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Pregnancy outcome relies on the maintenance of immune and metabolic homeostasis at the maternal fetal interface. Maternal and perinatal morbidity and mortality is associated with impaired placental development. Multiple regulatory effects of the endogenous-produced vasoactive intestinal peptide (VIP) on vascular, metabolic and immune functions at the maternal-fetal interface have been reported. Here we studied the involvement of the two primary high affinity receptors for VIP (VPAC1 and VPAC2) on maternal immune response, placental homeostasis and pregnancy outcome. Targeted disruption of each receptor gene led to altered placental structure, vascular and trophoblast functional markers and shaped the functional profiles of macrophages and neutrophils towards a proinflammatory state. Several changes in pregnant mice were receptor specific: ROS production elicited by VIP on neutrophils was selectively dependent on the presence of VPAC1 whereas apoptosis rate was associated with the VPAC2 deletion. In peritoneal macrophages from pregnant mice, levels of MHC-II, TLR2, and IL-10 were selectively altered in VPAC2 receptor-deficient mice, whereas IL-6 gene expression was reduced only in mice lacking VPAC1 receptors. Additionally, MMP9 mRNA in isolated TGCs was reduced in VPAC2 receptor deleted mice, while the percentage of IL-12 cells in post-phagocytosis macrophage cultures was selectively reduced in VPAC2 receptor deficient mice. The results indicate that manipulation of VPAC1 and VPAC2 receptor affects immune, vascular and metabolic environment at the maternal fetal interface. These mouse models offer new approaches to study pregnancy complications adding new perspectives to the development of VPAC receptor-selective drugs.

Trauma can cause dysfunctional fear regulation leading some people to develop disorders, such as post-traumatic stress disorder (PTSD). The amygdala regulates fear, whereas PACAP (pituitary adenylate activating peptide) and PAC1 receptors are linked to PTSD symptom severity at genetic/epigenetic levels, with a strong link in females with PTSD. We discovered a PACAPergic projection from the basomedial amygdala (BMA) to the medial intercalated cells (mICCs) in adult mice. In vivo optogenetic stimulation of this pathway increased CFOS expression in mICCs, decreased fear recall, and increased fear extinction. Selective deletion of PAC1 receptors from the mICCs in females reduced fear acquisition, but enhanced fear generalization and reduced fear extinction in males. Optogenetic stimulation of the BMA-mICC PACAPergic pathway produced EPSCs in mICC neurons, which were enhanced by the PAC1 receptor antagonist, PACAP 6-38. Our findings show that mICCs modulate contextual fear in a dynamic and sex-dependent manner via a microcircuit containing the BMA and mICCs, and in a manner that was dependent on behavioral state.SIGNIFICANCE STATEMENT Traumatic stress can affect different aspects of fear behaviors, including fear learning, generalization of learned fear to novel contexts, how the fear of the original context is recalled, and how fear is reduced over time. While the amygdala has been studied for its role in regulation of different aspects of fear, the molecular circuitry of this structure is quite complex. In addition, aspects of fear can be modulated differently in males and females. Our findings show that a specific circuitry containing the neuropeptide PACAP and its receptor, PAC1, regulates various aspects of fear, including acquisition, generalization, recall, and extinction in a sexually dimorphic manner, characterizing a novel pathway that modulates traumatic fear.

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.

The sympathetic nervous system (SNS) serves to maintain homeostasis of vital organ systems throughout the body, and its dysfunction plays a major role in human disease. The SNS also links the central nervous system to the immune system during different types of stress via innervation of the lymph nodes, spleen, thymus, and bone marrow. Previous studies have shown that pituitary adenylate cyclase-activating polypeptide (PACAP, gene name adcyap1) exhibits anti-inflammatory properties in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Because PACAP is known to regulate SNS function, we hypothesized that part of the immunoprotective action of PACAP is due to its neuromodulatory effects on sympathetic neurons. To examine this, we used an inducible, targeted approach to conditionally disrupt not only the PACAP-preferring PAC1 receptor gene (adcyap1r1) in dopamine β-hydroxylase-expressing cells, which includes postganglionic sympathetic neurons, but also catecholaminergic neurons in the brain and adrenomedullary chromaffin cells. In contrast to our previous EAE studies using PACAP global knockout mice which developed severe and prolonged EAE, we found that mice with conditional loss of PAC1 receptors in catecholaminergic cells developed a delayed time course of EAE with reduced helper T cell type 1 (Th1) and Th17 and enhanced Th2 cell polarization. At later time points, similar to mice with global PACAP loss, mice with conditional loss of PAC1 exhibited more severe clinical disease than controls. The latter was associated with a reduction in the abundance of thymic regulatory T cells (Tregs). These studies indicate that PAC1 receptor signaling acts in catecholaminergic cells in a time-dependent manner. At early stages of disease development, it enhances the ability of the SNS to polarize the Th response towards a more inflammatory state. Then, after disease is established, it enhances the ability of the SNS to dampen the inflammatory response via Tregs. The lack of concordance in results between global PACAP KO mice and mice with the PAC1 deletion targeted to catecholaminergic cells during early EAE may be explained by the fact that PACAP acts to regulate inflammation via multiple receptor subtypes and multiple targets, including inflammatory cells.

