RESUMO
Astrocytes, the predominant glial cells in the central nervous system, play essential roles in maintaining brain function. Reprogramming induced pluripotent stem cells (iPSCs) to become astrocytes through overexpression of the transcription factors, NFIB and SOX9, is a rapid and efficient approach for studying human neurological diseases and identifying therapeutic targets. However, the precise differentiation path and molecular signatures of induced astrocytes remain incompletely understood. Accordingly, we performed single-cell RNA sequencing analysis on 64,736 cells to establish a comprehensive atlas of NFIB/SOX9-directed astrocyte differentiation from human iPSCs. Our dataset provides detailed information about the path of astrocyte differentiation, highlighting the stepwise molecular changes that occur throughout the differentiation process. This dataset serves as a valuable reference for dissecting uncharacterized transcriptomic features of NFIB/SOX9-induced astrocytes and investigating lineage progression during astrocyte differentiation. Moreover, these findings pave the way for future studies on neurological diseases using the NFIB/SOX9-induced astrocyte model.
Assuntos
Astrócitos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição NFI , Fatores de Transcrição SOX9 , Transcriptoma , Fatores de Transcrição SOX9/genética , Astrócitos/metabolismo , Astrócitos/citologia , Humanos , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Análise de Célula ÚnicaRESUMO
During embryonic development of the retina of the eye, astrocytes, a type of glial cell, migrate over the retinal surface and form a dynamic mesh. This mesh then serves as scaffolding for blood vessels to form the retinal vasculature network that supplies oxygen and nutrients to the inner portion of the retina. Astrocyte spreading proceeds in a radially symmetric manner over the retinal surface. Additionally, astrocytes mature from astrocyte precursor cells (APCs) to immature perinatal astrocytes (IPAs) during this embryonic stage. We extend a previously-developed continuum model that describes tension-driven migration and oxygen and growth factor influenced proliferation and differentiation. Comparing numerical simulations to experimental data, we identify model equation components that can be removed via model reduction using approximate Bayesian computation (ABC). Our results verify experimental studies indicating that the choroid oxygen supply plays a negligible role in promoting differentiation of APCs into IPAs and in promoting IPA proliferation, and the hyaloid artery oxygen supply and APC apoptosis play negligible roles in astrocyte spreading and differentiation.
Assuntos
Astrócitos , Teorema de Bayes , Diferenciação Celular , Movimento Celular , Simulação por Computador , Conceitos Matemáticos , Modelos Biológicos , Retina , Astrócitos/citologia , Astrócitos/fisiologia , Movimento Celular/fisiologia , Animais , Diferenciação Celular/fisiologia , Retina/citologia , Retina/embriologia , Proliferação de Células/fisiologia , Oxigênio/metabolismo , CamundongosRESUMO
Calcium imaging has become a popular way to probe astrocyte activity, but few techniques holistically capture discrete calcium signals occurring across the astrocyte domain. Here, we introduce STARDUST, a pipeline for the spatio-temporal analysis of regional dynamics and unbiased sorting of transients from fluorescence recordings of astrocytes. We describe steps for installing software, detecting active pixel patches, obtaining region of activity (ROA) maps, and extracting time series from ROAs. We then detail procedures for extracting signal features using custom-made code.
Assuntos
Astrócitos , Sinalização do Cálcio , Cálcio , Software , Astrócitos/metabolismo , Astrócitos/citologia , Cálcio/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Análise Espaço-Temporal , CamundongosRESUMO
Mosaic Analysis with Double Markers (MADM) is a powerful genetic method typically used for lineage tracing and to disentangle cell autonomous and tissue-wide roles of candidate genes with single cell resolution. Given the relatively sparse labeling, depending on which of the 19 MADM chromosomes one chooses, the MADM approach represents the perfect opportunity for cell morphology analysis. Various MADM studies include reports of morphological anomalies and phenotypes in the central nervous system (CNS). MADM for any candidate gene can easily incorporate morphological analysis within the experimental workflow. Here, we describe the methods of morphological cell analysis which we developed in the course of diverse recent MADM studies. This chapter will specifically focus on methods to quantify aspects of the morphology of neurons and astrocytes within the CNS, but these methods can broadly be applied to any MADM-labeled cells throughout the entire organism. We will cover two analyses-soma volume and dendrite characterization-of physical characteristics of pyramidal neurons in the somatosensory cortex, and two analyses-volume and Sholl analysis-of astrocyte morphology.
