RESUMEN
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, resulting in the loss of dystrophin, a large cytosolic protein that links the cytoskeleton to extracellular matrix receptors in skeletal muscle. Aside from progressive muscle damage, many patients with DMD also have neurological deficits of unknown etiology. To investigate potential mechanisms for DMD neurological deficits, we assessed postnatal oligodendrogenesis and myelination in the Dmdmdx mouse model. In the ventricular-subventricular zone (V-SVZ) stem cell niche, we found that oligodendrocyte progenitor cell (OPC) production was deficient, with reduced OPC densities and proliferation, despite a normal stem cell niche organization. In the Dmdmdx corpus callosum, a large white matter tract adjacent to the V-SVZ, we also observed reduced OPC proliferation and fewer oligodendrocytes. Transmission electron microscopy further revealed significantly thinner myelin, an increased number of abnormal myelin structures and delayed myelin compaction, with hypomyelination persisting into adulthood. Our findings reveal alterations in oligodendrocyte development and myelination that support the hypothesis that changes in diffusion tensor imaging seen in patients with DMD reflect developmental changes in myelin architecture.
Asunto(s)
Ratones Endogámicos mdx , Distrofia Muscular de Duchenne , Vaina de Mielina , Oligodendroglía , Animales , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/patología , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/genética , Proliferación Celular , Distrofina/metabolismo , Distrofina/deficiencia , Distrofina/genética , Cuerpo Calloso/patología , Cuerpo Calloso/metabolismo , Ratones Endogámicos C57BL , Ratones , Células Precursoras de Oligodendrocitos/metabolismo , Células Precursoras de Oligodendrocitos/patología , Ventrículos Laterales/patología , Ventrículos Laterales/metabolismo , Modelos Animales de Enfermedad , Diferenciación Celular , MasculinoRESUMEN
Multiple Sclerosis (MS) is a chronic disease characterized by immune-mediated destruction of myelinating oligodendroglia in the central nervous system. Loss of myelin leads to neurological dysfunction and, if myelin repair fails, neurodegeneration of the denuded axons. Virtually all treatments for MS act by suppressing immune function, but do not alter myelin repair outcomes or long-term disability. Excitingly, the diabetes drug metformin, a potent activator of the cellular "energy sensor" AMPK complex, has recently been reported to enhance recovery from demyelination. In aged mice, metformin can restore responsiveness of oligodendrocyte progenitor cells (OPCs) to pro-differentiation cues, enhancing their ability to differentiate and thus repair myelin. However, metformin's influence on young oligodendroglia remains poorly understood. Here we investigated metformin's effect on the temporal dynamics of differentiation and metabolism in young, healthy oligodendroglia and in oligodendroglia following myelin damage in young adult mice. Our findings reveal that metformin accelerates early stages of myelin repair following cuprizone-induced myelin damage. Metformin treatment of both isolated OPCs and oligodendrocytes altered cellular bioenergetics, but in distinct ways, suppressing oxidative phosphorylation and enhancing glycolysis in OPCs, but enhancing oxidative phosphorylation and glycolysis in both immature and mature oligodendrocytes. In addition, metformin accelerated the differentiation of OPCs to oligodendrocytes in an AMPK-dependent manner that was also dependent on metformin's ability to modulate cell metabolism. In summary, metformin dramatically alters metabolism and accelerates oligodendroglial differentiation both in health and following myelin damage. This finding broadens our knowledge of metformin's potential to promote myelin repair in MS and in other diseases with myelin loss or altered myelination dynamics.
RESUMEN
Once believed to be part of the nervenkitt or "nerve glue" network in the central nervous system (CNS), oligodendroglial cells now have established roles in key neurological functions such as myelination, neuroprotection, and motor learning. More recently, oligodendroglia has become the subject of intense investigations aimed at understanding the contributions of its energetics to CNS physiology and pathology. In this review, we discuss the current understanding of oligodendroglial metabolism in regulating key stages of oligodendroglial development and health, its role in providing energy to neighboring cells such as neurons, as well as how alterations in oligodendroglial bioenergetics contribute to disease states. Importantly, we highlight how certain inputs can regulate oligodendroglial metabolism, including extrinsic and intrinsic mediators of cellular signaling, pharmacological compounds, and even dietary interventions. Lastly, we discuss emerging studies aimed at discovering the therapeutic potential of targeting components within oligodendroglial bioenergetic pathways.
