RESUMEN
EPM1 is the most common form of Progressive Myoclonus Epilepsy characterized by late-childhood onset, ever-worsening and disabling myoclonus, seizures, ataxia, psychiatric disease, and shortened lifespan. EPM1 is caused by expansions of a dodecamer repeat sequence in the promoter of CSTB (cystatin B), which dramatically reduces, but does not eliminate, gene expression. The relatively late onset and consistent presence of a minimal amount of protein product makes EPM1 a favorable target for gene replacement therapy. If treated early, these children's normally developed brains could be rescued from the neurodegeneration that otherwise follows, and their cross-reactive immunological material (CRIM) positive status greatly reduces transgene related toxicity. We performed a proof-of-concept CSTB gene replacement study in Cstb knockout mice by introducing full-length human CSTB driven by the CBh promoter packaged in AAV9 and administered at postnatal days 21 and 60. Mice were sacrificed at 2 or 9 months of age, respectively. We observed significant improvements in expression levels of neuroinflammatory pathway genes and cerebellar granule cell layer apoptosis, as well as amelioration of motor impairment. The data suggest that gene replacement is a promising therapeutic modality for EPM1 and could spare affected children and families the ravages of this otherwise severe neurodegenerative disease.
Asunto(s)
Cistatina B , Terapia Genética , Ratones Noqueados , Enfermedades Neuroinflamatorias , Animales , Ratones , Terapia Genética/métodos , Cistatina B/genética , Enfermedades Neuroinflamatorias/terapia , Enfermedades Neuroinflamatorias/genética , Humanos , Ataxia/genética , Ataxia/terapia , Epilepsias Mioclónicas Progresivas/genética , Epilepsias Mioclónicas Progresivas/terapia , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificaciónRESUMEN
The ERK1/2 signaling pathway promotes myelin wrapping during development and remyelination, and sustained ERK1/2 activation in the oligodendrocyte (OL) lineage results in hypermyelination of the CNS. We therefore hypothesized that increased ERK1/2 signaling in the OL lineage would 1) protect against immune-mediated demyelination due to increased baseline myelin thickness and/or 2) promote enhanced remyelination and thus functional recovery after experimental autoimmune encephalomyelitis (EAE) induction. Cnp-Cre;Mek1DD-eGFP/+ mice that express a constitutively active form of MEK1 (the upstream activator of ERK1/2) in the OL lineage, exhibited a significant decrease in EAE clinical severity compared to controls. However, experiments using tamoxifen-inducible Plp-CreERT;Mek1DD-eGFP/+ or Pdgfrα-CreERT;Mek1DD-eGFP mice revealed this was not solely due to a protective or reparative effect resulting from MEK1DD expression specifically in the OL lineage. Because EAE is an immune-mediated disease, we examined Cnp-Cre;Mek1DD-eGFP/+ splenic immune cells for recombination. Surprisingly, GFP+ recombined CD19+ B-cells, CD11b+ monocytes, and CD3+ T-cells were noted when Cre expression was driven by the Cnp promoter. While ERK1/2 signaling in monocytes and T-cells is associated with proinflammatory activation, fewer studies have examined ERK1/2 signaling in B-cell populations. After in vitro stimulation, MEK1DD-expressing B-cells exhibited a 3-fold increase in CD138+ plasmablasts and a 5-fold increase in CD5+CD1dhi B-cells compared to controls. Stimulated MEK1DD-expressing B-cells also exhibited an upregulation of IL-10, known to suppress the initiation of EAE when produced by CD5+CD1dhi regulatory B-cells. Taken together, our data support the conclusion that sustained ERK1/2 activation in B-cells suppresses immune-mediated demyelination via increasing activation of regulatory B10 cells.
Asunto(s)
2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/biosíntesis , Linfocitos B/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/prevención & control , Sistema de Señalización de MAP Quinasas/fisiología , Regiones Promotoras Genéticas/fisiología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/inmunología , Animales , Linfocitos B/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Astrocytes have been implicated in regulating oligodendrocyte development and myelination in vitro, although their functions in vivo remain less well defined. Using a novel approach to locally ablate GFAP+ astrocytes, we demonstrate that astrocytes are required for normal CNS myelin compaction during development, and for maintaining myelin integrity in the adult. Transient ablation of GFAP+ astrocytes in the mouse spinal cord during the first postnatal week reduced the numbers of mature oligodendrocytes and inhibited myelin formation, while prolonged ablation resulted in myelin that lacked compaction and structural integrity. Ablation of GFAP+ astrocytes in the adult spinal cord resulted in the rapid, local loss of myelin integrity and regional demyelination. The loss of myelin integrity induced by astrocyte ablation was greatly reduced by NMDA receptor antagonists, both in vitro and in vivo, suggesting that myelin stability was affected by elevation of local glutamate levels following astrocyte ablation. Furthermore, targeted delivery of glutamate into adult spinal cord white matter resulted in reduction of myelin basic protein expression and localized disruption of myelin compaction which was also reduced by NMDA receptor blockade. The pathology induced by localized astrocyte loss and elevated exogenous glutamate, supports the concept that astrocytes are critical for maintenance of myelin integrity in the adult CNS and may be primary targets in the initiation of demyelinating diseases of the CNS, such as Neuromyelitis Optica (NMO).
