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The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is a major risk factor for the development of Parkinson's disease (PD). LRRK2, although ubiquitously expressed, is highly abundant in cells of the innate immune system. Given the importance of central and peripheral immune cells in the development of PD, we sought to investigate the consequences of the G2019S mutation on microglial and monocyte transcriptome and function. We have generated large-scale transcriptomic profiles of isogenic human induced microglial cells (iMGLs) and patient derived monocytes carrying the G2019S mutation under baseline culture conditions and following exposure to the proinflammatory factors IFNγ and LPS. We demonstrate that the G2019S mutation exerts a profound impact on the transcriptomic profile of these myeloid cells, and describe corresponding functional differences in iMGLs. The G2019S mutation led to an upregulation in lipid metabolism and phagolysosomal pathway genes in untreated and LPS/IFNγ stimulated iMGLs, which was accompanied by an increased phagocytic capacity of myelin debris. We also identified dysregulation of cell cycle genes, with a downregulation of the E2F4 regulon. Transcriptomic characterization of human-derived monocytes carrying the G2019S mutation confirmed alteration in lipid metabolism associated genes. Altogether, these findings reveal the influence of G2019S on the dysregulation of the myeloid cell transcriptome under proinflammatory conditions.
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Microglia, the innate immune cells of the central nervous system, have been genetically implicated in multiple neurodegenerative diseases. We previously mapped the genetic regulation of gene expression and mRNA splicing in human microglia, identifying several loci where common genetic variants in microglia-specific regulatory elements explain disease risk loci identified by GWAS. However, identifying genetic effects on splicing has been challenging due to the use of short sequencing reads to identify causal isoforms. Here we present the isoform-centric microglia genomic atlas (isoMiGA) which leverages the power of long-read RNA-seq to identify 35,879 novel microglia isoforms. We show that the novel microglia isoforms are involved in stimulation response and brain region specificity. We then quantified the expression of both known and novel isoforms in a multi-ethnic meta-analysis of 555 human microglia short-read RNA-seq samples from 391 donors, the largest to date, and found associations with genetic risk loci in Alzheimer's disease and Parkinson's disease. We nominate several loci that may act through complex changes in isoform and splice site usage.
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Genome-wide association studies (GWAS) have identified more than 40 loci associated with Alzheimer's disease (AD), but the causal variants, regulatory elements, genes and pathways remain largely unknown, impeding a mechanistic understanding of AD pathogenesis. Previously, we showed that AD risk alleles are enriched in myeloid-specific epigenomic annotations. Here, we show that they are specifically enriched in active enhancers of monocytes, macrophages and microglia. We integrated AD GWAS with myeloid epigenomic and transcriptomic datasets using analytical approaches to link myeloid enhancer activity to target gene expression regulation and AD risk modification. We identify AD risk enhancers and nominate candidate causal genes among their likely targets (including AP4E1, AP4M1, APBB3, BIN1, MS4A4A, MS4A6A, PILRA, RABEP1, SPI1, TP53INP1, and ZYX) in twenty loci. Fine-mapping of these enhancers nominates candidate functional variants that likely modify AD risk by regulating gene expression in myeloid cells. In the MS4A locus we identified a single candidate functional variant and validated it in human induced pluripotent stem cell (hiPSC)-derived microglia and brain. Taken together, this study integrates AD GWAS with multiple myeloid genomic datasets to investigate the mechanisms of AD risk alleles and nominates candidate functional variants, regulatory elements and genes that likely modulate disease susceptibility.
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Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad/genética , Genómica , Células Mieloides , Secuencias Reguladoras de Ácidos Nucleicos/genética , Alelos , Enfermedad de Alzheimer/metabolismo , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos , Microglía/metabolismo , TranscriptomaRESUMEN
Recent human genetic studies suggest that cells of the innate immune system have a primary role in the pathogenesis of neurodegenerative diseases. However, the results from these studies often do not elucidate how the genetic variants affect the biology of these cells to modulate disease risk. Here, we applied a tensor decomposition method to uncover disease associated gene networks linked to distal genetic variation in stimulated human monocyte and macrophage gene expression profiles. We report robust evidence that some disease associated genetic variants affect the expression of multiple genes in trans. These include a Parkinson's disease locus influencing the expression of genes mediated by a protease that controls lysosomal function, and Alzheimer's disease loci influencing the expression of genes involved in type 1 interferon signaling, myeloid phagocytosis, and complement cascade pathways. Overall, we uncover gene networks in induced innate immune cells linked to disease associated genetic variants, which may help elucidate the underlying biology of disease.
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Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad , Modelos Genéticos , Enfermedad de Parkinson/genética , Sitios de Carácter Cuantitativo/inmunología , Enfermedad de Alzheimer/inmunología , Línea Celular , Mapeo Cromosómico , Conjuntos de Datos como Asunto , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/inmunología , Variación Genética/inmunología , Estudio de Asociación del Genoma Completo , Humanos , Inmunidad Innata/genética , Interferón gamma/inmunología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedad de Parkinson/inmunologíaRESUMEN
Alzheimer's disease (AD) is a highly heritable complex disease with no current effective prevention or treatment. The majority of drugs developed for AD focus on the amyloid cascade hypothesis, which implicates Aß plaques as a causal factor in the disease. However, it is possible that other underexplored disease-associated pathways may be more fruitful targets for drug development. Findings from gene network analyses implicate immune networks as being enriched in AD; many of the genes in these networks fall within genomic regions that contain common and rare variants that are associated with increased risk of developing AD. Of these genes, several (including CR1, SPI1, the MS4As, TREM2, ABCA7, CD33, and INPP5D) are expressed by microglia, the resident immune cells of the brain. We summarize the gene network and genetics findings that implicate that these microglial genes are involved in AD, as well as several studies that have looked at the expression and function of these genes in microglia and in the context of AD. We propose that these genes are contributing to AD in a non-Aß-dependent fashion.
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Enfermedad de Alzheimer/genética , Redes Reguladoras de Genes/genética , Predisposición Genética a la Enfermedad/genética , Microglía/patología , Edad de Inicio , Enfermedad de Alzheimer/inmunología , Animales , Redes Reguladoras de Genes/inmunología , Humanos , Microglía/inmunología , Factores de RiesgoRESUMEN
There is great need to develop more predictive drug discovery tools to identify new therapies to treat diseases of the central nervous system (CNS). Current nonpluripotent stem cell-based models often utilize non-CNS immortalized cell lines and do not enable the development of personalized models of disease. In this review, we discuss why in vitro models are necessary for translational research and outline the unique advantages of induced pluripotent stem cell (iPSC)-based models over those of current systems. We suggest that iPSC-based models can be patient specific and isogenic lines can be differentiated into many neural cell types for detailed comparisons. iPSC-derived cells can be combined to form small organoids, or large panels of lines can be developed that enable new forms of analysis. iPSC and embryonic stem cell-derived cells can be readily engineered to develop reporters for lineage studies or mechanism of action experiments further extending the utility of iPSC-based systems. We conclude by describing novel technologies that include strategies for the development of diversity panels, novel genomic engineering tools, new three-dimensional organoid systems, and modified high-content screens that may bring toxicology into the 21st century. The strategic integration of these technologies with the advantages of iPSC-derived cell technology, we believe, will be a paradigm shift for toxicology and drug discovery efforts.
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Fármacos del Sistema Nervioso Central/farmacología , Evaluación Preclínica de Medicamentos/métodos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Neuronas/citologíaRESUMEN
Niemann-Pick disease, type C1 (NPC1) is a familial disorder that has devastating consequences on postnatal development with multisystem effects, including neurodegeneration. There is no Food and Drug Administration-approved treatment option for NPC1; however, several potentially therapeutic compounds have been identified in assays using yeast, rodent models, and NPC1 human fibroblasts. Although these discoveries were made in fibroblasts from NPC1 subjects and were in some instances validated in animal models of the disease, testing these drugs on a cell type more relevant for NPC1 neurological disease would greatly facilitate both study of the disease and identification of more relevant therapeutic compounds. Toward this goal, we have generated an induced pluripotent stem cell line from a subject homozygous for the most frequent NPC1 mutation (p.I1061T) and subsequently created a stable line of neural stem cells (NSCs). These NSCs were then used to create neurons as an appropriate disease model. NPC1 neurons display a premature cell death phenotype, and gene expression analysis of these cells suggests dysfunction of important signaling pathways, including calcium and WNT. The clear readout from these cells makes them ideal candidates for high-throughput screening and will be a valuable tool to better understand the development of NPC1 in neural cells, as well as to develop better therapeutic options for NPC1.
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Señalización del Calcio , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Vía de Señalización Wnt , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/patología , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutación , Neuronas/patología , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/genéticaRESUMEN
Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC-derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell-type specificity, this study demonstrates the feasibility of this NSC-driven platform for higher-throughput neurotoxicity screens.
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Astrocitos/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Células-Madre Neurales/efectos de los fármacos , Pruebas de Toxicidad/métodos , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , RatasRESUMEN
INTRODUCTION: Since the initial discoveries of human embryonic and induced pluripotent stem cells, many strategies have been developed to utilize the potential of these cells for translational research and disease modeling. The success of these aims and the development of future applications in this area will depend on the ability to generate high-quality and large numbers of differentiated cell types that genetically, epigenetically, and functionally mimic the cells found in the body. AREAS COVERED: In this review, we highlight the current strategies used to maintain stem cell pluripotency (a measure of stem cell quality), as well as provide an overview of the various differentiation strategies being used to generate cells from all three germ lineages. We also discuss the particular considerations that must be addressed when utilizing these cells for translational therapy, and provide an example of a cell type currently used in clinical trials. EXPERT OPINION: The major challenge in regenerative medicine and disease modeling will be in generating functional cells of sufficient quality that are physiologically and epigenetically similar to the diverse cells that they are modeled after. By meeting these criteria, these differentiated products can be successfully used in disease modeling, drug/toxicology screens, and cellular replacement therapy.
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Diferenciación Celular , Linaje de la Célula/fisiología , Células Madre Embrionarias/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Madre Embrionarias/citología , Humanos , Medicina Regenerativa/métodos , Células Madre/fisiologíaRESUMEN
Astrocytes are the most abundant cell type in the central nervous system (CNS) and have a multitude of functions that include maintenance of CNS homeostasis, trophic support of neurons, detoxification, and immune surveillance. It has only recently been appreciated that astrocyte dysfunction is a primary cause of many neurological disorders. Despite their importance in disease very little is known about global gene expression for human astrocytes. We have performed a microarray expression analysis of human fetal astrocytes to identify genes and signaling pathways that are important for astrocyte development and maintenance. Our analysis confirmed that the fetal astrocytes express high levels of the core astrocyte marker GFAP and the transcription factors from the NFI family which have been shown to play important roles in astrocyte development. A group of novel markers were identified that distinguish fetal astrocytes from pluripotent stem cell-derived neural stem cells (NSCs) and NSC-derived neurons. As in murine astrocytes, the Notch signaling pathway appears to be particularly important for cell fate decisions between the astrocyte and neuronal lineages in human astrocytes. These findings unveil the repertoire of genes expressed in human astrocytes and serve as a basis for further studies to better understand astrocyte biology, especially as it relates to disease.