RESUMO
Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. It is a primary astrocyte disease with a pathological hallmark of Rosenthal fibres within astrocytes. AxD astrocytes show several abnormal phenotypes. Our previous study showed that AxD astrocytes in model mice exhibit aberrant Ca2+ signals that induce AxD aetiology. Here, we show that microglia have unique phenotypes with morphological and functional alterations, which are related to the pathogenesis of AxD. Immunohistochemical studies of 60TM mice (AxD model) showed that AxD microglia exhibited highly ramified morphology. Functional changes in microglia were assessed by Ca2+ imaging using hippocampal brain slices from Iba1-GCaMP6-60TM mice and two-photon microscopy. We found that AxD microglia showed aberrant Ca2+ signals, with high frequency Ca2+ signals in both the processes and cell bodies. These microglial Ca2+ signals were inhibited by pharmacological blockade or genetic knockdown of P2Y12 receptors but not by tetrodotoxin, indicating that these signals are independent of neuronal activity but dependent on extracellular ATP from non-neuronal cells. Our single-cell RNA sequencing data showed that the expression level of Entpd2, an astrocyte-specific gene encoding the ATP-degrading enzyme NTPDase2, was lower in AxD astrocytes than in wild-type astrocytes. In situ ATP imaging using the adeno-associated virus vector GfaABC1D ATP1.0 showed that exogenously applied ATP was present longer in 60TM mice than in wild-type mice. Thus, the increased ATP level caused by the decrease in its metabolizing enzyme in astrocytes could be responsible for the enhancement of microglial Ca2+ signals. To determine whether these P2Y12 receptor-mediated Ca2+ signals in AxD microglia play a significant role in the pathological mechanism, a P2Y12 receptor antagonist, clopidogrel, was administered. Clopidogrel significantly exacerbated pathological markers in AxD model mice and attenuated the morphological features of microglia, suggesting that microglia play a protective role against AxD pathology via P2Y12 receptors. Taken together, we demonstrated that microglia sense AxD astrocyte dysfunction via P2Y12 receptors as an increase in extracellular ATP and alter their morphology and Ca2+ signalling, thereby protecting against AxD pathology. Although AxD is a primary astrocyte disease, our study may facilitate understanding of the role of microglia as a disease modifier, which may contribute to the clinical diversity of AxD.
Assuntos
Doença de Alexander , Camundongos , Animais , Doença de Alexander/metabolismo , Doença de Alexander/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Astrócitos/metabolismo , Microglia/metabolismo , Clopidogrel/metabolismo , Cálcio/metabolismo , Progressão da Doença , Trifosfato de Adenosina/metabolismoRESUMO
Microglia are the specialized macrophages of the central nervous system and play an important role in neural circuit development, modulating neurotransmission, and maintaining brain homeostasis. Microglia in normal brain is quiescent and show ramified morphology with numerous branching processes. They constantly survey their surrounding microenvironment through the extension and retraction of their processes and interact with neurons, astrocytes, and blood vessels using these processes. Microglia respond quickly to any pathological event in the brain by assuming ameboid morphology devoid of branching processes and restore homeostasis. However, when there is chronic inflammation, microglia may lose their homeostatic functions and secrete various proinflammatory cytokines and mediators that initiate neural dysfunction and neurodegeneration. In this article, we review the role of microglia in the normal brain and in various pathological brain conditions, such as Alzheimer's disease and multiple sclerosis. We describe strategies to manipulate microglia, focusing on depletion, repopulation, and replacement, and we discuss their therapeutic potential.