Assuntos
Astrócitos , Neuroglia , Neurônios , Animais , Neurônios/citologia , Neurônios/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Camundongos , Mosaicismo , Biomarcadores , Dendritos/metabolismo , Córtex Somatossensorial/citologiaRESUMO
Understanding the intracellular dynamics of brain cells entails performing three-dimensional molecular simulations incorporating ultrastructural models that can capture cellular membrane geometries at nanometer scales. While there is an abundance of neuronal morphologies available online, e.g. from NeuroMorpho.Org, converting those fairly abstract point-and-diameter representations into geometrically realistic and simulation-ready, i.e. watertight, manifolds is challenging. Many neuronal mesh reconstruction methods have been proposed; however, their resulting meshes are either biologically unplausible or non-watertight. We present an effective and unconditionally robust method capable of generating geometrically realistic and watertight surface manifolds of spiny cortical neurons from their morphological descriptions. The robustness of our method is assessed based on a mixed dataset of cortical neurons with a wide variety of morphological classes. The implementation is seamlessly extended and applied to synthetic astrocytic morphologies that are also plausibly biological in detail. Resulting meshes are ultimately used to create volumetric meshes with tetrahedral domains to perform scalable in silico reaction-diffusion simulations for revealing cellular structure-function relationships. Availability and implementation: Our method is implemented in NeuroMorphoVis, a neuroscience-specific open source Blender add-on, making it freely accessible for neuroscience researchers.
Assuntos
Simulação por Computador , Neurônios , Neurônios/ultraestrutura , Neurônios/citologia , Modelos Neurológicos , Humanos , Animais , Astrócitos/citologia , Astrócitos/ultraestruturaRESUMO
Alzheimer's disease (AD) is marked by the aggregation of extracellular amyloid-ß (Aß) and astrocyte dysfunction. For Aß oligomers or aggregates to be formed, there must be Aß monomers present; however, the roles of monomeric Aß (mAß) and oligomeric Aß (oAß) in astrocyte pathogenesis are poorly understood. We cultured astrocytes in a brain-mimicking three-dimensional (3D) extracellular matrix and revealed that both mAß and oAß caused astrocytic atrophy and hyper-reactivity, but showed distinct Ca2+ changes in astrocytes. This 3D culture evolved into a microfluidic glymphatics-on-chip model containing astrocytes and endothelial cells with the interstitial fluid (ISF). The glymphatics-on-chip model not only reproduced the astrocytic atrophy, hyper-reactivity, and Ca2+ changes induced by mAß and oAß, but recapitulated that the components of the dystrophin-associated complex (DAC) and aquaporin-4 (AQP4) were properly maintained by the ISF, and dysregulated by mAß and oAß. Collectively, mAß and oAß cause distinct AD pathophysiological characteristics in the astrocytes.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Astrócitos , Dispositivos Lab-On-A-Chip , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/citologia , Peptídeos beta-Amiloides/metabolismo , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sistema Glinfático/metabolismo , Sistema Glinfático/patologia , Cálcio/metabolismo , Células Cultivadas , Aquaporina 4/metabolismoRESUMO
Astrocytes are responsible for maintaining homoeostasis and cognitive functions through calcium signalling, a process that is altered in brain diseases. Current bioelectronic tools are designed to study neurons and are not suitable for controlling calcium signals in astrocytes. Here, we show that electrical stimulation of astrocytes using electrodes coated with graphene oxide and reduced graphene oxide induces respectively a slow response to calcium, mediated by external calcium influx, and a sharp one, exclusively due to calcium release from intracellular stores. Our results suggest that the different conductivities of the substrate influence the electric field at the cell-electrolyte or cell-material interfaces, favouring different signalling events in vitro and ex vivo. Patch-clamp, voltage-sensitive dye and calcium imaging data support the proposed model. In summary, we provide evidence of a simple tool to selectively control distinct calcium signals in brain astrocytes for straightforward investigations in neuroscience and bioelectronic medicine.
