Endocytosis of NBD-sphingolipids in neurons: Exclusion from degradative compartments and transport to the Golgi complex

Sphingolipids are abundant constituents of neuronal membranes that have bee n implicated in intracellular signaling, neurite outgrowth and differentiat ion. Differential localization and trafficking of lipids to membrane domain s contribute to the specialized functions. In non-neuronal cultured cell li nes, plasma membrane short-chain sphingomyelin and glucosylceramide are rec ycled via endosomes or sorted to degradative compartments. However, dependi ng on cell type and lipid membrane composition, short-chain glucosylceramid e can also be diverted to the Golgi complex. Here, we show that NBD-labeled glucosylceramide and sphingomyelin are transported from the plasma membran e to the Golgi complex in cultured rat hippocampal neurons Irrespective of the stage of neuronal differentiation. Golgi complex localization was confi rmed by colocalization and Golgi disruption studies, and importantly did no t result from conversion of NBD-glucosylceramide or NBD-sphingomyelin to NB D-ceramide. Double-labeling experiments with transferrin or wheat-germ aggl utinin showed that NBD-sphingolipids are first internalized to early/recycl ing endosomes, and subsequently transported to the Golgi complex. The inter nalization of these two sphingolipid analogs was energy and temperature dep endent, and their intracellular transport was insensitive to the NBD fluore scence quencher sodium dithionite. These results indicate that vesicles med iate the transport of internalized NBD-glucosylceramide and NBD-sphingomyel in to the Golgi complex.