Karen M. Braas

Post-traumatic stress disorder is associated with PACAP and the PAC1 receptor

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to broadly regulate the cellular stress response. In contrast, it is unclear if the PACAP–PAC1 receptor pathway has a role in human psychological stress responses, such as post-traumatic stress disorder (PTSD). Here we find, in heavily traumatized subjects, a sex-specific association of PACAP blood levels with fear physiology, PTSD diagnosis and symptoms in females. We examined 44 single nucleotide polymorphisms (SNPs) spanning the PACAP (encoded by ADCYAP1) and PAC1 (encoded by ADCYAP1R1) genes, demonstrating a sex-specific association with PTSD. A single SNP in a putative oestrogen response element within ADCYAP1R1, rs2267735, predicts PTSD diagnosis and symptoms in females only. This SNP also associates with fear discrimination and with ADCYAP1R1 messenger RNA expression in human brain. Methylation of ADCYAP1R1 in peripheral blood is also associated with PTSD. Complementing these human data, ADCYAP1R1 mRNA is induced with fear conditioning or oestrogen replacement in rodent models. These data suggest that perturbations in the PACAP–PAC1 pathway are involved in abnormal stress responses underlying PTSD. These sex-specific effects may occur via oestrogen regulation of ADCYAP1R1. PACAP levels and ADCYAP1R1 SNPs may serve as useful biomarkers to further our mechanistic understanding of PTSD.

Chronic stress increases pituitary adenylate cyclase-activating peptide (PACAP) and brain-derived neurotrophic factor (BDNF) mRNA expression in the bed nucleus of the stria terminalis (BNST): Roles for PACAP in anxiety-like behavior

Exposure to chronic stress has been argued to produce maladaptive anxiety-like behavioral states, and many of the brain regions associated with stressor responding also mediate anxiety-like behavior. Pituitary adenylate cyclase activating polypeptide (PACAP) and its specific G protein-coupled PAC(1) receptor have been associated with many of these stress- and anxiety-associated brain regions, and signaling via this peptidergic system may facilitate the neuroplasticity associated with pathological affective states. Here we investigated whether chronic stress increased transcript expression for PACAP, PAC(1) receptor, brain-derived neurotrophic factor (BDNF), and tyrosine receptor kinase B (TrkB) in several nuclei. In rats exposed to a 7 days chronic variate stress paradigm, chronic stress enhanced baseline startle responding induced by handling and exposure to bright lights. Following chronic stress, quantitative transcript assessments of brain regions demonstrated dramatic increases in PACAP and PAC(1) receptor, BDNF, and TrkB receptor mRNA expression selectively in the dorsal aspect of the anterolateral bed nucleus of the stria terminalis (dBNST). Related vasoactive intestinal peptide (VIP) and VPAC receptor, and other stress peptide transcript levels were not altered compared to controls. Moreover, acute PACAP38 infusion into the dBNST resulted in a robust dose-dependent anxiogenic response on baseline startle responding that persisted for 7 days. PACAP/PAC(1) receptor signaling has established trophic functions and its coordinate effects with chronic stress-induced dBNST BDNF and TrkB transcript expression may underlie the maladaptive BNST remodeling and plasticity associated with anxiety-like behavior.

Pituitary adenylate cyclase-activating polypeptides directly stimulate sympathetic neuron neuropeptide Y release through PAC(1) receptor isoform activation of specific intracellular signaling pathways.

Pituitary adenylate cyclase-activating polypeptides (PACAP) have potent regulatory and neurotrophic activities on superior cervical ganglion (SCG) sympathetic neurons with pharmacological profiles consistent for the PACAP-selective PAC1 receptor. Multiple PAC1 receptor isoforms are suggested to determine differential peptide potency and receptor coupling to multiple intracellular signaling pathways. The current studies examined rat SCG PAC1 receptor splice variant expression and coupling to intracellular signaling pathways mediating PACAP-stimulated peptide release. PAC1 receptor mRNA was localized in over 90% of SCG neurons, which correlated with the cells expressing receptor protein. The neurons expressed the PAC1(short)HOP1 receptor but not VIP/PACAP-nonselective VPAC1 receptors; low VPAC2 receptor mRNA levels were restricted to ganglionic nonneuronal cells. PACAP27 and PACAP38 potently and efficaciously stimulated both cAMP and inositol phosphate production; inhibition of phospholipase C augmented PACAP-stimulated cAMP production, but inhibition of adenylyl cyclase did not alter stimulated inositol phosphate production. Phospholipase C inhibition blunted neuron peptide release, suggesting that the phosphatidylinositol pathway was a prominent component of the secretory response. These studies demonstrate preferential sympathetic neuron expression of PACAP-selective receptor variants contributing to regulation of autonomic function.

