RESUMEN
Neuron-derived molecules are potent regulators of oligodendrocyte differentiation and myelination during brain development and upon demyelination. Their analysis will thus contribute to understanding remyelination failure in demyelinating diseases, such as multiple sclerosis (MS). Previously, we have identified neuronal galectin-4 as a novel negative soluble regulator in the timing of developmental myelination. Here, we investigated whether galectin-4 is re-expressed in axons upon demyelination to regulate the timing of remyelination. Our findings revealed that galectin-4 is transiently localized to axons in demyelinated areas upon cuprizone-induced demyelination. In contrast, in chronic demyelinated MS lesions, where remyelination fails, galectin-4 is permanently present on axons. Remarkably, microglia/macrophages in cuprizone-demyelinated areas also harbor galectin-4, as also observed in activated microglia/macrophages that are present in active MS lesions and in inflammatory infiltrates in chronic-relapsing experimental autoimmune encephalomyelitis. In vitro analysis showed that galectin-4 is effectively endocytosed by macrophages, and may scavenge galectin-4 from oligodendrocytes, and that endogenous galectin-4 levels are increased in alternatively interleukin-4-activated macrophages and microglia. Hence, similar to developmental myelination, the (re)expressed galectin-4 upon demyelination may act as factor in the timing of oligodendrocyte differentiation, while the persistent presence of galectin-4 on demyelinated axons may disrupt this fine-tuning of remyelination.
Asunto(s)
Axones/metabolismo , Encéfalo/patología , Galectina 4/metabolismo , Macrófagos/efectos de los fármacos , Microglía/patología , Esclerosis Múltiple/patología , Animales , Encéfalo/metabolismo , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Cuprizona/toxicidad , Citocinas/metabolismo , Modelos Animales de Enfermedad , Galectina 4/genética , Regulación de la Expresión Génica/efectos de los fármacos , Lectinas/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Inhibidores de la Monoaminooxidasa/toxicidad , Esclerosis Múltiple/inducido químicamente , Glicoproteína Mielina-Oligodendrócito/toxicidad , Fragmentos de Péptidos/toxicidad , ARN Mensajero/metabolismo , Ratas , Factores de TiempoRESUMEN
Remyelination following central nervous system demyelination is essential to prevent axon degeneration. However, remyelination ultimately fails in demyelinating diseases such as multiple sclerosis. This failure of remyelination is likely mediated by many factors, including changes in the extracellular signalling environment. Here, we examined the expression of the extracellular matrix molecule fibronectin on demyelinating injury and how this affects remyelination by oligodendrocytes progenitors. In toxin-induced lesions undergoing efficient remyelination, fibronectin expression was transiently increased within demyelinated areas and declined as remyelination proceeded. Fibronectin levels increased both by leakage from the blood circulation and by production from central nervous system resident cells. In chronically demyelinated multiple sclerosis lesions, fibronectin expression persisted in the form of aggregates, which may render fibronectin resistant to degradation. Aggregation of fibronectin was similarly observed at the relapse phase of chronic experimental autoimmune encephalitis, but not on toxin-induced demyelination, suggesting that fibronectin aggregation is mediated by inflammation-induced demyelination. Indeed, the inflammatory mediator lipopolysaccharide induced fibronectin aggregation by astrocytes. Most intriguingly, injection of astrocyte-derived fibronectin aggregates in toxin-induced demyelinated lesions inhibited oligodendrocyte differentiation and remyelination, and fibronectin aggregates are barely expressed in remyelinated multiple sclerosis lesions. Therefore, these findings suggest that fibronectin aggregates within multiple sclerosis lesions contribute to remyelination failure. Hence, the inhibitory signals induced by fibronectin aggregates or factors that affect fibronectin aggregation could be potential therapeutic targets for promoting remyelination.
Asunto(s)
Enfermedades Desmielinizantes/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Fibronectinas/metabolismo , Esclerosis Múltiple/metabolismo , Vaina de Mielina/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Axones/metabolismo , Axones/patología , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Células Cultivadas , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/fisiopatología , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Humanos , Esclerosis Múltiple/patología , Esclerosis Múltiple/fisiopatología , Vaina de Mielina/patología , Regeneración Nerviosa/fisiología , Oligodendroglía/metabolismo , Oligodendroglía/patología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Médula Espinal/patología , Médula Espinal/fisiopatologíaRESUMEN
Myelination of axons by oligodendrocytes (OLGs) is essential for proper saltatory nerve conduction, i.e., rapid transmission of nerve impulses. Among others, extracellular matrix (ECM) molecules, neuronal signaling, and axonal adhesion regulate the biogenesis and maintenance of myelin membranes, driven by polarized transport of myelin-specific proteins and lipids. Galectin-4, a tandem-repeat-type lectin with affinity to sulfatide and nonsialylated termini of N-glycans, has the ability to regulate adhesion of cells to ECM components and is also involved in polarized membrane trafficking. We, therefore, anticipated that galectin-4 might play a role in myelination. Here, we show that in developing postnatal rat brains galectin-4 expression is downregulated just before the onset of myelination. Intriguingly, when immature OLGs were treated with galectin-4, OLG maturation was retarded, while a subset of the immature OLGs reverted to a morphologically less complex progenitor stage, displaying concomitantly an increase in proliferation. Similarly, myelination was inhibited when galectin-4 or anti-galectin-4 antibodies were added to co-cultures of dorsal root ganglion neurons and OLGs. Neurons and OLGs were identified as a possible source of galectin-4, both in vitro and in vivo. In culture, neurons but not OLGs released galectin-4. Interestingly, in co-cultures, a reduced release of endogenous galectin-4 correlated with the onset of myelination. Moreover, galectin-4-reactive sites are transiently expressed on processes of premyelinating primary OLGs, but not on neurons. Taken together, these results identify neuronal galectin-4 as a candidate for a soluble regulator of OLG differentiation and, hence, myelination. © 2012 Wiley Periodicals, Inc.
Asunto(s)
Galectina 4/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de la Mielina/metabolismo , Vaina de Mielina/metabolismo , Neuronas/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Encéfalo/citología , Bromodesoxiuridina/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Galectina 4/genética , Galectina 4/inmunología , Galectina 4/farmacología , Ganglios Espinales/citología , Gangliósidos/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Proteína Básica de Mielina/fisiología , Neuronas/fisiología , Oligodendroglía , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Transducción GenéticaRESUMEN
The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells.