Neuropharmacology 110 (2016) 458e469

Contents lists available at ScienceDirect

Neuropharmacology

journal homepage: www.elsevier.com/locate/neuropharm

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

Naohiro Hashimoto 1, Ikiru Matsumoto 1, Hiromasa Takahashi, Hitomi Ashikawa, * Hiroyuki Nakamura , Toshihiko Murayama

Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan article info abstract

Article history: such as and (GSLs) derived from glucosylceramide Received 1 February 2016 (GlcCer), in addition to cholesterol, accumulate in cells/neurons in Niemann-Pick disease type C (NPC). Received in revised form The activities of acid sphingomyelinase and lysosomal (GCase), which degrade 10 August 2016 sphingomyelin and GlcCer, respectively, are down-regulated in NPC cells, however, changes in GlcCer Accepted 12 August 2016 synthase activity have not yet been elucidated. We herein demonstrated for the first time that GlcCer Available online 15 August 2016 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. Keywords: (/) fl Niemann-Pick disease type C In NBD-ceramide-labeled NPC1 cells, NBD- uorescence preferentially accumulated in the Golgi Cholesterol complex and vesicular specks in the cytoplasm 40 and 150 min, respectively, after labeling, while a Glucosylceramide synthase treatment for 48 h with the GlcCer synthase inhibitors, N-butyldeoxynojirimycin (NB-DNJ) and 1-phenyl- Sphingomyelin 2-palmitoylamino-3-morpholino-1-propanol, accelerated the appearance of vesicular specks emitting Vesicular trafficking 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 depen- dent 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. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Abbreviations: NPC, Niemann-Pick disease type C; SM, sphingomyelin; GSLs, glycosphingolipids; GlcCer, glucosylceramide; LacCer, lactosylceramide; GCase, Most cases of Niemann-Pick disease type C (NPC) result from (/) glucocerebrosidase; NB-DNJ, N-butyldeoxynojirimycin; NPC1 , NPC1-null; NBD, mutations in the NPC1 encoding the NPC1 protein, which is 4-nitrobenzo-2-oxa-1,3-diazole; NBD-ceramide, 6-((N-(7-nitrobenz-2-oxa-1,3- involved in the transport and/or processing of unesterified diazol-4-yl)amino)hexanoyl); BODIPY-LacCer, boron dipyrromethene difluoride-labeled lactosylceramide; FBS, fetal bovine serum; LPDS, lipoprotein- cholesterol. In addition to cholesterol, sphingolipids including deficient serum; PPMP, 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol; sphingomyelin (SM) and glycosphingolipids (GSLs) such as gluco- HPbCD, 2-hydroxypropyl-b-cyclodextrin; CHO, Chinese hamster ovary; NBD- sylceramide (GlcCer), lactosylceramide (LacCer), and fl specks, specks emitting NBD- uorescence; NB-DGJ, N- accumulate in the late endosomes/lysosomes of cells in the livers butyldeoxygalactonojirimycin. * Corresponding author. Laboratory of Chemical Pharmacology, Graduate School and spleens of NPC patients and animal models (Vanier, 1983, 2010; of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba Mukherjee and Maxfield, 2004; Vance and Karten, 2014). GlcCer 260-8675, Japan. Tel.: þ81 43 226 2876; fax: þ81 43 226 2875. levels are known to be markedly higher than those of LacCer and E-mail address: [email protected] (H. Nakamura). gangliosides in the liver and spleen, and, among GSLs, GlcCer is 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neuropharm.2016.08.011 0028-3908/© 2016 Elsevier Ltd. All rights reserved. N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469 459 stored at high levels in these tissues in NPC patients, 10e40-fold bovine serum (FBS) from Thermo Trace Ltd. (Nobel Park, Australia); more than those in controls (Vanier, 2010, 2015). Cellular levels of lipoprotein-deficient serum (LPDS) from fetal calf, cholesterol and GlcCer are directly controlled by the activities of GlcCer synthase conduritol B-epoxide were from Sigma-Aldrich (St. Louis, MO); NB- and glucocerebrosidases (GCases, b-glucosidases). Complex GSLs DNJ hydrochloride was from Enzo Life Sciences (Farmingdale, NY); derived from GlcCer are the main storage lipids in the brains of NPC U18666A from Cayman Chemical (Ann Arbor, MI); 1-phenyl-2- patients and animal models, whereas the accumulation of choles- palmitoylamino-3-morpholino-1-propanol (PPMP, Biomol, Ply- terol and SM is limited (Elleder, 1989; Vanier, 1999, 2010; Zervas mouth Meeting, PA). NB-DNJ hydrochoride, U18666A, and PPMP et al., 2001a; Taniguchi et al., 2001; Stein et al., 2012). GSLs are were used at similar concentrations as those in previous reports essential components of the pre- and post-synaptic machineries including ours (Platt et al., 1994; Delgado et al., 2006; Tada et al., regulated by neurons and glial cells in the brain. A treatment with 2010; Nakamura et al., 2012). NBD-ceramide and PPMP were dis- ® N-butyldeoxynojirimycin (NB-DNJ, miglustat, BRAZAVES , an in- solved in dimethyl sulfoxide, and the reagents were diluted in hibitor of GlcCer synthase) has been shown to decrease the accu- medium prior to experiments. The final concentration of dimethyl mulation of gangliosides in the brain, such as in the cerebral cortex sulfoxide in culture dishes was less than 0.5%, and the vehicle had and cerebellum, delay the onset of neurological dysfunction, and no effect on the NBD-ceramide or trafficking of NBD- increase the average life span of NPC patients and animal models fluorescence derived from metabolites. For preparing a complex (Zervas et al., 2001b; Patterson et al., 2010; Stein et al., 2012). Thus, of cholesterol and 2-hydroxypropyl-b-cyclodextrin (HPbCD), changes in GSL levels appear to be involved in neuronal dysfunction cholesterol dissolved in chloroform:methanol (2:1) was dried un- in NPC; however, the exact roles of GSLs in the neuropathology of der nitrogen. An appropriate volume of 100 mM HPbCD was added NPC currently remain unknown (Lee et al., 2014; Marques et al., to the dried film at a molar ratio of 10:1 (HPbCD:cholesterol) and 2015). The accumulation of complex GSLs in NPC cells has been, vortexed to suspend the film. Suspensions were bath sonicated for at least partially, attributed to decreases in the activity of lysosomal 20 min, then incubated at 37 C with shaking. Finally, suspensions GCase (Besley and Moss, 1983; Salvioli et al., 2004). In contrast, a were filtered through 0.2 mm filters to clarify solutions. recent study reported that the activity and expression of a non- lysosomal GCase were increased in the brains of NPC1 / mice 2.2. Cell cultures (NPC1-null mice, Marques et al., 2015); therefore, the roles of GCase in the accumulation of GSLs including GlcCer in NPC cells have yet A control Chinese hamster ovary (CHO) cell line (CHOeK1 cells to be determined. To the best of our knowledge, changes in GlcCer named JP17 cells) and cells lacking NPC1 (NPC1( / ) cells named synthase activity have not been examined in NPC cells. A101 cells) were established and kindly provided by Professor H. The NPC1 protein is essential for the transport of cholesterol Ninomiya (Tottori University). These two cell lines have been from late endosomes/lysosomes to the endoplasmic reticulum. In shown to be a useful tool to study the regulation of cellular NPC1( / ) cells cultured with normal serum, cholesterol accumu- cholesterol homeostasis and the pathogenesis of NPC (Higaki et al., lated in various endosomes including late and recycling endosomes 2001; Sugimoto et al., 2001). Cells were cultured in Ham's F12 (Salvioli et al., 2004; Wojtanik and Liscum, 2003; Pipalia et al., medium containing 10% FBS as described previously (Nakamura 2007; Sztolsztener et al., 2012). Furthermore, NPC1 and choles- et al., 2012). In order to measure NBD-ceramide metabolites in terol have been reported to regulate vesicular trafficking for the cells, control and NPC1( / ) cells in 6-well plate at approximately sorting of cargo including GSLs bi-directionally between late 50e60% confluence were further cultured in Ham's F12 medium endosomes/lysosomes, and the recycling of cargo to various cellular with 10% FBS or 10% LPDS for 1 or 2 days. In some experiments, organelles including the plasma membrane via many types of 2 mM U18666A was further supplemented in the medium. endosomes (Puri et al., 1999; Choudhury et al., 2002; Cheng et al., Regarding fluorescence microscopy in living cells, cells were seeded 2006). However, the effects of NPC1 and cholesterol on the trans- on the cover slips (12 mm in diameter) of glass-bottomed dishes port of sphingolipids such as SM and GlcCer endogenously formed (Iwaki, Tokyo, Japan), and images of fluorescence derived from in the Golgi complex have not been elucidated in detail. In addition, NBD-ceramide and BODIPY-LacCer were examined when cells the effects of the inhibition of GlcCer synthase on the trafficking of achieved 60% confluence. sphingolipids formed in the Golgi complex in NPC1( / ) cells remain unknown. In the present study, we focused on changes in 2.3. Measurement of NBD-ceramide metabolites in cells GlcCer synthase activity in NPC1( / ) cells and the trafficking of 4- nitrobenzo-2-oxa-1,3-diazole (NBD)-fluorescence derived from the Cells were labeled with 10 mM NBD-ceramide at 37 C for metabolites of NBD-labeled C6-ceramide (NBD-ceramide) in 30 min. Labeled cells were washed with NBD-ceramide-free Hank's NPC1( / ) cells treated with and without GlcCer synthase in- solution, and further incubated at 37 C for 10 min or 120 min with hibitors. We demonstrated for the first time that the formation of Ham's F12 medium. Ceramide metabolites were extracted by the NBD-GlcCer was enhanced in cells and cell homogenate without addition of chloroform and methanol, and were analyzed on a TLC changing the protein levels of GlcCer synthase in NPC1( / ) cells, silica gel-60 plate (#105724, Merk, Darmstadt, Germany) using 1- suggesting an upregulation of synthase activity. The possible butanol:acetic acid:water (3:1:1) as the mobile phase, as cellular roles for enhanced GlcCer synthesis and the effects of described previously (Tada et al., 2010; Makiyama et al., 2015) with GlcCer synthase inhibitors on the trafficking of ceramide metabo- minor modifications. For a quantitative analysis, various amounts of lites in NPC1( / ) cells have been discussed. standard NBD-ceramide (0.1e4 pmol) were spotted in the upper area of the plate after separation by TLC. The fluorescence intensity 2. Materials and methods was linear to 10 pmol NBD-ceramide, as previously reported (Tada et al., 2010). We previously confirmed that more than 80% of NBD- 2.1. Materials fluorescence was native NBD-ceramide and approximately 5e10% of NBD-fluorescence was derived from NBD-SM and NBD-GlcCer, The materials used in this study and their sources were as fol- respectively, 30e40 min after NBD-ceramide labeling in various lows: NBD-ceramide from Molecular Probes (Eugene, OR, USA); cells including CHO cells (Tada et al., 2010; Makiyama et al., 2015). boron dipyrromethene difluoride-labeled LacCer (BODIPY-LacCer) The levels of NBD-labeled molecules in cells 150 min after labeling and BODIPY-TR-ceramide were from Invitrogen (Carlsbad, CA); fetal were as follows; 40e55% in NBD-GlcCer, 20e25% in NBD-SM, 460 N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469

3e17% in NBD-caproic acid, and 15e30% in native NBD-ceramide, 2.7. Assays for cholesterol and SM levels depending on the experiments. In a typical experiment, the abso- lute levels of NBD-labeled molecules (pmol/well) in control cells Cells were seeded onto glass-bottomed dishes at a density of and NPC1( / ) cells were as follows; 226 and 280 in NBD-GlcCer, 1.5 104 cells/well for filipin staining. Cells were fixed in 3.7% 96.4 and 112 in NBD-SM, 14.8 and 19.8 in NBD-caproic acid, 135 paraformaldehyde and permeabilized by an incubation with 0.05% and 109 in NBD-ceramide, and 472 and 520 in sum total NBD- Nonidet P-40 in PBS at room temperature for 10 min as described ceramide metabolites, respectively. Although NBD-ceramide is (Nakamura et al., 2012). Cells were then incubated with 100 mg/mL converted to NBD-ceramide-1-phosphate by ceramide kinase, its filipin at room temperature for 30 min, and fluorescence images level was quite low at less than 1%, as reported previously (Tada were taken with a fluorescence microscope. Increases in and/or the et al., 2010; Makiyama et al., 2015). NBD-GlcCer levels were accumulation of filipin staining in vesicular specks have been greater in NPC1( / ) cells than in control cells, and the formation of correlated with cholesterol levels in CHO cells (Nakamura et al., LacCer and gangliosides emitting NBD-fluorescence was not 2012) and other cells (Sillence et al., 2002; te Vruchte et al., detected within 150 min of labeling, as previously reported (Lipsky 2004). Cells were seeded on 12-well plates at a density of and Pagano, 1983; Tada et al., 2010). 2 104 cells/well, and cultured for 2 days for the SM assay. Cells at 70e80% confluence were rinsed three times with PBS, and lipids 2.4. GlcCer synthase activity in vitro containing SM were extracted by the Bligh and Dyer method as previously reported (Nakamura et al., 2015). SM levels were Cells at 80% confluence were scraped and homogenized with determined by the Bartlett phosphorus method, and the lower limit ice-cold lysis buffer (10 mM HEPES, 2 mM EGTA, 40 mM KCl, and of detection was 0.1 pmol with linear detector response extended to protease inhibitors (10 mg/mL leupeptin, 10 mg/mL aprotinin, and approximately 10 pmol in this method (Alvarez and Ludmir, 1993). 100 mM phenylmethylsulfonyl fluoride), pH 7.4). After centrifuga- Briefly, dried samples were dissolved in 10 mL of chlor- tion (1000 g, 10 min), equal amounts of proteins (100e150 mg/ oform:methanol (1:1) and analyzed on TLC silica gel-60 plates us- tube,100 mL) in the soluble fractions of lysates were used as enzyme ing chloroform:methanol:water (65:25:4). The plates were heated preparations. In the assay, 100 mL of the reaction mixture containing at 150 C on a hot plate after spraying with 47% sulfuric acid and the 10 mM NBD-ceramide, 40 mM HEPES (pH 7.4), 1 mM dithiothreitol, intensity of staining was measured using LAS1000 (Fuji-Film). 0.7 mM EDTA, 350 mM UDP-glucose, 10% glycerol, 2.5 mM con- duritol B-epoxide, and 100 mL of the enzyme preparations was 2.8. Statistics mixed and incubated at 37 C for 30 min. Lipids including NBD- GlcCer were extracted and analyzed on TLC silica gel-60 plates. Values are the means ± S.D. of three to four independent ex- Under our assay conditions, the formation of NBD-GlcCer was linear periments performed in duplicate or triplicate. In the case of for 30 min depending on the amounts of proteins from 100 to multiple comparisons, the significance of differences was deter- 200 mg per assay tube, and more than 95% of NBD-ceramide added mined using a one-way analysis of variance by Dunnett's or Tukey's existed as a native NBD-ceramide after 30-min incubation. test. The Student's two-tailed t-test was used for pairwise com- parisons. P values < 0.05 were considered to be significant. 2.5. Western blotting 3. Results Cells were scraped and homogenized with ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris/HCl, 5 mM EDTA, 10 mM NaF, 10 mM 3.1. Increased formation of NBD-GlcCer in NPC1( / ) cells Na2H2P2O7, 1% Nonidet P-40, 0.1% SDS, 0.5% Na-deoxycholate, and protease inhibitors, pH 8.0). After centrifugation (1000 g, 10 min), Control CHO cells and cells lacking NPC1, NPC1( / ) cells, were equal amounts of proteins (50e70 mg/lane) in the soluble fractions used in the present study. NBD-ceramide has been shown to of lysates were separated by SDS-PAGE and electrotransferred onto directly reach the Golgi complex within 30 min at room temper- PVDF membranes (Bio-Rad, Hercules, CA). GlcCer synthase was ature (Pagano et al., 1989; Fukasawa et al., 1999), and be converted detected using an anti-GlcCer synthase antibody (ab124296, into four metabolites: NBD-GlcCer, NBD-SM, NBD-ceramide-1- 1:1000, Abcam, Tokyo) followed by an anti-rabbit horseradish phosphate, and NBD-caproic acid (C6-fatty acid and a counter- peroxidase antibody (Amersham, Buckinghamshire, UK). Immu- part of sphingosine) in various cells including CHO cells (Graf et al., noreactive bands were visualized by enhanced chemiluminescence. 2008; Tada et al., 2010; Makiyama et al., 2015). Control cells and Results were analyzed using a ChemiDoc-XRS system equipped NPC1( / ) cells cultured in medium with 10% FBS for 48 h were with Image Lab software (BIO-RAD), and the intensity of chem- labeled with 10 mM NBD-ceramide for 30 min, and washed cells iluminescence was measured using NIH ImageJ software. were incubated further at 37 C for 2 h. NBD-GlcCer levels were significantly higher in NPC1( / ) cells than in control cells (Fig. 1A 2.6. Fluorescence microscopy in living cells and Exp. I in Table 1). The amounts of NBD-ceramide, NBD-SM, and NBD-caproic acid after 2-hr incubation were similar in control and Cells were labeled by cultivation with Ham's F12 medium con- NPC1( / ) cells (Table 1). The formation of NBD-GlcCer in control taining 0.1% fatty acid free-albumin, 1 mM NBD-ceramide or 50 nM cells and its enhancement in NPC1( / ) cells were detected in cells BODIPY-LacCer, and 10 mM HEPES (pH 7.4) at 37 C for 30 min. immediately after labeling for 30 min: NBD-GlcCer levels in Labeled cells were washed with Hank's solution, and further NPC1( / ) cells were 1.83 ± 0.17-fold those of the control (P < 0.05, incubated at 37 C for 10 min (a total of 40 min after labeling) or n ¼ 3). NBD-GlcCer levels 6 h after labeling were approximately 2- 120 min (a total of 150 min) with Ham's F12 medium. Images of fold higher in the NPC1( / ) cells than in control cells in a typical NBD- and BODIPY-fluorescence in living cells were obtained using a experiment. The accumulation of cholesterol in cellular organelles laser-scanning confocal microscope (Olympus, Tokyo). The cellular was monitored by filipin staining. We previously reported that the localization of fluorescence 40 min and 150 min after labeling was cultivation of NPC1( / ) cellsfor48hinmediumcontaining10% analyzed at a normal range (PMT voltage, 450e500) and enhanced FBS led to higher cholesterol levels and more intense filipin intensity (650e700), respectively. Data were from a representative staining than those in control cells, whereas no significant differ- experiment repeated more than three times with similar results. ences were observed between NPC1( / ) cells cultured with LPDS N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469 461