Roles for pituitary adenylate cyclase-activating peptide (PACAP) expression and signaling in the bed nucleus of the stria terminalis (BNST) in mediating the behavioral consequences of chronic stress.

Anxiety disorders are frequently long-lasting and debilitating for more than 40 million American adults. Although stressor exposure plays an important role in the etiology of some anxiety disorders, the mechanisms by which exposure to stressful stimuli alters central circuits that mediate anxiety-like emotional behavior are still unknown. Substantial evidence has implicated regions of the central extended amygdala, including the bed nucleus of the stria terminalis (BNST) and the central nucleus of the amygdala as critical structures mediating fear- and anxiety-like behavior in both humans and animals. These areas organize coordinated fear- and anxiety-like behavioral responses as well as peripheral stress responding to threats via direct and indirect projections to the paraventricular nucleus of the hypothalamus and brainstem regions (Walker et al. Eur J Pharmacol 463:199–216, 2003, Prog Neuropsychopharmacol Biol Psychiatry 33(8):1291–1308, 2009; Ulrich-Lai and Herman Nat Rev Neurosci 10:397–409, 2009). In particular, the BNST has been argued to mediate these central and peripheral responses when the perceived threat is of long duration (Waddell et al. Behav Neurosci 120:324–336, 2006) and/or when the anxiety-like response is sustained (Walker and Davis Brain Struct Funct 213:29–42, 2008); hence, the BNST may mediate pathological anxiety-like states that result from exposure to chronic stress. Indeed, chronic stress paradigms result in enhanced BNST neuroplasticity that has been associated with pathological anxiety-like states (Vyas et al. Brain Res 965:290–294, 2003; Pego et al. Eur J Neurosci 27:1503–1516, 2008). Here we review evidence that suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) and corticotropin-releasing hormone (CRH) work together to modulate BNST function and increase anxiety-like behavior. Moreover, we have shown that BNST PACAP as well as its cognate PAC1 receptor is substantially upregulated following chronic stress, particularly in the BNST oval nucleus where PACAP-containing neurons closely interact with CRH-containing neurons (Kozicz et al. Brain Res 767:109–119, 1997; Hammack et al. Psychoneuroendocrinology 34:833–843, 2009). We describe how interactions between PACAP and CRH in the BNST may mediate stress-associated behaviors, including anorexia and anxiety-like behavior. These studies have the potential to define specific mechanisms underlying anxiety disorders, and may provide important therapeutic strategies for stress and anxiety management.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression

Abstract: Two forms of pituitary adenylate cyclase‐activating polypeptide (PACAP), the 38‐ and 27‐amino‐acid forms (PACAP38 and PACAP27, respectively), which share amino acid sequence homology with vasoactive intestinal peptide (VIP), were evaluated for their abilities to regulate sympathetic neuron catecholamine and neuropeptide Y (NPY) expression. PACAP38 and PACAP27 potently and efficaciously stimulated NPY and catecholamine secretion in primary cultured superior cervical ganglion (SCG) neurons; 100‐ to 1,000‐fold higher concentrations of VIP were required to modulate secretion, suggesting that SCG neurons express the PACAP‐selective type I receptor. PACAP38 elicited a sustained seven‐ to ninefold increase in the rate of NPY secretion and three‐fold stimulation in the rate of catecholamine release. PACAP38 and PACAP27 produced parallel neuronal NPY and catecholamine release, but cellular levels of NPY and catecholamines were differentially regulated. Sympathetic neuron NPY content was decreased, whereas cellular total catecholamine levels were elevated by the PACAP peptides; total NPY and catecholamine levels (secreted plus cellular content) were increased. In concert with the increased total peptide and transmitter production, pro‐NPY and tyrosine hydroxylase mRNA levels were elevated. Furthermore, PACAP38 was more efficacious than PACAP27 in regulating pro‐NPY and tyrosine hydroxylase mRNA. SCG neuronal expression of mRNA encoding the type I PACAP receptor further supported the studies demonstrating that sympathetic neuronal levels of NPY and catecholamine content and secretion and mRNA are differentially regulated by the PACAP peptides.

