RESUMO
The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Difosfato de Adenosina/metabolismo , Doença de Alzheimer/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores Imunológicos/metabolismo , Receptores Purinérgicos/metabolismoRESUMO
Variations in many genes linked to sporadic Alzheimer's disease (AD) show abundant expression in microglia, but relationships among these genes remain largely elusive. Here, we establish isogenic human ESC-derived microglia-like cell lines (hMGLs) harboring AD variants in CD33, INPP5D, SORL1, and TREM2 loci and curate a comprehensive atlas comprising ATAC-seq, ChIP-seq, RNA-seq, and proteomics datasets. AD-like expression signatures are observed in AD mutant SORL1 and TREM2 hMGLs, while integrative multi-omic analysis of combined epigenetic and expression datasets indicates up-regulation of APOE as a convergent pathogenic node. We also observe cross-regulatory relationships between SORL1 and TREM2, in which SORL1R744X hMGLs induce TREM2 expression to enhance APOE expression. AD-associated SORL1 and TREM2 mutations also impaired hMGL Aß uptake in an APOE-dependent manner in vitro and attenuated Aß uptake/clearance in mouse AD brain xenotransplants. Using this modeling and analysis platform for human microglia, we provide new insight into epistatic interactions in AD genes and demonstrate convergence of microglial AD genes at the APOE locus.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Apolipoproteínas E/genética , Variação Genética , Células-Tronco Embrionárias Humanas/metabolismo , Microglia/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/patologia , Diferenciação Celular , Linhagem Celular , Cromatina/metabolismo , Epigênese Genética , Redes Reguladoras de Genes , Marcação de Genes , Loci Gênicos , Humanos , Camundongos Transgênicos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Fagocitose , Proteoma/metabolismo , Transdução de Sinais , Transcriptoma/genética , Transplante Heterólogo , Regulação para Cima/genéticaRESUMO
iPSC-derived microglia offer a powerful tool to study microglial homeostasis and disease-associated inflammatory responses. Yet, microglia are highly sensitive to their environment, exhibiting transcriptomic deficiencies when kept in isolation from the brain. Furthermore, species-specific genetic variations demonstrate that rodent microglia fail to fully recapitulate the human condition. To address this, we developed an approach to study human microglia within a surrogate brain environment. Transplantation of iPSC-derived hematopoietic-progenitors into the postnatal brain of humanized, immune-deficient mice results in context-dependent differentiation into microglia and other CNS macrophages, acquisition of an ex vivo human microglial gene signature, and responsiveness to both acute and chronic insults. Most notably, transplanted microglia exhibit robust transcriptional responses to Aß-plaques that only partially overlap with that of murine microglia, revealing new, human-specific Aß-responsive genes. We therefore have demonstrated that this chimeric model provides a powerful new system to examine the in vivo function of patient-derived and genetically modified microglia.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Diferenciação Celular , Expressão Gênica , Microglia/metabolismo , Placa Amiloide/genética , Quimeras de Transplante , Animais , Encéfalo/citologia , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Camundongos , Camundongos Transgênicos , Microglia/citologia , Trombopoetina/genéticaRESUMO
BACKGROUND: Microglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS: iPSCs are first differentiated toward a mesodermal, hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGFß-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGFß signaling (IDE1) can be used to replace recombinant TGFß1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered. CONCLUSION: We anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.
Assuntos
Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Microglia/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Citocinas/metabolismo , HumanosRESUMO
Exposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson's disease (PD). Therapies based on PQ's presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology. Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor.