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The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) and its specific receptor (PAC1R) are widely present in the central nervous system (CNS), and PACAP/PAC1R signaling has been implicated in anxiety-related behaviors. The locus coeruleus (LC), with its extensive noradrenergic (NA) projections throughout the CNS, is also implicated in anxiety. Although the LC exhibits a high expression of PAC1R, the precise role of PACAP/PAC1R signaling in the LC's involvement in anxiety remains unclear. Histochemical analysis confirmed high levels of PAC1R mRNA in the LC and showed that PAC1R gene transcripts were highly localized to NA neurons. Targeted deletion of PAC1R from these cells led to a hyperactive/low anxiety phenotype in the open field and elevated-plus maze tests. Retrograde neurocircuit tracing indicated PACAP neurons from the anterior insular cortex (aIC) and a few other regions projected axons to the LC. The selective activation of PACAP neurons in the aIC led to significantly increased anxiety behavior without a change in overall locomotor activity. Moreover, shRNA PACAP knockdown in the aIC in wild-type mice led to a selective decrease in anxiety. The present results identify an aIC to LC neurocircuit controlling anxiety that critically requires PACAP/PAC1R signaling., Competing Interests: Declaration of competing interest The authors declare no competing financial interests or personal relationships that could have influenced the current work., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

PACAP (pituitary adenylate cyclase activating polypeptide) is a widespread neuropeptide with cytoprotective and anti-inflammatory effects. It plays a role in innate and adaptive immunity, but data are limited about gut-associated lymphoid tissue. We aimed to reveal differences in Peyer's patches between wild-type (WT) and PACAP-deficient (KO) mice. Peyer's patch morphology from young (3-months-old) and aging (12-15-months-old) mice was examined, along with flow cytometry to assess immune cell populations, expression of checkpoint molecules (PD-1, PD-L1, TIM-3, Gal-9) and functional markers (CD69, granzyme B, perforin) in CD3+, CD4+, and CD8+ T cells. We found slight differences between aging, but not in young, WT, and KO mice. In WT mice, aging reduced CD8+ T cell numbers frequency and altered checkpoint molecule expression (higher TIM-3, granzyme B; lower Gal-9, CD69). CD4+ T cell frequency was higher with similar checkpoint alterations, indicating a regulatory shift. In PACAP KO mice, aging did not change cell population frequencies but led to higher TIM-3, granzyme B and lower PD-1, PD-L1, Gal-9, and CD69 expression in CD4+ and CD8+ T cells, with reduced overall T cell activity. Thus, PACAP deficiency impacts immune dysfunction by altering checkpoint molecules and T cell functionality, particularly in CD8+ T cells, suggesting complex immune responses by PACAP, highlighting its role in intestinal homeostasis and potential implications for inflammatory bowel diseases.

Objectives: To explore the relationship between sensitization points of the body surface and the expression of pituitary adenylate cyclase activating polypeptides (PACAP) in myocardial ischemia (MI) mice, so as to reveal the underlying mechanisms of acupoint sensitization from the perspective of molecular biology., Methods: Male C57BL/6J mice were randomly divided into control and model groups (28 mice/group). The MI-induced visceral pain model was established by intraperitoneal injection of isoprenaline (ISO, 160 mg/kg). The mice of the control group received intraperitoneal injection of the same dose of normal saline. Six days after modeling, the Evans blue (EB) dye was injected into the tail vein of mice to observe the distribution and quantity of the plasma extravasated EB points at the body surface. Meanwhile, the mechanical pain threshold (MPT) was measured to evaluate the level of pain sensitivity in the activated area on their body surface and left forelimb and hindlimb, respectively. Hematoxylin-eosin (H.E.) staining was used to evaluate the morphologic and pathological changes of the heart tissue in the two groups. Then, the expressions of PACAP in the thoracic (T)1-T5 dorsal root ganglia (DRGs), spinal cord and skin in the dominant area of body surface were detected by Western blot and immunofluorescence staining, respectively., Results: Compared with the control group, the heart tissue of the model group was hypertrophic and the myocardial tissue showed obvious inflammatory cell infiltration and fibrosis. In addition to these pathologic changes, the number of EB exudation points on the body surface was significantly increased ( P <0.01), and was mainly distributed in the innervated region of T1-T5 segments of the spinal cord, and the MPT of these EB exudation points was lower than that of non-exudation points ( P <0.01). At the same time, the MPTs of left forelimb and hindlimb were significantly decreased in the model group ( P <0.001). More importantly, the level of protein and positive expression of PACAP were significantly higher in the model group than those in the control group, which was observed in the innervated body surface, spinal cord and its DRG neurons of T1-T5 segments ( P <0.01, P <0.001, P <0.05)., Conclusions: ISO injection resulted in histological lesions and cardiogenic referred pain on the body surface after the formation of MI in mice. The expression of PACAP in the body surface of the sensitization points, the corresponding T1-T5 segments of spinal cord and DRG neurons were significantly increased, which may partly explain the reason for acupoint sensitization in the animal model of visceral pain.

The physiological processes in animal production are regulated through biologically active molecules like peptides, proteins, and hormones identified through the development of the fundamental sciences and their application. One of the main polypeptides that plays an essential role in regulating physiological responses is the pituitary adenylate cyclase-activating polypeptide (PACAP). PACAP belongs to the glucagon/growth hormone-releasing hormone (GHRH)/vasoactive intestinal proteins (VIP) family and regulates feed intake, stress, and immune response in birds. Most of these regulations occur after PACAP stimulates the cAMP signaling pathway, which can regulate the expression of genes like MuRF1, FOXO1, Atrogin 1, and other ligases that are essential members of the ubiquitin system. On the other hand, PACAP stimulates the secretion of CRH in response to stress, activating the ubiquitin signaling pathway that plays a vital role in protein degradation and regulates oxidative stress and immune responses. Many studies conducted on rodents, mammals, and other models confirm the regulatory effects of PACAP, cAMP, and the ubiquitin pathway; however, there are no studies testing whether PACAP-induced cAMP signaling in poultry regulates the ubiquitin pathway. Besides, it would be interesting to investigate if PACAP can regulate ubiquitin signaling during stress response via CRH altered by HPA axis stimulation. Therefore, this review highlights a summary of research studies that indicate the potential interaction of the PACAP and ubiquitin signaling pathways on different molecular and physiological parameters in poultry species through the cAMP and stress signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Obesity and obesity-related insulin resistance have been a research hotspot. Pituitary adenylate cyclase activating polypeptide (PACAP) has emerged as playing a significant role in energy metabolism, holding promising potential for attenuating insulin resistance. However, the precise mechanism is not fully understood. Palmitic acid and a high-fat diet (HFD) were used to establish insulin resistance model in Alpha mouse liver 12 cell line and C57BL/6 mice, respectively. Subsequently, we assessed the effects of PACAP both in vivo and in vitro. Lentivirus vectors were used to explore the signaling pathway through which PACAP may ameliorate insulin resistance. PACAP was found to selectively bind to the PACAP type I receptor receptor and ameliorate insulin resistance, which was characterized by increased glycogen synthesis and the suppression of gluconeogenesis in the insulin-resistant cell model and HFD-fed mice. These effects were linked to the activation of the Fas apoptotic inhibitory molecule/rapamycin-insensitive companion of mammalian target of rapamycin/RAC-alpha serine/threonine-protein kinase (FAIM/Rictor/AKT) axis. Furthermore, PACAP ameliorated insulin resistance by increasing solute carrier family 2, facilitated glucose transporter members 2/4 and inhibiting gluconeogenesis-related proteins glucose 6-phosphatase catalytic subunit 1 and phosphoenolpyruvate carboxykinase 2 expression. Meanwhile, the phosphorylation of hepatic AKT/glycogen synthase kinase 3β was promoted both in vivo and in vitro by PACAP. Additionally, PACAP treatment decreased body weight, food intake and blood glucose levels in obese mice. Our study shows that PACAP ameliorated insulin resistance through the FAIM/Rictor/AKT axis, presenting it as a promising drug candidate for the treatment of obesity-related insulin resistance., (© 2024 Federation of European Biochemical Societies.)