The original version of this article unfortunately contained mistakes. The captured article title and corresponding author were incorrect.

Medulloblastoma is a brain tumor that arises predominantly in infants and children. It is the most common pediatric brain malignancy. Around 25% of medulloblastomas are driven by constitutive activation of the Hedgehog signaling pathway. Hedgehog-driven medulloblastoma is often studied in the laboratory using genetic mouse models with overactive Hedgehog signaling, which recapitulate many of the pathological features of human Hedgehog-dependent tumors. However, we show here that on a molecular level the human and mouse HH-dependent MB are quite distinct, with human, but not mouse, tumors characterized by the presence of markers of increased oxidative phosphorylation and mitochondrial biogenesis. The latter suggests that, unlike for many other types of tumors, a switch to glycolytic metabolism might not be co-opted by human SHH-MB to perpetuate their survival and growth. This needs to be taken into consideration and could potentially be exploited in the design of therapies.

The discovery that intravenous (IV) infusions of the neuropeptide PACAP-38 (pituitary adenylyl cyclase activating peptide-38) induced delayed migraine-like headaches in a large majority of migraine patients has resulted in considerable excitement in headache research. In addition to suggesting potential therapeutic targets for migraine, the finding provides an opportunity to better understand the pathological events from early events (aura) to the headache itself. Although PACAP-38 and the closely related peptide VIP (vasoactive intestinal peptide) are well-known as vasoactive molecules, the dilation of cranial blood vessels per se is no longer felt to underlie migraine headaches. Thus, more recent research has focused on other possible PACAP-mediated mechanisms, and has raised some important questions. For example, (1) are endogenous sources of PACAP (or VIP) involved in the triggering and/or propagation of migraine headaches?; (2) which receptor subtypes are involved in migraine pathophysiology?; (3) can we identify specific anatomical circuit(s) where PACAP signaling is involved in the features of migraine? The purpose of this review is to discuss the possibility, and supportive evidence, that PACAP acts to induce migraine-like symptoms not only by directly modulating nociceptive neural circuits, but also by indirectly regulating the production of inflammatory mediators. We focus here primarily on postulated extra-dural sites because potential mechanisms of PACAP action in the dura are discussed in detail elsewhere (see X, this edition).

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with a strong neuroinflammatory component. Current treatments principally target the immune system but fail to preserve long‐term myelin health and do not prevent neurological decline. Studies over the past two decades have shown that the structurally related neuropeptides VIP and PACAP (vasoactive intestinal peptide and pituitary adenylate cyclase‐activating polypeptide, respectively) exhibit pronounced anti‐inflammatory activities and reduce clinical symptoms in MS disease models, largely via actions on their bivalent VIP receptor type 1 and 2. Here, using the cuprizone demyelination model, we demonstrate that PACAP and VIP, and strikingly the PACAP‐selective receptor PAC1 agonist maxadilan, prevented locomotor deficits in the horizontal ladder and open field tests. Moreover, only PACAP and maxadilan were able to prevent myelin deterioration, as assessed by a reduction in the expression of the myelin markers proteolipid protein 1, oligodendrocyte transcription factor 2, quaking‐7 (APC) and Luxol Fast Blue staining. Furthermore, PACAP and maxadilan (but not VIP), prevented striatal synaptic loss and diminished astrocyte and microglial activation in the corpus callosum of cuprizone‐fed mice. In vitro, PACAP or maxadilan prevented lipopolysaccharide (LPS)‐induced polarisation of primary astrocytes at 12–24 h, an effect that was not seen with maxadilan in LPS‐stimulated microglia. Taken together, our data demonstrates for the first time that PAC1 agonists provide distinctive protective effects against white matter deterioration, neuroinflammation and consequent locomotor dysfunctions in the cuprizone model. The results indicate that targeting the PAC1 receptor may provide a path to treat myelin‐related diseases in humans. [ABSTRACT FROM AUTHOR]