RESUMEN
UNLABELLED: Growing evidence shows that mechanisms controlling CNS plasticity extend beyond the synapse and that alterations in myelin can modify conduction velocity, leading to changes in neural circuitry. Although it is widely accepted that newly generated oligodendrocytes (OLs) produce myelin in the adult CNS, the contribution of preexisting OLs to functional myelin remodeling is not known. Here, we show that sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in preexisting OLs of adult mice is sufficient to drive increased myelin thickness, faster conduction speeds, and enhanced hippocampal-dependent emotional learning. Although preexisting OLs do not normally contribute to remyelination, we show that sustained activation of ERK1/2 renders them able to do so. These data suggest that strategies designed to push mature OLs to reinitiate myelination may be beneficial both for enhancing remyelination in demyelinating diseases and for increasing neural plasticity in the adult CNS. SIGNIFICANCE STATEMENT: Myelin is a crucial regulator of CNS plasticity, function, and repair. Although it is generally accepted that new myelin production in the adult CNS is initiated by newly generated oligodendrocytes (OLs), great interest remains in additionally driving mature preexisting OLs to make myelin. The ability to induce myelination by the larger population of preexisting OLs carries the potential for enhanced remyelination in demyelinating diseases and increased neural plasticity in the adult CNS. Here, we show that sustained activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway is sufficient to drive mature OLs in the adult mouse CNS to reinitiate myelination, leading to new myelin wraps and functional changes.
Asunto(s)
Sistema Nervioso Central/fisiología , Enfermedades Desmielinizantes/patología , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Vaina de Mielina/metabolismo , Oligodendroglía/fisiología , Análisis de Varianza , Animales , Condicionamiento Psicológico/fisiología , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/fisiopatología , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Conducta Exploratoria/fisiología , Miedo/fisiología , Galactosilceramidas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Lisofosfolipasa/toxicidad , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Proteína Quinasa 3 Activada por Mitógenos/genética , Fuerza Muscular/genética , Proteína Proteolipídica de la Mielina/genética , Proteína Proteolipídica de la Mielina/metabolismo , Vaina de Mielina/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/ultraestructura , Desempeño Psicomotor/fisiología , ARN Mensajero/metabolismo , Reconocimiento en Psicología/fisiología , Espectrometría de Masas en TándemRESUMEN
The restoration of myelin sheaths on demyelinated axons remains a major obstacle in the treatment of multiple sclerosis (MS). Currently approved therapies work by modulating the immune system to reduce the number and rate of lesion formation but are only partially effective since they are not able to restore lost myelin. In the healthy CNS, myelin continues to be generated throughout life and spontaneous remyelination occurs readily in response to insults. In patients with MS, however, remyelination eventually fails, at least in part as a result of a failure of oligodendrocyte precursor cell (OPC) differentiation and the subsequent production of new myelin. A better understanding of the molecular mechanisms and signaling pathways that drive the process of myelin sheath formation is therefore important in order to speed the development of novel therapeutics designed to target remyelination. Here we review data supporting critical roles for three highly conserved intracellular signaling pathways: Wnt/ß-catenin, PI3K/AKT/mTOR, and ERK/MAPK in the regulation of OPC differentiation and myelination both during development and in remyelination. Potential points of crosstalk between the three pathways and important areas for future research are also discussed.
Asunto(s)
Sistema Nervioso Central/citología , Vaina de Mielina/fisiología , Regeneración Nerviosa/fisiología , Transducción de Señal/fisiología , Animales , Diferenciación Celular/fisiología , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/fisiopatología , Humanos , Oligodendroglía/fisiologíaRESUMEN
Successful myelin repair in the adult CNS requires the robust and timely production of myelin proteins to generate new myelin sheaths. The underlying regulatory mechanisms and complex molecular basis of myelin regeneration, however, remain poorly understood. Here, we investigate the role of ERK MAP kinase signaling in this process. Conditional deletion of Erk2 from cells of the oligodendrocyte lineage resulted in delayed remyelination following demyelinating injury to the adult mouse corpus callosum. The delayed repair occurred as a result of a specific deficit in the translation of the major myelin protein, MBP. In the absence of ERK2, activation of the ribosomal protein S6 kinase (p70S6K) and its downstream target, ribosomal protein S6 (S6RP), was impaired at a critical time when premyelinating oligodendrocytes were transitioning to mature cells capable of generating new myelin sheaths. Thus, we have described an important link between the ERK MAP kinase signaling cascade and the translational machinery specifically in remyelinating oligodendrocytes in vivo. These results suggest an important role for ERK2 in the translational control of MBP, a myelin protein that appears critical for ensuring the timely generation of new myelin sheaths following demyelinating injury in the adult CNS.
Asunto(s)
Cuerpo Calloso/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Básica de Mielina/metabolismo , Vaina de Mielina/metabolismo , Animales , Cuerpo Calloso/citología , Cuerpo Calloso/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Básica de Mielina/genética , Proteína S6 Ribosómica/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de SeñalRESUMEN
Oligodendrocytes, the myelin-forming cells of the CNS, exquisitely tailor the thickness of individual myelin sheaths to the diameter of their target axons to maximize the speed of action potential propagation, thus ensuring proper neuronal connectivity and function. Following demyelinating injuries to the adult CNS, newly formed oligodendrocytes frequently generate new myelin sheaths. Following episodes of demyelination such as those that occur in patients with multiple sclerosis, however, the matching of myelin thickness to axon diameter fails leaving remyelinated axons with thin myelin sheaths potentially compromising function and leaving axons vulnerable to damage. How oligodendrocytes determine the appropriate thickness of myelin for an axon of defined size during repair is unknown and identifying the signals that regulate myelin thickness has obvious therapeutic implications. Here, we show that sustained activation of extracellular-regulated kinases 1 and 2 (ERK1/2) in oligodendrocyte lineage cells results in accelerated myelin repair after injury, and is sufficient for the generation of thick myelin sheaths around remyelinated axons in the adult mouse spinal cord. Our findings suggest a model where ERK1/2 MAP kinase signaling acts as a myelin thickness rheostat that instructs oligodendrocytes to generate axon-appropriate quantities of myelin.
Asunto(s)
Sistema Nervioso Central/patología , Enfermedades Desmielinizantes/patología , Sistema de Señalización de MAP Quinasas/fisiología , Vaina de Mielina/patología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Vaina de Mielina/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Oligodendroglía/metabolismo , Polisacáridos/toxicidad , Médula Espinal/metabolismo , Médula Espinal/patología , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Wrapping of the myelin sheath around axons by oligodendrocytes is critical for the rapid conduction of electrical signals required for the normal functioning of the CNS. Myelination is a multistep process where oligodendrocytes progress through a well coordinated differentiation program regulated by multiple extracellular growth and differentiation signals. The intracellular transduction of the extracellular signals that regulate myelination is poorly understood. Here we demonstrate a critical role for two important signaling molecules, extracelluar signal-regulated protein kinases 1 and 2 (ERK1/ERK2), downstream mediators of mitogen-activated protein kinases, in the control of CNS myelin thickness. We generated and analyzed two lines of mice lacking both ERK1/ERK2 function specifically in oligodendrocyte-lineage cells. In the absence of ERK1/ERK2 signaling NG2⺠oligodendrocyte progenitor cells proliferated and differentiated on schedule. Mutant oligodendrocytes also ensheathed axons normally and made a few wraps of compact myelin. However, the subsequent increase in myelination that correlated myelin thickness in proportion to the axon caliber failed to occur. Furthermore, although the numbers of differentiated oligodendrocytes in the adult mutants were unchanged, they showed an inability to upregulate the transcription of major myelin genes that normally occurs during active myelination. Similarly, in vitro ERK1/ERK2-deficient oligodendrocytes differentiated normally but failed to form typical myelin-like membrane sheets. None of these effects were observed in single ERK1 or ERK2 mutants. These studies suggest that the predominant role of ERK1/ERK2 signaling in vivo is in promoting rapid myelin growth to increase its thickness, subsequent to oligodendrocyte differentiation and the initiation of myelination.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Vaina de Mielina/fisiología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa , Factores de Edad , Animales , Antígenos/genética , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Proteína Quinasa 1 Activada por Mitógenos/deficiencia , Proteína Quinasa 3 Activada por Mitógenos/deficiencia , Mutación/genética , Proteína Básica de Mielina/metabolismo , Proteína Proteolipídica de la Mielina/genética , Proteína Proteolipídica de la Mielina/metabolismo , Vaina de Mielina/genética , Vaina de Mielina/ultraestructura , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo , Neuronas/ultraestructura , Oligodendroglía/metabolismo , Oligodendroglía/ultraestructura , Técnicas de Cultivo de Órganos , Hidrolasas Diéster Fosfóricas/genética , Fosforilación , Proteoglicanos/genética , ARN Mensajero/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Oligodendrocyte development is tightly controlled by a variety of extracellular growth and differentiation factors. The mitogen-activated protein kinases (MAPKs), ERK1 and ERK2, are critical intracellular signaling molecules important for transducing these extracellular signals. The extracellular signal-regulated kinases (ERKs) are ubiquitously expressed, coordinately regulated, and highly similar, but Erk2 deletion in mice is embryonic lethal whereas Erk1 deletion is not. Several studies have suggested that MAPK signaling is important for oligodendrocyte differentiation, although specific roles for the two ERK isoforms have not been investigated. In this study, we deleted Erk2 in the developing mouse cortex from GFAP-expressing radial glia that generate neurons and oligodendrocytes. In vitro analysis revealed that loss of ERK2 resulted in fewer galactocerebroside-expressing mature oligodendrocytes in cortical cultures. In vivo, a delay in the expression of the myelin protein MBP was observed in the corpus callosum at postnatal day 10 (P10). In contrast, Erk1 deletion did not affect oligodendrocyte differentiation. By P21, MBP expression was restored to wild-type levels, demonstrating that the loss of ERK2 results in a delay but not a complete arrest in the appearance of differentiated oligodendrocytes in vivo. Importantly, both the proliferation and total number of oligodendrocyte progenitor cells (OPCs) appeared normal in the Erk2 conditional knock-out cortex, demonstrating that ERK2 plays a specific role in the timing of forebrain myelination but is not critical for the proliferation or survival of OPCs. Oligodendrocyte-specific deletion of Erk2 also resulted in decreased levels of MBP, indicating a cell-autonomous effect of ERK2 in the oligodendrocyte lineage.
Asunto(s)
Diferenciación Celular/fisiología , Corteza Cerebral/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Animales , Western Blotting , Células Cultivadas , Corteza Cerebral/citología , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Neuronas/metabolismo , Oligodendroglía/citología , Células Madre/metabolismoRESUMEN
Multiple sclerosis (MS) is considered an autoimmune-mediated demyelinating disease that targets the central nervous system (CNS). Despite considerable research efforts over multiple decades, our understanding of the basic biological processes that are targeted in the disease and the mechanisms of pathogenesis are poorly understood. Consequently, current therapies directed at controlling the progression of the disease are limited in their effectiveness. Historically, the primary focus of MS research has been to define the cellular and molecular basis of the immunological pathogenic mechanisms. Recently, however, it has become clear that long-term functional recovery in MS will require the development of strategies that facilitate myelin repair in lesion areas. The emerging evidence that the adult vertebrate CNS retains the capacity to regenerate neural cells that have been lost to disease or damage has provoked intensive research focused on defining the mechanisms of myelin repair. Unfortunately, the existing animal models of MS are poorly equipped to assess myelin repair, and new validated strategies to identify therapeutics targeted at promoting myelin repair are badly needed. This Commentary will review established murine models of MS, and discuss emerging technologies that promise to provide insights into the mechanisms of myelin repair.
Asunto(s)
Modelos Animales de Enfermedad , Esclerosis Múltiple/patología , Cicatrización de Heridas , Animales , Ratones , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Vaina de Mielina/patologíaRESUMEN
Rett syndrome (RTT) is characterized by specific motor, cognitive, and behavioral deficits. Because several of these abnormalities occur in other disease states associated with alterations in aminergic neurotransmitters, we investigated the contribution of such alterations to RTT pathogenesis. We found that both individuals with RTT and Mecp2-null mice have lower-than-normal levels of aminergic metabolites and content. Deleting Mecp2 from either TH-positive dopaminergic and noradrenergic neurons or PET1-positive serotonergic neurons in mice decreased corresponding neurotransmitter concentration and specific phenotypes, likely through MeCP2 regulation of rate-limiting enzymes involved in aminergic neurotransmitter production. These data support a cell-autonomous, MeCP2-dependent mechanism for the regulation of aminergic neurotransmitter synthesis contributing to unique behavioral phenotypes.