RESUMO
Nicotine dependence and schizophrenia are two mental health disorders with remarkably high comorbidity. Cigarette smoking is particularly prevalent amongst schizophrenic patients and it is hypothesised to comprise a form of self-medication for relieving cognitive deficits in these patients. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin in the modulation of drug addiction, as well as schizophrenia symptomology; however, the underlying mechanism remains unclear. Therefore, we sought to investigate the effects of chronic nicotine administration on oxytocin receptor (OTR) binding in the brain of a transgenic mouse model of schizophrenia that carries a bacterial artificial chromosome of the human G72/G30 locus (G72Tg). Female wild-type (WT) and heterozygous G72 transgenic CD-1 mice were treated with a chronic nicotine regimen (24 mg/kg/day, osmotic minipumps for 14 days) and quantitative autoradiographic mapping of oxytocin receptors was carried out in brains of these animals. OTR binding levels were higher in the cingulate cortex (CgCx), nucleus accumbens (Acb), and central amygdala (CeA) of saline treated G72Tg mice compared to WT control mice. Chronic nicotine administration reversed this upregulation in the CgCx and CeA. Interestingly, chronic nicotine administration induced an increase in OTR binding in the CeA of solely WT mice. These results indicate that nicotine administration normalises the dysregulated central oxytocinergic system of this mouse model of schizophrenia and may contribute towards nicotine's ability to modulate cognitive deficits which are common symptoms of schizophrenia.
Assuntos
Encéfalo/metabolismo , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Nicotina/administração & dosagem , Receptores de Ocitocina/metabolismo , Esquizofrenia/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Transgênicos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Ocitocina/genética , Esquizofrenia/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Cerebral malaria is a severe and often fatal complication of Plasmodium falciparum infection. It is characterized by parasite sequestration, a breakdown of the blood-brain barrier, and a strong inflammation in the brain. We investigated the role of the cannabinoid receptor 2 (CB2), an important modulator of neuroinflammatory responses, in experimental cerebral malaria (ECM). Strikingly, mice with a deletion of the CB2-encoding gene (Cnr2(-/-)) inoculated with Plasmodium berghei ANKA erythrocytes exhibited enhanced survival and a diminished blood-brain barrier disruption. Therapeutic application of a specific CB2 antagonist also conferred increased ECM resistance in wild type mice. Hematopoietic derived immune cells were responsible for the enhanced protection in bone marrow (BM) chimeric Cnr2(-/-) mice. Mixed BM chimeras further revealed that CB2-expressing cells contributed to ECM development. A heterogeneous CD11b(+) cell population, containing macrophages and neutrophils, expanded in the Cnr2(-/-) spleen after infection and expressed macrophage mannose receptors, arginase-1 activity, and IL-10. Also in the Cnr2(-/-) brain, CD11b(+) cells that expressed selected anti-inflammatory markers accumulated, and expression of inflammatory mediators IFN-γ and TNF-α was reduced. Finally, the M2 macrophage chemokine CCL17 was identified as an essential factor for enhanced survival in the absence of CB2, because CCL17 × Cnr2 double-deficient mice were fully susceptible to ECM. Thus, targeting CB2 may be promising for the development of alternative treatment regimes of ECM.
Assuntos
Barreira Hematoencefálica/imunologia , Quimiocina CCL17/imunologia , Malária Cerebral/imunologia , Plasmodium berghei/imunologia , Receptor CB2 de Canabinoide/imunologia , Animais , Arginase/genética , Arginase/imunologia , Barreira Hematoencefálica/parasitologia , Barreira Hematoencefálica/patologia , Quimiocina CCL17/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Interleucina-10/genética , Interleucina-10/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Malária Cerebral/genética , Malária Cerebral/patologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/patologia , Receptor CB2 de Canabinoide/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologiaRESUMO
The CC chemokine ligand 17 (CCL17) and its cognate CC chemokine receptor 4 (CCR4) are known to control leukocyte migration, maintenance of TH17 cells, and regulatory T cell (Treg) expansion in vivo. In this study we characterized the expression and functional role of CCL17 in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). Using a CCL17/EGFP reporter mouse model, we could show that CCL17 expression in the CNS can be found in a subset of classical dendritic cells (DCs) that immigrate into the CNS during the effector phase of MOG-induced EAE. CCL17 deficient (CCL17-/-) mice exhibited an ameliorated disease course upon MOG-immunization, associated with reduced immigration of IL-17 producing CD4+ T cells and peripheral DCs into the CNS. CCL17-/- DCs further showed equivalent MHC class II and costimulatory molecule expression and an equivalent capacity to secrete IL-23 and induce myelin-reactive TH17 cells when compared to wildtype DCs. In contrast, their transmigration in an in vitro model of the blood-brain barrier was markedly impaired. In addition, peripheral Treg cells were enhanced in CCL17-/- mice at peak of disease pointing towards an immunoregulatory function of CCL17 in EAE. Our study identifies CCL17 as a unique modulator of EAE pathogenesis regulating DC trafficking as well as peripheral Treg cell expansion in EAE. Thus, CCL17 operates at distinct levels and on different cell subsets during immune response in EAE, a property harboring therapeutic potential for the treatment of CNS autoimmunity.
Assuntos
Quimiocina CCL17/metabolismo , Células Dendríticas/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Movimento Celular , Quimiocina CCL17/genética , Feminino , Interleucina-23/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/imunologia , Baço/fisiopatologia , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismoRESUMO
Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na(+) current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendritic INaP is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic INaP causes augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy. SIGNIFICANCE STATEMENT: In this paper, we describe a novel mechanism that causes increased dendritic persistent Na(+) current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na(+) currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.
Assuntos
Região CA1 Hipocampal/patologia , Dendritos/fisiologia , Regulação para Baixo/fisiologia , Canais de Sódio/fisiologia , Espermina/metabolismo , Estado Epiléptico/patologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Análise de Variância , Animais , Dendritos/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Agonistas Muscarínicos/toxicidade , Pilocarpina/toxicidade , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Estatísticas não Paramétricas , Estado Epiléptico/induzido quimicamente , Sinaptofisina/metabolismo , Tetrodotoxina/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Dendritic cells (DCs) are pivotal for the development of experimental autoimmune encephalomyelitis (EAE). However, the mechanisms by which they control disease remain to be determined. This study demonstrates that expression of CC chemokine receptor 4 (CCR4) by DCs is required for EAE induction. CCR4(-/-) mice presented enhanced resistance to EAE associated with a reduction in IL-23 and GM-CSF expression in the CNS. Restoring CCR4 on myeloid cells in bone marrow chimeras or intracerebral microinjection of CCR4-competent DCs, but not macrophages, restored EAE in CCR4(-/-) mice, indicating that CCR4(+) DCs are cellular mediators of EAE development. Mechanistically, CCR4(-/-) DCs were less efficient in GM-CSF and IL-23 production and also T(H)-17 maintenance. Intraspinal IL-23 reconstitution restored EAE in CCR4(-/-) mice, whereas intracerebral inoculation using IL-23(-/-) DCs or GM-CSF(-/-) DCs failed to induce disease. Thus, CCR4-dependent GM-CSF production in DCs required for IL-23 release in these cells is a major component in the development of EAE. Our study identified a unique role for CCR4 in regulating DC function in EAE, harboring therapeutic potential for the treatment of CNS autoimmunity by targeting CCR4 on this specific cell type.
Assuntos
Células Dendríticas/citologia , Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-23/metabolismo , Receptores CCR4/fisiologia , Animais , Células da Medula Óssea/citologia , Encefalomielite Autoimune Experimental/metabolismo , Inflamação , Ligantes , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Receptores CCR4/metabolismoRESUMO
The modulation of synaptic plasticity by NMDA receptor (NMDAR)-mediated processes is essential for many forms of learning and memory. Activation of NMDARs by glutamate requires the binding of a coagonist to a regulatory site of the receptor. In many forebrain regions, this coagonist is d-serine. Here, we show that experimental epilepsy in rats is associated with a reduction in the CNS levels of d-serine, which leads to a desaturation of the coagonist binding site of synaptic and extrasynaptic NMDARs. In addition, the subunit composition of synaptic NMDARs changes in chronic epilepsy. The desaturation of NMDARs causes a deficit in hippocampal long-term potentiation, which can be rescued with exogenously supplied d-serine. Importantly, exogenous d-serine improves spatial learning in epileptic animals. These results strongly suggest that d-serine deficiency is important in the amnestic symptoms of temporal lobe epilepsy. Our results point to a possible clinical utility of d-serine to alleviate these disease manifestations.
Assuntos
Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/patologia , Epilepsia do Lobo Temporal/patologia , Agonistas de Aminoácidos Excitatórios/uso terapêutico , Hipocampo/metabolismo , Serina/uso terapêutico , Transmissão Sináptica/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Transtornos Cognitivos/etiologia , D-Aminoácido Oxidase/genética , D-Aminoácido Oxidase/metabolismo , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/complicações , Epilepsia do Lobo Temporal/dietoterapia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Agonistas Muscarínicos/toxicidade , Pilocarpina/toxicidade , Ligação Proteica/efeitos dos fármacos , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Escopolamina/toxicidade , Serina/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Significant progress in elucidating the genetic etiology of anxiety and depression has been made during the last decade through a combination of human and animal studies. In this study, we aimed to discover genetic loci linked with anxiety as well as depression in order to reveal new candidate genes. Therefore, we initially tested the behavioral sensitivity of 543 F2 animals derived from an intercross of C57BL/6J and C3H/HeJ mice in paradigms for anxiety and depression. Next, all animals were genotyped with 269 microsatellite markers with a mean distance of 5.56 cM. Finally, a Quantitative Trait Loci (QTL) analysis was carried out, followed by selection of candidate genes. The QTL analysis revealed several new QTL on chromosome 5 with a common core interval of 19 Mb. We further narrowed this interval by comparative genomics to a region of 15 Mb. A database search and gene prioritization revealed Enoph1 as the most significant candidate gene on the prioritization list for anxiety and also for depression fulfilling our selection criteria. The Enoph1 gene, which is involved in polyamine biosynthesis, is differently expressed in parental strains, which have different brain spermidine levels and show distinct anxiety and depression-related phenotype. Our result suggests a significant role in polyamines in anxiety and depression-related behaviors.
Assuntos
Ansiedade/genética , Depressão/genética , Complexos Multienzimáticos/genética , Monoéster Fosfórico Hidrolases/genética , Estresse Psicológico/genética , Animais , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Cromossomos de Mamíferos/genética , Depressão/metabolismo , Depressão/fisiopatologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/metabolismo , Fenótipo , Monoéster Fosfórico Hidrolases/metabolismo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Especificidade da Espécie , Espermidina/metabolismo , Estresse Psicológico/metabolismo , Estresse Psicológico/fisiopatologiaRESUMO
Genetic studies have linked the evolutionary novel, anthropoid primate-specific gene locus G72/G30 in the etiology of schizophrenia and other psychiatric disorders. However, the function of the protein encoded by this locus, LG72, is currently controversially discussed. Some studies have suggested that LG72 binds to and regulates the activity of the peroxisomal enzyme D-amino-acid-oxidase, while others proposed an alternative role of this protein due to its mitochondrial location in vitro. Studies with transgenic mice expressing LG72 further suggested that high levels of LG72 lead to an impairment of mitochondrial functions with a concomitant increase in reactive oxygen species production. In the present study, we now performed extensive interaction analyses and identified the mitochondrial methionine-R-sulfoxide reductase B2 (MSRB2) as a specific interaction partner of LG72. MSRB2 belongs to the MSR protein family and functions in mitochondrial oxidative stress defense. Based on our results, we propose that LG72 is involved in the regulation of mitochondrial oxidative stress.
Assuntos
Proteínas de Transporte/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Mitocôndrias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas dos Microfilamentos , Ligação ProteicaRESUMO
The regulation of gene expression by light enables the versatile, spatiotemporal manipulation of biological function in bacterial and mammalian cells. Optoribogenetics extends this principle by molecular RNA devices acting on the RNA level whose functions are controlled by the photoinduced interaction of a light-oxygen-voltage photoreceptor with cognate RNA aptamers. Here light-responsive ribozymes, denoted optozymes, which undergo light-dependent self-cleavage and thereby control gene expression are described. This approach transcends existing aptamer-ribozyme chimera strategies that predominantly rely on aptamers binding to small molecules. The optozyme method thus stands to enable the graded, non-invasive, and spatiotemporally resolved control of gene expression. Optozymes are found efficient in bacteria and mammalian cells and usher in hitherto inaccessible optoribogenetic modalities with broad applicability in synthetic and systems biology.
Assuntos
RNA Catalítico , RNA , Animais , Motivos de Nucleotídeos , RNA/genética , RNA Catalítico/química , RNA Catalítico/genética , RNA Catalítico/metabolismo , Bactérias/metabolismo , Expressão Gênica , Mamíferos/metabolismoRESUMO
The CRISPR/Cas technology has been successfully used to stimulate the integration of small DNA sequences in a target locus to produce gene mutations. However, many applications require homologous recombination using large gene-targeting constructs. Here we address the potential of CRISPR/Cas-mediated double-strand breaks to enhance the genetic engineering of large target sequences using a construct for "humanizing" the mouse Cnr2 gene locus. We designed a small-guide RNA that directs the induction of double strand breaks by Cas9 in the Cnr2 coding exon. By co-transfection of the CRISPR/Cas system with the 10 kb targeting construct we were able to boost the recombination frequency more than 200-fold from 0.27% to 67%. This simple technology can thus be used for the homologous integration of large gene fragments and should greatly enhance our ability to generate any kind of genetically altered mouse models.
Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Engenharia Genética/métodos , Receptor CB2 de Canabinoide/genética , Recombinação Genética/genética , Animais , Sequência de Bases , Éxons , Loci Gênicos , Humanos , Camundongos , Dados de Sequência Molecular , RNA/genética , TransfecçãoRESUMO
The primate-specific G72/G30 gene locus has been associated with major psychiatric disorders, such as schizophrenia and bipolar disorder. We have previously generated transgenic mice which carry the G72/G30 locus and express the longest G72 splice variant (LG72) protein encoded by this locus with schizophrenia-related symptoms. Here, we used a multi-omics approach, including quantitative proteomics and metabolomics to investigate molecular alterations in the hippocampus of G72/G30 transgenic (G72Tg) mice. Our proteomics analysis revealed decreased expression of myelin-related proteins and NAD-dependent protein deacetylase sirtuin-2 (Sirt2) as well as increased expression of the scaffolding presynaptic proteins bassoon (Bsn) and piccolo (Pclo) and the cytoskeletal protein plectin (Plec1) in G72Tg compared to wild-type (WT) mice. Metabolomics analysis indicated decreased levels of nicotinate in G72Tg compared to WT hippocampi. Decreased hippocampal protein expression for selected proteins, namely myelin oligodentrocyte glycoprotein (Mog), Cldn11 and myelin proteolipid protein (Plp), was confirmed with Western blot in a larger population of G72Tg and WT mice. The identified molecular pathway alterations shed light on the hippocampal function of LG72 protein in the context of neuropsychiatric phenotypes.
RESUMO
Peroxisomes are organelles with main functions in the metabolism of lipids and of reactive oxygen species. Within the testis, they have different functional profiles depending on the cell types. A dysfunction of peroxisomes interferes with regular spermatogenesis and can lead to infertility due to spermatogenic arrest. However, so far only very little is known about the functions of peroxisomes in germ cells. We have therefore analyzed the peroxisomal compartment in germ cells and its alterations during spermatogenesis by fluorescence and electron microscopy as well as by expression profiling of peroxisome-related genes in purified cell populations isolated from mouse testis. We could show that peroxisomes are present in all germ cells of the germinal epithelium. During late spermiogenesis, the peroxisomes form large clusters that are segregated from the spermatozoa into the residual bodies upon release from the germinal epithelium. Germ cells express genes for proteins involved in numerous metabolic pathways of peroxisomes. Based on the expression profile, we conclude that newly identified functions of germ cell peroxisomes are the synthesis of plasmalogens as well as the metabolism of retinoids, polyunsaturated fatty acids and polyamines. Thus, germ cell peroxisomes are involved in the regulation of the homeostasis of signaling molecules regulating spermatogenesis and they contribute to the protection of germ cells against oxidative stress.
Assuntos
Células Germinativas/metabolismo , Estresse Oxidativo , Peroxissomos/genética , Espermatogênese , Testículo/citologia , Animais , Diferenciação Celular , Perfilação da Expressão Gênica , Células Germinativas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Estresse Oxidativo/genética , Peroxissomos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatogênese/genética , Testículo/metabolismoRESUMO
Combined application of DNA recombinases Cre and FLP enables tightly controlled independent and/or sequential gene regulations. However, in practice, such dual recombinase strategies are hampered by the comparably low efficiency of the FLP recombinase. Here, we present the engineering of a recombinant cell-permeant FLP protein (TAT-FLP) that induces recombination in >75% of fibroblasts and mouse as well as human embryonic stem (ES) cells. We show that TAT-FLP ideally complements the strength of cell-permeant Cre recombinase for genetic engineering as exemplified by FLP-ON-Cre-OFF, an inducible transgene expression cassette that enables tightly controlled expression in a reversible manner. We exemplify this concept by conditional overexpression of LacZ and the caudal-related homeobox transcription factor CDX2. We expect our FLP transduction system to become widely useful for numerous genetic interventions addressing complex biological questions and the generation of transgene-free therapeutically applicable ES cell-derived cells.
Assuntos
Permeabilidade da Membrana Celular , DNA Nucleotidiltransferases/genética , Células-Tronco Embrionárias/metabolismo , Produtos do Gene tat/genética , Engenharia Genética/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Animais , Desdiferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Permeabilidade da Membrana Celular/genética , Células-Tronco Embrionárias/citologia , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Transdução Genética/métodosRESUMO
Cannabinoid CB2 receptor (CB2) agonists are potential analgesics void of psychotropic effects. Peripheral immune cells, neurons and glia express CB2; however, the involvement of CB2 from these cells in neuropathic pain remains unresolved. We explored spontaneous neuropathic pain through on-demand self-administration of the selective CB2 agonist JWH133 in wild-type and knockout mice lacking CB2 in neurons, monocytes or constitutively. Operant self-administration reflected drug-taking to alleviate spontaneous pain, nociceptive and affective manifestations. While constitutive deletion of CB2 disrupted JWH133-taking behavior, this behavior was not modified in monocyte-specific CB2 knockouts and was increased in mice defective in neuronal CB2 knockouts suggestive of increased spontaneous pain. Interestingly, CB2-positive lymphocytes infiltrated the injured nerve and possible CB2transfer from immune cells to neurons was found. Lymphocyte CB2depletion also exacerbated JWH133 self-administration and inhibited antinociception. This work identifies a simultaneous activity of neuronal and lymphoid CB2that protects against spontaneous and evoked neuropathic pain.
Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Neuralgia/tratamento farmacológico , Substâncias Protetoras/farmacologia , Receptores de Canabinoides/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Distribuição Aleatória , AutoadministraçãoRESUMO
Neuropathic pain can develop after nerve injury, leading to a chronic condition with spontaneous pain and hyperalgesia. Pain is typically restricted to the side of the injured nerve, but may occasionally spread to the contralateral side, a condition that is often referred to as mirror-image pain. Mechanisms leading to mirror-image pain are not completely understood, but cannabinoid CB2 receptors have been implicated. In this study, we use genetic mouse models to address the question if CB2 receptors on neurons or on microglia/macrophages are involved. First, we show that a GFP reporter protein under control of the CB2 promoter is induced upon partial sciatic nerve ligation in spinal cord, dorsal root ganglia, and highest in sciatic nerve macrophages, but not in neurons. Mice which lack CB2 receptors specifically on myeloid cells (microglia, macrophages) developed a mirror-image allodynia [treatment F1,48 = 45.69, p < 0.0001] similar to constitutive CB2 receptor knockout mice [treatment F1,70 = 92.41, p < 0.0001]. Such a phenotype was not observed after the deletion of CB2 from neurons [treatment F1,70 = 0.1315, p = 0.7180]. This behavioral pain phenotype was accompanied by an increased staining of microglia in the dorsal horn of the spinal cord, as evidenced by an enhanced Iba 1 expression [CB2KO, p = 0.0175; CB2-LysM, p = 0.0425]. Similarly, myeloid-selective knockouts showed an increased expression of the leptin receptor in the injured ipsilateral sciatic nerve, thus further supporting the notion that leptin signaling contributes to the increased neuropathic pain responses of CB2 receptor knockout mice. We conclude that CB2 receptors on microglia and macrophages, but not on neurons, modulate neuropathic pain responses.
Assuntos
Hiperalgesia/metabolismo , Macrófagos/metabolismo , Neuralgia/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Deleção de Genes , Hiperalgesia/fisiopatologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Microglia/fisiologia , Neuralgia/fisiopatologia , Neurônios/metabolismo , Neurônios/fisiologia , Receptor CB2 de Canabinoide/genética , Receptores para Leptina/metabolismoRESUMO
BACKGROUND: It has been shown that the brain regulates bone remodelling through sympathetic and parasympathetic nerve fibres. However, it is unclear if signals from the skeleton also influence brain functions and animal behaviours. METHODS: Bone formation was conditionally disrupted by daily injections of aciclovir (10 mg/kg) to transgenic mice expressing a herpes-simplex-virus thymidine kinase under the control of the osteoblast-specific promoter of the Bglap gene. Behavioural studies were conducted after 10 weeks of treatment. RESULTS: Transgenic mice receiving aciclovir injections showed a reduced number of osteoblasts with a concomitantly reduced trabecular bone volume density, when compared to wild-type controls that were treated identically. The general health of the animals was not severely affected, as indicated by a similar increase in body weight, similar activity profiles and similar social behaviours. However, transgenic mice showed significantly increased despair behaviour and increased adrenal gland weights. CONCLUSIONS: Specific animal behaviours can be modulated by a selective disruption of bone formation. The increased despair behaviour observed in transgenic animals indicates that these animals may be more prone to depression-related phenotypes. These findings are important in the context of the well-established clinical association between depression and reduced bone mass.
Assuntos
Osso e Ossos/fisiologia , Osteogênese/fisiologia , Animais , Diferenciação Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/fisiologia , Osteoblastos/fisiologiaRESUMO
BACKGROUND: Disruption of the endocannabinoid system through pharmacological or genetic invalidation of cannabinoid CB1 receptors has been linked to depression in humans and depression-like behaviors in mice. The two main endogenous cannabinoids, anandamide and 2-arachidonoyl glycerol (2-AG), are produced on demand from phospholipids. The pathways and enzymes involved in endocannabinoid biosynthesis thus play a major role in regulating the activity of this system. This study investigates the role of the main 2-AG producing enzyme diacylglycerol lipase α (DAGL-α). METHODS: We generated and used knockout mice lacking DAGL-α (Dagla(-/-)) to assess the behavioral consequences of reduced endocannabinoid levels in the brain. We performed different behavior tests to determine anxiety- and depression-related behavioral changes in Dagla(-/-) mice. We also analyzed expression of genes related to the endocannabinoid system via real-time polymerase chain reaction and used the mitotic marker 5-bromo-2'-deoxyuridine to analyze adult neurogenesis. RESULTS: Dagla(-/-) animals show an 80% reduction of brain 2-AG levels but also a reduction in cortical and amygdalar anandamide. The behavioral changes induced by Dagla deletion include a reduced exploration of the central area of the open field, a maternal neglect behavior, a fear extinction deficit, increased behavioral despair, increased anxiety-related behaviors in the light/dark box, and reduced hippocampal neurogenesis. Some of these behavioral changes resemble those observed in animals lacking the CB1 receptor. CONCLUSIONS: Our findings demonstrate that the deletion of Dagla adversely affects the emotional state of animals and results in enhanced anxiety, stress, and fear responses.
Assuntos
Ansiedade/metabolismo , Endocanabinoides/metabolismo , Medo/fisiologia , Lipase Lipoproteica/deficiência , Estresse Psicológico/metabolismo , Animais , Encéfalo/metabolismo , Estudos de Coortes , Comportamento Exploratório/fisiologia , Extinção Psicológica/fisiologia , Feminino , Lipase Lipoproteica/genética , Masculino , Comportamento Materno/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Neurogênese/fisiologia , Comportamento SocialRESUMO
Endocannabinoids (eCBs) exert major control over neuronal activity by activating cannabinoid receptors (CBRs). The functionality of the eCB system is primarily ascribed to the well-documented retrograde activation of presynaptic CB1Rs. We find that action potential-driven eCB release leads to a long-lasting membrane potential hyperpolarization in hippocampal principal cells that is independent of CB1Rs. The hyperpolarization, which is specific to CA3 and CA2 pyramidal cells (PCs), depends on the activation of neuronal CB2Rs, as shown by a combined pharmacogenetic and immunohistochemical approach. Upon activation, they modulate the activity of the sodium-bicarbonate co-transporter, leading to a hyperpolarization of the neuron. CB2R activation occurred in a purely self-regulatory manner, robustly altered the input/output function of CA3 PCs, and modulated gamma oscillations in vivo. To conclude, we describe a cell type-specific plasticity mechanism in the hippocampus that provides evidence for the neuronal expression of CB2Rs and emphasizes their importance in basic neuronal transmission.
Assuntos
Endocanabinoides/metabolismo , Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Receptor CB2 de Canabinoide/metabolismo , Sinapses/metabolismo , Potenciais de Ação/fisiologia , Animais , Moduladores de Receptores de Canabinoides/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Camundongos , Células Piramidais/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos Transgênicos , Receptor CB2 de Canabinoide/genética , Animais , Linfócitos B/metabolismo , Encéfalo/metabolismo , Proteínas de Fluorescência Verde/sangue , Proteínas de Fluorescência Verde/genética , Camundongos , Regiões Promotoras Genéticas , Receptor CB2 de Canabinoide/sangue , Receptor CB2 de Canabinoide/metabolismo , Baço/metabolismo , Timo/metabolismoRESUMO
Pxmp2 is the most abundant peroxisomal membrane protein in higher eukaryotes. Its expression is tissue-specific with highest levels of expression in liver, kidney and heart tissue. We have analysed the 5'-flanking genomic region of the murine Pxmp2 gene and we found, that the first exon of the gene encoding the DNA polymerase epsilon (PoleI) was localized adjacent to the first exon of the Pxmp2 gene in head to head orientation. Both genes were separated by only 393 bp containing a CpG island with numerous binding sites for Sp1. A TATA box, however, was lacking. Northern blot analysis revealed that both genes were expressed differently, indicating that their expression was regulated independently. We have analysed the promoter activity of the small genomic fragment separating the Pxmp2 and PoleI genes using luciferase as a reporter molecule in transient transfection assays. The small genomic fragment was a functional promoter, controlling gene expression regardless of its orientation. Promoter activity was 60-70% compared with the activity of the strong CMV promoter. The Pxmp2 and PoleI genes were also linked on the human and rat genome. Furthermore, the sequence of the intergenic fragment was highly conserved among these species. Thus, the small intergenic fragment is probably the common basic element of two independently regulated promoters.