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1.
Science ; 372(6540)2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33888612

RESUMO

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets.


Assuntos
Astrócitos/fisiologia , Comunicação Celular , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Microglia/fisiologia , Esclerose Múltipla/fisiopatologia , Análise de Célula Única , Animais , Antígenos CD/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Sistema Nervoso Central/fisiopatologia , Encefalomielite Autoimune Experimental/patologia , Efrina-B3/metabolismo , Herpesvirus Suídeo 1/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Esclerose Múltipla/patologia , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Receptor EphB3/antagonistas & inibidores , Receptor EphB3/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforinas/metabolismo , Transdução de Sinais , Linfócitos T/fisiologia , Serina-Treonina Quinases TOR/metabolismo
2.
Nat Commun ; 12(1): 2265, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859199

RESUMO

Nerve-glia (NG2) glia or oligodendrocyte precursor cells (OPCs) are distributed throughout the gray and white matter and generate myelinating cells. OPCs in white matter proliferate more than those in gray matter in response to platelet-derived growth factor AA (PDGF AA), despite similar levels of its alpha receptor (PDGFRα) on their surface. Here we show that the type 1 integral membrane protein neuropilin-1 (Nrp1) is expressed not on OPCs but on amoeboid and activated microglia in white but not gray matter in an age- and activity-dependent manner. Microglia-specific deletion of Nrp1 compromised developmental OPC proliferation in white matter as well as OPC expansion and subsequent myelin repair after acute demyelination. Exogenous Nrp1 increased PDGF AA-induced OPC proliferation and PDGFRα phosphorylation on dissociated OPCs, most prominently in the presence of suboptimum concentrations of PDGF AA. These findings uncover a mechanism of regulating oligodendrocyte lineage cell density that involves trans-activation of PDGFRα on OPCs via Nrp1 expressed by adjacent microglia.


Assuntos
Doenças Desmielinizantes/patologia , Microglia/fisiologia , Neuropilina-1/metabolismo , Células Precursoras de Oligodendrócitos/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Remielinização , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Corpo Caloso/citologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/crescimento & desenvolvimento , Corpo Caloso/patologia , Doenças Desmielinizantes/induzido quimicamente , Modelos Animais de Doenças , Feminino , Humanos , Lisofosfatidilcolinas/administração & dosagem , Lisofosfatidilcolinas/toxicidade , Masculino , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos Animais , Bainha de Mielina/metabolismo , Neuropilina-1/genética , Oligodendroglia/fisiologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Cultura Primária de Células
3.
Neuron ; 109(7): 1100-1117.e10, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33606969

RESUMO

Aging results in gray and white matter degeneration, but the specific microglial responses are unknown. Using single-cell RNA sequencing from white and gray matter separately, we identified white matter-associated microglia (WAMs), which share parts of the disease-associated microglia (DAM) gene signature and are characterized by activation of genes implicated in phagocytic activity and lipid metabolism. WAMs depend on triggering receptor expressed on myeloid cells 2 (TREM2) signaling and are aging dependent. In the aged brain, WAMs form independent of apolipoprotein E (APOE), in contrast to mouse models of Alzheimer's disease, in which microglia with the WAM gene signature are generated prematurely and in an APOE-dependent pathway similar to DAMs. Within the white matter, microglia frequently cluster in nodules, where they are engaged in clearing degenerated myelin. Thus, WAMs may represent a potentially protective response required to clear degenerated myelin accumulating during white matter aging and disease.


Assuntos
Microglia/fisiologia , Substância Branca/citologia , Substância Branca/crescimento & desenvolvimento , Envelhecimento/fisiologia , Doença de Alzheimer/genética , Animais , Apolipoproteínas E/genética , Doenças Desmielinizantes/patologia , Regulação da Expressão Gênica , Substância Cinzenta/citologia , Substância Cinzenta/crescimento & desenvolvimento , Imuno-Histoquímica , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/ultraestrutura , Bainha de Mielina/metabolismo , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Análise de Sequência de RNA , Transdução de Sinais/fisiologia , Análise de Célula Única
4.
Nat Genet ; 53(3): 392-402, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33589840

