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The role that astrocytes play in central nervous system (CNS) myelination is poorly understood. We investigated the contribution of astrocyte-derived factors to myelination and revealed a substantial overlap in the secretomes of human and rat astrocytes. Using in vitro myelinating co-cultures of primary retinal ganglion cells and cortical oligodendrocyte precursor cells, we discovered that factors secreted by resting astrocytes, but not reactive astrocytes, facilitated myelination. Soluble brevican emerged as a new enhancer of developmental myelination in vivo, CNS and its absence was linked to remyelination deficits following an immune-mediated damage in an EAE mouse model. The observed reduction of brevican expression in reactive astrocytes and human MS lesions suggested a potential link to the compromised remyelination characteristic of neurodegenerative diseases. Our findings suggested brevican's role in myelination may be mediated through interactions with binding partners such as contactin-1 and tenascin-R. Proteomic analysis of resting versus reactive astrocytes highlighted a shift in protein expression profiles, pinpointing candidates that either facilitate or impede CNS repair, suggesting that depending on their reactivity state, astrocytes play a dual role during myelination.
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The study of adult neural cell production has concentrated on neurogenesis. The mechanisms controlling adult gliogenesis are still poorly understood. Here, we provide evidence for a homeostatic process that maintains the population of glial cells in the Drosophila adult brain. Flies lacking microRNA miR-31a start adult life with a normal complement of glia, but transiently lose glia due to apoptosis. miR-31a expression identifies a subset of predominantly gliogenic adult neural progenitor cells. Failure to limit expression of the predicted E3 ubiquitin ligase, Rchy1, in these cells results in glial loss. After an initial decline in young adults, glial numbers recovered due to compensatory overproduction of new glia by adult progenitor cells, indicating an unexpected plasticity of the Drosophila nervous system. Experimentally induced ablation of glia was also followed by recovery of glia over time. These studies provide evidence for a homeostatic mechanism that maintains the number of glia in the adult fly brain.
Assuntos
Encéfalo/citologia , Drosophila/fisiologia , Homeostase , MicroRNAs/metabolismo , Neuroglia/fisiologia , Animais , MicroRNAs/genética , MutaçãoRESUMO
Development, physiological functions, and pathologies of the brain depend on tight interactions between neurons and different types of glial cells, such as astrocytes, microglia, oligodendrocytes, and oligodendrocyte precursor cells. Assessing the relative contribution of different glial cell types is required for the full understanding of brain function and dysfunction. Over the recent years, several technological breakthroughs were achieved, allowing "glio-scientists" to address new challenging biological questions. These technical developments make it possible to study the roles of specific cell types with medium or high-content workflows and perform fine analysis of their mutual interactions in a preserved environment. This review illustrates the potency of several cutting-edge experimental approaches (advanced cell cultures, induced pluripotent stem cell (iPSC)-derived human glial cells, viral vectors, in situ glia imaging, opto- and chemogenetic approaches, and high-content molecular analysis) to unravel the role of glial cells in specific brain functions or diseases. It also illustrates the translation of some techniques to the clinics, to monitor glial cells in patients, through specific brain imaging methods. The advantages, pitfalls, and future developments are discussed for each technique, and selected examples are provided to illustrate how specific "gliobiological" questions can now be tackled.
Assuntos
Astrócitos , Neuroglia , Humanos , Microglia , Neurônios , OligodendrogliaRESUMO
Disruption of the blood-brain barrier (BBB) leads to various neurovascular diseases. Development of therapeutics required to cross the BBB is difficult due to a lack of relevant in vitro models. We have developed a three-dimensional (3D) microfluidic BBB chip (BBBC) to study cell interactions in the brain microvasculature and to test drug candidates of neurovascular diseases. We isolated primary brain microvascular endothelial cells (ECs), pericytes, and astrocytes from neonatal rats and cocultured them in the BBBC. To mimic the 3D in vivo BBB structure, we used type I collagen hydrogel to pattern the microchannel via viscous finger patterning technique to create a matrix. ECs, astrocytes, and pericytes were cocultured in the collagen matrix. The fluid flow in the BBBC was controlled by a pump-free strategy utilizing gravity as driving force and resistance in a paper-based flow resistor. The primary cells cultured in the BBBC expressed high levels of junction proteins and formed a tight endothelial barrier layer. Addition of tumor necrosis factor alpha to recapitulate neuroinflammatory conditions compromised the BBB functionality. To mitigate the neuroinflammatory stimulus, we treated the BBB model with the glucocorticoid drug dexamethasone, and observed protection of the BBB. This BBBC represents a new simple, cost-effective, and scalable in vitro platform for validating therapeutic drugs targeting neuroinflammatory conditions.
Assuntos
Barreira Hematoencefálica , Técnicas de Cocultura/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Dispositivos Lab-On-A-Chip , Animais , Anti-Inflamatórios/farmacologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Células Cultivadas , Técnicas de Cocultura/métodos , Desenho de Equipamento , Inflamação/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Pericitos/citologia , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.
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Astrócitos/metabolismo , Proteínas de Membrana/metabolismo , Vias Neurais/metabolismo , Fagocitose , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sinapses/metabolismo , Animais , Astrócitos/citologia , Encéfalo/citologia , Técnicas In Vitro , Núcleos Laterais do Tálamo/citologia , Núcleos Laterais do Tálamo/metabolismo , Aprendizagem/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Vias Neurais/citologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Retina/fisiologia , c-Mer Tirosina QuinaseRESUMO
In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.
Assuntos
Astrócitos/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Glipicanas/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Animais , Astrócitos/citologia , Cerebelo/citologia , Cerebelo/metabolismo , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Feminino , Glipicanas/deficiência , Glipicanas/farmacologia , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/patologiaRESUMO
Expansion of astrocyte populations in the central nervous system is characteristic of evolutionarily more complex organisms. However, regulation of mammalian astrocyte precursor proliferation during development remains poorly understood. Here, we used Aldh1L1-GFP to identify two morphologically distinct types of proliferative astrocyte precursors: radial glia (RG) in the ventricular zone and a second cell type we call an 'intermediate astrocyte precursor' (IAP) located in the mantle region of the spinal cord. Astrogenic RG and IAP cells proliferated in a progressive ventral-to-dorsal fashion in a tight window from embryonic day 13.5 until postnatal day 3, which correlated precisely with the pattern of active ERK signalling. Conditional loss of BRAF function using BLBP-cre resulted in a 20% decrease in astrocyte production, whereas expression of activated BRAFV600E resulted in astrocyte hyperproliferation. Interestingly, BRAFV600E mitogenic effects in astrocytes were restricted, in part, by the function of p16INK4A-p19(ARF), which limited the temporal epoch for proliferation. Together, these findings suggest that astrocyte precursor proliferation involves distinct RG and IAP cells; is subjected to temporal and spatial control; and depends in part on BRAF signalling at early stages of mammalian spinal cord development.
Assuntos
Astrócitos/citologia , Medula Espinal/citologia , Aldeído Desidrogenase/metabolismo , Animais , Astrócitos/metabolismo , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Medula Espinal/embriologiaRESUMO
Alzheimer's disease (AD) is a neurodegenerative disorder with complex pathological manifestations and is the leading cause of cognitive decline and dementia in elderly individuals. A major goal in AD research is to identify new therapeutic pathways by studying the molecular and cellular changes in the disease, either downstream or upstream of the pathological hallmarks. In this study, we present a comprehensive investigation of cellular heterogeneity from the temporal cortex region of 40 individuals, comprising healthy donors and individuals with differing tau and amyloid burden. Using single-nucleus transcriptome analysis of 430,271 nuclei from both gray and white matter of these individuals, we identified cell type-specific subclusters in both neuronal and glial cell types with varying degrees of association with AD pathology. In particular, these associations are present in layer specific glutamatergic (excitatory) neuronal types, along with GABAergic (inhibitory) neurons and glial subtypes. These associations were observed in early as well as late pathological progression. We extended this analysis by performing multiplexed in situ hybridization using the CARTANA platform, capturing 155 genes in 13 individuals with varying levels of tau pathology. By modeling the spatial distribution of these genes and their associations with the pathology, we not only replicated key findings from our snRNA data analysis, but also identified a set of cell type-specific genes that show selective enrichment or depletion near pathological inclusions. Together, our findings allow us to prioritize specific cell types and pathways for targeted interventions at various stages of pathological progression in AD.
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Reactive astrogliosis is characterized by a profound change in astrocyte phenotype in response to all CNS injuries and diseases. To better understand the reactive astrocyte state, we used Affymetrix GeneChip arrays to profile gene expression in populations of reactive astrocytes isolated at various time points after induction using two mouse injury models, ischemic stroke and neuroinflammation. We find reactive gliosis consists of a rapid, but quickly attenuated, induction of gene expression after insult and identify induced Lcn2 and Serpina3n as strong markers of reactive astrocytes. Strikingly, reactive astrocyte phenotype strongly depended on the type of inducing injury. Although there is a core set of genes that is upregulated in reactive astrocytes from both injury models, at least 50% of the altered gene expression is specific to a given injury type. Reactive astrocytes in ischemia exhibited a molecular phenotype that suggests that they may be beneficial or protective, whereas reactive astrocytes induced by LPS exhibited a phenotype that suggests that they may be detrimental. These findings demonstrate that, despite well established commonalities, astrocyte reactive gliosis is a highly heterogeneous state in which astrocyte activities are altered to respond to the specific injury. This raises the question of how many subtypes of reactive astrocytes exist. Our findings provide transcriptome databases for two subtypes of reactive astrocytes that will be highly useful in generating new and testable hypotheses of their function, as well as for providing new markers to detect different types of reactive astrocytes in human neurological diseases.
Assuntos
Astrócitos/metabolismo , Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Gliose/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/metabolismo , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Genoma/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteoma/genéticaRESUMO
The metabolic enzyme for folate, Aldh1L1, has been shown to be expressed robustly in astrocytes of the brain. It is now well accepted that astrocytes in certain regions of the adult brain also serve as neural stem cells. Here, we examined whether Aldh1L1 is also expressed in postnatal neural stem cells. In vitro, cells in neural stem cell culture conditions have robust Aldh1L1 promoter activity. In vivo, in the adult brain, astroctyes in neurogenic regions express Aldh1L1 in a pattern consistent with inclusion in neural stem cells, and analysis of Aldh1L1+ cell transcriptome profiles from neurogenic regions reveal a robust enrichment of known regulators of neurogenesis. Genetic fate mapping with Aldh1L1 BAC Cre animals reveals adult-born neuroblasts of the rostral migratory stream are derived from Aldh1L1 expressing cells, as are sporadic neurons in other regions of the brain. Combining these lines of evidence from transgenic animals, cell culture, transcriptome profiling, and fate mapping, we conclude that Aldh1L1 is also expressed in neural stem cells in the brain. These findings may influence the future design of experiments utilizing Aldh1L1 genetic tools, and also suggest existing Aldh1L1 bacTRAP mice may be of use for further experiments profiling neural stem cells in vivo.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Isoenzimas/metabolismo , Células-Tronco Neurais/metabolismo , Retinal Desidrogenase/metabolismo , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Encéfalo/citologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Isoenzimas/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/efeitos dos fármacos , Retinal Desidrogenase/genética , Nicho de Células-Tronco/efeitos dos fármacos , Nicho de Células-Tronco/genética , Fosfatase Ácida Resistente a Tartarato , Fatores de Transcrição/metabolismoRESUMO
Multiple sclerosis (MS) is the most common demyelinating autoimmune disease of the central nervous system (CNS). Immune-mediated myelin and axonal damage that is accompanied by chronic axonal loss causing destruction of the myelin sheaths are hallmarks of MS. While great strides have been made in understanding the molecular underpinnings of re-/myelination, currently no remyelination therapy is available for MS. As myelination is a complex process that is not fully understood, we sought to develop a systematic, reliable, automated and quantitative higher throughput screening method. We aimed to quantitate myelin sheaths in vitro with high sensitivity at the single cell level suitable for testing small compound libraries. To this end, we miniaturised in vitro retinal ganglion cell-oligodendrocyte precursor cell (RGC-OPC) co-cultures into a multi-well plate format. This allowed us to maintain the reciprocal interaction of live axons and oligodendrocytes (OLs) to ensure compact myelin formation. To quantify our co-cultures, we developed a novel computer vision algorithm to precisely measure myelination. We demonstrated efficacy of our system with known pro-differentiating compounds BQ3020 and XAV939 which exhibited robust, efficient, and dose dependent effects on myelination. Through this combination of experimental and technical advances, we have developed a method allowing systematic and reliable testing of remyelinating compound efficacy.
Assuntos
Esclerose Múltipla , Bainha de Mielina , Humanos , Avaliação Pré-Clínica de Medicamentos , Fluxo de Trabalho , Algoritmos , AxôniosRESUMO
To date, most expression quantitative trait loci (eQTL) studies, which investigate how genetic variants contribute to gene expression, have been performed in heterogeneous brain tissues rather than specific cell types. In this study, we performed an eQTL analysis using single-nuclei RNA sequencing from 192 individuals in eight brain cell types derived from the prefrontal cortex, temporal cortex and white matter. We identified 7,607 eGenes, a substantial fraction (46%, 3,537/7,607) of which show cell-type-specific effects, with strongest effects in microglia. Cell-type-level eQTLs affected more constrained genes and had larger effect sizes than tissue-level eQTLs. Integration of brain cell type eQTLs with genome-wide association studies (GWAS) revealed novel relationships between expression and disease risk for neuropsychiatric and neurodegenerative diseases. For most GWAS loci, a single gene co-localized in a single cell type, providing new clues into disease etiology. Our findings demonstrate substantial contrast in genetic regulation of gene expression among brain cell types and reveal potential mechanisms by which disease risk genes influence brain disorders.
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Estudo de Associação Genômica Ampla , Doenças do Sistema Nervoso , Encéfalo , Predisposição Genética para Doença/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genéticaRESUMO
Glia cells have a crucial role in the central nervous system and are involved in the majority of neurological diseases. While glia isolation techniques are well established for rodent brain, only recent advances in isolating glial cells from human brain enabled analyses of human-specific glial-cell profiles. Immunopanning that is the prospective purification of cells using cell type-specific antibodies, has been successfully established for isolating glial cells from human fetal brain or from tissue obtained during brain surgeries. Here, we describe an immunopanning protocol to acutely isolate glial cells from post-mortem human brain tissue for e.g. transcriptome and proteome analyses. We enriched for microglia, oligodendrocytes and astrocytes from cortical gray matter tissue from three donors. For each enrichment, we assessed the presence of known glia-specific markers at the RNA and protein levels. In this study we show that immunopanning can be employed for acute isolation of glial cells from human post-mortem brain, which allows characterization of glial phenotypes depending on age, disease and brain regions.
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Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate neural cell types. Here we describe methods for the prospective isolation and purification of astrocytes, neurons, and oligodendrocytes from developing and mature mouse forebrain. We used FACS (fluorescent-activated cell sorting) to isolate astrocytes from transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of an S100beta promoter. Using Affymetrix GeneChip Arrays, we then created a transcriptome database of the expression levels of >20,000 genes by gene profiling these three main CNS neural cell types at various postnatal ages between postnatal day 1 (P1) and P30. This database provides a detailed global characterization and comparison of the genes expressed by acutely isolated astrocytes, neurons, and oligodendrocytes. We found that Aldh1L1 is a highly specific antigenic marker for astrocytes with a substantially broader pattern of astrocyte expression than the traditional astrocyte marker GFAP. Astrocytes were enriched in specific metabolic and lipid synthetic pathways, as well as the draper/Megf10 and Mertk/integrin alpha(v)beta5 phagocytic pathways suggesting that astrocytes are professional phagocytes. Our findings call into question the concept of a "glial" cell class as the gene profiles of astrocytes and oligodendrocytes are as dissimilar to each other as they are to neurons. This transcriptome database of acutely isolated purified astrocytes, neurons, and oligodendrocytes provides a resource to the neuroscience community by providing improved cell-type-specific markers and for better understanding of neural development, function, and disease.
