RESUMO
BACKGROUND: Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. METHODS: We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45+ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. RESULTS: CD4+ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7+ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)+ regulatory CD4+ T cells. The proportion of central memory CD4+ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4+ T cells into effector CD4+ T cells and that from naive CD4+ T cells into central memory CD4+ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4+ T cells. CONCLUSIONS: For the first time, we revealed single cell-level characteristics of CD4+ T cells in patients with ACS. CD4+ T cells could be therapeutic targets of ACS. REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.
Assuntos
Síndrome Coronariana Aguda , Linfócitos T CD4-Positivos , Placa Aterosclerótica , Análise de Célula Única , Humanos , Síndrome Coronariana Aguda/imunologia , Síndrome Coronariana Aguda/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Masculino , Pessoa de Meia-Idade , Feminino , Idoso , RNA-Seq , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Vasos Coronários/imunologia , Vasos Coronários/patologia , Análise de Sequência de RNA , Doença da Artéria Coronariana/imunologia , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , FenótipoRESUMO
Despite the importance of lipid mediators in stress and depression and their link to inflammation, the influence of stress on these mediators and their role in inflammation is not fully understood. This study used RNA-seq, LC-MS/MS, and flow cytometry analyses in a mouse model subjected to chronic social defeat stress to explore the effects of acute and chronic stress on lipid mediators, gene expression, and cell population in the bone marrow and spleen. In the bone marrow, chronic stress induced a sustained transition from lymphoid to myeloid cells, accompanied by corresponding changes in gene expression. This change was associated with decreased levels of 15-deoxy-d12,14-prostaglandin J2, a lipid mediator that inhibits inflammation. In the spleen, chronic stress also induced a lymphoid-to-myeloid transition, albeit transiently, alongside gene expression changes indicative of extramedullary hematopoiesis. These changes were linked to lower levels of 12-HEPE and resolvins, both critical for inhibiting and resolving inflammation. Our findings highlight the significant role of anti-inflammatory and pro-resolving lipid mediators in the immune responses induced by chronic stress in the bone marrow and spleen. This study paves the way for understanding how these lipid mediators contribute to the immune mechanisms of stress and depression.
Assuntos
Medula Óssea , Baço , Camundongos , Animais , Baço/metabolismo , Medula Óssea/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Inflamação/metabolismo , Lipídeos , Expressão GênicaRESUMO
Microglia are crucial for tissue homeostasis and its disturbance. However, microglial heterogeneity and its relationship with microglial activation in physiological conditions remain elusive. Using single-cell RNA sequencing, we identified microglial subpopulations with distinct transcriptome signatures in the resting brain. The distribution of two major, continuous subpopulations varied across brain regions, especially between cerebral cortices and the hypothalamus. Lipopolysaccharide and chronic social defeat stress, both of which involve the innate immune receptor TLR4, upregulate the marker genes of selective microglial subpopulations. These findings suggest that microglial subpopulations contribute to the heterogeneity of microglial transcriptome and responsiveness within and across brain regions.
Assuntos
Microglia , Transcriptoma , Animais , Camundongos , Microglia/fisiologia , Encéfalo , Lipopolissacarídeos , HomeostaseRESUMO
N-acetylcysteine (NAC) is an antioxidant that prevents tumor necrosis factor (TNF)-α-induced cell death, but it also acts as a pro-oxidant, promoting reactive oxygen species independent apoptosis. Although there is plausible preclinical evidence for the use of NAC in the treatment of psychiatric disorders, deleterious side effects are still of concern. Microglia, key innate immune cells in the brain, play an important role in inflammation in psychiatric disorders. This study aimed to investigate the beneficial and deleterious effects of NAC on microglia and stress-induced behavior abnormalities in mice, and its association with microglial TNF-α and nitric oxide (NO) production. The microglial cell line MG6 was stimulated by Escherichia coli lipopolysaccharide (LPS) using NAC at varying concentrations for 24 h. NAC inhibited LPS-induced TNF-α and NO synthesis, whereas high concentrations (≥30 mM) caused MG6 mortality. Intraperitoneal injections of NAC did not ameliorate stress-induced behavioral abnormalities in mice, but high-doses induced microglial mortality. Furthermore, NAC-induced mortality was alleviated in microglial TNF-α-deficient mice and human primary M2 microglia. Our findings provide ample evidence for the use of NAC as a modulating agent of inflammation in the brain. The risk of side effects from NAC on TNF-α remains unclear and merits further mechanistic investigations.
Assuntos
Acetilcisteína , Inflamação , Microglia , Fator de Necrose Tumoral alfa , Animais , Humanos , Camundongos , Acetilcisteína/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The evolution of mass spectrometry (MS) and analytical techniques has led to the demand for proteome analysis with high proteome coverage in single-shot measurements. Focus has been placed on data-independent acquisition (DIA)-MS and ion mobility spectrometry as techniques for deep proteome analysis. We aimed to expand the proteome coverage by single-shot measurements using optimizing high-field asymmetric waveform ion mobility spectrometry parameters in DIA-MS. With our established proteome analysis system, more than 10,000 protein groups were identified from HEK293 cell digests within 120 min of MS measurement time. Additionally, we applied our approach to the analysis of host proteins in mouse feces and detected as many as 892 host protein groups (771 upregulated/121 downregulated proteins) in a mouse model of repeated social defeat stress (R-SDS) used in studying depression. Interestingly, 285 proteins elevated by R-SDS were related to mental disorders. The fecal host protein profiling by deep proteome analysis may help us understand mental illness pathologies noninvasively. Thus, our approach will be helpful for an in-depth comparison of protein expression levels for biological and medical research because it enables the analysis of highly proteome coverage in a single-shot measurement.
Assuntos
Espectrometria de Mobilidade Iônica , Proteoma , Proteômica , Animais , Fezes/química , Células HEK293 , Humanos , Espectrometria de Massas , Camundongos , Proteoma/análise , Proteômica/métodosRESUMO
Microglia have diverse physiological and pathological functions. However, the transcriptional mechanisms remain elusive. Here we sought new transcription factors relevant to microglial functions from the microglial transcriptome of stressed mice and evaluated their roles in primary microglia. TLR2 and TLR4 agonists increased Rel, Atf3, and Cebpb and decreased Hhex in primary microglia as repeated social defeat stress. Although Hhex was not studied in microglia, TLR2 and TLR4 agonists decreased Hhex, and Hhex overexpression attenuated TLR4-increased expression of inflammation-related genes. These findings suggest that Hhex negatively regulates inflammation-related genes in microglia and that TLR2/4 activation reduces Hhex, facilitating TLR4-mediated neuroinflammation.
Assuntos
Proteínas de Homeodomínio , Microglia , Fatores de Transcrição , Animais , Proteínas de Homeodomínio/metabolismo , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Major depressive disorder (MDD) is associated with repeated exposure to environmental stress. Autophagy is activated under various stress conditions that are associated with several diseases in the brain. This study was aimed at elucidating the autophagy signaling changes in the prefrontal cortex (PFC) under repeated social defeat (RSD) to investigate the involvement of microglial autophagy in RSD-induced behavioral changes. We found that RSD stress, an animal model of MDD, significantly induced initial autophagic signals followed by increased transcription of autophagy-related genes (Atg6, Atg7, and Atg12) in the PFC. Similarly, significantly increased transcripts of ATGs (Atg6, Atg7, Atg12, and Atg5) were confirmed in the postmortem PFC of patients with MDD. The protein levels of the prefrontal cortical LC3B were significantly increased, whereas p62 was significantly decreased in the resilient but not in susceptible mice and patients with MDD. This indicates that enhanced autophagic flux may alleviate stress-induced depression. Furthermore, we identified that FKBP5, an early-stage autophagy regulator, was significantly increased in the PFC of resilient mice at the transcript and protein levels. In addition, the resilient mice exhibited enhanced autophagic flux in the prefrontal cortical microglia, and the autophagic deficiency in microglia aggravated RSD-induced social avoidance, indicating that microglial autophagy involves stress-induced behavioral changes.
Assuntos
Transtorno Depressivo Maior , Microglia , Animais , Autofagia , Transtorno Depressivo Maior/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Derrota Social , Estresse Psicológico/metabolismoRESUMO
The mobilization efficiency of hematopoietic stem/progenitor cells from bone marrow (BM) to circulation by granulocyte colony-stimulating factor (G-CSF) is dramatically dispersed in humans and mice with no mechanistic lead for poor mobilizers. The regulatory mechanism for mobilization efficiency by dietary fat was assessed in mice. Fat-free diet (FFD) for 2 weeks greatly increased mobilization compared to normal diet (ND). The BM mRNA level of peroxisome proliferator-activated receptor δ (PPARδ), a receptor for lipid mediators, was markedly up-regulated by G-CSF in mice fed with ND and displayed strong positive correlation with widely scattered mobilization efficiency. It was hypothesized that BM fat ligand for PPARδ might inhibit mobilization. The PPARδ agonist inhibited mobilization in mice fed with ND and enhanced mobilization by FFD. Treatment with the PPARδ antagonist and chimeric mice with PPARδ+/- BM showed enhanced mobilization. Immunohistochemical staining and flow cytometry revealed that BM PPARδ expression was enhanced by G-CSF mainly in mature/immature neutrophils. BM lipid mediator analysis revealed that G-CSF treatment and FFD resulted in the exhaustion of ω3-polyunsaturated fatty acids such as eicosapentaenoic acid (EPA). EPA induced the up-regulation of genes downstream of PPARδ, such as carnitine palmitoyltransferase-1α and angiopoietin-like protein 4 (Angptl4), in mature/immature neutrophils in vitro and inhibited enhanced mobilization in mice fed with FFD in vivo. Treatment of wild-type mice with the anti-Angptl4 antibody enhanced mobilization together with BM vascular permeability. Collectively, PPARδ signaling in BM mature/immature neutrophils induced by dietary fatty acids negatively regulates mobilization, at least partially, via Angptl4 production.
Assuntos
Medula Óssea , PPAR delta , Animais , Células da Medula Óssea , Fator Estimulador de Colônias de Granulócitos , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Camundongos , PPAR delta/genéticaRESUMO
Autophagy plays an essential role in intracellular degradation and maintenance of cellular homeostasis in all cells, including neurons. Although a recent study reported a copy number variation of Ulk2, a gene essential for initiating autophagy, associated with a case of schizophrenia (SZ), it remains to be studied whether Ulk2 dysfunction could underlie the pathophysiology of the disease. Here we show that Ulk2 heterozygous (Ulk2+/-) mice have upregulated expression of sequestosome-1/p62, an autophagy-associated stress response protein, predominantly in pyramidal neurons of the prefrontal cortex (PFC), and exhibit behavioral deficits associated with the PFC functions, including attenuated sensorimotor gating and impaired cognition. Ulk2+/- neurons showed imbalanced excitatory-inhibitory neurotransmission, due in part to selective down-modulation of gamma-aminobutyric acid (GABA)A receptor surface expression in pyramidal neurons. Genetically reducing p62 gene dosage or suppressing p62 protein levels with an autophagy-inducing agent restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Moreover, expressing a short peptide that specifically interferes with the interaction of p62 and GABAA receptor-associated protein, a protein that regulates endocytic trafficking of GABAA receptors, also restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Thus, the current study reveals a novel mechanism linking deregulated autophagy to functional disturbances of the nervous system relevant to SZ, through regulation of GABAA receptor surface presentation in pyramidal neurons.
Assuntos
Autofagia/genética , Proteínas Serina-Treonina Quinases/genética , Esquizofrenia/genética , Proteína Sequestossoma-1/genética , Animais , Variações do Número de Cópias de DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Peptídeos/genética , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Transporte Proteico/genética , Células Piramidais/metabolismo , Células Piramidais/patologia , Receptores de GABA-A/genética , Esquizofrenia/fisiopatologia , Transmissão Sináptica/genéticaRESUMO
Prolonged or excessive stress may induce emotional and cognitive disturbances, and is a risk factor for mental illnesses. Using rodent chronic stress models of depression, roles of multiple lipid mediators related to inflammation have been revealed in chronic stress-induced emotional alterations. Prostaglandin (PG) E2, an arachidonic acid (AA)-derived lipid mediator, and its receptor subtype EP1 mediate depression-like behavior induced by repeated social defeat stress through attenuating prefrontal dopaminergic activity. Repeated social defeat stress activates microglia through innate immune receptors, and induces PGE2 synthesis through cyclooxygenase-1, a prostaglandin synthase enriched in microglia. PGD2, another AA-derived lipid mediator, has been implicated in depression induced by chronic stress, although either pro-depressive or anti-depressive actions have been reported. Chronic stress up-regulates hippocampal expression of 5-lipoxygenase, hence synthesis of cysteinyl leukotrienes, thereby inducing depression through their receptors. Consistent with beneficial effects of n-3 fatty acids in the diet of depressive patients, resolvins-a novel class of pro-resolving lipid mediators-in the brain attenuate neuroinflammation-associated depression. These findings in animal models of depression offer lipid mediators and related molecules as novel therapeutic targets for treating depression. To translate these findings into clinics, translational biomarkers to visualize lipid mediator profiles in depressive patients need to be established.
Assuntos
Depressão/tratamento farmacológico , Depressão/metabolismo , Dinoprostona/metabolismo , Dinoprostona/uso terapêutico , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/metabolismo , Animais , HumanosRESUMO
Iso-α-acids (IAAs) are hop-derived bitter acids of beer. Epidemiologic studies suggest that moderate alcohol consumption is beneficial for cognitive function, but they do not show the ingredients in alcoholic beverages. Previously, we reported that long-term consumption of IAAs prevents inflammation and Alzheimer pathologies in mice, but their effects on cognitive function have not been evaluated. In the present study, we demonstrated that the consumption of IAAs improves spatial and object recognition memory functions not only in normal Crl:CD1(ICR) male mice but also in mice with pharmacologically induced amnesia. IAA consumption increased the total and extracellular levels of dopamine in the hippocampus of mice and Sprague-Dawley male rats, respectively. Dopamine D1 receptor antagonist treatment and knockdown of dopamine D1 receptor expression in the hippocampus attenuated IAA-induced spatial memory improvement. Furthermore, vagotomy attenuated the effects of IAAs in improving spatial and object recognition memory functions and increasing the total level of dopamine in the hippocampus. These results suggest that the consumption of IAAs activates dopamine D1 receptor-signaling in the hippocampus in a vagus nerve-dependent manner and, consequently, improves spatial and object recognition memory functions. Vagal activation with food components including IAAs may be an easy and safe approach to improve cognitive functions.-Ano, Y., Hoshi, A., Ayabe, T., Ohya, R., Uchida, S., Yamada, K., Kondo, K., Kitaoka, S., Furuyashiki, T. Iso-α-acids, the bitter components of beer, improve hippocampus-dependent memory through vagus nerve activation.
Assuntos
Ácidos/uso terapêutico , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Memória/efeitos dos fármacos , Nervo Vago/efeitos dos fármacos , Animais , Cerveja , Masculino , Memória/fisiologia , Memória Episódica , Camundongos , Camundongos Endogâmicos ICR , Microdiálise , Ratos , Ratos Sprague-Dawley , Nervo Vago/fisiologiaRESUMO
As daily lifestyle is closely associated with mental illnesses, diet-based preventive approaches are receiving attention. Supplementation with hop bitter acids such as iso-α-acids (IAA) and mature hop bitter acids (MHBA) improves mood states in healthy older adults. However, the underlying mechanism remains unknown. Since acute oral consumption with IAA increases dopamine levels in hippocampus and improves memory impairment via vagal nerve activation, here we investigated the effects of chronic administration of hop bitter acids on the dopaminergic activity associated with emotional disturbance in a mouse model of repeated social defeat stress (R-SDS). Chronic administration of IAA and MHBA significantly increased dopaminergic activity based on the dopamine metabolite to dopamine ratio in the hippocampus and medial prefrontal cortex following R-SDS. Hippocampal dopaminergic activity was inversely correlated with the level of R-SDS-induced social avoidance with or without IAA administration. Therefore, chronic treatment with hop bitter acids enhances stress resilience-related hippocampal dopaminergic activity.
Assuntos
Cicloexenos/administração & dosagem , Dopamina/metabolismo , Hipocampo/metabolismo , Humulus/química , Extratos Vegetais/administração & dosagem , Derrota Social , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/metabolismo , Terpenos/administração & dosagem , Sintomas Afetivos/tratamento farmacológico , Animais , Comportamento Animal/efeitos dos fármacos , Cicloexenos/química , Modelos Animais de Doenças , Isomerismo , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Extratos Vegetais/química , Interação Social/efeitos dos fármacos , Terpenos/químicaRESUMO
Granulocyte colony-stimulating factor (G-CSF) is widely used for peripheral blood stem/progenitor mobilization. G-CSF causes low-grade fever that is ameliorated by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting the activation of arachidonic acid (AA) cascade. How G-CSF regulated this reaction was assessed. G-CSF treatment in mice resulted in fever, which was canceled in prostaglandin E synthase (mPGES-1)-deficient mice. Mobilization efficiency was twice as high in chimeric mice lacking mPGES-1, specifically in hematopoietic cells, suggesting that prostaglandin E2 (PGE2) from hematopoietic cells modulated the bone marrow (BM) microenvironment. Neutrophils from steady-state BM constitutively expressed mPGES-1 and significantly enhanced PGE2 production in vitro by ß-adrenergic stimulation, but not by G-CSF, which was inhibited by an NSAID. Although neutrophils expressed all ß-adrenergic receptors, only ß3-agonist induced this phenomenon. Liquid chromatography-tandem mass spectrometry traced ß-agonist-induced PGE2 synthesis from exogenous deuterium-labeled AA. Spontaneous PGE2 production was highly efficient in Gr-1high neutrophils among BM cells from G-CSF-treated mice. In addition to these in vitro data, the in vivo depletion of Gr-1high neutrophils disrupted G-CSF-induced fever. Furthermore, sympathetic denervation eliminated both neutrophil priming for PGE2 production and fever during G-CSF treatment. Thus, sympathetic tone-primed BM neutrophils were identified as one of the major PGE2 producers. PGE2 upregulated osteopontin, specifically in preosteoblasts, to retain progenitors in the BM via EP4 receptor. Thus, the sympathetic nervous system regulated neutrophils as an indispensable PGE2 source to modulate BM microenvironment and body temperature. This study provided a novel mechanistic insight into the communication of the nervous system, BM niche components, and hematopoietic cells.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Dinoprostona/metabolismo , Febre/induzido quimicamente , Fator Estimulador de Colônias de Granulócitos/farmacologia , Neutrófilos/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Febre/genética , Deleção de Genes , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/metabolismo , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Receptores Adrenérgicos beta/metabolismoRESUMO
Stress caused by adverse and demanding conditions, a risk factor for mental illnesses, induces adaptive or maladaptive neural and behavioral consequences, depending on the conditions. Studies using rodent stress models have revealed multiple mechanisms related to dopamine and inflammation for stress-induced neural and behavioral changes. Thus, repeated stress alters activities of ventral tegmental area dopamine neurons projecting to the nucleus accumbens and the medial prefrontal cortex in distinct manners. In the nucleus accumbens, repeated stress decreases activities of D1 receptor-expressing neurons. In the medial prefrontal cortex, single stress increases dopamine D1 receptor signaling, leading to dendritic hypertrophy of excitatory neurons and stress resilience. These changes are attenuated with repetition of stress via prostaglandin E2 , an inflammation-related lipid mediator. Repeated stress activates microglia in the medial prefrontal cortex and the hippocampus. Innate immune receptors, such as the toll-like receptor 2/4 and P2X7, are crucial for repeated stress-induced microglial activation, leading to neural and behavioral changes through proinflammatory cytokines. In addition, repeated stress induces monocyte infiltration to the brain, and impairs the blood-brain barrier in the nucleus accumbens, leading to cytokine leakage to the brain. These monocyte-derived responses are involved in stress-induced behavioral changes. These findings show crucial roles of the accumbal and prefrontal dopamine pathways and inflammatory responses in the brain and body to direct adaptive and maladaptive consequences of stress, and pave the way for identifying a neural origin of stress and understanding the stress-related pathology of mental illnesses.
Assuntos
Adaptação Psicológica , Dopamina , Inflamação/patologia , Estresse Psicológico/patologia , Estresse Psicológico/psicologia , Animais , Humanos , Receptores DopaminérgicosRESUMO
Involvement of reactive oxygen species (ROS) has been suggested in the development of psychiatric disorders. NOX1 is a nonphagocytic form of NADPH oxidase whose expression in the nervous system is negligible compared with other NOX isoforms. However, NOX1-derived ROS increase inflammatory pain and tolerance to opioid analgesia. To clarify the role of NOX1 in the brain, we examined depressive-like behaviors in mice deficient in Nox1 (Nox1-/Y). Depressive-like behaviors induced by chronic social defeat stress or administration of corticosterone (CORT) were significantly ameliorated in Nox1-/Y Generation of ROS was significantly elevated in the prefrontal cortex (PFC) of mice administrated with CORT, while NOX1 mRNA was upregulated only in the ventral tegmental area (VTA) among brain areas responsible for emotional behaviors. Delivery of miRNA against NOX1 to VTA restored CORT-induced depressive-like behaviors in wild-type (WT) littermates. Administration of CORT to WT, but not to Nox1-/Y, significantly reduced transcript levels of brain-derived neurotrophic factor (bdnf), with a concomitant increase in DNA methylation of the promoter regions in bdnf Delivery of miRNA against NOX1 to VTA restored the level of BDNF mRNA in WT PFC. Redox proteome analyses demonstrated that NMDA receptor 1 (NR1) was among the molecules redox regulated by NOX1. In cultured cortical neurons, hydrogen peroxide significantly suppressed NMDA-induced upregulation of BDNF transcripts in NR1-expressing cells but not in cells harboring mutant NR1 (C744A). Together, these findings suggest a key role of NOX1 in depressive-like behaviors through NR1-mediated epigenetic modification of bdnf in the mesoprefrontal projection.SIGNIFICANCE STATEMENT NADPH oxidase is a source of reactive oxygen species (ROS) that have been implicated in the pathogenesis of various neurological disorders. We presently showed the involvement of a nonphagocytic type of NADPH oxidase, NOX1, in major depressive disorders, including behavioral, biochemical, and anatomical changes in mice. The oxidation of NR1 by NOX1-derived ROS was demonstrated in prefrontal cortex (PFC), which may be causally linked to the downregulation of BDNF, promoting depressive-like behaviors. Given that NOX1 is upregulated only in VTA but not in PFC, mesocortical projections appear to play a crucial role in NOX1-dependent depressive-like behaviors. Our study is the first to present the potential molecular mechanism underlying the development of major depression through the NOX1-induced oxidation of NR1 and epigenetic modification of bdnf.
Assuntos
Transtorno Depressivo/metabolismo , NADH NADPH Oxirredutases/deficiência , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Transtorno Depressivo/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , NADPH Oxidases/deficiência , Proteínas do Tecido Nervoso/genética , Oxirredução , Córtex Pré-Frontal , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Azole antifungals directly inhibit enzymes for ergosterol biosynthesis, and this direct action is thought to underlie antifungal actions of these drugs. Recent studies showed that azoles alter expression of genes for various cellular functions. However, transcription factors regulated by azoles and their roles in antifungal actions remain poorly characterized. Using luciferase assay, we found that miconazole increased luciferase activity under the promoter containing the cAMP response element (CRE) motif. This azole-induced activation of CRE reporter was abolished in Atf1-deficient cells, suggesting that azoles induce Atf1 activation. As Atf1 is activated by stress-activated MAP kinase Sty1 upon various stressors, we examined its involvement. Azoles increased phosphorylation of Sty1 for its activation, and Sty1 deletion impaired azole-induced CRE reporter activation. In contrast, deletion of Pyp1, a tyrosine phosphatase which negatively regulates Sty1, increased CRE reporter activation. In addition, cells deficient in Atf1 and stress-activated MAP kinase pathway showed resistance to azoles, whereas cells lacking Pyp1 increased azole susceptibility, suggesting a critical role for azole-induced activation of MAP kinase-Atf1 pathway in antifungal actions of azoles. Collectively, these results suggest that azoles activate stress-activated MAP kinase pathway, thereby facilitating Atf1-mediated transcription for antifungal effects.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Fatores Ativadores da Transcrição/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de SinaisRESUMO
Prostaglandin E(2) (PGE(2)) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE(2) signaling remains unclear. Here, we identified EP4 as a receptor activated by PGE(2) to regulate HSPCs. Knockdown of Ep4 in HSPCs reduced long-term reconstitution capacity, whereas an EP4-selective agonist induced phosphorylation of GSK3ß and ß-catenin and enhanced long-term reconstitution capacity. Next, we analyzed the niche-mediated effect of PGE(2) in HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation of MPCs with PGE(2) significantly increased their ability to support HSPC colony formation. Among the EP receptor agonists, only an EP4 agonist facilitated the formation of HSPC colonies after the coculture with MPCs. PGE(2) up-regulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE(2)/EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE(2) or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil (5-FU)-induced myelosuppression, indicating a role for PGE(2)/EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE(2)-mediated direct and indirect regulation of HSPCs.
Assuntos
Dinoprostona/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Receptores de Prostaglandina E Subtipo EP4/fisiologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Células Cultivadas , Dinoprostona/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/farmacologia , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP2/fisiologia , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores de Prostaglandina E Subtipo EP4/metabolismoRESUMO
Receptor-activator of NF-kappaB ligand (TNFSF11, also known as RANKL, OPGL, TRANCE and ODF) and its tumour necrosis factor (TNF)-family receptor RANK are essential regulators of bone remodelling, lymph node organogenesis and formation of a lactating mammary gland. RANKL and RANK are also expressed in the central nervous system. However, the functional relevance of RANKL/RANK in the brain was entirely unknown. Here we report that RANKL and RANK have an essential role in the brain. In both mice and rats, central RANKL injections trigger severe fever. Using tissue-specific Nestin-Cre and GFAP-Cre rank(floxed) deleter mice, the function of RANK in the fever response was genetically mapped to astrocytes. Importantly, Nestin-Cre and GFAP-Cre rank(floxed) deleter mice are resistant to lipopolysaccharide-induced fever as well as fever in response to the key inflammatory cytokines IL-1beta and TNFalpha. Mechanistically, RANKL activates brain regions involved in thermoregulation and induces fever via the COX2-PGE(2)/EP3R pathway. Moreover, female Nestin-Cre and GFAP-Cre rank(floxed) mice exhibit increased basal body temperatures, suggesting that RANKL and RANK control thermoregulation during normal female physiology. We also show that two children with RANK mutations exhibit impaired fever during pneumonia. These data identify an entirely novel and unexpected function for the key osteoclast differentiation factors RANKL/RANK in female thermoregulation and the central fever response in inflammation.
Assuntos
Regulação da Temperatura Corporal/efeitos dos fármacos , Regulação da Temperatura Corporal/fisiologia , Febre/induzido quimicamente , Febre/metabolismo , Ligante RANK/farmacologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Caracteres Sexuais , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Criança , Dinoprostona/metabolismo , Feminino , Febre/complicações , Perfilação da Expressão Gênica , Humanos , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/complicações , Pneumonia/metabolismo , Ligante RANK/administração & dosagem , Ligante RANK/antagonistas & inibidores , Ligante RANK/metabolismo , Ratos , Ratos Wistar , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP3RESUMO
mDia is an actin nucleator and polymerization factor regulated by the small GTPase Rho and consists of three isoforms. Here, we found that mice lacking mDia1 and mDia3, two isoforms expressed in the brain, in combination (mDia-DKO mice) show impaired left-right limb coordination during locomotion and aberrant midline crossing of axons of corticospinal neurons and spinal cord interneurons. Given that mice lacking Ephrin-B3-EphA4 signaling show a similar impairment in locomotion, we examined whether mDia is involved in Ephrin-B3-EphA4 signaling for axon repulsion. In primary cultured neurons, mDia deficiency impairs growth cone collapse and axon retraction induced by chemo-repellants including EphA ligands. In mDia-DKO mice, the Ephrin-B3-expressing midline structure in the spinal cord is disrupted, and axons aberrantly cross the spinal cord midline preferentially through the region devoid of Ephrin-B3. Therefore, mDia plays multiple roles in the proper formation of the neural network in vivo.