RESUMO

Genome-wide association studies have discovered numerous genomic loci associated with Alzheimer's disease (AD); yet the causal genes and variants are incompletely identified. We performed an updated genome-wide AD meta-analysis, which identified 37 risk loci, including new associations near CCDC6, TSPAN14, NCK2 and SPRED2. Using three SNP-level fine-mapping methods, we identified 21 SNPs with >50% probability each of being causally involved in AD risk and others strongly suggested by functional annotation. We followed this with colocalization analyses across 109 gene expression quantitative trait loci datasets and prioritization of genes by using protein interaction networks and tissue-specific expression. Combining this information into a quantitative score, we found that evidence converged on likely causal genes, including the above four genes, and those at previously discovered AD loci, including BIN1, APH1B, PTK2B, PILRA and CASS4.


Assuntos
Doença de Alzheimer/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Mapeamento Cromossômico , Proteínas do Citoesqueleto/genética , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Desequilíbrio de Ligação , Microglia/fisiologia , Proteínas Oncogênicas/genética , Polimorfismo de Nucleotídeo Único , Mapas de Interação de Proteínas/genética , Locos de Características Quantitativas , Fatores de Risco , Tetraspaninas/genética
5.
Neuron ; 109(5): 767-777.e5, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472038

RESUMO

Tau is a major driver of neurodegeneration and is implicated in over 20 diseases. Tauopathies are characterized by synaptic loss and neuroinflammation, but it is unclear if these pathological events are causally linked. Tau binds to Synaptogyrin-3 on synaptic vesicles. Here, we interfered with this function to determine the role of pathogenic Tau at pre-synaptic terminals. We show that heterozygous knockout of synaptogyrin-3 is benign in mice but strongly rescues mutant Tau-induced defects in long-term synaptic plasticity and working memory. It also significantly rescues the pre- and post-synaptic loss caused by mutant Tau. However, Tau-induced neuroinflammation remains clearly upregulated when we remove the expression of one allele of synaptogyrin-3. Hence neuroinflammation is not sufficient to cause synaptic loss, and these processes are separately induced in response to mutant Tau. In addition, the pre-synaptic defects caused by mutant Tau are enough to drive defects in cognitive tasks.


Assuntos
Transtornos da Memória/fisiopatologia , Microglia/fisiologia , Terminações Pré-Sinápticas/fisiologia , Sinaptogirinas/fisiologia , Proteínas tau/fisiologia , Animais , Encefalite/fisiopatologia , Feminino , Hipocampo/fisiopatologia , Hipocampo/ultraestrutura , Masculino , Camundongos Knockout , Plasticidade Neuronal , Terminações Pré-Sinápticas/ultraestrutura , Sinaptogirinas/genética
6.
Nat Protoc ; 16(2): 1013-1033, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33424025

RESUMO

Microglia are critically involved in complex neurological disorders with a strong genetic component, such as Alzheimer's disease, Parkinson's disease and frontotemporal dementia. Although mouse microglia can recapitulate aspects of human microglia physiology, they do not fully capture the human genetic aspects of disease and do not reproduce all human cell states. Primary cultures of human microglia or microglia derived from human induced pluripotent stem cells (PSCs) are difficult to maintain in brain-relevant cell states in vitro. Here we describe MIGRATE (microglia in vitro generation refined for advanced transplantation experiments, which provides a combined in vitro differentiation and in vivo xenotransplantation protocol to study human microglia in the context of the mouse brain. This article details an accurate, step-by-step workflow that includes in vitro microglia differentiation from human PSCs, transplantation into the mouse brain and quantitative analysis of engraftment. Compared to current differentiation and xenotransplantation protocols, we present an optimized, faster and more efficient approach that yields up to 80% chimerism. To quantitatively assess engraftment efficiency by flow cytometry, access to specialized flow cytometry is required. Alternatively, the percentage of chimerism can be estimated by standard immunohistochemical analysis. The MIGRATE protocol takes ~40 d to complete, from culturing PSCs to engraftment efficiency assessment.


Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Microglia/citologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Microglia/metabolismo , Microglia/fisiologia , Células-Tronco Pluripotentes/citologia , Gravidez
7.
Nat Neurosci ; 24(1): 19-23, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33318667

RESUMO

Microglial surveillance is a key feature of brain physiology and disease. Here, we found that Gi-dependent microglial dynamics prevent neuronal network hyperexcitability. By generating MgPTX mice to genetically inhibit Gi in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, Gi-dependent microglia dynamics may prevent hyperexcitability in neurological diseases.


