RESUMEN
Neuron-glial antigen 2-positive (NG2(+)) glial cells are the most proliferative glia type in the adult CNS, and their tile-like arrangement in adult gray matter is under tight regulation. However, little is known about the cues that govern this unique distribution. To this end, using a NG2(+) glial cell ablation model in mice, we examined the repopulation dynamics of NG2(+) glial cells in the mature and aged mice gray matter. We found that some resident NG2(+) glial cells that escaped depletion rapidly enter the cell cycle to repopulate the cortex with altered spatial distribution. We reveal that netrin-1 signaling is involved in the NG2(+) glial cell early proliferative, late repopulation, and distribution response after ablation in the gray matter. However, ablation of NG2(+) glial cell in older animals failed to stimulate a similar repopulation response, possibly because of a decrease in the sensitivity to netrin-1. Our findings indicate that endogenous netrin-1 plays a role in NG2(+) glial cell homeostasis that is distinct from its role in myelination.
Asunto(s)
Envejecimiento , Antígenos/metabolismo , Sustancia Gris/citología , Homeostasis/fisiología , Factores de Crecimiento Nervioso/metabolismo , Neuroglía/metabolismo , Proteoglicanos/metabolismo , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Anticuerpos/farmacología , Antígenos/genética , Bromodesoxiuridina/metabolismo , Recuento de Células , Homeostasis/efectos de los fármacos , Inmunoprecipitación , Antígeno Ki-67/metabolismo , Ratones Transgénicos , Microscopía Confocal , Factores de Crecimiento Nervioso/inmunología , Netrina-1 , Neuroglía/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteoglicanos/genética , ARN no Traducido/genética , ARN no Traducido/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
UNLABELLED: The identification of the molecular network that supports oligodendrocyte (OL) regeneration under demyelinating conditions has been a primary goal for regenerative medicine in demyelinating disorders. We recently described an essential function for TACE/ADAM17 in regulating oligodendrogenesis during postnatal myelination, but it is unknown whether this protein also plays a role in OL regeneration and remyelination under demyelinating conditions. By using genetic mouse models to achieve selective gain- or loss-of-function of TACE or EGFR in OL lineage cells in vivo, we found that TACE is critical for EGFR activation in OLs following demyelination, and therefore, for sustaining OL regeneration and CNS remyelination. TACE deficiency in oligodendrocyte progenitor cells following demyelination disturbs OL lineage cell expansion and survival, leading to a delay in the remyelination process. EGFR overexpression in TACE deficient OLs in vivo restores OL development and postnatal CNS myelination, but also OL regeneration and CNS remyelination following demyelination. Our study reveals an essential function of TACE in supporting OL regeneration and CNS remyelination that may contribute to the design of new strategies for therapeutic intervention in demyelinating disorders by promoting oligodendrocyte regeneration and myelin repair. SIGNIFICANCE STATEMENT: Oligodendrocyte (OL) regeneration has emerged as a promising new approach for the treatment of demyelinating disorders. By using genetic mouse models to selectively delete TACE expression in oligodendrocyte progenitors cells (OPs), we found that TACE/ADAM17 is required for supporting OL regeneration following demyelination. TACE genetic depletion in OPs abrogates EGFR activation in OL lineage cells, and perturbs cell expansion and survival, blunting the process of CNS remyelination. Moreover, EGFR overexpression in TACE-deficient OPs in vivo overcomes the defects in OL development during postnatal development but also OL regeneration during CNS remyelination. Our study identifies TACE as an essential player in OL regeneration that may provide new insights in the development of new strategies for promoting myelin repair in demyelinating disorders.
Asunto(s)
Proteínas ADAM/metabolismo , Sistema Nervioso Central/patología , Enfermedades Desmielinizantes/patología , Regulación de la Expresión Génica/fisiología , Esclerosis Múltiple/patología , Oligodendroglía/fisiología , Regeneración/fisiología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/genética , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/metabolismo , Proteína ADAM17 , Animales , Antígenos/genética , Antígenos/metabolismo , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Oligodendroglía/efectos de los fármacos , Oligodendroglía/ultraestructura , Proteoglicanos/genética , Proteoglicanos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Regeneración/efectos de los fármacosRESUMEN
Specialized cellular microenvironments, or 'niches', modulate stem cell properties, including cell number, self-renewal and fate decisions. In the adult brain, niches that maintain a source of neural stem cells (NSCs) and neural progenitor cells (NPCs) are the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus of the hippocampus. The size of the NSC population of the SVZ at any time is the result of several ongoing processes, including self-renewal, cell differentiation, and cell death. Maintaining the balance between NSCs and NPCs in the SVZ niche is critical to supply the brain with specific neural populations, both under normal conditions or after injury. A fundamental question relevant to both normal development and to cell-based repair strategies in the central nervous system is how the balance of different NSC and NPC populations is maintained in the niche. EGFR (epidermal growth factor receptor) and Notch signalling pathways have fundamental roles during development of multicellular organisms. In Drosophila and in Caenorhabditis elegans these pathways may have either cooperative or antagonistic functions. In the SVZ, Notch regulates NSC identity and self-renewal, whereas EGFR specifically affects NPC proliferation and migration. This suggests that interplay of these two pathways may maintain the balance between NSC and NPC numbers. Here we show that functional cell-cell interaction between NPCs and NSCs through EGFR and Notch signalling has a crucial role in maintaining the balance between these cell populations in the SVZ. Enhanced EGFR signalling in vivo results in the expansion of the NPC pool, and reduces NSC number and self-renewal. This occurs through a non-cell-autonomous mechanism involving EGFR-mediated regulation of Notch signalling. Our findings define a novel interaction between EGFR and Notch pathways in the adult SVZ, and thus provide a mechanism for NSC and NPC pool maintenance.
Asunto(s)
Receptores ErbB/metabolismo , Neuronas/citología , Receptores Notch/metabolismo , Transducción de Señal , Células Madre/citología , Animales , Recuento de Células , División Celular , Receptores ErbB/genética , Humanos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Receptor Notch1/metabolismo , Nicho de Células Madre/citología , UbiquitinaciónRESUMEN
Research on myelination has focused on identifying molecules capable of inducing oligodendrocyte (OL) differentiation in an effort to develop strategies that promote functional myelin regeneration in demyelinating disorders. Here, we show that transforming growth factor ß (TGFß) signaling is crucial for allowing oligodendrocyte progenitor (OP) cell cycle withdrawal, and therefore, for oligodendrogenesis and postnatal CNS myelination. Enhanced oligodendrogenesis and subcortical white matter (SCWM) myelination was detected after TGFß gain of function, while TGFß receptor II (TGFß-RII) deletion in OPs prevents their development into mature myelinating OLs, leading to SCWM hypomyelination in mice. TGFß signaling modulates OP cell cycle withdrawal and differentiation through the transcriptional modulation of c-myc and p21 gene expression, mediated by the interaction of SMAD3/4 with Sp1 and FoxO1 transcription factors. Our study is the first to demonstrate an autonomous and crucial role of TGFß signaling in OL development and CNS myelination, and may provide new avenues in the treatment of demyelinating diseases.
Asunto(s)
Ciclo Celular/fisiología , Sistema Nervioso Central/metabolismo , Vaina de Mielina/metabolismo , Oligodendroglía/fisiología , Transducción de Señal/fisiología , Células Madre/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Benzamidas/farmacología , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Sistema Nervioso Central/citología , Dioxoles/farmacología , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligodendroglía/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína smad3/genética , Proteína smad3/metabolismo , Células Madre/efectos de los fármacosRESUMEN
Several studies have elucidated the significance of a disintegrin and metalloproteinase proteins (ADAMs) in PNS myelination, but there is no evidence if they also play a role in oligodendrogenesis and CNS myelination. Our study identifies ADAM17, also called tumor necrosis factor-α converting enzyme (TACE), as a novel key modulator of oligodendrocyte (OL) development and CNS myelination. Genetic deletion of TACE in oligodendrocyte progenitor cells (OPs) induces premature cell cycle exit and reduces OL cell survival during postnatal myelination of the subcortical white matter (SCWM). These cellular and molecular changes lead to deficits in SCWM myelination and motor behavior. Mechanistically, TACE regulates oligodendrogenesis by modulating the shedding of EGFR ligands TGFα and HB-EGF and, consequently, EGFR signaling activation in OL lineage cells. Constitutive TACE depletion in OPs in vivo leads to similar alterations in CNS myelination and motor behavior as to what is observed in the EGFR hypofunctional mouse line EgfrWa2. EGFR overexpression in TACE-deficient OPs restores OL survival and development. Our study reveals an essential function of TACE in oligodendrogenesis, and demonstrates how this molecule modulates EGFR signaling activation to regulate postnatal CNS myelination.
Asunto(s)
Proteínas ADAM/metabolismo , Encéfalo/metabolismo , Vaina de Mielina/metabolismo , Neurogénesis , Oligodendroglía/metabolismo , Proteínas ADAM/genética , Proteína ADAM17 , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Línea Celular , Linaje de la Célula , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Locomoción , Ratones , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Oligodendroglía/citología , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
Discrete cellular microenvironments regulate stem cell pools and their development, as well as function in maintaining tissue homeostasis. Although the signaling elements modulating neural progenitor cells (NPCs) of the adult subventricular zone (SVZ) niche are fairly well understood, the pathways activated following injury and the resulting outcomes, are less clear. In the present study, we used mouse models of demyelination and proteomics analysis to identify molecular cues present in the adult SVZ niche during injury, and analyzed their role on NPCs in the context of promoting myelin repair. Proteomic analysis of SVZ tissue from mice with experimental demyelination identified several proteins that are known to play roles in NPC proliferation, adhesion, and migration. Among the proteins found to be upregulated were members of the N-cadherin signaling pathway. During the onset of demyelination in the subcortical white matter (SCWM), activation of epidermal growth factor receptor (EGFR) signaling in SVZ NPCs stimulates the interaction between N-cadherin and ADAM10. Upon cleavage and activation of N-cadherin signaling by ADAM10, NPCs undergo cytoskeletal rearrangement and polarization, leading to enhanced migration out of the SVZ into demyelinated lesions of the SCWM. Genetically disrupting either EGFR signaling or ADAM10 inhibits this pathway, preventing N-cadherin regulated NPC polarization and migration. Additionally, in vivo experiments using N-cadherin gain- and loss-of-function approaches demonstrated that N-cadherin enhances the recruitment of SVZ NPCs into demyelinated lesions. Our data revealed that EGFR-dependent N-cadherin signaling physically initiated by ADAM10 cleavage is the response of the SVZ niche to promote repair of the injured brain.
Asunto(s)
Cadherinas/metabolismo , Movimiento Celular/fisiología , Regulación de la Expresión Génica/fisiología , Ventrículos Laterales/citología , Células-Madre Neurales/fisiología , Recuperación de la Función/fisiología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/genética , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/metabolismo , Animales , Antígenos/genética , Antígenos/metabolismo , Cadherinas/genética , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Movimiento Celular/efectos de los fármacos , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Transgénicos , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Técnicas de Cultivo de Órganos , Proteoglicanos/genética , Proteoglicanos/metabolismo , Proteómica , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Diffuse white matter injury (DWMI) caused by hypoxia is associated with permanent neurodevelopmental disabilities in preterm infants. The cellular and molecular mechanisms producing DWMI are poorly defined. Using a mouse model of neonatal hypoxia, we demonstrate a biphasic effect on oligodendrocyte development, resulting in hypomyelination. Oligodendrocyte death and oligodendrocyte progenitor cell (OPC) proliferation during the week after hypoxia were followed by delayed oligodendrocyte differentiation and abnormal myelination, as demonstrated by electron microscopy. Cdk2 activation was essential for the regenerative OPC response after hypoxia and was accompanied by reduced FoxO1-dependent p27(Kip1) expression. p27(Kip1) was also reduced in OPCs in human infant white matter lesions after hypoxia. The negative effects of hypoxia on oligodendrogenesis and myelination were more pronounced in p27(Kip1)-null mice; conversely, overexpression of FoxO1 or p27(Kip1) in OPCs after hypoxia promoted oligodendrogenesis. Our studies demonstrate for the first time that neonatal hypoxia affects the Foxo1/p27(Kip1) pathway during white matter development. We also show that molecular manipulation of this pathway enhances oligodendrocyte regeneration during a critical developmental time window after DWMI. Thus, FoxO1 and p27(Kip1) may serve as promising target molecules for promoting timely oligodendrogenesis in neonatal DWMI.
