RESUMEN
The ability to spatially map multiple layers of the omics information over different time points allows for exploring the mechanisms driving brain development, differentiation, arealization, and alterations in disease. Herein we developed and applied spatial tri-omic sequencing technologies, DBiT ARP-seq (spatial ATAC-RNA-Protein-seq) and DBiT CTRP-seq (spatial CUT&Tag-RNA-Protein-seq) together with multiplexed immunofluorescence imaging (CODEX) to map spatial dynamic remodeling in brain development and neuroinflammation. A spatiotemporal tri-omic atlas of the mouse brain was obtained at different stages from postnatal day P0 to P21, and compared to the regions of interest in the human developing brains. Specifically, in the cortical area, we discovered temporal persistence and spatial spreading of chromatin accessibility for the layer-defining transcription factors. In corpus callosum, we observed dynamic chromatin priming of myelin genes across the subregions. Together, it suggests a role for layer specific projection neurons to coordinate axonogenesis and myelination. We further mapped the brain of a lysolecithin (LPC) neuroinflammation mouse model and observed common molecular programs in development and neuroinflammation. Microglia, exhibiting both conserved and distinct programs for inflammation and resolution, are transiently activated not only at the core of the LPC lesion, but also at distal locations presumably through neuronal circuitry. Thus, this work unveiled common and differential mechanisms in brain development and neuroinflammation, resulting in a valuable data resource to investigate brain development, function and disease.
RESUMEN
Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single-cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single-cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease-associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single-cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Médula Espinal , Animales , Humanos , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Médula Espinal/metabolismo , Médula Espinal/patología , Ratones , Análisis de Expresión Génica de una Sola Célula , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Neuroglía/metabolismo , Neuroglía/patologíaRESUMEN
OBJECTIVE: To measure the impact of JHU-083, a novel prodrug of the glutamine antagonist 6-diazo-5-oxo-l-norleucine, on immune cell proliferation and activation, along with physical and cognitive impairments associated with the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. METHODS: Splenic-derived T cells and bone marrow-derived dendritic cells (DCs) were cultured, activated, and treated daily with vehicle or JHU-083. Proliferation and activation were measured via flow cytometry and IncuCyte live cell analysis. C57BL/6 mice were immunized for EAE. Vehicle or JHU-083 was administered orally every other day either from the time of immunization in the prevention paradigm or from the time of disease onset in the treatment paradigm. Disease scores and body weight were monitored. In the treatment paradigm, cognition was evaluated using the Barnes maze test. RESULTS: JHU-083 selectively inhibits T-cell proliferation and decreases T-cell activation, with no effect on DCs. In vivo, orally administered JHU-083 significantly decreases EAE severity in both prevention and treatment paradigms and reverses EAE-induced cognitive impairment. CONCLUSIONS: JHU-083, a well-tolerated, brain penetrable glutamine antagonist, is a promising novel treatment for both the physical and cognitive deficits of MS.
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Compuestos Azo/farmacología , Caproatos/farmacología , Disfunción Cognitiva/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Esclerosis Múltiple/tratamiento farmacológico , Animales , Conducta Animal/efectos de los fármacos , Células Cultivadas , Disfunción Cognitiva/etiología , Encefalomielitis Autoinmune Experimental/complicaciones , Femenino , Glutamina , Masculino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/complicacionesRESUMEN
Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.
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Axones/metabolismo , Enfermedades Desmielinizantes/patología , Oligodendroglía/citología , Células Madre/citología , Animales , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Fluorescencia , RatasRESUMEN
Mesenchymal stem cells (MSCs) are pleiotropic cells with potential therapeutic benefits for a wide range of diseases. Because of their immunomodulatory properties they have been utilized to treat autoimmune diseases such as multiple sclerosis (MS), which is characterized by demyelination. The microenvironment surrounding MSCs is thought to affect their differentiation and phenotype, which could in turn affect the efficacy. We thus sought to dissect the potential for differential impact of MSCs on central nervous system (CNS) disease in T cell mediated and non-T cell mediated settings using the MOG35-55 experimental autoimmune encephalomyelitis (EAE) and cuprizone-mediated demyelination models, respectively. As the pathogeneses of MS and EAE are thought to be mediated by IFNγ-producing (TH1) and IL-17A-producing (TH17) effector CD4+ T cells, we investigated the effect of MSCs on the development of these two key pathogenic cell groups. Although MSCs suppressed the activation and effector function of TH17 cells, they did not affect TH1 activation, but enhanced TH1 effector function and ultimately produced no effect on EAE. In the non- T cell mediated cuprizone model of demyelination, MSC administration had a positive effect, with an overall increase in myelin abundance in the brain of MSC-treated mice compared to controls. These results highlight the potential variability of MSCs as a biologic therapeutic tool in the treatment of autoimmune disease and the need for further investigation into the multifaceted functions of MSCs in diverse microenvironments and the mechanisms behind the diversity.