The structurally related neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in stress regulation and learning and memory. Several bodies of research have shown the impact of the PACAP specific receptor PAC1 on fear memory, but the roles of other PACAP receptors in regulating fear stress responses remain to be elucidated. Here we aimed to investigate the effects of genetic deletion of VIPR2 encoding the VPAC2 receptor, which binds both VIP and PACAP, on fear-related memory and on dendritic morphology in the brain regions of the fear circuitry. Male VPAC2 receptor knockout (VPAC2-KO) and littermate wild-type control mice were subjected to Pavlovian fear conditioning paradigm. VPAC2-KO mice displayed normal acquisition of fear conditioning, contextual and cued fear memory, but impaired extinction of cued fear memory. Morphological analyses revealed reductions in cell body size and total branch number and length of apical and basal dendrites of prelimbic cortex neurons in VPAC2-KO mice. In addition, Sholl analysis indicated that the amount of dendritic material distal to the soma was decreased, while proximal dendritic material was increased. In the infralimbic cortex, the amount of apical dendritic material proximal to the soma was increased in VPAC2-KO mice, while other indices of morphology did not differ. Finally, there were no differences in dendritic morphology in basolateral amygdala neurons between genotypes. These findings suggest that the VPAC2 receptor plays an important role in the fear extinction processes and the regulation of the dendritic morphology in the prelimbic and infralimbic cortices.

New neurons are born throughout the life of mammals in germinal zones of the brain known as neurogenic niches: the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampus. These niches contain a subpopulation of cells known as adult neural progenitor cells (aNPCs), which self-renew and give rise to new neurons and glia. aNPCs are regulated by many factors present in the niche, including the extracellular matrix (ECM). We show that the neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) affects subventricular zone-derived aNPCs by increasing their surface adhesion. Gene array and reconstitution assays indicate that this effect can be attributed to the regulation of ECM components and ECM-modifying enzymes in aNPCs by PACAP. Our work suggests that PACAP regulates a bidirectional interaction between the aNPCs and their niche: PACAP modifies ECM production and remodeling, in turn the ECM regulates progenitor cell adherence. We speculate that PACAP may in this manner help restrict adult neural progenitors to the stem cell niche in vivo, with potential significance for aNPC function in physiological and pathological states.

Pituitary adenylate cyclase-activating polypeptide (PACAP, gene name Adcyap1) regulates a wide variety of neurological and physiological functions, including metabolism and cognition, and plays roles in of multiple forms of stress. Because of its preferential expression in nerve fibers, it has often been difficult to trace and identify the endogenous sources of the peptide in specific populations of neurons. Here, we introduce a transgenic mouse line that harbors in its genome a bacterial artificial chromosome containing an enhanced green fluorescent protein (EGFP) expression cassette inserted upstream of the PACAP ATG translation initiation codon. Analysis of expression in brain sections of these mice using a GFP antibody reveals EGFP expression in distinct neuronal perikarya and dendritic arbors in several major brain regions previously reported to express PACAP from using a variety of approaches, including radioimmunoassay, in situ hybridization, and immunohistochemistry with and without colchicine. EGFP expression in neuronal perikarya was modulated in a manner similar to PACAP gene expression in motor neurons after peripheral axotomy in the ipsilateral facial motor nucleus in the brainstem, providing an example in which the transgene undergoes proper regulation in vivo. These mice and the high-resolution map obtained are expected to be useful in understanding the anatomical patterns of PACAP expression and its plasticity in the mouse. J. Comp. Neurol. 524:3827-3848, 2016. © 2016 Wiley Periodicals, Inc.

Fear is an important emotional reaction in response to threatening stimuli and is important for survival. However, when fear occurs in inappropriate circumstances, it can lead to pathological conditions including an increased vulnerability for developing anxiety disorders such as posttraumatic stress disorder (PTSD). Patients with PTSD generalize fear to contexts or to environments that are not associated with the trauma. We sought to explore if increasing the level of dissimilarity relative to the context in which mice learn fear results in changes in the level of fear responding to the new context. We also determined with this procedure if the number of cells expressing the immediate early gene cfos changes with the corresponding level of expressed fear within brain regions known to be important in modulating fear, including the basolateral amygdala (BLA) and hippocampus. Our results indicate that mice that were tested in increasingly different contexts showed significantly different levels of fear responses. Freezing level was higher in the context most similar to the acquisition context than the one that was highly different. The level of cfos within the BLA, but not hippocampus, was also significantly different between the test contexts, with higher levels in the somewhat similar compared with the most different context. Overall, these results highlight the BLA as a critical region in the node of fear circuitry for modulating fear generalization. © 2016 Wiley Periodicals, Inc.

Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide that belongs to the secretin-glucagon superfamily of peptides and has 68 % homology with PACAP. VIP is abundantly expressed in the central and peripheral nervous system and in the gastrointestinal tract, where it exercises several physiological functions. Previously, it has been reported that VIP regulates feeding behavior centrally in different species of vertebrates such as goldfishes, chicken and rodents. Additional studies are necessary to analyze the role of endogenous VIP on the regulation of appetite/satiety, feeding behavior, metabolic hormones, body mass composition and energy balance. The aim of the study was to elucidate the physiological pathways by which VIP regulates appetite/satiety, feeding behavior, metabolic hormones, and body mass composition. VIP deficient (VIP -/-) and age-matched wild-type (WT) littermates were weekly monitored from 5 to 22 weeks of age using a whole body composition EchoMRI analyzer. Food intake and feeding behavior were analyzed using the BioDAQ automated monitoring system. Plasma levels of metabolic hormones including active-ghrelin, GLP-1, leptin, PYY, pancreatic polypeptide (PP), adiponectin, and insulin were measured in fasting as well as in postprandial conditions. The genetic lack of VIP led to a significant reduction of body weight and fat mass and to an increase of lean mass as the mice aged. Additionally, VIP-/- mice had a disrupted pattern of circadian feeding behavior resulting in an abolished regular nocturnal/diurnal feeding. These changes were associated with an altered secretion of adiponectin, GLP-1, leptin, PYY and insulin in VIP-/- mice. Our data demonstrates that endogenous VIP is involved in the control of appetite/satiety, feeding behavior, body mass composition and in the secretion of six different key regulatory metabolic hormones. VIP plays a key role in the regulation of body phenotype by significantly enhancing body weight and fat mass accumulation. Therefore, VIP signaling is critical for the modulation of appetite/satiety and body mass phenotype and is a potential target for future treatment of obesity.

RationaleAn abundance of genetic and epidemiologic evidence as well as longitudinal neuroimaging data point to developmental origins for schizophrenia and other mental health disorders. Recent clinical studies indicate that microduplications of VIPR2, encoding the vasoactive intestinal peptide (VIP) receptor VPAC2, confer significant risk for schizophrenia and autism spectrum disorder. Lymphocytes from patients with these mutations exhibited higher VIPR2 gene expression and VIP responsiveness (cAMP induction), but mechanisms by which overactive VPAC2 signaling may lead to these psychiatric disorders are unknown.ObjectivesWe subcutaneously administered the highly selective VPAC2 receptor agonist Ro 25-1553 to C57BL/6 mice from postnatal day 1 (P1) to P14 to determine if overactivation of VPAC2 receptor signaling during postnatal brain maturation affects synaptogenesis and selected behaviors.ResultsWestern blot analyses on P21 revealed significant reductions of synaptophysin and postsynaptic density protein 95 (PSD-95) in the prefrontal cortex, but not in the hippocampus in Ro 25-1553-treated mice. The same postnatally restricted treatment resulted in a disruption in prepulse inhibition of the acoustic startle measured in adult mice. No effects were observed in open-field locomotor activity, sociability in the three-chamber social interaction test, or fear conditioning or extinction.ConclusionOveractivation of the VPAC2 receptor in the postnatal mouse results in a reduction in synaptic proteins in the prefrontal cortex and selective alterations in prepulse inhibition. These findings suggest that the VIPR2-linkage to mental health disorders may be due in part to overactive VPAC2 receptor signaling during a critical time of synaptic maturation.

