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When adult mice are repeatedly exposed to a particular visual stimulus for as little as 1 h per day for several days while their visual cortex (V1) is in the high-gain state produced by locomotion, that specific stimulus elicits much stronger responses in V1 neurons for the following several weeks, even when measured in anesthetized animals. Such stimulus-specific enhancement (SSE) is not seen if locomotion is prevented. The effect of locomotion on cortical responses is mediated by vasoactive intestinal peptide (VIP) positive interneurons, which can release both the peptide and the inhibitory neurotransmitter GABA. Previous studies have examined the role of VIP-ergic interneurons, but none have distinguished the individual roles of peptide from GABA release. Here, we used genetic ablation to determine which of those molecules secreted by VIP-ergic neurons is responsible for SSE. SSE was not impaired by VIP deletion but was prevented by compromising release of GABA from VIP cells. This finding suggests that SSE may result from Hebbian mechanisms that remain present in adult V1.NEW & NOTEWORTHY Many neurons package and release a peptide along with a conventional neurotransmitter. The conventional view is that such peptides exert late, slow effects on plasticity. We studied a form of cortical plasticity that depends on the activity of neurons that express both vasoactive intestinal peptide (VIP) and the inhibitory neurotransmitter GABA. GABA release accounted for their action on plasticity, with no effect of deleting the peptide on this phenomenon.
Assuntos
Interneurônios , Peptídeo Intestinal Vasoativo , Córtex Visual , Ácido gama-Aminobutírico , Animais , Peptídeo Intestinal Vasoativo/metabolismo , Interneurônios/metabolismo , Interneurônios/fisiologia , Ácido gama-Aminobutírico/metabolismo , Camundongos , Córtex Visual/metabolismo , Córtex Visual/fisiologia , Camundongos Endogâmicos C57BL , Masculino , Estimulação Luminosa , FemininoRESUMO
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.
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Group 3 innate lymphoid cells (ILC3s) control the formation of intestinal lymphoid tissues and play key roles in intestinal defense. They express neuropeptide vasoactive intestinal peptide (VIP) receptor 2 (VPAC2), through which VIP modulates their function, but whether VIP exerts other effects on ILC3 remains unclear. We show that VIP promotes ILC3 recruitment to the intestine through VPAC1 independent of the microbiota or adaptive immunity. VIP is also required for postnatal formation of lymphoid tissues as well as the maintenance of local populations of retinoic acid (RA)-producing dendritic cells, with RA up-regulating gut-homing receptor CCR9 expression by ILC3s. Correspondingly, mice deficient in VIP or VPAC1 suffer a paucity of intestinal ILC3s along with impaired production of the cytokine IL-22, rendering them highly susceptible to the enteric pathogen Citrobacter rodentium This heightened susceptibility to C. rodentium infection was ameliorated by RA supplementation, adoptive transfer of ILC3s, or by recombinant IL-22. Thus, VIP regulates the recruitment of intestinal ILC3s and formation of postnatal intestinal lymphoid tissues, offering protection against enteric pathogens.
Assuntos
Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Linfócitos/imunologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Células Dendríticas/imunologia , Microbioma Gastrointestinal/imunologia , Interleucinas/análise , Tecido Linfoide/citologia , Tecido Linfoide/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR/biossíntese , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Tretinoína/metabolismo , Peptídeo Intestinal Vasoativo/genética , Interleucina 22RESUMO
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.
Assuntos
Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Sequência de Aminoácidos/genética , Animais , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Encéfalo/imunologia , Encéfalo/metabolismo , Morte Celular/efeitos dos fármacos , Simulação por Computador , Bases de Dados Genéticas , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neuropeptídeos/metabolismo , Filogenia , Transdução de Sinais/fisiologiaRESUMO
Chronic inflammation drives synaptic loss in multiple sclerosis (MS) and is also commonly observed in other neurodegenerative diseases. Clinically approved treatments for MS provide symptomatic relief but fail to halt neurodegeneration and neurological decline. Studies in animal disease models have demonstrated that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) exhibits anti-inflammatory, neuroprotective and regenerative properties. Anti-inflammatory actions appear to be mediated primarily by two receptors, VPAC1 and VPAC2, which also bind vasoactive intestinal peptide (VIP). Pharmacological experiments indicate that another receptor, PAC1 (ADCYAP1R1), which is highly selective for PACAP, provides protection to neurons, although genetic evidence and other mechanistic information is lacking. To determine if PAC1 receptors protect neurons in a cell-autonomous manner, we used adeno-associated virus (AAV2) to deliver Cre recombinase to the retina of mice harboring floxed PAC1 alleles. Mice were then subjected to chronic experimental autoimmune encephalomyelitis (EAE), a disease model that recapitulates major clinical and pathological features of MS and associated optic neuritis. Unexpectedly, deletion of PAC1 in naïve mice resulted in a deficit of retinal ganglionic neurons (RGNs) and their dendrites, suggesting a homeostatic role of PAC1. Moreover, deletion of PAC1 resulted in increased EAE-induced loss of a subpopulation of RGNs purported to be vulnerable in animal models of glaucoma. Increased axonal pathology and increased secondary presence of microglia/macrophages was also prominently seen in the optic nerve. These findings demonstrate that neuronal PAC1 receptors play a homeostatic role in protecting RGNs and directly protects neurons and their axons against neuroinflammatory challenge. SIGNIFICANCE STATEMENT: Chronic inflammation is a major component of neurodegenerative diseases and plays a central role in multiple sclerosis (MS). Current treatments for MS do not prevent neurodegeneration and/or neurological decline. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to have anti-inflammatory, neuroprotective and regenerative properties but the cell type- and receptor-specific mechanisms are not clear. To test whether the protective effects of PACAP are direct on the PAC1 receptor subtype on neurons, we delete PAC1 receptors from neurons and investigate neuropathologigical changes in an animal model of MS. The findings demonstrate that PAC1 receptors on neurons play a homeostatic role in maintaining neuron health and can directly protect neurons and their axons during neuroinflammatory disease.
Assuntos
Axônios/metabolismo , Morte Celular/fisiologia , Esclerose Múltipla/metabolismo , Neurite Óptica/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Neurônios Retinianos/metabolismo , Animais , Axônios/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Neurite Óptica/genética , Neurite Óptica/patologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genéticaRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers Iba1 and iNOS (### p < 0.001), as well as the pro-inflammatory mediators IL-1ß, IL-6, Itgam and CD68 (### p < 0.001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs. 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped somata (48.41% vs. 31.36% in LPS-treated cells). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities.
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Microglia/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Fenótipo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
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.
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Espinhas Dendríticas/metabolismo , MicroRNAs/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Morfogênese/fisiologia , Neurogênese/fisiologia , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
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).
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Mediadores da Inflamação/metabolismo , Transtornos de Enxaqueca/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , HumanosRESUMO
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.
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Dendritos , Extinção Psicológica/fisiologia , Medo/fisiologia , Córtex Pré-Frontal/fisiologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/fisiologia , Animais , Condicionamento Clássico , Sinais (Psicologia) , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Córtex Pré-Frontal/citologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genéticaRESUMO
BACKGROUND: Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two highly homologous neuropeptides. In vitro and ex vivo experiments repeatedly demonstrate that these peptides exert pronounced immunomodulatory (primarily anti-inflammatory) actions which are mediated by common VPAC1 and VPAC2 G protein-coupled receptors. In agreement, we have shown that mice deficient in PACAP ligand or VPAC2 receptors exhibit exacerbated experimental autoimmune encephalomyelitis (EAE). However, we observed that VIP-deficient mice are unexpectedly resistant to EAE, suggesting a requirement for this peptide at some stage of disease development. Here, we investigated the involvement of VPAC1 in the development of EAE using a VPAC1-deficient mouse model. METHODS: EAE was induced in wild-type (WT) and VPAC1 knockout (KO) mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55), and clinical scores were assessed continuously over 30 days. Immune responses in the spinal cords were determined by histology, real-time PCR and immunofluorescence, and in the draining lymph nodes by antigen-recall assays. The contribution of VPAC1 expression in the immune system to the development of EAE was evaluated by means of adoptive transfer and bone marrow chimera experiments. In other experiments, VPAC1 receptor analogs were given to WT mice. RESULTS: MOG35-55-induced EAE was ameliorated in VPAC1 KO mice compared to WT mice. The EAE-resistant phenotype of VPAC1 KO mice correlated with reduced central nervous system (CNS) histopathology and cytokine expression in the spinal cord. The immunization phase of EAE appeared to be unimpaired because lymph node cells from EAE-induced VPAC1 KO mice stimulated in vitro with MOG exhibited robust proliferative and Th1/Th17 responses. Moreover, lymph node and spleen cells from KO mice were fully capable of inducing EAE upon transfer to WT recipients. In contrast, WT cells from MOG-immunized mice did not transfer the disease when administered to VPAC1 KO recipients, implicating a defect in the effector phase of the disease. Bone marrow chimera studies suggested that the resistance of VPAC1-deficient mice was only minimally dependent on the expression of this receptor in the immunogenic/hematopoietic compartment. Consistent with this, impaired spinal cord inductions of several chemokine mRNAs were observed in VPAC1 KO mice. Finally, treatment of WT mice with the VPAC1 receptor antagonist PG97-269 before, but not after, EAE induction mimicked the clinical phenotype of VPAC1 KO mice. CONCLUSIONS: VPAC1 gene loss impairs the development of EAE in part by preventing an upregulation of CNS chemokines and invasion of inflammatory cells into the CNS. Use of VPAC1 antagonists in WT mice prior to EAE induction also support a critical role for VPAC1 signaling for the development of EAE.
Assuntos
Citocinas/metabolismo , Encefalomielite Autoimune Experimental/complicações , Encefalomielite Autoimune Experimental/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/deficiência , Transferência Adotiva , Animais , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Citocinas/genética , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Adjuvante de Freund/toxicidade , Laminina/metabolismo , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/toxicidade , RNA Mensageiro/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Medula Espinal/metabolismo , Medula Espinal/patologia , Células Th1/metabolismo , Células Th1/patologia , Células Th17/metabolismo , Células Th17/patologia , Fatores de TempoRESUMO
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.
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Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/metabolismo , Medo/psicologia , Generalização Psicológica , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Condicionamento Psicológico , Hipocampo/citologia , Hipocampo/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos de Estresse Pós-Traumáticos/psicologiaRESUMO
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.
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Canais de Cálcio/metabolismo , Sinalização do Cálcio , Receptores Nucleares Órfãos/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Íons/metabolismo , Camundongos , Fatores de Transcrição NFATC/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Proteína ORAI1 , Receptores Nucleares Órfãos/deficiência , Receptores Nucleares Órfãos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Elementos de Resposta , Bibliotecas de Moléculas Pequenas , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th2/citologia , Células Th2/imunologia , Células Th2/metabolismoRESUMO
The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock-in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet-1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate-based screening and identified BMP type I receptor kinase inhibitors LDN-193189 and DMH1 as activators of luciferase. LDN-193189 induced ES cells to express the genes encoding Pet-1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF-ß receptor inhibitor SB-431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN-193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF-ß target gene Lefty, whereas the opposite effect was observed with SB-431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF-ß receptor signaling are implicated in serotonergic differentiation. Candidate-based screening for serotonergic induction using a rapid assay in mouse embryonic stem cells revealed that the bone morphogenetic protein (BMP) type I receptor kinase inhibitors selectively induce serotonergic differentiation, whereas the TGF-ß receptor inhibitor SB-431542 inhibits the differentiation. These results suggest that inhibition of BMP type I receptors and concomitant activation of transforming growth factor-ß (TGF-ß) receptor signaling are involved in the early trajectory of serotonergic differentiation.
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Receptores de Proteínas Morfogenéticas Ósseas Tipo I/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Neurônios Serotoninérgicos/fisiologia , Animais , Benzamidas/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/antagonistas & inibidores , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Dioxóis/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Técnicas de Introdução de Genes/métodos , Camundongos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Neurônios Serotoninérgicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
OBJECTIVES: Vasoactive intestinal peptide (VIP) is an immunomodulatory neuropeptide with therapeutic properties in multiple murine models of inflammatory disease including the trinitrobenzene-sulfonic acid (TNBS)-colitis model of Crohn's disease. Understanding the spectrum of biological actions of endogenously produced VIP may help us dissect the complex and multifactorial pathogenesis of such inflammatory diseases. Our goal was to determine the contribution of endogenously produced VIP to TNBS-colitis by using VIP knockout (KO) mice. METHODS: TNBS was intracolonically administered to wild-type (WT) and VIP KO mice, and weight loss and colitis were assessed over time. Colon histopathological changes and myeloperoxidase activities were analyzed and the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in colon and serum quantified. The proliferative response in vitro of splenocytes from TNBS WT and VIP KO administered mice to anti-CD3 and anti-CD28 was determined. RESULTS: VIP KO mice did not exhibit the predicted exacerbated response to TNBS. Instead, they developed a milder clinical profile than WT mice, with lower TNF-α and IL-6 levels. Such potential defects seem selective, because other parameters such as the histopathological scores and the cytokine levels in the colon did not differ between the two strains of mice. Moreover, splenocytes from TNBS-treated VIP KO mice exhibited an enhanced proliferative response to anti-CD3/CD28 stimulation in vitro. CONCLUSION: Chronic loss of VIP in mice leads to a disruption of certain but not all immunological compartments, corroborating recent findings that VIP KO mice exhibit reduced mortality in the lipopolysaccharide-induced endotoxemia model and attenuated clinical development of experimental autoimmune encephalomyelitis while developing robust T-cell responses.
Assuntos
Colite/induzido quimicamente , Colite/patologia , Colo/patologia , Ácido Trinitrobenzenossulfônico/toxicidade , Peptídeo Intestinal Vasoativo/deficiência , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Colo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Fatores de Tempo , Peptídeo Intestinal Vasoativo/genéticaRESUMO
During corticogenesis, pituitary adenylate cyclase-activating polypeptide (PACAP; ADCYAP1) may contribute to proliferation control by activating PAC1 receptors of neural precursors in the embryonic ventricular zone. PAC1 receptors, specifically the hop and short isoforms, couple differentially to and activate distinct pathways that produce pro- or anti-mitogenic actions. Previously, we found that PACAP was an anti-mitogenic signal from embryonic day 13.5 (E13.5) onward both in culture and in vivo and activated cAMP signaling through the short isoform. However, we now find that mice deficient in PACAP exhibited a decrease in the BrdU labeling index (LI) in E9.5 cortex, suggesting that PACAP normally promotes proliferation at this stage. To further define mechanisms, we established a novel culture model in which the viability of very early cortical precursors (E9.5 mouse and E10.5 rat) could be maintained. At this stage, we found that PACAP evoked intracellular calcium fluxes and increased phospho-PKC levels, as well as stimulated G1 cyclin mRNAs and proteins, S-phase entry, and proliferation without affecting cell survival. Significantly, expression of hop receptor isoform was 24-fold greater than the short isoform at E10.5, a ratio that was reversed at E14.5 when short expression was 15-fold greater and PACAP inhibited mitogenesis. Enhanced hop isoform expression, elicited by in vitro treatment of E10.5 precursors with retinoic acid, correlated with sustained pro-mitogenic action of PACAP beyond the developmental switch. Conversely, depletion of hop receptor using short-hairpin RNA abolished PACAP mitogenic stimulation at E10.5. These observations suggest that PACAP elicits temporally specific effects on cortical proliferation via developmentally regulated expression of specific receptor isoforms.
Assuntos
Córtex Cerebral/citologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Mitógenos/farmacologia , Neurônios/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Isoformas de RNA/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Fatores Etários , Animais , Bromodesoxiuridina/metabolismo , Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Córtex Cerebral/embriologia , Ventrículos Cerebrais/citologia , Ventrículos Cerebrais/embriologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Marcação In Situ das Extremidades Cortadas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitógenos/deficiência , Mitógenos/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Gravidez , Proteína Quinase C/metabolismo , Piridinas/farmacologia , Pirrolidinonas/farmacologia , Isoformas de RNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Tretinoína/farmacologiaRESUMO
UNLABELLED: Hepatic ischemia and reperfusion injury (IRI), an exogenous antigen-independent local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The immune system and the nervous system maintain extensive communication and mount a variety of integrated responses to danger signals through intricate chemical messengers. This study examined the function and potential therapeutic potential of neuropeptide pituitary adenylate cyclase-activating polypeptides (PACAP) in a murine model of partial liver "warm" ischemia (90 minutes) followed by reperfusion. Liver IRI readily triggered the expression of intrinsic PACAP and its receptors, whereas the hepatocellular damage was exacerbated in PACAP-deficient mice. Conversely, PACAP27, or PACAP38 peptide monotherapy, which elevates intracellular cyclic adenosine monophosphate/protein kinase A (cAMP-PKA) signaling, protected livers from IRI, as evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture. The liver protection rendered by PACAP peptides was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and selectively augmented hepatic interleukin (IL)-10 expression. Strikingly, PKA inhibition readily restored liver damage in otherwise IR-resistant, PACAP-conditioned mice. In vitro, PACAP treatment not only diminished macrophage tumor necrosis factor alpha/IL-6/IL-12 levels in a PKA-dependent manner, but also prevented necrosis and apoptosis in primary mouse hepatocyte cultures. CONCLUSION: Our novel findings document the importance of PACAP-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 manage liver inflammation and IRI in transplant patients.
Assuntos
Fatores Imunológicos/metabolismo , Hepatopatias/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/imunologia , Animais , Apoptose/imunologia , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Homeostase/imunologia , Fatores Imunológicos/imunologia , Hepatopatias/imunologia , Hepatopatias/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
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.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fígado/patologia , Traumatismo por Reperfusão/patologia , Peptídeo Intestinal Vasoativo/química , Animais , Apoptose , Caspase 3/metabolismo , Citometria de Fluxo/métodos , Hepatócitos/citologia , Hepatócitos/metabolismo , Homeostase , Sistema Imunitário , Inflamação , Interleucina-10/metabolismo , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Neutrófilos/citologia , Peroxidase/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: Pathological 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. METHODS: Postmortem 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. RESULTS: Activated 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)ß-1 in microglia, which in healthy controls decreased with myelin maturation in an age-dependent manner. TGFß-1 directly and dose-dependently inhibited the maturation of purified oligodendrocyte progenitors, and pharmacological inhibition of TGFß-1 signaling in vivo reversed the delay in myelin development. Factors secreted from STAT3-deficient astrocytes promoted elevated TGFß-1 production in cultured microglia compared to wild-type astrocytes. INTERPRETATION: These 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 TGFß-1 in microglia and the impairment of oligodendrocyte maturation.
Assuntos
Astrócitos/metabolismo , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Gliose/etiologia , Bainha de Mielina/metabolismo , Fator de Transcrição STAT3/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Astrócitos/química , Astrócitos/efeitos dos fármacos , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Dioxóis/farmacologia , Dioxóis/uso terapêutico , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mudanças Depois da Morte , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Fator de Transcrição STAT3/deficiência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad2/metabolismo , Células-Tronco/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The neuropeptide vasoactive intestinal peptide (VIP) has been shown to inhibit macrophage proinflammatory actions, promote a positive Th2/Th1 balance, and stimulate regulatory T-cell production. The fact that this peptide is highly efficacious in animal models of inflammatory diseases such as collagen-induced arthritis and experimental autoimmune encephalomyelitis (EAE) suggests that the endogenous peptide might normally provide protection against such pathologies. We thus studied the response of VIP-deficient (i.e., VIP KO) mice to myelin oligodendrocyte protein-induced EAE. Surprisingly, VIP KO mice were almost completely resistant to EAE, with delayed onset and mild or absent clinical profile. Despite this, flow cytometric analyses and antigen-rechallenge experiments indicated that myelin oligodendrocyte protein-treated VIP KO mice exhibited robust Th1/Th17 cell inductions and antigen-specific proliferation and cytokine responses. Moreover, adoptive transfer of lymphocytes from immunized VIP KO mice to WT recipients resulted in full-blown EAE, supporting their encephalitogenic potential. In contrast, transfer of encephalitogenic WT cells to VIP KO hosts did not produce EAE, suggesting that loss of VIP specifically affected the effector phase of the disease. Histological analyses indicated that CD4 T cells entered the meningeal and perivascular areas of VIP-deficient mice, but that parenchymal infiltration was strongly impaired. Finally, VIP pretreatment of VIP KO mice before immunization was able to restore their sensitivity to EAE. These results indicate that VIP plays an unanticipated permissive and/or proinflammatory role in the propagation of the inflammatory response in the CNS, a finding with potential therapeutic relevance in autoimmune neuroinflammatory diseases such as multiple sclerosis.
Assuntos
Movimento Celular/imunologia , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T/imunologia , Peptídeo Intestinal Vasoativo/deficiência , Peptídeo Intestinal Vasoativo/imunologia , Animais , Doenças Autoimunes/etiologia , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/etiologia , Inflamação/etiologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Glicoproteína Associada a Mielina/farmacologia , Células Th1 , Células Th17RESUMO
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.