Pituitary Adenylyl Cyclase-activating Polypeptide Research Articles

Canonical transient receptor potential channels and hypothalamic control of homeostatic functions.

Recent molecular biological and electrophysiological studies have identified multiple transient receptor potential (TRP) channels in hypothalamic neurons as critical modulators of homeostatic functions. In particular, the canonical transient receptor potential channels (TRPCs) are expressed in hypothalamic neurons that are vital for the control of fertility and energy homeostasis. Classical neurotransmitters such as serotonin and glutamate and peptide neurotransmitters such as kisspeptin, neurokinin B and pituitary adenylyl cyclase-activating polypeptide signal through their cognate G protein-coupled receptors to activate TPRC 4, 5 channels, which are essentially ligand-gated calcium channels. In addition to neurotransmitters, circulating hormones like insulin and leptin signal through insulin receptor (InsR) and leptin receptor (LRb), respectively, to activate TRPC 5 channels in hypothalamic arcuate nucleus pro-opiomelanocortin (POMC) and kisspeptin (arcuate Kiss1 [Kiss1ARH]) neurons to have profound physiological (excitatory) effects. Besides its overt depolarizing effects, TRPC channels conduct calcium ions into the cytoplasm, which has a plethora of downstream effects. Moreover, not only the expression of Trpc5 mRNA but also the coupling of receptors to TRPC 5 channel opening are regulated in different physiological states. In particular, the mRNA expression of Trpc5 is highly regulated in kisspeptin neurons by circulating estrogens, which ultimately dictates the firing pattern of kisspeptin neurons. In obesity states, InsRs are "uncoupled" from opening TRPC 5 channels in POMC neurons, rendering them less excitable. Therefore, in this review, we will focus on the critical role of TRPC 5 channels in regulating the excitability of Kiss1ARH and POMC neurons in different physiological and pathological states.

Neuropeptides and the Nodes of Ranvier in Cranial Headaches.

The trigeminovascular system (TGV) comprise of the trigeminal ganglion with neurons and satellite glial cells, with sensory unmyelinated C-fibers and myelinated Aδ-fibers picking up information from different parts of the head and sending signals to the brainstem and the central nervous system. In this review we discuss aspects of signaling at the distal parts of the sensory fibers, the extrasynaptic signaling between C-fibers and Aδ-fibers, and the contact between the trigeminal fibers at the nerve root entry zone where they transit into the CNS. We also address the possible role of the neuropeptides calcitonin gene-related peptide (CGRP), the neurokinin family and pituitary adenylyl cyclase-activating polypeptide 38 (PACAP-38), all found in the TGV system together with their respective receptors. Elucidation of the expression and localization of neuropeptides and their receptors in the TGV system may provide novel ways to understand their roles in migraine pathophysiology and suggest novel ways for treatment of migraine patients.

The PACAP/PAC1 Receptor System and Feeding.

Pituitary adenylyl cyclase activating polypeptide (PACAP) belongs to the vasoactive intestinal polypeptide (VIP)/secretin/glucagon superfamily. PACAP is present in two forms (PACAP-38 and PACAP-27) and binds to three guanine-regulatory (G) protein-coupled receptors (PAC1, VPAC1, and VPAC2). PACAP is expressed in the central and peripheral nervous systems, with high PACAP levels found in the hypothalamus, a brain region involved in feeding and energy homeostasis. PAC1 receptors are high-affinity and PACAP-selective receptors, while VPAC1 and VPAC2 receptors show a comparable affinity to PACAP and VIP. PACAP and its receptors are expressed in the central and peripheral nervous systems with moderate to high expression in the hypothalamus, amygdala, and other limbic structures. Consistent with their expression, PACAP is involved in several physiological responses and pathological states. A growing body of literature suggests that PACAP regulates food intake in laboratory animals. However, there is no comprehensive review of the literature on this topic. Thus, the purpose of this article is to review the literature regarding the role of PACAP and its receptors in food intake regulation and to synthesize how PACAP exerts its anorexic effects in different brain regions. To achieve this goal, we searched PubMed and reviewed 68 articles regarding the regulatory action of PACAP on food intake. Here, we present the literature regarding the effect of exogenous PACAP on feeding and the role of endogenous PACAP in this process. We also provide evidence regarding the effect of PACAP on the homeostatic and hedonic aspects of food intake, the neuroanatomical sites where PACAP exerts its regulatory action, which PACAP receptors may be involved, and the role of various signaling pathways and neurotransmitters in hypophagic effects of PACAP.

Endogenous Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Plays a Protective Effect Against Noise-Induced Hearing Loss.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide (VIP)-the secretin-glucagon family of neuropeptides. They act through two classes of receptors: PACAP type 1 (PAC1) and type 2 (VPAC1 and VPAC2). Among their pleiotropic effects throughout the body, PACAP functions as neuromodulators and neuroprotectors, rescuing neurons from apoptosis, mostly through the PAC1 receptor. To explore the potential protective effect of endogenous PACAP against Noise-induced hearing loss (NIHL), we used a knockout mouse model lacking PAC1 receptor expression (PACR1−/−) and a transgenic humanized mouse model expressing the human PAC1 receptor (TgHPAC1R). Based on complementary approaches combining electrophysiological, histochemical, and molecular biological evaluations, we show PAC1R expression in spiral ganglion neurons and in cochlear apical cells of the organ of Corti. Wild-type (WT), PAC1R−/−, and TgHPAC1R mice exhibit similar auditory thresholds. For most of the frequencies tested after acute noise damage, however, PAC1R−/− mice showed a larger elevation of the auditory threshold than did their WT counterparts. By contrast, in a transgene copy number-dependent fashion, TgHPAC1R mice showed smaller noise-induced elevations of auditory thresholds compared to their WT counterparts. Together, these findings suggest that PACAP could be a candidate for endogenous protection against noise-induced hearing loss.

Attenuation of relaxing response induced by pituitary adenylate cyclase-activating polypeptide in bronchial smooth muscle of experimental asthma.

Bronchomotor tone is regulated by contraction and relaxation of airway smooth muscle (ASM). A weakened ASM relaxation might be a cause of airway hyperresponsiveness (AHR), a characteristic feature of bronchial asthma. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is known as a mediator that causes ASM relaxation. To date, whether or not the PACAP responsiveness is changed in asthmatic ASM is unknown. The current study examined the hypothesis that relaxation induced by PACAP is reduced in bronchial smooth muscle (BSM) of allergic asthma. The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. Tension study showed a BSM hyperresponsiveness to acetylcholine in the OA-challenged mice. Both quantitative RT-PCR and immunoblot analyses revealed a significant decrease in PAC1 receptor expression in BSMs of the diseased mice. Accordingly, in the antigen-challenged group, the PACAP-induced PAC1 receptor-mediated BSM relaxation was significantly attenuated, whereas the relaxation induced by vasoactive intestinal polypeptide was not changed. These findings suggest that the relaxation induced by PACAP is impaired in BSMs of experimental asthma due to a downregulation of its binding partner PAC1 receptor. Impaired BSM responsiveness to PACAP might contribute to the AHR in asthma.

ClC-2-like Chloride Current Alterations in a Cell Model of Spinal and Bulbar Muscular Atrophy, a Polyglutamine Disease.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by expansions of a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. SBMA is associated with the progressive loss of lower motor neurons, together with muscle weakness and atrophy. PolyQ-AR is converted to a toxic species upon binding to its natural ligands, testosterone, and dihydrotestosterone (DHT). Our previous patch-clamp studies on a motor neuron-derived cell model of SBMA showed alterations in voltage-gated ion currents. Here, we identified and characterized chloride currents most likely belonging to the chloride channel-2 (ClC-2) subfamily, which showed significantly increased amplitudes in the SBMA cells. The treatment with the pituitary adenylyl cyclase-activating polypeptide (PACAP), a neuropeptide with a proven protective effect in a mouse model of SBMA, recovered chloride channel current alterations in SBMA cells. These observations suggest that the CIC-2 currents are affected in SBMA, an alteration that may contribute and potentially determine the pathophysiology of the disease.

The role of pituitary adenylyl cyclase activating polypeptide in affective signs of nicotine withdrawal.

Recent evidence implicates endogenous pituitary adenylyl cyclase activating polypeptide (PACAP) in the aversive effect of nicotine. In the present study, we assessed if nicotine-induced conditioned place preference (CPP) or affective signs of nicotine withdrawal would be altered in the absence of PACAP and if there were any sex-related differences in these responses. Male and female mice lacking PACAP and their wild-type controls were tested for baseline place preference on day 1, received conditioning with saline or nicotine (1mg/kg) on alternate days for 6days and were then tested for CPP the next day. Mice were then exposed to four additional conditioning and were tested again for nicotine-induced CPP 24hr later. Controls were conditioned with saline in both chambers and tested similarly. All mice were then, 96hr later, challenged with mecamylamine (3mg/kg), and tested for anxiety-like behaviors 30min later. Mice were then, 2hr later, forced to swim for 15min and then tested for depression-like behaviors 24hr later. Our results showed that male but not female mice lacking PACAP expressed a significant CPP that was comparable to their wild-type controls. In contrast, male but not female mice lacking PACAP exhibited reduced anxiety- and depression-like behaviors compared to their wild-type controls following the mecamylamine challenge. These results suggest that endogenous PACAP is involved in affective signs of nicotine withdrawal, but there is a sex-related difference in this response.