RESUMEN
Healthy myelin is essential for proper brain function. When the myelin sheath is damaged, fast saltatory impulse conduction is lost and neuronal axons become vulnerable to degeneration. Thus, regeneration of the myelin sheath by encouraging oligodendrocyte lineage cells to remyelinate the denuded axons is a promising therapeutic target for demyelinating diseases such as multiple sclerosis. Ex vivo organotypic cerebellar slice cultures are a useful model to study developmental myelination, demyelination, remyelination and remyelination failure. In these cultures, the cerebellum's three-dimensional architecture and various cell populations remain largely intact, providing a realistic and relatively cost-efficient model that can be easily manipulated by the addition of viral vectors, pharmaceuticals or (transgenic) cells to augment or replace resident cell populations. Moreover, slice cultures can be treated with lysolecithin or polyinosinic:polycytidylic acid to induce demyelination and mimic efficient as well as inefficient remyelination. It can be challenging to set up slice cultures for the first time, as in our experience, seemingly minor differences in technique and materials can make a great difference to the quality of the cultures. Therefore, this report provides an in-depth description for the generation and maintenance of ex vivo organotypic cerebellar cultures for demyelination-remyelination studies with a focus on practical tips for scientists that are new to this technique.
Asunto(s)
Enfermedades Desmielinizantes , Remielinización , Cerebelo , Enfermedades Desmielinizantes/tratamiento farmacológico , Humanos , Vaina de Mielina , Remielinización/fisiología , Flujo de TrabajoRESUMEN
Laminin α2 gene (LAMA2)-related Congenital Muscular Dystrophy (CMD) was distinguished by a defining central nervous system (CNS) abnormality-aberrant white matter signals by MRI-when first described in the 1990s. In the past 25 years, researchers and clinicians have expanded our knowledge of brain involvement in LAMA2-related CMD, also known as Congenital Muscular Dystrophy Type 1A (MDC1A). Neurological changes in MDC1A can be structural, including lissencephaly and agyria, as well as functional, including epilepsy and intellectual disability. Mouse models of MDC1A include both spontaneous and targeted LAMA2 mutations and range from a partial loss of LAMA2 function (e.g., dy2J/dy2J ), to a complete loss of LAMA2 expression (dy 3K/dy 3K). Diverse cellular and molecular changes have been reported in the brains of MDC1A mouse models, including blood-brain barrier dysfunction, altered neuro- and gliogenesis, changes in synaptic plasticity, and decreased myelination, providing mechanistic insight into potential neurological dysfunction in MDC1A. In this review article, we discuss selected studies that illustrate the potential scope and complexity of disturbances in brain development in MDC1A, and as well as highlight mechanistic insights that are emerging from mouse models.
RESUMEN
Aerobic exercise is known to influence brain function, e.g., enhancing executive function in both children and adults, with many of these influences being attributed to alterations in neurogenesis and neuronal function. Yet oligodendroglia in adult brains have also been reported to be highly responsive to exercise, including in the prefrontal cortex (PFC), a late myelinating region implicated in working memory. However, whether exercise affects oligodendroglia or myelination in juveniles, either in the PFC or in other brain regions, remains unknown. To address this, both juvenile and young adult mice were provided free access to running wheels for four weeks followed by an analysis of oligodendrocyte development and myelination in the PFC and the corpus callosum, a major white matter tract. Working memory and PFC NG2+ cell development were both affected by exercise in juvenile mice, yet surprisingly these exercise-mediated effects were distinct in juveniles and young adults. In the PFC, NG2+ cell proliferation was increased in exercising juveniles, but not young adults, whereas newly-born oligodendrocyte production was increased in exercising young adults, but not juveniles. Although no overall changes in myelin genes were found, elevated levels of Monocarboxylate Transporter 1, a glial lactate transporter important during active myelination, were found in the PFC of exercising young adults. Overall our findings reveal that long-term exercise modulates PFC glial development and does so differentially in juvenile and young adult mice, providing insight into the cellular responses that may underlie cognitive benefits to teenagers and young adults in response to exercise. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 687-700, 2018.
