RESUMEN
Frosty pod rot, caused by Moniliophthora roreri, is the most damaging disease of cacao in Latin America and, to better comprehend its epidemiology, we must understand its dissemination and proliferation. However, we do not know how M. roreri spores loads fluctuate in time and space due to the lack of a reliable technique to quantify M. roreri spores in the fields. Therefore, we developed a method that relies on spore traps and qPCR to detect and quantify M. roreri spore loads. This study demonstrated that the qPCR protocol can detect down to 0.025 ng of M. roreri DNA and quantify between 0.006 ng and 60 ng. Moreover, it demonstrated that qPCR protocol can detect and quantify DNA extracted from spore suspension and spore traps containing at least 2.9 × 104 M. roreri spores. However, the variability of the estimates for spore samples was high. Finally, we described a spore-trap device designed to carry spore traps in the field. The qPCR protocol and spore-trap device here developed will help in the understanding of the M. roreri dissemination patterns since they can be used to assess the environmental loads of M. roreri spore in cacao fields.
RESUMEN
CDK5 plays an important role in neurotransmission and synaptic plasticity in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. In a previous study, we demonstrated that RNAi knock down of CDK5 reduced the formation of neurofibrillary tangles (NFT) and prevented neuronal loss in triple transgenic Alzheimer's mice. Here, we report that CDK5 RNAi protected against glutamate-mediated excitotoxicity using primary hippocampal neurons transduced with adeno-associated virus 2.5 viral vector eGFP-tagged scrambled or CDK5 shRNA-miR during 12 days. Protection was dependent on a concomitant increase in p35 and was reversed using p35 RNAi, which affected the down-stream Rho GTPase activity. Furthermore, p35 over-expression and constitutively active Rac1 mimicked CDK5 silencing-induced neuroprotection. In addition, 3xTg-Alzheimer's disease mice (24 months old) were injected in the hippocampus with scrambled or CDK5 shRNA-miR, and spatial learning and memory were performed 3 weeks post-injection using 'Morris' water maze test. Our data showed that CDK5 knock down induced an increase in p35 protein levels and Rac activity in triple transgenic Alzheimer's mice, which correlated with the recovery of cognitive function; these findings confirm that increased p35 and active Rac are involved in neuroprotection. In summary, our data suggest that p35 acts as a mediator of Rho GTPase activity and contributes to the neuroprotection induced by CDK5 RNAi.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Animales , Western Blotting , Quinasa 5 Dependiente de la Ciclina/genética , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Hipocampo/metabolismo , Hipocampo/patología , Ratones , Ratones Transgénicos , Neuronas/patología , ARN Interferente Pequeño , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción Genética , TransfecciónRESUMEN
Neurodegeneration is one of the greatest public health challenges for the 21st century. Among neurodegenerative diseases, Alzheimer's disease (AD) is the most prevalent and best characterized. Nevertheless, despite the large investment in AD research, currently there is no effective therapeutic option. In the present review, we highlight a novel alternative, which takes advantage of the biotechnological outbreak deployed by the discovery of the RNA interference-based gene silencing mechanism, and its application as a tool for neurodegeneration treatment. Here, we highlight cyclin-dependent kinase 5 (CDK5) as a key candidate target for therapeutic gene silencing. Unlike other members of the cyclin-dependent kinase family, CDK5 does not seem to play a crucial role in cell cycle regulation. By contrast, CDK5 participates in multiple functions during nervous system development and has been established as a key mediator of Tau hyperphosphorylation and neurofibrillary pathology, thus serving as an optimal candidate for targeted therapy in the adult nervous system. We propose that the use of RNA interference for CDK5 silencing presents an attractive and specific therapeutic alternative for AD and perhaps against other tauopathies.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/terapia , Quinasa 5 Dependiente de la Ciclina/metabolismo , Interferencia de ARN/fisiología , Animales , Humanos , Modelos Biológicos , Enfermedades Neurodegenerativas/enzimología , FosforilaciónRESUMEN
Alzheimer's disease is a major cause of dementia for which treatments remain unsatisfactory. Cyclin-dependent kinase 5 (CDK5) is a relevant kinase that has been hypothesized to contribute to the tau pathology. Several classes of chemical inhibitors for CDK5 have been developed, but they generally lack the specificity to distinguish among various ATP-dependent kinases. Therefore, the efficacy of these compounds when tested in animal models cannot definitively be attributed to an effect on CDK5. However, RNA interference (RNAi) targeting of CDK5 is specific and can be used to validate CDK5 as a possible treatment target. We delivered a CDK5 RNAi by lentiviral or adenoassociated viral vectors and analyzed the results in vitro and in vivo. Silencing of CDK5 reduces the phosphorylation of tau in primary neuronal cultures and in the brain of wild-type C57BL/6 mice. Furthermore, the knockdown of CDK5 strongly decreased the number of neurofibrillary tangles in the hippocampi of triple-transgenic mice (3×Tg-AD mice). Our data suggest that this downregulation may be attributable to the reduction of the CDK5 availability in the tissue, without affecting the CDK5 kinase activity. In summary, our findings validate CDK5 as a reasonable therapeutic target for ameliorating tau pathology.
Asunto(s)
Enfermedad de Alzheimer/genética , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/fisiología , Ovillos Neurofibrilares/genética , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/patología , Animales , Anticuerpos Monoclonales , Western Blotting , Región CA1 Hipocampal/metabolismo , Técnica del Anticuerpo Fluorescente , Silenciador del Gen , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovillos Neurofibrilares/patología , Neuronas/metabolismo , Fosforilación , Plásmidos/genética , Interferencia de ARN/fisiología , Ratas , Ratas Wistar , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Several studies have linked estrogens with sphingosine kinase (SphK) activity, enzyme responsible of sphingosine-1-phosphate synthesis (S-1P), however their possible interaction in the nervous system is not documented yet. In the present study, we developed a glutamate toxicity model in SH-SY5Y cells to evaluate the possible effect of the inhibition of SphK activity on the protective capability of 17ß-estradiol (E2). Glutamate induced cytoskeletal actin changes associated to cytotoxic stress, significant increase of apoptotic-like nuclear fragmentation, Tau hyperphosphorylation and increase of p25/p35 cleavage. These effects were prevented by E2 pre-treatment during 24 h. Although the inhibition of SphK did not block this protective effect, significantly increased Tau hyperphosphorylation by glutamate, in a way that was not reverted by E2. Our results suggest that the decrease of glutamate-induced Tau hyperphosphorylation by 17ß-estradiol requires SphK.
Asunto(s)
Estradiol/farmacología , Ácido Glutámico/toxicidad , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Proteínas tau/metabolismo , Actinas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuroblastoma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Introducción: Un componente molecular predominante en el estudio de las enfermedades neurodegenerativas es la presencia del complejo Tau-GSK3β y su asociación con agregados proteicos al interior de la célula. Evidencias considerables muestran que GSK3β es el principal causante de la hiperfosforilación de Tau. Sin embargo, son poco claros los eventos moleculares que gobiernan este complejo. Objetivo: Determinar el efecto del 17 β-estradiol en la expresión y asociación de las quinasas responsables de la hiperfosforilación de Tau. Métodos: Se realizaron tratamientos con 17 β-estradiol en hipocampo de rata Wistar adulta ovariectomizada y en cultivos primarios de hipocampo de rata tratados con b-amiloide. Se evaluó la asociación de complejos proteicos por co-inmunoprecipitación, ensayo de toxicidad por liberación LDH y cambios morfológicos celulares por microscopía confocal. Resultados: Este estudio mostró evidencias de que el estradiol disocia complejos macromoleculares como Tau/GSK3β, Tau/GluR2/3, Tau/FAK, Tau/Fyn en hipocampo de rata adulta. Ademßs, disminuyó la expresión de GSK3β-ptyr por el tratamiento hormonal y éste reguló la defosforilación de Tau en un modelo de excitoxicidad poráβ-amiloide. Conclusiones: Lo anterior sugiere, nuevos blancos que contribuyen al estudio de la neuroprotección y plasticidad neuronal mediada por el estrógeno.
Introduction: A predominant molecular component analyzed in the study of neurodegenerative diseases is the presence of the Tau-GSK3β complex and its association with protein aggregation into the cell. Several evidences show that GSK3β has an important role in abnormal pattern of the phosphorylation of Tau. However, the molecular events that are governing this complex are unknown. Aim: To determine the effect of 17 β-estradiol treatment on the expression and association of Tau hyperphosphorylation responsible kinases. Methods: 17 β-estradiol treatments were realized in the hippocampus of ovariectomized adult wistar rats and in hippocampal primary cultures treated with β-amiloid. Protein complex association was assessed by co-immunoprecipitation, toxicity assay by LDH release and cell morphologic changes by confocal microscopy. Results: Our results show that 17β-estradiol produced dissociation of macromolecular complexes like Tau/GSK3β, Tau /GluR2/3, Tau/FAK, and Tau/Fyn in hippocampus of adult rat. In addition the expression of GSK3β-ptyr was decreased by the hormonal treatment and this one regulated the defosforilation of Tau in an excitotoxicity model by β-amiloid. Conclusions: It suggests new targets that will contribute to neuroprotection and neuronal plasticity studies mediated by the estrogen.
Asunto(s)
Amiloide , Estradiol , Plasticidad Neuronal , FosfotransferasasRESUMEN
Among Latin American countries, Colombia is considered a low-risk area for multiple sclerosis (MS) and no studies on MS prevalence have been conducted in any of the country's large urban settings. To fill this gap and assess the prevalence of MS in Bogotá as of December 31, 2002, this study reviewed the clinical records of patients diagnosed with MS in most Bogotá hospitals. This review produced a sample of 296 patients with an MS diagnosis whose reliability was verified by a neurologist with expertise in MS. The total prevalence rate identified for December 2002 was 4.41/100,000 inhabitants (95% CI 3.9-4.9), including a rate of 5.98/100,000 (95% CI 5.2-6.8) for women and 2.71/100,000 (95% CI 2.2-3.3) for men (differences measured at p < 0.001). The prevalence estimates for Bogotá, confirm the city's status as a low-risk area for MS.
Asunto(s)
Esclerosis Múltiple/epidemiología , Adolescente , Adulto , Distribución por Edad , Anciano , Colombia/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/diagnóstico , Prevalencia , Medición de Riesgo , Distribución por Sexo , Salud UrbanaRESUMEN
Historia de las vicisitudes sufridas por un investigador que se adentró en el averno de la ignorancia, de la propia: De cómo un hombre se vio envuelto en los vericuetos de la técnica, y cómo se desenvolvió, cuando aprendió del empirismo. De cómo una comunidad aprehendió al investigador y cómo fue resuelto el misterio de su liberación por medio de la concertación abierta. ¡Lean ustedes, y sabrán quién es el asesino misterioso ... o si es simple confusión. Todavía se puede rescatar la ciencia y entregarla viva al servicio de las comunidades.
History of the vicissitudes undergone by an investigator who inside in the hell of the ignorance, the own one: Of how a man were itself surrounded in the rough road of the technique, and how unfolded himself, when he learned about the empircism. Of how a community kidnapped the investigator and how the mystery of its liberation by means of the open agreement were solved. You read, and you will know who is the mysterious assassin... or if it is simple confusion. Still science can be rescued and to give it lives to the service of the communities.