RESUMEN
Huntington's Disease is a neurodegenerative condition caused by a polyglutamine expansion in the huntingtin (Htt) protein, which aggregates and also causes neuronal dysfunction. Pathogenic N-terminal htt fragments perturb axonal transport in vitro. To determine whether this occurs in vivo and to elucidate how transport is affected, we expressed htt exon 1 with either pathogenic (HttEx1Q93) or non-pathogenic (HttEx1Q20) polyglutamine tracts in Drosophila. We found that HttEx1Q93 expression causes axonal accumulation of GFP-tagged fast axonal transport vesicles in vivo and leads to aggregates within larval motor neuron axons. Time-lapse video microscopy, shows that vesicle velocity is unchanged in HttEx1Q93-axons compared to HttEx1Q20-axons, but vesicle stalling occurs to a greater extent. Whilst HttEx1Q93 expression did not affect locomotor behaviour, external heat stress unveiled a locomotion deficit in HttEx1Q93 larvae. Therefore vesicle transport abnormalities amidst axonal htt aggregation places a cumulative burden upon normal neuronal function under stressful conditions.
Asunto(s)
Transporte Axonal/genética , Axones/metabolismo , Sistema Nervioso Central/metabolismo , Drosophila/metabolismo , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animales , Axones/patología , Temperatura Corporal/genética , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Drosophila/genética , Femenino , Trastornos Neurológicos de la Marcha/genética , Trastornos Neurológicos de la Marcha/metabolismo , Trastornos Neurológicos de la Marcha/fisiopatología , Trastornos de Estrés por Calor/genética , Trastornos de Estrés por Calor/metabolismo , Trastornos de Estrés por Calor/fisiopatología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/fisiopatología , Masculino , Neuronas Motoras/patología , Péptidos/genética , Péptidos/metabolismo , Estrés Fisiológico/genética , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/patologíaRESUMEN
Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of beta-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of beta-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of beta-catenin.
Asunto(s)
Proteínas Portadoras , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores , Transcripción Genética , Proteínas de Pez Cebra , Proteínas Adaptadoras Transductoras de Señales , Animales , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Litio/farmacología , Luciferasas/genética , Microscopía Fluorescente , Presenilina-1 , Transducción de Señal , Fracciones Subcelulares/metabolismo , Proteínas Wnt , beta CateninaRESUMEN
Hyperphosphorylated tau is a pathological hallmark of Alzheimer's disease, but the mechanisms that lead to its formation are poorly understood. To investigate what effect deafferentation of the hippocampus has on the phosphorylation state of tau, we lesioned the entorhinal cortex in rats and looked for hyperphosphorylated tau in the hippocampus at various days post lesioning. After 7 and 21 days, small AT8-positive 'granules' appeared in the molecular layer of the dentate gyrus on the lesioned side. No such staining was seen in the animals injected with saline. This study shows that deafferentation leads to induction of hyperphosphorylated tau. The AT8 positive 'granules' seen resemble the argyrophilic grains that characterize Argyrophilic Grain disease suggesting that lesioning the perforant pathway may serve as a useful model for inducing argyrophilic grains in vivo.
Asunto(s)
Corteza Entorrinal/metabolismo , Hipocampo/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Corteza Entorrinal/lesiones , Hipocampo/lesiones , Vía Perforante/lesiones , Vía Perforante/metabolismo , Fosforilación , RatasRESUMEN
Although hyperphosphorylated tau is an established feature of Alzheimer's Disease, its role in the disease process is poorly understood, partly because of lack of suitable animal models. We describe the use of living slices of rat hippocampal formation to study tau phosphorylation. Using the AT8 antibody in an ELISA, phosphorylated tau was detected in freshly frozen slices and it increased significantly in slices that were incubated in an electrophysiological recording chamber; the amount detected was greatest when the homogenisation buffer contained phosphatase and kinase inhibitors. The phosphorylated tau content of the slices increased significantly after exposure to the phosphatase 1 and 2A inhibitor okadaic acid (OA) - 1.5 microM. Electrophysiological recordings confirmed that slices were alive and that OA had no acute toxic effect. In control slices phosphorylated tau, detected immunohistochemically, was mainly in the somatodendritic compartment of neurones; in OA treated slices, there was an apparent decrease in somatodendritic AT8 staining and an increase in neuropil staining. Our system enables the induction of hyperphosphorylated tau within living slices, in an experimental environment that can be used to study the biological consequences of such a change, and may therefore help further our understanding of the significance of hyperphosphorylated tau in Alzheimer's Disease.
Asunto(s)
Hipocampo/química , Hipocampo/enzimología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Bicuculina/farmacología , Química Encefálica/efectos de los fármacos , Química Encefálica/fisiología , Tampones (Química) , Electrofisiología , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Antagonistas del GABA/farmacología , Ionóforos/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ácido Ocadaico/farmacología , Técnicas de Cultivo de Órganos , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 1 , Ratas , Ratas WistarRESUMEN
The pathological hallmarks of Alzheimer's disease include neurofibrillary tangles, neuropil threads and neuritic plaques. Neurofibrillary tangles and neuropil threads are comprised of paired helical filaments which are themselves composed of a hyperphosphorylated form of the microtubule-associated protein tau. Neuritic plaques are extracellular deposits of aggregated beta amyloid associated with neurites containing hyperphosphorylated tau. The mechanisms by which the neurofibrillary tangles and neuritic plaques develop in Alzhemier's disease are not clear but it is hypothesized that sulphated glycosaminoglycans are important in their formation. This impression is based on the finding that the glycosaminoglycan, heparan sulphate, is found associated with neurofibrillary tangles, neuritic plaques and neuropil threads while dermatan sulphate, chondroitin sulphate and keratan sulphate immunoreactivity is found around neuritic plaques in brains of Alzheimer's disease patients. Furthermore, in vitro studies demonstrate that sulphated glycosaminoglycans such as heparan sulphate and the closely related molecule heparin interact with tau and potentiate its phosphorylation by a number of serine/threonine kinases, reduce its ability to bind to microtubules and induce paired helical filament formation, all properties associated with tau isolated from Alzheimer's disease brain. Thus, we were interested to learn whether intracerebral injection of the sulphated glycosaminoglycan heparin would give rise to alterations in the cytoskeletal protein tau in the rat brain. Although no cytoskeletal changes were observed, to our considerable surprise we found that the intrahippocampal injection of heparin gave rise to seizures. We have investigated this unexpected effect further in vivo and by using in vitro electrophysiological techniques.