RESUMO
The inhibitory GABAergic system in the brain is involved in the etiology of various psychiatric problems, including autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD) and others. These disorders are influenced not only by genetic but also by environmental factors, such as preterm birth, although the underlying mechanisms are not known. In a translational hyperoxia model, exposing mice pups at P5 to 80% oxygen for 48â h to mimic a steep rise of oxygen exposure caused by preterm birth from in utero into room air, we documented a persistent reduction of cortical mature parvalbumin-expressing interneurons until adulthood. Developmental delay of cortical myelin was observed, together with decreased expression of oligodendroglial glial cell-derived neurotrophic factor (GDNF), a factor involved in interneuronal development. Electrophysiological and morphological properties of remaining interneurons were unaffected. Behavioral deficits were observed for social interaction, learning and attention. These results demonstrate that neonatal oxidative stress can lead to decreased interneuron density and to psychiatric symptoms. The obtained cortical myelin deficit and decreased oligodendroglial GDNF expression indicate that an impaired oligodendroglial-interneuronal interplay contributes to interneuronal damage.
Assuntos
Lesões Encefálicas/metabolismo , Neurônios GABAérgicos/metabolismo , Hiperóxia/metabolismo , Interneurônios/metabolismo , Parvalbuminas/metabolismo , Nascimento Prematuro/metabolismo , Roedores/metabolismo , Animais , Linhagem Celular , Cognição/fisiologia , Modelos Animais de Doenças , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligodendroglia/metabolismo , Comportamento SocialRESUMO
Microglia modulate synaptic refinement in the central nervous system (CNS). We have previously shown that a mouse model with innate high anxiety-related behavior (HAB) displays higher CD68+ microglia density in the key regions of anxiety circuits compared to mice with normal anxiety-related behavior (NAB) in males, and that minocycline treatment attenuated the enhanced anxiety of HAB male. Given that a higher prevalence of anxiety is widely reported in females compared to males, little is known concerning sex differences at the cellular level. Herein, we address this by analyzing microglia heterogeneity and function in the HAB and NAB brains of both sexes. Single-cell RNA sequencing revealed ten distinct microglia clusters varied by their frequency and gene expression profile. We report striking sex differences, especially in the major microglia clusters of HABs, indicating a higher expression of genes associated with phagocytosis and synaptic engulfment in the female compared to the male. On a functional level, we show that female HAB microglia engulfed a greater amount of hippocampal vGLUT1+ excitatory synapses compared to the male. We moreover show that female HAB microglia engulfed more synaptosomes compared to the male HAB in vitro. Due to previously reported effects of minocycline on microglia, we finally administered oral minocycline to HABs of both sexes and showed a significant reduction in the engulfment of synapses by female HAB microglia. In parallel to our microglia-specific findings, we further showed an anxiolytic effect of minocycline on female HABs, which is complementary to our previous findings in the male HABs. Our study, therefore, identifies the altered function of synaptic engulfment by microglia as a potential avenue to target and resolve microglia heterogeneity in mice with innate high anxiety.
Assuntos
Ansiedade , Microglia , Minociclina , Caracteres Sexuais , Animais , Minociclina/farmacologia , Microglia/metabolismo , Microglia/efeitos dos fármacos , Feminino , Ansiedade/metabolismo , Ansiedade/tratamento farmacológico , Masculino , Camundongos , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Modelos Animais de Doenças , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Fagocitose/efeitos dos fármacosRESUMO
Microglia are increasingly recognized to contribute to brain health and disease. Preclinical studies using laboratory rodents are essential to advance our understanding of the physiological and pathophysiological roles of these cells in the central nervous system. Rodents are nocturnal animals, and they are mostly maintained in a defined light-dark cycle within animal facilities, with many laboratories investigating the molecular and functional profiles of microglia exclusively during the animals' light (sleep) phase. However, only a few studies have considered possible differences in microglial functions between the active and sleep phases. Based on initial evidence suggesting that microglial intrinsic clock genes can affect their phenotypes, we sought to investigate differences in transcriptional, proteotype and functional profiles of microglia between light (sleep) and dark (active) phases, and how these changes are affected in pathological models. We found marked transcriptional and proteotype differences between microglia harvested from male mice during the light or dark phase. Amongst others, these differences related to genes and proteins associated with immune responses, motility, and phagocytosis, which were reflected by functional alterations in microglial synaptic pruning and response to bacterial stimuli. Possibly accounting for such changes, we found RNA and protein regulation in SWI/SNF and NuRD chromatin remodeling complexes between light and dark phases. Importantly, we also show that the time of microglial sample collection influences the nature of microglial transcriptomic changes in a model of immune-mediated neurodevelopmental disorders. Our findings emphasize the importance of considering diurnal factors in studying microglial cells and indicate that implementing a circadian perspective is pivotal for advancing our understanding of their physiological and pathophysiological roles in brain health and disease.
Assuntos
Ritmo Circadiano , Microglia , Animais , Microglia/metabolismo , Masculino , Camundongos , Ritmo Circadiano/fisiologia , Camundongos Endogâmicos C57BL , Fotoperíodo , Encéfalo/metabolismo , Adaptação Fisiológica/fisiologia , Sono/fisiologia , LuzRESUMO
Neuroligin-4 (NLGN4) loss-of-function mutations are associated with monogenic heritable autism spectrum disorder (ASD) and cause alterations in both synaptic and behavioral phenotypes. Microglia, the resident CNS macrophages, are implicated in ASD development and progression. Here we studied the impact of NLGN4 loss in a mouse model, focusing on microglia phenotype and function in both male and female mice. NLGN4 depletion caused lower microglia density, less ramified morphology, reduced response to injury and purinergic signaling specifically in the hippocampal CA3 region predominantly in male mice. Proteomic analysis revealed disrupted energy metabolism in male microglia and provided further evidence for sexual dimorphism in the ASD associated microglial phenotype. In addition, we observed impaired gamma oscillations in a sex-dependent manner. Lastly, estradiol application in male NLGN4-/- mice restored the altered microglial phenotype and function. Together, these results indicate that loss of NLGN4 affects not only neuronal network activity, but also changes the microglia state in a sex-dependent manner that could be targeted by estradiol treatment.
Assuntos
Transtorno do Espectro Autista , Masculino , Feminino , Animais , Camundongos , Transtorno do Espectro Autista/genética , Microglia , Camundongos Knockout , Proteômica , Neurônios/fisiologiaRESUMO
Neuroinflammation is discussed to play a role in specific subgroups of different psychiatric disorders, including anxiety disorders. We have previously shown that a mouse model of trait anxiety (HAB) displays enhanced microglial density and phagocytic activity in key regions of anxiety circuits compared to normal-anxiety controls (NAB). Using minocycline, we provided causal evidence that reducing microglial activation within the dentate gyrus (DG) attenuated enhanced anxiety in HABs. Besides pharmacological intervention, "positive environmental stimuli", which have the advantage of exerting no side-effects, have been shown to modulate inflammation-related markers in human beings. Therefore, we now investigated whether environmental enrichment (EE) would be sufficient to modulate upregulated neuroinflammation in high-anxiety HABs. We show for the first time that EE can indeed attenuate enhanced trait anxiety, even when presented as late as adulthood. We further found that EE-induced anxiolysis was associated with the attenuation of enhanced microglial density (using Iba-1 as the marker) in the DG and medial prefrontal cortex. Additionally, EE reduced Iba1 + CD68+ microglia density within the anterior DG. Hence, the successful attenuation of trait anxiety by EE was associated in part with the normalization of neuro-inflammatory imbalances. These results suggest that pharmacological and/or positive behavioral therapies triggering microglia-targeted anti-inflammatory effects could be promising as novel alternatives or complimentary anxiolytic therapeutic approaches in specific subgroups of individuals predisposed to trait anxiety.
Assuntos
Ansiedade , Microglia , Animais , Camundongos , Humanos , Adulto , Ansiedade/tratamento farmacológico , Transtornos de Ansiedade , Modelos Animais de Doenças , Minociclina/farmacologia , Minociclina/uso terapêutico , HipocampoRESUMO
Microglia are the immune cells of the brain and become activated during any type of brain injury. In the middle cerebral artery occlusion (MCAo) model, a mouse model for ischemic stroke, we have previously shown that microglia and invaded monocytes upregulate the expression of the muscarinic acetylcholine receptor 3 (M3R) in the ischemic lesion. Here we tested whether this upregulation has an impact on the pathogenesis of MCAo. We depleted the m3R receptor in microglia, but not in circulating monocytes by giving tamoxifen to CX3CR1-CreERT+/+M3Rflox/flox (M3RKOmi) animals 3 weeks prior to MCAo. We found that M3RKOmi male mice had bigger lesions, more pronounced motor deficits after one week and cognitive deficits after about one month compared to control males. The density of Iba1+ cells was lower in the lesions of M3RKO male mice in the early, but not in the late disease phase. In females, these differences were not significant. By giving tamoxifen 1 week prior to MCAo, we depleted m3R in microglia and in circulating monocytes (M3RKOmi/mo). Male M3RKOmi/mo did not differ in lesion size, but had a lower survival rate, showed motor deficits and a reduced accumulation of Iba1+ positive cells into the lesion site. In conclusion, our data suggest that the upregulation of m3R in microglia and monocytes in stroke has a beneficial effect on the clinical outcome in male mice.
Assuntos
Isquemia Encefálica , Microglia , Receptor Muscarínico M3/genética , Acidente Vascular Cerebral , Animais , Encéfalo , Modelos Animais de Doenças , Feminino , Infarto da Artéria Cerebral Média , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
As the immune-competent cells of the brain, microglia play an increasingly important role in maintaining normal brain function. They invade the brain early in development, transform into a highly ramified phenotype, and constantly screen their environment. Microglia are activated by any type of pathologic event or change in brain homeostasis. This activation process is highly diverse and depends on the context and type of the stressor or pathology. Microglia can strongly influence the pathologic outcome or response to a stressor due to the release of a plethora of substances, including cytokines, chemokines, and growth factors. They are the professional phagocytes of the brain and help orchestrate the immunological response by interacting with infiltrating immune cells. We describe here the diversity of microglia phenotypes and their responses in health, aging, and disease. We also review the current literature about the impact of lifestyle on microglia responses and discuss treatment options that modulate microglial phenotypes.
Assuntos
Encéfalo/imunologia , Microglia/imunologia , Microglia/fisiologia , Envelhecimento/imunologia , Envelhecimento/fisiologia , Animais , Encéfalo/fisiologia , Humanos , Fagócitos/imunologia , Fagócitos/fisiologiaRESUMO
In recent years schizophrenia has been recognized as a neurodevelopmental disorder likely involving a perinatal insult progressively affecting brain development. The poly I:C maternal immune activation (MIA) rodent model is considered as a neurodevelopmental model of schizophrenia. Using this model we and others demonstrated the association between neuroinflammation in the form of altered microglia and a schizophrenia-like endophenotype. Therapeutic intervention using the anti-inflammatory drug minocycline affected altered microglia activation and was successful in the adult offspring. However, less is known about the effect of preventive therapeutic strategies on microglia properties. Previously we found that deep brain stimulation of the medial prefrontal cortex applied pre-symptomatically to adolescence MIA rats prevented the manifestation of behavioral and structural deficits in adult rats. We here studied the effects of deep brain stimulation during adolescence on microglia properties in adulthood. We found that in the hippocampus and nucleus accumbens, but not in the medial prefrontal cortex, microglial density and soma size were increased in MIA rats. Pro-inflammatory cytokine mRNA was unchanged in all brain areas before and after implantation and stimulation. Stimulation of either the medial prefrontal cortex or the nucleus accumbens normalized microglia density and soma size in main projection areas including the hippocampus and in the area around the electrode implantation. We conclude that in parallel to an alleviation of the symptoms in the rat MIA model, deep brain stimulation has the potential to prevent the neuroinflammatory component in this disease.
Assuntos
Estimulação Encefálica Profunda/métodos , Microglia/efeitos dos fármacos , Animais , Comportamento Animal/fisiologia , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Minociclina/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Poli I-C/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Gravidez , Complicações Infecciosas na Gravidez , Efeitos Tardios da Exposição Pré-Natal/imunologia , Ratos , Ratos Wistar , Esquizofrenia/imunologia , Esquizofrenia/terapiaRESUMO
Cerebral infection with the parasite Toxoplasma gondii is followed by activation of resident cells and recruitment of immune cells from the periphery to the CNS. In this study, we show that a subset of myeloid cells, namely Ly6C(high)CCR2(+) inflammatory monocytes that infiltrate the brain upon chronic T. gondii infection, plays a decisive role in host defense. Depletion of this monocyte subset resulted in elevated parasite load and decreased survival of infected mice, suggesting their crucial role. Notably, Ly6C(high)CCR2(+) monocytes governed parasite control due to production of proinflammatory mediators, such as IL-1α, IL-1ß, IL-6, inducible NO synthase, TNF, and reactive oxygen intermediate. Interestingly, Ly6C(high)CCR2(+) monocytes were also able to produce the regulatory cytokine IL-10, revealing their dual feature. Moreover, we confirmed by adoptive transfer that the recruited monocytes further develop into two distinct subpopulations contributing to parasite control and profound host defense. The differentiated Ly6C(int)CCR2(+)F4/80(int) subset upregulated MHC I and MHC II molecules, suggesting dendritic cell properties such as interaction with T cells, whereas the Ly6C(neg)F4/80(high) cell subset displayed elevated phagocytic capacity while upregulating triggering receptor expressed on myeloid cells-2. Finally, we have shown that the recruitment of Ly6C(high) monocytes to the CNS is regulated by P-selectin glycoprotein ligand-1. These results indicate the critical importance of recruited Ly6C(high) monocytes upon cerebral toxoplasmosis and reveal the behavior of further differentiated myeloid-derived mononuclear cell subsets in parasite control and immune regulation of the CNS.
Assuntos
Antígenos Ly/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Toxoplasmose Cerebral/imunologia , Transferência Adotiva , Animais , Quimiotaxia de Leucócito/imunologia , Doença Crônica , Citocinas/biossíntese , Modelos Animais de Doenças , Feminino , Imunofenotipagem , Glicoproteínas de Membrana/metabolismo , Camundongos , Microglia/imunologia , Microglia/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Fagocitose/imunologia , Fenótipo , Receptores CCR2/metabolismo , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Cerebral/patologiaRESUMO
BACKGROUND: Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. METHODS AND RESULTS: We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3(floxed/KO) and Tie2-Cre(ERT2) mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. CONCLUSIONS: Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.
Assuntos
Endotélio Vascular/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Neovascularização Fisiológica/fisiologia , Plasticidade Neuronal/fisiologia , Fator de Transcrição STAT3/fisiologia , Proteínas ADAM/biossíntese , Proteínas ADAM/genética , Proteína ADAMTS9 , Animais , Axônios/fisiologia , Encéfalo/patologia , Microambiente Celular , Circulação Cerebrovascular , Convalescença , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Infarto da Artéria Cerebral Média/patologia , Camundongos , Camundongos Knockout , Microglia/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Processamento de Proteína Pós-Traducional , Recuperação de Função Fisiológica , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologiaRESUMO
Glioblastoma (GBM) is the most aggressive brain tumor in adults. It is strongly infiltrated by microglia and peripheral monocytes that support tumor growth. In the present study we used RNA sequencing to compare the expression profile of CD11b(+) human glioblastoma-associated microglia/monocytes (hGAMs) to CD11b(+) microglia isolated from non-tumor samples. Hierarchical clustering and principal component analysis showed a clear separation of the two sample groups and we identified 334 significantly regulated genes in hGAMs. In comparison to human control microglia hGAMs upregulated genes associated with mitotic cell cycle, cell migration, cell adhesion, and extracellular matrix organization. We validated the expression of several genes associated with extracellular matrix organization in samples of human control microglia, hGAMs, and the hGAMs-depleted fraction via qPCR. The comparison to murine GAMs (mGAMs) showed that both cell populations share a significant fraction of upregulated transcripts compared with their respective controls. These genes were mostly related to mitotic cell cycle. However, in contrast to murine cells, human GAMs did not upregulate genes associated to immune activation. Comparison of human and murine GAMs expression data to several data sets of in vitro-activated human macrophages and murine microglia showed that, in contrast to mGAMs, hGAMs share a smaller overlap to these data sets in general and in particular to cells activated by proinflammatory stimulation with LPS + INFγ or TNFα. Our findings provide new insights into the biology of human glioblastoma-associated microglia/monocytes and give detailed information about the validity of murine experimental models. GLIA 2016 GLIA 2016;64:1416-1436.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Microglia/metabolismo , Monócitos/metabolismo , Animais , Antígeno CD11b/metabolismo , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , TranscriptomaRESUMO
The capsaicin receptor (TRPV1, transient receptor potential vanilloid type 1) was first discovered in the peripheral nervous system as a detector of noxious chemical and thermal stimuli including the irritant chili pepper. Recently, there has been increasing evidence of TRPV1 expression in the central nervous system. Here, we show that TRPV1 is expressed in neural precursor cells (NPCs) during postnatal development, but not in the adult. However, expression of TRPV1 is induced in the adult in paradigms linked to an increase in neurogenesis, such as spatial learning in the Morris water maze or voluntary exercise. Loss of TRPV1 expression in knockout mice leads to an increase in NPC proliferation. Functional TRPV1 expression has been confirmed in cultured NPCs. Our results indicate that TRPV1 expression influences both postnatal and activity-induced neurogenesis in adulthood.
Assuntos
Capsaicina/metabolismo , Proliferação de Células/fisiologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Neurônios/citologia , Canais de Cátion TRPV/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Células Cultivadas , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/metabolismoRESUMO
Recently, neurotransmitters/neurohormones have been identified as factors controlling the function of microglia, the immune competent cells of the central nervous system. In this study, we compared the responsiveness of microglia to neurotransmitters/neurohormones. We freshly isolated microglia from healthy adult C57Bl/6 mice and found that only a small fraction (1-20%) responded to the application of endothelin, histamine, substance P, serotonin, galanin, somatostatin, angiotensin II, vasopressin, neurotensin, dopamine, or nicotine. In cultured microglia from neonatal and adult mice, a similarly small population of cells responded to these neurotransmitters/neurohormones. To induce a proinflammatory phenotype, we applied lipopolysaccaride (LPS) or interferon-gamma (IFN-γ) to the cultures for 24 h. Several of the responding populations increased; however, there was no uniform pattern when comparing adult with neonatal microglia or LPS with IFN-γ treatment. IL-4 as an anti-inflammatory substance increased the histamine-, substance P-, and somatostatin-sensitive populations only in microglia from adult, but not in neonatal cells. We also found that the expression of different receptors was not strongly correlated, indicating that there are many different populations of microglia with a distinct set of receptors. Our results demonstrate that microglial cells are a heterogeneous population with respect to their sensitivity to neurotransmitters/neurohormones and that they are more responsive in defined activation states.
Assuntos
Interferon gama/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurotransmissores/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The invasiveness of malignant gliomas is one of the major obstacles in glioma therapy and the reason for the poor survival of patients. Glioma cells infiltrate into the brain parenchyma and thereby escape surgical resection. Glioma associated microglia/macrophages support glioma infiltration into the brain parenchyma by increased expression and activation of extracellular matrix degrading proteases such as matrix metalloprotease (MMP) 2, MMP9 and membrane-type 1 MMP. In this work we demonstrate that, MMP9 is predominantly expressed by glioma associated microglia/macrophages in mouse and human glioma tissue but not by the glioma cells. Supernatant from glioma cells induced the expression of MMP9 in cultured microglial cells. Using mice deficient for different Toll-like receptors we identified Toll-like receptor 2/6 as the signaling pathway for the glioma induced upregulation of microglial MMP9. Also in an experimental mouse glioma model, Toll-like receptor 2 deficiency attenuated the upregulation of microglial MMP9. Moreover, glioma supernatant triggered an upregulation of Toll-like receptor 2 expression in microglia. Both, the upregulation of MMP9 and Toll-like receptor 2 were attenuated by the antibiotic minocycline and a p38 mitogen-activated protein kinase antagonist in vitro. Minocycline also extended the survival rate of glioma bearing mice when given to the drinking water. Thus glioma cells change the phenotype of glioma associated microglia/macrophages in a complex fashion using Toll-like receptor 2 as an important signaling pathway and minocycline further proved to be a potential candidate for adjuvant glioma therapy.
Assuntos
Antibacterianos/farmacologia , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Minociclina/farmacologia , Receptor 2 Toll-Like/metabolismo , Animais , Western Blotting , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Modelos Animais de Doenças , Citometria de Fluxo , Imunofluorescência , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Técnicas Imunoenzimáticas , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/genética , Células Tumorais CultivadasRESUMO
We previously reported that glioma cells induce the expression of membrane-type 1 metalloproteinase (MT1-MMP or MMP-14) in tumor-associated microglia/macrophages and promote tumor growth, whereas MMP-14 expression in microglia under physiological conditions is very low. Here, we show that the increase in MMP-14 expression is also found in microglia/macrophages associated with neurodegenerative and neuroinflammatory pathologies in mouse models as well as in human biopsies or post-mortem tissue. We found that microglial/macrophage MMP-14 expression was upregulated in Alzheimer's disease tissue, in active lesions of multiple sclerosis, and in tissue from stage II stroke as well as in the corresponding mouse models for the human diseases. In contrast, we observed no upregulation for MMP-14 in microglia/macrophages in the early phase of stroke or in the corresponding mouse model, in human amyotrophic lateral sclerosis (ALS) tissue or in a mouse model of ALS as well as in human cases of acute brain trauma. These data indicate that MMP-14 expression is not a general marker for activated microglia/macrophages but is upregulated in defined stages of neuroinflammatory and neurodegenerative diseases and that there is generally a good match between mouse models and human brain pathologies.
Assuntos
Encéfalo/patologia , Encefalite/patologia , Macrófagos/enzimologia , Metaloproteinase 14 da Matriz/metabolismo , Microglia/enzimologia , Doenças Neurodegenerativas/patologia , Regulação para Cima/fisiologia , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/complicações , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Encefalite/etiologia , Glioma/complicações , Glioma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos , Doenças Neurodegenerativas/etiologia , Ferimentos Perfurantes/complicações , Ferimentos Perfurantes/patologiaRESUMO
Adult neurogenesis in the hippocampus is impaired in schizophrenic patients and in an animal model of schizophrenia. Amongst a plethora of regulators, the immune system has been shown repeatedly to strongly modulate neurogenesis under physiological and pathological conditions. It is well accepted, that schizophrenic patients have an aberrant peripheral immune status, which is also reflected in the animal model. The microglia as the intrinsic immune competent cells of the brain have recently come into focus as possible therapeutic targets in schizophrenia. We here used a maternal immune stimulation rodent model of schizophrenia in which polyinosinic-polycytidilic acid (Poly I:C) was injected into pregnant rats to mimic an anti-viral immune response. We identified microglia IL-1ß and TNF-α increase constituting the factors correlating best with decreases in net-neurogenesis and impairment in pre-pulse inhibition of a startle response in the Poly I:C model. Treatment with the antibiotic minocycline (3mg/kg/day) normalized microglial cytokine production in the hippocampus and rescued neurogenesis and behavior. We could also show that enhanced microglial TNF-α and IL-1ß production in the hippocampus was accompanied by a decrease in the pro-proliferative TNFR2 receptor expression on neuronal progenitor cells, which could be attenuated by minocycline. These findings strongly support the idea to use anti-inflammatory drugs to target microglia activation as an adjunctive therapy in schizophrenic patients.
Assuntos
Antibacterianos/farmacologia , Microglia/efeitos dos fármacos , Minociclina/farmacologia , Neurogênese/efeitos dos fármacos , Esquizofrenia/fisiopatologia , Filtro Sensorial/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Citocinas/biossíntese , Modelos Animais de Doenças , Masculino , Microglia/imunologia , Poli I-C/farmacologia , Ratos , Ratos Wistar , Esquizofrenia/imunologia , Esquizofrenia/metabolismoRESUMO
Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry. In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.
Assuntos
Citometria de Fluxo , Microglia , Sinapses , Animais , Microglia/citologia , Microglia/metabolismo , Camundongos , Sinapses/fisiologia , Sinapses/química , Citometria de Fluxo/métodos , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/análise , Sinaptossomos/metabolismo , Encéfalo/citologiaRESUMO
High-grade gliomas are the most common primary brain tumors. Their malignancy is promoted by the complex crosstalk between different cell types in the central nervous system. Microglia/brain macrophages infiltrate high-grade gliomas and contribute to their progression. To identify factors that mediate the attraction of microglia/macrophages to malignant brain tumors, we established a glioma cell encapsulation model that was applied in vivo. Mouse GL261 glioma cell line and human high-grade glioma cells were seeded into hollow fibers (HF) that allow the passage of soluble molecules but not cells. The glioma cell containing HF were implanted into one brain hemisphere and simultaneously HF with non-transformed fibroblasts (controls) were introduced into the contralateral hemisphere. Implanted mouse and human glioma- but not fibroblast-containing HF attracted microglia and up-regulated immunoreactivity for GFAP, which is a marker of astrogliosis. In this study, we identified GDNF as an important factor for microglial attraction: (1) GL261 and human glioma cells secret GDNF, (2) reduced GDNF production by siRNA in GL261 in mouse glioma cells diminished attraction of microglia, (3) over-expression of GDNF in fibroblasts promoted microglia attraction in our HF assay. In vitro migration assays also showed that GDNF is a strong chemoattractant for microglia. While GDNF release from human or mouse glioma had a profound effect on microglial attraction, the glioma-induced astrogliosis was not affected. Finally, we could show that injection of GL261 mouse glioma cells with GDNF knockdown by shRNA into mouse brains resulted in reduced tumor expansion and improved survival as compared to injection of control cells.
Assuntos
Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Glioblastoma/metabolismo , Gliose/metabolismo , Microglia/metabolismo , Animais , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Quimiotaxia , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Glioblastoma/genética , Glioblastoma/patologia , Gliose/genética , Gliose/patologia , Humanos , Camundongos , Microglia/patologia , Transplante de NeoplasiasRESUMO
Microglia are the immune effector cells of the central nervous system (CNS) and react to pathologic events with a complex process including the release of nitric oxide (NO). NO is a free radical, which is toxic for all cells at high concentrations. To target an exaggerated NO release, we tested a library of 16 544 chemical compounds for their effect on lipopolysaccharide (LPS)-induced NO release in cell line and primary neonatal microglia. We identified a compound (C1) which significantly reduced NO release in a dose-dependent manner, with a low IC50 (252 nM) and no toxic side effects in vitro or in vivo. Target finding strategies such as in silico modelling and mass spectroscopy hint towards a direct interaction between C1 and the nitric oxide synthase making C1 a great candidate for specific intra-cellular interaction with the NO producing machinery.
Assuntos
Microglia , Óxido Nítrico , Recém-Nascido , Humanos , Microglia/metabolismo , Óxido Nítrico/metabolismo , Doenças Neuroinflamatórias , Óxido Nítrico Sintase Tipo II/metabolismo , Linhagem Celular , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismoRESUMO
In chronic autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS) clinical signs of cognitive dysfunction have been associated with structural changes in the hippocampus. Moreover, experimental studies indicate that inflammatory responses within the CNS modulate the homeostasis of newborn cells in the adult dentate gyrus (DG). However, it remained open whether such changes happen regardless of the primary immunological target or whether a CNS antigen-directed T lymphocyte-mediated autoimmune response may exert a specific impact. We therefore induced experimental autoimmune encephalomyelitis (EAE), a common model of MS serving as a paradigm for a CNS-specific immune response, by immunizing C57BL/6 mice with encephalitogenic myelin oligodendrocyte glycoprotein (MOG) p35-55. In EAE animals, we found enhanced de novo generation and survival of doublecortin (DCX)-positive immature neurons when compared with controls immunized with CNS-irrelevant antigen (ovalbumine). However, despite activation of neurogenesis, we observed a reduced capacity of these cells to generate mature neurons. Moreover, the high number of newly born cells retained the expression of the glial marker GFAP. These effects were associated with downregulation of pro-neurogenic factors Neurogenin1 and Neurogenin2 and dysregulation of Notch, ß-catenin, Sonic Hedgehog (Shh) signaling as suggested by altered gene expression of effector molecules. Thus, a CNS antigen-specific immune response leads to an aberrant differentiation of neural precursors associated with dysbalance of signaling pathways relevant for adult hippocampal neurogenesis. These results may further extend our understanding of disturbed regeneration in the course of chronic inflammatory CNS diseases such as MS.