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

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

Allosteric inhibition of phosphodiesterase 4D induces biphasic memory-enhancing effects associated with learning-activated signaling pathways.

RATIONALE: Phosphodiesterase 4D negative allosteric modulators (PDE4D NAMs) enhance memory and cognitive function in animal models without emetic-like side effects. However, the relationship between increased cyclic adenosine monophosphate (cAMP) signaling and the effects of PDE4D NAM remains elusive. OBJECTIVE: To investigate the roles of hippocampal cAMP metabolism and synaptic activation in the effects of D159687, a PDE4D NAM, under baseline and learning-stimulated conditions. RESULTS: At 3 mg/kg, D159687 enhanced memory formation and consolidation in contextual fear conditioning; however, neither lower (0.3 mg/kg) nor higher (30 mg/kg) doses induced memory-enhancing effects. A biphasic (bell-shaped) dose-response effect was also observed in a scopolamine-induced model of amnesia in the Y-maze, whereas D159687 dose-dependently caused an emetic-like effect in the xylazine/ketamine anesthesia test. At 3 mg/kg, D159687 increased cAMP levels in the hippocampal CA1 region after conditioning in the fear conditioning test, but not in the home-cage or conditioning cage (i.e., context only). By contrast, 30 mg/kg of D159687 increased hippocampal cAMP levels under all conditions. Although both 3 and 30 mg/kg of D159687 upregulated learning-induced Fos expression in the hippocampal CA1 30 min after conditioning, 3 mg/kg, but not 30 mg/kg, of D159687 induced phosphorylation of synaptic plasticity-related proteins such as cAMP-responsive element-binding protein, synaptosomal-associated protein 25 kDa, and the N-methyl-D-aspartate receptor subunit NR2A. CONCLUSIONS: Our findings suggest that learning-stimulated conditions can alter the effects of a PDE4D NAM on hippocampal cAMP levels and imply that a PDE4D NAM exerts biphasic memory-enhancing effects associated with synaptic plasticity-related signaling activation.

Mouse maternal odontogenic infection with Porphyromonas gingivalis induces cognitive decline in offspring.

Introduction: Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, causes intrauterine infection/inflammation. Offspring exposed to intrauterine infection/inflammation have an increased risk of neurological disorders, regardless of gestational age. However, the relationship between maternal periodontitis and offspring functional/histological changes in the brain has not yet been elucidated. Methods: In this study, we used a gestational mouse model to investigate the effects of maternal odontogenic infection of P. gingivalis on offspring behavior and brain tissue. Results: The step-through passive avoidance test showed that the latency of the acquisition trial was significantly shorter in the P. gingivalis group (p < 0.05), but no difference in spontaneous motor/exploratory parameters by open-field test. P. gingivalis was diffusely distributed throughout the brain, especially in the hippocampus. In the hippocampus and amygdala, the numbers of neuron cells and cyclic adenosine monophosphate response element binding protein-positive cells were significantly reduced (p < 0.05), whereas the number of ionized calcium binding adapter protein 1-positive microglia was significantly increased (p < 0.05). In the hippocampus, the number of glial fibrillary acidic protein-positive astrocytes was also significantly increased (p < 0.05). Discussion: The offspring of P. gingivalis-infected mothers have reduced cognitive function. Neurodegeneration/neuroinflammation in the hippocampus and amygdala may be caused by P. gingivalis infection, which is maternally transmitted. The importance of eliminating maternal P. gingivalis-odontogenic infection before or during gestation in maintenance healthy brain function in offspring should be addressed in near future.

The VIPR2-selective antagonist KS-133 changes macrophage polarization and exerts potent anti-tumor effects as a single agent and in combination with an anti-PD-1 antibody.

We have previously demonstrated that KS-133 is a specific and potent antagonist of vasoactive intestinal peptide receptor 2 (VIPR2). We have also shown that vasoactive intestinal peptide-VIPR2 signaling affects the polarity and activation of tumor-associated macrophages, which is another strategy for cancer immunotherapy apart from the activation of effector T cells. In this study, we aimed to examine whether the selective blockade of VIPR2 by KS-133 changes the polarization of macrophages and induces anti-tumor effects. In the presence of KS-133, genetic markers indicative of tumor-aggressive M1-type macrophages were upregulated, and conversely, those of tumor-supportive M2-type macrophages were downregulated. Daily subcutaneous administration of KS-133 tended to suppress the growth of CT26 tumors (murine colorectal cancer-derived cells) implanted subcutaneously in Balb/c mice. To improve the pharmacological efficacy and reduce the number of doses, we examined a nanoformulation of KS-133 using the US Food and Drug Administration-approved pharmaceutical additive surfactant Cremophor® EL. KS-133 nanoparticles (NPs) were approximately 15 nm in size and stable at 4°C after preparation. Meanwhile, KS-133 was gradually released from the NPs as the temperature was increased. Subcutaneous administration of KS-133 NPs once every 3 days had stronger anti-tumor effects than daily subcutaneous administration of KS-133. Furthermore, KS-133 NPs significantly enhanced the pharmacological efficacy of an immune checkpoint-inhibiting anti-PD-1 antibody. A pharmacokinetic study suggested that the enhancement of anti-tumor activity was associated with improvement of the pharmacokinetic profile of KS-133 upon nanoformulation. Our data have revealed that specific blockade of VIPR2 by KS-133 has therapeutic potential for cancer both alone and in combination with immune checkpoint inhibitors.

[Development of the vasoactive intestinal peptide receptor 2 (VIPR2) antagonist peptide for the treatment of schizophrenia].

Schizophrenia affects approximately 24 million people worldwide. Existing medications for the treatment of schizophrenia work primarily by improving positive symptoms such as agitation, hallucinations, delusions, and aggression. They possess common mechanism of action (MOA), blocking to neurotransmitter receptors such as dopamine, serotonin, and adrenaline receptors. Although multiple agents are available for the treatment of schizophrenia, the majority do not address negative symptoms or cognitive dysfunction. In other cases, patients have drug-related adverse effects. The vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2 receptor) might be an attractive drug target for the treatment of schizophrenia because both clinical and preclinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Despite these backgrounds, the proof-of-concept of VIPR2 inhibitors has not been examined clinically. A reason might be that VIPR2 belongs to class-B GPCRs, and the discovery of small-molecule drugs against class-B GPCRs is generally difficult. We have developed a bicyclic peptide KS-133, which shows VIPR2 antagonist activity and suppresses cognitive decline in a mouse model relevant to schizophrenia. KS-133 has a different MOA from current therapeutic drugs and exhibits high selectivity for VIPR2 and potent inhibitory activity against a single-target molecule. Therefore, it may contribute to both the development of a novel drug candidate for the treatment of psychiatric disorders such as schizophrenia and acceleration of basic studies on VIPR2.

Suppressive effect of black tea polyphenol theaflavins in a mouse model of ovalbumin-induced food allergy.

Food allergy is recognized as a global medical problem with increasing prevalence in recent years. Currently, the treatment of food allergy mainly involves avoidance of allergens and allergen-specific immunotherapy. Barring the spontaneous resolution of food allergy during the growth process, this disease is difficult to treat fundamentally. In recent years, the use of functional food ingredients derived from natural products has been attracting attention for their prophylactic use in food allergy. Theaflavins, i.e., black tea polyphenols, are potent antioxidants that have inhibitory effects on a variety of diseases. However, little is known about the preventive effect of theaflavins on food allergy. In this study, we designed a mouse model of food allergy and examined the effect of theaflavins using the severity of diarrhea, a symptom of food allergy, as an indicator. The administration of a black tea extract rich in theaflavins or theaflavin 1 (subgroup of theaflavins) to mice reduced the severity of diarrhea when compared with a normal diet. A reduction in malondialdehyde levels, a key marker of lipid peroxidation, was also observed. Overall, these data suggest that theaflavins may potentially inhibit food allergy by alleviating oxidative stress in the colon and can be a potential food material for prevention of food allergy.

Overexpression of VIPR2 in mice results in microencephaly with paradoxical increased white matter volume.