Asunto(s)
Diferenciación Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Factores de Transcripción Forkhead/fisiología , Regulación del Desarrollo de la Expresión Génica , Hipoxia Encefálica/metabolismo , Regeneración Nerviosa/fisiología , Oligodendroglía/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proteína Forkhead Box O1 , Humanos , Hipoxia Encefálica/patología , Lactante , Recién Nacido , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oligodendroglía/citologíaRESUMEN
The mechanisms that regulate neural stem cell (NSC) lineage progression and maintain NSCs within different domains of the adult neural stem cell niche, the subventricular zone are not well defined. Quiescent NSCs are arranged at the apical ventricular wall, while mitotically activated NSCs are found in the basal, vascular region of the subventricular zone. Here, we found that ADAM10 (a disintegrin and metalloproteinase 10) is essential in NSC association with the ventricular wall, and via this adhesion to the apical domain, ADAM10 regulates the switch from quiescent and undifferentiated NSC to an actively proliferative and differentiating cell state. Processing of JAMC (junctional adhesion molecule C) by ADAM10 increases Rap1GAP activity. This molecular machinery promotes NSC transit from the apical to the basal compartment and subsequent lineage progression. Understanding the molecular mechanisms responsible for regulating the proper positioning of NSCs within the subventricular zone niche and lineage progression of NSCs could provide new targets for drug development to enhance the regenerative properties of neural tissue.
RESUMEN
Cellular strategies for oligodendrocyte regeneration and remyelination involve characterizing endogenous neural progenitors that are capable of generating oligodendrocytes during normal development and after demyelination, and identifying the molecular signals that enhance oligodendrogenesis from these progenitors. Using both gain- and loss-of-function approaches, we explored the role of epidermal growth factor receptor (EGFR) signaling in adult myelin repair and in oligodendrogenesis. We show that 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter-driven overexpression of human EGFR (hEGFR) accelerated remyelination and functional recovery following focal demyelination of mouse corpus callosum. Lesion repopulation by Cspg4+ (also known as NG2) Ascl1+ (also known as Mash1) Olig2+ progenitors and functional remyelination were accelerated in CNP-hEGFR mice compared with wild-type mice. EGFR overexpression in subventricular zone (SVZ) and corpus callosum during early postnatal development also expanded this NG2+Mash1+Olig2+ progenitor population and promoted SVZ-to-lesion migration, enhancing oligodendrocyte generation and axonal myelination. Analysis of hypomorphic EGFR-mutant mice confirmed that EGFR signaling regulates oligodendrogenesis and remyelination by NG2+Mash1+Olig2+ progenitors. EGFR targeting holds promise for enhancing oligodendrocyte regeneration and myelin repair.
Asunto(s)
Enfermedades Desmielinizantes/fisiopatología , Receptores ErbB/fisiología , Vaina de Mielina/fisiología , Regeneración Nerviosa/fisiología , Oligodendroglía/fisiología , Transducción de Señal/fisiología , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa , Células Madre Adultas/fisiología , Animales , Animales Recién Nacidos , Bromodesoxiuridina/metabolismo , Proliferación Celular , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisofosfatidilcolinas , Ratones , Ratones Mutantes , Ratones Transgénicos , Microscopía Electrónica de Transmisión/métodos , Vaina de Mielina/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/ultraestructura , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismoRESUMEN
In the postnatal brain, oligodendrocyte progenitor cells (OPCs) arise from the subventricular zone (SVZ) and migrate into the developing white matter, where they differentiate into oligodendrocytes and myelinate axons. The mechanisms regulating OPC migration and differentiation are not fully defined. The present study demonstrates that endothelin-1 (ET-1) is an astrocyte-derived signal that regulates OPC migration and differentiation. OPCs in vivo and in culture express functional ET(A) and ET(B) receptors, which mediate ET-1-induced ERK (extracellular signal-regulated kinase) and CREB (cAMP response element-binding protein) phosphorylation. ET-1 exerts both chemotactic and chemokinetic effects on OPCs to enhance cell migration; it also prevents lineage progression from the O4(+) to the O1(+) stage without affecting cell proliferation. Astrocyte-conditioned medium stimulates OPC migration in culture through ET receptor activation, whereas multiphoton time-lapse imaging shows that selective ET receptor antagonists or anti-ET-1 antibodies inhibit OPC migration from the SVZ. Inhibition of ET receptor activity also derepresses OPC differentiation in the corpus callosum in slice cultures. Our findings indicate that ET-1 is a soluble astrocyte-derived signal that regulates OPC migration and differentiation during development.
Asunto(s)
Endotelina-1/metabolismo , Oligodendroglía/fisiología , Células Madre/fisiología , Animales , Astrocitos/metabolismo , Autoanticuerpos , Encéfalo/fisiología , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Quimiotaxis/fisiología , Medios de Cultivo Condicionados , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Antagonistas de los Receptores de la Endotelina A , Antagonistas de los Receptores de la Endotelina B , Endotelina-1/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Ratas , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , PorcinosRESUMEN
Neuronal progenitor cells of the anterior subventricular zone (SVZa) migrate along the rostral migratory stream (RMS) to the olfactory bulb, where they exit the cell cycle and differentiate. The molecular mechanisms that regulate SVZa progenitor proliferation and cell-cycle exit are largely undefined. We investigated the role of p27(KIP1) in regulating cell proliferation and survival in the RMS and olfactory bulb between postnatal day 1 (P1) and P14, the peak period of olfactory bulb neuron generation. A large proportion of cells in the RMS and the olfactory bulb express cytoplasmic p27(KIP1), but a small percentage display high nuclear p27(KIP1) immunostaining, which exhibit a caudal(low)-rostral(high) gradient: lowest in the SVZa and highest in the glomerular layer of the olfactory bulb. p27(KIP1) is also present in the nucleus and/or the cytoplasm of neuron-specific type III beta-tubulin(+) cells. Cells with strong nuclear p27(KIP1) expression are BrdU(-) and Ki67(-). The percentage of BrdU(+) cells in the SVZa, RMS, and olfactory bulb is higher in p27(KIP1) null than wild-type (WT) mice at all ages analyzed. Consistent with these findings, p27(KIP1) overexpression in cultured p27(KIP1) null and WT SVZ cells reduced cell proliferation and self-renewal. Finally, in p27(KIP1) null mice, the diameter of the horizontal limb of the RMS is larger than in WT mice, and development of the olfactory bulb granule cell layer is delayed, together with increased apoptotic cell density. Our results indicate that in the postnatal brain, p27(KIP1) regulates the proliferation and survival of neuronal cells in the RMS and olfactory bulb.
Asunto(s)
Animales Recién Nacidos/fisiología , Movimiento Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Bulbo Olfatorio/fisiología , Animales , Antimetabolitos , Western Blotting , Bromodesoxiuridina , Proteína Quinasa CDC2/antagonistas & inhibidores , Proliferación Celular , Supervivencia Celular , Proteínas de Unión al ADN , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Mesencéfalo/citología , Mesencéfalo/crecimiento & desarrollo , Mesencéfalo/fisiología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Bulbo Olfatorio/citología , Bulbo Olfatorio/crecimiento & desarrolloRESUMEN
The subventricular zone (SVZ) is a source of neural progenitors throughout brain development. The identification and purification of these progenitors and the analysis of their lineage potential are fundamental issues for future brain repair therapies. We demonstrate that early postnatal NG2-expressing (NG2+) progenitor cells located in the SVZ self-renew in vitro and display phenotypic features of transit-amplifier type C-like multipotent cells. NG2+ cells in the SVZ are highly proliferative and express the epidermal growth factor receptor, the transcription factors Dlx, Mash1, and Olig2, and the Lewis X (LeX) antigen. We show that grafted early postnatal NG2+ cells generate hippocampal GABAergic interneurons that propagate action potentials and receive functional glutamatergic synaptic inputs. Our work identifies Dlx+/Mash1+/LeX+/NG2+/GFAP-negative cells of the SVZ as a new class of postnatal multipotent progenitor cells that may represent a specific cellular reservoir for renewal of postnatal and adult inhibitory interneurons in the hippocampus.
Asunto(s)
Antígenos/metabolismo , Diferenciación Celular/genética , Hipocampo/metabolismo , Interneuronas/metabolismo , Ventrículos Laterales/metabolismo , Proteoglicanos/metabolismo , Células Madre/metabolismo , Potenciales de Acción/genética , Animales , Animales Recién Nacidos , Antígenos/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , División Celular/genética , Movimiento Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Receptores ErbB/metabolismo , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Interneuronas/citología , Ventrículos Laterales/citología , Ventrículos Laterales/crecimiento & desarrollo , Antígeno Lewis X/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Inhibición Neural/genética , Factor de Transcripción 2 de los Oligodendrocitos , Proteoglicanos/genética , Trasplante de Células Madre , Células Madre/citología , Transmisión Sináptica/genética , Factores de Transcripción/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan-positive progenitor cells that express the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.
Asunto(s)
Antígenos/metabolismo , Diferenciación Celular/genética , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Células Madre Multipotentes/metabolismo , Proteínas del Tejido Nervioso , Neuronas/metabolismo , Proteoglicanos/metabolismo , 2',3'-Nucleótido Cíclico Fosfodiesterasas/genética , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Potenciales de Acción/genética , Animales , Animales Recién Nacidos , Antígenos/genética , Astrocitos/citología , Astrocitos/metabolismo , Células Cultivadas , Giro Dentado/citología , Giro Dentado/crecimiento & desarrollo , Giro Dentado/metabolismo , Hipocampo/citología , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Transgénicos , Modelos Animales , Células Madre Multipotentes/citología , Nestina , Vías Nerviosas/citología , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/metabolismo , Neuronas/citología , Oligodendroglía/citología , Oligodendroglía/metabolismo , Fenotipo , Regiones Promotoras Genéticas/genética , Proteoglicanos/genética , Proteínas Recombinantes de Fusión , Esferoides Celulares/citología , Esferoides Celulares/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Adult neural stem cells (NSCs) reside in a specialized microenvironment, the subventricular zone (SVZ), which provides them with unique signaling cues to control their basic properties and prevent their exhaustion. While the signaling mechanisms that regulate NSC lineage progression are well characterized, the molecular mechanisms that trigger the activation of quiescent NSCs during homeostasis and tissue repair are still unclear. Here, we uncovered that the NSC quiescent state is maintained by Rho-GTPase Cdc42, a downstream target of non-canonical Wnt signaling. Mechanistically, activation of Cdc42 induces expression of molecules involved in stem cell identity and anchorage to the niche. Strikingly, during a demyelination injury, downregulation of non-canonical Wnt-dependent Cdc42 activity is necessary to promote activation and lineage progression of quiescent NSCs, thereby initiating the process of tissue repair.
Asunto(s)
Enfermedades Desmielinizantes , Homeostasis , Células-Madre Neurales/citología , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Microarray analysis of oligodendrocyte lineage cells purified by fluorescence-activated cell sorting (FACS) from 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mice revealed Sox17 (SRY-box containing gene 17) gene expression to be coordinately regulated with that of four myelin genes during postnatal development. In CNP-EGFP-positive (CNP-EGFP+) cells, Sox17 mRNA and protein levels transiently increased between postnatal days 2 and 15, with white matter O4+ preoligodendrocytes expressing greater Sox17 levels than Nkx2.2+ (NK2 transcription factor related, locus 2) NG2+, or GalC+ (galactocerebroside) cells. In spinal cord, Sox17 protein expression was undetectable in the primary motor neuron domain between embryonic days 12.5 and 15.5 but was evident in Nkx2.2+ and CC1+ cells. In cultured oligodendrocyte progenitor cells (OPCs), Sox17 levels were maximal in O4+ cells and peaked during the phenotypic conversion from bipolar to multipolar. Parallel increases in Sox17 and p27 occurred before MBP protein expression, and Sox17 upregulation was prevented by conditions inhibiting differentiation. Sox17 downregulation with small interfering RNAs increased OPC proliferation and decreased lineage progression after mitogen withdrawal, whereas Sox17 overexpression in the presence of mitogen had opposite effects. Sox17 overexpression enhanced myelin gene expression in OPCs and directly stimulated MBP gene promoter activity. These findings support important roles for Sox17 in controlling both oligodendrocyte progenitor cell cycle exit and differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas HMGB/fisiología , Oligodendroglía/citología , Oligodendroglía/fisiología , Factores de Transcripción/fisiología , Animales , Células COS , Ciclo Celular/fisiología , Linaje de la Célula , Células Cultivadas , Chlorocebus aethiops , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio , Humanos , Ratones , Ratones Transgénicos , Proteínas Nucleares , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Factores de Transcripción SOXFRESUMEN
Oligodendrocyte maturation has been defined based on expression of developmentally regulated antigens. However, transitions at early stages of the lineage have not been functionally characterized fully in situ. Combining 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-promoter driven enhanced green fluorescent protein expression and whole-cell capacitance measurements permitted a reliable distinction between subcortical white matter NG2+ oligodendrocyte progenitors (OPs) and O4+ preoligodendrocytes (pre-OLs) in situ. We focused on K+ channels because their expression has been associated previously with the proliferation and differentiation potential of OPs. Using whole-cell patch clamp, we observed a downregulation of the delayed outward-rectifying current (IKDR) between the NG2+ and O4+ stages but no significant changes in transient K+-channel current (IKA) amplitude. Tyrosine kinase inhibition in NG2+ cells reduced IKDR amplitude with no effect on IKA, which mimicked the endogenous changes observed between OPs and pre-OLs. Tyrosine kinase inhibition also reduced the proliferative capacity of NG2+ OPs in slice cultures. Conversely, acute platelet-derived growth factor receptor-alpha (PDGFR-alpha) activation caused an increase of IKDR in NG2+ but not in O4+ cells. Consistent with this finding, PDGFR-alpha immunoreactivity was confined to NG2+ cells with undetectable levels in O4+ cells, suggesting that PDGFR-alpha signaling is absent in pre-OLs in situ. Importantly, the PDGF-induced increase of IKDR in NG2+ cells was prevented by tyrosine kinase inhibition. Together, these data indicate that PDGFR-alpha and tyrosine kinase activity act via a common pathway that influences functional expression of K+ channels and proliferative capacity of OPs in situ.
