RESUMEN
Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo , Técnicas Analíticas Microfluídicas , Retina/citología , Análisis de la Célula Individual , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Análisis de Secuencia de ARNRESUMEN
A hallmark of autoimmunity in murine models of lupus is the formation of germinal centers (GCs) in lymphoid tissues where self-reactive B cells expand and differentiate. In the host response to foreign antigens, follicular dendritic cells (FDCs) maintain GCs through the uptake and cycling of complement-opsonized immune complexes. Here, we examined whether FDCs retain self-antigens and the impact of this process in autoantibody secretion in lupus. We found that FDCs took up and retained self-immune complexes composed of ribonucleotide proteins, autoantibody, and complement. This uptake, mediated through CD21, triggered endosomal TLR7 and led to the secretion of interferon (IFN) α via an IRF5-dependent pathway. Blocking of FDC secretion of IFN-α restored B cell tolerance and reduced the amount of GCs and pathogenic autoantibody. Thus, FDCs are a critical source of the IFN-α driving autoimmunity in this lupus model. This pathway is conserved in humans, suggesting that it may be a viable therapeutic target in systemic lupus erythematosus.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos B/inmunología , Células Dendríticas Foliculares/inmunología , Lupus Eritematoso Sistémico/inmunología , Animales , Autoantígenos/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Interferón-alfa/biosíntesis , Interferón-alfa/inmunología , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Receptor Toll-Like 7/inmunología , TranscriptomaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Systemic lupus erythematosus (SLE) is an incurable autoimmune disease characterized by autoantibody deposition in tissues such as kidney, skin and lungs. Notably, up to 75% of patients with SLE experience neuropsychiatric symptoms that range from anxiety, depression and cognitive impairment to seizures and, in rare cases, psychosis-collectively this is referred to as central nervous system (CNS) lupus. In some cases, certain autoantibodies, such as anti-NMDAR or anti-phospholipid antibodies, promote CNS lupus. However, in most patients, the mechanisms that underlie these symptoms are unknown. CNS lupus typically presents at lupus diagnosis or within the first year, suggesting that early factors contributing to peripheral autoimmunity may promote CNS lupus symptoms. Here we report behavioural phenotypes and synapse loss in lupus-prone mice that are prevented by blocking type I interferon (IFN) signalling. Furthermore, we show that type I IFN stimulates microglia to become reactive and engulf neuronal and synaptic material in lupus-prone mice. These findings and our observation of increased type I IFN signalling in post-mortem hippocampal brain sections from patients with SLE may instruct the evaluation of ongoing clinical trials of anifrolumab, a type I IFN-receptor antagonist. Moreover, identification of IFN-driven microglia-dependent synapse loss, along with microglia transcriptome data, connects CNS lupus with other CNS diseases and provides an explanation for the neurological symptoms observed in some patients with SLE.
Asunto(s)
Interferón Tipo I/inmunología , Vasculitis por Lupus del Sistema Nervioso Central/inmunología , Vasculitis por Lupus del Sistema Nervioso Central/patología , Microglía/inmunología , Microglía/patología , Sinapsis/patología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Conducta Animal , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Interferón Tipo I/antagonistas & inhibidores , Vasculitis por Lupus del Sistema Nervioso Central/psicología , Masculino , Ratones , Microglía/metabolismo , Fenotipo , Transducción de Señal , Sinapsis/inmunología , TranscriptomaRESUMEN
Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia's strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.
Asunto(s)
Complemento C4/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Esquizofrenia/genética , Alelos , Secuencia de Aminoácidos , Animales , Axones/metabolismo , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/patología , Complemento C4/química , Vía Clásica del Complemento , Dendritas/metabolismo , Dosificación de Gen/genética , Regulación de la Expresión Génica/genética , Haplotipos/genética , Humanos , Complejo Mayor de Histocompatibilidad/genética , Ratones , Modelos Animales , Plasticidad Neuronal/genética , Plasticidad Neuronal/fisiología , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Factores de Riesgo , Esquizofrenia/patología , Sinapsis/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by motor neuron (MN) death. Lipid dysregulation manifests during disease; however, it is unclear whether lipid homeostasis is adversely affected in the in the spinal cord gray matter (GM), and if so, whether it is because of an aberrant increase in lipid synthesis. Moreover, it is unknown whether lipid dysregulation contributes to MN death. Here, we show that cholesterol ester (CE) and triacylglycerol levels are elevated several-fold in the spinal cord GM of male sporadic ALS patients. Interestingly, HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, was reduced in the spinal cord GM of ALS patients. Increased cytosolic phospholipase A2 activity and lyso-phosphatidylcholine (Lyso-PC) levels in ALS patients suggest that CE accumulation was driven by acyl group transfer from PC to cholesterol. Notably, Lyso-PC, a byproduct of CE synthesis, was toxic to human MNs in vitro Elevations in CE, triacylglycerol, and Lyso-PC were also found in the spinal cord of SOD1G93A mice, a model of ALS. Similar to ALS patients, a compensatory downregulation of cholesterol synthesis occurred in the spinal cord of SOD1G93A mice; levels of sterol regulatory element binding protein 2, a transcriptional regulator of cholesterol synthesis, progressively declined. Remarkably, overexpressing sterol regulatory element binding protein 2 in the spinal cord of normal mice to model CE accumulation led to ALS-like lipid pathology, MN death, astrogliosis, paralysis, and reduced survival. Thus, spinal cord lipid dysregulation in ALS likely contributes to neurodegeneration and developing therapies to restore lipid homeostasis may lead to a treatment for ALS.SIGNIFICANCE STATEMENT Neurons that control muscular function progressively degenerate in patients with amyotrophic lateral sclerosis (ALS). Lipid dysregulation is a feature of ALS; however, it is unclear whether disrupted lipid homeostasis (i.e., lipid cacostasis) occurs proximal to degenerating neurons in the spinal cord, what causes it, and whether it contributes to neurodegeneration. Here we show that lipid cacostasis occurs in the spinal cord gray matter of ALS patients. Lipid accumulation was not associated with an aberrant increase in synthesis or reduced hydrolysis, as enzymatic and transcriptional regulators of lipid synthesis were downregulated during disease. Last, we demonstrated that genetic induction of lipid cacostasis in the CNS of normal mice was associated with ALS-like lipid pathology, astrogliosis, neurodegeneration, and clinical features of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Metabolismo de los Lípidos , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Muerte Celular , Ésteres del Colesterol/metabolismo , Sustancia Gris/metabolismo , Humanos , Lisofosfatidilcolinas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/patología , Receptores Acoplados a Proteínas G/genética , Receptores de Fosfolipasa A2/metabolismo , Médula Espinal/metabolismo , Superóxido Dismutasa-1/genética , Triglicéridos/metabolismoRESUMEN
Recent discoveries implicate the classical complement cascade in normal brain development and in disease. Complement proteins C1q, C3, and C4 participate in synapse elimination, tagging inappropriate synaptic connections between neurons for removal by phagocytic microglia that exist in a special, highly phagocytic state during the synaptic pruning period. Several neurodevelopmental disorders, such as schizophrenia and autism, are thought to be caused by an imbalance in synaptic pruning, and recent studies suggest that dysregulation of complement could promote this synaptic pruning imbalance. Moreover, in the mature brain, complement can be aberrantly activated in early stages of neurodegenerative diseases to stimulate synapse loss. Similar pathways can also be activated in response to inflammation, as in West Nile Virus infection or in lupus, where peripheral inflammation can promote microglia-mediated synapse loss. Whether synapse loss in disease is a true reactivation of developmental synaptic pruning programs remains unclear; nonetheless, complement proteins represent potential therapeutic targets for both neurodevelopmental and neurodegenerative diseases.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Red Nerviosa/inmunología , Neurogénesis/inmunología , Plasticidad Neuronal/inmunología , Sinapsis/inmunología , Animales , Trastorno Autístico/genética , Trastorno Autístico/inmunología , Trastorno Autístico/patología , Proteínas del Sistema Complemento/genética , Epilepsia/genética , Epilepsia/inmunología , Epilepsia/patología , Regulación del Desarrollo de la Expresión Génica , Humanos , Microglía/inmunología , Microglía/patología , Red Nerviosa/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Neurogénesis/genética , Plasticidad Neuronal/genética , Neuronas/inmunología , Neuronas/patología , Esquizofrenia/genética , Esquizofrenia/inmunología , Esquizofrenia/patología , Sinapsis/genética , Sinapsis/patología , Transmisión SinápticaRESUMEN
Mutations in GPR56, a member of the adhesion G protein-coupled receptor family, cause a human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Magnetic resonance imaging (MRI) of BFPP brains reveals myelination defects in addition to brain malformation. However, the cellular role of GPR56 in oligodendrocyte development remains unknown. Here, we demonstrate that loss of Gpr56 leads to hypomyelination of the central nervous system in mice. GPR56 levels are abundant throughout early stages of oligodendrocyte development, but are downregulated in myelinating oligodendrocytes. Gpr56-knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation and diminished levels of active RhoA, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum and optic nerves. Conditional ablation of Gpr56 in OPCs leads to a reduced number of mature oligodendrocytes as seen in constitutive knockout of Gpr56. Together, our data define GPR56 as a cell-autonomous regulator of oligodendrocyte development.
Asunto(s)
Regulación de la Expresión Génica , Oligodendroglía/citología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Axones/metabolismo , Encéfalo/metabolismo , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Cuerpo Calloso/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Malformaciones del Desarrollo Cortical/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Mutación , Vaina de Mielina/química , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Nervio Óptico/metabolismo , Transducción de Señal , Tamoxifeno/química , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Immune molecules, including complement proteins C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement localizes to synapses and refines the developing visual system through C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. Previous work implicated an astrocyte-derived factor in regulating neuronal C1q expression. Here we identify retinal transforming growth factor (TGF)-ß as a key regulator of neuronal C1q expression and synaptic pruning in the developing visual system. Mice lacking TGF-ß receptor II (TGFßRII) in retinal neurons had reduced C1q expression in RGCs and reduced synaptic localization of complement, and phenocopied refinement defects observed in complement-deficient mice, including reduced eye-specific segregation and microglial engulfment of RGC inputs. These data implicate TGF-ß in regulating neuronal C1q expression to initiate complement- and microglia-mediated synaptic pruning.
Asunto(s)
Complemento C1q/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Ganglionares de la Retina/metabolismo , Transducción de Señal/fisiología , Sinapsis/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Recuento de Células , Células Cultivadas , Corteza Cerebral/citología , Complemento C1q/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroglía/química , Neuroglía/fisiología , Proteínas Serina-Treonina Quinasas/deficiencia , Ratas , Ratas Sprague-Dawley , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Retina/citología , Retina/efectos de los fármacos , Retina/crecimiento & desarrollo , Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
Glia cells are uniquely positioned at synapses, contacting pre- and post-synaptic terminals. At the Drosophila neuromuscular junction, a novel glia-derived TGF-ß ligand has been found that modulates a retrograde synaptogenic signal.