Neuropharmacology 110 (2016) 458e469
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Neuropharmacology
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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
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: Sphingolipids such as sphingomyelin and glycosphingolipids (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 glucocerebrosidase (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 gene 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)sphingosine; 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 gangliosides 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 metabolism 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