RESUMEN
Genome-wide studies have identified genetic variants linked to neurologic diseases. Environmental factors also play important roles, but no methods are available for their comprehensive investigation. We developed an approach that combines genomic data, screens in a novel zebrafish model, computational modeling, perturbation studies, and multiple sclerosis (MS) patient samples to evaluate the effects of environmental exposure on CNS inflammation. We found that the herbicide linuron amplifies astrocyte pro-inflammatory activities by activating signaling via sigma receptor 1, inositol-requiring enzyme-1α (IRE1α), and X-box binding protein 1 (XBP1). Indeed, astrocyte-specific shRNA- and CRISPR/Cas9-driven gene inactivation combined with RNA-seq, ATAC-seq, ChIP-seq, and study of patient samples suggest that IRE1α-XBP1 signaling promotes CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, MS. In summary, these studies define environmental mechanisms that control astrocyte pathogenic activities and establish a multidisciplinary approach for the systematic investigation of the effects of environmental exposure in neurologic disorders.
Asunto(s)
Astrocitos/metabolismo , Sistema Nervioso Central/metabolismo , Animales , Sistema Nervioso Central/inmunología , Biología Computacional/métodos , Encefalomielitis Autoinmune Experimental/inmunología , Endorribonucleasas/metabolismo , Ambiente , Exposición a Riesgos Ambientales/efectos adversos , Genoma , Genómica , Humanos , Inflamación/metabolismo , Linurona/efectos adversos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores sigma/efectos de los fármacos , Receptores sigma/metabolismo , Transducción de Señal , Proteína 1 de Unión a la X-Box/metabolismo , Pez CebraRESUMEN
Metabolism has been shown to control peripheral immunity, but little is known about its role in central nervous system (CNS) inflammation. Through a combination of proteomic, metabolomic, transcriptomic, and perturbation studies, we found that sphingolipid metabolism in astrocytes triggers the interaction of the C2 domain in cytosolic phospholipase A2 (cPLA2) with the CARD domain in mitochondrial antiviral signaling protein (MAVS), boosting NF-κB-driven transcriptional programs that promote CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis. cPLA2 recruitment to MAVS also disrupts MAVS-hexokinase 2 (HK2) interactions, decreasing HK enzymatic activity and the production of lactate involved in the metabolic support of neurons. Miglustat, a drug used to treat Gaucher and Niemann-Pick disease, suppresses astrocyte pathogenic activities and ameliorates EAE. Collectively, these findings define a novel immunometabolic mechanism that drives pro-inflammatory astrocyte activities, outlines a new role for MAVS in CNS inflammation, and identifies candidate targets for therapeutic intervention.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Astrocitos/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Fosfolipasas A2 Secretoras/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , 1-Desoxinojirimicina/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Hexoquinasa/metabolismo , Humanos , Ácido Láctico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Fosfolipasas A2 Secretoras/genéticaRESUMEN
Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.
Asunto(s)
Astrocitos , Encefalomielitis Autoinmune Experimental , Memoria Epigenética , Esclerosis Múltiple , Animales , Femenino , Humanos , Masculino , Ratones , Acetilcoenzima A/metabolismo , Astrocitos/enzimología , Astrocitos/metabolismo , Astrocitos/patología , ATP Citrato (pro-S)-Liasa/metabolismo , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Secuenciación de Inmunoprecipitación de Cromatina , Sistemas CRISPR-Cas , Encefalomielitis Autoinmune Experimental/enzimología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Inflamación/enzimología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Esclerosis Múltiple/enzimología , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Análisis de Expresión Génica de una Sola Célula , Transposasas/metabolismoRESUMEN
Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.
Asunto(s)
Astrocitos , Encefalomielitis Autoinmune Experimental , Microfluídica , Esclerosis Múltiple , Ácidos Nucleicos , Análisis de Expresión Génica de una Sola Célula , Animales , Humanos , Ratones , Astrocitos/metabolismo , Astrocitos/patología , Regulación de la Expresión Génica , Ratones Noqueados , Esclerosis Múltiple/patología , Microfluídica/métodos , Análisis de Expresión Génica de una Sola Célula/métodos , Ácidos Nucleicos/análisis , Edición GénicaRESUMEN
Astrocytes are glial cells that are abundant in the central nervous system (CNS) and that have important homeostatic and disease-promoting functions1. However, little is known about the homeostatic anti-inflammatory activities of astrocytes and their regulation. Here, using high-throughput flow cytometry screening, single-cell RNA sequencing and CRISPR-Cas9-based cell-specific in vivo genetic perturbations in mice, we identify a subset of astrocytes that expresses the lysosomal protein LAMP12 and the death receptor ligand TRAIL3. LAMP1+TRAIL+ astrocytes limit inflammation in the CNS by inducing T cell apoptosis through TRAIL-DR5 signalling. In homeostatic conditions, the expression of TRAIL in astrocytes is driven by interferon-γ (IFNγ) produced by meningeal natural killer (NK) cells, in which IFNγ expression is modulated by the gut microbiome. TRAIL expression in astrocytes is repressed by molecules produced by T cells and microglia in the context of inflammation. Altogether, we show that LAMP1+TRAIL+ astrocytes limit CNS inflammation by inducing T cell apoptosis, and that this astrocyte subset is maintained by meningeal IFNγ+ NK cells that are licensed by the microbiome.
Asunto(s)
Astrocitos/inmunología , Microbioma Gastrointestinal/inmunología , Inflamación/prevención & control , Interferón gamma/inmunología , Células Asesinas Naturales/inmunología , Proteínas de Membrana de los Lisosomas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Apoptosis , Astrocitos/metabolismo , Biomarcadores , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Femenino , Homeostasis , Humanos , Inflamación/inmunología , Meninges/citología , Meninges/inmunología , Ratones , Ratones Endogámicos C57BL , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.
Asunto(s)
Astrocitos/patología , Sistema Nervioso Central/patología , Inflamación/patología , Factor de Transcripción MafG/genética , Proteínas Represoras/genética , Animales , Antioxidantes/metabolismo , Astrocitos/metabolismo , Sistema Nervioso Central/metabolismo , Metilación de ADN , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inflamación/genética , Masculino , Metionina Adenosiltransferasa/genética , Ratones , Esclerosis Múltiple/genética , Esclerosis Múltiple/patología , Factor 2 Relacionado con NF-E2/genética , Análisis de Secuencia de ARN , Transducción de Señal , Transcripción GenéticaRESUMEN
Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the CNS. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 Th17 cells can interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we used single cell RNA sequencing, flow cytometry and immunofluorescence studies on CNS tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM) in multiple sclerosis, in experimental autoimmune encephalomyelitis and in controls, although their regulation differs between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of MCAM but not of ALCAM at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live imaging, immunofluorescence and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking ALCAM but not MCAM limited prolonged interactions between human primary oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferring significant protection of oligodendrocytic processes. In conclusion, we showed that human oligodendrocytes express MCAM and ALCAM, which are differently modulated by inflammation and T cell contact. We found that ALCAM is a ligand for Th17-polarized cells, contributing to their capacity to adhere and induce damage to human oligodendrocytes, and therefore could represent a relevant target for neuroprotection in multiple sclerosis.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Humanos , Ratones , Animales , Linfocitos T CD4-Positivos/metabolismo , Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Adhesión Celular , Oligodendroglía/metabolismoRESUMEN
The trafficking of autoreactive leucocytes across the blood-brain barrier endothelium is a hallmark of multiple sclerosis pathogenesis. Although the blood-brain barrier endothelium represents one of the main CNS borders to interact with the infiltrating leucocytes, its exact contribution to neuroinflammation remains understudied. Here, we show that Mcam identifies inflammatory brain endothelial cells with pro-migratory transcriptomic signature during experimental autoimmune encephalomyelitis. In addition, MCAM was preferentially upregulated on blood-brain barrier endothelial cells in multiple sclerosis lesions in situ and at experimental autoimmune encephalomyelitis disease onset by molecular MRI. In vitro and in vivo, we demonstrate that MCAM on blood-brain barrier endothelial cells contributes to experimental autoimmune encephalomyelitis development by promoting the cellular trafficking of TH1 and TH17 lymphocytes across the blood-brain barrier. Last, we showcase ST14 as an immune ligand to brain endothelial MCAM, enriched on CD4+ T lymphocytes that cross the blood-brain barrier in vitro, in vivo and in multiple sclerosis lesions as detected by flow cytometry on rapid autopsy derived brain tissue from multiple sclerosis patients. Collectively, our findings reveal that MCAM is at the centre of a pathological pathway used by brain endothelial cells to recruit pathogenic CD4+ T lymphocyte from circulation early during neuroinflammation. The therapeutic targeting of this mechanism is a promising avenue to treat multiple sclerosis.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Humanos , Barrera Hematoencefálica/patología , Encéfalo/patología , Antígeno CD146/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/metabolismo , Endotelio/metabolismo , Endotelio/patología , Esclerosis Múltiple/patología , Enfermedades NeuroinflamatoriasRESUMEN
AIMS: Axonal injury in multiple sclerosis (MS) and experimental models is most frequently detected in acutely demyelinating lesions. We recently reported a compensatory neuronal response, where mitochondria move to the acutely demyelinated axon and increase the mitochondrial content following lysolecithin-induced demyelination. We termed this homeostatic phenomenon, which is also evident in MS, the axonal response of mitochondria to demyelination (ARMD). The aim of this study is to determine whether ARMD is consistently evident in experimental demyelination and how its perturbation relates to axonal injury. METHODS: In the present study, we assessed axonal mitochondrial content as well as axonal mitochondrial respiratory chain complex IV activity (cytochrome c oxidase or COX) of axons and related these to axonal injury in nine different experimental disease models. We used immunofluorescent histochemistry as well as sequential COX histochemistry followed by immunofluorescent labelling of mitochondria and axons. RESULTS: We found ARMD a consistent and robust phenomenon in all experimental disease models. The increase in mitochondrial content within demyelinated axons, however, was not always accompanied by a proportionate increase in complex IV activity, particularly in highly inflammatory models such as experimental autoimmune encephalomyelitis (EAE). Axonal complex IV activity inversely correlated with the extent of axonal injury in experimental disease models. CONCLUSIONS: Our findings indicate that ARMD is a consistent and prominent feature and emphasise the importance of complex IV activity in the context of ARMD, especially in autoimmune inflammatory demyelination, paving the way for the development of novel neuroprotective therapies.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Esclerosis Múltiple/patología , Axones/patología , Encefalomielitis Autoinmune Experimental/patología , Neuronas/patología , Mitocondrias/patologíaRESUMEN
B cells have been implicated in the pathogenesis of multiple sclerosis, but the mechanisms that guide B cell activation in the periphery and subsequent migration to the CNS remain incompletely understood. We previously showed that systemic inflammation induces an accumulation of B cells in the spleen in a CCR6/CCL20-dependent manner. In this study, we evaluated the role of CCR6/CCL20 in the context of myelin oligodendrocyte glycoprotein (MOG) protein-induced (B cell-dependent) experimental autoimmune encephalomyelitis (EAE). We found that CCR6 is upregulated on murine B cells that migrate into the CNS during neuroinflammation. In addition, human B cells that migrate across CNS endothelium in vitro were found to be CCR6+, and we detected CCL20 production by activated CNS-derived human endothelial cells as well as a systemic increase in CCL20 protein during EAE. Although mice that lack CCR6 expression specifically on B cells exhibited an altered germinal center reaction in response to MOG protein immunization, CCR6-deficient B cells did not exhibit any competitive disadvantage in their migration to the CNS during EAE, and the clinical and pathological presentation of EAE induced by MOG protein was unaffected. These data, to our knowledge, provide new information on the role of B cell-intrinsic CCR6 expression in a B cell-dependent model of neuroinflammation.
Asunto(s)
Linfocitos B/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Centro Germinal/inmunología , Inmunización/métodos , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Receptores CCR6/deficiencia , Animales , Linfocitos B/metabolismo , Donantes de Sangre , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Quimiocina CCL20/metabolismo , Encefalomielitis Autoinmune Experimental/inducido químicamente , Células Endoteliales/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/genética , Receptores CCR6/genética , Proteínas Recombinantes/administración & dosificaciónRESUMEN
Early multiple sclerosis lesions feature relative preservation of oligodendrocyte cell bodies with dying back retraction of their myelinating processes. Cell loss occurs with disease progression. Putative injury mediators include metabolic stress (low glucose/nutrient), pro-inflammatory mediators (interferon γ and tumour necrosis factor α), and excitotoxins (glutamate). Our objective was to compare the impact of these disease relevant mediators on the injury responses of human mature oligodendrocytes. In the current study, we determined the effects of these mediators on process extension and survival of human brain derived mature oligodendrocytes in vitro and used bulk RNA sequencing to identify distinct effector mechanisms that underlie the responses. All mediators induced significant process retraction of the oligodendrocytes in dissociated cell culture. Only metabolic stress (low glucose/nutrient) conditions resulted in delayed (4-6 days) non-apoptotic cell death. Metabolic effects were associated with induction of the integrated stress response, which can be protective or contribute to cell injury dependent on its level and duration of activation. Addition of Sephin1, an agonist of the integrated stress response induced process retraction under control conditions and further enhanced retraction under metabolic stress conditions. The antagonist ISRIB restored process outgrowth under stress conditions, and if added to already stressed cells, reduced delayed cell death and prolonged the period in which recovery could occur. Inflammatory cytokine functional effects were associated with activation of multiple signalling pathways (including Jak/Stat-1) that regulate process outgrowth, without integrated stress response induction. Glutamate application produced limited transcriptional changes suggesting a contribution of effects directly on cell processes. Our comparative studies indicate the need to consider both the specific injury mediators and the distinct cellular mechanisms of responses to them by human oligodendrocytes to identify effective neuroprotective therapies for multiple sclerosis.
Asunto(s)
Esclerosis Múltiple , Humanos , Esclerosis Múltiple/patología , Oligodendroglía/metabolismo , Encéfalo/patología , Muerte Celular , Glucosa/metabolismo , Células CultivadasRESUMEN
BACKGROUND: Resolution of inflammation is an active and regulated process that leads to the clearance of cell debris and immune cells from the challenged tissue, facilitating the recovery of homeostasis. This physiological response is coordinated by endogenous bioactive lipids known as specialized pro-resolving mediators (SPMs). When resolution fails, inflammation becomes uncontrolled leading chronic inflammation and tissue damage, as occurs in multiple sclerosis (MS). METHODS: SPMs and the key biosynthetic enzymes involved in SPM production were analysed by metabololipidomics and qPCR in active brain lesions, serum and peripheral blood mononuclear cells (PBMC) of MS patients as well as in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE). We also tested the therapeutic actions of the SPM coined Maresin-1 (MaR1) in EAE mice and studied its impact on inflammation by doing luminex and flow cytometry analysis. RESULTS: We show that levels of MaR1 and other SPMs were below the limit of detection or not increased in the spinal cord of EAE mice, whereas the production of pro-inflammatory eicosanoids was induced during disease progression. Similarly, we reveal that SPMs were undetected in serum and active brain lesion samples of MS patients, which was linked to impaired expression of the enzymes involved in the biosynthetic pathways of SPMs. We demonstrate that exogenous administration of MaR1 in EAE mice suppressed the protein levels of various pro-inflammatory cytokines and reduced immune cells counts in the spinal cord and blood. MaR1 also decreased the numbers of Th1 cells but increased the accumulation of regulatory T cells and drove macrophage polarization towards an anti-inflammatory phenotype. Importantly, we provide clear evidence that administration of MaR1 in mice with clinical signs of EAE enhanced neurological outcomes and protected from demyelination. CONCLUSIONS: This study reveals that there is an imbalance in the production of SPMs in MS patients and in EAE mice, and that increasing the bioavailability of SPMs, such as MaR1, minimizes inflammation and mediates therapeutic actions. Thus, these data suggest that immunoresolvent therapies, such as MaR1, could be a novel avenue for the treatment of MS.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Animales , Antiinflamatorios/farmacología , Encefalomielitis Autoinmune Experimental/metabolismo , Humanos , Inflamación/metabolismo , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos C57BL , Médula Espinal/patologíaRESUMEN
Oncostatin M (OSM) is an IL-6 family member which exerts neuroprotective and remyelination-promoting effects after damage to the central nervous system (CNS). However, the role of OSM in neuro-inflammation is poorly understood. Here, we investigated OSM's role in pathological events important for the neuro-inflammatory disorder multiple sclerosis (MS). We show that OSM receptor (OSMRß) expression is increased on circulating lymphocytes of MS patients, indicating their elevated responsiveness to OSM signalling. In addition, OSM production by activated myeloid cells and astrocytes is increased in MS brain lesions. In experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, OSMRß-deficient mice exhibit milder clinical symptoms, accompanied by diminished T helper 17 (Th17) cell infiltration into the CNS and reduced BBB leakage. In vitro, OSM reduces BBB integrity by downregulating the junctional molecules claudin-5 and VE-cadherin, while promoting secretion of the Th17-attracting chemokine CCL20 by inflamed BBB-endothelial cells and reactive astrocytes. Using flow cytometric fluorescence resonance energy transfer (FRET) quantification, we found that OSM-induced endothelial CCL20 promotes activation of lymphocyte function-associated antigen 1 (LFA-1) on Th17 cells. Moreover, CCL20 enhances Th17 cell adhesion to OSM-treated inflamed endothelial cells, which is at least in part ICAM-1 mediated. Together, these data identify an OSM-CCL20 axis, in which OSM contributes significantly to BBB impairment during neuro-inflammation by inducing permeability while recruiting Th17 cells via enhanced endothelial CCL20 secretion and integrin activation. Therefore, care should be taken when considering OSM as a therapeutic agent for treatment of neuro-inflammatory diseases such as MS.
Asunto(s)
Barrera Hematoencefálica , Encefalomielitis Autoinmune Experimental , Oncostatina M , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Oncostatina M/metabolismo , Oncostatina M/farmacología , Subunidad beta del Receptor de Oncostatina M/biosíntesis , Subunidad beta del Receptor de Oncostatina M/genética , Células Th17/metabolismo , Células Th17/patologíaRESUMEN
The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions and that these are reflected in its posttranslational modifications. We found that in post mortem human brain, including in the Substantia nigra, parkin is largely insoluble after age 40 years; this transition is linked to its oxidation, such as at residues Cys95 and Cys253. In mice, oxidative stress induces posttranslational modifications of parkin cysteines that lower its solubility in vivo. Similarly, oxidation of recombinant parkin by hydrogen peroxide (H2O2) promotes its insolubility and aggregate formation, and in exchange leads to the reduction of H2O2. This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. In prkn-null mice, H2O2 levels are increased under oxidative stress conditions, such as acutely by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin exposure or chronically due to a second, genetic hit; H2O2 levels are also significantly increased in parkin-deficient human brain. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at the primate-specific residue Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation in vitro. By probing sections of adult, human midbrain from control individuals with epitope-mapped, monoclonal antibodies, we found specific and robust parkin reactivity that co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these complementary redox effects may augment oxidative stress during ageing in dopamine-producing cells of mutant PRKN allele carriers, thereby enhancing the risk of Parkinson's-linked neurodegeneration.