Assuntos
Receptor Quinase 1 Acoplada a Proteína G/fisiologia , Microglia/fisiologia , Rede Nervosa/fisiologia , Animais , Sinalização do Cálcio , Movimento Celular , Convulsivantes , Eletroencefalografia , Vigilância Imunológica , Camundongos , Microglia/enzimologia , Microglia/ultraestrutura , Doenças do Sistema Nervoso/fisiopatologia , Fenômenos Fisiológicos do Sistema Nervoso , Pilocarpina , Convulsões/fisiopatologia , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo
8.
Nat Neurosci ; 24(1): 47-60, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33349711

RESUMO

The repair of inflamed, demyelinated lesions as in multiple sclerosis (MS) necessitates the clearance of cholesterol-rich myelin debris by microglia/macrophages and the switch from a pro-inflammatory to an anti-inflammatory lesion environment. Subsequently, oligodendrocytes increase cholesterol levels as a prerequisite for synthesizing new myelin membranes. We hypothesized that lesion resolution is regulated by the fate of cholesterol from damaged myelin and oligodendroglial sterol synthesis. By integrating gene expression profiling, genetics and comprehensive phenotyping, we found that, paradoxically, sterol synthesis in myelin-phagocytosing microglia/macrophages determines the repair of acutely demyelinated lesions. Rather than producing cholesterol, microglia/macrophages synthesized desmosterol, the immediate cholesterol precursor. Desmosterol activated liver X receptor (LXR) signaling to resolve inflammation, creating a permissive environment for oligodendrocyte differentiation. Moreover, LXR target gene products facilitated the efflux of lipid and cholesterol from lipid-laden microglia/macrophages to support remyelination by oligodendrocytes. Consequently, pharmacological stimulation of sterol synthesis boosted the repair of demyelinated lesions, suggesting novel therapeutic strategies for myelin repair in MS.


Assuntos
Doenças Desmielinizantes/patologia , Microglia/fisiologia , Esteróis/biossíntese , Animais , Colesterol/metabolismo , Desmosterol/metabolismo , Encefalomielite Autoimune Experimental , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos , Receptores X do Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Esclerose Múltipla , Oligodendroglia/metabolismo , Fagocitose , Esqualeno/metabolismo
9.
J Ethnopharmacol ; 264: 113246, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32781257

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (Cyperaceae) is a widespread herbal in China and widely used in Traditional Chinese Medicine for multiple effects such as anti-arthritic, anti-genotoxic, anti-mutagenic, anti-bacterial effects, and analgesic. α-Cyperone is an active compound in Cyperus rotundus and has analgesic effects, but the exact molecular mechanisms require further investigations. MATERIALS AND METHODS: Tumor-derived DNA isolated from Lewis cell lines was transfected into microglia, and analyzed for stimulator of interferon genes (STING) effects. The downstream protein, such as interferon regulatory factor 3 (IRF3) and p65 nuclear factor-κB (NF-κB) were treated with STING siRNA and 5,6-dimethyllxanthenone-4-acetic acid (DMXAA) in microglia. The α-Cyperone effect on microglia was also investigated. RESULTS: Tumor-derived DNA activate microglia by upregulation of STING and downstream proteins. STING siRNA was reduced to its downstream expression and neuroinflammation inhibition was caused by tumor-derived DNA. However, DMXAA reversed the STING siRNA effect and increased neuroinflammation. α-Cyperone takes inhibitory effects on tumor-derived DNA that trigger microglia by STING pathway. CONCLUSIONS: α-Cyperone inhibition by tumor-derived DNA activated microglial to neuroinflammation in STING signaling pathway.