¡ ¡ Fig. 1. Increased formation of NBD-GlcCer in NPC1( / ) cells. In A, C and D, control (WT) or NPC1( / ) (NPC1*) cells were cultured with 10% FBS for 24 h. In B, cells were cultured with 10% FBS or 10% LPDS for 48 h. In A, culture medium was further supplemented with vehicle or 2 mM U18666A. In C, WT cells were treated with the complex of HPbCD and cholesterol in the indicated concentrations for 1 h. Cells were then labeled with 10 mM NBD-ceramide for 30 min, and washed cells were incubated further for 2 h. In D, inhibitors of GlcCer synthase (200 mM NB-DNJ and 5 mM PPMP) and vehicle were added during the labeling and incubation periods. Cellular lipids were extracted and separated by TLC. In A ~ D, typical data are shown in the upper panels and quantitative data are shown as bar charts in the lower panels. NBD-GlcCer levels were expressed as fold changes from those in control cells cultured with FBS. Data are means ± S.D. of 3 independent experiments. *P < 0.05, significantly different from the respective compared groups.

(cholesterol-reduced serum) and control cells (Nakamura et al., (/) Table 1 2012). The cultivation of NPC1 cells for 48 h with 10% LPDS Enhanced formation of NBD-GlcCer in NPC1( / ) cells and U18666A-treated control significantly reduced the formation of NBD-GlcCer to the same cells. levels as those in control cells (Fig. 1B). U18666A, a cholesterol GlcCer SM Caproic acid Ceramide transport-inhibiting reagent, has been shown to induce an NPC phenotype in cells by inducing the accumulation of cholesterol NBD-labeled lipids (fold change from the control) (Lange et al., 2000; te Vruchte et al., 2004). The treatment of Experiment I control cells with 2 mM U18666A for 24 h markedly increased fil- Control cells 1 1 1 1 With U18666A 1.24 ± 0.05a 0.88 ± 0.14 0.67 ± 0.05 1.10 ± 0.09 ipin staining in vesicular specks in cells (data not shown), as NPC1( / ) cells 1.57 ± 0.23a 0.90 ± 0.15 0.95 ± 0.11 1.14 ± 0.07 shown in L929 fibroblast cells (Nakamura et al., 2012). The With U18666A 1.77 ± 0.14 0.72 ± 0.15 0.74 ± 0.15 1.21 ± 0.18 response induced by 1 mM U18666A was variable, and treatment with 5 mM U18666A caused cell detachment approximately 20% in Experiment II both cell types, thus we used the reagent at 2 mM. The treatment of Control cells 1 1 1 1 (/) With LPDS 1.05 ± 0.11 0.94 ± 0.16 0.87 ± 0.21 1.03 ± 0.02 control cells, but not NPC1 cells, with U18666A enhanced the NPC1( / ) cells 1.37 ± 0.05a 0.91 ± 0.16 0.82 ± 0.11 1.31 ± 0.13 formation of NBD-GlcCer (Fig. 1A). The treatment with U18666A With LPDS 1.11 ± 0.06b 0.86 ± 0.15 0.80 ± 0.07 1.17 ± 0.21 slightly decreased the formations of NBD-SM and NBD-caproic In Experiment I, control and NPC1( / ) cells were cultured with 10% FBS for 24 h. The acid and increased the levels of remaining NBD-ceramide in both culture medium was further supplemented with vehicle or 2 mM U18666A. In cells types (Exp. I in Table 1). Next, we used the complex of HPbCD Experiment II, cells were cultured with 10% FBS or 10% LPDS for 48 h. Cells were then and cholesterol to increase the cellular cholesterol levels. Treat- m labeled with 10 M NBD-ceramide for 30 min, and washed cells were incubated ment with the HPbCD/cholesterol complex significantly enhanced further for 120 min. Lipids in cells and the medium were extracted and analyzed by TLC. Data were expressed as fold changes from the respective values in control cells, the formation of NBD-GlcCer (Fig. 1C). We next examined the ef- and are means ± S.D. of three independent experiments. fects of NB-DNJ and PPMP, GlcCer synthase inhibitors, on NBD- 462 N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469 ceramide metabolism. NB-DNJ at 200 mM has been shown to inhibit approximately 90% of GlcCer synthase activity in vitro (Platt et al., 1994; Delgado et al., 2006). A co-treatment with 200 mMNB- DNJ for NBD-ceramide labeling lead to a more than 90% reduction in the formation of NBD-GlcCer in both cell types (Fig. 1D), and the reduction induced by 100 mM NB-DNJ was variable from 30% to 60% depending on experiments. Previously, we reported that 10 mM PPMP completely inhibited the formation of NBD-GlcCer in CHO cells (Tada et al., 2010). In cells cultured with 10 mMPPMPfor 24 and 48 h, greater than 30e50% of cells were detached from wells in both cell types, and we could not analyze. Thus, we used 5 mM PPMP in this study, and the reagent effectively reduced the formation of NBD-GlcCer in both cell types (Fig. 1D) without showing cell detachment after 48-hr treatment. These results suggest that the formation of NBD-GlcCer is increased in NPC1( / ) cells via NB-DNJ/PPMP-sensitive GlcCer synthase in a cholesterol- dependent manner. NBD-ceramide metabolites, except for NBD- GlcCer, showed similar values in both cell types treated with and without LPDS (Exp. II in Table 1). In the present study, we focused on the formation of GlcCer in NPC1( / ) cells.

3.2. Increased activity of GlcCer synthase without changes to its protein levels in NPC1( / ) cells

We investigated GlcCer synthase activity in vitro using crude cytoplasm preparations from NPC1( / ) cells. In the presence of conduritol B-epoxide, a lysosomal GCase inhibitor (Hayashi et al., 2007), the formation of NBD-GlcCer in NPC1( / ) cells was 2-fold that of control cells (Fig. 2A). In the case of NPC1( / ) cells cultured with LPDS for 48 h, the activity of GlcCer synthase was the same as that of control cells cultured with FBS or LPDS. The pre- treatment of lysates from both cell types for 20 min with 200 mM NB-DNJ or 5 mM PPMP markedly decreased GlcCer synthase activity in vitro (data not shown). The levels of GlcCer synthase protein were not changed in NPC1( / ) cells (Fig. 2B).

3.3. Cholesterol-dependent formation of vesicular specks emitting NBD-fluorescence (NBD-specks) in NPC1( / ) cells

Ceramide metabolism, specifically the formation of GlcCer (and resulting GSLs) and SM, mainly occurs in the Golgi complex. fi Therefore, changes in the intracellular traf cking of NBD-ceramide in vitro (¡/¡) Fig. 2. GlcCer synthase activity and its protein level in NPC1 cells.InA, ( / ) metabolites were investigated in control and NPC1 cells. Cells control (WT) and NPC1( / ) (NPC1*) cells were cultured with 10% FBS or LPDS for 48 h. were labeled with 10 mM NBD-ceramide at 37 C for 30 min, and, The cytoplasm preparations from both cells were incubated with NBD-ceramide for after washing, these cells were incubated further at 37 C for 10 min 30 min in the presence of 1.25 mM conduritol B-epoxide, and the lipids were extracted. (/) or 120 min. The cellular distribution of NBD-fluorescence was then In B, control and NPC1 cells were cultured with 10% FBS for 48 h. The levels of (/) GlcCer synthase (UDP-glucose:ceramide glucosyltransferase, UGCG) were then observed. In control and NPC1 cells 40 min after NBD-ceramide measured by immunoblotting. In A and B, typical data are shown in the upper panels labeling, NBD-fluorescence was mainly localized in the perinuclear and quantitative data are shown as bar charts in the lower panels. NBD-GlcCer levels region and/or at one pole of the nucleus (Fig. 3A), which is char- were expressed as fold changes from those in control cells with FBS. The ratio of GlcCer acteristic of the Golgi complex. The localization of NBD- synthase to GAPDH was calculated, and data were normalized to the control. Data are means ± S.D. of 3e5 independent experiments. *P < 0.05, significantly different from fluorescence in the perinuclear region was consistent with that of the control. other Golgi markers including BODIPY-TR-ceramide in both cell types (data not shown), as reported previously (Makiyama et al., 2015). In control and NPC1( / ) cells, the intensity of NBD- then investigated the effects of the accumulation of cholesterol on fluorescence was markedly decreased 150 min after labeling; the formation of vesicular NBD-specks in NPC1( / ) cells. The thus, images of the cellular distribution of fluorescence were accumulation of cholesterol monitored by filipin staining was analyzed at an enhanced intensity. At 150 min, NBD-fluorescence observed in NPC1( / ) cells cultured for 48 h with 10% FBS, but not mainly accumulated in the Golgi complex in control cells, while a in cells cultured with 10% LPDS (Fig. 3B, upper panels). In NBD- large number of small vesicular specks emitting NBD-fluorescence ceramide-labeled NPC1( / ) cells cultured with LPDS, NBD- (NBD-specks) were observed in the cytoplasm of NPC1( / ) cells. fluorescence preferentially accumulated in the Golgi complex The percentages of cells with small vesicular NBD-specks were 40 min (data not shown) and 150 min after labeling (Fig. 3B, lower 0e10% and 90e100% in control and NPC1( / ) cells, respectively, at panels): the percentage of cells having vesicular NBD-specks was 150 min. Similarly, the percentages of cells showing the accumu- lower than 10% at 150 min. Thus, the formation of vesicular NBD- lation of NBD-fluorescence in the Golgi complex were more than specks in NPC1( / ) cells appeared to be dependent on choles- 90% and 15e30% in control and NPC1( / ) cells, respectively. We terol. The accumulation of NBD-fluorescence in the Golgi complex N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469 463

after 40 min in control and NPC1( / ) cells was not affected by the endocytic process of BODIPY-LacCer in control and NPC1( / ) cells. LPDS treatment (data not shown). Cells were labeled with 50 nM BODIPY-LacCer at 37 C for 30 min, and, after washing, the labeled cells were incubated further at 37 C 3.4. Acceleration of formation of vesicular NBD-specks in NPC1( / ) for 10 min. The fluorescence of BODIPY-LacCer accumulated in the cells cultured with NB-DNJ/PPMP Golgi complex in control cells, but not in NPC1( / ) cells (Fig. 5A), as previously reported (te Vruchte et al., 2004). In NPC1( / ) cells, In the present study, we found that GlcCer synthase activity BODIPY-LacCer accumulated in small vesicular specks in the cyto- increased in NPC1( / ) cells, as shown in Figs. 1 and 2. We then plasm at 40 min, while the NB-DNJ/PPMP treatment for 48 h had no investigated the effects of the GlcCer synthase inhibitors, NB-DNJ effect on this response. The accumulation of BODIPY-LacCer in ve- and PPMP, on the intracellular trafficking of NBD-ceramide me- sicular specks was detected in NPC1( / ) cells 10 min after labeling, tabolites and cholesterol accumulation in NPC1( / ) cells. Treat- and the NB-DNJ/PPMP treatment did not change this response. In ment with 200 mM NB-DNJ and 5 mM PPMP did not cause cell control cells, BODIPY-LacCer accumulated in the Golgi complex detachment within 48 h in both cell types, as described above. The with and without the NB-DNJ/PPMP treatment. The treatment of treatment of NPC1( / ) cells with 200 mM NB-DNJ for 48 h markedly another NPC1-null CHO type for 5 days with N-butyl deoxy- decreased the formation of NBD-GlcCer 2 h after NBD-ceramide galactonojirimycin (NB-DGJ), a GlcCer synthase inhibitor, restored labeling to approximately 40e45% that of cells not treated with the preferential accumulation of BODIPY-LacCer in the Golgi com- NB-DNJ; the absolute levels of NBD-GlcCer in a typical experiment plex (te Vruchte et al., 2004), and the treatment of control RAW were 87.6 (pmol/well) and 211 in NPC1( / ) cells with and without macrophages with NB-DGJ for 24 h led to the accumulation of NB-DNJ. The treatment of NPC1( / ) cells with NB-DNJ slightly BODIPY-LacCer in vesicular specks and/or late endosomes (Sillence increased the levels of NBD-SM and NBD-ceramide and decreased et al., 2002). Differences in the inhibitors used, treatment periods, that of NBD-caproic acid, although the treatment did not change and cell types may explain the discrepancy between these findings sum total of NBD-ceramide metabolites (data not shown). Thus, the and our results. In NPC1( / ) cells cultured with LPDS, BODIPY- NB-DNJ treatment appeared to effectively inhibit GlcCer synthase LacCer mainly accumulated in the Golgi complex, and the treat- activity in NPC1( / ) cells 48 h after its addition; however, the ment with 200 mM NB-DNJ for 48 h did not change this response extent of this inhibition was less than that observed with the short- (Fig. 5B). The treatment of control and NPC1( / ) cells with NB-DNJ term NB-DNJ treatment (Fig. 1D). In NBD-ceramide-labeled NPC1( / did not change the uptake of BODIPY-LacCer under our conditions, ) cells cultured with NB-DNJ/PPMP for 48 h, a large number of whereas the treatment of CHOeK1 cells with GlcCer synthase in- vesicular NBD-specks were observed in the cytoplasm 40 min after hibitors reduced its uptake (Cheng et al., 2006). labeling (Fig. 3C, upper panels); the number of NPC1( / ) cells with vesicular NBD-specks was more than 70% and less than 10% with 4. Discussion and without NB-DNJ, respectively. Many vesicular NBD-specks were observed in NPC1( / ) cells treated with and without NB- 4.1. Increased activity of GlcCer synthase in NPC1( / ) cells DNJ at 150 min (lower panels). NBD-SM and remaining NBD- ceramide levels were greater in NB-DNJ-treated NPC1( / ) cells In the present study, we showed for the first time that the ac- than in cells not treated with NB-DNJ, while the sum total of NBD- tivity of GlcCer synthase was increased in NPC1( / ) cells in a ceramide metabolites were not modified by NB-DNJ treatment cholesterol-dependent manner. Due to the presence of conduritol (data not shown). The treatment of control cells with NB-DNJ for B-epoxide-resistant GCases (Hayashi et al., 2007; Ridley et al., 2013) 48 h did not change the preferential accumulation of NBD- and increases in the activity of non-lysosomal GCase in the brains of fluorescence in the Golgi complex 40 min and 150 min after la- NPC1( / ) mice (Marques et al., 2015), the net increase observed in beling (Fig. 3D). In NPC1( / ) cells pretreated with NB-DNJ for GlcCer synthase activity in NPC1( / ) cells appears to have been 30 min, in which the formation of NBD-GlcCer was effectively greater than 2-fold that of the control. The expression of the tran- decreased (Fig. 1D), NBD-fluorescence accumulated in the Golgi scription factor Sp1, which increases expression of GlcCer synthase complex 40 min after labeling (Fig. 3E). mRNA (Uchida et al., 2004), was increased in the livers of mice In NPC1( / ) cells cultured for 48 h with NB-DNJ in the presence exhibiting cholesterol accumulation (Wu et al., 2013). However, of LPDS, the formation of vesicular NBD-specks was not observed GlcCer synthase protein levels in NPC1( / ) cells were the same as 40 min and 150 min after labeling (Fig. 3F): the preferential local- those in control cells (Fig. 2B). A post-transcriptional regulation of ization of NBD-fluorescence was observed in the Golgi complex of GlcCer synthase was reported (Boldin and Futerman, 2000) and c- NPC1( / ) cells with and without the NB-DNJ treatment. Similarly, fos protein activated the enzyme in vitro (Crespo et al., 2008), the PPMP treatment-induced formation of vesicular NBD-specks at whereas the expression of the c-fos protein in the brain was 40 min was markedly decreased in NPC1( / ) cells cultured with markedly lower in NPC1( / ) mice than in control mice (Byun et al., LPDS (data not shown). We then investigated the effects of the NB- 2013). Although AMP-activated protein kinase suppressed synthe- DNJ treatment on the endogenous levels of cholesterol and SM in sis of GlcCer by phosphorylation and activation of the UDP-glucose NPC1( / ) cells. Treatment with NB-DNJ for 48 h further enhanced pyrophosphatase Nudt14, direct modification of GlcCer synthase the accumulation of cholesterol, whereas cholesterol did not protein including phosphorylation has not been reported (Ishibashi accumulate in NB-DNJ-treated NPC1( / ) cells cultured with LPDS and Hirabayashi, 2015). In our studies, cholesterol determined the (Fig. 4A). SM levels in NPC1( / ) cells, which were approximately GlcCer synthase activity in NPC1( / ) cells (Figs. 1B and 2A). GCase 1.5-fold those in control cells, were not changed by the NB-DNJ protein levels were decreased in NPC fibroblasts because of the treatment (Fig. 4B). These results showed that the formation of enhanced processing of the enzyme through the cholesterol- vesicular NBD-specks in NPC1( / ) cells treated with NB-DNJ were induced physical dissociation of GCase and its activating/stabiliz- dependent on LDL-derived cholesterol. ing factors (Salvioli et al., 2004). The activity of acid sphingomye- linase was decreased without changes in enzyme protein levels in 3.5. Endocytosis of BODIPY-LacCer in NPC1( / ) cells cultured with cells including NPC1-defective CHO cells (Reagan et al., 2000; NB-DNJ/PPMP and/or LPDS Devlin et al., 2010), and regulated by the surrounding lipid condi- tions including cholesterol (Qiu et al., 2003; Oninla et al., 2014). We subsequently examined the effects of NB-DNJ/PPMP on the Thus, cholesterol may regulate GlcCer synthase activity physically 464 N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469

