RESUMEN
Our previous studies showed that the intracerebral injection of apotransferrin (aTf) attenuates white matter damage and accelerates the remyelination process in a neonatal rat model of cerebral hypoxia-ischemia (HI) injury. However, the intracerebral injection of aTf might not be practical for clinical treatments. Therefore, the development of less invasive techniques capable of delivering aTf to the central nervous system would clearly aid in its effective clinical use. In this work, we have determined whether intranasal (iN) administration of human aTf provides neuroprotection to the neonatal mouse brain following a cerebral hypoxic-ischemic event. Apotransferrin was infused into the naris of neonatal mice and the HI insult was induced by right common carotid artery ligation followed by exposure to low oxygen concentration. Our results showed that aTf was successfully delivered into the neonatal HI brain and detected in the olfactory bulb, forebrain and posterior brain 30 min after inhalation. This treatment successfully reduced white matter damage, neuronal loss and astrogliosis in different brain regions and enhanced the proliferation and survival of oligodendroglial progenitor cells (OPCs) in the subventricular zone and corpus callosum (CC). Additionally, using an in vitro hypoxic model, we demonstrated that aTf prevents oligodendrocyte progenitor cell death by promoting their differentiation. In summary, these data suggest that iN administration of aTf has the potential to be used for clinical treatment to protect myelin and to induce remyelination in demyelinating hypoxic-ischemic events in the neonatal brain.
Asunto(s)
Apoproteínas/administración & dosificación , Lesiones Encefálicas/prevención & control , Hipoxia-Isquemia Encefálica/patología , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fármacos Neuroprotectores/administración & dosificación , Transferrina/administración & dosificación , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/genética , Administración Intranasal , Factores de Edad , Animales , Animales Recién Nacidos , Antígenos/metabolismo , Proteínas Relacionadas con la Autofagia , Lesiones Encefálicas/etiología , Bromodesoxiuridina/metabolismo , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Colchicina/farmacología , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/patología , Citocalasina B/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Hipoxia/tratamiento farmacológico , Hipoxia-Isquemia Encefálica/complicaciones , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Proteínas de Filamentos Intermediarios/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ventrículos Laterales/efectos de los fármacos , Ventrículos Laterales/fisiología , Masculino , Ratones , Ratones Transgénicos , Proteína Básica de Mielina/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neurogénesis/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteoglicanos/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factores de TiempoRESUMEN
We have previously demonstrated that the addition of apotransferrin (aTf) to oligodendroglial cell (OLGc) primary cultures accelerates their maturation. Cells treated with aTf developed a multipolar morphology and displayed increased expression of mature OLGc markers. In this work, we studied the effect of Tf overexpression in two OLGc lines, N19 and N20.1. The former cells exhibit characteristics of OLGc precursors (O2A), while N20.1 cells express markers of more mature OLGcs. Using the complete cDNA of the human Tf gene, we obtained clones overexpressing Tf in both cell lines. These clones were evaluated for the expression of OLGc differentiation markers. In agreement with our previous results, we found that in the cells overexpressing Tf, there was an increased O(4), GC, and MBP immunoreactivity. To study the myelinogenic potential of these cells, we co-cultured N19 and N20.1 Tf-transfected cells together with cortical neurons. There was a dramatic increase in the morphological differentiation of the OLGcs accompanied by enhanced GC and MBP expression. The OLGcs appeared to establish contact with neurites and extend their processes along them. Only two MBP isoforms were detected in Tf-overexpressing clones, while all the isoforms were present in the co-cultures, suggesting that there was a modulation of MBP expression by neurons. Concomitantly, we found an increase in several proteins involved in axon-glia interaction, such as MAG, N-CAM, and F3/Contactin. This co-culture system represents a potentially powerful tool to study neuron-glia interactions that occur during myelinogenesis and the role of Tf in this process.
Asunto(s)
Diferenciación Celular/genética , Oligodendroglía/metabolismo , Transferrina/genética , Transferrina/metabolismo , Animales , Biomarcadores/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Comunicación Celular/genética , Línea Celular , Sistema Nervioso Central/citología , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Células Clonales/citología , Células Clonales/metabolismo , Técnicas de Cocultivo/métodos , Contactinas , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Ratones , Proteína Básica de Mielina/metabolismo , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Glicoproteína Asociada a Mielina/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Oligodendroglía/citología , Ratas , Células Madre/citología , Células Madre/metabolismo , Transfección/métodosRESUMEN
We have previously shown that addition of apotransferrin (aTf) accelerates maturation of oligodendroglial cells (OLGcs) in primary cultures. In this work, we examined the effect of aTf on two conditionally immortalized cell lines: N19 and N20.1. These cells proliferate at 34 degrees C and differentiate into mature OLGcs at 39 degrees C. In vitro addition of aTf to both cell lines at the differentiation temperature for 7 days showed increased expression of galactocerebroside, O4, and myelin basic protein (MBP) and a drop in the percentage of BrdU+ cells. The effect on MBP expression was particularly interesting in the less mature N19 cells. These cells do not express either MBP mRNAs or proteins, so aTf induced, rather than modulated, MBP expression in this cell line. In addition, even though MBP mRNAs for all four isoforms were induced, only the 17 and 21.5 kDa appeared to be translated. OLGc differentiation has been shown to be stimulated by the cAMP-CREB pathway. In N19 cells, following a pulse of aTf, there was a 10-fold increase in cAMP levels accompanied by elevated levels of pCREB. In the more mature N20.1 cells, there were no changes in cAMP levels. We conclude that addition of aTf to immature OLGc lines can enhance their expression of differentiated markers, such as MBP. The action of aTf on MBP gene expression in the least mature line is likely to be mediated by the cAMP pathway. In the N20.1 cells, it appears that different signals and/or mechanisms are involved in modulating myelin lipid and MBP expression. The results suggest that aTf can influence OLGc gene expression and differentiation through multiple mechanisms depending on the maturation of the cell.