RESUMEN
Spinal cord injury results in neural loss and consequently motor and sensory impairment below the injury. Reactive astrocytes contribute to formation of glial scar, thus impeding axonal regeneration, through secretion of extracellular matrix molecules, chondroitin sulfate proteoglycans (CSPGs). In this study, we analyze lesion site tissue to reveal the possible mechanism underlying the functional recovery after cell transplantation of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cell (OPC) and motoneuron progenitors (MP) and propose that transplanted cells increase astrogliosis through the regenerative signaling pathways activated in the host tissue that may crucial for restoring locomotor ability. We show that the transplantation of hESC-derived OPC and MP promotes astrogliosis, through activation of Jagged1-dependent Notch and Jak/STAT signaling that support axonal survival. The transplanted cells in synergism with reactive astrocytes create permissive environment in which the expression of detrimental genes (Cspg, Tenascins, and genes involved in SLIT/ROBO signaling) was significantly decreased while expression of beneficial ones (Laminins and Fibronectin) was increased. According to our data, this mechanism is activated in all transplantation groups independently of the level of locomotor recovery. These results indicate that modifying the beneficial function of reactive astrocytes could be a feasible therapeutic strategy for spinal cord injury in future.
Asunto(s)
Astrocitos/metabolismo , Gliosis/genética , Transducción de Señal/genética , Traumatismos de la Médula Espinal , Trasplante de Células , Células Madre Embrionarias/metabolismo , Humanos , Neuronas Motoras/metabolismo , Regeneración Nerviosa , Oligodendroglía/citología , Oligodendroglía/metabolismo , Recuperación de la FunciónRESUMEN
Toll-like receptors play an important role in the innate immune response, although emerging evidence indicates their role in brain injury and neurodegeneration. Alcohol abuse induces brain damage and can sometimes lead to neurodegeneration. We recently found that ethanol can promote TLR4 signaling in glial cells by triggering the induction of inflammatory mediators and causing cell death, suggesting that the TLR4 response could be an important mechanism of ethanol-induced neuroinflammation. This study aims to establish the potential role of TLR4 in both ethanol-induced glial activation and brain damage. Here we report that TLR4 is critical for ethanol-induced inflammatory signaling in glial cells since the knockdown of TLR4, by using both small interfering RNA or cells from TLR4-deficient mice, abolished the activation of microtubule-associated protein kinase and nuclear factor-kappaB pathways and the production of inflammatory mediators by astrocytes. Our results demonstrate, for the first time, that whereas chronic ethanol intake upregulates the immunoreactive levels of CD11b (microglial marker) and glial fibrillary acidic protein (astrocyte marker), and also increases caspase-3 activity and inducible nitric oxide synthase, COX-2, and cytokine levels [interleukin (IL)-1beta, tumor necrosis factor-alpha, IL-6] in the cerebral cortex of female wild-type mice, TLR4 deficiency protects against ethanol-induced glial activation, induction of inflammatory mediators, and apoptosis. Our findings support the critical role of the TLR4 response in the neuroinflammation, brain injury, and possibly in the neurodegeneration induced by chronic ethanol intake.
Asunto(s)
Lesiones Encefálicas/inducido químicamente , Depresores del Sistema Nervioso Central/administración & dosificación , Encefalitis/inducido químicamente , Etanol/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Receptor Toll-Like 4/fisiología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Lesiones Encefálicas/sangre , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Caspasa 3/metabolismo , Depresores del Sistema Nervioso Central/sangre , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Encefalitis/sangre , Encefalitis/metabolismo , Encefalitis/patología , Etanol/sangre , Femenino , Proteínas Fluorescentes Verdes/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/efectos adversos , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Estadísticas no Paramétricas , Factores de Tiempo , Receptor Toll-Like 4/deficiencia , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
PURPOSE: The objective of this study was to characterize the demographic and clinical profile of RRMS patients receiving fingolimod in Spain, and to evaluate drug effectiveness and safety in clinical practice. METHODS: This observational, retrospective, multicentre, nationwide study was performed at 56 Spanish hospitals and involved 804 RRMS patients who received oral fingolimod (0.5 mg) since November 2011, with a minimum follow-up of 12 months. RESULTS: The mean annualized relapse rate (ARR) in the year before fingolimod was 1.08 and the median EDSS was 3; patients were exposed to fingolimod for 2.2 years as average; regarding magnetic resonance imaging (MRI) activity, more than half of the patients had >20 lesions at baseline. Patients were previously treated with first-line injectable DMTs (60.3%), or natalizumab (31.3%), and 8.3% were naïve patients. Overall, the ARR significantly decreased to 0.28, 0.22 and 0.17 (74.1%, 79.7% and 83.5% of relative reduction, respectively) after 12, 24 and 36 months of treatment, P<0.001. The ARR of patients who switched from natalizumab to fingolimod was stable over the study. Most of the patients (88.7%) were free from confirmed disability and MRI activity (67.3%) after 24 months. The persistence after 12 months on fingolimod was 93.9%. CONCLUSIONS: The subgroups of patients analysed showed differential baseline demographic and clinical characteristics. The analysis of patients who received fingolimod in routine clinical practice confirmed adequate efficacy and safety, even for long-term treatment. The present data also confirmed the positive benefit/risk balance with fingolimod in real-world clinical practice setting.
Asunto(s)
Clorhidrato de Fingolimod/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Adulto , Anciano , Personas con Discapacidad , Femenino , Clorhidrato de Fingolimod/metabolismo , Humanos , Inmunosupresores/uso terapéutico , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Natalizumab/uso terapéutico , Recurrencia , Estudios Retrospectivos , EspañaRESUMEN
The endogenous cannabinoid acylethanolamide AEA (arachidonoylethanolamide; also known as anandamide) participates in the neuroadaptations associated with chronic ethanol exposure. However, no studies have described the acute actions of ethanol on AEA production and degradation. In the present study, we investigated the time course of the effects of the intraperitoneal administration of ethanol (4 g/kg of body mass) on the endogenous levels of AEA in central and peripheral tissues. Acute ethanol administration decreased AEA in the cerebellum, the hippocampus and the nucleus accumbens of the ventral striatum, as well as in plasma and adipose tissue. Parallel decreases of a second acylethanolamide, PEA (palmitoylethanolamide), were observed in the brain. Effects were observed 45-90 min after ethanol administration. In vivo studies revealed that AEA decreases were associated with a remarkable inhibition of the release of both anandamide and glutamate in the nucleus accumbens. There were no changes in the expression and enzymatic activity of the main enzyme that degrades AEA, the fatty acid amidohydrolase. Acute ethanol administration did not change either the activity of N-acyltransferase, the enzyme that catalyses the synthesis of the AEA precursor, or the expression of NAPE-PLD (N-acylphosphatidylethanolamine-hydrolysing phospholipase D), the enzyme that releases AEA from membrane phospholipid precursors. These results suggest that receptor-mediated release of acylethanolamide is inhibited by the acute administration of ethanol, and that this effect is not derived from increased fatty acid ethanolamide degradation.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Encéfalo/metabolismo , Moduladores de Receptores de Cannabinoides/metabolismo , Etanol/farmacología , Alcamidas Poliinsaturadas/metabolismo , Alcoholismo/metabolismo , Animales , Encéfalo/efectos de los fármacos , Endocannabinoides , Etanol/administración & dosificación , Inyecciones Intraperitoneales , Masculino , Modelos Animales , Ratas , Ratas WistarRESUMEN
Endocannabinoid signaling has recently been implicated in ethanol-seeking behavior. We analyzed the expression of endocannabinoid-related genes in key brain regions of reward and dependence, and compared them between the alcohol-preferring AA (Alko Alcohol) and nonpreferring ANA (Alko Non-Alcohol) rat lines. A decreased expression of fatty acid amidohydrolase (FAAH), the main endocannabinoid-degrading enzyme, was found in prefrontal cortex (PFC) of AA rats, and was accompanied by decreased enzyme activity in this region. Binding of the endocannabinoid-cannabinoid 1 (CB1) receptor ligand (3)[H]SR141716A, and [35S]GTPgammaS incorporation stimulated by the CB1 agonist WIN 55,212-2 were downregulated in the same area. Together, this suggests an overactive endocannabinoid transmission in the PFC of AA animals, and a compensatory downregulation of CB1 signaling. The functional role of impaired FAAH function for alcohol self-administration was validated in two independent ways. The CB1 antagonist SR141716A potently and dose-dependently suppressed self-administration in AA rats when given systemically, or locally into the PFC, but not in the striatum. Conversely, intra-PFC injections of the competitive FAAH inhibitor URB597 increased ethanol self-administration in nonselected Wistar rats. These results show for the first time that impaired FAAH function may confer a phenotype of high voluntary alcohol intake, and point to a FAAH both as a potential susceptibility factor and a therapeutic target.
Asunto(s)
Consumo de Bebidas Alcohólicas/genética , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Expresión Génica/fisiología , Corteza Prefrontal/metabolismo , Receptor Cannabinoide CB1/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Analgésicos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Benzamidas/farmacología , Benzoxazinas , Química Encefálica/efectos de los fármacos , Química Encefálica/genética , Carbamatos/farmacología , Depresores del Sistema Nervioso Central/administración & dosificación , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Etanol/administración & dosificación , Expresión Génica/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Hibridación in Situ/métodos , Masculino , Morfolinas/farmacología , Naftalenos/farmacología , Piperidinas/farmacocinética , Corteza Prefrontal/efectos de los fármacos , Pirazoles/farmacocinética , ARN Mensajero/metabolismo , Ratas , Receptor Cannabinoide CB1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Rimonabant , Autoadministración/métodosRESUMEN
Here we show that the activation of cannabinoid CB2 receptors improved glucose tolerance after a glucose load. Blockade of cannabinoid CB2 receptors counteracted this effect, leading to glucose intolerance. Since blockade of cannabinoid CB1 receptors mimics the actions of cannabinoid CB2 receptor agonists, we propose that the endocannabinoid system modulates glucose homeostasis through the coordinated actions of cannabinoid CB1 and CB2 receptors. We also describe the presence of both cannabinoid CB1 and CB2 receptor immunoreactivity in rat pancreatic beta- and non-beta-cells, adding the endocrine pancreas to adipose tissue and the liver as potential sites for endocannabinoid regulation of glucose homeostasis.
Asunto(s)
Glucosa/metabolismo , Homeostasis , Receptor Cannabinoide CB2/fisiología , Animales , Moduladores de Receptores de Cannabinoides/fisiología , Inmunohistoquímica , Islotes Pancreáticos/química , Masculino , Piperidinas/farmacología , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/análisis , Receptor Cannabinoide CB2/análisisRESUMEN
Recent reports have described the presence of cannabinoid CB1 receptors in pancreatic islets. Here we show that administration of the endogenous cannabinoid anandamide or the selective cannabinoid CB1 receptor agonist Arachidonyl-2'-chloroethylamide (ACEA) results in glucose intolerance after a glucose load. This effect is reversed by the selective cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251). These results suggest that targeting cannabinoid CB1 receptors may serve as new therapeutic alternatives for metabolic disorders such as diabetes.
Asunto(s)
Intolerancia a la Glucosa/fisiopatología , Receptor Cannabinoide CB1/fisiología , Animales , Ácidos Araquidónicos/farmacología , Glucemia/metabolismo , Moduladores de Receptores de Cannabinoides/farmacología , Endocannabinoides , Intolerancia a la Glucosa/inducido químicamente , Intolerancia a la Glucosa/prevención & control , Masculino , Piperidinas/farmacología , Alcamidas Poliinsaturadas , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidoresRESUMEN
Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro, increase our understanding of human embryonic development, and provide clinically relevant cell types for transplantation, drug testing, and toxicology studies. Since their discovery, numerous advances have been made in order to eliminate issues such as vector integration into the host genome, low reprogramming efficiency, incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally, we illustrate methods by which to validate pluripotency of the resulting stem cell population.
Asunto(s)
Reprogramación Celular , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , ARN/genética , Virus Sendai/genética , Transgenes , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Nutrientes/citología , Células Nutrientes/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Modelos Biológicos , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
New subventricular zone (SVZ)-derived neuroblasts that migrate via the rostral migratory stream are continuously added to the olfactory bulb (OB) of the adult rodent brain. Anosmin-1 (A1) is an extracellular matrix protein that binds to FGF receptor 1 (FGFR1) to exert its biological effects. When mutated as in Kallmann syndrome patients, A1 is associated with severe OB morphogenesis defects leading to anosmia and hypogonadotropic hypogonadism. Here, we show that A1 over-expression in adult mice strongly increases proliferation in the SVZ, mainly with symmetrical divisions, and produces substantial morphological changes in the normal SVZ architecture, where we also report the presence of FGFR1 in almost all SVZ cells. Interestingly, for the first time we show FGFR1 expression in the basal body of primary cilia in neural progenitor cells. Additionally, we have found that A1 over-expression also enhances neuroblast motility, mainly through FGFR1 activity. Together, these changes lead to a selective increase in several GABAergic interneuron populations in different OB layers. These specific alterations in the OB would be sufficient to disrupt the normal processing of sensory information and consequently alter olfactory memory. In summary, this work shows that FGFR1-mediated A1 activity plays a crucial role in the continuous remodelling of the adult OB.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Ventrículos Laterales/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neurogénesis , Bulbo Olfatorio/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , División Celular , Movimiento Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Humanos , Interneuronas/metabolismo , Interneuronas/fisiología , Ventrículos Laterales/metabolismo , Ventrículos Laterales/ultraestructura , Memoria a Corto Plazo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiología , Vías Nerviosas/ultraestructura , Odorantes , Bulbo Olfatorio/metabolismo , Percepción Olfatoria/fisiologíaRESUMEN
The in vitro generation of neural cells from human embryonic stem cells is a powerful tool to acquire better knowledge of the cellular and molecular events involved in early human neural and brain development under physiological and pathological conditions. Prenatal alcohol exposure can induce important anomalies in the developing brain, the embryogenesis being an important critical period for the craniofacial defects and mental disabilities associated with fetal alcohol syndrome. Here, we report the generation of neural progenitors (NPs) from human embryonic stem cells. Neuroepithelial progenitors display the morphological and functional characteristics of their embryonic counterparts and the proper timing of neurons and glia cells generation. Immunocytochemical and real time (RT)-polymerase chain reaction analyses reveal that cells appeared as clusters during neuroepithelial cell proliferation and that the genes associated with the neuroectodermal (Pax-6) and the endodermic (α-fetoprotein) lineages decreased in parallel to the upregulation of the genes of NPs (nestin and Tuj1), followed by their differentiation into neurons (MAP-2+, GABA+), oligodendrocytes [galactocerebroside (GalC+)], and astrocytes (GFAP+). We further demonstrate, for the first time, that human NPs express the endocannabinoid receptors (CB1 and CB2) and the enzymes involved in endocannabinoids synthesis (NAPE-PLD) and degradation (FAAH). Using this in vitro culture, we demonstrate that ethanol exposure impairs NPs survival, affects the differentiation of NPs into neurons and astrocytes, disrupts the actin cytoskeleton, and affects the expression of different genes associated with neural differentiation. The results provide new insights into the effects of ethanol on human embryogenesis and neuroprogenitors and offer an opportunity to delineate potential therapeutic strategies to restore early ethanol-induced brain damage.