Large scale studies in populations of European and Han Chinese ancestry found a series of rare gain-of-function microduplications in VIPR2, encoding VPAC2, a receptor that binds vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide with high affinity, that were associated with an up to 13-fold increased risk for schizophrenia. To address how VPAC2 receptor overactivity might affect brain development, we used a well-characterized Nestin-Cre mouse strain and a knock-in approach to overexpress human VPAC2 in the central nervous system. Mice that overexpressed VPAC2 were found to exhibit a significant reduction in brain weight. Magnetic resonance imaging analysis confirmed a decrease in brain size, a specific reduction in the hippocampus grey matter volume and a paradoxical increase in whole-brain white matter volume. Sex-specific changes in behavior such as impaired prepulse inhibition and contextual fear memory were observed in VPAC2 overexpressing mice. The data indicate that the VPAC2 receptor may play a critical role in brain morphogenesis and suggest that overactive VPAC2 signaling during development plays a mechanistic role in some forms of schizophrenia.

Vasoactive intestinal peptide receptor 2 signaling promotes breast cancer cell proliferation by enhancing the ERK pathway.

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a class B G protein-coupled receptor with the neuropeptide VIP as a ligand. Increased VIPR2 mRNA expression and/or VIPR2 gene copy number has been documented in several cancers including breast carcinoma. However, the pathophysiological role of increased VIPR2 in the proliferation of breast cancer cells remains largely unknown. In this study, we found that VIPR2 overexpression in MCF-7 and MDA-MB-231 cells, human breast cancer cell lines, promoted cell proliferation. Increased VIPR2 also exacerbated intraperitoneal proliferation of breast cancer MDA-MB-231 cells in a tumor nude mouse model in vivo. Treatment with KS-133, a VIPR2-selective antagonist peptide, significantly inhibited VIP-induced cell proliferation in VIPR2-overexpressing MCF-7 and MDA-MB-231 cells. Overexpressed VIPR2 caused increases in the levels of cAMP and phosphorylated extracellular signal-regulated kinase (ERK), which involves a VIPR2 signaling pathway through Gs protein. Additionally, phosphorylation of vasodilator-stimulated phosphoprotein (Ser157) and cAMP response element binding protein (Ser133) in VIPR2-overexpressing MCF-7 cells was greater than that in control cells, suggesting the increased PKA activity. Moreover, an inhibitor of mitogen-activated protein kinase kinase, U0126, attenuated tumor proliferation in exogenous VIPR2-expressing MCF-7 and MDA-MB-231 cells at the same level as observed in EGFP-expressing cells treated with U0126. Together, these findings suggest that VIPR2 controls breast tumor growth by regulating the cAMP/PKA/ERK signaling pathway, and the excessive expression of VIPR2 may lead to an exacerbation of breast carcinoma.

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

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

Vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration.

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 Rac-mediated 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.

Phospholipase C-related catalytically inactive protein enhances cisplatin-induced apoptotic cell death.

Cisplatin is one of the most widely used chemotherapeutic agents and induces caspase-9-mediated apoptosis. Here, we examined whether phospholipase C-related catalytically inactive protein (PRIP) enhances cisplatin-induced apoptosis of breast cancer cells. PRIP depletion increased expression of X-linked inhibitor of apoptosis protein (XIAP) by inhibiting protein degradation, which is downstream of the phosphatidylinositol 3-kinase/AKT pathway and inhibits apoptotic signaling by blocking caspase-9 activation. Conversely, the viability of MCF-7 cells transfected with Prip1 was significantly lower than that of control cells in the presence of cisplatin. The number of apoptotic nuclei and expression levels of cleaved caspase-9 and downstream cleaved caspase-7 and poly-ADP ribose polymerase were greater in PRIP1-expressing MCF-7 cells treated with cisplatin than in control cells. XIAP was decreased by expression of pleckstrin homology domain of PRIP1 (PRIP1-PH domain) that blocked phosphatidylinositol 4,5 bisphosphate metabolism. In an orthotopic transplantation model, combined administration of PRIP1-PH domain-containing liposomes and cisplatin reduced the size of MCF-7 tumors compared with cisplatin alone. Our findings demonstrate that PRIP promotes XIAP degradation by inhibiting PI(3,4,5)P3/AKT signaling and enhances cisplatin-induced apoptotic cell death. Therefore, we propose that PRIP1-PH liposomes are a novel agent to avoid cisplatin resistance.