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Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/terapia , Encefalomielitis Autoinmune Experimental/terapia , Células Madre Mesenquimatosas/citología , Animales , Linfocitos T CD4-Positivos/inmunología , Muerte Celular , Proliferación Celular , Cuerpo Calloso/patología , Cuprizona , Citocinas/biosíntesis , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Subgrupos Linfocitarios/inmunología , Trasplante de Células Madre Mesenquimatosas , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/inmunología , Oligodendroglía/patología , Fragmentos de Péptidos/inmunologíaRESUMEN
Multiple sclerosis (MS) is a demyelinating disease of the CNS characterized by inflammation and neurodegeneration. Animal models that enable the study of remyelination in the context of ongoing inflammation are greatly needed for the development of novel therapies that target the pathological inhibitory cues inherent to the MS plaque microenvironment. We report the development of an innovative animal model combining cuprizone-mediated demyelination with transfer of myelin-reactive CD4(+) T cells. Characterization of this model reveals both Th1 and Th17 CD4(+) T cells infiltrate the CNS of cuprizone-fed mice, with infiltration of Th17 cells being more efficient. Infiltration correlates with impaired spontaneous remyelination as evidenced by myelin protein expression, immunostaining, and ultrastructural analysis. Electron microscopic analysis further reveals that demyelinated axons are preserved but reduced in caliber. Examination of the immune response contributing to impaired remyelination highlights a role for peripheral monocytes with an M1 phenotype. This study demonstrates the development of a novel animal model that recapitulates elements of the microenvironment of the MS plaque and reveals an important role for T cells and peripheral monocytes in impairing endogenous remyelination in vivo. This model could be useful for testing putative MS therapies designed to enhance remyelination in the setting of active inflammation, and may also facilitate modeling the pathophysiology of denuded axons, which has been a challenge in rodents because they typically remyelinate very quickly.
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Sistema Nervioso Central/patología , Cuprizona/toxicidad , Enfermedades Desmielinizantes/terapia , Inhibidores de la Monoaminooxidasa/toxicidad , Vaina de Mielina/metabolismo , Células Th17/fisiología , Traslado Adoptivo , Animales , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Sistema Nervioso Central/ultraestructura , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Adyuvante de Freund/toxicidad , Interleucina-17/metabolismo , Antígenos Comunes de Leucocito/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/patología , Monocitos/ultraestructura , Proteínas de la Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidad , Infiltración Neutrófila , Fragmentos de Péptidos/toxicidad , Regeneración/efectos de los fármacos , Células Th17/ultraestructura , Factores de TiempoRESUMEN
Nerve conduction within the mammalian central nervous system is made efficient by oligodendrocyte-derived myelin. Historically, thyroid hormones have a well described role in regulating oligodendrocyte differentiation and myelination during development; however, it remains unclear which thyroid hormone receptors are required to drive these effects. This is a question with clinical relevance since nonspecific thyroid receptor stimulation can produce deleterious side-effects. Here we report that GC-1, a thyromimetic with selective thyroid receptor ß action and a potentially limited side-effect profile, promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. In addition, we used in vivo genetic fate tracing of oligodendrocyte progenitor cells via PDGFαR-CreER;Rosa26-eYFP double-transgenic mice to examine the effect of GC-1 on cellular fate and find that treatment with GC-1 during developmental myelination promotes oligodendrogenesis within the corpus callosum, occipital cortex and optic nerve. GC-1 was also observed to enhance the expression of the myelin proteins MBP, CNP and MAG within the same regions. These results indicate that a ß receptor selective thyromimetic can enhance oligodendrocyte differentiation in vitro and during developmental myelination in vivo and warrants further study as a therapeutic agent for demyelinating models.
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Acetatos/farmacología , Fármacos del Sistema Nervioso Central/farmacología , Neurogénesis/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Oligodendroglía/fisiología , Fenoles/farmacología , Receptores beta de Hormona Tiroidea/agonistas , Adolescente , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiopatología , Encéfalo/cirugía , Células Cultivadas , Niño , Preescolar , Epilepsia/fisiopatología , Epilepsia/cirugía , Sustancia Gris/fisiopatología , Sustancia Gris/cirugía , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Nervio Óptico/efectos de los fármacos , Nervio Óptico/crecimiento & desarrollo , Nervio Óptico/fisiología , Ratas Sprague-Dawley , Receptores beta de Hormona Tiroidea/metabolismo , Adulto JovenRESUMEN
Engraftment of human embryonic stem cell (hESC)-derived OPCs in animal models of demyelination results in remyelination and clinical recovery, supporting the feasibility of cell replacement therapies in promoting repair of damaged neural tissue. A critical gap in our understanding of the mechanisms associated with repair revolves around the effects of the local microenvironment on transplanted cell survival. We have determined that treatment of human ESC-derived OPCs with the pleiotropic cytokine IFN-γ promotes apoptosis that is associated with mitochondrial cytochrome c released into the cytosol with subsequent caspase 3 activation. IFN-γ-induced apoptosis is mediated, in part, by secretion of the CXC chemokine ligand 10 (CXCL10) from IFN-γ-treated cells. Signaling through the chemokine receptor CXCR2 by the ligand CXCL1 functions in a tonic manner by muting apoptosis and this is associated with reduced levels of cytosolic cytochrome c and impaired cleavage of caspase 3. These findings support a role for both IFN-γ and CXCL10 in contributing to neuropathology by promoting OPC apoptosis. In addition, these data suggest that hOPCs used for therapeutic treatment for human neurologic disease/damage are susceptible to death through exposure to local inflammatory cytokines present within the inflammatory milieu.
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Apoptosis , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Interferón gamma/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Receptores de Interleucina-8B/metabolismo , Transducción de Señal , Línea Celular , Quimiocina CXCL10/metabolismo , Citocromos c/metabolismo , Humanos , Receptores de Interleucina-8B/genéticaRESUMEN
Canonical Wnt/ß-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here, we show that SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4, Sox2, and Nanog. Furthermore, Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers, and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge, SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months.