Noncompensation in peptide/receptor gene expression and distinct behavioral phenotypes in VIP- and PACAP-deficient mice

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are closely related neurotrophic peptides of the secretin/glucagon family. The two peptides are derived from a common ancestral gene and share many functional attributes in neuronal development/regeneration which occur not only from overlapping receptor subtype signaling but also through common mechanisms regulating their expression. Although PACAP or VIP null mice have been generated for study, it is unclear whether the expression of the complementary peptide or their receptor systems are altered in a compensatory manner during nervous system development. By radioimmunoassay and quantitative PCR measurements, we first show that PACAP and VIP have very different temporal patterns of expression in developing postnatal mouse brain. In wild‐type animals, PACAP transcript and peptide levels increased rapidly 2‐ and 5‐fold, respectively, within 1 week of age. These levels at 1 week of age were maintained through adulthood. VIP transcript and peptide levels, by contrast, increased 25‐ and 50‐fold, respectively, over a later time course. In parallel studies of development, there were no apparent compensatory increases in brain VIP expression in the PACAP knockout animals, PACAP expression in the VIP‐deficient animals, or receptor mRNA levels in either genotype. To the contrary, there was evidence for developmental delays in the expression of peptide and receptor transcripts in the knockout animals. A series of behavioral and neurological tests demonstrated differences between the knockout genotypes, revealing some functional distinctions between the two genes. These results suggest that the PACAP and VIP have evolved to possess distinct biological activities and intimate that the respective knockout phenotypes represent deficits unmitigated by the actions of the complementary related peptide.

Bradykinin analogues: differential agonist and antagonist activities suggesting multiple receptors

Bradykinin analogues with specific antagonist activity in several bioassays were evaluated for effects on [3H]‐bradykinin receptor binding sites and inositol phosphate production in neuroblastoma N1E‐115 cells. The analogues varied in their affinities for bradykinin receptors in guinea‐pig ileum and N1E‐115 cell membranes, in their effects on uterine and ileal contractions and in their agonist or antagonist activity on phosphoinositide turnover in N1E‐115 cells. These tissue specific effects suggest the presence of multiple bradykinin receptor subtypes.

Pituitary adenylate cyclase activating polypeptide (PACAP) expression in sympathetic preganglionic projection neurons to the superior cervical ganglion

Pituitary adenylate cyclase activating polypeptides (PACAP27 and PACAP38) are members of the VIP/secretin/glucagon family of peptides and have diverse neuroregulatory effects in sympathoadrenal cell development and function. PACAP peptides regulate rat superior cervical ganglion (SCG) neuron catecholamine and neuropeptide Y content and secretion, and promote sympathoneuroblast survival through activation of specific PACAP1 receptor isoforms. In examining the potential sources of PACAP regulating the SCG, PACAP expression was identified in rat preganglionic neurons in the intermediolateral cell column (IML) of the thoracic spinal cord which provide primary afferent projections to this sympathetic ganglion. Thoracic spinal cord segments (T1-4) contained approximately 17 pmol PACAP38 immunoreactivity/g tissue wet weight. Reverse-transcription polymerase chain reaction of cDNA from microdissected thoracic spinal cord using primers specific for rat neuronal proPACAP identified proPACAP mRNA expression in the IML; the results correlated with neurons labeled for proPACAP mRNA by in situ hybridization histochemistry and implicated PACAP biosynthesis in IML neurons. To demonstrate directly proPACAP transcript expression in preganglionic projection neurons to the SCG, the ganglion was decentralized and the sympathetic trunk immersed in fluorogold to identify sympathetic preganglionic neurons by retrograde labeling. Cryosections of spinal cord segments containing preganglionic neuron fluorogold labeled neurons were processed subsequently for in situ hybridization histochemical localization of proPACAP mRNA using a digoxigenin-labeled riboprobe; IML neurons were examined for fluorogold and digoxigenin/alkaline phosphatase product dual labeling. More than half of the preganglionic projection neurons to the SCG expressed PACAP mRNA, consistent with the postulate that PACAP peptides released from a subpopulation of thoracic IML preganglionic neurons may be physiological anterograde modulators of sympathetic SCG function.

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways Akt ACTIVATION THROUGH PHOSPHATIDYLINOSITOL 3-KINASE γ AND VESICLE ENDOCYTOSIS FOR NEURONAL SURVIVAL

MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC1 receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC1HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC1HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC1HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on Gαq/phosphatidylinositol 3-kinase γ activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase γ-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC1HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways.

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC1 receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC1HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC1HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC1HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on Gαq/phosphatidylinositol 3-kinase γ activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase γ-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC1HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways.