RESUMEN
Fate and behavior of neural progenitor cells are tightly regulated during mammalian brain development. Metabolic pathways, such as glycolysis and oxidative phosphorylation, that are required for supplying energy and providing molecular building blocks to generate cells govern progenitor function. However, the role of de novo lipogenesis, which is the conversion of glucose into fatty acids through the multienzyme protein fatty acid synthase (FASN), for brain development remains unknown. Using Emx1Cre-mediated, tissue-specific deletion of Fasn in the mouse embryonic telencephalon, we show that loss of FASN causes severe microcephaly, largely due to altered polarity of apical, radial glia progenitors and reduced progenitor proliferation. Furthermore, genetic deletion and pharmacological inhibition of FASN in human embryonic stem cell-derived forebrain organoids identifies a conserved role of FASN-dependent lipogenesis for radial glia cell polarity in human brain organoids. Thus, our data establish a role of de novo lipogenesis for mouse and human brain development and identify a link between progenitor-cell polarity and lipid metabolism.
Asunto(s)
Encéfalo/metabolismo , Ácido Graso Sintasas/metabolismo , Lipogénesis/fisiología , Animales , Tipificación del Cuerpo , Encéfalo/crecimiento & desarrollo , Encéfalo/patología , Acido Graso Sintasa Tipo I , Ácido Graso Sintasas/genética , Humanos , Metabolismo de los Lípidos , Lipogénesis/genética , Ratones , Ratones Noqueados , Células-Madre Neurales/metabolismo , Neuroglía/metabolismo , Neuroglía/patología , TranscriptomaRESUMEN
The division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. Here, we developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo. Analyzing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT can provide novel insights into stem cell behavior. Further, we use single-cell RNA sequencing (scRNA-seq) of iCOUNT-labeled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify functionally relevant molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a tool to characterize the molecular consequences of repeated cell divisions of stem cells that allows an analysis of the cellular principles underlying tissue formation, homeostasis, and repair.
Asunto(s)
Células-Madre Neurales , Animales , Encéfalo , División Celular , Proliferación Celular , Ratones , Organoides , Análisis de Secuencia de ARNRESUMEN
Neural stem cells (NSCs) generate neurons throughout life in the hippocampal dentate gyrus. With advancing age, levels of neurogenesis sharply drop, which has been associated with a decline in hippocampal memory function. However, cell-intrinsic mechanisms mediating age-related changes in NSC activity remain largely unknown. Here, we show that the nuclear lamina protein lamin B1 (LB1) is downregulated with age in mouse hippocampal NSCs, whereas protein levels of SUN-domain containing protein 1 (SUN1), previously implicated in Hutchinson-Gilford progeria syndrome (HGPS), increase. Balancing the levels of LB1 and SUN1 in aged NSCs restores the strength of the endoplasmic reticulum diffusion barrier that is associated with segregation of aging factors in proliferating NSCs. Virus-based restoration of LB1 expression in aged NSCs enhances stem cell activity in vitro and increases progenitor cell proliferation and neurogenesis in vivo. Thus, we here identify a mechanism that mediates age-related decline of neurogenesis in the mammalian hippocampus.
Asunto(s)
Envejecimiento , Lamina Tipo B , Células-Madre Neurales , Progeria , Animales , Hipocampo/citología , Ratones , NeurogénesisRESUMEN
Neural stem cells (NSCs) generate neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. We used two-photon microscopy and followed NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI family zinc finger 1 (Gli1) or achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny, we identify a population of Gli1-targeted NSCs showing long-term self-renewal in the adult hippocampus. In contrast, once activated, Ascl1-targeted NSCs undergo limited proliferative activity before they become exhausted. Using single-cell RNA sequencing, we show that Gli1- and Ascl1-targeted cells have highly similar yet distinct transcriptional profiles, supporting the existence of heterogeneous NSC populations with diverse behavioral properties. Thus, we here identify long-term self-renewing NSCs that contribute to the generation of new neurons in the adult hippocampus.
Asunto(s)
Hipocampo/crecimiento & desarrollo , Células-Madre Neurales/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linaje de la Célula , Femenino , Perfilación de la Expresión Génica , Hipocampo/citología , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Microscopía Intravital , Masculino , Metalotioneína 3 , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía de Fluorescencia por Excitación Multifotónica , Regeneración Nerviosa , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Análisis de la Célula Individual , Proteína con Dedos de Zinc GLI1/biosíntesis , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Currently, researchers rely on generalized methods to quantify transposable element (TE) RNA expression, such as RT-qPCR and RNA-seq, that do not distinguish between TEs expressed from their own promoter (bona fide) and TEs that are transcribed from a neighboring gene promoter such as within an intron or exon. This distinction is important owing to the differing functional roles of TEs depending on whether they are independently transcribed. Here we report a simple strategy to examine bona fide TE expression, termed BonaFide-TEseq. This approach can be used with any template-switch based library such as Smart-seq2 or the single-cell 5' gene expression kit from 10x, extending its utility to single-cell RNA-sequencing. This approach does not require TE-specific enrichment, enabling the simultaneous examination of TEs and protein-coding genes. We show that TEs identified through BonaFide-TEseq are expressed from their own promoter, rather than captured as internal products of genes. We reveal the utility of BonaFide-TEseq in the analysis of single-cell data and show that short-interspersed nuclear elements (SINEs) show cell type-specific expression profiles in the mouse hippocampus. We further show that, in response to a brief exposure of home-cage mice to a novel stimulus, SINEs are activated in dentate granule neurons in a time course that is similar to that of protein-coding immediate early genes. This work provides a simple alternative approach to assess bona fide TE transcription at single-cell resolution and provides a proof-of-concept using this method to identify SINE activation in a context that is relevant for normal learning and memory.
Asunto(s)
Hipocampo/metabolismo , RNA-Seq , Elementos de Nucleótido Esparcido Corto , Análisis de la Célula Individual , Transcripción Genética , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Hipocampo/citología , Hipocampo/fisiología , Ratones , Regiones Promotoras GenéticasRESUMEN
This protocol presents a plate-based workflow to perform RNA sequencing analysis of single cells/nuclei using Smart-seq2. We describe (1) the dissociation procedures for cell/nucleus isolation from the mouse brain and human organoids, (2) the flow sorting of single cells/nuclei into 384-well plates, and (3) the preparation of libraries following miniaturization of the Smart-seq2 protocol using a liquid-handling robot. This pipeline allows for the reliable, high-throughput, and cost-effective preparation of mouse and human samples for full-length deep single-cell/nucleus RNA sequencing. For complete details on the use and execution of this protocol, please refer to Bowers et al. (2020).
Asunto(s)
Análisis de Secuencia de ARN/instrumentación , Análisis de la Célula Individual/instrumentación , Análisis de la Célula Individual/métodos , Animales , Secuencia de Bases/genética , Encéfalo/citología , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Separación Celular/métodos , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Ratones , Miniaturización , ARN/genética , Análisis de Secuencia de ARN/métodos , Transcriptoma/genética , Secuenciación del Exoma/métodos , Flujo de TrabajoRESUMEN
Altered neural stem/progenitor cell (NSPC) activity and neurodevelopmental defects are linked to intellectual disability. However, it remains unclear whether altered metabolism, a key regulator of NSPC activity, disrupts human neurogenesis and potentially contributes to cognitive defects. We investigated links between lipid metabolism and cognitive function in mice and human embryonic stem cells (hESCs) expressing mutant fatty acid synthase (FASN; R1819W), a metabolic regulator of rodent NSPC activity recently identified in humans with intellectual disability. Mice homozygous for the FASN R1812W variant have impaired adult hippocampal NSPC activity and cognitive defects because of lipid accumulation in NSPCs and subsequent lipogenic ER stress. Homozygous FASN R1819W hESC-derived NSPCs show reduced rates of proliferation in embryonic 2D cultures and 3D forebrain regionalized organoids, consistent with a developmental phenotype. These data from adult mouse models and in vitro models of human brain development suggest that altered lipid metabolism contributes to intellectual disability.
Asunto(s)
Metabolismo de los Lípidos , Células-Madre Neurales , Animales , Proliferación Celular , Ácido Graso Sintasas , Hipocampo , Trastornos de la Memoria , Ratones , NeurogénesisRESUMEN
Autism spectrum disorder (ASD) is thought to emerge during early cortical development. However, the exact developmental stages and associated molecular networks that prime disease propensity are elusive. To profile early neurodevelopmental alterations in ASD with macrocephaly, we monitored subject-derived induced pluripotent stem cells (iPSCs) throughout the recapitulation of cortical development. Our analysis revealed ASD-associated changes in the maturational sequence of early neuron development, involving temporal dysregulation of specific gene networks and morphological growth acceleration. The observed changes tracked back to a pathologically primed stage in neural stem cells (NSCs), reflected by altered chromatin accessibility. Concerted over-representation of network factors in control NSCs was sufficient to trigger ASD-like features, and circumventing the NSC stage by direct conversion of ASD iPSCs into induced neurons abolished ASD-associated phenotypes. Our findings identify heterochronic dynamics of a gene network that, while established earlier in development, contributes to subsequent neurodevelopmental aberrations in ASD.
Asunto(s)
Trastorno del Espectro Autista/genética , Redes Reguladoras de Genes , Potenciales Postsinápticos Inhibidores/fisiología , Red Nerviosa/fisiopatología , Neuronas/fisiología , Trastorno del Espectro Autista/patología , Trastorno del Espectro Autista/fisiopatología , Humanos , Células Madre Pluripotentes Inducidas/patología , Células-Madre Neurales/patología , Neuronas/patologíaRESUMEN
Activity-induced remodeling of neuronal circuits is critical for memory formation. This process relies in part on transcription, but neither the rate of activity nor baseline transcription is equal across neuronal cell types. In this study, we isolated mouse hippocampal populations with different activity levels and used single nucleus RNA-seq to compare their transcriptional responses to activation. One hour after novel environment exposure, sparsely active dentate granule (DG) neurons had a much stronger transcriptional response compared to more highly active CA1 pyramidal cells and vasoactive intestinal polypeptide (VIP) interneurons. Activity continued to impact transcription in DG neurons up to 5 h, with increased heterogeneity. By re-exposing the mice to the same environment, we identified a unique transcriptional signature that selects DG neurons for reactivation upon re-exposure to the same environment. These results link transcriptional heterogeneity to functional heterogeneity and identify a transcriptional correlate of memory encoding in individual DG neurons.
Asunto(s)
Giro Dentado/metabolismo , Regulación de la Expresión Génica , Memoria , Neuronas/metabolismo , Transcripción Genética , Animales , Región CA1 Hipocampal/citología , Gránulos Citoplasmáticos , Femenino , Perfilación de la Expresión Génica , Interneuronas , Ratones , Ratones Endogámicos C57BL , Modelos Neurológicos , Neurogénesis , Plasticidad Neuronal , Células Piramidales/metabolismo , Procesos Estocásticos , Factores de Tiempo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Mitochondria are a major target for aging and are instrumental in the age-dependent deterioration of the human brain, but studying mitochondria in aging human neurons has been challenging. Direct fibroblast-to-induced neuron (iN) conversion yields functional neurons that retain important signs of aging, in contrast to iPSC differentiation. Here, we analyzed mitochondrial features in iNs from individuals of different ages. iNs from old donors display decreased oxidative phosphorylation (OXPHOS)-related gene expression, impaired axonal mitochondrial morphologies, lower mitochondrial membrane potentials, reduced energy production, and increased oxidized proteins levels. In contrast, the fibroblasts from which iNs were generated show only mild age-dependent changes, consistent with a metabolic shift from glycolysis-dependent fibroblasts to OXPHOS-dependent iNs. Indeed, OXPHOS-induced old fibroblasts show increased mitochondrial aging features similar to iNs. Our data indicate that iNs are a valuable tool for studying mitochondrial aging and support a bioenergetic explanation for the high susceptibility of the brain to aging.
Asunto(s)
Envejecimiento/patología , Reprogramación Celular , Metabolómica , Mitocondrias/metabolismo , Neuronas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Células Cultivadas , Niño , Preescolar , Fibroblastos/citología , Regulación de la Expresión Génica , Genes Mitocondriales , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Fosforilación Oxidativa , Fenotipo , Donantes de Tejidos , Adulto JovenRESUMEN
Flavonoids are a class of plant-derived dietary polyphenols that have attracted attention for their pro-cognitive and anti-inflammatory effects. The diversity of flavonoids and their extensive in vivo metabolism suggest that a variety of cellular targets in the brain are likely to be impacted by flavonoid consumption. Initially characterized as antioxidants, flavonoids are now believed to act directly on neurons and glia via the interaction with major signal transduction cascades, as well as indirectly via interaction with the blood-brain barrier and cerebral vasculature. This review discusses potential mechanisms of flavonoid action in the brain, with a focus on two critical transcription factors: cAMP response element-binding protein (CREB) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). To advance beyond current understanding of cellular targets, critical bioavailability studies need to be performed to verify the identity and concentration of flavonoid metabolites reaching the brain after ingestion and to validate that these metabolites are produced not just in rodent models but also in humans. Recent advances in human induced pluripotent stem cell (iPSC) differentiation protocols to generate human neuronal and glial cell types could also provide a unique tool for clinically relevant in vitro investigation of the mechanisms of action of bioavailable flavonoid metabolites in humans.
Asunto(s)
Dieta , Flavonoides/farmacología , Inflamación/patología , Neuronas/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Humanos , Neuronas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1ß or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1ß. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration.
Asunto(s)
Astrocitos/citología , Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Células Madre/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Glutámico/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Interleucina-1beta/farmacología , Factor Inhibidor de Leucemia/farmacología , Microscopía Fluorescente , Neuronas/citología , Neuronas/metabolismo , Análisis de Componente Principal , ARN/química , ARN/aislamiento & purificación , ARN/metabolismo , Análisis de Secuencia de ARN , Células Madre/metabolismo , Transcriptoma , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Microglia play essential roles in central nervous system (CNS) homeostasis and influence diverse aspects of neuronal function. However, the transcriptional mechanisms that specify human microglia phenotypes are largely unknown. We examined the transcriptomes and epigenetic landscapes of human microglia isolated from surgically resected brain tissue ex vivo and after transition to an in vitro environment. Transfer to a tissue culture environment resulted in rapid and extensive down-regulation of microglia-specific genes that were induced in primitive mouse macrophages after migration into the fetal brain. Substantial subsets of these genes exhibited altered expression in neurodegenerative and behavioral diseases and were associated with noncoding risk variants. These findings reveal an environment-dependent transcriptional network specifying microglia-specific programs of gene expression and facilitate efforts to understand the roles of microglia in human brain diseases.
Asunto(s)
Ambiente , Redes Reguladoras de Genes/fisiología , Microglía/citología , Microglía/fisiología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatología , Células Cultivadas , Epilepsia/genética , Epilepsia/fisiopatología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo.
Asunto(s)
Neuronas/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma , Animales , Núcleo Celular/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Giro Dentado/citología , Giro Dentado/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Ontología de Genes , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Inmunohistoquímica , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Cells of the immune system have evolved various molecular mechanisms to sense their environment and react to alterations of self. NK cells are lymphocytes with effector and regulatory functions, which are remarkably adaptable to changes in self. In a study published in this issue of the JCI, Tarek and colleagues report the clinical benefits of manipulating NK cell adaptation to self in an innovative mAb-based therapy against neuroblastoma (NB). This novel therapeutic strategy should stimulate further research on NK cell therapies.
RESUMEN
A major challenge for the immune system is to control pathogens and stressed cells, such as infected or tumors cells, while sparing healthy self-cells. To achieve this tolerance to self, immune cells must recognize and differentiate "self" versus "nonself" and "self" versus "altered self." In the absence of self-tolerance, cells of the adaptive immune system attack healthy cells and cause autoimmune diseases such as lupus, psoriasis, and type I diabetes. Mechanisms at work to ensure tolerance in the innate immune system are still poorly understood. Natural killer cells are innate immune lymphocytes, which have the capacity to kill cellular targets and produce cytokines without prior specific sensitization. Because of these intrinsic effector capacities, tolerance mechanisms must exist to prevent autoreactivity. Herein, we will review the present knowledge on NK cell tolerance.
Asunto(s)
Células Asesinas Naturales/inmunología , Autotolerancia/fisiología , Animales , Humanos , Complejo Mayor de Histocompatibilidad/fisiología , Ratones , Modelos Inmunológicos , Receptores Inmunológicos/fisiologíaRESUMEN
Natural killer (NK) cells are bone marrow (BM)-derived granular lymphocytes involved in immune defense against microbial infections and tumors. In an N-ethyl N-nitrosourea (ENU) mutagenesis strategy, we identified a mouse mutant with impaired NK cell reactivity both in vitro and in vivo. Dissection of this phenotype showed that mature neutrophils were required both in the BM and in the periphery for proper NK cell development. In mice lacking neutrophils, NK cells displayed hyperproliferation and poor survival and were blocked at an immature stage associated with hyporesponsiveness. The role of neutrophils as key regulators of NK cell functions was confirmed in patients with severe congenital neutropenia and autoimmune neutropenia. In addition to their direct antimicrobial activity, mature neutrophils are thus endowed with immunoregulatory functions that are conserved across species. These findings reveal novel types of cooperation between cells of the innate immune system and prompt examination of NK cell functional deficiency in patients suffering from neutropenia-associated diseases.