Tracking sphingosine metabolism and transport in sphingolipidoses: NPC1 deficiency as a test case

The late endosomal/lysosomal compartment (LE/LY) plays a key role in sphingolipid breakdown, with the last degradative step catalyzed by acid ceramidase. The released sphingosine can be converted to ceramide in the ER and transported by ceramide transfer protein (CERT) to the Golgi for conversion to sphingomyelin. The mechanism by which sphingosine exits LE/LY is unknown but Niemann-Pick C1 protein (NPC1) has been suggested to be involved. Here, we used sphingomyelin, ceramide and sphingosine labeled with [(3)H] in carbon-3 of the sphingosine backbone and targeted them to LE/LY in low-density lipoprotein (LDL) particles. These probes traced LE/LY sphingolipid degradation and recycling as suggested by (1) accumulation of [(3)H]-sphingomyelin-derived [(3)H]-ceramide and depletion of [(3)H]-sphingosine upon acid ceramidase depletion, and (2) accumulation of [(3)H]-sphingosine-derived [(3)H]-ceramide and attenuation of [(3)H]-sphingomyelin synthesis upon CERT depletion. NPC1 silencing did not result in the accumulation of [(3)H]-sphingosine derived from [(3)H]-sphingomyelin/LDL or [(3)H]-ceramide/LDL. Additional evidence against NPC1 playing a significant role in LE/LY sphingosine export was obtained in experiments using the [(3)H]-sphingolipids or a fluorescent sphingosine derivative in NPC1 knock-out cells. Instead, NPC1-deficient cells displayed an increased affinity for sphingosine independently of protein-mediated lipid transport. This likely contributes to the increased sphingosine content of NPC1 cells.