones elevating cAMP in perfused pituitary . It is an ancient and well-conserved peptide that exists in a 38-amino acid and a 27-amino acid form processed from a prohormone precursor . Processing of PACAP to its 27- and 38 forms (the latter predominates), occurs through the processing enzymes PC1 and 2 and the amidating enzyme PAM [5, 6]except in gonads where PC4 is used . An analog of PACAP in Drosophila has been shown by Zhang, Feany and others to function both in the brain and at the neuromuscular junction through a dual signaling pathway involving both ion channel opening and cAMP [8, 9], and this dual signaling paradigm characterizes PACAP signaling in mammalian nervous system as well . PACAP is a neurotrophic factor, promoting the survival of cortical neuronal progenitor cells , cortical neurons , dorsal root ganglion cells , cerebellar granule cells , and peripheral sympathetic neurons . PACAP elicits both PC12 cell  and pluripotent stem cell differentiation . It is also neuroprotective in ischemia in vivo and in response to oxidative stress in granule cell culture , and increases cell survival in response to multiple stressors including environmental toxins, hypoxia, and excitotoxins in various cell culture systems [13, 18, 19]. PACAP is also cardioprotective in response to cardiotoxin and ischemic insult . Arimura remarked at a meeting of the Editorial Board of J. Mol. Neurosci. New Orleans several years ago that the 10-fold higher levels of PACAP in brains of the diving turtle , a species chronically exposed to brain ischemia, was his inspiration for the investigation of PACAP action as a neuroprotective agent in stroke (vide infra).
PACAP was shown to potently activate catecholamine secretion and biosynthesis in the adrenal medulla (, and references therein) and later to be present in neurons innervating the adrenal medulla [23, 24]. Hamelink et al.  demonstrated how PACAP acts as an emergency response peptide in paraphysiological situations. PACAP, present at the adrenomedullary synapse, is absolutely required for survival from prolonged hypoglycemia—mice without PACAP do not survive insulin administration, while wild-type mice become comatose but recover. This is because activation of tyrosine hydroxylase to provide enhanced catecholamine synthesis and release to allow gluconeogenesis to reestablish euglycemia (Figure 1) does not occur in PACAP-deficient mice. Induction of TH and PNMT mRNA after insulin is also PACAP-dependent (Stroth et al., in preparation), and PACAP-dependent induction of mRNAs encoding TH and PNMT also occurs in adrenal gland during restraint stress (Figure 2; from ). It is noteworthy that effects of stress on TH and PNMT are time-dependent—occurring only after six hours although catecholamine release begins much earlier, and is prolonged, during immobilization .
In order to determine if PACAP induces additional cellular plasticity associated with stress transduction at the adrenomedullary synapse, we performed microarray experiments in cultured chromaffin cells exposed to PACAP for six hours, corresponding to the period of prolonged stress in vivo causing up-regulation of TH and PNMT mRNA. We wished in particular to test the hypothesis that prolonged neuronal stress transduction, itself potentially stressful to neurons due to prolonged calcium elevation and metabolic demand during prolonged episodes of secretion, is accompanied by induction of calcium-neuroprotective proteins in the post-synaptic cell. A number of growth factors, cytokines and neuropeptides are induced by prolonged exposure of chromaffin cells to PACAP . Among these is stanniocalcin-1 (Stc1). Stc1 is a 56 kD protein which interacts with a mitochondrial receptor to enhance mitochondrial calcium uptake and protect cardiomyocytes and neurons against toxicity associated with excessive calcium uptake [29-34].
PC12 cells have been a convenient model for gathering sufficient numbers of a pure population of cells to perform combined pharmacological, biochemical, and microarray analysis of signaling pathways underlying PACAP action, and tie together PACAP-initiated cellular signaling pathways, inducible genes, target genes, and cellular outcomes (see Figure 3). In particular, this is a model system in which the transitional transcription (gene expression that occurs during the transition from state A to state B), often difficult to define in vivo, can be systematically analyzed. Thus, in previous studies, we determined that PACAP expression leading to a full neuritogenic response of PC12 cells can be obtained by PACAP exposure for 6 hours , and chose this time point for initial work, while also performing time courses to establish the temporal order of gene transcriptional changes occurring during the process of differentiation . The pharmacology of neuritogenesis was defined using inhibitors of PKA, ERK, and other putative nodes in PACAP signaling pathways (Figure 3). This in turn to allow categorization of induced genes based on their pharmacological profile, as potential actors in PACAP-initiated processes including proliferation, neurite extension, cell size, growth arrest, survival following serum withdrawal, as well as further alterations in the downstream transcriptome of the cell itself, including those encoding late or end-stage differentiation-associated proteins, both pan-neuronal and specific to particular sublineages such as noradrenergic, cholinergic, etc. This strategy allowed the characterization of Egr1 as a PACAP-dependent gene required for neuritogenesis .