¡ ¡ Fig. 3. Localization of NBD-fluorescence in NBD-ceramide-labeled NPC1( / ) cells treated with GlcCer synthase inhibitors for 48 h. In A, control (WT) and NPC1( / ) (NPC*) cells were labeled with 10 mM NBD-ceramide for 30 min, and washed cells were incubated further for 10 min and 120 min. Images of NBD-fluorescence were analyzed at the same intensity (a, b, d, and e; PMT voltage, 500) 40 min (a and d) and 150 min (b and e) after NBD-ceramide labeling. In c and f, images (b and e) were analyzed at an enhanced intensity (PMT voltage, 700). In B, NPC1( / ) cells were cultured with 10% FBS or LPDS for 48 h. The accumulation of cholesterol was examined by filipin staining (upper panels) and images of N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469 465

¡ ¡ Fig. 4. Effects of the NB-DNJ treatment for 48 h on cholesterol accumulation and SM levels in NPC1( / ) cells. NPC1( / ) cells were cultured with 10% FBS or 10% LPDS in the presence and absence of 200 mM NB-DNJ for 48 h. In A, the accumulation of cholesterol was analyzed by filipin staining. Typical images and quantitative data were shown in the upper and lower panels, respectively. The total intensity of filipin fluorescence in 120 cells per experiment (40 cells in a field and three different fields in the same experiment) was counted. Quantitative data were expressed as fold changes from those in NPC( / ) cells without NB-DNJ and were from four independent experiments. In B, SM levels were expressed as fold changes from those without NB-DNJ, and data are the means ± S.D. of four experiments.

or by the modification of molecules interacting with the enzyme in to extracellular spaces within 150 min (Lipsky and Pagano, 1983; NPC1( / ) cells. van Helvoort et al., 1997; Tettamanti, 2004; Halter et al., 2007). GlcCer and SM are mainly transported by vesicular transport to various organelles (Tettamanti, 2004; Halter et al., 2007). NBD- fi 4.2. Abnormal traf cking of NBD-ceramide metabolites fluorescence accumulated in the Golgi complex of control cells (/) endogenously formed in the Golgi complex in NPC1 cells and its labeled with NBD-ceramide 150 min after labeling (Fig. 3A); acceleration by the NB-DNJ/PPMP treatment therefore, the NBD-labeled metabolites that formed in the Golgi complex appeared to be transported again to the complex by NBD-labeled metabolites derived from NBD-ceramide appeared recycling and/or endocytic pathways. These results were consistent to diffuse into the cytoplasm, other organelles, and/or be exported

NBD-fluorescence were analyzed at 150 min (lower panels). In C and D, NPC1( / ) (NPC*, C) and control (WT, D) cells were cultured with 10% FBS in the presence and absence of 200 mM NB-DNJ or 5 mM PPMP for 48 h. Images of NBD-fluorescence were analyzed 40 min (upper panels) and 150 min (lower panels). In E, NPC1( / ) cells cultured with 10% FBS for 48 h were further incubated with 200 mM NB-DNJ for 30 min. Images of NBD-fluorescence were analyzed 40 min after labeling. In F, NPC1( / ) cells were cultured with 10% FBS or LPDS for 48 h in the presence and absence of 200 mM NB-DNJ. Images of NBD-fluorescence were analyzed 40 min (upper panels) and 150 min (lower panels) after labeling. In A-F, images are from a typical experiment and similar results were obtained three times. 466 N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469

¡ ¡ Fig. 5. Endocytosis of BODIPY-LacCer in NPC1( / ) cells treated with NB-DNJ/PPMP. In A, control (WT) and NPC1( / ) (NPC1*) cells were cultured with 10% FBS in the presence and absence of 200 mM NB-DNJ or 5 mM PPMP for 48 h. Cells were labeled with 50 nM BODIPY-LacCer for 30 min, and washed cells were incubated further for 10 min. Images of BODIPY-fluorescence were then analyzed. In B, NPC1( / ) cells were cultured with 10% FBS or LPDS for 48 h in the presence and absence of NB-DNJ. Images of BODIPY-fluorescence were analyzed 40 min after labeling. In A and B, images are from a typical experiment and similar results were obtained three times. with previous work reporting that exogenous NBD-SM and NBD- Rab protein family (Zhang et al., 2001; Ganley and Pfeffer, 2006). GlcCer accumulated in the Golgi complex in various cells (Puri Thus, the excess accumulation of cholesterol appears to be involved et al., 1999, 2001; Babia et al., 2001; Cheng et al., 2006). In in the accelerated appearance of vesicular NBD-specks in NPC1( / ) NPC1( / ) cells that showed a greater activity of GlcCer synthase, cells cultured with NB-DNJ/PPMP. NBD-fluorescence accumulated in the Golgi complex after 40 min, SM interacts directly with cholesterol (Rog and Pasenkiewicz- but formation of vesicular NBD-specks was observed after 150 min Gierula, 2006; Abdul-Hammed et al., 2010), and both lipids are after labeling. The potential mechanism responsible for the for- accumulated in intracellular vesicles in various NPC cells (Chen mation of vesicular NBD-specks in NPC1( / ) cells may involve the et al., 2005; Devlin et al., 2010; Sztolsztener et al., 2012). However, abnormal trafficking of vesicles emitting NBD-fluorescence. In the cellular levels of SM in NPC1( / ) cells were not affected by the NPC1( / ) cells cultured with NB-DNJ for 48 h, in which the for- NB-DNJ treatment (Fig. 4B). The mechanisms responsible for the mation of GlcCer (and resulting GSLs) was decreased, NBD- further accumulation of cholesterol in NPC1( / ) cells cultured with fluorescence was detected mainly in small vesicular specks, not in NB-DNJ/PPMP require further study. SM and GSLs including LacCer the Golgi complex after 40 min (Fig. 3C). These results may be and gangliosides were reported to be essential for vesicle biogenesis contradictory to each other, but the following may explain the in and its trafficking from the Golgi complex (Cheng et al., 2006; observations. Sillence et al. (2002) reported that both the increase Duran et al., 2012), and distinct vesicles enriched with either SM of GlcCer by inhibition of glucocerebrosidase and decrease of or GlcCer have been reported (Maier and Hoekstra, 2003; Moore GlcCer by inhibition of GlcCer synthase altered sorting of BODIPY- et al., 2008; Duran et al., 2012). Thus, an increase in vesicle forma- LacCer in endocytotic pathway. A deviation from the normal tion and/or the formation of different types of vesicles from the range of GSLs in vesicles (and/or cells), both the increases and de- Golgi complex may explain the appearance of vesicular NBD-specks creases, may disturb the normal trafficking of vesicles having NBD- in NPC1( / ) cells. However, an increase in vesicle formation did not ceramide metabolites. The treatment of NPC1( / ) cells with NB- appear to occur in the NB-DNJ-treated NPC1( / ) cells because of the DNJ for 48 h further enhanced the accumulation of cholesterol decrease in GlcCer (and resulting GSLs) and constant level of SM. In (Fig. 4A), as shown in NB-DGJ-treated human fibroblasts (Sillence NPC1( / ) cells treated for 30 min with NB-DNJ, in which the for- et al., 2002) and HepG2 cells (Bijl et al., 2008). The formation of mation of NBD-GlcCer was markedly reduced (Fig. 1D), and, thus, vesicular NBD-specks in NPC1( / ) cells was dependent on NBD-SM was the main fluorescent material. In these cells, NBD- cholesterol in FBS (Fig. 3B and F). The excessive application of fluorescence mainly accumulated in the Golgi complex 40 min af- cholesterol disturbed the trafficking of vesicles containing sphin- ter labeling (Fig. 3E), thus levels of NBD-SM formed for 40 min did golipids including BODIPY-LacCer by modulating the activity of the not appear to determine the accelerated formation of vesicular NBD- N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469 467

specks in NB-DNJ/PPMP-treated NPC1( / ) cells.

4.3. Characteristics of vesicles containing NBD-ceramide metabolites and endocytic vesicles

BODIPY-LacCer taken up by endocytosis ultimately accumulates in the Golgi complex in normal cells; however, the abnormal accumulation of this lipid in vesicular specks occurs in storage diseases including NPC (Puri et al., 2001; Choundhury et al., 2004; Cheng et al., 2006). We revealed that the transport pathways carrying NBD-ceramide metabolites formed in the Golgi complex and BODIPY-LacCer taken up by endocytosis exhibited different sensitivities to the NB-DNJ treatment for 48 h in NPC1( / ) cells; the former remained normal for 40 min and was disturbed by the NB- DNJ treatment, while the latter was already disturbed within 40 min. Cholesterol levels in the plasma membranes of NPC cells did not appear to be decreased; levels were the same in several cells (Lange et al., 2000), but were increased in NPC fibroblasts (Miersch et al., 2008) and hepatocytes (Vainio et al., 2005). In contrast, the depletion of functional cholesterol in the Golgi complex in NPC cells (Coxey et al., 1993) and a reduction in cholesterol levels in fractions containing the Golgi complex from NPC1 mutant CHO cells (Reverter et al., 2014) were reported. Cholesterol deposits were not detected in the Golgi complex in NPC fibroblasts by a high-sensitive visualization method (Kwiatkowska et al., 2014). Thus, cholesterol levels in vesicles may explain differences in the trafficking of BODIPY-LacCer and NBD-ceramide metabolites 40 min after label- Fig. 6. A proposed model for the abnormal trafficking of NBD-ceramide metabo- (¡/¡) (/) ing in NPC1( / ) cells; the accumulation of BODIPY-LacCer in ve- lites in NPC1 cells cultured with NB-DNJ for 48 h. In NPC1 cells (NPC1*), the sicular specks with large amounts of cholesterol, and the accumulation of cholesterol increased GlcCer synthase activity, and the increased formation of GlcCer (and probable GSLs) may have a feed-back inhibitory effect on the accumulation of NBD-SM/GlcCer in the Golgi complex possibly accumulation of cholesterol. Under these conditions, the trafficking of NBD-SM/GlcCer reflecting the normal trafficking of vesicles with low levels of endogenously formed was normal within 40 min, but was disturbed 150 min after cholesterol. At 150 min after labeling, the fusion of vesicles may NBD-ceramide labeling. Cultivation with NB-DNJ for 48 h appeared to decrease GlcCer have masked initial differences in the levels of cholesterol in vesi- and GSLs, and the trafficking of NBD-SM was disturbed within 40 min in these cells because of an additional increase in cholesterol levels. The increased formation of cles derived from the Golgi complex and endocytic vesicles. GlcCer (and probable GSLs) in NPC cells may have a positive effect on the normal trafficking of NBD-ceramide metabolites in NPC1( / ) cells. 4.4. A proposed model and future challenges

A model for the probable role of increases in the activity of about the involvement of cholesterol in the regulation of GlcCer GlcCer synthase on the abnormal trafficking of NBD-SM/GlcCer in synthase activity in future. NPC1( / ) cells has been proposed (Fig. 6). The increases in GlcCer (and resulting GSLs) may have two effects in NPC1( / ) cells, i) an inhibitory effect on cholesterol accumulation, and ii) maintenance 5. Conclusions of the normal vesicular trafficking of NBD-SM/GlcCer formed in the Golgi complex, at least for 40 min. These two effects of GlcCer/GSLs In the present study, we showed that 1) the activity of GlcCer (/) may be lost in NPC1 cells cultured with NB-DNJ for 48 h, synthase was increased in NPC1( / ) cells without affecting protein resulting in the further accumulation of cholesterol, such that the levels, and 2) the cultivation of cells with NB-DNJ enhanced the fi vesicular traf cking of NBD-ceramide metabolites was disturbed accumulation of cholesterol and accelerated the appearance of the within 40 min of NBD-ceramide labeling. We could not specify the abnormal trafficking of endogenously formed NBD-ceramide me- nature of NBD-ceramide metabolites in vesicular NBD-specks and tabolites in the Golgi complex. Although treatment with NB-DNJ is in the Golgi complex in this study. The absolute levels of the useful for NPC patients by inhibition of formation of GlcCer (and respective species of GSLs, SM, and cholesterol need to be deter- GSLs) in neurons in CNS, the treatment appeared to disturb intra- mined in organelle- and vesicle-dependent manners in the future. cellular trafficking of ceramide metabolites in the present study. Treatment with an iminosugar type inhibitor of GlcCer synthase The effects of NB-DNJ on the intracellular trafficking of GSLs and induced synthesis of cholesterol via -dependent cholesterol levels in the tissues including the liver and spleen need fi manner (Bijl et al., 2008). In addition to modi cation of cellular to be considered in NB-DNJ-treated NPC patients. distribution of cholesterol, U18666A treatment regulates various of cholesterol metabolism, and thus changes levels of cholesterol metabolites including cholesterol-5,6-epoxides, mod- Authorship contributions ulators of gene expression (Chapuy-Regaud et al., 2013). Precise mechanisms for cholesterol accumulation in NPC1( / ) cells with Participated in Research Design: Nakamura, and Murayama. and without NB-DNJ treatment remain to be solved. In this study, Conducted experiments: Hashimoto, Matsumoto, Takahashi, treatment of WT cells with the HPbCD/cholesterol complex signif- Ashikawa, and Nakamura. icantly enhanced the formation of NBD-GlcCer (Fig. 1C), however, Performed data analysis: Nakamura, and Murayama. treatment of NPC1( / ) cells with CD did not alter the formation of Wrote or contributed to the writing of the manuscript: Nakamura, NBD-GlcCer (data not shown). We should clarify the mechanism and Murayama. 468 N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469

Conflict of interest Fukasawa, M., Nishijima, M., Hanada, K., 1999. Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphin- gomyelin synthesis in Chinese hamster ovary cells. J. Cell Biol. 144, 673e685. The authors declare that there are no conflicts of interest. Ganley, I.G., Pfeffer, S.R., 2006. Cholesterol accumulation sequesters Rab9 and dis- turbs late endosome function in NPC1-deficient cells. J. Biol. Chem. 281, 17890e17899. Financial support Graf, C., Klumpp, M., Habig, M., Rovina, P., Billich, A., Baumruker, T., Oberhauser, B., Bornancin, F., 2008. Targeting ceramide metabolism with a potent and specific These studies were partially supported by Grants-in-Aid ceramide kinase inhibitor. Mol. Pharmacol. 74, 925e932. Halter, D., Neumann, S., van Dijk, S.M., Wolthoorn, J., de Maziere, A.M., Vieira, O.V., (24790066, 26460060 to H.N. and 26460093 to T.M.) from the Mattjus, P., Klumperman, J., van Meer, G., Sprong, H., 2007. Pre- and post-Golgi Ministry of Education, Culture, Sports, Science, and Technology of translocation of glucosylceramide in synthesis. J. Cell Biol. Japan. 179, 101e115. Hayashi, Y., Okino, N., Kakuta, Y., Shikanai, T., Tani, M., Narimatsu, H., Ito, M., 2007. Klotho-related protein is a novel cytosolic neutral b-glycosylceramidase. J. Biol. Acknowledgments Chem. 282, 30889e30900. Higaki, K., Ninomiya, H., Sugimoto, Y., Suzuki, T., Taniguchi, M., Niwa, H., Pentchev, P.G., Vanier, M.T., Ohno, K., 2001. Isolation of NPC1-deficient Chinese We thank Prof. A. Nishida (Chiba University, Japan) for providing hamster ovary cell mutants by gene trap mutagenesis. J. Biochem. 129, NBD-ceramide. 875e880. Ishibashi, Y., Hirabayashi, Y., 2015. AMP-activated protein kinase suppresses biosynthesis of glucosylceramide by reducing intracellular sugar nucleotides. References J. Biol. Chem. 290, 18245e18260. Kwiatkowska, K., Marszałek-Sadowska, E., Traczyk, G., Koprowski, P., Musielak, M., Abdul-Hammed, M., Breiden, B., Adebayo, M.A., Babalola, J.O., Schwarzmann, G., Ługowska, A., Kulma, M., Grzelczyk, A., Sobota, A., 2014. Visualization of Sandhoff, K., 2010. Role of endosomal membrane lipids and NPC2 in cholesterol cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using transfer and membrane fusion. J. Lipid Res. 51, 1747e1760. recombinant perfringolysin O. Orphanet. J. Rare Dis. 9, 64. Alvarez, J.G., Ludmir, J., 1993. Semiautomated multisample analysis of amniotic fluid Lange, Y., Ye, J., Rigney, M., Steck, T., 2000. Cholesterol movement in Niemann-Pick lipids by high-performance thin-layer chromatography-reflectance spec- type C cells and in cells treated with amphiphiles. J. Biol. Chem. 275, trodensitometry. J. Chromatogr. 615, 142e147. 17468e17475. Babia, T., Ledesma, M.D., Saffrich, R., Kok, J.W., Dotti, C.G., Egea, G., 2001. Endocytosis Lee, H., Lee, J.K., Bae, Y.C., Yang, S.H., Okino, N., Schuchman, E.H., Yamashita, T., of NBD-sphingolipids in neurons: exclusion from degradative compartments Bae, J.S., Jin, H.K., 2014. Inhibition of GM3 symtgase attenuates neuropathology and transport to the Golgi complex. Traffic 2, 395e4-5. of Niemann-Pick disease type C by affecting sphingolipid metabolism. Mol. Cells Besley, G.T., Moss, S.E., 1983. Studies on sphingomyelinase and b-glucosidase ac- 37, 161e171. tivities in Niemann-Pick disease variants: phosphodiesterase activities Lipsky, N.G., Pagano, R.E., 1983. Sphingolipid metabolism in cultured fibroblasts: measured with natural and artificial substrates. Biochim. Biophys. Acta 752, microscopic and biochemical studies employing a fluorescent ceramide 54e64. analogue. Proc. Natl. Acad. Sci. U. S. A. 80, 2608e2612. Bijl, N., Scheij, S., Houten, S., Boot, R.G., Groen, A.K., Aerts, J.M., 2008. The gluco- Maier, O., Hoekstra, D., 2003. Trans-Golgi network and subapical compartment of sylceramide synthase inhibitor N-(5-adamantane-1-yl-methoxy-pentyl)-deox- GepG2 cells display different properties in sorting and exiting of sphingolipids. ynojirimycin induces sterol regulatory element-binding protein-regulated gene J. Biol. Chem. 278, 164e173. expression and cholesterol synthesis in HepG2 cells. J. Pharmacol. Exp. Ther. Makiyama, T., Nakamura, H., Nagasaka, N., Yamashita, H., Honda, T., Yamaguchi, N., 326, 849e855. Nishida, A., Murayama, T., 2015. Trafficking of acetyl-C16-ceramide-NBD with Boldin, S.A., Futerman, A.H., 2000. Up-regulation of glucosylceramide synthesis long-term stability and no cytotoxicity into the Golgi complex. Traffic16, upon stimulation of axonal growth by basic fibroblast growth factor: evidence 476e492. for post-translational modification of glucosylceramide synthase. J. Biol. Chem. Marques, A.R., Aten, J., Ottenhoff, R., van Roomen, C.P., Herrera Moro, D., 275, 9905e9909. Claessen, N., Vinueza Veloz, M.F., Zhou, K., Lin, Z., Mirzaian, M., Boot, R.G., De Byun, K., Kim, D., Bayarsaikhan, E., Oh, J., Kim, J., Kwak, G., Jeong, G.B., Jo, S.M., Zeeuw, C.I., Overkleeft, H.S., Yildiz, Y., Aerts, J.M., 2015. Reducing GBA2 activity Lee, B., 2013. Changes of calcium binding proteins, c-Fos and COX in hippo- ameliorates neuropathology in Niemann-Pick type C mice. PLoS One 10, campal formation and cerebellum of Niemann-Pick, type C mouse. J. Chem. e0135889. Neuroanat. 52, 1e8. Miersch, S., Espey, M.G., Chaube, R., Akarca, A., Tweten, R., Ananvoranich, S., Chapuy-Regaud, S., Subra, C., Requena, M., de Medina, P., Amara, S., Delton- Mutus, B., 2008. Plasma membrane cholesterol content affects nitric oxide Vandenbroucke, I., Payre, B., Cazabat, M., Carriere, F., Izopet, J., Poirot, M., diffusion dynamics and signaling. J. Biol. Chem. 283, 18513e18521. Record, M., 2013. Progesterone and a phospholipase inhibitor increase the Moore, E.R., Fischer, E.R., Mead, D.J., Hackstadt, T., 2008. The chlamudial inclusion endosomal bis(monoacylglycero)phosphate concent and block HIV viral parti- preferentially intercepts basolaterally directed sphingomyelin-containing exo- cle intercellular transmission. Biochimie 95, 1677e1688. cytic vacuoles. Traffic9,2130e2140. Chen, F.W., Gordon, R.E., Ioannou, Y.A., 2005. NPC1 late endosomes contain elevated Mukherjee, S., Maxfield, F.R., 2004. Lipid and cholesterol trafficking in NPC. Biochim. levels of non-esterified (‘free’) fatty acids and an abnormally glucosylated form Biophys. Acta 1685, 28e37. of the NPC2 protein. Biochem. J. 390, 549e561. Nakamura, H., Yasufuku, K., Makiyama, T., Matsumoto, I., Fujino, H., Murayama, T., Cheng, Z.J., Singh, R.D., Sharma, D.K., Holicky, E.L., Hanada, K., Marks, D.L., 2012. Arachidonic acid metabolism via cytosolic phospholipase A2a induces Pagano, R.E., 2006. Distinct mechanisms of clathrin-independent endocytosis cytotoxicity in Niemann-Pick disease type C cells. J. Cell. Physiol. 227, have unique sphingolipid requirements. Mol. Biol. Cell 17, 3197e3210. 2847e2855. Choudhury, A., Dominguez, M., Puri, V., Sharma, D.K., Narita, K., Wheatley, C.L., Nakamura, H., Wakita, S., Yasufuku, K., Makiyama, T., Waraya, M., Hashimoto, N., Marks, D.L., Pagano, R.E., 2002. Rab proteins mediate Golgi transport of caveola- Murayama, T., 2015. Sphongomyelin regulates the activity of secretory phos- internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C pholipase A2 in the plasma membrane. J. Cell. Biochem. 116, 1898e1907. cells. J. Clin. Investig. 109, 1541e1550. Oninla, V.O., Breiden, B., Babalola, J.O., Sandhoff, K., 2014. Acid sphingomyelinase Choundhury, A., Sharma, D.K., Marks, D.L., Pagano, R.E., 2004. Elevated endosomal activity is regulated by membrane lipids and facilitates cholesterol transfer by cholesterol levels in Niemann-Pick cells inhibit rab4 and perturb membrane NPC2. J. Lipid Res. 55, 2606e2619. recycling. Mol. Biol. Cell 15, 4500e4511. Pagano, R.E., Sepanski, M.A., Martin, O.C., 1989. Molecular trapping of a fluorescent Coxey, R.A., Pentchev, P.G., Campbell, G., Blanchette-Mackie, E.J., 1993. Differential ceramide analogue at the Golgi apparatus of fixed cells: interaction with accumulation of cholesterol in Golgi compartments of normal and Niemann- endogenous lipids provides a trans-Golgi markers doe both light and electron Pick type C fibroblasts incubated with LDL: a cytochemical freeze-fracture microscopy. J. Cell Biol. 109, 2067e2079. study. J. Lipid Res. 34, 1165e1176. Patterson, M.C., Vecchio, D., Jacklin, E., Abel, L., Chadha-Boreham, H., Luzy, C., Crespo, P.M., Silvestre, D.C., Gil, G.A., Maccioni, H.J., Daniotti, J., Caputto, B.L., 2008. Giorgino, R., Wraith, J.E., 2010. Long-term miglustat therapy in children with C-Fos activates glucosylceramide synthase and synthesis in PC12 Niemann-Pick disease type C. J. Child. Neirol 25, 300e305. cells. J. Biol. Chem. 283, 31163e31171. Pipalia, N.H., Hao, M., Mukherjee, S., Maxfield, F.R., 2007. Sterol, protein and lipid Delgado, A., Casas, J., Llebaria, A., Abad, J.L., Fabrias, G., 2006. Inhibitors of sphin- trafficking in Chinease hamster ovary cells with Niemann-Pick type C1 defect. golipid metabolism enzymes. Biochim. Biophys. Acta 1758, 1957e1977. Traffic8,130e141. Devlin, C., Pipalia, N.H., Liao, X., Schuchman, E.H., Maxfield, F.R., Tabas, I., 2010. Platt, F.M., Neises, G.R., Dwek, R.A., Butters, T.D., 1994. N-Butyldeoxynojirimycin is a Improvement in lipid and protein trafficking in Niemann-Pick C1 cells by novel inhibitor of glycolipid biosynthesis. J. Biol. Chem. 269, 8362e8365. correction of a secondary enzyme defect. Traffic 11, 601e615. Puri, V., Watanabe, R., Dominguez, M., Sun, X., Wheatley, C.L., Marks, D.L., Duran, J.M., Campelo, F., van Galen, J., Sachsenheimer, T., Sot, J., Egorov, M.V., Pagano, R.E., 1999. Cholesterol modulates membrane traffic along the endocytic Rentero, C., Enrich, C., Polishchuk, R.S., Goni,~ F.M., Brügger, B., Wieland, F., pathway in sphingolipid-storage diseases. Nat. Cell Biol. 1, 386e388. Malhotra, V., 2012. Sphingomyelin organization is required for vesicle biogen- Puri, V., Watanabe, R., Singh, R.D., Dominguez, M., Brown, J.C., Wheatley, C.L., esis at the Golgi complex. EMBO J. 31, 4535e4546. Marks, D.L., Pagano, R.E., 2001. Clathrin-dependent and -independent inter- Elleder, M., 1989. Niemann-Pick disease. Pathol. Res. Pract. 185, 293e328. nalization of plasma membrane sphingolipids initiates two Golgi targeting N. Hashimoto et al. / Neuropharmacology 110 (2016) 458e469 469

pathways. J. Cell Biol. 154, 535e547. te Vruchte, D., Lloyd-Evans, E., Veldman, R.J., Neville, D.C., Dwek, R.A., Platt, F.M., van Qiu, H., Edmunds, T., Baker-Malcolm, J., Karey, K.P., Estes, S., Schwarz, C., Hughes, H., Blitterswijk, W.J., Sillence, D.J., 2004. Accumulation of glycosphingolipids in Van Patten, S.M., 2003. Activation of human acid sphingomyelinase through Niemann-Pick C disease disrupts endosomal transport. J. Biol. Chem. 279, modification or deletion of C-terminal cysteine. J. Biol. Chem. 278, 26167e26175. 32744e32752. Uchida, Y., Itoh, M., Taguchi, Y., Yamaoka, S., Umehara, H., Ichikawa, S., Ridley, C.M., Thur, K.E., Shanahan, J., Thillaiappan, N.B., Shen, A., Uhl, K., Hirabayashi, Y., Holleran, W.M., Okazaki, T., 2004. Ceramide reduction and Walden, C.M., Rahim, A.A., Waddington, S.N., Platt, F.M., van der Spoel, A.C., transcriptional up-regulation of glucosylceramide synthase through 2013. b-Glucosidase 2 (GBA2) activity and imino sugar pharmacology. J. Biol. doxorubicin-activated Sp1 in drug-resistant HL-60/ADR cells. Cancer Res. 64, Chem 288, 26052e26066. 6271e6279. Reagan, J.W., Hubbert, M.L., Shelness, G.S., 2000. Posttranslational regulation of acid Vainio, S., Bykov, I., Hermansson, M., Jokitalo, E., Somerharju, P., Ikonen, E., 2005. sphingomyelinase in Niemann-Pick type C1 fibroblasts and free cholesterol- Defective insulin receptor activation and altered lipids rafts in Niemann-Pick enriched Chinese hamster ovary cells. J. Biol. Chem. 275, 38104e38110. type C disease hepatocytes. Biochem. J. 391, 465e472. Reverter, M., Rentero, C., Garcia-Melero, A., Hoque, M., de Muga, S.V., Alvarez- Vance, J.E., Karten, B., 2014. Niemann-Pick C disease and mobilization of lysosomal Guaita, A., Conway, J.R., Wood, P., Cairns, R., Lykopoulou, L., Grinberg, D., cholesterol by cyclodextrin. J. Lipid Res. 55, 1609e1621. Vilageliu, L., Bosch, M., Heeren, J., Blasi, J., Timpson, P., Pol, A., Tebar, F., van Helvoort, A., Giudici, M.L., Thielemans, M., van Meer, G., 1997. Transport of Murray, R.Z., Grewal, T., Enrich, C., 2014. Cholesterol regulates syntaxin 6 traf- sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, ficking at trans-Golgi network endosomal boundaries. Cell Rep. 7, 883e897. where C6-NBD-sphingomyelin is translocated across the plasma membrane by a Rog, T., Pasenkiewicz-Gierula, M., 2006. Cholesterol-sphingomyelin interactions: a multidrug transpoter activity. J. Cell Sci. 110, 75e83. molecular dynamics stimulation study. Biophys. J. 91, 3756e3767. Vanier, M.T., 1983. Biochemical studies in Niemann-Pick disease. Biochim. Biophys. Salvioli, R., Scarpa, S., Ciaffoni, F., Tatti, M., Ramoni, C., Vanier, M.T., Vaccaro, A.M., Acta 750, 178e184. 2004. Glucosylceramidase mass and subcellular localization are modulated by Vanier, M.T., 1999. Lipid changes in Niemann-Pick disease type C brain: personal cholesterol in Niemann-Pick disease type C. J. Biol. Chem. 279, 17674e17680. experience and review of the literature. Neurochem. Res. 24, 481e489. Sillence, D.J., Puri, V., Marks, D.L., Butters, T.D., Dwek, R.A., Pagano, R.E., Platt, F.M., Vanier, M.T., 2010. Niemann-Pick disease type C. Orphanet J. Rare Dis. 5, 16. 2002. Glucosylceramide modulates membrane traffic along the endocytic Vanier, M.T., 2015. Complex lipid trafficking in Niemann-Pick disease type C. pathway. J. Lipid Res. 43, 1837e1845. J. Inherit. Metab. Dis. 38, 187e199. Sugimoto, Y., Ninomiya, H., Ohsaki, Y., Higaki, K., Davies, J.P., Ioannou, Y.A., Ohno, K., Wojtanik, K.M., Liscum, L., 2003. The transport of low density lipoprotein-derived 2001. Accumulation of cholera toxin and GM1 in the early endo- cholesterol to the plasma membrane is defective in NPC1 cells. J. Biol. Chem. some of Niemann-Pick C1-deficient cells. Proc. Natl. Acad. Sci. U. S. A. 98, 278, 14850e14856. 12392e12396. Wu, N., Sarna, L.K., Hwang, S.Y., Zhu, Q., Wang, P., Siow, Y.L., Karmin, O., 2013. Sztolsztener, M.E., Dobrzyn, A., Pikula, S., Tylki-Szymanska, A., Bandorowicz- Activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase Pikula, J., 2012. Impaired dynamics of the late endosome/lysosome compart- during high fat diet feeding. Biochim. Biophys. Acta 1832, 1560e1568. ment in human Niemann-Pick type C skin fibroblasts carrying mutation in Zervas, M., Dobrenis, K., Walkley, S.U., 2001a. Neurons in Niemann-Pick disease type NPC1 gene. Mol. Biosyst. 8, 1197e1205. C accumulate gangliosides as well as unesterified cholesterol and undergo Tada, E., Toyomura, K., Nakamura, H., Sasaki, H., Saito, T., Kaneko, M., Okuma, Y., dendritic and axonal alterations. J. Neuropath. Exp. Neurol. 60, 49e64. Murayama, T., 2010. Activation of and ceramide kinase by vanadate Zervas, M., Somers, K.L., Thrall, M.A., Walkley, S.U., 2001b. Critical role for glyco- via a tyrosine kinase-mediated pathway. J. Pharmacol. Sci. 114, 420e432. sphingolipids in Niemann-Pick disease type C. Curr. Biol. 11, 1283e1287. Taniguchi, M., Shinoda, Y., Ninomiya, H., Vanier, M.T., Ohno, K., 2001. Sites and Zhang, M., Dwyer, N.K., Neufeld, E.B., Love, D.C., Cooney, A., Comly, M., Patel, S., temporal changes of gangliosides GM1/GM2 storage in the Niemann-Pick dis- Watari, H., Strauss III, J.F., Pentchev, P.G., Hanover, J.A., Blanchette-Mackie, E.J., ease type C mouse brain. Brain Dev. 23, 414e421. 2001. Sterol-modulated glycolipid sorting occurs in Niemann-Pick C1 late Tettamanti, G., 2004. Ganglioside/glycosphingolipid turnover: new concepts. Gly- endosomes. J. Biol. Chem. 276, 3417e3425. coconj. J. 20, 301e317.