Assuntos
Astrócitos , Encéfalo , Sinalização do Cálcio , Estimulação Elétrica , Eletrodos , Grafite , Astrócitos/metabolismo , Astrócitos/citologia , Grafite/química , Grafite/farmacologia , Animais , Encéfalo/metabolismo , Encéfalo/citologia , Cálcio/metabolismo , Células Cultivadas , Ratos , CamundongosAssuntos
Astrócitos , Sinalização do Cálcio , Eletrodos , Grafite , Grafite/química , Astrócitos/metabolismo , Astrócitos/citologia , Animais , Cálcio/metabolismoRESUMO
Despite recent technological advances in drug discovery, the success rate for neurotherapeutics remains alarmingly low compared to treatments for other areas of the body. One of the biggest challenges for delivering therapeutics to the central nervous system (CNS) is the presence of the blood-brain barrier (BBB). In vitro blood-brain barrier models with high predictability are essential to aid in designing parameters for new therapeutics, assess their ability to cross the BBB, and investigate therapeutic strategies that can be employed to enhance transport. Here, we demonstrate the development of a 3D printable hydrogel blood-brain barrier model that mimics the cellular composition and structure of the blood-brain barrier with human brain endothelial cells lining the surface, pericytes in direct contact with the endothelial cells on the abluminal side of the endothelium, and astrocytes in the surrounding printed bulk matrix. We introduce a simple, static printed hemi-cylinder model to determine design parameters such as media selection, co-culture ratios, and cell incorporation timing in a resource-conservative and high-throughput manner. Presence of cellular adhesion junction, VE-Cadherin, efflux transporters, P-glycoprotein (P-gp) and Breast cancer resistance protein (BCRP), and receptor-mediated transporters, Transferrin receptor (TfR) and low-density lipoprotein receptor-related protein 1 (LRP1) were confirmed via immunostaining demonstrating the ability of this model for screening in therapeutic strategies that rely on these transport systems. Design parameters determined in the hemi-cylinder model were translated to a more complex, perfusable vessel model to demonstrate its utility for determining barrier function and assessing permeability to model therapeutic compounds. This 3D-printed blood-brain barrier model represents one of the first uses of projection stereolithography to fabricate a perfusable blood-brain barrier model, enabling the patterning of complex vessel geometries and precise arrangement of cell populations. This model demonstrates potential as a new platform to investigate the delivery of neurotherapeutic compounds and drug delivery strategies through the blood-brain barrier, providing a useful in vitro screening tool in central nervous system drug discovery and development.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Impressão Tridimensional , Barreira Hematoencefálica/metabolismo , Humanos , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Técnicas de Cocultura , Hidrogéis/química , Modelos Biológicos , Astrócitos/metabolismo , Astrócitos/citologia , Pericitos/metabolismo , Pericitos/citologiaRESUMO
Intravascularly transplanted bone marrow cells, including bone marrow mononuclear cells (BM-MNC) and mesenchymal stem cells, transfer water-soluble molecules to cerebral endothelial cells via gap junctions. After transplantation of BM-MNC, this fosters hippocampal neurogenesis and enhancement of neuronal function. Herein, we report the impact of transplanted BM-MNC on neural stem cells (NSC) in the brain. Surprisingly, direct transfer of water-soluble molecules from transplanted BM-MNC and peripheral mononuclear cells to NSC in the hippocampus was observed already 10 min after cell transplantation, and transfer from BM-MNC to GFAP-positive cortical astrocytes was also observed. In vitro investigations revealed that BM-MNC abolish the expression of hypoxia-inducible factor-1α in astrocytes. We suggest that the transient and direct transfer of water-soluble molecules between cells in circulation and NSC in the brain may be one of the biological mechanisms underlying the repair of brain function.
Assuntos
Astrócitos , Células da Medula Óssea , Transplante de Medula Óssea , Hipocampo , Células-Tronco Neurais , Animais , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/transplante , Hipocampo/metabolismo , Hipocampo/citologia , Camundongos , Astrócitos/metabolismo , Astrócitos/citologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Água/metabolismo , Solubilidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos Endogâmicos C57BL , Células CultivadasRESUMO
Alzheimer's disease is the leading cause of dementia worldwide, but the cellular pathways that underlie its pathological progression across brain regions remain poorly understood1-3. Here we report a single-cell transcriptomic atlas of six different brain regions in the aged human brain, covering 1.3 million cells from 283 post-mortem human brain samples across 48 individuals with and without Alzheimer's disease. We identify 76 cell types, including region-specific subtypes of astrocytes and excitatory neurons and an inhibitory interneuron population unique to the thalamus and distinct from canonical inhibitory subclasses. We identify vulnerable populations of excitatory and inhibitory neurons that are depleted in specific brain regions in Alzheimer's disease, and provide evidence that the Reelin signalling pathway is involved in modulating the vulnerability of these neurons. We develop a scalable method for discovering gene modules, which we use to identify cell-type-specific and region-specific modules that are altered in Alzheimer's disease and to annotate transcriptomic differences associated with diverse pathological variables. We identify an astrocyte program that is associated with cognitive resilience to Alzheimer's disease pathology, tying choline metabolism and polyamine biosynthesis in astrocytes to preserved cognitive function late in life. Together, our study develops a regional atlas of the ageing human brain and provides insights into cellular vulnerability, response and resilience to Alzheimer's disease pathology.
Assuntos
Doença de Alzheimer , Encéfalo , Perfilação da Expressão Gênica , Análise de Célula Única , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Camundongos , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Astrócitos/classificação , Astrócitos/citologia , Astrócitos/metabolismo , Astrócitos/patologia , Autopsia , Encéfalo/anatomia & histologia , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Colina/metabolismo , Cognição/fisiologia , Redes Reguladoras de Genes , Interneurônios/classificação , Interneurônios/citologia , Interneurônios/metabolismo , Interneurônios/patologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Inibição Neural , Neurônios/classificação , Neurônios/citologia , Neurônios/metabolismo , Neurônios/patologia , Poliaminas/metabolismo , Proteína Reelina , Transdução de Sinais , Tálamo/citologia , Tálamo/metabolismo , Tálamo/patologia , TranscriptomaRESUMO
Adult neural stem cells (NSCs) reside in the dentate gyrus of the hippocampus, and their capacity to generate neurons and glia plays a role in learning and memory. In addition, neurodegenerative diseases are known to be caused by a loss of neurons and glial cells, resulting in a need to better understand stem cell fate commitment processes. We previously showed that NSC fate commitment toward a neuronal or glial lineage is strongly influenced by extracellular matrix stiffness, a property of elastic materials. However, tissues in vivo are not purely elastic and have varying degrees of viscous character. Relatively little is known about how the viscoelastic properties of the substrate impact NSC fate commitment. Here, we introduce a polyacrylamide-based cell culture platform that incorporates mismatched DNA oligonucleotide-based cross-links as well as covalent cross-links. This platform allows for tunable viscous stress relaxation properties via variation in the number of mismatched base pairs. We find that NSCs exhibit increased astrocytic differentiation as the degree of stress relaxation is increased. Furthermore, culturing NSCs on increasingly stress-relaxing substrates impacts cytoskeletal dynamics by decreasing intracellular actin flow rates and stimulating cyclic activation of the mechanosensitive protein RhoA. Additionally, inhibition of motor-clutch model components such as myosin II and focal adhesion kinase partially or completely reverts cells to lineage distributions observed on elastic substrates. Collectively, our results introduce a unique system for controlling matrix stress relaxation properties and offer insight into how NSCs integrate viscoelastic cues to direct fate commitment.
Assuntos
Diferenciação Celular , Células-Tronco Neurais , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Astrócitos/fisiologia , Camundongos , Resinas Acrílicas/química , Proteína rhoA de Ligação ao GTP/metabolismo , Células Cultivadas , Neurônios/metabolismo , Neurônios/fisiologia , Neurônios/citologia , Matriz Extracelular/metabolismo , Estresse MecânicoRESUMO
BACKGROUND: In the intricate pathological milieu post-spinal cord injury (SCI), neural stem cells (NSCs) frequently differentiate into astrocytes rather than neurons, significantly limiting nerve repair. Hence, the utilization of biocompatible hydrogel scaffolds in conjunction with exogenous factors to foster the differentiation of NSCs into neurons has the potential for SCI repair. METHODS: In this study, we engineered a 3D-printed porous SilMA hydrogel scaffold (SM) supplemented with pH-/temperature-responsive paclitaxel nanoparticles (PTX-NPs). We analyzed the biocompatibility of a specific concentration of PTX-NPs and its effect on NSC differentiation. We also established an SCI model to explore the ability of composite scaffolds for in vivo nerve repair. RESULTS: The physical adsorption of an optimal PTX-NPs dosage can simultaneously achieve pH/temperature-responsive release and commendable biocompatibility, primarily reflected in cell viability, morphology, and proliferation. An appropriate PTX-NPs concentration can steer NSC differentiation towards neurons over astrocytes, a phenomenon that is also efficacious in simulated injury settings. Immunoblotting analysis confirmed that PTX-NPs-induced NSC differentiation occurred via the MAPK/ERK signaling cascade. The repair of hemisected SCI in rats demonstrated that the composite scaffold augmented neuronal regeneration at the injury site, curtailed astrocyte and fibrotic scar production, and enhanced motor function recovery in rat hind limbs. CONCLUSION: The scaffold's porous architecture serves as a cellular and drug carrier, providing a favorable microenvironment for nerve regeneration. These findings corroborate that this strategy amplifies neuronal expression within the injury milieu, significantly aiding in SCI repair.
Assuntos
Diferenciação Celular , Hidrogéis , Nanopartículas , Células-Tronco Neurais , Neurônios , Paclitaxel , Ratos Sprague-Dawley , Traumatismos da Medula Espinal , Alicerces Teciduais , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Paclitaxel/farmacologia , Alicerces Teciduais/química , Diferenciação Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Nanopartículas/química , Hidrogéis/química , Hidrogéis/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Porosidade , Ratos , Regeneração Nervosa/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/citologiaRESUMO
Toll-like receptors 3 (TLR3) are innate immune receptors expressed on a wide range of cell types, including glial cells. Inflammatory responses altered by hyperglycemia highlight the need to explore the molecular underpinnings of these changes in cellular models. Therefore, here we estimated TLR3-mediated response of astrocytes cultured at normal (NG, 5 mM) and high (HG, 22.5 mM) glucose concentrations for 48 h before stimulation with polyinosinic:polycytidylic acid Poly(I:C) (PIC) for 6 h. Seahorse Extracellular Flux Analyzer (Seahorse XFp) was used to estimate the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Although adaptation to HG affected ECAR and OCR, the stimulation of cells with PIC had no effect on ECAR. PIC reduced maximal OCR, but this effect disappeared upon adaptation to HG. PIC-stimulated release of cytokines IL-1ß, IL-10 was reduced, and that of IL-6 and iNOS was increased in the HG model. Adaptation to HG reduced PIC-stimulated synthesis of COX-derived oxylipins measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Adaptation to HG did not alter PIC-stimulated p38 activity, ERK mitogen-activated protein kinase, STAT3 and ROS production. Metformin exhibited anti-inflammatory activity, reducing PIC-stimulated synthesis of cytokines and oxylipins. Cell adaptation to high glucose concentration altered the sensitivity of astrocytes to TLR3 receptor activation, and the hypoglycemic drug metformin may exert anti-inflammatory effects under these conditions.
Assuntos
Astrócitos , Glucose , Metformina , Poli I-C , Receptor 3 Toll-Like , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/citologia , Metformina/farmacologia , Receptor 3 Toll-Like/metabolismo , Glucose/metabolismo , Animais , Poli I-C/farmacologia , Células Cultivadas , Oxilipinas/farmacologia , Oxilipinas/metabolismo , Camundongos , Consumo de Oxigênio/efeitos dos fármacos , Citocinas/metabolismoRESUMO
Astrocytes in the cerebrum play important roles such as the regulation of synaptic functions, homeostasis, water transport, and the blood-brain barrier. It has been proposed that astrocytes in the cerebrum acquired diversity and developed functionally during evolution. Here, we show that like human astrocytes, ferret astrocytes in the cerebrum exhibit various morphological subtypes which mice do not have. We found that layer 1 of the ferret cerebrum contained not only protoplasmic astrocytes but also pial interlaminar astrocytes and subpial interlaminar astrocytes. Morphologically polarized astrocytes, which have a long unbranched process, were found in layer 6. Like human white matter, ferret white matter exhibited four subtypes of astrocytes. Furthermore, our quantification showed that ferret astrocytes had a larger territory size and a longer radius length than mouse astrocytes. Thus, our results indicate that, similar to the human cerebrum, the ferret cerebrum has a well-developed diversity of astrocytes. Ferrets should be useful for investigating the molecular and cellular mechanisms leading to astrocyte diversity, the functions of each astrocyte subtype and the involvement of different astrocyte subtypes in various neurological diseases.
Assuntos
Astrócitos , Furões , Animais , Astrócitos/metabolismo , Astrócitos/citologia , Cérebro/anatomia & histologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/genética , Camundongos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Substância Branca/citologia , Substância Branca/anatomia & histologiaRESUMO
As the main players in the central nervous system (CNS), neurons dominate most life activities. However, after accidental trauma or neurodegenerative diseases, neurons are unable to regenerate themselves. The loss of this important role can seriously affect the quality of life of patients, ranging from movement disorders to disability and even death. There is no suitable treatment to prevent or reverse this process. Therefore, the regeneration of neurons after loss has been a major clinical problem and the key to treatment. Replacing the lost neurons by transdifferentiation of other cells is the only viable approach. Although much progress has been made in stem cell therapy, ethical issues, immune rejection, and limited cell sources still hinder its clinical application. In recent years, somatic cell reprogramming technology has brought a new dawn. Among them, astrocytes, as endogenously abundant cells homologous to neurons, have good potential and application value for reprogramming into neurons, having been reprogrammed into neurons in vitro and in vivo in a variety of ways.
Assuntos
Astrócitos , Reprogramação Celular , Neurônios , Humanos , Astrócitos/metabolismo , Astrócitos/fisiologia , Astrócitos/citologia , Animais , Neurônios/fisiologia , Neurônios/metabolismo , Neurônios/citologia , Reprogramação Celular/fisiologia , Transdiferenciação Celular/fisiologiaRESUMO
Thyroid hormone (TH) is a critical regulator of cellular function and cell fate. The circulating TH level is relatively stable, while tissue TH action fluctuates according to cell type-specific mechanisms. Here, we focused on identifying mechanisms that regulate TH action through the type 2 deiodinase (D2) in glial cells. Dio2 mRNA has an unusually long 3'UTR where we identified multiple putative MSI1 binding sites for Musashi-1 (MSI1), a highly conserved RNA-binding cell cycle regulator. Binding to these sites was confirmed through electrophoretic mobility shift assay. In H4 glioma cells, shRNA-mediated MSI1 knockdown increased endogenous D2 activity, whereas MSI1 overexpression in HEK293T cells decreased D2 expression. This latter effect could be prevented by the deletion of a 3.6 kb region of the 3'UTR of Dio2 mRNA containing MSI1 binding sites. MSI1 immunoreactivity was observed in 2 mouse Dio2-expressing cell types, that is, cortical astrocytes and hypothalamic tanycytes, establishing the anatomical basis for a potential in vivo interaction of Dio2 mRNA and MSl1. Indeed, increased D2 expression was observed in the cortex of mice lacking MSI1 protein. Furthermore, MSI1 knockdown-induced D2 expression slowed down cell proliferation by 56% in primary cultures of mouse cortical astrocytes, establishing the functionality of the MSI1-D2-T3 pathway. In summary, Dio2 mRNA is a target of MSI1 and the MSI1-D2-T3 pathway is a novel regulatory mechanism of astrocyte proliferation with the potential to regulate the pathogenesis of human glioblastoma.
Assuntos
Astrócitos , Proliferação de Células , Iodotironina Desiodinase Tipo II , Proteínas do Tecido Nervoso , Proteínas de Ligação a RNA , Animais , Humanos , Camundongos , Regiões 3' não Traduzidas , Astrócitos/metabolismo , Astrócitos/citologia , Linhagem Celular Tumoral , Células HEK293 , Iodeto Peroxidase/metabolismo , Iodeto Peroxidase/genética , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/genéticaRESUMO
The Drosophila melanogaster brain is a complex organ with various cell types, orchestrating the development, physiology, and behaviors of the fly. While each cell type in Drosophila brain is known to express a unique gene set, their complete genetic profile is still unknown. Advances in the RNA sequencing techniques at single-cell resolution facilitate identifying novel cell type markers and/or re-examining the specificity of the available ones. In this study, exploiting a single-cell RNA sequencing data of Drosophila optic lobe, we categorized the cells based on their expression pattern for known markers, then the genes with enriched expression in astrocytes were identified. CG11000 was identified as a gene with a comparable expression profile to the Eaat1 gene, an astrocyte marker, in every individual cell inside the Drosophila optic lobe and midbrain, as well as in the entire Drosophila brain throughout its development. Consistent with our bioinformatics data, immunostaining of the brains dissected from transgenic adult flies showed co-expression of CG11000 with Eaat1 in a set of single cells corresponding to the astrocytes in the Drosophila brain. Physiologically, inhibiting CG11000 through RNA interference disrupted the normal development of male D. melanogaster, while having no impact on females. Expression suppression of CG11000 in adult flies led to decreased locomotion activity and also shortened lifespan specifically in astrocytes, indicating the gene's significance in astrocytes. We designated this gene as 'deathstar' due to its crucial role in maintaining the star-like shape of glial cells, astrocytes, throughout their development into adult stage.
Assuntos
Astrócitos , Proteínas de Drosophila , Drosophila melanogaster , Locomoção , Longevidade , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Astrócitos/metabolismo , Astrócitos/citologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Longevidade/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 1 de Aminoácido Excitatório/genética , Masculino , Feminino , Encéfalo/metabolismo , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimentoRESUMO
Astrocytes, the most abundant glial cell type in the brain, play crucial roles in maintaining homeostasis within the central nervous system (CNS). Impairment or abnormalities of typical astrocyte functions in the CNS serve as a causative or contributing factor in numerous neurodevelopmental, neurodegenerative, and neuropsychiatric disorders. Currently, disease-modeling and drug-screening approaches, primarily focused on human astrocytes, rely on human pluripotent stem cell (hPSC)-derived astrocytes. However, it is important to acknowledge that these hPSC-derived astrocytes exhibit notable differences across studies and when compared to their in vivo counterparts. These differences may potentially compromise translational outcomes if not carefully accounted for. This review aims to explore state-of-the-art in vitro models of human astrocyte development, focusing on the developmental processes, functional maturity, and technical aspects of various hPSC-derived astrocyte differentiation protocols. Additionally, it summarizes their successful application in modeling neurological disorders. The discussion extends to recent advancements in the large-scale production of human astrocytes and their application in developing high-throughput assays conducive to therapeutic drug discovery.
Assuntos
Astrócitos , Diferenciação Celular , Doenças do Sistema Nervoso , Células-Tronco Pluripotentes , Humanos , Astrócitos/metabolismo , Astrócitos/citologia , Doenças do Sistema Nervoso/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Pesquisa Translacional Biomédica , AnimaisRESUMO
Astrocytes are ubiquitous in the brain and spinal cord and display a complex morphology important for the local interactions with neighboring cells, resulting in the modulation of circuit function. Thus, studies focusing on astrocyte physiology in the healthy and diseased brain generally present analyses of astrocytic structure. The labeling method used to visualize the astrocytic structure defines the morphological level to observe and may vary depending on the anatomical sub-regions. The method choice may significantly affect our understanding of their structural diversity. The main goal of this work was to identify a straightforward and efficient protocol for labeling and reconstructing a detailed astrocytic structure to apply and validate in different brain tissue preparations across laboratories. For that, we explored different tissue processing protocols before GFAP labeling to determine the most effective method for reconstructing astrocytic backbones in the mouse hippocampus. Our results show that the reconstruction of astrocytic structure in vibratome sections labeled by free-floating immunofluorescence protocol provides a more practical method to achieve a higher level of detail and arbor complexity in astrocyte backbone reconstruction. Free-floating immunofluorescence labeling is the most reliable method for obtaining better antibody penetration and more detailed astrocyte structure. Finally, we also show that introducing an antigen retrieval step appears useful for visualizing more complete structural details.