The signaling pathway through which Egr1 gene induction occurs in response to PAC1 receptor activation is a unique cAMP-initiated, but protein kinase A-independent one, first characterized in bovine chromaffin cells . Egr1 transcription is also up-regulated in the adrenal gland after restraint stress . We have identified additional genes, including Stc1 and galanin (GAL), that are elevated in bovine chromaffin cells and in stress and also regulated by the non-canonical cAMP pathway as determined by resistance to H89 inhibition and blocked by U0126 (Stroth et al., in preparation). In contrast, available evidence suggests that PACAP regulation of CRH biosynthesis at the level of the hypothalamus involves canonical (PKA-dependent) cAMP signaling .
PACAP-deficient mice show aberrant locomotor activity and lack of adaptation to novelty in exploratory behavior, and PAC1 receptor-deficient mice show impaired social investigation reminiscent of autistic behaviors in humans [40, 41]. We have also shown that PACAP is neuroprotective in ischemic insult ([42, 43]; Figure 4) in mice, as previously demonstrated by Reglodi et al. in the rat . Furthermore, lack of PACAP expression in cerebral cortex increases the size of the ischemic lesion after middle cerebral artery occlusion (MCAO), a mouse model for stroke, and enhances the neurological damage caused by MCAO, while treatment with PACAP (even when administered an hour after the ischemic event) significantly decreases lesion size and improves neurological outcome . Ohta et al. have also determined that lack of endogenous PACAP exacerbates tissue damage after stroke, and further, that the protective effects of PACAP seem to require patent regulation of IL-6 gene expression, suggesting that induction of IL-6 by PACAP may contribute to its neuroprotective actions . Because PACAP does not induce IL-6 in cortical neuronal cultures (Holighaus, unpublished observations), it is most likely that PACAP’s effects on IL-6 induction are mediated through astrocytes or perhaps even microglial cells in the brain in situ. However, PACAP does induce neuroprotective genes, including BDNF , and the neuro- and cardioprotective protein Stc1 (Holighaus unpublished observations), directly in cultured cortical neurons, as first observed in PC12 cells expressing physiological levels of the PAC1hop receptor . Indeed PACAP regulation of Stc1 occurs in cerebrocortical neuronal cultures, and appears to use the non-canonical cAMP pathway identified in bovine chromaffin cells for regulation of Stc1, GAL and other genes (Holighaus, unpublished observations).
The importance of PACAP at the splanchnicoadrenomedullary has been documented , as summarized in Figure 1. At the same time, PACAP deficiency does not affect elevation of CORT levels 2 hours after induction of hypoglycemia by administration of insulin (i.e. conditions under which PACAP is absolutely required for full epinephrine secretion, maintenance of blood glucose, and survival) . It was thus initially surprising to us to find that following restraint stress in mice, induction of immediate early genes in hypothalamus; elevation of ACTH secretion; induction of steroidogenic enzyme mRNA in adrenal cortex; and elevation of plasma CORT were all (Figure 5) significantly attenuated in PACAP-deficient mice (; Stroth et al., in preparation).
Induction of CRH mRNA in hypothalamus by restraint stress, as shown in Figure 5, is completely dependent on the presence of PACAP. It is not yet known whether PACAP release from neurons directly innervating CRH neurons of the paraventricular nucleus controls CRH gene transcription in stress, although several anatomical lines of evidence suggest this is the case [46-48]. In addition, reports of PACAP expression in the parvocellular neurons themselves, suggest that PACAP in addition to regulating CRH biosynthesis, could be co-released with CRH to regulate ACTH secretion directly [49, 50]. It will be of particular interest to elucidate the roles of PACAP in promoting HPA axis activation in acute and chronic (prolonged) stress. Our initial observations suggest that initial (acute) HPA responses are relatively independent of PACAP, while prolonged stress responses are sustained by PACAP-dependent mechanisms (see Figure 5C), possibly reflecting adequacy of pre-stored CRH for acute responses, and a need for stimulus-secretion-synthesis coupling to sustain HPA axis activation in response to stress.