Generation of KS-133 as a Novel Bicyclic Peptide with a Potent and Selective VIPR2 Antagonist Activity that Counteracts Cognitive Decline in a Mouse Model of Psychiatric Disorders.

Worldwide, more than 20 million people suffer from schizophrenia, but effective and definitive new therapeutic drugs/treatments have not been established. Vasoactive intestinal peptide receptor 2 (VIPR2) might be an attractive drug target for the treatment of schizophrenia because both preclinical and clinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Nevertheless, VIPR2-targeting drugs are not yet available. VIPR2 is a class-B G protein-coupled receptor that possesses high structural homology to its subtypes, vasoactive intestinal peptide receptor 1 (VIPR1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). These biological and structural properties have made it difficult to discover small molecule drugs against VIPR2. In 2018, cyclic peptide VIpep-3, a VIPR2-selective antagonist, was reported. The aim of this study was to generate a VIpep-3 derivative for in vivo experiments. After amino acid substitution and structure optimization, we successfully generated KS-133 with 1) a VIPR2-selective and potent antagonistic activity, 2) at least 24 h of stability in plasma, and 3) in vivo pharmacological efficacies in a mouse model of psychiatric disorders through early postnatal activation of VIPR2. To the best of our knowledge, this is the first report of a VIPR2-selective antagonistic peptide that counteracts cognitive decline, a central feature of schizophrenia. KS-133 may contribute to studies and development of novel schizophrenia therapeutic drugs that target VIPR2.

Probing the VIPR2 Microduplication Linkage to Schizophrenia in Animal and Cellular Models.

Pituitary adenylate cyclase-activating polypeptide (PACAP, gene name ADCYAP1) is a multifunctional neuropeptide involved in brain development and synaptic plasticity. With respect to PACAP function, most attention has been given to that mediated by its specific receptor PAC1 (ADCYAP1R1). However, PACAP also binds tightly to the high affinity receptors for vasoactive intestinal peptide (VIP, VIP), called VPAC1 and VPAC2 (VIPR1 and VIPR2, respectively). Depending on innervation patterns, PACAP can thus interact physiologically with any of these receptors. VPAC2 receptors, the focus of this review, are known to have a pivotal role in regulating circadian rhythms and to affect multiple other processes in the brain, including those involved in fear cognition. Accumulating evidence in human genetics indicates that microduplications at 7q36.3, containing VIPR2 gene, are linked to schizophrenia and possibly autism spectrum disorder. Although detailed molecular mechanisms have not been fully elucidated, recent studies in animal models suggest that overactivation of the VPAC2 receptor disrupts cortical circuit maturation. The VIPR2 linkage can thus be potentially explained by inappropriate control of receptor signaling at a time when neural circuits involved in cognition and social behavior are being established. Alternatively, or in addition, VPAC2 receptor overactivity may disrupt ongoing synaptic plasticity during processes of learning and memory. Finally, in vitro data indicate that PACAP and VIP have differential activities on the maturation of neurons via their distinct signaling pathways. Thus perturbations in the balance of VPAC2, VPAC1, and PAC1 receptors and their ligands may have important consequences in brain development and plasticity.

The pivotal role of pituitary adenylate cyclase-activating polypeptide for lactate production and secretion in astrocytes during fear memory.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an essential role in the modulation of astrocyte functions. Although lactate secretion from astrocytes contributes to many forms of neuronal plasticity in the central nervous system, including fear learning and memory, the role of PACAP in lactate secretion from astrocytes is unclear. METHODS: The amygdala and hippocampus of PACAP (+ / +) and PACAP (-/-) mice were acquired 1 h after memory acquisition and recall in the passive avoidance test. The concentration of glycogen and lactate in these regions was measured. The concentration of lactate in the hippocampus's extracellular fluid was also measured by microdialysis during memory acquisition or intracerebroventricular administration of PACAP. RESULTS: We observed that memory acquisition caused a significant decrease in glycogen concentration and increased lactate concentration in the PACAP (+ / +) mice's hippocampus. However, memory acquisition did not increase in the lactate concentration in PACAP (-/-) mice's hippocampus. Further, memory retrieval evoked lactate production in the amygdala and the hippocampus of PACAP (+ / +) mice. Still, there was no significant increase in lactate concentration in the same regions of PACAP (-/-) mice. In vivo microdialysis in rats revealed that the hippocampus's extracellular lactate concentration increased after a single PACAP intracerebroventricular injection. Additionally, the hippocampus's extracellular lactate concentration increased with the memory acquisition in PACAP (+ / +) mice, but not in PACAP (-/-) mice. CONCLUSIONS: PACAP may enhance lactate production and secretion in astrocytes during the acquisition and recall of fear memories.

Lipocalin-type prostaglandin D synthase regulates light-induced phase advance of the central circadian rhythm in mice.

We previously showed that mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) exhibit attenuated light-induced phase shift. To explore the underlying mechanisms, we performed gene expression analysis of laser capture microdissected suprachiasmatic nuclei (SCNs) and found that lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is involved in the impaired response to light stimulation in the late subjective night in PACAP-deficient mice. L-PGDS-deficient mice also showed impaired light-induced phase advance, but normal phase delay and nonvisual light responses. Then, we examined the receptors involved in the response and observed that mice deficient for type 2 PGD2 receptor DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells) show impaired light-induced phase advance. Concordant results were observed using the selective DP2/CRTH2 antagonist CAY10471. These results indicate that L-PGDS is involved in a mechanism of light-induced phase advance via DP2/CRTH2 signaling.

Activation of the VPAC2 Receptor Impairs Axon Outgrowth and Decreases Dendritic Arborization in Mouse Cortical Neurons by a PKA-Dependent Mechanism.

Clinical studies have shown that microduplications at 7q36.3, containing VIPR2, confer significant risk for schizophrenia and autism spectrum disorder (ASD). VIPR2 gene encodes the VPAC2 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Lymphocytes from patients with these mutations exhibited higher VIPR2 gene expression and VIP-induced cAMP responsiveness, but mechanisms by which overactive VPAC2 signaling may lead to these psychiatric disorders are unknown. We have previously found that repeated administration of a selective VPAC2 receptor agonist Ro25-1553 in the mouse during early postnatal development caused synaptic alterations in the prefrontal cortex and sensorimotor gating deficits. In this study, we aimed to clarify the effects of VPAC2 receptor activation on neurite outgrowth in cultured primary mouse cortical neurons. Ro25-1553 and VIP caused reductions in total numbers and lengths of both neuronal dendrites and axons, while PACAP38 facilitated elongation of dendrites, but not axons. These effects of Ro25-1553 and VIP were blocked by a VPAC2 receptor antagonist PG99-465 and abolished in VPAC2 receptor-deficient mice. Additionally, Ro25-1553-induced decreases in axon and dendritic outgrowth in wild-type mice were blocked by a protein kinase A (PKA) inhibitor H89, but not by a PKC inhibitor GF109203X or a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor U0126. PACAP38- induced facilitation of dendritic outgrowth was blocked by U0126. These results suggest that activation of the VPAC2 receptor impairs neurite outgrowth and decreases branching of cortical neurons by a PKA-dependent mechanism. These findings also imply that the VIPR2-linkage to mental health disorders may be due in part to deficits in neuronal maturation induced by VPAC2 receptor overactivation.

