RESUMEN
BACKGROUND: The axonal tau protein is a tubulin-binding protein, which plays important roles in the formation and stability of the microtubule. Mutations in the tau gene are associated with familial forms of frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Paired helical filaments of tau and extracellular plaques containing beta-amyloid are found in the brain of Alzheimer's disease (AD) patients. RESULTS: Transgenic models, including those of zebrafish, have been employed to elucidate the mechanisms by which tau protein causes neurodegeneration. In this study, a transient expression system was established to express GFP fusion proteins of zebrafish and human tau under the control of a neuron-specific HuC promoter. Approximately ten neuronal cells expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording, in order to evaluate the neurotoxicity induced by tau-GFP proteins. Expression of tau-GFP was observed to cause high levels of neuronal death. However, multiple signaling factors, such as Bcl2-L1, Nrf2, and GDNF, were found to effectively protect neuronal cells expressing tau-GFP from death. Treatment with chemical compounds that exert anti-oxidative or neurotrophic effects also resulted in a similar protective effect and maintained human tau-GFP protein in a phosphorylated state, as detected by antibodies pT212 and AT8. CONCLUSIONS: The novel finding of this study is that we established an expression system expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording to evaluate the neurotoxicity induced by tau-GFP proteins. This system may serve as an efficient in vivo imaging platform for the discovery of novel drugs against tauopathy.
Asunto(s)
Demencia Frontotemporal/metabolismo , Neuronas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Proteínas tau/metabolismo , Animales , Animales Modificados Genéticamente , Muerte Celular , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 17/metabolismo , Modelos Animales de Enfermedad , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Humanos , Neuronas/patología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas tau/genéticaRESUMEN
Layer-by-layer deposition of polyelectrolyte multilayer (PEM) membranes has recently been applied successfully to a number of biomedical applications. This simple and versatile technique provides a broad surface modification platform, for example, for the display of biomolecules such as cell-adhesion peptides. In this work, we investigated the effects of PEM coatings on RGD-immobilization and osteoblast cell culture. RGD-containing peptides were conjugated to the amino groups of poly(allylamine hydrochloride) (PAH), and then adsorbed on top of 10-layer PAH/poly(acrylic acid) (PAA) multilayer membranes that were assembled at either pH 2.0 or pH 6.5. MG63 osteoblast-like cells were then seeded and cultured on the RGD-conjugated surfaces. We found that the cells adhered to and grew better on the RGD-conjugated PEM membranes. Furthermore, the cells grew better on the RGD-conjugated PEM coatings assembled at pH 6.5 than those assembled at pH 2.0. On the other hand, MG63 cells exhibited better differentiated phenotype on the pH 2.0 coatings compared to the pH 6.5 coatings with respect to alkaline phosphatase activity and calcium deposition, while cells did not express osteoblast phenotype on the PAH surfaces. These results clearly show that the base PEM membranes play an important role in RGD-immobilization and osteoblast functions.
Asunto(s)
Materiales Biocompatibles Revestidos/química , Oligopéptidos/química , Osteoblastos/citología , Osteoblastos/metabolismo , Calcio/metabolismo , Adhesión Celular , Línea Celular , Humanos , Membranas Artificiales , Andamios del Tejido/químicaRESUMEN
Layer-by-layer deposition of polyelectrolyte multilayer (PEM) thin films has recently been applied to biomaterial applications. This simple and versatile technique provides a wide variety of potential utilization by insertion of biomolecules such as cell adhesion peptides. In this work dual peptides containing RGD (a cell-binding domain) and LHRRVKI (a heparin-binding domain) were immobilized onto polystyrene by the PEM technique and the effects on osteoblast cell culture were investigated. These peptides were conjugated to the amino groups of poly(allylamine hydrochloride) and then adsorbed onto the top of a 10 layer poly(allylamine hydrochloride)/poly(acrylic acid) film assembled at either pH 2.0 or pH 6.5. Osteoblasts, isolated from neonatal rat calvariae, were then seeded and cultured on the peptide-conjugated surfaces. We found that the cells adhered and grew better on the RGD-conjugated PEM films. The osteoblasts exhibited a better differentiated phenotype on the pH 2.0 films than the pH 6.5 films with respect to calcium deposition. The incorporation of LHRRVKI did not support cell adhesion, growth and matrix mineral deposition. Our results showed that the efficacy of RGD conjugation on osteoblast behavior was affected by the base PEM film.