Several conclusions about PACAP involvement in stress transduction and neuroprotection during stress can be inferred from the data obtained so far, both from our own laboratory and that of others. However, several questions remain to be addressed, as enumerated below, before the role of PACAP in these functions emerges with sufficient clarity to exploit therapeutically in traumatic brain injury, post-traumatic stress disorder and other anxiety disorders.
First, PACAP regulates stress hormone biosynthesis in adrenal gland via up-regulation of at least two enzymes involved in epinephrine biosynthesis, TH and PNMT. Second, PACAP acts at the level of the hypothalamus to control induction, though not basal levels, of CRH mRNA. Third, PACAP control of stress hormone biosynthesis appears to be specialized for prolonged versus acute stress axis activation. Fourth, at least in its effects on the chromaffin cell of the adrenal medulla, and possibility also in cerebrocortical neurons, PACAP utilizes a novel ERK-mediated cAMP-dependent/PKA-dependent signaling pathway to activate a ‘stress response transcriptome’ that includes other neuropeptides, neuroprotective factors, and prohormone convertases. Finally, it is noteworthy that PACAP modulation of stress response in the brain may extend even beyond the HPA and HSA axes. May and colleagues have characterized PACAP induction of a cohort of neuropeptides in cultured post-ganglionic sympathetic neurons in culture [14, 51]. These results imply an action of PACAP to mediate stress responses specific to the sympathetic nervous system (SNS) although these have not yet been investigated in vivo despite the critical role of SNS activation in acute and chronic homeostatic and allostatic responses to a wide range of stressful stimuli [27, 52-54]. Hammack and colleagues have observed that PACAP expression in the bed nucleus of the stria terminalis is greatly enhanced by prolonged stress, and have postulated a potential anxiogenic role for PACAP in the limbic system [55, 56]. The effects of PACAP at these different levels of stress response are summarized in Figure 6.
As a master regulator of stress responses throughout the neuroaxis, PACAP may represent only a ‘jack of all trades’ in mediating neuroprotective, neurotransmission, and gating functions in hypothalamic, hippocampal, amygdalar, and peripheral nervous transduction of homeostatic and allostatic stress responses. GAL, CRH, adenosine, and other neuropeptides may play more specialized roles at particular neuroanatomical locations and under specific stress conditions. PACAP does, however, deserve further attention as a major multi-level stress regulator whose modulation could affect deleterious sustained stress leaving intact the acute (fight or flight) stress response helpful for survival.
In summary, PACAP is paradigmatic as a neuropeptide regulator in stress, and some more general conclusions can also be drawn about microarray analysis of neuropeptide-regulated genes from the accumulating information about PACAP signaling. Microarray analysis reveals--in an unbiased way—gene targets related to mechanisms, biomarkers, and potential drug targets for stress-related disease. These may overlap, and may also function to reinforce each other in translational and reverse-translational approaches to understanding and treatment of stress disorders (Figure 7). Signaling pathways activated in stress may be dissected pharmacologically to allow manipulation of individual components, e.g. neuroprotection, stress hormone biosynthesis, neurotrophin production. Finally, deciding when neuropeptide agonists or antagonists should be sought as stress management therapeutics remains a key question, and one that can be addressed in part by microarray analysis of neuropeptide signaling to the nucleus in a variety of physiological contexts.
Work described in this review was carried out under NIMH-IRP Projects Z01- MH002386-21, Z01- MH002386-22, Z01- MH002386-23, and Z01- MH002386-24. We thank our colleagues Abdel Elkahloun (NHGRI-NIMH-NINDS Microarray Core), Eberhard Weihe (Philipps University, Marburg) and members of the Section on Molecular Neuroscience (Tomris Mustafa) for critical reading of the manuscript.
This contribution summarizes the State-of-the-Art Lecture “Signaling in Stress: The ‘emergency response’ transcriptome” presented by L.E.E. at The 7th International Congress of Neuroendocrinology, July 11-15, 2010, Rouen, France.
1Glutamate and acetylcholine are released from SSVs and act as both fast and slow transmitters depending on receptor interaction; DA is released from both SSVs and LDCVs and acts as a slow transmitter in both cases. Neuropeptides are uniquely released from LDCVs, in part because their prohormones and processing enzymes do not traffic to SSVs.