RESUMEN
BACKGROUND: Although several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity. METHODS: Tau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors. RESULTS: We observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients. CONCLUSIONS: This study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Agregación Patológica de Proteínas/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Epítopos , Células HEK293 , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Unión Proteica , Proteínas Recombinantes , Sonicación , Espectroscopía Infrarroja por Transformada de Fourier , Proteínas tau/genética , Proteínas tau/ultraestructuraRESUMEN
The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated- and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular- and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice- and postmortem human AD brain.
Asunto(s)
Enfermedad de Alzheimer/patología , Anticuerpos Monoclonales/metabolismo , Encéfalo/metabolismo , Proteínas tau/inmunología , Animales , Mapeo Epitopo , Femenino , Células HEK293 , Humanos , Inmunización Pasiva , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Resonancia por Plasmón de Superficie , Proteínas tau/deficiencia , Proteínas tau/genéticaRESUMEN
Este trabalho objetivou avaliar o conhecimento sobre a regulamentação e a utilização das terapias comple-mentares pelos fisioterapeutas. Foram entrevistados 30 fisioterapeutas, que estavam em plena atividade e que trabalhavam em clínicas, hospitais, faculdades, no período de agosto a novembro de 2007, na Faculdade Estácio do Ceará. Aplicou-se um questionário a respeito da utilização, conhecimento, interesse e importância dessas terapias complementares pelo profissional de Fisioterapia e se o mesmo tinha conhecimento sobre as respectivas regulamentações. Evidenciamos que os fisioterapeutas consideram importante em seu plano de tratamento a utilização das terapias complementares (87%), porém ainda são poucos os que utilizam essas terapias em suas condutas (43%), e os que utilizam, não conhecem a regulamentação das terapias complemen-tares (67%). Em relação ao conhecimento específico de cada resolução e portaria dos 18 fisioterapeutas que relataram ter conhecimento sobre alguma regulamentação, 94,44% conheciam a resolução da Acupuntura. Constatou-se a importância que as terapias complementares vêm conquistando na área da Fisioterapia, porém se faz necessário maior utilização das mesmas pelos profissionais e ampliação do conhecimento dos mesmos sobre as regulamentações que regem as terapias, para que tenham maior abrangência e aceitação no mercado de trabalho