RESUMEN
Sphingolipid metabolites are involved in the regulation of autophagy, a degradative recycling process that is required to prevent neuronal degeneration. Drosophila blue cheese mutants neurodegenerate due to perturbations in autophagic flux, and consequent accumulation of ubiquitinated aggregates. Here, we demonstrate that blue cheese mutant brains exhibit an elevation in total ceramide levels; surprisingly, however, degeneration is ameliorated when the pool of available ceramides is further increased, and exacerbated when ceramide levels are decreased by altering sphingolipid catabolism or blocking de novo synthesis. Exogenous ceramide is seen to accumulate in autophagosomes, which are fewer in number and show less efficient clearance in blue cheese mutant neurons. Sphingolipid metabolism is also shifted away from salvage toward de novo pathways, while pro-growth Akt and MAP pathways are down-regulated, and ER stress is increased. All these defects are reversed under genetic rescue conditions that increase ceramide generation from salvage pathways. This constellation of effects suggests a possible mechanism whereby the observed deficit in a potentially ceramide-releasing autophagic pathway impedes survival signaling and exacerbates neuronal death.
Asunto(s)
Autofagia , Ceramidas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Mutación/genética , Proteínas del Tejido Nervioso/genética , Transducción de Señal , Estrés Fisiológico , Animales , Células Cultivadas , Ceramidasas/metabolismo , Regulación hacia Abajo , Drosophila melanogaster/enzimología , Técnicas de Silenciamiento del Gen , Metabolismo de los Lípidos , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Degeneración Nerviosa/patología , Neuronas/metabolismo , Fagosomas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingolípidos/metabolismo , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Fatty acids are abundant constituents of all biological systems, and their metabolism is important for normal function at all levels of an organism. Aberrations in fatty acid metabolism are associated with pathological states and have become a focus of current research, particularly due to the interest in metabolic overload diseases. Here we present a click-chemistry-based method that allows tracing of fatty acid metabolism in virtually any biological system. It combines high sensitivity with excellent linearity and fast sample turnover. Since it is free of radioactivity, it can be combined with any other modern analysis technology and can be used in high-throughput applications. Using the new method, we provide for the first time an analysis of cellular fatty metabolism with high time resolution and a comprehensive comparison of utilization of a broad spectrum of fatty acids in hepatoma and adipose cell lines.