Cholesterol-dependent increases in glucosylceramide synthase activity in Niemann-Pick disease type C model cells: Abnormal trafficking of endogenously formed ceramide metabolites by inhibition of the enzyme

- In NPC cells, GluCer synthase activity was increased depending on cholesterol.
- NPC cells showed an abnormal vesicular trafficking of ceramide metabolites.
- NB-DNJ treatment enhanced the abnormal trafficking with cholesterol increase.
- GlcCer (increase/decrease) and cholesterol regulate the trafficking in NPC cells.
- For taking NB-DNJ, cellular cholesterol/vesicular trafficking should be monitored.

Sphingolipids such as sphingomyelin and glycosphingolipids (GSLs) derived from glucosylceramide (GlcCer), in addition to cholesterol, accumulate in cells/neurons in Niemann-Pick disease type C (NPC). The activities of acid sphingomyelinase and lysosomal glucocerebrosidase (GCase), which degrade sphingomyelin and GlcCer, respectively, are down-regulated in NPC cells, however, changes in GlcCer synthase activity have not yet been elucidated. We herein demonstrated for the first time that GlcCer synthase activity for the fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide) increased in intact NPC1(−/−) cells and cell lysates without affecting the protein levels. In NBD-ceramide-labeled NPC1(−/−) cells, NBD-fluorescence preferentially accumulated in the Golgi complex and vesicular specks in the cytoplasm 40 and 150 min, respectively, after labeling, while a treatment for 48 h with the GlcCer synthase inhibitors, N-butyldeoxynojirimycin (NB-DNJ) and 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, accelerated the appearance of vesicular specks emitting NBD-fluorescence within 40 min. The treatment of NPC1(−/−) cells with NB-DNJ for 48 h additionally increased the levels of cholesterol, but not those of sphingomyelin. Increases in the activity of GlcCer synthase and formation of vesicular specks emitting NBD-fluorescence in NPC1(−/−) cells were dependent on cholesterol. LacCer taken up by endocytosis, which accumulated in the Golgi complex in normal cells, accumulated in vesicular specks after 10 and 40 min in NPC1(−/−) cells, and this response was not accelerated by the NB-DNJ treatment, but was restored by the depletion of cholesterol. The cellular roles for enhanced GlcCer synthesis and increased levels of cholesterol in the trafficking of NBD-ceramide metabolites in NPC1(−/−) cells have been discussed.