RESUMEN
RNA-binding proteins (RNA-BPs) play critical roles in development and disease to regulate gene expression. However, genome-wide identification of their targets in primary human cells has been challenging. Here, we applied a modified CLIP-seq strategy to identify genome-wide targets of the FMRP translational regulator 1 (FMR1), a brain-enriched RNA-BP, whose deficiency leads to Fragile X Syndrome (FXS), the most prevalent inherited intellectual disability. We identified FMR1 targets in human dorsal and ventral forebrain neural progenitors and excitatory and inhibitory neurons differentiated from human pluripotent stem cells. In parallel, we measured the transcriptomes of the same four cell types upon FMR1 gene deletion. We discovered that FMR1 preferentially binds long transcripts in human neural cells. FMR1 targets include genes unique to human neural cells and associated with clinical phenotypes of FXS and autism. Integrative network analysis using graph diffusion and multitask clustering of FMR1 CLIP-seq and transcriptional targets reveals critical pathways regulated by FMR1 in human neural development. Our results demonstrate that FMR1 regulates a common set of targets among different neural cell types but also operates in a cell type-specific manner targeting distinct sets of genes in human excitatory and inhibitory neural progenitors and neurons. By defining molecular subnetworks and validating specific high-priority genes, we identify novel components of the FMR1 regulation program. Our results provide new insights into gene regulation by a critical neuronal RNA-BP in human neurodevelopment.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Trastorno Autístico/genética , Línea Celular , Secuenciación de Inmunoprecipitación de Cromatina , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Eliminación de Gen , Redes Reguladoras de Genes , Humanos , Masculino , Células-Madre Neurales/citología , Neurogénesis , Células Madre Pluripotentes/citología , Prosencéfalo/citología , Prosencéfalo/metabolismo , TranscriptomaRESUMEN
Astrocytes display diverse morphologies in different regions of the central nervous system. Whether astrocyte diversity is attributable to developmental processes and bears functional consequences, especially in humans, is unknown. RNA-seq of human pluripotent stem cell-derived regional astrocytes revealed distinct transcript profiles, suggesting differential functional properties. This was confirmed by differential calcium signaling as well as effects on neurite growth and blood-brain barrier formation. Distinct transcriptional profiles and functional properties of human astrocytes generated from regionally specified neural progenitors under the same conditions strongly implicate the developmental impact on astrocyte diversity. These findings provide a rationale for renewed examination of regional astrocytes and their role in the pathogenesis of psychiatric and neurological disorders.
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Astrocitos/fisiología , Diferenciación Celular/genética , Neurogénesis/genética , Células Madre Pluripotentes/fisiología , Transcriptoma , Secuencia de Bases , Biomarcadores/análisis , Biomarcadores/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Células-Madre Neurales/fisiología , Especificidad de Órganos/genética , Prosencéfalo/citología , Prosencéfalo/metabolismo , Análisis de Secuencia de ARNRESUMEN
The analysis of animal models of neurological disease has been instrumental in furthering our understanding of neurodevelopment and brain diseases. However, animal models are limited in revealing some of the most fundamental aspects of development, genetics, pathology, and disease mechanisms that are unique to humans. These shortcomings are exaggerated in disorders that affect the brain, where the most significant differences between humans and animal models exist, and could underscore failures in targeted therapeutic interventions in affected individuals. Human pluripotent stem cells have emerged as a much-needed model system for investigating human-specific biology and disease mechanisms. However, questions remain regarding whether these cell-culture-based models are sufficient or even necessary. In this review, we summarize human-specific features of neurodevelopment and the most common neurodevelopmental disorders, present discrepancies between animal models and human diseases, demonstrate how human stem cell models can provide meaningful information, and discuss the challenges that exist in our pursuit to understand distinctively human aspects of neurodevelopment and brain disease. This information argues for a more thoughtful approach to disease modeling through consideration of the valuable features and limitations of each model system, be they human or animal, to mimic disease characteristics.
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Trastornos del Neurodesarrollo/patología , Animales , Encéfalo/patología , Técnicas de Cultivo de Célula , Humanos , Células Madre Pluripotentes Inducidas/patología , Modelos BiológicosRESUMEN
Progress in addressing the origins of intellectual and developmental disabilities accelerated with the establishment 50 years ago of the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health and associated Intellectual and Developmental Disabilities Research Centers. Investigators at these Centers have made seminal contributions to understanding human brain and behavioral development and defining mechanisms and treatments of disorders of the developing brain. ANN NEUROL 2019;86:332-343.
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Academias e Institutos/historia , Discapacidades del Desarrollo , Discapacidad Intelectual , National Institute of Child Health and Human Development (U.S.)/historia , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Estados UnidosRESUMEN
Human patient-derived induced pluripotent stem cells (hiPSCs) provide unique opportunities for disease modeling and drug development. However, adapting hiPSCs or their differentiated progenies to high throughput assays for phenotyping or drug screening has been challenging. Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and a major genetic cause of autism. FXS is caused by mutational trinucleotide expansion in the FMR1 gene leading to hypermethylation and gene silencing. One potential therapeutic strategy is to reactivate the silenced FMR1 gene, which has been attempted using both candidate chemicals and cell-based screening. However, molecules that effectively reactivate the silenced FMR1 gene are yet to be identified; therefore, a high throughput unbiased screen is needed. Here we demonstrate the creation of a robust FMR1-Nluc reporter hiPSC line by knocking in a Nano luciferase (Nluc) gene into the endogenous human FMR1 gene using the CRISPR/Cas9 genome editing method. We confirmed that luciferase activities faithfully report FMR1 gene expression levels and showed that neural progenitor cells derived from this line could be optimized for high throughput screening. The FMR1-Nluc reporter line is a good resource for drug screening as well as for testing potential genetic reactivation strategies. In addition, our data provide valuable information for the generation of knockin human iPSC reporter lines for disease modeling, drug screening, and mechanistic studies. Stem Cells 2017;35:158-169.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Genes Reporteros , Neuronas/metabolismo , Activación Transcripcional , Azacitidina/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Humanos , Luciferasas/metabolismo , Nanopartículas/química , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Activación Transcripcional/efectos de los fármacosRESUMEN
Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism. The causal mutation in FXS is a trinucleotide CGG repeat expansion in the FMR1 gene that leads to human specific epigenetic silencing and loss of Fragile X Mental Retardation Protein (FMRP) expression. Human pluripotent stem cells (PSCs), including human embryonic stem cells (ESCs) and particularly induced PSCs (iPSCs), offer a model system to reveal cellular and molecular events underlying human neuronal development and function in FXS. Human FXS PSCs have been established and have provided insight into the epigenetic silencing of the FMR1 gene as well as aspects of neuronal development.
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Epigénesis Genética , Síndrome del Cromosoma X Frágil/patología , Células Madre Pluripotentes/metabolismo , Técnicas de Cultivo de Célula/métodos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Silenciador del Gen , Humanos , Modelos Biológicos , Células Madre Pluripotentes/citologíaRESUMEN
Down syndrome (trisomy 21) is the most common genetic cause of intellectual disability, but the precise molecular mechanisms underlying impaired cognition remain unclear. Elucidation of these mechanisms has been hindered by the lack of a model system that contains full trisomy of chromosome 21 (Ts21) in a human genome that enables normal gene regulation. To overcome this limitation, we created Ts21-induced pluripotent stem cells (iPSCs) from two sets of Ts21 human fibroblasts. One of the fibroblast lines had low level mosaicism for Ts21 and yielded Ts21 iPSCs and an isogenic control that is disomic for human chromosome 21 (HSA21). Differentiation of all Ts21 iPSCs yielded similar numbers of neurons expressing markers characteristic of dorsal forebrain neurons that were functionally similar to controls. Expression profiling of Ts21 iPSCs and their neuronal derivatives revealed changes in HSA21 genes consistent with the presence of 50% more genetic material as well as changes in non-HSA21 genes that suggested compensatory responses to oxidative stress. Ts21 neurons displayed reduced synaptic activity, affecting excitatory and inhibitory synapses equally. Thus, Ts21 iPSCs and neurons display unique developmental defects that are consistent with cognitive deficits in individuals with Down syndrome and may enable discovery of the underlying causes of and treatments for this disorder.
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Síndrome de Down/genética , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Cromosomas Humanos Par 21/genética , Fibroblastos/citología , Perfilación de la Expresión Génica , Genotipo , Humanos , Hibridación Fluorescente in Situ , Células Madre Pluripotentes Inducidas/citología , Mosaicismo , Neuronas/citología , Neuronas/fisiología , Estrés Oxidativo , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Potenciales Sinápticos/genéticaRESUMEN
Probing how the human neural networks operate is hindered by the lack of reliable human neural tissues amenable for dynamic functional assessment of neural circuits. We developed a 3D bioprinting platform to assemble tissues with defined human neural cell types in a desired dimension using a commercial bioprinter. The printed neuronal progenitors differentiate to neurons and form functional neural circuits in and between tissue layers with specificity within weeks, evidenced by the cortical-to-striatal projection, spontaneous synaptic currents and synaptic response to neuronal excitation. Printed astrocyte progenitors develop into mature astrocytes with elaborated processes and form functional neuron-astrocyte networks, indicated by calcium flux and glutamate uptake in response to neuronal excitation under physiological and pathological conditions. These designed human neural tissues will likely be useful for understanding the wiring of human neural networks, modeling pathological processes, and serving as platforms for drug testing.
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Introduction: Down syndrome, caused by trisomy 21, is a complex developmental disorder associated with intellectual disability and reduced growth of multiple organs. Structural pathologies are present at birth, reflecting embryonic origins. A fundamental unanswered question is how an extra copy of human chromosome 21 contributes to organ-specific pathologies that characterize individuals with Down syndrome, and, relevant to the hallmark intellectual disability in Down syndrome, how trisomy 21 affects neural development. We tested the hypothesis that trisomy 21 exerts effects on human neural development as early as neural induction. Methods: Bulk RNA sequencing was performed on isogenic trisomy 21 and euploid human induced pluripotent stem cells (iPSCs) at successive stages of neural induction: embryoid bodies at Day 6, early neuroectoderm at Day 10, and differentiated neuroectoderm at Day 17. Results: Gene expression analysis revealed over 1,300 differentially expressed genes in trisomy 21 cells along the differentiation pathway compared to euploid controls. Less than 5% of the gene expression changes included upregulated chromosome 21 encoded genes at every timepoint. Genes involved in specific growth factor signaling pathways (WNT and Notch), metabolism (including oxidative stress), and extracellular matrix were altered in trisomy 21 cells. Further analysis uncovered heterochronic expression of genes. Conclusion: Trisomy 21 impacts discrete developmental pathways at the earliest stages of neural development. The results suggest that metabolic dysfunction arises early in embryogenesis in trisomy 21 and may affect development and function more broadly.
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Probing how human neural networks operate is hindered by the lack of reliable human neural tissues amenable to the dynamic functional assessment of neural circuits. We developed a 3D bioprinting platform to assemble tissues with defined human neural cell types in a desired dimension using a commercial bioprinter. The printed neuronal progenitors differentiate into neurons and form functional neural circuits within and between tissue layers with specificity within weeks, evidenced by the cortical-to-striatal projection, spontaneous synaptic currents, and synaptic response to neuronal excitation. Printed astrocyte progenitors develop into mature astrocytes with elaborated processes and form functional neuron-astrocyte networks, indicated by calcium flux and glutamate uptake in response to neuronal excitation under physiological and pathological conditions. These designed human neural tissues will likely be useful for understanding the wiring of human neural networks, modeling pathological processes, and serving as platforms for drug testing.
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Bioimpresión , Tejido Nervioso , Humanos , Neuronas/metabolismo , Astrocitos/metabolismo , Ingeniería de TejidosRESUMEN
The human genome has many short tandem repeats, yet the normal functions of these repeats are unclear. The 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene contains polymorphic CGG repeats, the length of which has differing effects on FMR1 expression and human health, including the neurodevelopmental disorder fragile X syndrome. We deleted the CGG repeats in the FMR1 gene (0CGG) in human stem cells and examined the effects on differentiated neurons. 0CGG neurons have altered subcellular localization of FMR1 mRNA and protein, and differential expression of cellular stress proteins compared with neurons with normal repeats (31CGG). In addition, 0CGG neurons have altered responses to glucocorticoid receptor (GR) activation, including FMR1 mRNA localization, GR chaperone HSP90α expression, GR localization, and cellular stress protein levels. Therefore, the CGG repeats in the FMR1 gene are important for the homeostatic responses of neurons to stress signals.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Neuronas , ARN Mensajero , Humanos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Neuronas/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Estrés Fisiológico/genética , Regiones no Traducidas 5'/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Repeticiones de Trinucleótidos/genética , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Human brain organoid models have emerged as a promising tool for studying human brain development and function. These models preserve human genetics and recapitulate some aspects of human brain development, while facilitating manipulation in an in vitro setting. Despite their potential to transform biology and medicine, concerns persist about their fidelity. To fully harness their potential, it is imperative to establish reliable analytic methods, ensuring rigor and reproducibility. Here, we review current analytical platforms used to characterize human forebrain cortical organoids, highlight challenges, and propose recommendations for future studies to achieve greater precision and uniformity across laboratories.
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Encéfalo , Organoides , Humanos , Organoides/citología , Organoides/metabolismo , Encéfalo/citología , Reproducibilidad de los Resultados , Prosencéfalo/citologíaRESUMEN
Injury to adult mammalian central nervous system (CNS) axons results in limited regeneration. Rodent studies have revealed a developmental switch in CNS axon regenerative ability, yet whether this is conserved in humans is unknown. Using human fibroblasts from 8 gestational-weeks to 72 years-old, we performed direct reprogramming to transdifferentiate fibroblasts into induced neurons (Fib-iNs), avoiding pluripotency which restores cells to an embryonic state. We found that early gestational Fib-iNs grew longer neurites than all other ages, mirroring the developmental switch in regenerative ability in rodents. RNA-sequencing and screening revealed ARID1A as a developmentally-regulated modifier of neurite growth in human neurons. These data suggest that age-specific epigenetic changes may drive the intrinsic loss of neurite growth ability in human CNS neurons during development. One-Sentence Summary: Directly-reprogrammed human neurons demonstrate a developmental decrease in neurite growth ability.
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Fragile X messenger ribonucleoprotein 1 protein (FMRP) deficiency leads to fragile X syndrome (FXS), an autism spectrum disorder. The role of FMRP in prenatal human brain development remains unclear. Here, we show that FMRP is important for human and macaque prenatal brain development. Both FMRP-deficient neurons in human fetal cortical slices and FXS patient stem cell-derived neurons exhibit mitochondrial dysfunctions and hyperexcitability. Using multiomics analyses, we have identified both FMRP-bound mRNAs and FMRP-interacting proteins in human neurons and unveiled a previously unknown role of FMRP in regulating essential genes during human prenatal development. We demonstrate that FMRP interaction with CNOT1 maintains the levels of receptor for activated C kinase 1 (RACK1), a species-specific FMRP target. Genetic reduction of RACK1 leads to both mitochondrial dysfunctions and hyperexcitability, resembling FXS neurons. Finally, enhancing mitochondrial functions rescues deficits of FMRP-deficient cortical neurons during prenatal development, demonstrating targeting mitochondrial dysfunction as a potential treatment.
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Trastorno del Espectro Autista , Síndrome del Cromosoma X Frágil , Enfermedades Mitocondriales , Humanos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Trastorno del Espectro Autista/metabolismo , Neuronas/metabolismo , Neurogénesis , Enfermedades Mitocondriales/metabolismo , Receptores de Cinasa C Activada/genética , Receptores de Cinasa C Activada/metabolismo , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.
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Investigación con Células Madre , Humanos , Reproducibilidad de los ResultadosRESUMEN
Down syndrome (DS) is the most common chromosomal abnormality and leads to intellectual disability, increased risk of cardiac defects, and an altered immune response. Individuals with DS have an extra full or partial copy of chromosome 21 (trisomy 21) and are more likely to develop early-onset Alzheimer's disease (AD) than the general population. Changes in expression of human chromosome 21 (Hsa21)-encoded genes, such as amyloid precursor protein (APP), play an important role in the pathogenesis of AD in DS (DS-AD). However, the mechanisms of DS-AD remain poorly understood. To date, several mouse models with an extra copy of genes syntenic to Hsa21 have been developed to characterise DS-AD-related phenotypes. Nonetheless, due to genetic and physiological differences between mouse and human, mouse models cannot faithfully recapitulate all features of DS-AD. Cells differentiated from human-induced pluripotent stem cells (iPSCs), isolated from individuals with genetic diseases, can be used to model disease-related cellular and molecular pathologies, including DS. In this review, we will discuss the limitations of mouse models of DS and how these can be addressed using recent advancements in modelling DS using human iPSCs and iPSC-mouse chimeras, and potential applications of iPSCs in preclinical studies for DS-AD.
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Neurodevelopmental impairment contributes to the hallmark cognitive disability in individuals with Down syndrome (DS, trisomy 21, T21). The appearance of cognitive deficits in infancy suggests that alterations emerge during the earliest stages of neural development and continue throughout the lifespan in DS. Neural correlates of intellectual and language function include cortical structures, specifically temporal and frontal lobes that are smaller in DS. Yet, despite increased understanding of the DS cognitive-behavioral phenotype in childhood, there is very little structural and histological information to help explain the deficits. Consequently, attempts to effectively design therapeutic targets or interventions are limited. We present a systematic review of published research on cortical development in DS that reveals a paucity of studies that rigorously identify cellular features that may underlie the gross morphological deficits of the developing DS brain. We assessed 115 published reports retrieved through PubMed and other sources and found that only 23 reported histological and/or immunohistochemical data to define cell composition affected in DS post-mortem brain. Further, our analysis reveals that many reports have limited samples sizes and few DS samples, making it difficult to draw conclusions that are generally applicable to the DS population. Thus, the lack of replication and limited number of studies indicate that more developmentally focused research, ideally using equal numbers of age-matched samples in analyses, is needed to elucidate the cellular nature of smaller brain size in DS.
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Malfunction of autophagy contributes to the progression of many chronic age-associated diseases. As such, improving normal proteostatic mechanisms is an active target for biomedical research and a key focal area for aging research. Endoplasmic reticulum (ER)-based acetylation has emerged as a mechanism that ensures proteostasis within the ER by regulating the induction of ER specific autophagy. ER acetylation is ensured by two ER-membrane bound acetyltransferases, ATase1 and ATase2. Here, we show that ATase inhibitors can rescue ongoing disease manifestations associated with the segmental progeria-like phenotype of AT-1 sTg mice. We also describe a pipeline to reliably identify ATase inhibitors with promising druggability properties. Finally, we show that successful ATase inhibitors can rescue the proteopathy of a mouse model of Alzheimer's disease. In conclusion, our study proposes that ATase-targeting approaches might offer a translational pathway for many age-associated proteopathies affecting the ER/secretory pathway.
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Retículo Endoplásmico , Vías Secretoras , Acetilación , Acetiltransferasas/metabolismo , Animales , Autofagia/genética , Retículo Endoplásmico/metabolismo , Ratones , Vías Secretoras/genéticaRESUMEN
Modeling age-related neurodegenerative disorders with human stem cells are difficult due to the embryonic nature of stem cell-derived neurons. We developed a chemical cocktail to induce senescence of iPSC-derived neurons to address this challenge. We first screened small molecules that induce embryonic fibroblasts to exhibit features characteristic of aged fibroblasts. We then optimized a cocktail of small molecules that induced senescence in fibroblasts and cortical neurons without causing DNA damage. The utility of the "senescence cocktail" was validated in motor neurons derived from ALS patient iPSCs which exhibited protein aggregation and axonal degeneration substantially earlier than those without cocktail treatment. Our "senescence cocktail" will likely enhance the manifestation of disease-related phenotypes in neurons derived from iPSCs, enabling the generation of reliable drug discovery platforms.
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Neuronas Motoras/metabolismo , Enfermedades Neurodegenerativas/genética , Diferenciación Celular , Humanos , FenotipoRESUMEN
Individuals with Down syndrome (DS; Ts21), the most common genetic cause of intellectual disability, have smaller brains that reflect fewer neurons at pre- and post-natal stages, implicating impaired neurogenesis during development. Our stereological analysis of adult DS cortex indicates a reduction of calretinin-expressing interneurons. Using Ts21 human induced pluripotent stem cells (iPSCs) and isogenic controls, we find that Ts21 progenitors generate fewer COUP-TFII+ progenitors with reduced proliferation. Single-cell RNA sequencing of Ts21 progenitors confirms the altered specification of progenitor subpopulations and identifies reduced WNT signaling. Activation of WNT signaling partially restores the COUP-TFII+ progenitor population in Ts21, suggesting that altered WNT signaling contributes to the defective development of cortical interneurons in DS.