Visuospatial working memory (vsWM), which is impaired in schizophrenia (SZ), is mediated by multiple cortical regions including the primary (V1) and association (V2) visual, posterior parietal (PPC) and dorsolateral prefrontal (DLPFC) cortices. In these regions, parvalbumin (PV) or somatostatin (SST) GABA neurons are altered in SZ as reflected in lower levels of activity-regulated transcripts. As PV and SST neurons receive excitatory inputs from neighboring pyramidal neurons, we hypothesized that levels of activity-regulated transcripts are also lower in pyramidal neurons in these regions. Thus, we quantified levels of four activity-regulated, pyramidal neuron-selective transcripts, namely adenylate cyclase-activating polypeptide-1 (ADCYAP1), brain-derived neurotrophic factor (BDNF), neuronal pentraxin-2 (NPTX2) and neuritin-1 (NRN1) mRNAs, in V1, V2, PPC and DLPFC from unaffected comparison and SZ individuals. In SZ, BDNF and NPTX2 mRNA levels were lower across all four regions, whereas ADCYAP1 and NRN1 mRNA levels were lower in V1 and V2. The regional pattern of deficits in BDNF and NPTX2 mRNAs was similar to that in transcripts in PV and SST neurons in SZ. These findings suggest that lower activity of pyramidal neurons expressing BDNF and/or NPTX2 mRNAs might contribute to alterations in PV and SST neurons across the vsWM network in SZ., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024. Published by Elsevier B.V.)

Inflammation with expression of interleukin 6 (IL-6) in the central nervous system (CNS) occurs in several neurodegenerative/neuroinflammatory conditions and may cause neurochemical changes to endogenous neuroprotective systems. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two neuropeptides with well-established protective and anti-inflammatory properties. Yet, whether PACAP and VIP levels are altered in mice with CNS-restricted, astrocyte-targeted production of IL-6 (GFAP-IL6) remains unknown. In this study, PACAP/VIP levels were assessed in the brain of GFAP-IL6 mice. In addition, we utilised bi-genic GFAP-IL6 mice carrying the human sgp130-Fc transgene (termed GFAP-IL6/sgp130Fc mice) to determine whether trans-signalling inhibition rescued PACAP/VIP changes in the CNS. Transcripts and protein levels of PACAP and VIP, as well as their receptors PAC1, VPAC1 and VPAC2, were significantly increased in the cerebrum and cerebellum of GFAP-IL6 mice vs. wild type (WT) littermates. These results were paralleled by a robust activation of the JAK/STAT3, NF-κB and ERK1/2MAPK pathways in GFAP-IL6 mice. In contrast, co-expression of sgp130Fc in GFAP-IL6/sgp130Fc mice reduced VIP expression and activation of STAT3 and NF-κB pathways, but it failed to rescue PACAP, PACAP/VIP receptors and Erk1/2MAPK phosphorylation. We conclude that forced expression of IL-6 in astrocytes induces the activation of the PACAP/VIP neuropeptide system in the brain, which is only partly modulated upon IL-6 trans-signalling inhibition. Increased expression of PACAP/VIP neuropeptides and receptors may represent a homeostatic response of the CNS to an uncontrolled IL-6 synthesis and its neuroinflammatory consequences.

Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1β and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1β protein from previously accumulated transcripts or the cleavage of accumulated IL-1β precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Orexin A and B (OXA and OXB) and their receptors are expressed in the majority of retinal neurons in humans, rats, and mice. Orexins modulate signal transmission between the different layers of the retina. The suprachiasmatic nucleus (SCN) and the retina are central and peripheral components of the body's biological clocks; respectively. The SCN receives photic information from the retina through the retinohypothalamic tract (RHT) to synchronize bodily functions with environmental changes. In present study, we aimed to investigate the impact of inhibiting retinal orexin receptors on the expression of retinal Bmal1 and c-fos, as well as hypothalamic c-fos, Bmal1, Vip, and PACAP at four different time-points (Zeitgeber time; ZT 3, 6, 11, and ZT-0). The intravitreal injection (IVI) of OX1R antagonist (SB-334867) and OX2R antagonist (JNJ-10397049) significantly up-regulated c-fos expression in the retina. Additionally, compared to the control group, the combined injection of SB-334867 and JNJ-10397049 showed a greater increase in retinal expression of this gene. Moreover, the expression of hypothalamic Vip and PACAP was significantly up-regulated in both the SB-334867 and JNJ-10397049 groups. In contrast, the expression of Bmal1 was down-regulated. Furthermore, the expression of hypothalamic c-fos was down-regulated in all groups treated with SB-334867 and JNJ-10397049. Additionally, the study demonstrated that blocking these receptors in the retina resulted in alterations in circadian rhythm parameters such as mesor, amplitude, and acrophase. Finally, it affected the phase of gene expression rhythms in both the retina and hypothalamus, as identified through cosinor analysis and the zero-amplitude test. This study represents the initial exploration of how retinal orexin receptors influence expression of rhythmic genes in the retina and hypothalamus. These findings could provide new insights into how the retina regulates the circadian rhythm in both regions and illuminate the role of the orexinergic system expression within the retina., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Objective: Hypothalamic signals potently stimulate energy expenditure by engaging peripheral mechanisms to restore energy homeostasis. Previous studies have identified several critical hypothalamic sites (e.g. preoptic area (POA) and ventromedial hypothalamic nucleus (VMN)) that could be part of an interconnected neurocircuit that controls tissue thermogenesis and essential for body weight control. However, the key neurocircuit that can stimulate energy expenditure has not yet been established., Methods: Here, we investigated the downstream mechanisms by which VMN neurons stimulate adipose tissue thermogenesis. We manipulated subsets of VMN neurons acutely as well as chronically and studied its effect on tissue thermogenesis and body weight control, using Sf1 Cre and Adcyap1 Cre mice and measured physiological parameters under both high-fat diet and standard chow diet conditions. To determine the node efferent to these VMN neurons, that is involved in modulating energy expenditure, we employed electrophysiology and optogenetics experiments combined with measurements using tissue-implantable temperature microchips., Results: Activation of the VMN neurons that express the steroidogenic factor 1 (Sf1; VMN Sf1 neurons) reduced body weight, adiposity and increased energy expenditure in diet-induced obese mice. This function is likely mediated, at least in part, by the release of the pituitary adenylate cyclase-activating polypeptide (PACAP; encoded by the Adcyap1 gene) by the VMN neurons, since we previously demonstrated that PACAP, at the VMN, plays a key role in energy expenditure control. Thus, we then shifted focus to the subpopulation of VMN Sf1 neurons that contain the neuropeptide PACAP (VMN PACAP neurons). Since the VMN neurons do not directly project to the peripheral tissues, we traced the location of the VMN PACAP neurons' efferents. We identified that VMN PACAP neurons project to and activate neurons in the caudal regions of the POA whereby these projections stimulate tissue thermogenesis in brown and beige adipose tissue. We demonstrated that selective activation of caudal POA projections from VMN PACAP neurons induces tissue thermogenesis, most potently in negative energy balance and activating these projections lead to some similar, but mostly unique, patterns of gene expression in brown and beige tissue. Finally, we demonstrated that the activation of the VMN PACAP neurons' efferents that lie at the caudal POA are necessary for inducing tissue thermogenesis in brown and beige adipose tissue., Conclusions: These data indicate that VMN PACAP connections with the caudal POA neurons impact adipose tissue function and are important for induction of tissue thermogenesis. Our data suggests that the VMN PACAP → caudal POA neurocircuit and its components are critical for controlling energy balance by activating energy expenditure and body weight control., Competing Interests: Declaration of competing interest Jonathan N. Flak and Travis S. Johnson have received research support from Eli Lilly and Company, United States for separate projects in their respective laboratories., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The global aquaculture industry has significant losses each year due to disease outbreaks. Antibiotics are one of the common methods to treat fish infections, but prolonged use can lead to the emergence of resistant strains. Aeromonas spp. Infections are a common and problematic disease in fish, and members of this genera can produce antibiotic resistant strains. Antimicrobial peptides (AMPs) have emerged as an alternative method to treat and prevent infections and pituitary adenylate cyclase activating polypeptide (PACAP) is a prominent member of this family. The objective of this research was to study PACAP's direct antimicrobial activity and its toxicity in fish cells. Four synthetic variants of the natural PACAP from Clarias gariepinus were tested in addition to the natural variant. The experimental results show a different antimicrobial activity against A. salmonicida and A. hydrophila of each PACAP variant, and for the first time show dependence on the culture broth used. Furthermore, the results suggest that the underlying mechanism of PACAP antimicrobial activity includes a bacterial membrane permeabilizing effect, classifying PACAP as a membrane disruptive AMP. This study also demonstrated that the five PACAP variants evaluated showed low toxicity in vitro, at concentrations relevant for in vivo applications. Therefore, PACAP could be a promising alternative to antibiotics in the aquaculture sector., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Perineural invasion (PNI) has emerged as a key pathological feature and be considered as a poor prognostic factor in cervical cancer. However, the underlying molecular mechanisms are largely unknown. Here, PNI status of 269 cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) samples were quantified by using whole-slide diagnostic images obtained from The Cancer Genome Atlas. Integrated analyses revealed that PNI was an indicative marker of poorer disease-free survival for CESC patients. Among the differentially expressed genes, ADCYAP1 were identified. Clinical specimens supported that high expression of PACAP (encoded by ADCYAP1) contributed to PNI in CESC. Mechanistically, PACAP, secreted from cervical cancer cells, reversed myelin differentiation of Schwann cells (SCs). Then, dedifferentiated SCs promoted PNI by producing chemokine FGF17 and by degrading extracellular matrix through secretion of Cathepsin S and MMP-12. In conclusion, this study identified PACAP was associated with PNI in cervical cancer and suggested that tumour-derived PACAP reversed myelin differentiation of SCs to aid PNI., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The midbrain periaqueductal gray (PAG) region is a critical anatomical regulator of fear-related species-specific defensive reactions (SSDRs). Pituitary adenylate-cyclase-activating polypeptide (PACAP), and its main receptor PAC1, play an important role in fear-related behavior and anxiety disorders. However, the function of the PACAP-PAC1 system within the PAG with regards to SSDRs has received little attention. To address this gap, we used transgenic PAC1 flox/flox mice to examine both conditional and unconditional defensive reactions. We performed conditional PAC1 gene deletion within the ventrolateral(vl)PAG of PAC1 flox/flox mice using an adeno-associated virus (AAV) coding for Cre recombinase. Following viral expression, we used a white noise fear conditioning preparation that produces both an unconditional activity burst to the onset of noise that is followed by conditional freezing. On Day 1, mice received five white noise foot-shock pairings, whereas on Day 2, they were exposed to white noise five times without shock and we scored the activity burst and freezing to the white noise. Following behavioral testing, histology for immunofluorescent analysis was conducted in order to identify PACAP positive cells and stress-induced c-fos activity respectively. We found that PAC1 deletion in vlPAG increased the unconditional activity burst response but disrupted conditional freezing. PAC1 deletion was accompanied by higher c-fos activity following the behavioral experiments. Furthermore, a significant portion of PACAP-EGFP positive cells showed overlapping expression with VGAT, indicating their association with inhibitory neurons. The findings suggested that intact PACAP-PAC1 mechanisms are essential for SSDRs in vlPAG. Therefore, midbrain PACAP contributes to the underlying molecular mechanisms regulating fear responses., (© 2023 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Background: Induction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE -/- ) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE -/- mouse model of atherosclerosis and under the influence of a high-fat diet., Methods: For this purpose, PACAP -/- /ApoE -/- , PAC1 -/- /ApoE -/- , and ApoE -/- mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1 + ], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified., Results: ApoE -/- mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1 -/- /ApoE -/- mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE -/- mice. In contrast, PACAP deficiency in ApoE -/- mice did not affect the chondrogenic signature under either diet., Conclusions: Our data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis., (© 2023. The Author(s).)