Asunto(s)
Regulación hacia Abajo/fisiología , Oligodendroglía/citología , Oligodendroglía/enzimología , Canales de Potasio/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/fisiología , Animales , Animales Recién Nacidos , Regulación hacia Abajo/efectos de los fármacos , Ratones , Ratones Transgénicos , Oligodendroglía/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Approaches to successful cell transplantation therapies for the injured brain involve selecting the appropriate neural progenitor type and optimizing the efficiency of the cell engraftment. Here we show that epidermal growth factor receptor (EGFR) expression enhances postnatal neural progenitor migration in vitro and in vivo. Migratory NG2-expressing (NG2+) progenitor cells of the postnatal subventricular zone (SVZ) express higher EGFR levels than nonmigratory, cortical NG2+ cells. The higher endogenous EGFR expression in SVZ NG2+ cells is causally related with their migratory potential in vitro as well as in vivo after cell engraftment. EGFR overexpression in cortical NG2+ cells by transient transfection converted these cells to a migratory phenotype in vitro and in vivo. Finally, cortical NG2+ cells purified from a transgenic mouse in which the EGFR is overexpressed under the CNP promoter exhibited enhanced migratory capability. These findings reveal a new role for EGFR in the postnatal brain and open new avenues to optimize cell engraftment for brain repair.
Asunto(s)
Animales Recién Nacidos/fisiología , Movimiento Celular/fisiología , Receptores ErbB/fisiología , Neuronas/fisiología , Células Madre/fisiología , Animales , Antígenos/genética , Antígenos/metabolismo , Antígenos/fisiología , Encéfalo/citología , Corteza Cerebral/citología , Ventrículos Cerebrales , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Proteínas de la Matriz Extracelular/fisiología , Humanos , Ratones , Ratones Transgénicos , Péptido Natriurético Tipo-C/metabolismo , Neuronas/metabolismo , Neuronas/trasplante , Fenotipo , Proteoglicanos/genética , Proteoglicanos/metabolismo , Proteoglicanos/fisiología , Trasplante de Células Madre , Células Madre/metabolismo , TransfecciónRESUMEN
We used a 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mouse to study postnatal subventricular zone (SVZ) progenitor fate, with a focus on the olfactory bulb (OB). The postnatal OB of the CNP-EGFP mouse contained EGFP+ interneurons and oligodendrocytes. In the anterior SVZ, the majority of EGFP+ progenitors were NG2+. These NG2+/EGFP+ progenitors expressed the OB interneuron marker Er81, the neuroblast markers doublecortin (DC) and Distalless-related homeobox (DLX), or the oligodendrocyte progenitor marker Nkx2.2. In the rostral migratory stream (RMS), EGFP+ cells displayed a migrating phenotype. A fraction of these cells were either NG2-/Er81+/DC+/DLX+ or NG2+/Nkx2.2+. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) injection into the lateral ventricle (LV) of early postnatal mice demonstrated that NG2+/EGFP+ progenitors migrate from the SVZ through the RMS into the OB. Moreover, fluorescence-activated cell-sorting-purified NG2+/CNP-EGFP+ or NG2+/beta-actin-enhanced yellow fluorescent protein-positive (EYFP+) progenitors transplanted into the early postnatal LV displayed extensive rostral and caudal migration. EYFP+ or EGFP+ graft-derived cells within the RMS were DLX+/Er81+ or Nkx2.2+, migrated to the OB, and differentiated to interneurons and oligodendrocytes. In the subcortical white matter (SCWM), grafted cells differentiated to either oligodendrocytes or astrocytes. Transplantation of NG2+/EYFP+ progenitors selectively purified from the SVZ showed that these cells were migratory and generated glia and neurons in the OB, hippocampus, and striatum. In contrast, cortical, OB, or cerebellar NG2+ cells had a very limited migratory potential and gave rise to glia in the SCWM and striatum. Our findings indicate region-specific differences between NG2+ progenitor cells and show that NG2+ cells can migrate throughout the RMS and contribute to both gliogenesis and neurogenesis in the postnatal OB.