Asunto(s)
Antígenos/metabolismo , Oligodendroglía/metabolismo , Corteza Prefrontal/crecimiento & desarrollo , Corteza Prefrontal/metabolismo , Proteoglicanos/metabolismo , Carrera/fisiología , Animales , Proliferación Celular/fisiología , Femenino , Aprendizaje por Laberinto/fisiología , Ratones Endogámicos C57BL , Transportadores de Ácidos Monocarboxílicos/metabolismo , Oligodendroglía/citología , Corteza Prefrontal/citología , Carrera/psicología , Simportadores/metabolismoRESUMEN
The regulatory mechanisms that control neural stem cell (NSC) activation in the adult ventricular-subventricular zone (V-SVZ) stem cell niche have been the focus of intense investigation, yet how the niche first develops and organizes is poorly understood. Here, we examined matrix metalloproteinases (MMPs) for potential roles in V-SVZ stem cell niche development. MMP12 was found to promote appropriate niche cellular arrangements, the formation of specialized niche extracellular matrix, and the translational planar cell polarity of ependymal cells that surround and support niche NSCs. Surprisingly, ependymal cells were found to have an intracellular pool of MMP12 that promoted ependymal cell ciliogenesis by upregulating FOXJ1. In addition, both extracellular and intracellular MMP12 were found to regulate V-SVZ niche output by promoting NSC quiescence. These findings reveal that extracellular and intracellular MMP12 have both unique and overlapping roles that help orchestrate the development of the adult V-SVZ stem cell niche.
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Matriz Extracelular/metabolismo , Ventrículos Laterales/metabolismo , Ventrículos Laterales/fisiología , Metaloproteinasa 12 de la Matriz/metabolismo , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Nicho de Células Madre/fisiología , Animales , Polaridad Celular/fisiología , Epéndimo/metabolismo , Epéndimo/fisiología , Matriz Extracelular/fisiología , Factores de Transcripción Forkhead/metabolismo , Ratones , Ratones Endogámicos C57BL , Regulación hacia Arriba/fisiologíaRESUMEN
Multiciliated cells of the airways, brain ventricles, and female reproductive tract provide the motive force for mucociliary clearance, cerebrospinal fluid circulation, and ovum transport. Despite their clear importance to human biology and health, the molecular mechanisms underlying multiciliated cell differentiation are poorly understood. Prior studies implicate the distal appendage/transition fiber protein CEP164 as a central regulator of primary ciliogenesis; however, its role in multiciliogenesis remains unknown. In this study, we have generated a novel conditional mouse model that lacks CEP164 in multiciliated tissues and the testis. These mice show a profound loss of airway, ependymal, and oviduct multicilia and develop hydrocephalus and male infertility. Using primary cultures of tracheal multiciliated cells as a model system, we found that CEP164 is critical for multiciliogenesis, at least in part, via its regulation of small vesicle recruitment, ciliary vesicle formation, and basal body docking. In addition, CEP164 is necessary for the proper recruitment of another distal appendage/transition fiber protein Chibby1 (Cby1) and its binding partners FAM92A and FAM92B to the ciliary base in multiciliated cells. In contrast to primary ciliogenesis, CEP164 is dispensable for the recruitment of intraflagellar transport (IFT) components to multicilia. Finally, we provide evidence that CEP164 differentially controls the ciliary targeting of membrane-associated proteins, including the small GTPases Rab8, Rab11, and Arl13b, in multiciliated cells. Altogether, our studies unravel unique requirements for CEP164 in primary versus multiciliogenesis and suggest that CEP164 modulates the selective transport of membrane vesicles and their cargoes into the ciliary compartment in multiciliated cells. Furthermore, our mouse model provides a useful tool to gain physiological insight into diseases associated with defective multicilia.