Assuntos
DNA de Neoplasias/antagonistas & inibidores , DNA de Neoplasias/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Microglia/efeitos dos fármacos , Naftalenos/farmacologia , Animais , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/genética , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Camundongos , Microglia/fisiologia , Naftalenos/uso terapêutico
10.
J Pharmacol Sci ; 144(4): 212-217, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33070840

RESUMO

The hippocampus is a brain region well-known to exhibit structural and functional changes in temporal lobe epilepsy. Studies analyzing the brains of patients with epilepsy and those from animal models of epilepsy have revealed that microglia are excessively activated, especially in the hippocampus. These findings suggest that microglia may contribute to the onset and aggravation of epilepsy; however, direct evidence for microglial involvement or the underlying mechanisms by which this occurs remain to be fully discovered. To date, neuron-microglia interactions have been vigorously studied in adult epilepsy models; such studies have clarified microglial responses to excessive synchronous firing of neurons. In contrast, the role of microglia in the postnatal brain of patients with epileptic seizures remain largely unclear. Some early-life seizures, such as complex febrile seizures, have been shown to cause structural and functional changes in the brain, which is a risk factor for future development of epilepsy. Because brain structure and function are actively modulated by microglia in both health and disease, it is essential to clarify the role of microglia in early-life seizures and its impact on epileptogenesis.


Assuntos
Comunicação Celular , Epilepsia do Lobo Temporal/etiologia , Epilepsia do Lobo Temporal/patologia , Hipocampo/citologia , Hipocampo/patologia , Microglia/patologia , Microglia/fisiologia , Fatores Etários , Idade de Início , Animais , Giro Denteado/citologia , Giro Denteado/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Neurônios/fisiologia , Ratos , Fatores de Risco , Convulsões Febris/etiologia , Convulsões Febris/patologia , Sinapses/fisiologia
11.
J Pharmacol Sci ; 144(3): 102-118, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32921391

RESUMO

Chronic microglial activation is associated with the pathogenesis of several CNS disorders. Microglia show phenotypic diversity and functional complexity in diseased CNS. Thus, understanding the pathology-specific heterogeneity of microglial behavior is crucial for the future development of microglia-modulating therapy for variety of CNS disorders. This review summarizes up-to-date knowledge on how microglia contribute to CNS homeostasis during development and throughout adulthood. We discuss the heterogeneity of microglial phenotypes in the context of CNS disorders with an emphasis on neurodegenerative diseases, demyelinating diseases, CNS trauma, and epilepsy. We conclude this review with a discussion about the disease-specific heterogeneity of microglial function and how it could be exploited for therapeutic intervention.


Assuntos
Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/terapia , Microglia/patologia , Microglia/fisiologia , Doenças do Sistema Nervoso Central/patologia , Doenças Desmielinizantes/etiologia , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/terapia , Epilepsia/etiologia , Epilepsia/patologia , Epilepsia/terapia , Homeostase , Humanos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Fenótipo
12.
Life Sci ; 260: 118470, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32950573

RESUMO

Microglial phenotypic polarization, divided into pro-inflammatory "M1" phenotype and anti-inflammatory "M2" phenotype, played a crucial role in the pathogenesis of Alzheimer's disease (AD). Facilitating microglial polarization from M1 to M2 phenotype was shown to alleviate AD-associate pathologic damage, and modulator of the microglial phenotype has become a promising therapeutic approach for the treatment of AD. Previous little evidence showed that DHCR24 (3-ß-hydroxysteroid-Δ-24-reductase), also known as seladin-1 (selective Alzheimer's disease indicator-1), exerted potential anti-inflammatory property, however, the link between DHCR24 and microglial polarization has never been reported. Thus, the role of DHCR24 in microglial polarization in amyloid-beta 25-35 (Aß25-35) treated BV-2 cells was evaluated in this study. Our results demonstrated that Aß25-35 aggravated inflammatory response and facilitated the transition of microglia phenotype from M2 to M1 in BV-2 cells, by upregulating M1 marker (i-NOS, IL-1ß and TNF-α) and downregulating M2 marker (arginase-1, IL-4 and TGF-ß). DHCR24 overexpression by lentivirus transfection could significantly reverse these effects, meanwhile, activated Akt/GSK3ß signaling pathway via increasing the protein expression of P-Akt and P-GSK3ß. Furthermore, when co-treated with Akt inhibitor MK2206, the effect of DHCR24 was obviously reversed. The study exhibited the neuroprotective function of DHCR24 in AD-related inflammatory injury and provided a novel therapeutic target for AD in the future.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteínas do Tecido Nervoso/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , Microglia/citologia , Microglia/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Am J Physiol Cell Physiol ; 319(3): C605-C610, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32783655

RESUMO

Epileptic seizures are the manifestation of hypersynchronous and excessive neuronal excitation. While the glutamatergic and GABAergic neurons play major roles in shaping fast neuronal excitation/inhibition homeostasis, it is well illustrated that astrocytes profoundly regulate neuronal excitation by controlling glutamate, GABA, cannabinoids, adenosine, and concentration of K+ around neurons. However, little is known about whether microglia take part in the regulation of acute neuronal excitation and ongoing epileptic behaviors. We proposed that if microglia are innately ready to respond to epileptic overexcitation, depletion of microglia might alter neuronal excitability and severity of acute epileptic seizures. We found that microglia depletion by plx3397, an inhibitor of CSF1R, exacerbates seizure severity and excitotoxicity-induced neuronal degeneration, indicating that microglia are rapidly responsive to the change of excitation/inhibition homeostasis and participate in the protection of neurons from overexcitation.


Assuntos
Astrócitos/metabolismo , Hipocampo/fisiologia , Microglia/fisiologia , Convulsões/fisiopatologia , Animais , Ácido Glutâmico/metabolismo , Camundongos , Neurônios/fisiologia , Convulsões/metabolismo
14.
PLoS One ; 15(8): e0237773, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822378

RESUMO

Microglial cells play a key role in brain homeostasis from development to adulthood. Here we show the involvement of a site-specific phosphorylation of Presenilin 1 (PS1) in microglial development. Profiles of microglia-specific transcripts in different temporal stages of development, combined with multiple systematic transcriptomic analysis and quantitative determination of microglia progenitors, indicate that the phosphorylation of PS1 at serine 367 is involved in the temporal dynamics of microglial development, specifically in the developing brain rudiment during embryonic microgliogenesis. We constructed a developing brain-specific microglial network to identify transcription factors linked to PS1 during development. Our data showed that PS1 functional connections appear through interaction hubs at Pu.1, Irf8 and Rela-p65 transcription factors. Finally, we showed that the total number of microglia progenitors was markedly reduced in the developing brain rudiment of embryos lacking PS1 phosphorylation compared to WT. Our work identifies a novel role for PS1 in microglial development.


Assuntos
Redes Reguladoras de Genes , Microglia/fisiologia , Presenilina-1/metabolismo , Células-Tronco/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Presenilina-1/genética , Células-Tronco/citologia , Transcriptoma
15.
J Vis Exp ; (162)2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32831314

RESUMO

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/fisiologia , Cálcio/metabolismo , Córtex Cerebral/fisiologia , Microglia/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Animais , Camundongos , Imagem Molecular/métodos
16.
Anticancer Res ; 40(8): 4719-4727, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727798

RESUMO

BACKGROUND/AIM: We investigated whether mastication affects microglia, whose activity is thought to be associated with cognition and brain tumor progression. MATERIALS AND METHODS: We kept mice by feeding either a hard or soft diet for 2, 4 or 8 months. After each period, we removed the whole brains and isolated microglia. The total RNA extracted from each brain's microglia was subjected to DNA microarray analysis. RESULTS: Many genes were found to be significantly differentially expressed between hard- and soft-diet-fed mice in each group of the same feeding period. The expression of several genes involved in the regulation of actin cytoskeleton was down-regulated in the soft-diet-fed mice. CONCLUSION: Mastication may affect microglia's roles in cognition as well as their neuroimmune activity through their activity of patrolling the brain.


Assuntos
Mastigação/fisiologia , Microglia/fisiologia , Transcriptoma/fisiologia , Animais , Encéfalo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H
18.
Cell Mol Life Sci ; 77(20): 4081-4091, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32405721

RESUMO

In most vertebrates, the yolk sac (YS) represents the very first tissue where blood cells are detected. Therefore, it was thought for a long time that it generated all the blood cells present in the embryo. This model was challenged using different animal models, and we now know that YS hematopoietic precursors are mostly transient although their contribution to the adult system cannot be excluded. In this review, we aim at properly define the different waves of blood progenitors that are produced by the YS and address the fate of each of them. Indeed, in the last decade, many evidences have emphasized the role of the YS in the emergence of several myeloid tissue-resident adult subsets. We will focus on the development of microglia, the resident macrophages in the central nervous system, and try to untangle the recent controversy about their origin.


Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Saco Vitelino/fisiologia , Animais , Humanos , Macrófagos/fisiologia , Microglia/fisiologia , Células Mieloides/fisiologia
19.
Inflamm Res ; 69(7): 697-710, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32350570

RESUMO

OBJECTIVE: Sepsis-associated encephalopathy (SAE) is a major cause of mortality worldwide. Oxidative stress, inflammatory response and apoptosis participate in the pathogenesis of SAE. Nuclear factor erythroid 2-related factor 2 (Nrf2) and nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) pathway is involved in oxidative stress and inflammatory response. We reported that hydrogen gas protected against sepsis in wild-type (WT) but not Nrf2 knockout (KO) mice. Therefore, it is vital to identify the underlying cause of hydrogen gas treatment of sepsis-associated encephalopathy. METHODS: SAE was induced in WT and Nrf2 KO mice by cecal ligation and puncture (CLP). As a NLRP3 inflammasome inhibitor, MCC950 (50 mg/kg) was administered by intraperitoneal (i.p.) injection before operation. Hydrogen gas (H2)-rich saline solution (5 mL/kg) was administered by i.p. injection at 1 h and 6 h after sham and CLP operations. Brain tissue was collected to assess the NLRP3 and Nrf2 pathways by western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. RESULTS: SAE increased NLRP3 and Nrf2 expression in microglia. MCC950 inhibited SAE-induced NLRP3 expression, interleukin (IL)-1ß and IL-18 cytokine release, neuronal apoptosis and mitochondrial dysfunction. SAE increased NLRP3 and caspase-1 expression in WT mice compared to Nrf2 KO mice. Hydrogen increased Nrf2 expression and inhibited the SAE-induced expression of NLRP3, caspase-1, cytokines IL-1ß and IL-18, neuronal apoptosis, and mitochondrial dysfunction in WT mice but not Nrf2 KO mice. CONCLUSION: SAE increased NLRP3 and Nrf2 expression in microglia. Hydrogen alleviated inflammation, neuronal apoptosis and mitochondrial dysfunction via inhibiting Nrf2-mediated NLRP3 pathway.


Assuntos
Hidrogênio/administração & dosagem , Fator 2 Relacionado a NF-E2/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Encefalopatia Associada a Sepse/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Química Encefálica , Ceco , Córtex Cerebral/ultraestrutura , Citocinas/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Masculino , Camundongos , Camundongos Knockout , Microglia/fisiologia , Mitocôndrias/fisiologia , Fator 2 Relacionado a NF-E2/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/análise , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Punções , Encefalopatia Associada a Sepse/patologia , Sulfonas/farmacologia
20.
Nat Med ; 26(5): 769-780, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284590

RESUMO

Our understanding of Alzheimer's disease (AD) pathophysiology remains incomplete. Here we used quantitative mass spectrometry and coexpression network analysis to conduct the largest proteomic study thus far on AD. A protein network module linked to sugar metabolism emerged as one of the modules most significantly associated with AD pathology and cognitive impairment. This module was enriched in AD genetic risk factors and in microglia and astrocyte protein markers associated with an anti-inflammatory state, suggesting that the biological functions it represents serve a protective role in AD. Proteins from this module were elevated in cerebrospinal fluid in early stages of the disease. In this study of >2,000 brains and nearly 400 cerebrospinal fluid samples by quantitative proteomics, we identify proteins and biological processes in AD brains that may serve as therapeutic targets and fluid biomarkers for the disease.


Assuntos
Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Líquido Cefalorraquidiano/metabolismo , Metabolismo Energético , Microglia/metabolismo , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/patologia , Animais , Astrócitos/patologia , Astrócitos/fisiologia , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Líquido Cefalorraquidiano/química , Estudos de Coortes , Progressão da Doença , Feminino , Redes Reguladoras de Genes/fisiologia , Humanos , Masculino , Espectrometria de Massas , Redes e Vias Metabólicas , Camundongos , Microglia/patologia , Microglia/fisiologia , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Proteômica/métodos , Tamanho da Amostra , Fatores de Tempo
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