VIP is highly expressed in the colon and regulates motility, vasodilatation, and sphincter relaxation. However, its role in the development and progress of colitis is still controversial. Our aim was to determine the participation of VIP on dextran sodium sulfate (DSS)-induced colonic mucosal inflammation using VIP(-/-) and WT mice treated with VIP antagonists. Colitis was induced in 32 adult VIP(-/-) and 14 age-matched WT litter-mates by giving 2.5 % DSS in the drinking water. DSS-treated WT mice were injected daily with VIP antagonists, VIPHyb (n = 22), PG 97-269 (n = 9), or vehicle (n = 31). After euthanasia, colons were examined; colonic cytokines mRNA were quantified. VIP(-/-) mice were remarkably resistant to DSS-induced colitis compared to WT. Similarly, DSS-treated WT mice injected with VIPHyb (1 μM) or PG 97-269 (1 nM) had significantly reduced clinical signs of colitis. Furthermore, colonic expression of IL-1ϐ, TNF-α, and IL-6 was significantly lower in VIP(-/-) and VIPHyb or PG 97-269 compared to vehicle-treated WT. Genetic deletion of VIP or pharmacological inhibition of VIP receptors resulted in resistance to colitis. These data demonstrate a pro-inflammatory role for VIP in murine colitis and suggest that VIP antagonists may be an effective clinical treatment for human inflammatory bowel diseases.

Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

Hepatic ischemia/reperfusion injury (IRI), an exogenous, antigen-independent, local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The nervous system maintains extensive crosstalk with the immune system through neuropeptide and peptide hormone networks. This study examined the function and therapeutic potential of the vasoactive intestinal peptide (VIP) neuropeptide in a murine model of liver warm ischemia (90 minutes) followed by reperfusion. Liver ischemia/reperfusion (IR) triggered an induction of gene expression of intrinsic VIP; this peaked at 24 hours of reperfusion and coincided with a hepatic self-healing phase. Treatment with the VIP neuropeptide protected livers from IRI; this was evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture and was associated with elevated intracellular cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling. The hepatocellular protection rendered by VIP was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and increased hepatic interleukin-10 (IL-10) expression. Strikingly, PKA inhibition restored liver damage in otherwise IR-resistant VIP-treated mice. In vitro, VIP not only diminished macrophage tumor necrosis factor α/IL-6/IL-12 expression in a PKA-dependent manner but also prevented necrosis/apoptosis in primary mouse hepatocyte cultures. In conclusion, our findings document the importance of VIP neuropeptide-mediated cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. Because the enhancement of neural modulation differentially regulates local inflammation and prevents hepatocyte death, these results provide the rationale for novel approaches to managing liver IRI in transplant patients.

ObjectivePathological findings in neonatal brain injury associated with preterm birth include focal and/or diffuse white matter injury (WMI). Despite the heterogeneous nature of this condition, reactive astrogliosis and microgliosis are frequently observed. Thus, molecular mechanisms by which glia activation contribute to WMI were investigated.MethodsPostmortem brains of neonatal brain injury were investigated to identify molecular features of reactive astrocytes. The contribution of astrogliosis to WMI was further tested in a mouse model in genetically engineered mice.ResultsActivated STAT3 signaling in reactive astrocytes was found to be a common feature in postmortem brains of neonatal brain injury. In a mouse model of neonatal WMI, conditional deletion of STAT3 in astrocytes resulted in exacerbated WMI, which was associated with delayed maturation of oligodendrocytes. Mechanistically, the delay occurred in association with overexpression of transforming growth factor (TGF)β-1 in microglia, which in healthy controls decreased with myelin maturation in an age-dependent manner. TGFβ-1 directly and dose-dependently inhibited the maturation of purified oligodendrocyte progenitors, and pharmacological inhibition of TGFβ-1 signaling in vivo reversed the delay in myelin development. Factors secreted from STAT3-deficient astrocytes promoted elevated TGFβ-1 production in cultured microglia compared to wild-type astrocytes.InterpretationThese results suggest that myelin development is regulated by a mechanism involving crosstalk between microglia and oligodendrocyte progenitors. Reactive astrocytes may modify this signaling in a STAT3-dependent manner, preventing the pathological expression of TGFβ-1 in microglia and the impairment of oligodendrocyte maturation.

Studies in humans and animal models link maternal infection and imbalanced levels of inflammatory mediators in the foetal brain to the aetiology of neuropsychiatric disorders. In a number of animal models, it was shown that exposure to viral or bacterial agents during a period that corresponds to the second trimester in human gestation triggers brain and behavioural abnormalities in the offspring. However, little is known about the early cellular and molecular events elicited by inflammation in the foetal brain shortly after maternal infection has occurred. In this study, maternal infection was mimicked by two consecutive intraperitoneal injections of 200 μg of LPS (lipopolysaccharide)/kg to timed-pregnant rats at GD15 (gestational day 15) and GD16. Increased thickness of the CP (cortical plate) and hippocampus together with abnormal distribution of immature neuronal markers and decreased expression of markers for neural progenitors were observed in the LPS-exposed foetal forebrains at GD18. Such effects were accompanied by decreased levels of reelin and the radial glial marker GLAST (glial glutamate transporter), and elevated levels of pro-inflammatory cytokines in maternal serum and foetal forebrains. Foetal inflammation elicited by maternal injections of LPS has discrete detrimental effects on brain development. The early biochemical and morphological changes described in this work begin to explain the sequelae of early events that underlie the neurobehavioural deficits reported in humans and animals exposed to prenatal insults.