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Astrócitos/fisiologia , Encéfalo , Perfilação da Expressão Gênica , Neurônios/fisiologia , Oligodendroglia/fisiologia , Transcrição Gênica , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
Microfluidic platforms have recently become popular as in vitro models because of their superiority in recapitulating microenvironments compared with conventional in vitro models. By providing various biochemical and biomechanical cues, healthy and diseased models at the organ level can be applied to disease progression and treatment studies. Microfluidic technologies are especially suitable for modeling biological barriers because the flow in the microchannels mimics the blood flow and body fluids at the interfaces of crucial organs, such as lung, intestine, liver, kidney, brain, and skin. These barriers have similar structures and can be studied with similar approaches for the testing of pharmaceutical compounds. Here, we review recent developments in microfluidic platforms for modeling biological barriers in the circulatory system.
Assuntos
Sistema Cardiovascular/fisiopatologia , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Animais , Biomimética/métodos , Humanos , Modelos BiológicosRESUMO
Neuronal and glial progenitor cells exist in the adult Drosophila brain. The primarily glial progenitor cells rely on a microRNA, mir-31a, to inhibit the expression of a predicted E3 ubiquitin ligase, CG16947. Erroneous inheritance of CG16947 by the progeny when the neural progenitor cell divides leads to death of the progeny, however how CG16947 achieves glial cell death is unknown. I have identified the interacting partner of CG16947 to be cdk9. I show that reduction of cdk9 expression in glia causes glial loss; highlighting the importance of cdk9 in mediating the survival of glia. Further, glial loss observed in mir-31a mutants was prevented with adult-specific expression of cdk9 in glia. I provide biochemical evidence that the binding of CG16947 to cdk9 causes its degradation. Taken together, this data shows that cdk9 plays a role in the survival of adult glia in the Drosophila brain. Thus, a fine balance exists between mir-31a and CG16947 expression in the progenitor cells that in turn regulates the levels of cdk9 in the progeny. This serves to allow the progenitor cells to regulate the number of glia in the adult brain.
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Quinase 9 Dependente de Ciclina/metabolismo , Proteínas de Drosophila/genética , Neuroglia/metabolismo , Animais , Quinase 9 Dependente de Ciclina/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Neuroglia/citologiaRESUMO
Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100ß immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development.
Assuntos
Astrócitos/fisiologia , Proteínas Morfogenéticas Ósseas/biossíntese , Receptores ErbB/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Astrócitos/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Receptores ErbB/genética , Camundongos , Fosforilação , Ratos , Transdução de SinaisRESUMO
For years, studies of neural-glial interactions have relied on the use of astrocytes derived from the extended culture of immature precursor cells isolated from the neonatal rodent brain. Although the astrocytes cultured under these selective cell survival conditions have been important tools for understanding astrocyte behavior, they do not necessarily reflect the behavior and function of mature astrocytes. We have developed methods for acute, prospective isolation and culture of mature astrocytes from rodent brains in a serum-free, defined medium. These immunopanning-based methods facilitate the study of astrocyte biology and function.
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Astrócitos/fisiologia , Encéfalo/citologia , Animais , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Meios de Cultura Livres de Soro/química , Camundongos , RatosRESUMO
The purification of astrocytes by fluorescence-activated cell sorting (FACS) requires that an astrocyte-specific promoter drive the expression of the green fluorescent protein (GFP). Our laboratory uses FACS to acutely isolate astrocytes from young and old tissue as well as to isolate GFP-negative neurons at the end of the FACS sorting to conduct comparative unbiased, large-scale gene expression studies. Because of the relatively harsh nature of FACS sorting, few astrocytes or neurons survive long enough after the sort to be cultured.
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Astrócitos/metabolismo , Citometria de Fluxo/métodos , Coloração e Rotulagem/métodos , Animais , Astrócitos/fisiologia , Sobrevivência Celular , Perfilação da Expressão Gênica/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
We describe the use of immunopanning to purify rodent astrocytes. Immunopanning of astrocytes permits the prospective isolation of astrocytes from the rodent brain. Prospective isolation refers to the direct selection of cells without multiple steps that extend over a few days, thereby permitting the selection of a representative proportion of the astrocytes from the cortex. Because immunopanning is a very gentle process, the cells are viable at the end of the preparation and can be cultured in a serum-free medium containing heparin-binding EGF-like growth factor (HBEGF), the critical survival factor of astrocytes in vitro, for at least 2 wk. This protocol was initially established and verified with rats, but modifications for the purification of mouse cells are also described here.