Valproic acid attenuates CCR2-dependent tumor infiltration of monocytic myeloid-derived suppressor cells, limiting tumor progression.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that promote tumor progression by inhibiting anti-tumor immunity and may be the cause of patient resistance to immune checkpoint inhibitors (ICIs). Therefore, MDSCs are a promising target for cancer immunotherapy, especially in combination with ICIs. Previous studies have shown that the anticonvulsant drug valproic acid (VPA) has additional anti-cancer and immunoregulatory activities due to its inhibition of histone deacetylases. We have previously shown that VPA can attenuate the immunosuppressive function of differentiated MDSCs in vitro. In the present study, we utilized anti-PD-1-sensitive EL4 and anti-PD-1-resistant B16-F10 tumor-bearing mouse models and investigated the effects of VPA on MDSCs with the aim of enhancing the anti-cancer activity of an anti-PD-1 antibody. We showed that VPA could inhibit EL4 and B16-F10 tumor progression, which was dependent on the immune system. We further demonstrated that VPA down-regulated the expression of CCR2 on monocytic (M)-MDSCs, leading to the reduced infiltration of M-MDSCs into tumors. Importantly, we demonstrated that VPA could relieve the immunosuppressive action of MDSCs on CD8+ T-cell and NK cell proliferation and enhance their activation in tumors. We also observed that the combination of VPA plus an anti-PD-1 antibody was more effective than either agent alone in both the EL4 and B16-F10 tumor models. These results suggest that VPA can effectively relieve the immunosuppressive tumor microenvironment by reducing tumor infiltration of M-MDSCs, resulting in tumor regression. Our findings also show that VPA in combination with an immunotherapeutic agent could be a potential new anti-cancer therapy.

Pathogenic POGZ mutation causes impaired cortical development and reversible autism-like phenotypes.

Pogo transposable element derived with ZNF domain (POGZ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD.

Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation.

Alterations in pituitary adenylate cyclase-activating polypeptide (PACAP), a multifunctional neuropeptide, and its receptors have been identified as risk factors for certain psychiatric disorders, including schizophrenia. Increasing evidence from human genetic and animal model studies suggest an association between various psychiatric disorders and altered dendritic spine morphology. In the present study, we investigated the role of exogenous and endogenous PACAP in spine formation and maturation. PACAP modified the density and morphology of PSD-95-positive spines in primary cultured hippocampal neurons. Notably, PACAP increased the levels of microRNA (miR)-132 and decreased expression of corresponding miR-132 target genes and protein expression of p250GAP, a miR-132 effector known to be involved in spine morphology regulation. In corroboration, PSD-95-positive spines were reduced in PACAP-deficient (PACAP-/-) mice versus WT mice. Golgi staining of hippocampal CA1 neurons revealed a reduced spine densities and atypical morphologies in the male PACAP-/- mice. Furthermore, viral miR-132 overexpression reversed the reduction in hippocampal spinal density in the male PACAP-/- mice. These results indicate that PACAP signaling plays a critical role in spine morphogenesis possibly via miR-132. We suggest that dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through its effects on spine formation.SIGNIFICANCE STATEMENT Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling dysfunction and dendritic spine morphology alterations have recently been suggested as important pathophysiological mechanisms underlying several psychiatric and neurological disorders. In this study, we investigated whether PACAP regulates dendritic spine morphogenesis. In a combination of pharmacological and viral gain- and loss-of-function approaches in vitro and in vivo experiments, we found PACAP to increase the size and density of dendritic spines via miR-132 upregulation. Together, our data suggest that a dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through abnormal spine formation.

The Robo4-TRAF7 complex suppresses endothelial hyperpermeability in inflammation.

Roundabout guidance receptor 4 (Robo4) is an endothelial cell-specific receptor that stabilizes the vasculature in pathological angiogenesis. Although Robo4 has been shown to suppress vascular hyperpermeability induced by vascular endothelial growth factor (VEGF) in angiogenesis, the role of Robo4 in inflammation is poorly understood. In this study, we investigated the role of Robo4 in vascular hyperpermeability during inflammation. Endotoxemia models using Robo4-/- mice showed increased mortality and vascular leakage. In endothelial cells, Robo4 suppressed tumor necrosis factor α (TNFα)-induced hyperpermeability by stabilizing VE-cadherin at cell junctions, and deletion assays revealed that the C-terminus of Robo4 was involved in this suppression. Through binding and localization assays, we demonstrated that in endothelial cells, Robo4 binds to TNF receptor-associated factor 7 (TRAF7) through interaction with the C-terminus of Robo4. Gain- and loss-of-function studies of TRAF7 with or without Robo4 expression showed that TRAF7 is required for Robo4-mediated suppression of hyperpermeability. Taken together, our results demonstrate that the Robo4-TRAF7 complex is a novel negative regulator of inflammatory hyperpermeability. We propose this complex as a potential future target for protection against inflammatory diseases.