Feeding-dependent VIP neuron-ILC3 circuit regulates the intestinal barrier.

The intestinal mucosa serves both as a conduit for the uptake of food-derived nutrients and microbiome-derived metabolites, and as a barrier that prevents tissue invasion by microorganisms and tempers inflammatory responses to the myriad contents of the lumen. How the intestine coordinates physiological and immune responses to food consumption to optimize nutrient uptake while maintaining barrier functions remains unclear. Here we show in mice how a gut neuronal signal triggered by food intake is integrated with intestinal antimicrobial and metabolic responses that are controlled by type-3 innate lymphoid cells (ILC3)1-3. Food consumption rapidly activates a population of enteric neurons that express vasoactive intestinal peptide (VIP)4. Projections of VIP-producing neurons (VIPergic neurons) in the lamina propria are in close proximity to clusters of ILC3 that selectively express VIP receptor type 2 (VIPR2; also known as VPAC2). Production of interleukin (IL)-22 by ILC3, which is upregulated by the presence of commensal microorganisms such as segmented filamentous bacteria5-7, is inhibited upon engagement of VIPR2. As a consequence, levels of antimicrobial peptide derived from epithelial cells are reduced but the expression of lipid-binding proteins and transporters is increased8. During food consumption, the activation of VIPergic neurons thus enhances the growth of segmented filamentous bacteria associated with the epithelium, and increases lipid absorption. Our results reveal a feeding- and circadian-regulated dynamic neuroimmune circuit in the intestine that promotes a trade-off between innate immune protection mediated by IL-22 and the efficiency of nutrient absorption. Modulation of this pathway may therefore be effective for enhancing resistance to enteropathogens2,3,9 and for the treatment of metabolic diseases.

Blockade of vasoactive intestinal peptide receptor 2 (VIPR2) signaling suppresses cyclin D1-dependent cell-cycle progression in MCF-7 cells.

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a G protein-coupled receptor that binds to Gαs, Gαi, and Gαq proteins to regulate various downstream signaling molecules, such as protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), and phospholipase C. In this study, we examined the role of VIPR2 in cell cycle progression. KS-133, a newly developed VIPR2-selective antagonist peptide, attenuated VIP-induced cell proliferation in MCF-7 cells. The percentage of cells in the S-M phase was decreased in MCF-7 cells treated with KS-133. KS-133 in the presence of VIP decreased the phosphorylation of extracellular signal-regulated kinase (ERK), AKT, and glycogen synthase kinase-3ß (GSK3ß), resulting in a decrease in cyclin D1 levels. In MCF-7 cells stably-expressing VIPR2, KS-133 decreased PI3K activity and cAMP levels. Treatment with the ERK-specific kinase (MEK) inhibitor U0126 and the class I PI3K inhibitor ZSTK474 decreased the percentage of cells in the S phase. KS-133 reduced the percentage of cells in the S phase more than treatment with U0126 or ZSTK474 alone and did not affect the effect of the mixture of these inhibitors. Our findings suggest that VIPR2 signaling regulates cyclin D1 levels through the cAMP/PKA/ERK and PI3K/AKT/GSK3ß pathways, and mediates the G1/S transition to control cell proliferation.

VPAC2 receptor mediates VIP-potentiated insulin secretion via ion channels in rat pancreatic ß cells.

Vasoactive intestinal peptide (VIP), a small neuropeptide composing of 28 amino acids, functions as a neuromodulator with insulinotropic effect on pancreatic ß cells, in which it is of vital importance in regulating the levels of blood glucose. VIP potently agonizes VPAC2 receptor (VPAC2-R). Agonists of VPAC2-R stimulate glucose-dependent insulin secretion. The purpose of this study was to further investigate the possible ion channel mechanisms in VPAC2-R-mediated VIP-potentiated insulin secretion. The results of insulin secretion experiments showed that VIP augmented insulin secretion in a glucose-dependent manner. The insulinotropic effect was mediated by VPAC2-R rather than VPAC1 receptor (VPAC1-R), through the adenylyl cyclase (AC)/protein kinase A (PKA) signalling pathway. The calcium imaging analysis demonstrated that VIP increased intracellular Ca2+ concentration ([Ca2+]i). In addition, in the whole-cell voltage-clamp mode, we found that VIP blocked the voltage-dependent potassium (Kv) channel currents, while this effect was reversed by inhibiting the VPAC2-R, AC or PKA respectively. Taken together, these findings suggest that VIP stimulates insulin secretion by inhibiting the Kv channels, activating the Ca2+ channels, and increasing [Ca2+]i through the VPAC2-R and AC/PKA signalling pathway. These findings provide theoretical basis for the research of VPAC2-R as a novel therapeutic target.

Vasoactive intestinal peptide promotes host defense against enteric pathogens by modulating the recruitment of group 3 innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) control the formation of intestinal lymphoid tissues and play key roles in intestinal defense. They express neuropeptide vasoactive intestinal peptide (VIP) receptor 2 (VPAC2), through which VIP modulates their function, but whether VIP exerts other effects on ILC3 remains unclear. We show that VIP promotes ILC3 recruitment to the intestine through VPAC1 independent of the microbiota or adaptive immunity. VIP is also required for postnatal formation of lymphoid tissues as well as the maintenance of local populations of retinoic acid (RA)-producing dendritic cells, with RA up-regulating gut-homing receptor CCR9 expression by ILC3s. Correspondingly, mice deficient in VIP or VPAC1 suffer a paucity of intestinal ILC3s along with impaired production of the cytokine IL-22, rendering them highly susceptible to the enteric pathogen Citrobacter rodentium This heightened susceptibility to C. rodentium infection was ameliorated by RA supplementation, adoptive transfer of ILC3s, or by recombinant IL-22. Thus, VIP regulates the recruitment of intestinal ILC3s and formation of postnatal intestinal lymphoid tissues, offering protection against enteric pathogens.

Differential Expression of PACAP/VIP Receptors in the Post-Mortem CNS White Matter of Multiple Sclerosis Donors.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two neuroprotective and anti-inflammatory molecules of the central nervous system (CNS). Both bind to three G protein-coupled receptors, namely PAC1, VPAC1 and VPAC2, to elicit their beneficial effects in various CNS diseases, including multiple sclerosis (MS). In this study, we assessed the expression and distribution of PACAP/VIP receptors in the normal-appearing white matter (NAWM) of MS donors with a clinical history of either relapsing-remitting MS (RRMS), primary MS (PPMS), secondary progressive MS (SPMS) or in aged-matched non-MS controls. Gene expression studies revealed MS-subtype specific changes in PACAP and VIP and in the receptors' levels in the NAWM, which were partly corroborated by immunohistochemical analyses. Most PAC1 immunoreactivity was restricted to myelin-producing cells, whereas VPAC1 reactivity was diffused within the neuropil and in axonal bundles, and VPAC2 in small vessel walls. Within and around lesioned areas, glial cells were the predominant populations showing reactivity for the different PACAP/VIP receptors, with distinctive patterns across MS subtypes. Together, these data identify the differential expression patterns of PACAP/VIP receptors among the different MS clinical entities. These results may offer opportunities for the development of personalized therapeutic approaches to treating MS and/or other demyelinating disorders.

VPAC1 and VPAC2 receptors mediate tactile hindpaw hypersensitivity and carotid artery dilatation induced by PACAP38 in a migraine relevant mouse model.

BACKGROUND: Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide pivotal in migraine pathophysiology and is considered a promising new migraine drug target. Although intravenous PACAP triggers migraine attacks and a recent phase II trial with a PACAP-inhibiting antibody showed efficacy in migraine prevention, targeting the PACAP receptor PAC1 alone has been unsuccessful. The present study investigated the role of three PACAP receptors (PAC1, VPAC1 and VPAC2) in inducing migraine-relevant hypersensitivity in mice. METHODS: Hindpaw hypersensitivity was induced by repeated PACAP38 injections. Tactile sensitivity responses were quantified using von Frey filaments in three knockout (KO) mouse strains, each lacking one of the PACAP-receptors (Ntotal = 160). Additionally, ex vivo wire myography was used to assess vasoactivity of the carotid artery, and gene expression of PACAP receptors was examined by qPCR. RESULTS: PACAP38 induced hypersensitivity in WT controls (p < 0.01) that was diminished in VPAC1 and VPAC2 KO mice (p < 0.05). In contrast, PAC1 KO mice showed similar responses to WT controls (p > 0.05). Myograph experiments supported these findings showing diminished vasoactivity in VPAC1 and VPAC2 KO mice. We found no upregulation of the non-modified PACAP receptors in KO mice. CONCLUSIONS: This study assessed all three PACAP receptors in a migraine mouse model and suggests a significant role of VPAC receptors in migraine pathophysiology. The lack of hypersensitivity reduction in PAC1 KO mice suggests the involvement of other PACAP receptors or compensatory mechanisms. The results indicate that targeting only individual PACAP receptors may not be an effective migraine treatment.

Outdoor time influences VIPR2 polymorphism rs2071623 to regulate axial length in Han Chinese children.

Clinical relevance: Identification of individuals with a higher risk of developing refractive error under specific gene and environmental backgrounds, especially myopia, could enable more personalized myopic control advice for patients. Background: Refractive error is a common disease that affects visual quality and ocular health worldwide. Its mechanisms have not been elaborated, although both genes and the environment are known to contribute to the process. Interactions between genes and the environment have been shown to exert effects on the onset of refractive error, especially myopia. Axial length elongation is the main characteristic of myopia development and could indicate the severity of myopia. Thus, the purpose of the study was to investigate the interaction between environmental factors and genetic markers of VIPR2 and their impact on spherical equivalence and axial length in a population of Han Chinese children. Methods: A total of 1825 children aged 13~15 years in the Anyang Childhood Eye Study (ACES) were measured for cycloplegic autorefraction, axial length, and height. Saliva DNA was extracted for genotyping three single-nucleotide polymorphisms (SNPs) in the candidate gene (VIPR2). The median outdoor time (2 h/day) was used to categorize children into high and low exposure groups, respectively. Genetic quality control and linear and logistic regressions were performed. Generalized multifactor dimensional reduction (GMDR) was used to investigate gene-environment interactions. Results: There were 1391 children who passed genetic quality control. Rs2071623 of VIPR2 was associated with axial length (T allele, ß=-0.11 se=0.04 p=0.006), while SNP nominally interacted with outdoor time (T allele, ß=-0.17 se=0.08 p=0.029). Rs2071623 in children with high outdoor exposure had a significant interaction effect on axial length (p=0.0007, ß=-0.19 se=0.056) compared to children with low outdoor exposure. GMDR further suggested the existence of an interaction effect between outdoor time and rs2071623. Conclusions: Rs2071623 within VIPR2 could interact with outdoor time in Han Chinese children. More outdoor exposure could enhance the protective effect of the T allele on axial elongation.

Dysfunction of VIPR2 leads to myopia in humans and mice.

BACKGROUND: Myopia is the leading cause of refractive errors. As its pathogenesis is poorly understood, we determined if the retinal VIP-VIPR2 signalling pathway axis has a role in controlling signalling output that affects myopia development in mice. METHODS: Association analysis meta-study, single-cell transcriptome, bulk RNA sequencing, pharmacological manipulation and VIPR2 gene knockout studies were used to clarify if changes in the VIP-VIPR2 signalling pathway affect refractive development in mice. RESULTS: The SNP rs6979985 of the VIPR2 gene was associated with high myopia in a Chinese Han cohort (randomceffect model: p=0.013). After either 1 or 2 days' form deprivation (FD) retinal VIP mRNA expression was downregulated. Retinal single-cell transcriptome sequencing showed that VIPR2 was expressed mainly by bipolar cells. Furthermore, the cAMP signalling pathway axis was inhibited in some VIPR2+ clusters after 2 days of FD. The selective VIPR2 antagonist PG99-465 induced relative myopia, whereas the selective VIPR2 agonist Ro25-1553 inhibited this response. In Vipr2 knockout (Vipr2-KO) mice, refraction was significantly shifted towards myopia (p<0.05). The amplitudes of the bipolar cell derived b-waves in 7-week-old Vipr2-KO mice were significantly larger than those in their WT littermates (p<0.05). CONCLUSIONS: Loss of VIPR2 function likely compromises bipolar cell function based on presumed changes in signal transduction due to altered signature electrical wave activity output in these mice. As these effects correspond with increases in form deprivation myopia (FDM), the VIP-VIPR2 signalling pathway axis is a viable novel target to control the development of this condition.

Development of a New Enzyme-Linked Immunosorbent Assay (ELISA) for Measuring the Content of PACAP in Mammalian Tissue and Plasma.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring neuropeptide found in both the central and peripheral nervous systems of vertebrates. Recent studies have revealed the presence of PACAP and its corresponding receptors, namely, the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R), vasoactive intestinal peptide receptor 1 (VIPR1), and vasoactive intestinal peptide receptor 2 (VIPR2), in various structures implicated in migraine pathophysiology, including sensory trigeminal neurons. Human studies have demonstrated that when infused, PACAP can cause dilation of cranial vessels and result in delayed migraine-like attacks. In light of this, we present a novel ELISA assay that has been validated for quantifying PACAP in tissue extracts and human plasma. Using two well characterized antibodies specifically targeting PACAP, we successfully developed a sandwich ELISA assay, capable of detecting and accurately quantifying PACAP without any cross-reactivity to closely related peptides. The quantification range was between 5.2 pmol/L and 400 pmol/L. The recovery in plasma ranged from 98.2% to 100%. The increasing evidence pointing to the crucial role of PACAP in migraine pathophysiology necessitates the availability of tools capable of detecting changes in the circulatory levels of PACAP and its potential application as a reliable biomarker.

Altered Hippocampal and Striatal Expression of Endothelial Markers and VIP/PACAP Neuropeptides in a Mouse Model of Systemic Lupus Erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the most common and severe manifestations of lupus; however, its pathogenesis is still poorly understood. While there is sparse evidence suggesting that the ongoing autoimmunity may trigger pathogenic changes to the central nervous system (CNS) microvasculature, culminating in inflammatory/ischemic damage, further evidence is still needed. In this study, we used the spontaneous mouse model of SLE (NZBWF1 mice) to investigate the expression of genes and proteins associated with endothelial (dys)function: tissue and urokinase plasminogen activators (tPA and uPA), intercellular and vascular adhesion molecules 1 (ICAM-1 and VCAM-1), brain derived neurotrophic factor (BDNF), endothelial nitric oxide synthase (eNOS) and Krüppel-like factor 4 (KLF4) and neuroprotection/immune modulation: pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), PACAP receptor (PAC1), VIP receptors 1 and 2 (VPAC1 and VPAC2). Analyses were carried out both in the hippocampus and striatum of SLE mice of two different age groups (2 and 7 months old), since age correlates with disease severity. In the hippocampus, we identified a gene/protein expression profile indicative of mild endothelial dysfunction, which increased in severity in aged SLE mice. These alterations were paralleled by moderate alterations in the expression of VIP, PACAP and related receptors. In contrast, we report a robust upregulation of endothelial activation markers in the striatum of both young and aged mice, concurrent with significant induction of the VIP/PACAP system. These data identify molecular signatures of endothelial alterations in the hippocampus and striatum of NZBWF1 mice, which are accompanied by a heightened expression of endogenous protective/immune-modulatory neuropeptides. Collectively, our results support the idea that NPSLE may cause alterations of the CNS micro-vascular compartment that cannot be effectively counteracted by the endogenous activity of the neuropeptides PACAP and VIP.

The Cell-Autonomous Clock of VIP Receptor VPAC2 Cells Regulates Period and Coherence of Circadian Behavior.

Circadian (approximately daily) rhythms pervade mammalian behavior. They are generated by cell-autonomous, transcriptional/translational feedback loops (TTFLs), active in all tissues. This distributed clock network is coordinated by the principal circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). Its robust and accurate time-keeping arises from circuit-level interactions that bind its individual cellular clocks into a coherent time-keeper. Cells that express the neuropeptide vasoactive intestinal peptide (VIP) mediate retinal entrainment of the SCN; and in the absence of VIP, or its cognate receptor VPAC2, circadian behavior is compromised because SCN cells cannot synchronize. The contributions to pace-making of other cell types, including VPAC2-expressing target cells of VIP, are, however, not understood. We therefore used intersectional genetics to manipulate the cell-autonomous TTFLs of VPAC2-expressing cells. Measuring circadian behavioral and SCN rhythmicity in these temporally chimeric male mice thus enabled us to determine the contribution of VPAC2-expressing cells (∼35% of SCN cells) to SCN time-keeping. Lengthening of the intrinsic TTFL period of VPAC2 cells by deletion of the CK1εTau allele concomitantly lengthened the period of circadian behavioral rhythms. It also increased the variability of the circadian period of bioluminescent TTFL rhythms in SCN slices recorded ex vivo Abrogation of circadian competence in VPAC2 cells by deletion of Bmal1 severely disrupted circadian behavioral rhythms and compromised TTFL time-keeping in the corresponding SCN slices. Thus, VPAC2-expressing cells are a distinct, functionally powerful subset of the SCN circuit, contributing to computation of ensemble period and maintenance of circadian robustness. These findings extend our understanding of SCN circuit topology.

AlphaFold version 2.0 elucidates the binding mechanism between VIPR2 and KS-133, and reveals an S-S bond (Cys25-Cys192) formation of functional significance for VIPR2.

The vasoactive intestinal peptide receptor 2 (VIPR2) has attracted attention as a drug target for the treatment of mental disorders, cancer, and immune diseases. In 2021, we identified the peptide KS-133 as a VIPR2-selective antagonist. In this study, we aimed to elucidate the binding mechanism between VIPR2 and KS-133. To this end, VIPR2/KS-133 and VIPR2/vasoactive intestinal peptide (VIP) complex models were constructed through AlphaFold version 2.0 and molecular dynamic simulations. Our models revealed that: (i) both KS-133 and VIP have helical structures, (ii) the interaction residues on VIPR2 for both peptides are similar, and (iii) the orientation of their helices upon their binding to VIPR2 are different by ∼45°. Interestingly, in the process of constructing the aforementioned models, an S-S bond formation between Cys25 and Cys192 of the human VIPR2 was identified. Although these two Cys residues are highly conserved among species (i.e., corresponding to Cys24 and Cys191 in the mouse), no previous reports regarding this S-S bond formation exist. In order to clarify the potential role of this S-S bond in the VIPR2 has functional consequences, a cell line expressing the mouse VIPR2(Cys24Ala, Cys191Ala) was generated. During the VIP stimulation of this cell line, the phosphorylation of AKT (a downstream signal marker of VIPR2) was found to be significantly attenuated, thereby suggesting that the S-S bond has a functional significance for VIPR2. Our study not only elucidates the VIPR2-binding mechanism of KS-133 for the first time, but also provides new insights into the structural biology of VIPR2.

Human CD4+CD45RA+ T Cells Behavior after In Vitro Activation: Modulatory Role of Vasoactive Intestinal Peptide.

Naїve CD4+ T cells, which suffer different polarizing signals during T cell receptor activation, are responsible for an adequate immune response. In this study, we aimed to evaluate the behavior of human CD4+CD45RA+ T cells after in vitro activation by anti-CD3/CD28 bead stimulation for 14 days. We also wanted to check the role of the VIP system during this process. The metabolic biomarker Glut1 was increased, pointing to an increase in glucose requirement whereas Hif-1α expression was higher in resting than in activated cells. Expression of Th1 markers increased at the beginning of activation, whereas Th17-associated biomarkers augmented after that, showing a pathogenic Th17 profile with a possible plasticity to Th17/1. Foxp3 mRNA expression augmented from day 4, but no parallel increases were observed in IL-10, IL-2, or TGFß mRNA expression, meaning that these potential differentiated Treg could not be functional. Both VIP receptors were located on the plasma membrane, and expression of VPAC2 receptor increased significantly with respect to the VPAC1 receptor from day 4 of CD4+CD45RA+ T activation, pointing to a shift in VPAC receptors. VIP decreased IFNγ and IL-23R expression during the activation, suggesting a feasible modulation of Th17/1 plasticity and Th17 stabilization through both VPAC receptors. These novel results show that, without polarizing conditions, CD4+CD45RA+ T cells differentiate mainly to a pathogenic Th17 subset and an unpaired Treg subset after several days of activation. Moreover, they confirm the important immunomodulatory role of VIP, also on naїve Th cells, stressing the importance of this neuropeptide on lymphocyte responses in different pathological or non-pathological situations.

Targeting VIP and PACAP Receptor Signaling: New Insights into Designing Drugs for the PACAP Subfamily of Receptors.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Vasoactive Intestinal Peptide (VIP) are neuropeptides involved in a diverse array of physiological and pathological processes through activating the PACAP subfamily of class B1 G protein-coupled receptors (GPCRs): VIP receptor 1 (VPAC1R), VIP receptor 2 (VPAC2R), and PACAP type I receptor (PAC1R). VIP and PACAP share nearly 70% amino acid sequence identity, while their receptors PAC1R, VPAC1R, and VPAC2R share 60% homology in the transmembrane regions of the receptor. PACAP binds with high affinity to all three receptors, while VIP binds with high affinity to VPAC1R and VPAC2R, and has a thousand-fold lower affinity for PAC1R compared to PACAP. Due to the wide distribution of VIP and PACAP receptors in the body, potential therapeutic applications of drugs targeting these receptors, as well as expected undesired side effects, are numerous. Designing selective therapeutics targeting these receptors remains challenging due to their structural similarities. This review discusses recent discoveries on the molecular mechanisms involved in the selectivity and signaling of the PACAP subfamily of receptors, and future considerations for therapeutic targeting.

VIP/VPAC Axis Expression in Immune-Mediated Inflammatory Disorders: Associated miRNA Signatures.

Few studies have considered immune-mediated inflammatory disorders (IMID) together, which is necessary to adequately understand them given they share common mechanisms. Our goal was to investigate the expression of vasoactive intestinal peptide (VIP) and its receptors VPAC1 and VPAC2 in selected IMID, analyze the effect of biological therapies on them, and identify miRNA signatures associated with their expression. Serum VIP levels and mRNA of VPAC and miRNA expression in peripheral blood mononuclear cells were analyzed from 52 patients with psoriasis, rheumatoid arthritis, Graves' disease, or spondyloarthritis and from 38 healthy subjects. IMID patients showed higher levels of VIP and increased expression of VPAC2 compared to controls (p < 0.0001 and p < 0.0192, respectively). Receiver operating characteristic curve analysis showed that the levels of VIP or VPAC2 expression were adequate discriminators capable of identifying IMID. Treatment of IMID patients with anti-TNFα and anti-IL12/23 significantly affected serum VIP levels. We identified miRNA signatures associated with levels of serum VIP and VPAC2 expression, which correlated with IMID diagnosis of the patients. The results indicate that the expression of VIP/VPAC2 is able of identify IMIDs and open up a line of research based on the association between the VIP/VPAC axis and miRNA signatures in immune-mediated diseases.

PACAP-38 Induces Transcriptomic Changes in Rat Trigeminal Ganglion Cells Related to Neuroinflammation and Altered Mitochondrial Function Presumably via PAC1/VPAC2 Receptor-Independent Mechanism.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a broadly expressed neuropeptide which has diverse effects in both the peripheral and central nervous systems. While its neuroprotective effects have been shown in a variety of disease models, both animal and human data support the role of PACAP in migraine generation. Both PACAP and its truncated derivative PACAP(6-38) increased calcium influx in rat trigeminal ganglia (TG) primary sensory neurons in most experimental settings. PACAP(6-38), however, has been described as an antagonist for PACAP type I (known as PAC1), and Vasoactive Intestinal Polypeptide Receptor 2 (also known as VPAC2) receptors. Here, we aimed to compare the signaling pathways induced by the two peptides using transcriptomic analysis. Rat trigeminal ganglion cell cultures were incubated with 1 µM PACAP-38 or PACAP(6-38). Six hours later RNA was isolated, next-generation RNA sequencing was performed and transcriptomic changes were analyzed to identify differentially expressed genes. Functional analysis was performed for gene annotation using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome databases. We found 200 common differentially expressed (DE) genes for these two neuropeptides. Both PACAP-38 and PACAP(6-38) treatments caused significant downregulation of NADH: ubiquinone oxidoreductase subunit B6 and upregulation of transient receptor potential cation channel, subfamily M, member 8. The common signaling pathways induced by both peptides indicate that they act on the same target, suggesting that PACAP activates trigeminal primary sensory neurons via a mechanism independent of the identified and cloned PAC1/VPAC2 receptor, either via another target structure or a different splice variant of PAC1/VPAC2 receptors. Identification of the target could help to understand key mechanisms of migraine.

Impaired cardiorespiratory responses to hypercapnia in neonatal mice lacking PAC1 but not VPAC2 receptors.

The evidence is mounting for a role for abnormal signaling of the stress peptide pituitary adenylate cyclase activating polypeptide (PACAP) and its canonical receptor PAC1 in the pathogenesis of sudden infant death syndrome. In this study, we investigated whether the PACAP receptors PAC1 or VPAC2 are involved in the neonatal cardiorespiratory response to hypercapnic stress. We used head-out plethysmography and surface ECG electrodes to assess cardiorespiratory responses to an 8% hypercapnic challenge in unanesthetized and spontaneously breathing 4-day-old PAC1 or VPAC2 knockout (KO) and wild-type mouse pups. We demonstrate that compared with WTs, breathing frequency (RR) and minute ventilation ([Formula: see text]) in PAC1 KO pups were significantly blunted in response to hypercapnia. Although heart rate was unaltered in PAC1 KO pups during hypercapnia, heart rate recovery posthypercapnia was impaired. In contrast, cardiorespiratory impairments in VPAC2 KO pups were limited to only an overall higher tidal volume (VT), independent of treatment. These findings suggest that PACAP signaling through the PAC1 receptor plays a more important role than signaling through the VPAC2 receptor in neonatal respiratory responses to hypercapnia. Thus deficits in PACAP signaling primarily via PAC1 may contribute to the inability of infants to mount an appropriate protective response to homeostatic stressors in childhood disorders such as SIDS.

Inhibition of PACAP/PAC1/VPAC2 signaling impairs the consolidation of social recognition memory and nitric oxide prevents this deficit.

Social recognition memory (SRM) forms the basis of social relationships of animals. It is essential for social interaction and adaptive behavior, reproduction and species survival. Evidence demonstrates that social deficits of psychiatric disorders such as autism and schizophrenia are caused by alterations in SRM processing by the hippocampus and amygdala. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptors PAC1, VPAC1 and VPAC2 are highly expressed in these regions. PACAP is a pleiotropic neuropeptide that modulates synaptic function and plasticity and is thought to be involved in social behavior. PACAP signaling also stimulates the nitric oxide (NO) production and targets outcomes to synapses. In the present work, we investigate the effect of the infusion of PACAP-38 (endogenous neuropeptide and potent stimulator of adenylyl cyclase), PACAP 6-38 (PAC1/VPAC2 receptors antagonist) and S-Nitroso-N-acetyl-DL-penicillamine (SNAP, NO donor) in the CA1 region of the hippocampus and in the basolateral amygdala (BLA) on the consolidation of SRM. For this, male Wistar rats with cannulae implanted in CA1 or in BLA were subjected to a social discrimination paradigm, which is based on the natural ability of rodents to investigate unfamiliar conspecifics more than familiar one. In the sample phase (acquisition), animals were exposed to a juvenile conspecific for 1 h. Immediately, 60 or 150 min after, animals received one of different pharmacological treatments. Twenty-four hours later, they were submitted to a 5 min retention test in the presence of the previously presented juvenile (familiar) and a novel juvenile. Animals that received infusions of PACAP 6-38 (40 pg/side) into CA1 immediately after the sample phase or into BLA immediately or 60 min after the sample phase were unable to recognize the familiar juvenile during the retention test. This impairment was abolished by the coinfusion of PACAP 6-38 plus SNAP (5 µg/side). These results show that the blockade of PACAP/PAC1/VPAC2 signaling in the CA1 and BLA during a restricted post-acquisition time window impairs the consolidation of SRM and that the SNAP is able to abolish this deficit. Findings like this could potentially be used in the future to influence studies of psychiatric disorders involving social behavior.

CFH and VIPR2 as susceptibility loci in choroidal thickness and pachychoroid disease central serous chorioretinopathy.

Central serous chorioretinopathy (CSC) is a common disease affecting younger people and may lead to vision loss. CSC shares phenotypic overlap with age-related macular degeneration (AMD). As recent studies have revealed a characteristic increase of choroidal thickness in CSC, we conducted a genome-wide association study on choroidal thickness in 3,418 individuals followed by TaqMan assays in 2,692 subjects, and we identified two susceptibility loci: CFH rs800292, an established AMD susceptibility polymorphism, and VIPR2 rs3793217 (P = 2.05 × 10-10 and 6.75 × 10-8, respectively). Case-control studies using patients with CSC confirmed associations between both polymorphisms and CSC (P = 5.27 × 10-5 and 5.14 × 10-5, respectively). The CFH rs800292 G allele is reportedly a risk allele for AMD, whereas the A allele conferred risk for thicker choroid and CSC development. This study not only shows that susceptibility genes for CSC could be discovered using choroidal thickness as a defining variable but also, deepens the understanding of differences between CSC and AMD pathophysiology.

Modulation of Prostanoids Profile and Counter-Regulation of SDF-1α/CXCR4 and VIP/VPAC2 Expression by Sitagliptin in Non-Diabetic Rat Model of Hepatic Ischemia-Reperfusion Injury.

Molecular mechanisms underlying the beneficial effect of sitagliptin repurposed for hepatic ischemia-reperfusion injury (IRI) are poorly understood. We aimed to evaluate the impact of IRI and sitagliptin on the hepatic profile of eicosanoids (LC-MS/MS) and expression/concentration (RTqPCR/ELISA) of GLP-1/GLP-1R, SDF-1α/CXCR4 and VIP/VPAC1, VPAC2, and PAC1 in 36 rats. Animals were divided into four groups and subjected to ischemia (60 min) and reperfusion (24 h) with or without pretreatment with sitagliptin (5 mg/kg) (IR and SIR) or sham-operated with or without sitagliptin pretreatment (controls and sitagliptin). PGI2, PGE2, and 13,14-dihydro-PGE1 were significantly upregulated in IR but not SIR, while sitagliptin upregulated PGD2 and 15-deoxy-12,14-PGJ2. IR and sitagliptin non-significantly upregulated GLP-1 while Glp1r expression was borderline detectable. VIP concentration and Vpac2 expression were downregulated in IR but not SIR, while Vpac1 was significantly downregulated solely in SIR. IRI upregulated both CXCR4 expression and concentration, and sitagliptin pretreatment abrogated receptor overexpression and downregulated Sdf1. In conclusion, hepatic IRI is accompanied by an elevation in proinflammatory prostanoids and overexpression of CXCR4, combined with downregulation of VIP/VPAC2. Beneficial effects of sitagliptin during hepatic IRI might be mediated by drug-induced normalization of proinflammatory prostanoids and upregulation of PGD2 and by concomitant downregulation of SDF-1α/CXCR4 and reinstating VIP/VCAP2 signaling.