Asunto(s)
Antígenos/biosíntesis , Interneuronas/citología , Bulbo Olfatorio/citología , Oligodendroglía/citología , Proteoglicanos/biosíntesis , Células Madre/citología , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/fisiología , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Ventrículos Cerebrales , Proteínas Fluorescentes Verdes/genética , Proteína Homeobox Nkx-2.2 , Inmunohistoquímica , Interneuronas/fisiología , Ratones , Ratones Transgénicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Bulbo Olfatorio/crecimiento & desarrollo , Oligodendroglía/fisiología , Trasplante de Células Madre , Células Madre/metabolismoRESUMEN
Proliferation of oligodendrocyte progenitor (OP) cells is a crucial process controlling myelination in the CNS. Previous studies demonstrated a correlation between OP proliferation rate and cyclin E/cyclin-dependent kinase-2 (cdk2) activity. To establish a causal link between cyclin E/cdk2 activity and OP proliferation, we selectively modulated cdk2 activity in vitro by transfection of cultured OP cells. Dominant-negative (Dn)-cdk2 overexpression inhibited mitogen-induced OP cell proliferation, whereas wild-type (wt)-cdk2 prevented cell cycle arrest caused by anti-mitotic signals. Dn-cdk2- or wt-cdk2-mediated regulation of G(1)/S transition, per se, did not influence initiation of OP differentiation. To study the function of cyclin E/cdk2 in OP cells during development in vivo, we analyzed cdk2 and cyclin E expression in cells acutely isolated from transgenic mice expressing the green fluorescent protein (GFP) under the control of the 2'-3'-cyclic nucleotide 3'-phosphodiesterase gene promoter. Both cyclin E/cdk2 protein levels and activity were decreased in GFP(+) oligodendrocyte lineage cells between postnatal days 4 and 30. Immunostaining of NG2(+)/GFP(+) OP cells in brain tissue sections showed a 90% decrease in overall cell proliferation and cdk2 expression between perinatal and adult cells. However, cdk2 expression within the proliferating (i.e., expressing the proliferating cell nuclear antigen) OP cell population was maintained throughout development. Our data indicate that: (1) cyclin E/cdk2 activity plays a pivotal function in OP cell cycle decisions occurring at G(1)/S checkpoint; (2) initiation of OP differentiation is independent of cyclinE/cdk2 checkpoint, and (3) intrinsic differences in cyclin E/cdk2 expression and activity may underlie the slowly proliferative state that characterizes so-called "quiescent" adult OP cells in vivo.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Oligodendroglía/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre/metabolismo , Animales , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula/fisiología , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/genética , Regulación hacia Abajo/fisiología , Activación Enzimática/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones , Ratones Transgénicos , Mitosis/fisiología , Oligodendroglía/citología , Proteínas Serina-Treonina Quinasas/genética , Ratas , Ratas Sprague-Dawley , Células Madre/citología , TransfecciónRESUMEN
NG2-expressing glia (NG2 glia) are a uniformly distributed and mitotically active pool of cells in the central nervous system (CNS). In addition to serving as progenitors of myelinating oligodendrocytes, NG2 glia might also fulfill physiological roles in CNS homeostasis, although the mechanistic nature of such roles remains unclear. Here, we report that ablation of NG2 glia in the prefrontal cortex (PFC) of the adult brain causes deficits in excitatory glutamatergic neurotransmission and astrocytic extracellular glutamate uptake and induces depressive-like behaviors in mice. We show in parallel that chronic social stress causes NG2 glia density to decrease in areas critical to Major Depressive Disorder (MDD) pathophysiology at the time of symptom emergence in stress-susceptible mice. Finally, we demonstrate that loss of NG2 glial secretion of fibroblast growth factor 2 (FGF2) suffices to induce the same behavioral deficits. Our findings outline a pathway and role for NG2 glia in CNS homeostasis and mood disorders.