Abstract Background Hedgehog (HH) signaling is critical for the expansion of granule neuron precursors (GNPs) within the external granular layer (EGL) during cerebellar development. Aberrant HH signaling within GNPs is thought to give rise to medulloblastoma (MB) - the most commonly-observed form of malignant pediatric brain tumor. Evidence in both invertebrates and vertebrates indicates that cyclic AMP-dependent protein kinase A (PKA) antagonizes HH signalling. Receptors specific for the neuropeptide pituitary adenylyl cyclase activating polypeptide (PACAP, gene name ADCYAP1) are expressed in GNPs. PACAP has been shown to protect GNPs from apoptosis in vitro, and to interact with HH signaling to regulate GNP proliferation. PACAP/ptch1 double mutant mice exhibit an increased incidence of MB compared to ptch1 mice, indicating that PACAP may regulate HH pathway-mediated MB pathogenesis. Methods Primary MB tumorsphere cultures were prepared from thirteen ptch1+/-/p53+/- double mutant mice and treated with the smoothened (SMO) agonist purmorphamine, the SMO antagonist SANT-1, the neuropeptide PACAP, the PKA activator forskolin, and the PKA inhibitor H89. Gene expression of gli1 and [3H]-thymidine incorporation were assessed to determine drug effects on HH pathway activity and proliferation, respectively. PKA activity was determined in cell extracts by Western blotting using a phospho-PKA substrate antibody. Results Primary tumor cells cultured for 1-week under serum-free conditions grew as tumorspheres and were found to express PAC1 receptor transcripts. Gli1 gene expression was significantly reduced by SANT-1, PACAP and forskolin, but was unaffected by purmorphamine. The attenuation of gli1 gene expression by PACAP was reversed by the PKA inhibitor H89, which also blocked PKA activation. Treatment of tumorsphere cultures with PACAP, forskolin, and SANT-1 for 24 or 48 hours reduced proliferation. Conclusions Primary tumorspheres derived from ptch1+/-/p53+/- mice exhibit constitutive HH pathway activity. PACAP antagonizes HH signalling in these cells in a manner blocked by the PKA antagonist H89. PACAP and pharmacological activation of PKA also inhibited proliferation. Our data suggests that regulation of HH signaling by PACAP/PKA signaling may provide an alternative to SMO inhibition for the treatment of MB.

Phosphoinositide metabolism is critically involved in human cancer cell migration and metastatic growth. The formation of lamellipodia at the leading edge of migrating cells is regulated by metabolism of the inositol phospholipid PI(4,5)P2 into PI(3,4,5)P3. The synthesized PI(3,4,5)P3 promotes the translocation of WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane and regulates guanine nucleotide exchange factor Racmediated actin filament remodeling. Here, we investigated if VIPR2, a receptor for vasoactive intestinal peptide (VIP), has a potential role in regulating cell migration via this pathway. We found that silencing of VIPR2 in MDA-MB-231 and MCF-7 human breast cancer cells inhibited VIP-induced cell migration. In contrast, stable expression of exogenous VIPR2 promoted VIP-induced tumor cell migration, an effect that was inhibited by the addition of a PI3-kinase (PI3K)γ inhibitor or a VIPR2-selective antagonist. VIPR2 stably-expressing cells exhibited increased PI3K activity. Membrane localization of PI(3,4,5)P3 was significantly attenuated by VIPR2-silencing. VIPR2-silencing in MDA-MB-231 cells suppressed lamellipodium extension; in VIPR2-overexpressing cells, VIPR2 accumulated in the cell membrane on lamellipodia and co-localized with WAVE2. Conversely, VIPR2-silencing reduced WAVE2 level on the cell membrane and inhibited the interaction between WAVE2, actin-related protein 3, and actin. These findings suggest that VIP–VIPR2 signaling controls cancer migration by regulating WAVE2-mediated actin nucleation and elongation for lamellipodium formation through the synthesis of PI(3,4,5)P3. [ABSTRACT FROM AUTHOR]