The role of endogenous pituitary adenylyl cyclase activating polypeptide (PACAP) in nicotine self-administration, reward and aversion

Pituitary adenylyl cyclase activating polypeptide (PACAP) and its receptors (PAC1, VPAC1, and VPAC2) are localized in brain regions implicated in stress response, reward seeking and aversive responses, raising the possibility that PACAP may be involved in motivational effects of nicotine. To test this hypothesis, we used two-bottle choice (TBC) and place conditioning paradigms and assessed if nicotine preference or conditioned place preference (CPP) or aversion (CPA) induced by nicotine would be altered in mice lacking PACAP compared to their wild-type controls. In the TBC paradigm, mice had access to two water bottles during the first week and then one of the water bottles was switched to nicotine solution (20, 40 and then 80 μg/mL). The volume of water and nicotine consumed was measured every day. In the place conditioning paradigm, mice were tested for baseline place preference on day 1, received conditioning with saline versus a low (0.25) or high (1 mg/kg) dose nicotine and, respectively, tested for CPP or CPA 24 h following the last conditioning. We discovered that mice lacking PACAP compared to their wild-type controls exhibited more preference for nicotine over water in the TBC paradigm, particularly at the two higher concentrations of nicotine. While the rewarding action of the low dose nicotine was not altered in mice lacking PACAP, the aversive effect of the high dose nicotine was blunted in these mice compared to their wild-type controls. The present results suggest that endogenous PACAP may play a functional role in nicotine preference and its aversive effect.

Pituitary Adenylyl Cyclase Activating Polypeptide (PACAP) Signaling and the Cell Cycle Machinery in Neurodegenerative Diseases.

Pituitary adenylyl cyclase activating polypeptide (PACAP) is a neuropeptide with great neuroprotective effects and remarkable therapeutic potential. PACAP activates several cellular pathways to exert its protective effects. Emerging evidence shows that PACAP can modify the levels and activity of cell cycle components involved in neurodegeneration to protect neurons from death. Cell cycle is a highly regulated process that controls the balance between proliferation, differentiation and death of every cell in the body. Aberrant expression and function of components of the cell cycle machinery have been linked to neurodegenerative diseases, in which different types of neuronal cells become dysfunctional and die in response to toxic insults. Since neurons are postmitotic cells, re-entry into the cell cycle has been shown to be pathological and contributes to the process of neurodegeneration. Moreover, an increasing number of studies highlight the importance of the role of cell cycle components outside the cell cycle and their involvement in neurodegenerative disorders. Here, we discuss the pleiotropic effects of PACAP on cell cycle machinery and the implication for the treatment of neurodegenerative diseases.

Emerging evidence for the role of pituitary adenylate cyclase-activating peptide in neuropsychiatric disorders.

Stress activates many brain nuclei and causes acute changes in several physiological and behavioral responses to restore homeostasis in affected organisms. While this response is protective, chronic stress exposure causes the sustained activation of these nuclei, leading to maladaptive physiological changes that underlie pathological mood and affective states. Hence, chronic stress may produce anxiety and mood disorders by promoting neuronal plasticity within these stress-responsive nuclei. A growing body of evidence attributes neuropeptide systems in mediating not only the physiological stress response but also pathological states that develop following chronic stress exposure. Recent preclinical data suggest that pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptors (PAC1, VPAC1, and VPAC2) play an important role in the behavioral and endocrine responses to stress, as well as in mood and affective disorders. Human studies also point out the significance of the PACAP/PAC1 receptor system in these disorders. For instance, PACAP through PAC1 receptor up-regulates the expression of DISC1 (disrupted in schizophrenia 1) and impedes its association with its interacting protein. Interestingly, the DISC1 gene mutation is linked to schizophrenia and depression. Moreover, a link between PACAP blood titer and fear physiology, post-traumatic stress disorder (PTSD) diagnosis and symptoms has been reported in heavily traumatized female patients. Additionally, in the peripheral blood, methylation of the gene encoding the PAC1 receptor is also associated with PTSD. This book chapter describes the emerging evidence that entails PACAP in the stress response and stress-mediated neuropsychiatric disorders.

A-to-I RNA editing enzyme ADAR2 regulates light-induced circadian phase-shift

In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus and it orchestrates peripheral clocks in the whole body to organize physiological and behavioral rhythms. Light-induced phase-shift of the SCN clock enables synchronization of the circadian clock system with 24-h environmental light/dark cycle. We previously found that adenosine deaminase acting on RNA 2 (Adar2), an A-to-I RNA editing enzyme catalyzing rhythmic A-to-I RNA editing, governs a wide range of mRNA rhythms in the mouse liver and regulates the circadian behavior. In brain, ADAR2-mediated A-to-I RNA editing was reported to occur in various transcripts encoding ion channels and neurotransmitter receptors, which could influence neuronal function of the SCN. Here we show that ADAR2 plays a crucial role for light-induced phase-shift of the circadian clock. Intriguingly, exposure of Adar2-knockout mice to a light pulse at late night caused an aberrant phase-advance of the locomotor rhythms. By monitoring the bioluminescence rhythms of the mutant SCN slices, we found that a phase-advance induced by treatment with pituitary adenylyl cyclase-activating polypeptide (PACAP) was markedly attenuated. The present study suggests that A-to-I RNA editing in the SCN regulates a proper phase response to light in the mouse circadian system.

Light-Triggerable Liposomes for Enhanced Endolysosomal Escape and Gene Silencing in PC12 Cells.

Liposomes are an effective gene and/or drug delivery system, widely used in biomedical applications including gene therapy and chemotherapy. Here, we designed a photo-responsive liposome (lipVP) loaded with a photosensitizer verteporfin (VP). This photosensitizer is clinically approved for photodynamic therapy (PDT). LipVP was employed as a DNA carrier for pituitary adenylyl cyclase-activating polypeptide (PACAP) receptor 1 (PAC1R) gene knockdown in PC12 cells. This has been done by incorporating PAC1R antisense oligonucleotides inside the lipVP cavity. Cells that have taken up the lipVP were exposed to light from a UV light source. As a result of this exposure, reactive oxygen species (ROS) were generated from VP, destabilizing the endolysosomal membranes and enhancing the liposomal release of antisense DNA into the cytoplasm. Endolysosomal escape of DNA was documented at different time points based on quantitative analysis of colocalization between fluorescently labeled DNA and endosomes and lysosomes. The released antisense oligonucleotides were found to silence PAC1R mRNA. The efficiency of this photo-induced gene silencing was demonstrated by a 74% ± 5% decrease in PAC1R fluorescence intensity. Following the light-induced DNA transfer into cells, cell differentiation with exposure to two kinds of PACAP peptides was observed to determine the cell phenotypic change after PAC1R gene knockdown.

Role of pituitary adenylyl cyclase-activating polypeptide in intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. The present study investigated whether stimulation of PACAP receptors (PACAPRs) induces responses in neurons and satellite cells of the superior cervical ganglia (SCG), with special reference to intracellular Ca2+ ([Ca2+]i) changes. The expression of PACAPRs in SCG was detected by reverse transcription-PCR. PACAP type 1 receptor (PAC1R), vasoactive intestinal peptide receptor type (VPAC)1R, and VPAC2R transcripts were expressed in SCG, with PAC1R showing the highest levels. Confocal microscopy analysis revealed that PACAP38 and PACAP27 induced an increase in [Ca2+]i in SCG, first in satellite cells and subsequently in neurons. Neither extracellular Ca2+ removal nor Ca2+ channel blockade affected the PACAP38-induced increase in [Ca2+]i in satellite cells; however, this was partly inhibited in neurons. U73122 or xestospongin C treatment completely and partly abrogated [Ca2+]i changes in satellite cells and in neurons, respectively, whereas VPAC1R and VPAC2R agonists increased [Ca2+]i in satellite cells only. This is the first report demonstrating the expression of PACAPRs specifically, VPAC1 and VPAC2 in SCG and providing evidence for PACAP38-induced [Ca2+]i changes in both satellite cells and neurons via Ca2+ mobilization.