Asunto(s)
Cilios/fisiología , Proteínas de Microtúbulos/fisiología , Animales , Cuerpos Basales/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Centriolos/metabolismo , Cilios/genética , Cilios/metabolismo , Células Epiteliales/citología , Femenino , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Transporte de Proteínas , Tráquea/citologíaRESUMEN
BACKGROUND: In Duchenne muscular dystrophy (DMD), the loss of the dystrophin component of the dystrophin-glycoprotein complex (DGC) compromises plasma membrane integrity in skeletal muscle, resulting in extensive muscle degeneration. In addition, many DMD patients exhibit brain deficits in which the cellular etiology remains poorly understood. We recently found that dystroglycan, a receptor component of the DGC that binds intracellularly to dystrophin, regulates the development of oligodendrocytes, the myelinating glial cells of the brain. RESULTS: We investigated whether dystrophin contributes to oligodendroglial function and brain myelination. We found that oligodendrocytes express up to three dystrophin isoforms, in conjunction with classic DGC components, which are developmentally regulated during differentiation and in response to extracellular matrix engagement. We found that mdx mice, a model of DMD lacking expression of the largest dystrophin isoform, have delayed myelination and inappropriate oligodendrocyte progenitor proliferation in the cerebral cortex. When we prevented the expression of all oligodendroglial dystrophin isoforms in cultured oligodendrocytes using RNA interference, we found that later stages of oligodendrocyte maturation were significantly delayed, similar to mdx phenotypes in the developing brain. CONCLUSIONS: We find that dystrophin is expressed in oligodendrocytes and influences developmental myelination, which provides new insight into potential cellular contributors to brain dysfunction associated with DMD.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Oligodendroglía/metabolismo , Animales , Encéfalo/patología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Oligodendroglía/patología , Isoformas de Proteínas , Interferencia de ARN , ARN Mensajero/metabolismo , Ratas Sprague-DawleyRESUMEN
While the extracellular matrix (ECM) is known to regulate neural stem cell quiescence in the adult subventricular zone (SVZ), the function of ECM in the developing SVZ remains unknown. Here, we report that the ECM receptor dystroglycan regulates a unique developmental restructuring of ECM in the early postnatal SVZ. Dystroglycan is furthermore required for ependymal cell differentiation and assembly of niche pinwheel structures, at least in part by suppressing Notch activation in radial glial cells, which leads to the increased expression of MCI, Myb, and FoxJ1, transcriptional regulators necessary for acquisition of the multiciliated phenotype. Dystroglycan also regulates perinatal radial glial cell proliferation and transition into intermediate gliogenic progenitors, such that either acute or constitutive loss of function in dystroglycan results in increased oligodendrogenesis. These findings reveal a role for dystroglycan in orchestrating both the assembly and function of the SVZ neural stem cell niche.
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Distroglicanos/genética , Ventrículos Laterales/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Nicho de Células Madre/genética , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Distroglicanos/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Ventrículos Laterales/crecimiento & desarrollo , Ratones , Neuroglía/metabolismo , Neuronas/metabolismo , Ratas , Receptores Notch/biosíntesis , Receptores Notch/genéticaRESUMEN
Many behavioral experiences are known to promote hippocampal neurogenesis. In contrast, the ability of behavioral experiences to influence the production of oligodendrocytes and myelin sheath formation remains relatively unknown. However, several recent studies indicate that voluntary exercise and environmental enrichment can positively influence both oligodendrogenesis and myelination, and that, in contrast, social isolation can negatively influence myelination. In this review we summarize studies addressing the influence of behavioral experiences on oligodendrocyte lineage cells and myelin, and highlight potential mechanisms including experience-dependent neuronal activity, metabolites, and stress effectors, as well as both local and systemic secreted factors. Although more study is required to better understand the underlying mechanisms by which behavioral experiences regulate oligodendrocyte lineage cells, this exciting and newly emerging field has already revealed that oligodendrocytes and their progenitors are highly responsive to behavioral experiences and suggest the existence of a complex network of reciprocal interactions among oligodendrocyte lineage development, behavioral experiences, and brain function. Achieving a better understanding of these relationships may have profound implications for human health, and in particular, for our understanding of changes in brain function that occur in response to experiences. This article is part of the Special Issue entitled 'Oligodendrocytes in Health and Disease'.
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Plasticidad de la Célula/fisiología , Ejercicio Físico/fisiología , Vaina de Mielina/fisiología , Oligodendroglía/fisiología , Células Madre/fisiología , Animales , Diferenciación Celular , Ambiente , Humanos , Aislamiento SocialRESUMEN
The cell surface receptor dystroglycan mediates interactions between oligodendroglia and laminin-211, an extracellular matrix protein that regulates timely oligodendroglial development. However, dystroglycan's precise role in oligodendroglial development and the potential mechanisms to regulate laminin-dystroglycan interactions remain unknown. Here we report that oligodendroglial dystroglycan is cleaved by metalloproteinases, thereby uncoupling oligodendroglia from laminin binding. Dystroglycan cleavage is selectively stimulated by oligodendrocyte progenitor cell attachment to laminin-211, but not laminin-111 or poly-D-lysine. In addition, dystroglycan cleavage occurs most prominently in oligodendrocyte progenitor cells, with limited dystroglycan cleavage observed in differentiating oligodendrocytes. When dystroglycan cleavage is blocked by metalloproteinase inhibitors, oligodendrocyte progenitor cell proliferation is substantially decreased. Conversely, expression of the intracellular portion of cleaved dystroglycan results in increased oligodendrocyte progenitor cell proliferation, suggesting that endogenous dystroglycan cleavage may promote oligodendrocyte progenitor cell cycle progression. Intriguingly, while matrix metalloproteinase-2 and/or -9 have been reported to be responsible for dystroglycan cleavage, we find that these two metalloproteinases are neither necessary nor sufficient for cleavage of oligodendroglial dystroglycan. In summary, laminin-211 stimulates metalloproteinase-mediated dystroglycan cleavage in oligodendrocyte progenitor cells (but not in differentiated oligodendrocytes), which in turn promotes oligodendrocyte progenitor cell proliferation. This novel regulation of oligodendroglial laminin-dystroglycan interactions may have important consequences for oligodendroglial differentiation, both during development and during disease when metalloproteinase levels become elevated.
Asunto(s)
Proliferación Celular/fisiología , Distroglicanos/metabolismo , Laminina/farmacología , Metaloproteasas/fisiología , Oligodendroglía/fisiología , Células Madre/fisiología , Animales , Animales Recién Nacidos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Ratones , Oligodendroglía/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-Dawley , Células Madre/efectos de los fármacosRESUMEN
Laminins are major constituents of the gliovascular basal lamina of the blood-brain barrier (BBB); however, the role of laminins in BBB development remains unclear. Here we report that Lama2(-/-) mice, lacking expression of the laminin α2 subunit of the laminin-211 heterotrimer expressed by astrocytes and pericytes, have a defective BBB in which systemically circulated tracer leaks into the brain parenchyma. The Lama2(-/-) vascular endothelium had significant abnormalities, including altered integrity and composition of the endothelial basal lamina, inappropriate expression of embryonic vascular endothelial protein MECA32, substantially reduced pericyte coverage, and tight junction abnormalities. Additionally, astrocytic endfeet were hypertrophic and lacked appropriately polarized aquaporin4 channels. Laminin-211 appears to mediate these effects at least in part by dystroglycan receptor interactions, as preventing dystroglycan expression in neural cells led to a similar set of BBB abnormalities and gliovascular disturbances, which additionally included perturbed vascular endothelial glucose transporter-1 localization. These findings provide insight into the cell and molecular changes that occur in congenital muscular dystrophies caused by Lama2 mutations or inappropriate dystroglycan post-translational modifications, which have accompanying brain abnormalities, including seizures. Our results indicate a novel role for laminin-dystroglycan interactions in the cooperative integration of astrocytes, endothelial cells, and pericytes in regulating the BBB.
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Barrera Hematoencefálica/crecimiento & desarrollo , Barrera Hematoencefálica/fisiología , Laminina/fisiología , Animales , Antígenos de Superficie/metabolismo , Acuaporina 4/metabolismo , Astrocitos/patología , Barrera Hematoencefálica/patología , Distroglicanos/metabolismo , Distroglicanos/fisiología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Laminina/genética , Ratones , Ratones Noqueados , Mutación , Neuronas/metabolismo , Uniones Estrechas/patologíaRESUMEN
The laminin family of extracellular matrix proteins are expressed broadly during embryonic brain development, but are enriched at ventricular and pial surfaces where laminins mediate radial glial attachment during corticogenesis. In the adult brain, however, laminin distribution is restricted, yet is found within the vascular basal lamina and associated fractones of the ventricular zone (VZ)-subventricular zone (SVZ) stem cell niche, where laminins regulate adult neural progenitor cell proliferation. It remains unknown, however, if laminins regulate the wave of oligodendrogenesis that occurs in the neonatal/early postnatal VZ-SVZ. Here we report that Lama2, the gene that encodes the laminin α2-subunit, regulates postnatal oligodendrogenesis. At birth, Lama2-/- mice had significantly higher levels of dying oligodendrocyte progenitor cells (OPCs) in the OPC germinal zone of the dorsal SVZ. This translated into fewer OPCs, both in the dorsal SVZ well as in an adjacent developing white matter tract, the corpus callosum. In addition, intermediate progenitor cells that give rise to OPCs in the Lama2-/- VZ-SVZ were mislocalized and proliferated nearer to the ventricle surface. Later, delays in oligodendrocyte maturation (with accompanying OPC accumulation), were observed in the Lama2-/- corpus callosum, leading to dysmyelination by postnatal day 21. Together these data suggest that prosurvival laminin interactions in the developing postnatal VZ-SVZ germinal zone regulate the ability, or timing, of oligodendrocyte production to occur appropriately.