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive pituitary adenylate cyclase-activating peptide (PACAP) knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type (WT) in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in WT mice that do not occur in knockout mice (aPRGs). Comparing constitutive PACAP knockout mice to a variety of temporally and regionally specific PACAP knockouts, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knockout mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Autophagy is vital in the pathogenesis of neurodegeneration. Thus far, no studies have specifically investigated the relationship between pituitary adenylate cyclase-activating polypeptide (PACAP) and autophagy, particularly in the context of Alzheimer's disease (AD). This study used in vitro and in vivo models, along with clinical samples, to explore interactions between PACAP and autophagy in AD., Methods: AD model mice were administered 6 μl of 0.1 mg/ml PACAP liquid intranasally for 4 weeks, then subjected to behavioral analyses to assess the benefits of PACAP treatment. The underlying mechanisms of PACAP-induced effects were investigated by methods including real-time quantitative polymerase chain reaction, RNA sequencing, immunofluorescence, and western blotting. Exosomes were extracted from human serum and subjected to enzyme-linked immunosorbent assays to examine autophagy pathways. The clinical and therapeutic implications of PACAP and autophagy were extensively investigated throughout the experiment., Results: Impaired autophagy was a critical step in amyloid β (Aβ) and Tau deposition; PACAP enhanced autophagy and attenuated cognitive impairment. RNA sequencing revealed three pathways that may be involved in AD progression: PI3K-AKT, mTOR, and AMPK. In vivo and in vitro studies showed that sirtuin3 knockdown diminished the ability of PACAP to restore normal autophagy function, resulting in phagocytosis dysregulation and the accumulation of pTau, Tau, and Aβ. Additionally, the autophagic biomarker MAP1LC3 demonstrated a positive association with PACAP in human serum., Conclusions: PACAP reverses AD-induced cognitive impairment through autophagy, using sirtuin3 as a key mediator. MAP1LC3 has a positive relationship with PACAP in humans. These findings provide insights regarding potential uses of intranasal PACAP and sirtuin3 agonists in AD treatment., Trial Registration: NCT04320368., (© 2023. The Author(s).)

Background: Severe peripheral nerve injury (PNI) often leads to significant movement disorders and intractable pain. Therefore, promoting nerve regeneration while avoiding neuropathic pain is crucial for the clinical treatment of PNI patients. However, established animal models for peripheral neuropathy fail to accurately recapitulate the clinical features of PNI. Additionally, researchers usually investigate neuropathic pain and axonal regeneration separately, leaving the intrinsic relationship between the development of neuropathic pain and nerve regeneration after PNI unclear. To explore the underlying connections between pain and regeneration after PNI and provide potential molecular targets, we performed single-cell RNA sequencing and functional verification in an established rat model, allowing simultaneous study of the neuropathic pain and axonal regeneration after PNI., Results: First, a novel rat model named spared nerve crush (SNC) was created. In this model, two branches of the sciatic nerve were crushed, but the epineurium remained unsevered. This model successfully recapitulated both neuropathic pain and axonal regeneration after PNI, allowing for the study of the intrinsic link between these two crucial biological processes. Dorsal root ganglions (DRGs) from SNC and naïve rats at various time points after SNC were collected for single-cell RNA sequencing (scRNA-seq). After matching all scRNA-seq data to the 7 known DRG types, we discovered that the PEP1 and PEP3 DRG neuron subtypes increased in crushed and uncrushed DRG separately after SNC. Using experimental design scRNA-seq processing (EDSSP), we identified Adcyap1 as a potential gene contributing to both pain and nerve regeneration. Indeed, repeated intrathecal administration of PACAP38 mitigated pain and facilitated axonal regeneration, while Adcyap1 siRNA or PACAP6-38, an antagonist of PAC1R (a receptor of PACAP38) led to both mechanical hyperalgesia and delayed DRG axon regeneration in SNC rats. Moreover, these effects can be reversed by repeated intrathecal administration of PACAP38 in the acute phase but not the late phase after PNI, resulting in alleviated pain and promoted axonal regeneration., Conclusions: Our study reveals that Adcyap1 is an intrinsic protective factor linking neuropathic pain and axonal regeneration following PNI. This finding provides new potential targets and strategies for early therapeutic intervention of PNI., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Previous evidence shows that rapid changes occur in the brain following spinal cord injury (SCI). Here, we interrogated the expression of the neuropeptides pituitary adenylyl cyclase-activating peptide (PACAP), vasoactive intestinal peptides (VIP), and their binding receptors in the rat brain 24 h following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebrate (SCI group); the other half underwent sham surgery (sham group). Twenty-four hours post-surgery, the hypothalamus, thalamus, amygdala, hippocampus (dorsal and ventral), prefrontal cortex, and periaqueductal gray were collected. PACAP, VIP, PAC1, VPAC1, and VPAC2 mRNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, PACAP expression was increased in the hypothalamus (104-141% vs sham) and amygdala (138-350%), but downregulated in the thalamus (35-95%) and periaqueductal gray (58-68%). VIP expression was increased only in the thalamus (175-385%), with a reduction in the amygdala (51-68%), hippocampus (40-75%), and periaqueductal gray (74-76%). The expression of the PAC1 receptor was the least disturbed by SCI, with decrease expression in the ventral hippocampus (63-68%) only. The expression levels of VPAC1 and VPAC2 receptors were globally reduced, with more prominent reductions of VPAC1 vs VPAC2 in the amygdala (21-70%) and ventral hippocampus (72-75%). In addition, VPAC1 downregulation also extended to the dorsal hippocampus (69-70%). These findings demonstrate that as early as 24 h post-SCI, there are region-specific disruptions of PACAP, VIP, and related receptor transcript and protein levels in supraspinal regions controlling higher cognitive functions., (© 2023. The Author(s).)

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread occurrence and diverse functions. It occurs in high levels in the gonads suggesting a potential central role in reproduction. The aim of our study was to assess the effect of PACAP treatment during embryo vitrification on the developmental rate and the expression of the heparin-binding EGF-like growth factor gene (Hbegf). Mouse embryos, obtained from superovulated females were allocated into the four treatment groups. In EM1 and EM2, the embryos were prepared for vitrification in an Equilibration Solution that was supplemented with 1 or 2 μM PACAP1-38, respectively. The embyos in groups CM1 and CM2 were not treated prior to vitrification but were cultured in a medium supplemented with 1 or 2 μM PACAP1-38 after thawing. The Vitrified Control group consisted of embryos vitrified and thawed then cultured without PACAP1-38 treatment. A non-vitrified, non-treated Fresh Control group was also used. After 24 h of culture, the developmental rate of the embryos, as well as the relative expression level of the Hbegf gene, as determined by qPCR, were compared among groups. Higher developmental rate and Hbegf gene expression level were found in the embryos treated with a higher concentration of PACAP. These results indicate that PACAP treatment has a beneficial effect on the survival and development of vitrified/thawed mouse embryos.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an important regulator of the stress response in mammals, influencing both the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). PACAP has been reported to influence energy homeostasis, including adaptive thermogenesis, an energy burning process in adipose tissue regulated by the SNS in response to cold stress and overfeeding. While research suggests PACAP acts centrally at the level of the hypothalamus, knowledge of PACAP's role within the sympathetic nerves innervating adipose tissues in response to metabolic stressors is limited. This work shows, for the first time, gene expression of PACAP receptors in stellate ganglia and highlights some differential expression with housing temperature. Additionally, we present our dissection protocol, analysis of tyrosine hydroxylase gene expression as a molecular biomarker for catecholamine producing tissue and recommend three stable reference genes for the normalization of quantitative real time-polymerase chain reaction (qRT-PCR) data when working with this tissue. This study adds to information about neuropeptide receptor expression in peripheral ganglia of the sympathetic nervous system innervating adipose tissue and provides insight into PACAP's role in the regulation of energy metabolism., (© 2023 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)

Migraine ranks among the most prevalent disorders worldwide, leading to disability and decreased quality of life in patients. Recently, neurogenic inflammation has been recognized as a potential underlying pathology contributing to the migraine pain pathway. Mast cells reside in the meninges and have been implicated in contributing to the pathophysiology of migraine. Here we report for the first time that the mouse Mas-Related G-protein-coupled Receptor B2 (MrgprB2), is expressed on meningeal connective tissue mast cells and contributes to Pituitary Adenylate Cyclase Activating Peptide (PACAP)-induced migraine-like pain behavior. We also found that PACAP was able to dose-dependently lead to enzyme release from human mast cells via activation of MRGPRX2; the human homolog of MrgprB2. Using a transgenic MRGPRX2 mouse, we observed significant increases in PACAP-induced migraine-like pain behavior in MRGPRX2 + mice vs mice lacking the receptor. These results reveal both MrgprB2 and MRGPRX2 as important contributors to neuropeptide-induced migraine pain., (© 2023. The Author(s).)

Numerous in vitro and in vivo models of Parkinson's disease (PD) demonstrate that pituitary adenylate cyclase-activating polypeptide (PACAP) conveys its strong neuroprotective actions mainly via its specific PAC1 receptor (PAC1R) in models of PD. We recently described the decrease in PAC1R protein content in the basal ganglia of macaques in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD that was partially reversed by levodopa therapy. In this work, we tested whether these observations occur also in the rotenone model of PD in the rat. The rotarod test revealed motor skill deterioration upon rotenone administration, which was reversed by benserazide/levodopa (B/L) treatment. The sucrose preference test suggested increased depression level while the open field test showed increased anxiety in rats rendered parkinsonian, regardless of the received B/L therapy. Reduced dopaminergic cell count in the substantia nigra pars compacta (SNpc) diminished the dopaminergic fiber density in the caudate-putamen (CPu) and decreased the peptidergic cell count in the centrally projecting Edinger-Westphal nucleus (EWcp), supporting the efficacy of rotenone treatment. RNAscope in situ hybridization revealed decreased PACAP mRNA ( Adcyap1 ) and PAC1R mRNA ( Adcyap1r1 ) expression in the CPu, globus pallidus, dopaminergic SNpc and peptidergic EWcp of rotenone-treated rats, but no remarkable downregulation occurred in the insular cortex. In the entopeduncular nucleus, only the Adcyap1r1 mRNA was downregulated in parkinsonian animals. B/L therapy attenuated the downregulation of Adcyap1 in the CPu only. Our current results further support the evolutionarily conserved role of the PACAP/PAC1R system in neuroprotection and its recruitment in the development/progression of neurodegenerative states such as PD.

The adipose-derived hormone leptin critically modulates reproductive function, such that its absence results in hypothalamic hypogonadism. Pituitary adenylate cyclase-activating polypeptide (PACAP)-expressing neurons are potential mediators of leptin's action on the neuroendocrine reproductive axis because they are leptin-sensitive and involved in both feeding behavior and reproductive function. In the complete absence of PACAP, male and female mice exhibit metabolic and reproductive abnormalities, yet there is some sexual dimorphism in the reproductive impairments. We tested whether PACAP neurons play a critical and/or sufficient role in mediating leptin's effects on reproductive function by generating PACAP-specific leptin receptor (LepR) knockout and rescue mice, respectively. We also generated PACAP-specific estrogen receptor alpha knockout mice to determine whether estradiol-dependent regulation of PACAP was critically involved in the control of reproductive function and whether it contributed to the sexually dimorphic effects of PACAP. We showed that LepR signaling in PACAP neurons is critically involved in the timing of female, but not male, puberty onset, but not fertility. Rescuing LepR-PACAP signaling in otherwise LepR-deficient mice was unable to rescue the reproductive deficits observed in LepR null mice but led to a marginal improvement in body weight and adiposity in females. Finally, PACAP-specific estrogen receptor alpha knockout did not lead to any changes in body weight or puberty onset compared with control mice. These data highlight that PACAP is a critical mediator of some of leptin's, but not estradiol's, influence on puberty onset in females, but is not critically involved in relaying leptin's effects in males or in adult females., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Age-related decline in cognitive functions is associated with reduced hippocampal neurogenesis caused by changes in the systemic inflammatory milieu. Mesenchymal stem cells (MSC) are known for their immunomodulatory properties. Accordingly, MSC are a leading candidate for cell therapy and can be applied to alleviate inflammatory diseases as well as aging frailty via systemic delivery. Akin to immune cells, MSC can also polarize into pro-inflammatory MSC (MSC1) and anti-inflammatory MSC (MSC2) following activation of Toll-like receptor 4 (TLR4) and TLR3, respectively. In the present study, we apply pituitary adenylate cyclase-activating peptide (PACAP) to polarize bone-marrow-derived MSC towards an MSC2 phenotype. Indeed, we found that polarized anti-inflammatory MSC were able to reduce the plasma levels of aging related chemokines in aged mice (18-months old) and increased hippocampal neurogenesis following systemic administration. Similarly, aged mice treated with polarized MSC displayed improved cognitive function in the Morris water maze and Y-maze assays compared with vehicle- and naïve-MSC-treated mice. Changes in neurogenesis and Y-maze performance were negatively and significantly correlated with sICAM, CCL2 and CCL12 serum levels. We conclude that polarized PACAP-treated MSC present anti-inflammatory properties that can mitigate age-related changes in the systemic inflammatory milieu and, as a result, ameliorate age related cognitive decline.