Assuntos
Encefalopatias , Microglia , Humanos , Microglia/patologia , Encéfalo/fisiologia , Sistema Nervoso Central , Macrófagos , Encefalopatias/patologiaRESUMO
Peripheral infection induces inflammation in peripheral tissues and the brain, impacting brain function. Glial cells are key players in this process. However, the effects of peripheral infection on glial activation and brain function remain unknown. Here, we showed that varying degrees of peripheral infection had different effects on the regulation of brain functions by microglia-dependent and -independent mechanisms. Acute mild infection (one-day LPS challenge: 1LPS) exacerbated middle cerebral artery occlusion (MCAO) injury, and severe infection (four-day LPS challenge: 4LPS) for one week suppressed it. MCAO injury was assessed by triphenyltetrazolium chloride staining. We observed early activation of microglia in the 1LPS and 4LPS groups. Depleting microglia with a colony-stimulating factor-1 receptor (CSF1R) antagonist had no effect on 1LPS-induced brain injury exacerbation but abolished 4LPS-induced protection, indicating microglial independence and dependence, respectively. Microglia-independent exacerbation caused by 1LPS involved peripheral immune cells including macrophages. RNA sequencing analysis of 4LPS-treated microglia revealed increased factors related to anti-inflammatory and neuronal tissue repair, suggesting their association with the protective effect. In conclusion, varying degrees of peripheral inflammation had contradictory effects (exacerbation vs. protection) on MCAO, which may be attributed to microglial dependence. Our findings highlight the significant impact of peripheral infection on brain function, particularly in relation to glial cells.
Assuntos
Lipopolissacarídeos , Microglia , Camundongos , Animais , Lipopolissacarídeos/toxicidade , Macrófagos , Encéfalo , Infarto da Artéria Cerebral Média , InflamaçãoRESUMO
Microglia are the resident immune cells of the brain, and play essential roles in neuronal development, homeostatic function, and neurodegenerative disease. Human microglia are relatively different from mouse microglia. However, most research on human microglia is performed in vitro, which does not accurately represent microglia characteristics under in vivo conditions. To elucidate the in vivo characteristics of human microglia, methods have been developed to generate and transplant induced pluripotent or embryonic stem cell-derived human microglia into neonatal or adult mouse brains. However, its widespread use remains limited by the technical difficulties of generating human microglia, as well as the need to use immune-deficient mice and conduct invasive surgeries. To address these issues, we developed a simplified method to generate induced pluripotent stem cell-derived human microglia and transplant them into the brain via a transnasal route in immunocompetent mice, in combination with a colony stimulating factor 1 receptor antagonist. We found that human microglia were able to migrate through the cribriform plate to different regions of the brain, proliferate, and become the dominant microglia in a region-specific manner by occupying the vacant niche when exogenous human cytokine is administered, for at least 60 days.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Transplante de Células-Tronco , Animais , Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Humanos , Camundongos , Microglia , Nariz , Transplante de Células-Tronco/métodosRESUMO
Metabotropic glutamate receptor 5 (mGluR5) in astrocytes is a key molecule for controlling synapse remodeling. Although mGluR5 is abundant in neonatal astrocytes, its level is gradually down-regulated during development and is almost absent in the adult. However, in several pathological conditions, mGluR5 re-emerges in adult astrocytes and contributes to disease pathogenesis by forming uncontrolled synapses. Thus, controlling mGluR5 expression in astrocyte is critical for several diseases, but the mechanism that regulates mGluR5 expression remains unknown. Here, we show that adenosine triphosphate (ATP)/adenosine-mediated signals down-regulate mGluR5 in astrocytes. First, in situ Ca2+ imaging of astrocytes in acute cerebral slices from post-natal day (P)7-P28 mice showed that Ca2+ responses evoked by (S)-3,5-dihydroxyphenylglycine (DHPG), a mGluR5 agonist, decreased during development, whereas those evoked by ATP or its metabolite, adenosine, increased. Second, ATP and adenosine suppressed expression of the mGluR5 gene, Grm5, in cultured astrocytes. Third, the decrease in the DHPG-evoked Ca2+ responses was associated with down-regulation of Grm5. Interestingly, among several adenosine (P1) receptor and ATP (P2) receptor genes, only the adenosine A2B receptor gene, Adora2b, was up-regulated in the course of development. Indeed, we observed that down-regulation of Grm5 was suppressed in Adora2b knockout astrocytes at P14 and in situ Ca2+ imaging from Adora2b knockout mice indicated that the A2B receptor inhibits mGluR5 expression in astrocytes. Furthermore, deletion of A2B receptor increased the number of excitatory synapse in developmental stage. Taken together, the A2B receptor is critical for down-regulation of mGluR5 in astrocytes, which would contribute to terminate excess synaptogenesis during development.
Assuntos
Astrócitos , Receptor A2B de Adenosina , Receptor de Glutamato Metabotrópico 5 , Adenosina/metabolismo , Adenosina/farmacologia , Animais , Astrócitos/metabolismo , Proteínas de Transporte/metabolismo , Camundongos , Receptor A2B de Adenosina/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismoRESUMO
We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs ('dual-acting lytic inhibitors') that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted 'Trp3') derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Lectinas/metabolismo , Calorimetria , Epitopos/genética , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Glicosilação , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
We previously reported a first-generation recombinant DAVEI construct, a dual action virus entry inhibitor composed of cyanovirin-N (CVN) fused to a membrane proximal external region or its derivative peptide Trp3. DAVEI exhibits potent and irreversible inactivation of HIV-1 (human immunodeficiency virus) viruses by dual engagement of gp120 and gp41. However, the promiscuity of CVN to associate with multiple glycosylation sites in gp120 and its multivalency limit current understanding of the molecular arrangement of the DAVEI molecules on trimeric spike. Here, we constructed and investigated the virolytic function of second-generation DAVEI molecules using a simpler lectin, microvirin (MVN). MVN is a monovalent lectin with a single glycan-binding site in gp120, is structurally similar to CVN and exhibits no toxicity or mitogenicity, both of which are liabilities with CVN. We found that, like CVN-DAVEI-L2-3Trp (peptide sequence DKWASLWNW), MVN-DAVEI2-3Trp exploits a similar mechanism of action for inducing HIV-1 lytic inactivation, but by more selective gp120 glycan engagement. By sequence redesign, we significantly increased the potency of MVN-DAVEI2-3Trp protein. Unlike CVN-DAVEI2-3Trp, re-engineered MVN-DAVEI2-3Trp(Q81K/M83R) virolytic activity and its interaction with gp120 were both competed by 2G12 antibody. That the lectin domain in DAVEIs can utilize MVN without loss of virolytic function argues that restricted HIV-1 Env (envelope glycoprotein) glycan engagement is sufficient for virolysis. It also shows that DAVEI lectin multivalent binding with gp120 is not required for virolysis. MVN-DAVEI2-3Trp(Q81K/M83R) provides an improved tool to elucidate productive molecular arrangements of Env-DAVEI enabling virolysis and also opens the way to form DAVEI fusions made up of gp120-binding small molecules linked to Trp3 peptide.
Assuntos
Fármacos Anti-HIV , Proteína gp120 do Envelope de HIV/metabolismo , Lectinas , Polissacarídeos/metabolismo , Proteínas Recombinantes de Fusão , Inativação de Vírus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Antivirais/química , Antivirais/metabolismo , Células HEK293 , Proteína gp120 do Envelope de HIV/química , HIV-1/química , HIV-1/metabolismo , HIV-1/fisiologia , Humanos , Lectinas/química , Lectinas/metabolismo , Modelos Moleculares , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais CultivadasRESUMO
Multiple sclerosis (MS) is a major neuroinflammatory demyelinating disease of the CNS. Current MS treatments, including immunomodulators and immunosuppressants, do not result in complete remission. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells derived from dental pulp. Both SHED and SHED-conditioned medium (SHED-CM) exhibit immunomodulatory and regenerative activities and have the potential to treat various diseases. In this study, we investigated the efficacy of SHED-CM in treating experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE mice treated with a single injection of SHED-CM exhibited significantly improved disease scores, reduced demyelination and axonal injury, and reduced inflammatory cell infiltration and proinflammatory cytokine expression in the spinal cord, which was associated with a shift in the microglia/macrophage phenotype from M1 to M2. SHED-CM also inhibited the proliferation of myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, as well as their production of proinflammatory cytokines in vitro. Treatment of EAE mice with the secreted ectodomain of sialic acid-binding Ig-like lectin-9, a major component of SHED-CM, recapitulated the effects of SHED-CM treatment. Our data suggest that SHED-CM and secreted ectodomain of sialic acid-binding Ig-like lectin-9 may be novel therapeutic treatments for autoimmune diseases, such as MS.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Meios de Cultivo Condicionados/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Macrófagos/imunologia , Células-Tronco Mesenquimais/fisiologia , Microglia/imunologia , Esclerose Múltipla/imunologia , Animais , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Dente Decíduo/fisiologia , Dente Decíduo/cirurgiaRESUMO