Asunto(s)
Diferenciación Celular/fisiología , Ventrículos Cerebrales/citología , Regulación del Desarrollo de la Expresión Génica/genética , Laminina/metabolismo , Oligodendroglía/fisiología , Células Madre/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Antígenos/metabolismo , Proliferación Celular , Supervivencia Celular/genética , Proteínas del Ojo/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Homeodominio/metabolismo , Etiquetado Corte-Fin in Situ , Laminina/deficiencia , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Fibras Nerviosas Mielínicas/fisiología , Oligodendroglía/ultraestructura , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteoglicanos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción SOXB1/genética , Células Madre/ultraestructuraRESUMEN
During central nervous system (CNS) development, individual oligodendrocytes myelinate multiple axons, thus requiring the outgrowth and extensive branching of oligodendroglial processes. Laminin (Lm)-deficient mice have a lower percentage of myelinated axons, which may indicate a defect in the ability to properly extend and branch processes. It remains unclear, however, to what extent extracellular matrix (ECM) receptors contribute to oligodendroglial process remodeling itself. In the current study, we report that the ECM receptor dystroglycan is necessary for Lm enhancement of filopodial formation, process outgrowth, and process branching in differentiating oligodendroglia. During early oligodendroglial differentiation, the disruption of dystroglycan-Lm interactions, via blocking antibodies or dystroglycan small interfering RNA (siRNA), resulted in decreased filopodial number and length, decreased process length, and decreased numbers of primary and secondary processes. Later in oligodendrocyte differentiation, dystroglycan-deficient cells developed fewer branches, thus producing less complex networks of processes as determined by Sholl analysis. In newly differentiating oligodendroglia, dystroglycan was localized in filopodial tips, whereas, in more mature oligodendrocytes, dystroglycan was enriched in focal adhesion kinase (FAK)-positive focal adhesion structures. These results suggest that dystroglycan-Lm interactions influence oligodendroglial process dynamics and therefore may regulate the myelination capacity of individual oligodendroglia.
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Diferenciación Celular/fisiología , Distroglicanos/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Seudópodos/fisiología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa , Actinas/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Distroglicanos/genética , Distroglicanos/inmunología , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunoprecipitación , Interferón beta/inmunología , Interferón beta/metabolismo , Laminina/farmacología , Oligodendroglía/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/metabolismo , Seudópodos/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Estadísticas no Paramétricas , Células Madre , Factores de Tiempo , TransfecciónRESUMEN
Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification (1-3). While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy & de Vellis (2) is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published (4-6), a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.
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Técnicas Citológicas/métodos , Neuronas/citología , Oligodendroglía/citología , Animales , Corteza Cerebral/citología , Técnicas de Cocultivo , Ratones , Microscopía Fluorescente/métodos , Vaina de Mielina/metabolismo , Neuroglía/citología , Neuronas/metabolismo , Oligodendroglía/metabolismoRESUMEN
The health and function of the nervous system relies on glial cells that ensheath neuronal axons with a specialized plasma membrane termed myelin. The molecular mechanisms by which glial cells target and enwrap axons with myelin are only beginning to be elucidated, yet several studies have implicated extracellular matrix proteins and their receptors as being important extrinsic regulators. This review provides an overview of the extracellular matrix proteins and their receptors that regulate multiple steps in the cellular development of Schwann cells and oligodendrocytes, the myelinating glia of the PNS and CNS, respectively, as well as in the construction and maintenance of the myelin sheath itself. The first part describes the relevant cellular events that are influenced by particular extracellular matrix proteins and receptors, including laminins, collagens, integrins, and dystroglycan. The second part describes the signaling pathways and effector molecules that have been demonstrated to be downstream of Schwann cell and oligodendroglial extracellular matrix receptors, including FAK, small Rho GTPases, ILK, and the PI3K/Akt pathway, and the roles that have been ascribed to these signaling mediators. Throughout, we emphasize the concept of extracellular matrix proteins as environmental sensors that act to integrate, or match, cellular responses, in particular to those downstream of growth factors, to appropriate matrix attachment.
Asunto(s)
Matriz Extracelular/fisiología , Vaina de Mielina/fisiología , Fibras Nerviosas Mielínicas/fisiología , Sistema Nervioso/citología , Sistema Nervioso/crecimiento & desarrollo , Neuroglía/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Sistema Nervioso/embriología , Vías Nerviosas/fisiología , Neuroglía/citologíaRESUMEN
The adhesion receptor dystroglycan positively regulates terminal differentiation of oligodendrocytes, but the mechanism by which this occurs remains unclear. Using primary oligodendrocyte cultures, we identified and examined a connection between dystroglycan and the ability of insulin-like growth factor-1 (IGF-1) to promote oligodendrocyte differentiation. Consistent with previous reports, treatment with exogenous IGF-1 caused an increase in MBP protein that was preceded by activation of PI3K (AKT) and MAPK (ERK) signaling pathways. The extracellular matrix protein laminin was further shown to potentiate the effect of IGF-1 on oligodendrocyte differentiation. Depletion of the laminin receptor dystroglycan using siRNA, however, blocked the ability of IGF-1 to promote oligodendrocyte differentiation of cells grown on laminin, suggesting a role for dystroglycan in IGF-1-mediated differentiation. Indeed, loss of dystroglycan led to a reduction in the ability of IGF-1 to activate MAPK, but not PI3K, signaling pathways. Pharmacological inhibition of MAPK signaling also prevented IGF-1-induced increases in myelin basic protein (MBP), indicating that MAPK signaling was necessary to drive IGF-1-mediated enhancement of oligodendrocyte differentiation. Using immunoprecipitation, we found that dystroglycan, the adaptor protein Grb2, and insulin receptor substrate-1 (IRS-1), were associated in a protein complex. Taken together, our results suggest that the positive regulatory effect of laminin on oligodendrocyte differentiation may be attributed, at least in part, to dystroglycan's ability to promote IGF-1-induced differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Distroglicanos/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Oligodendroglía/citología , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Laminina/metabolismo , Oligodendroglía/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Interferencia de ARN , Ratas , Transducción de Señal/fisiologíaRESUMEN
Oligodendrocyte progenitor cells first proliferate to generate sufficient cell numbers and then differentiate into myelin-producing oligodendrocytes. The signal transduction mediators that underlie these events, however, remain poorly understood. The tyrosine phosphatase Shp1 has been linked to oligodendrocyte differentiation as Shp1-deficient mice show hypomyelination. The Shp1 homolog, Shp2, has recently been shown to regulate astrogliogenesis, but its role in oligodendrocyte development remains unknown. Here, we report that Shp2 protein levels were developmentally regulated in oligodendrocytes, with Shp2 phosphorylation being promoted by oligodendroglial mitogens but suppressed by laminin, an extracellular matrix protein that promotes oligodendroglial differentiation. In contrast, oligodendrocyte progenitors were found to be unresponsive to mitogens following Shp2, but not Shp1, depletion. In agreement with previous studies, Shp1 depletion led to decreased levels of myelin basic protein in differentiating oligodendrocytes, as well as reduced outgrowth of myelin membrane sheets. Shp2 depletion in contrast did not prevent oligodendrocyte differentiation but promoted expanded myelin membrane outgrowth. Taken together these data suggest that Shp1 and Shp2 have distinct functions in oligodendrocyte development: Shp2 regulates oligodendrocyte progenitor proliferation and Shp1 regulates oligodendrocyte differentiation. Adhesion to laminin may additionally provide extrinsic regulation of Shp2 activity and thus promote the transition from progenitor to differentiating oligodendrocyte.