Effects of VPAC1 activation in nucleus ambiguus neurons

The pituitary adenylyl cyclase-activating polypeptide (PACAP) and its G protein-coupled receptors, PAC1, VPAC1 and VPAC2 form a system involved in a variety of biological processes. Although some sympathetic stimulatory effects of this system have been reported, its central cardiovascular regulatory properties are poorly characterized. VPAC1 receptors are expressed in the nucleus ambiguus (nAmb), a key center controlling cardiac parasympathetic tone. In this study, we report that selective VPAC1 activation in rhodamine-labeled cardiac vagal preganglionic neurons of the rat nAmb produces inositol 1,4,5-trisphosphate receptor-mediated Ca2+ mobilization, membrane depolarization and activation of P/Q-type Ca2+ channels. In vivo, this pathway converges onto transient reduction in heart rate of conscious rats. Therefore we demonstrate a VPAC1-dependent mechanism in the central parasympathetic regulation of the heart rate, adding to the complexity of PACAP-mediated cardiovascular modulation.

Adenylyl cyclase activating polypeptide reduces phosphorylation and toxicity of the polyglutamine-expanded androgen receptor in spinobulbar muscular atrophy.

Spinobulbar muscular atrophy (SBMA) is an X-linked neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. SBMA belongs to the family of polyQ diseases, which are fatal neurodegenerative disorders mainly caused by protein-mediated toxic gain-of-function mechanisms and characterized by deposition of misfolded proteins in the form of aggregates. The neurotoxicity of the polyQ proteins can be modified by phosphorylation at specific sites, thereby providing the rationale for the development of disease-specific treatments. We sought to identify signaling pathways that modulate polyQ-AR phosphorylation for therapy development. We report that cyclin-dependent kinase 2 (CDK2) phosphorylates polyQ-AR specifically at Ser96 Phosphorylation of polyQ-AR by CDK2 increased protein stabilization and toxicity and is negatively regulated by the adenylyl cyclase (AC)/protein kinase A (PKA) signaling pathway. To translate these findings into therapy, we developed an analog of pituitary adenylyl cyclase activating polypeptide (PACAP), a potent activator of the AC/PKA pathway. Chronic intranasal administration of the PACAP analog to knock-in SBMA mice reduced Ser96 phosphorylation, promoted polyQ-AR degradation, and ameliorated disease outcome. These results provide proof of principle that noninvasive therapy based on the use of PACAP analogs is a therapeutic option for SBMA.

VPAC1 receptor (Vipr1)-deficient mice exhibit ameliorated experimental autoimmune encephalomyelitis, with specific deficits in the effector stage.

BackgroundVasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two highly homologous neuropeptides. In vitro and ex vivo experiments repeatedly demonstrate that these peptides exert pronounced immunomodulatory (primarily anti-inflammatory) actions which are mediated by common VPAC1 and VPAC2 G protein-coupled receptors. In agreement, we have shown that mice deficient in PACAP ligand or VPAC2 receptors exhibit exacerbated experimental autoimmune encephalomyelitis (EAE). However, we observed that VIP-deficient mice are unexpectedly resistant to EAE, suggesting a requirement for this peptide at some stage of disease development. Here, we investigated the involvement of VPAC1 in the development of EAE using a VPAC1-deficient mouse model.MethodsEAE was induced in wild-type (WT) and VPAC1 knockout (KO) mice using myelin oligodendrocyte glycoprotein 35–55 (MOG35–55), and clinical scores were assessed continuously over 30 days. Immune responses in the spinal cords were determined by histology, real-time PCR and immunofluorescence, and in the draining lymph nodes by antigen-recall assays. The contribution of VPAC1 expression in the immune system to the development of EAE was evaluated by means of adoptive transfer and bone marrow chimera experiments. In other experiments, VPAC1 receptor analogs were given to WT mice.ResultsMOG35–55-induced EAE was ameliorated in VPAC1 KO mice compared to WT mice. The EAE-resistant phenotype of VPAC1 KO mice correlated with reduced central nervous system (CNS) histopathology and cytokine expression in the spinal cord. The immunization phase of EAE appeared to be unimpaired because lymph node cells from EAE-induced VPAC1 KO mice stimulated in vitro with MOG exhibited robust proliferative and Th1/Th17 responses. Moreover, lymph node and spleen cells from KO mice were fully capable of inducing EAE upon transfer to WT recipients. In contrast, WT cells from MOG-immunized mice did not transfer the disease when administered to VPAC1 KO recipients, implicating a defect in the effector phase of the disease. Bone marrow chimera studies suggested that the resistance of VPAC1-deficient mice was only minimally dependent on the expression of this receptor in the immunogenic/hematopoietic compartment. Consistent with this, impaired spinal cord inductions of several chemokine mRNAs were observed in VPAC1 KO mice. Finally, treatment of WT mice with the VPAC1 receptor antagonist PG97-269 before, but not after, EAE induction mimicked the clinical phenotype of VPAC1 KO mice.ConclusionsVPAC1 gene loss impairs the development of EAE in part by preventing an upregulation of CNS chemokines and invasion of inflammatory cells into the CNS. Use of VPAC1 antagonists in WT mice prior to EAE induction also support a critical role for VPAC1 signaling for the development of EAE.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0626-3) contains supplementary material, which is available to authorized users.

Cracking the code of neuronal apoptosis and survival.

Neuronal apoptosis and survival are tightly controlled processes that regulate cell fate during the development of the central nervous system and its homeostasis throughout adulthood. A new study in primary cultures of cerebellar granule neurons identified common transcriptional cascades during rescue from apoptosis by insulin-like growth factor-1 (Igf1) and pituitary adenylyl cyclase-activating polypeptide (Pacap), thus suggesting the existence of a high degree of conservation of cell survival pathways.

Igf1 and Pacap rescue cerebellar granule neurons from apoptosis via a common transcriptional program

A shift of the delicate balance between apoptosis and survival-inducing signals determines the fate of neurons during the development of the central nervous system and its homeostasis throughout adulthood. Both pathways, promoting or protecting from apoptosis, trigger a transcriptional program. We conducted whole-genome expression profiling to decipher the transcriptional regulatory elements controlling the apoptotic/survival switch in cerebellar granule neurons following the induction of apoptosis by serum and potassium deprivation or their rescue by either insulin-like growth factor-1 (Igf1) or pituitary adenylyl cyclase-activating polypeptide (Pacap). Although depending on different upstream signaling pathways, the survival effects of Igf1 and Pacap converged into common transcriptional cascades, thus suggesting the existence of a general transcriptional program underlying neuronal survival.

VPAC2 (vasoactive intestinal peptide receptor type 2) receptor deficient mice develop exacerbated experimental autoimmune encephalomyelitis with increased Th1/Th17 and reduced Th2/Treg responses.

Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two structurally-related neuropeptides with widespread expression in the central and peripheral nervous systems. Although these peptides have been repeatedly shown to exert potent anti-inflammatory actions when administered in animal models of inflammatory disease, mice deficient in VIP and PACAP were recently shown to exhibit different phenotypes (ameliorated and exacerbated, respectively) in response to experimental autoimmune encephalomyelitis (EAE). Therefore, elucidating what are the specific immunoregulatory roles played by each of their receptor subtypes (VPAC1, VPAC2, and PAC1) is critical. In this study, we found that mice with a genetic deletion of VIPR2, encoding the VPAC2 receptor, exhibited exacerbated (MOG35-55)-induced EAE compared to wild type mice, characterized by enhanced clinical and histopathological features, increased proinflammatory cytokines (TNF-α, IL-6, IFN-γ (Th1), and IL-17 (Th17)) and reduced anti-inflammatory cytokines (IL-10, TGFβ, and IL-4 (Th2)) in the CNS and lymph nodes. Moreover, the abundance and proliferative index of lymph node, thymus and CNS CD4(+)CD25(+)FoxP3(+) Tregs were strikingly reduced in VPAC2-deficient mice with EAE. Finally, the in vitro suppressive activity of lymph node and splenic Tregs from VPAC2-deficient mice was impaired. Overall, our results demonstrate critical protective roles for PACAP and the VPAC2 receptor against autoimmunity, promoting the expansion and maintenance of the Treg pool.

Pleiotropic Functions of Pituitary Adenylyl Cyclase-Activating Polypeptide on Retinal Ontogenesis: Involvement of KLF4 in the Control of Progenitor Cell Proliferation

We showed previously that the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) negatively regulates proliferation of postnatal rat retinal progenitor cells through the downregulation of cyclin D1 in a cAMP/protein kinase A dependent manner. In the present study, we describe by microarray analysis several putative PACAP targets regulated by different transcription factor families. One of these families is the Sp/Klf family of transcriptional factors capable of regulating cyclin D1, and among members, we demonstrate by immunocytochemistry that KLF4 is expressed throughout rat retinal development by retinal progenitor cells and in most differentiated cell types. Using retinal explants preparations, PACAP treatment can transiently increase Klf4 mRNA levels; from electrophoretic mobility shift assays, PACAP is also able to increase the nuclear KLF4 content. From these results, we suggest that KLF4 may be involved in the anti-proliferative effects of PACAP as one mechanism regulating progenitor cell transition from proliferation to differentiation throughout retinal development.