The chemokine CCL11 (also known as eotaxin-1) is a potent eosinophil chemoattractant that mediates allergic diseases such as asthma, atopic dermatitis, and inflammatory bowel diseases. Previous studies demonstrated that concentrations of CCL11 are elevated in the sera and cerebrospinal fluids (CSF) of patients with neuroinflammatory disorders, including multiple sclerosis. Moreover, the levels of CCL11 in plasma and CSF increase with age, and CCL11 suppresses adult neurogenesis in the central nervous system (CNS), resulting in memory impairment. However, the precise source and function of CCL11 in the CNS are not fully understood. In this study, we found that activated astrocytes release CCL11, whereas microglia predominantly express the CCL11 receptor. CCL11 significantly promoted the migration of microglia, and induced microglial production of reactive oxygen species by upregulating nicotinamide adenine dinucleotide phosphate-oxidase 1 (NOX1), thereby promoting excitotoxic neuronal death. These effects were reversed by inhibition of NOX1. Our findings suggest that CCL11 released from activated astrocytes triggers oxidative stress via microglial NOX1 activation and potentiates glutamate-mediated neurotoxicity, which may be involved in the pathogenesis of various neurological disorders.
Assuntos
Quimiocina CCL11/metabolismo , Ácido Glutâmico/toxicidade , Microglia/metabolismo , Neurônios/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Astrócitos/fisiologia , Antígeno CD11b/metabolismo , Morte Celular/fisiologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Proteínas de Ligação a DNA , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: The accumulation of activated microglia is a hallmark of various neurodegenerative diseases. Microglia may have both protective and toxic effects on neurons through the production of various soluble factors, such as chemokines. Indeed, various chemokines mediate the rapid and accurate migration of microglia to lesions. In the zebra fish, another well-known cellular migrating factor is fibroblast growth factor-2 (FGF-2). Although FGF-2 does exist in the mammalian central nervous system (CNS), it is unclear whether FGF-2 influences microglial function. METHODS: The extent of FGF-2 release was determined by ELISA, and the expression of its receptors was examined by immunocytochemistry. The effect of several drug treatments on a neuron and microglia co-culture system was estimated by immunocytochemistry, and the neuronal survival rate was quantified. Microglial phagocytosis was evaluated by immunocytochemistry and quantification, and microglial migration was estimated by fluorescence-activated cell sorting (FACS). Molecular biological analyses, such as Western blotting and promoter assay, were performed to clarify the FGF-2 downstream signaling pathway in microglia. RESULTS: Fibroblast growth factor-2 is secreted by neurons when damaged by glutamate or oligomeric amyloid ß 1-42. FGF-2 enhances microglial migration and phagocytosis of neuronal debris, and is neuroprotective against glutamate toxicity through FGFR3-extracellular signal-regulated kinase (ERK) signaling pathway, which is directly controlled by Wnt signaling in microglia. CONCLUSIONS: FGF-2 secreted from degenerating neurons may act as a 'help-me' signal toward microglia by inducing migration and phagocytosis of unwanted debris.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Microglia/metabolismo , Degeneração Neural/metabolismo , Neurônios/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Quimiocina CCL3/metabolismo , Embrião de Mamíferos , Ácido Glutâmico/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Fagocitose/fisiologiaRESUMO
Microglia are the immune cells of the central nervous system. They play an important role in maintaining brain homeostasis by constantly surveying their surrounding microenvironment. During pathological event in the brain, microglia respond quickly to restore homeostasis by clearing damaged cells and secreting various proinflammatory mediators. However, during chronic inflammation, their homeostatic functions is lost and they secrete various proinflammatory cytokines and mediators that induce neural dysfunction and neurodegeneration. These microglia mediated tissue damage plays an important role in pathogenesis of various neurological disorders like Alzheimer's disease and Parkinson's disease. Microglia require colony receptor factor 1 receptor (CSF1R)-mediated signals for their survival. Recently, CSF1R antagonist has been used to deplete microglia, reset microglia by forced depletion and repopulation or depletion followed by transplantation with new microglia as a therapeutic strategy for various neurological disorders. In this article, we describe the role of microglia in the in various neurological disorders, and discuss potential therapeutic strategy to manipulate microglia by depletion, resetting and transplantation.