Heat stroke is a life-threatening illness caused by exposure to high ambient temperatures and relative humidity. The incidence of heat stroke is expected to increase due to climate change. Although pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in thermoregulation, the role of PACAP on heat stress remains unclear. PACAP knockout (KO) and wild-type ICR mice were subjected to heat exposure at an ambient temperature of 36 °C and relative humidity of 99% for 30-150 min. After heat exposure, the PACAP KO mice had a greater survival rate and maintained a lower body temperature than the wild-type mice. Moreover, the gene expression and immunoreaction of c-Fos in the ventromedially preoptic area of the hypothalamus, which is known to harbor temperature-sensitive neurons, were significantly lower in PACAP KO mice than those in wild-type mice. In addition, differences were observed in the brown adipose tissue, the primary site of heat production, between PACAP KO and wild-type mice. These results suggest that PACAP KO mice are resistant to heat exposure. The heat production mechanism differs between PACAP KO and wild-type mice.

The hypothalamic gonadotropin-releasing hormone (GnRH)-kisspeptin neuronal network regulates fertility in all mammals. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide isolated from the hypothalamus that is involved in the regulation of several releasing hormones and trop hormones. It is well-known that PACAP influences fertility at central and peripheral levels. However, the effects of PACAP on GnRH and kisspeptin neurons are not well understood. The present study investigated the integrity of the estrous cycle in PACAP-knockout (KO) mice. The number and immunoreactivity of GnRH (GnRH-ir) neurons in wild-type (WT) and PACAP KO female mice were determined using immunohistochemistry. In addition, the number of kisspeptin neurons was measured by counting kisspeptin mRNA-positive cells in the rostral periventricular region of the third ventricle (RP3V) and arcuate nucleus (ARC) using the RNAscope technique. Finally, the mRNA and protein expression of estrogen receptor alpha (ERα) was also examined. Our data showed that the number of complete cycles decreased, and the length of each cycle was longer in PACAP KO mice. Furthermore, the PACAP KO mice experienced longer periods of diestrus and spent significantly less time in estrus. There was no difference in GnRH-ir or number of GnRH neurons. In contrast, the number of kisspeptin neurons was decreased in the ARC, but not in the R3PV, in PACAP KO mice compared to WT littermates. Furthermore, ERα mRNA and protein expression was decreased in the ARC, whereas in the R3PV region, ERα mRNA levels were elevated. Our results demonstrate that embryonic deletion of PACAP significantly changes the structure and presumably the function of the GnRH-kisspeptin neuronal network, influencing fertility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Barabás, Kovács, Vértes, Kövesdi, Faludi, Udvarácz, Pham, Reglődi, Abraham and Nagy.)

According to the three hit concept of depression, interaction of genetic predisposition altered epigenetic programming and environmental stress factors contribute to the disease. Earlier we demonstrated the construct and face validity of our three hit concept-based mouse model. In the present work, we aimed to examine the predictive validity of our model, the third willnerian criterion. Fluoxetine treatment was applied in chronic variable mild stress (CVMS)-exposed (environmental hit) CD1 mice carrying one mutated allele of pituitary adenylate cyclase-activating polypeptide gene (genetic hit) that were previously exposed to maternal deprivation (epigenetic hit) vs. controls. Fluoxetine reduced the anxiety level in CVMS-exposed mice in marble burying test, and decreased the depression level in tail suspension test if mice were not deprived maternally. History of maternal deprivation caused fundamental functional-morphological changes in response to CVMS and fluoxetine treatment in the corticotropin-releasing hormone-producing cells of the bed nucleus of the stria terminalis and central amygdala, in tyrosine-hydroxylase content of ventral tegmental area, in urocortin 1-expressing cells of the centrally projecting Edinger-Westphal nucleus, and serotonergic cells of the dorsal raphe nucleus. The epigenetic background of alterations was approved by altered acetylation of histone H3. Our findings further support the validity of both the three hit concept and that of our animal model. Reversal of behavioral and functional-morphological anomalies by fluoxetine treatment supports the predictive validity of the model. This study highlights that early life stress does not only interact with the genetic and environmental factors, but has strong influence also on therapeutic efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gaszner, Farkas, Kun, Ujvári, Berta, Csernus, Füredi, Kovács, Hashimoto, Reglődi, Kormos and Gaszner.)

The lateral habenula (LHb) is a small, bilateral, epithalamic nucleus which processes aversive information. While primarily glutamatergic, LHb neurons express genes coding for many neuropeptides, such as Adcyap1 the gene encoding pituitary adenylate cyclase-activating polypeptide (PACAP), which itself has been associated with anxiety and stress disorders. Using Cre-dependent viral vectors, we targeted and characterized these neurons based on their anatomical projections and found that they projected to both the raphe and rostromedial tegmentum but only weakly to ventral tegmental area. Using RiboTag to capture ribosomal-associated mRNA from these neurons and reanalysis of existing single cell RNA sequencing data, we did not identify a unique molecular phenotype that characterized these PACAP-expressing neurons in LHb. In order to understand the function of these neurons, we conditionally expressed hM 3 Dq DREADD selectively in LHb PACAP-expressing neurons and chemogenetically excited these neurons during behavioral testing in the open field test, contextual fear conditioning, sucrose preference, novelty suppressed feeding, and conditioned place preference. We found that Gq activation of these neurons produce behaviors opposite to what is expected from the LHb as a whole-they decreased anxiety-like and fear behavior and produced a conditioned place preference. In conclusion, PACAP-expressing neurons in LHb represents a molecularly diverse population of cells that oppose the actions of the remainder of LHb neurons by being rewarding or diminishing the negative consequences of aversive events., (© 2022 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Gs-coupled GPCR-stimulated neuritogenesis in PC12 and NS-1 - cells depends on activation of the MAP kinase ERK. Here, we examine changes in ERK activation (phosphorylation), and the time course of ERK-dependent gene induction, to seek transcriptional determinants for this process. Quenching of ERK activation by inhibition of MEK with U0126 at any time point for at least 24 h following addition of PACAP resulted in arrest of neurite formation. Changes in the transcriptome profile throughout this time period revealed at least two phases of gene induction: an early phase dominated by induction of immediate-early genes, and a later phase of gene induction after 4-6 h of exposure to PACAP with persistent elevation of phospho-ERK levels. Genes induced by PACAP in both phases consisted in those whose induction was dependent on ERK (i.e., blocked by U0126), and some whose induction was blocked by the protein kinase A inhibitor H89. ERK-dependent "late gene" transcripts included Gpr50, implicated earlier in facilitation of NGF-induced neurite formation in NS-1 cells. Gpr50 induction by PACAP, but not NGF, was dependent on the guanine nucleotide exchange factor RapGEF2, which has been shown to be required for PACAP-induced neuritogenesis in NS-1 cells. Expression of a Gpr50-directed shRNA lowered basal levels of Gpr50 mRNA and attenuated Gpr50 mRNA and GPR50 protein induction by PACAP, with a corresponding attenuation of PACAP-induced neuritogenesis. Gs-GPCR-stimulated neuritogenesis first requires immediate-early gene induction, including that of Egr1 (Zif268/NGF1A/Krox24) as previously reported. This early phase of gene induction, however, is insufficient to maintain the neuritogenic process without ERK-dependent induction of additional late genes, including Gpr50, upon continuous exposure to neurotrophic neuropeptide. Early (Egr1) and late (Gpr50) gene induction by NGF, like that for PACAP, was inhibited by U0126, but was independent of RapGEF2, confirming distinct modes of ERK activation by Gs-coupled GPCRs and neurotrophic tyrosine receptor kinases, converging on a final common ERK-dependent signaling pathway for neuritogenesis., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA.)

PAC1-R is a recognized preferential receptor for the neuropeptide of pituitary adenylate cyclase-activating polypeptide (PACAP), which mediates neuroprotective and nerve regenerative activities of PACAP. In this study, we found that in both PAC1R-CHO cells with high expression of PAC1R-eGFP and retinal ganglion cells (RGC-5) with the natural expression of PAC1-R, oligo-peptide PACAP(28-38) and the positively charged arginine-rich penetrating peptide TAT, as positive allosteric modulators of PAC1-R, significantly trigger the nuclear translocation of PAC1-R. The chromatin immunoprecipitation (ChIP)-PCR results show that the nuclear translocated PAC1-R binds with the promoter regions of PAC1-R and its specific ligand PACAP . The up-regulated promoter activities of PAC1-R and PACAP induced by PACAP(28-38) or TAT are positively correlative with the increase of the expression levels of PAC1-R and PACAP. Moreover, the nuclear translocation of PAC1-R induced by PACAP(28-38) or TAT is significantly inhibited by the mutation of PAC1-R on Cys25 and the palmitoylation inhibitor 2-bromopalmitate. Meanwhile, the increase in both PAC1-R and PACAP levels and the neuroprotective activities of PACAP(28-38) and TAT in MPP-induced cell model of Parkinson ' s disease are synchronously inhibited by 2-bromopalmitate, which are positively correlated with the nuclear translocation of PAC1-R induced by PACAP(28-38) or TAT. Bioinformatics analysis and motif enrichment analysis following ChIP-sequencing show that the transcription factors including SP1, Zic2, GATA1, REST and YY1 may be recruited by nuclear PAC1-R and involved in regulating the promoter activities of PAC1-R and PACAP . ChIP-sequencing and related bioinformatics analysis show that the downstream target genes regulated by the nuclear PAC1-R are mostly involved in the process of cellular stress and related to neuroprotection, neuronal genesis and development.

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the steroidogenesis and spermatogenesis in the testis through its receptors PAC1, VPAC1, and VPAC2. In this study, we investigated the seasonal expressions of PACAP, PAC1, VPAC1, VPAC2, luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), and CYP17A1 in the testis of the male muskrat during the breeding season and the non-breeding season. Histologically, we found the presence of Leydig cells, Sertoli cells and all kinds of germ cells in the testis during the breeding season but only Leydig cells, Sertoli cells, spermatogonia and primary spermatocyte during the non-breeding season. The immunohistochemical localizations of PACAP and VPAC1 were identified in the Leydig cells, spermatogonia and spermatozoa during the breeding season while only in Leydig cells and spermatogonia during the non-breeding season, and PAC1 and VPAC2 were localized in the Leydig cells in both seasons, in which LHR, StAR, 3β-HSD and CYP17A1 were also expressed. Meanwhile, protein and mRNA expression levels of PACAP, PAC1, VPAC1, VPAC2, LHR, FSHR, StAR, 3β-HSD and CYP17A1 in the testis during the breeding season were significantly higher than those during the non-breeding season. These results suggested that PACAP may involve in the regulation of, steroidogenesis and spermatogenesis via an endocrine, autocrine or paracrine manner in the testis of the muskrat.

The Hatschek's pit in the cephalochordate amphioxus, an invertebrate deuterostome basal to chordates is suggested to be the functional homolog structure of the vertebrate adenohypophysis based on anatomy and expression of homologous neuroendocrine genes. However, the endocrine potential of the cephalochordate Hatschek's pit remains to be demonstrated as well as the physiological actions of the secreted neuropeptides. In this study, we have explored the distribution and characterize the potential function of the amphioxus PACAP/GCG precursor, which is the ortholog of the hypothalamic PACAP neuropeptide in vertebrates. In amphioxi, two PACAP/GCG transcripts PACAP/GCGa and PACAP/GCGbc that are alternative isoforms of a single gene with different peptide coding potentials were isolated. Immunofluorescence staining detected their expression around the nucleus of Rohde, supporting that this structure may be homologous of the neurosecretory cells of the vertebrate hypothalamus where abundant PACAP is found. PACAP/GCGa was also detected in the infundibulum-like downgrowth approaching the Hatschek's pit, indicating diffusion of PACAP/GCGa from the CNS to the pit via the infundibulum-like downgrowth. Under a high salinity challenge, PACAP/GCGa was upregulated in amphioxi head and PACAP/GCGa treatment increased expression of GHl in Hatschek's pit in a dose-dependent manner, suggesting that PACAP/GCGa may be involved in the regulation of GHl via hypothalamic-pituitary (HP)-like axis similar as in the vertebrates. Our results support that the amphioxus Hatschek's pit is likely to be the functional homolog of pituitary gland in vertebrates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 On, Su, Shen, Arokiaraj, Cardoso, Li and Chow.)

A pharmacological and genetic blockade of the dopamine D3 receptor (D3R) has shown to be neuroprotective in models of Parkinson's disease (PD). The anxiolytic drug buspirone, a serotonin receptor 1A agonist, also functions as a potent D3R antagonist. To test if buspirone elicited neuroprotective activities, C57BL/6 mice were subjected to rotenone treatment (10mg/kg i.p for 21 days) to induce PD-like pathology and were co-treated with increasing dosages of buspirone (1, 3, or 10 mg/kg i.p.) to determine if the drug could prevent rotenone-induced damage to the central nervous system (CNS). We found that high dosages of buspirone prevented the behavioural deficits caused by rotenone in the open field test. Molecular and histological analyses confirmed that 10 mg/kg of buspirone prevented the degeneration of TH-positive neurons. Buspirone attenuated the induction of interleukin-1β and interleukin-6 expression by rotenone, and this was paralleled by the upregulation of arginase-1, brain-derived neurotrophic factor (BDNF), and activity-dependent neuroprotective protein (ADNP) in the midbrain, striatum, prefrontal cortex, amygdala, and hippocampus. Buspirone treatment also improved mitochondrial function and antioxidant activities. Lastly, the drug prevented the disruptions in the expression of two neuroprotective peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). These results pinpoint the neuroprotective efficacy of buspirone against rotenone toxicity, suggesting its potential use as a therapeutic agent in neurodegenerative and neuroinflammatory diseases, such as PD.

Since the advent of gene knock-out technology in 1987, insight into the role(s) of neuropeptides in centrally- and peripherally-mediated physiological regulation has been gleaned by examining altered physiological functioning in mammals, predominantly mice, after genetic editing to produce animals deficient in neuropeptides or their cognate G-protein coupled receptors (GPCRs). These results have complemented experiments involving infusion of neuropeptide agonists or antagonists systemically or into specific brain regions. Effects of gene loss are often interpreted as indicating that the peptide and its receptor(s) are required for the physiological or behavioral responses elicited in wild-type mice at the time of experimental examination. These interpretations presume that peptide/peptide receptor gene deletion affects only the expression of the peptide/receptor itself, and therefore impacts physiological events only at the time at which the experiment is conducted. A way to support 'real-time' interpretations of neuropeptide gene knock-out is to demonstrate that the wild-type transcriptome, except for the deliberately deleted gene(s), in tissues of interest, is preserved in the knock-out mouse. Here, we show that there is a cohort of genes (constitutively PACAP-Regulated Genes, or cPRGs) whose basal expression is affected by constitutive knock-out of the Adcyap1 gene in C57Bl6/N mice, and additional genes whose expression in response to physiological challenge, in adults, is altered or impaired in the absence of PACAP expression (acutely PACAP-Regulated Genes, or aPRGs). Distinguishing constitutive and acute transcriptomic effects of neuropeptide deficiency on physiological function and behavior in mice reveals alternative mechanisms of action, and changing functions of neuropeptides, throughout the lifespan., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

High-fat diet (HFD)-induced comorbid cognitive and behavioural impairments are thought to be the result of persistent low-grade neuroinflammation. Metformin, a first-line medication for the treatment of type-2 diabetes, seems to ameliorate these comorbidities, but the underlying mechanism(s) are not clear. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective peptides endowed with anti-inflammatory properties. Alterations to the PACAP/VIP system could be pivotal during the development of HFD-induced neuroinflammation. To unveil the pathogenic mechanisms underlying HFD-induced neuroinflammation and assess metformin's therapeutic activities, (1) we determined if HFD-induced proinflammatory activity was present in vulnerable brain regions associated with the development of comorbid behaviors, (2) investigated if the PACAP/VIP system is altered by HFD, and (3) assessed if metformin rescues such diet-induced neurochemical alterations. C57BL/6J male mice were divided into two groups to receive either standard chow (SC) or HFD for 16 weeks. A further HFD group received metformin (HFD + M) (300 mg/kg BW daily for 5 weeks) via oral gavage. Body weight, fasting glucose, and insulin levels were measured. After 16 weeks, the proinflammatory profile, glial activation markers, and changes within the PI3K/AKT intracellular pathway and the PACAP/VIP system were evaluated by real-time qPCR and/or Western blot in the hypothalamus, hippocampus, prefrontal cortex, and amygdala. Our data showed that HFD causes widespread low-grade neuroinflammation and gliosis, with regional-specific differences across brain regions. HFD also diminished phospho-AKT (Ser473) expression and caused significant disruptions to the PACAP/VIP system. Treatment with metformin attenuated these neuroinflammatory signatures and reversed PI3K/AKT and PACAP/VIP alterations caused by HFD. Altogether, our findings demonstrate that metformin treatment rescues HFD-induced neuroinflammation in vulnerable brain regions, most likely by a mechanism involving the reinstatement of PACAP/VIP system homeostasis. Data also suggests that the PI3K/AKT pathway, at least in part, mediates some of metformin's beneficial effects.

G protein-coupled receptor 55 (GPR55) is a recently deorphanized lipid- and peptide-sensing receptor. Its lipidic endogenous agonists belong to lysoglycerophospholipids, with lysophosphatidylinositol (LPI) being the most studied. Peptide agonists derive from fragmentation of pituitary adenylate cyclase-activating polypeptide (PACAP). Although GPR55 and its ligands were implicated in several physiological and pathological conditions, their biological function remains unclear. Thus, the aim of the study was to conduct a large-scale re-analysis of publicly available gene expression datasets to identify physiological and pathological conditions affecting the expression of GPR55 and the production of its ligands. The study revealed that regulation of GPR55 occurs predominantly in the context of immune activation pointing towards the role of the receptor in response to pathogens and in immune cell lineage determination. Additionally, it was revealed that there is almost no overlap between the experimental conditions affecting the expression of GPR55 and those modulating agonist production. The capacity to synthesize LPI was enhanced in various types of tumors, indicating that cancer cells can hijack the motility-related activity of GPR55 to increase aggressiveness. Conditions favoring accumulation of PACAP-derived peptides were different than those for LPI and were mainly related to differentiation. This indicates a different function of the two agonist classes and possibly the existence of a signaling bias.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having trophic and protective functions in neural tissues, including the retina. Previously, we have shown that intravitreal PACAP administration can maintain retinal structure in the animal model of retinopathy of prematurity (ROP). The purpose of this study is to examine the development of ROP in PACAP-deficient and wild-type mice to reveal the function of endogenous PACAP. Wild-type and PACAP-knockout (KO) mouse pups at postnatal day (PD) 7 were maintained at 75% oxygen for 5 consecutive days then returned to room air on PD12 to develop oxygen-induced retinopathy (OIR). On PD15, animals underwent electroretinography (ERG) to assess visual function. On PD16, eyes were harvested for either immunohistochemistry to determine the percentage of the central avascular retinal area or molecular analysis to assess angiogenesis proteins by array kit and anti-apoptotic protein kinase B (Akt) change by western blot. Retinas of PACAP-deficient OIR mice showed a greater central avascular area than that of the wild types. ERG revealed significantly decreased b-wave amplitude in PACAP KO compared to their controls. Several angiogenic proteins were upregulated due to OIR, and 11 different proteins markedly increased in PACAP-deficient mice, whereas western blot analysis revealed a reduction in Akt phosphorylation, suggesting an advanced cell death in the lack of PACAP. This is the first study to examine the endogenous effect of PACAP in the OIR model. Previously, we have shown the beneficial effect of exogenous local PACAP treatment in the rat OIR model. Together with the present findings, we suggest that PACAP could be a novel retinoprotective agent in ROP., (© 2021. The Author(s).)