Asunto(s)
Envejecimiento/metabolismo , Dopamina/metabolismo , Mesencéfalo/metabolismo , Degeneración Nerviosa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/patología , Animales , Niño , Preescolar , Femenino , Humanos , Masculino , Mesencéfalo/patología , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Degeneración Nerviosa/patología , Oxidación-Reducción , Adulto JovenRESUMEN
Axonal loss is the key pathological substrate of neurological disability in demyelinating disorders, including multiple sclerosis (MS). However, the consequences of demyelination on neuronal and axonal biology are poorly understood. The abundance of mitochondria in demyelinated axons in MS raises the possibility that increased mitochondrial content serves as a compensatory response to demyelination. Here, we show that upon demyelination mitochondria move from the neuronal cell body to the demyelinated axon, increasing axonal mitochondrial content, which we term the axonal response of mitochondria to demyelination (ARMD). However, following demyelination axons degenerate before the homeostatic ARMD reaches its peak. Enhancement of ARMD, by targeting mitochondrial biogenesis and mitochondrial transport from the cell body to axon, protects acutely demyelinated axons from degeneration. To determine the relevance of ARMD to disease state, we examined MS autopsy tissue and found a positive correlation between mitochondrial content in demyelinated dorsal column axons and cytochrome c oxidase (complex IV) deficiency in dorsal root ganglia (DRG) neuronal cell bodies. We experimentally demyelinated DRG neuron-specific complex IV deficient mice, as established disease models do not recapitulate complex IV deficiency in neurons, and found that these mice are able to demonstrate ARMD, despite the mitochondrial perturbation. Enhancement of mitochondrial dynamics in complex IV deficient neurons protects the axon upon demyelination. Consequently, increased mobilisation of mitochondria from the neuronal cell body to the axon is a novel neuroprotective strategy for the vulnerable, acutely demyelinated axon. We propose that promoting ARMD is likely to be a crucial preceding step for implementing potential regenerative strategies for demyelinating disorders.
Asunto(s)
Enfermedades Desmielinizantes/patología , Mitocondrias/patología , Esclerosis Múltiple/patología , Degeneración Nerviosa/patología , Neuroprotección/fisiología , Animales , Axones/patología , Humanos , Ratones , Biogénesis de OrganelosRESUMEN
Multiple sclerosis is a chronic inflammatory, demyelinating, and neurodegenerative disease affecting the brain, spinal cord and optic nerves. Neuronal damage is triggered by various harmful factors that engage diverse signalling cascades in neurons; thus, therapeutic approaches to protect neurons will need to focus on agents that can target multiple biological processes. We have therefore focused our attention on microRNAs: small non-coding RNAs that primarily function as post-transcriptional regulators that target messenger RNAs and repress their translation into proteins. A single microRNA can target many functionally related messenger RNAs making microRNAs powerful epigenetic regulators. Dysregulation of microRNAs has been described in many neurodegenerative diseases including multiple sclerosis. Here, we report that two microRNAs, miR-223-3p and miR-27a-3p, are upregulated in neurons in the experimental autoimmune encephalomyelitis mouse model of CNS inflammation and in grey matter-containing multiple sclerosis lesions. Prior work has shown peripheral blood mononuclear cell conditioned media causes sublethal degeneration of neurons in culture. We find overexpression of miR-27a-3p or miR-223-3p protects dissociated cortical neurons from condition media mediated degeneration. Introduction of miR-223-3p in vivo in mouse retinal ganglion cells protects their axons from degeneration in experimental autoimmune encephalomyelitis. In silico analysis revealed that messenger RNAs involved in glutamate receptor signalling are enriched as miR-27a-3p and miR-223-3p targets. We observe that antagonism of NMDA and AMPA type glutamate receptors protects neurons from condition media dependent degeneration. Our results suggest that miR-223-3p and miR-27a-3p are upregulated in response to inflammation to mediate a compensatory neuroprotective gene expression program that desensitizes neurons to glutamate by targeting messenger RNAs involved in glutamate receptor signalling.
Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , MicroARNs/genética , Neuronas/patología , Animales , Axones/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/metabolismo , Ácido Glutámico/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Ratones , MicroARNs/metabolismo , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/genética , Médula Espinal/patologíaRESUMEN
Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule found on blood-brain barrier endothelial cells (BBB-ECs) that was previously shown to be involved in leukocyte transmigration across the endothelium. In the present study, we found that ALCAM knockout (KO) mice developed a more severe myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE). The exacerbated disease was associated with a significant increase in the number of CNS-infiltrating proinflammatory leukocytes compared with WT controls. Passive EAE transfer experiments suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. In addition, phenotypic characterization of unimmunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further in vivo, in vitro, and molecular analysis confirmed that ALCAM is associated with tight junction molecule assembly at the BBB, explaining the increased permeability of CNS blood vessels in ALCAM KO animals. Collectively, our data point to a biologically important function of ALCAM in maintaining BBB integrity.
Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Células Endoteliales/metabolismo , Molécula de Adhesión Celular del Leucocito Activado/genética , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Encefalomielitis Autoinmune Experimental/patología , Femenino , Homeostasis , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos , Índice de Severidad de la Enfermedad , Médula Espinal/metabolismo , Proteínas de Uniones Estrechas/metabolismoRESUMEN
CD4+Foxp3+ T regulatory (Treg) cells provide a key defence against inflammatory disease, but also have an ability to produce pro-inflammatory cytokines. The evidence for these two possibilities in multiple sclerosis (MS) is controversial. However, this has largely been based on studies of circulating Treg cells derived from peripheral blood, rather than the central nervous system. We show that Foxp3+ cells in the brains of MS patients predominantly produce interleukin-10 (IL-10) and show high expression of the IL-33 receptor ST2 (associated with potent Treg function), indicating that Treg in the inflamed brain maintain their suppressive function.
Asunto(s)
Encéfalo/patología , Factores de Transcripción Forkhead/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-10/biosíntesis , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Animales , Antígenos CD4/metabolismo , Femenino , Humanos , Ratones Endogámicos C57BL , Linfocitos T Reguladores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Strategies for treating progressive multiple sclerosis (MS) remain limited. Here, we found that miR-145-5p is overabundant uniquely in chronic lesion tissues from secondary progressive MS patients. We induced both acute and chronic demyelination in miR-145 knockout mice to determine its contributions to remyelination failure. Following acute demyelination, no advantage to miR-145 loss could be detected. However, after chronic demyelination, animals with miR-145 loss demonstrated increased remyelination and functional recovery, coincident with altered presence of astrocytes and microglia within the corpus callosum relative to wild-type animals. This improved response in miR-145 knockout animals coincided with a pathological upregulation of miR-145-5p in wild-type animals with chronic cuprizone exposure, paralleling human chronic lesions. Furthermore, miR-145 overexpression specifically in oligodendrocytes (OLs) severely stunted differentiation and negatively impacted survival. RNAseq analysis showed altered transcriptome in these cells with downregulated major pathways involved in myelination. Our data suggest that pathological accumulation of miR-145-5p is a distinctive feature of chronic demyelination and is strongly implicated in the failure of remyelination, possibly due to the inhibition of OL differentiation together with alterations in other glial cells. This is mirrored in chronic MS lesions, and thus miR-145-5p serves as a potential relevant therapeutic target in progressive forms of MS.