Assuntos
Doença de Alzheimer , Doenças do Sistema Nervoso , Doença de Parkinson , Humanos , Microglia , Doenças do Sistema Nervoso/tratamento farmacológico , Sistema Nervoso Central , Doença de Parkinson/terapiaRESUMO
BACKGROUND: Microglia are resident macrophage-like cells in the central nervous system (CNS) and cause innate immune responses via the LPS receptors, Toll-like receptor (TLR) 4 and CD14, in a variety of neuroinflammatory disorders including bacterial infection, Alzheimer's disease, and amyotrophic lateral sclerosis. Granulocyte macrophage-colony stimulating factor (GM-CSF) activates microglia and induces inflammatory responses via binding to GM-CSF receptor complex composed of two different subunit GM-CSF receptor α (GM-CSFRα) and common ß chain (ßc). GM-CSF has been shown to be associated with neuroinflammatory responses in multiple sclerosis and Alzheimer's disease. However, the mechanisms how GM-CSF promotes neuroinflammation still remain unclear. METHODS: Microglia were stimulated with 20 ng/ml GM-CSF and the levels of TLR4 and CD14 expression were evaluated by RT-PCR and flowcytometry. LPS binding was analyzed by flowcytometry. GM-CSF receptor complex was analyzed by immunocytochemistry. The levels of IL-1ß, IL-6 and TNF-α in culture supernatant of GM-CSF-stimulated microglia and NF-κB nuclear translocation were determined by ELISA. Production of nitric oxide (NO) was measured by the Griess method. The levels of p-ERK1/2, ERK1/2, p-p38 and p38 were assessed by Western blotting. Statistically significant differences between experimental groups were determined by one-way ANOVA followed by Tukey test for multiple comparisons. RESULTS: GM-CSF receptor complex was expressed in microglia. GM-CSF enhanced TLR4 and CD14 expressions in microglia and subsequent LPS-binding to the cell surface. In addition, GM-CSF priming increased LPS-induced NF-κB nuclear translocation and production of IL-1ß, IL-6, TNF-α and NO by microglia. GM-CSF upregulated the levels of p-ERK1/2 and p-p38, suggesting that induction of TLR4 and CD14 expression by GM-CSF was mediated through ERK1/2 and p38, respectively. CONCLUSIONS: These results suggest that GM-CSF upregulates TLR4 and CD14 expression in microglia through ERK1/2 and p38, respectively, and thus promotes the LPS receptor-mediated inflammation in the CNS.
Assuntos
Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Receptores de Lipopolissacarídeos/metabolismo , Microglia/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Regulação para Cima/efeitos dos fármacos , Análise de Variância , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/genética , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Lipopolissacarídeos/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/química , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nitritos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Quinase Induzida por NF-kappaBRESUMO
BACKGROUND: Phosphatidylserine receptor is a key molecule that mediates the phagocytosis of apoptotic cells. Milk fat globule-EGF factor 8 (MFG-E8) is a phosphatidylserine receptor that is expressed on various macrophage lineage cells, including microglia in the central nervous system (CNS). Targeted clearance of degenerated neurons by microglia is essential to maintain healthy neural networks. We previously showed that the CX3C chemokine fractalkine is secreted from degenerated neurons and accelerates microglial clearance of neuronal debris via inducing the release of MFG-E8. However, the mechanisms by which microglia produce MFG-E8 and the precise functions of MFG-E8 are unknown. METHODS: The release of MFG-E8 from microglia treated with conditioned medium from neurons exposed to neurotoxic substances, glutamate or oligomeric amyloid ß (oA ß) was measured by ELISA. The neuroprotective effects of MFG-E8 and MFG-E8-induced microglial phagocytosis of oA ß were assessed by immunocytochemistry. The effects of MFG-E8 on the production of the anti-oxidative enzyme hemeoxygenase-1 (HO-1) were determined by ELISA and immunocytochemisty. RESULTS: MFG-E8 was induced in microglia treated with conditioned medium from neurons that had been exposed to neurotoxicants, glutamate or oA ß. MFG-E8 significantly attenuated oA ß-induced neuronal cell death in a primary neuron-microglia coculture system. Microglial phagocytosis of oA ß was accelerated by MFG-E8 treatment due to increased CD47 expression in the absence of neurotoxic molecule production, such as tumor necrosis factor-α, nitric oxide, and glutamate. MFG-E8-treated microglia induced nuclear factor E(2)-related factor 2 (Nrf2)-mediated HO-1 production, which also contributed to neuroprotection. CONCLUSIONS: These results suggest that microglia release MFG-E8 in response to signals from degenerated neurons and that MFG-E8 protects oA ß-induced neuronal cell death by promoting microglial phagocytic activity and activating the Nrf2-HO-1 pathway. Thus, MFG-E8 may have novel roles as a neuroprotectant in neurodegenerative conditions.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Antígenos de Superfície/fisiologia , Glicolipídeos/fisiologia , Glicoproteínas/fisiologia , Fármacos Neuroprotetores/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/toxicidade , Animais , Animais Recém-Nascidos , Morte Celular/fisiologia , Técnicas de Cocultura , Glicolipídeos/metabolismo , Glicolipídeos/uso terapêutico , Glicoproteínas/metabolismo , Glicoproteínas/uso terapêutico , Humanos , Gotículas Lipídicas , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Proteínas do Leite/metabolismo , Neurônios/metabolismo , Cultura Primária de CélulasRESUMO
Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.
Assuntos
Células-Tronco Pluripotentes Induzidas , Microglia , Animais , Encéfalo/cirurgia , Humanos , Macrófagos , CamundongosRESUMO
Activation of astrocytes has a profound effect on brain plasticity and is critical for the pathophysiology of several neurological disorders including neuropathic pain. Here, we show that metabotropic glutamate receptor 5 (mGluR5), which reemerges in astrocytes in a restricted time frame, is essential for these functions. Although mGluR5 is absent in healthy adult astrocytes, it transiently reemerges in astrocytes of the somatosensory cortex (S1). During a limited spatiotemporal time frame, astrocytic mGluR5 drives Ca2+ signals; upregulates multiple synaptogenic molecules such as Thrombospondin-1, Glypican-4, and Hevin; causes excess excitatory synaptogenesis; and produces persistent alteration of S1 neuronal activity, leading to mechanical allodynia. All of these events were abolished by the astrocyte-specific deletion of mGluR5. Astrocytes dynamically control synaptic plasticity by turning on and off a single molecule, mGluR5, which defines subsequent persistent brain functions, especially under pathological conditions.
Assuntos
Astrócitos , Dor Crônica , Animais , Astrócitos/metabolismo , Dor Crônica/patologia , Camundongos , Plasticidade Neuronal , Neurônios/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismoRESUMO
Interleukin-19 (IL-19) acts as a negative-feedback regulator to limit proinflammatory response of macrophages and microglia in autocrine/paracrine manners in various inflammatory diseases. Multiple sclerosis (MS) is a major neuroinflammatory disease in the central nervous system (CNS), but it remains uncertain how IL-19 contributes to MS pathogenesis. Here, we demonstrate that IL-19 deficiency aggravates experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by promoting IL-17-producing helper T cell (Th17 cell) infiltration into the CNS. In addition, IL-19-deficient splenic macrophages expressed elevated levels of major histocompatibility complex (MHC) class II, co-stimulatory molecules, and Th17 cell differentiation-associated cytokines such as IL-1ß, IL-6, IL-23, TGF-ß1, and TNF-α. These observations indicated that IL-19 plays a critical role in suppression of MS pathogenesis by inhibiting macrophage antigen presentation, Th17 cell expansion, and subsequent inflammatory responses. Furthermore, treatment with IL-19 significantly abrogated EAE. Our data suggest that IL-19 could provide significant therapeutic benefits in patients with MS.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Interleucinas/metabolismo , Animais , Biomarcadores , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Encefalomielite Autoimune Experimental/patologia , Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Imuno-Histoquímica , Imunofenotipagem , Interleucinas/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/efeitos adversos , Glicoproteína Mielina-Oligodendrócito/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Extracellular ATP, which is released from damaged cells after ischemia, activates P2 receptors. P2Y1 receptors (P2Y1R) have received considerable attention, especially in astrocytes, because their activation plays a central role in the regulation of neuron-to-glia communication. However, the functions or even existence of P2Y1R in microglia remain unknown, despite the fact that many microglial P2 receptors are involved in several brain diseases. Herein, we demonstrate the presence and functional capability of microglial P2Y1R to provide neuroprotective effects following ischemic stress. Cerebral ischemia resulted in increased microglial P2Y1R expression. The number of injured hippocampal neurons was significantly higher in P2Y1 R knockout (KO) mice than wildtype mice after forebrain ischemia. Propidium iodide (PI) uptake, a marker for dying cells, was significantly higher in P2Y1R KO hippocampal slices compared with wildtype hippocampal slices at 48 h after 40-min oxygen-glucose deprivation (OGD). Furthermore, increased PI uptake following OGD was rescued by ectopic overexpression of P2Y1R in microglia. In summary, these data suggest that microglial P2Y1R mediate neuroprotective effects against ischemic stress and OGD insult.
Assuntos
Microglia/química , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Receptores Purinérgicos P2Y1/fisiologia , Animais , Isquemia Encefálica , Morte Celular/efeitos dos fármacos , Glucose/deficiência , Hipocampo/metabolismo , Hipocampo/patologia , Hipóxia , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P2Y1/análiseRESUMO
Activated microglia can exert either neurotoxic or neuroprotective effects, and they play pivotal roles in the pathogenesis and progression of various neurological diseases. In this study, we used cDNA microarrays to show that interleukin-19 (IL-19), an IL-10 family cytokine, is markedly upregulated in activated microglia. Furthermore, we found that microglia are the only cells in the nervous system that express the IL-19 receptor, a heterodimer of the IL-20Rα and IL-20Rß subunits. IL-19 deficiency increased the production of such pro-inflammatory cytokines as IL-6 and tumor necrosis factor-α in activated microglia, and IL-19 treatment suppressed this effect. Moreover, in a mouse model of Alzheimer's disease, we observed upregulation of IL-19 in affected areas in association with disease progression. Our findings demonstrate that IL-19 is an anti-inflammatory cytokine, produced by activated microglia, that acts negatively on microglia in an autocrine manner. Thus, microglia may self-limit their inflammatory response by producing the negative regulator IL-19.
Assuntos
Comunicação Autócrina , Interleucina-10/metabolismo , Microglia/metabolismo , Animais , Comunicação Autócrina/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Progressão da Doença , Humanos , Mediadores da Inflamação/metabolismo , Interleucinas , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Receptores de Citocinas/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The blood-brain barrier (BBB) is composed of capillary endothelial cells, pericytes, and perivascular astrocytes, which regulate central nervous system homeostasis. Sonic hedgehog (SHH) released from astrocytes plays an important role in the maintenance of BBB integrity. BBB disruption and microglial activation are common pathological features of various neurologic diseases such as multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Interleukin-1ß (IL-1ß), a major pro-inflammatory cytokine released from activated microglia, increases BBB permeability. Here we show that IL-1ß abolishes the protective effect of astrocytes on BBB integrity by suppressing astrocytic SHH production. Astrocyte conditioned media, SHH, or SHH signal agonist strengthened BBB integrity by upregulating tight junction proteins, whereas SHH signal inhibitor abrogated these effects. Moreover, IL-1ß increased astrocytic production of pro-inflammatory chemokines such as CCL2, CCL20, and CXCL2, which induce immune cell migration and exacerbate BBB disruption and neuroinflammation. Our findings suggest that astrocytic SHH is a potential therapeutic target that could be used to restore disrupted BBB in patients with neurologic diseases.