title: 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.
abstract: 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.
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Hashimoto
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
authors.forename: Ikiru
authors.initials: I
authors.lastname: Matsumoto
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Ashikawa
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan. Electronic address: nakahiro@faculty.chiba-u.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakamura
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Murayama
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology*; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism*; D003412:Cricetulus; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D005963:Glucosylceramides / Q000378:metabolism*; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics; D052556:Niemann-Pick Disease, Type C / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism
chemical_list: D005963:Glucosylceramides; D008562:Membrane Glycoproteins; D013109:Sphingomyelins; D002784:Cholesterol; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase
keywords: Cholesterol; Glucosylceramide synthase; Niemann-Pick disease type C; Sphingomyelin; Vesicular trafficking