Biochem. J. (2008) 410, 81–92 (Printed in Great Britain)
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doi:10.1042/BJ20070976
Endosomes and lysosomes play distinct roles in sulfatide-induced neuroblastoma apoptosis: potential mechanisms contributing to abnormal sulfatide metabolism in related neuronal diseases Youchun ZENG*, Hua CHENG*, Xuntian JIANG* and Xianlin HAN*1 *Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Medicine, Washington University School of Medicine, Campus Box 8020, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.
Alterations in sulfatide metabolism, trafficking and homoeostasis are present at the earliest clinically recognizable stages of Alzheimer’s disease and are associated with metachromatic leukodystrophy. However, the role of sulfatide in these disease states remains unknown. In the present study, we investigated the sequelae of NB (neuroblastoma) cells upon sulfatide supplementation and the biochemical mechanisms contributing to the sulfatide-induced changes. By using shotgun lipidomics, we showed dramatic accumulations of sulfatide, ceramide and sphingosine in NB cells in a time- and dose-dependent manner. Further studies utilizing subcellular fractionation and shotgun lipidomics analyses demonstrated that most of the increased ceramide content was generated in the endosomal compartment, whereas sulfatides predominantly accumulated in lysosomes. In addition, we determined that the sulfatide-mediated increase in endosomal ceramide content mainly resulted from β-galacto-
sidase activity, which directly hydrolyses sulfatide to ceramide without a prior desulfation step. Substantial cell apoptosis occurred in parallel with the accumulation of sulfatides and ceramides, as revealed by mitochondrial membrane depolarization, by phosphatidylserine translocation and by the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay. These findings were also demonstrated with primary neuron cultures. Collectively, our results demonstrate that abnormal sulfatide metabolism can induce cell apoptosis due to endosome-mediated ceramide generation and the accumulation of cytotoxic levels of sulfatides in lysosomes.
INTRODUCTION
diseases. The accumulation of sulfatides caused by a defect in their degradation pathway (e.g. through a deficiency in sulfatidase) is responsible for metachromatic leukodystrophy [12]. Moreover, sulfatide accumulation has also been demonstrated in postmortem brain tissues from subjects with Parkinson’s disease [13] by a currently unknown process. An altered ratio of sulfatide to cerebroside, due to a galactosylceramidase deficiency, is a hallmark of globoid-cell leukodystrophy (i.e. Krabbe disease) [14]. Sulfatide deficiency and ceramide elevation are manifest in subjects with very mild AD (Alzheimer’s disease) [13,15–17]. However, the causes of sulfatide depletion, its relationship to disease pathogenesis and how sulfatide depletion and ceramide elevation are linked in AD remain to be determined. Although available evidence supports the conclusion that sulfatides are only synthesized in oligodendrocytes in the CNS [1,2], the presence of sulfatides in neurons and astrocytes (e.g. [18–20]) suggests the existence of a transport network and endocytotic pathways that are associated with sulfatide metabolism and homoeostasis. Recently, we have identified that ApoE (apolipoprotein E)-associated lipoprotein particles present in the cerebrospinal fluid contain large amounts of sulfatides and that ApoE plays an important role in modulating sulfatide homoeostasis in both the CNS and the PNS [21–23], probably through low-density lipoprotein (or its family members)-mediated endocytotic pathways. The accumulation of sulfatides caused by
Sulfatides are a class of sulfated GalCers (galactosylceramides) that are synthesized primarily in the oligodendrocytes in the CNS (central nervous system) and in the Schwann cells of the PNS (peripheral nervous system) [1,2]. Sulfatide molecular species differ mainly in the aliphatic chain attached to the amino group of the sphingosine backbone, although other minor sphingoid-based backbones are also present [1–3]. Sulfatide molecular species are also categorized into two subgroups based on the presence or absence of a hydroxyl moiety at the α-position of the fatty acyl amide [2,3]. Sulfatides play numerous essential roles in a variety of biological processes such as cell growth, protein trafficking, signal transduction, cell–cell recognition, neuronal plasticity and cell morphogenesis [1,2,4–7]. For example, severe myelin developmental abnormalities, myelin sheath degeneration and significant increases in deteriorating nodal/paranodal structures are manifest in sulfatide-deficient mice [6]. Mice deficient in sulfatides and GalCers (generated by knocking out a ceramide galactosyltransferase) generally die by 3 months of age and display many abnormalities including abnormal axonal function, dysmyelinosis and loss of axonal conduction velocity [8–11]. The importance of sulfatides in biological processes has also been underscored through their linkage with multiple human
Key words: Alzheimer’s disease (AD), ceramide, electrospray ionization MS (ESI–MS), metachromatic leukodystrophy, shotgun lipidomics, sulfatide.
Abbreviations used: AD, Alzheimer’s disease; ApoE, apolipoprotein E; BODIPY® , 4,4-difluoro-4-bora-3a,4a-diaza-s -indacene; CNS, central nervous system; EEA1, early endosome antigen 1; ESI–MS, electrospray ionization MS; GalCer, galactosylceramide; HB, homogenization buffer; LAMP2b, lysosome-associated membrane protein 2b; MEM, minimum essential medium; MTG, methyl β-D-thiogalactopyranoside; NB, neuroblastoma; nSMase, neutral sphingomyelinase; PNS, peripheral nervous system; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. 1 To whom correspondence should be addressed (email
[email protected]). c The Authors Journal compilation c 2008 Biochemical Society
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deficient lysosomal sulfatidase activity in neuronal cells in metachromatic leukodystrophy [12] further supports the importance of the endocytotic pathway of ApoE in sulfatide metabolism and homoeostasis. Moreover, it should be recognized that although glycosphingolipids (including sulfatide) in the plasma membrane are transported to lysosomes through vesicular membrane trafficking via different routes such as clathrin-coated pits or caveolae [24], the sulfatides carried by ApoE particles are probably transported to lysosomes through trafficking of vesicles containing ApoE [25]. To study further the metabolism and trafficking of sulfatides as well as to better understand their role in AD and metachromatic leukodystrophy, we treated NB (neuroblastoma) cells with different concentrations of sulfatides for various time intervals. ESI–MS (electrospray ionization MS)-based lipidomics analyses demonstrated the rapid internalization of sulfatides by NB cells through a phagocytotic process that occurred upon sulfatide supplementation. Exogenously supplied sulfatides initiated apoptosis as evidenced by mitochondrial membrane depolarization, by phosphatidylserine translocation and by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) staining. Moreover, quantification of the altered sulfatide, ceramide and sphingosine contents in NB cells induced by sulfatide supplementation showed the interrelationship between these metabolites in a time- and dosedependent manner. Shotgun lipidomics analyses of isolated subcellular membranes and organelles demonstrated that ceramides were primarily generated in the endosomes, whereas sulfatides predominantly accumulated in the lysosomal compartment. Further mechanistic studies demonstrated that the increase in endosomal ceramide content upon sulfatide supplementation primarily resulted from a β-galactosidase activity that directly hydrolyses sulfatide to ceramide without a prior desulfation step. Accumulation of sulfatide and ceramide as well as cell apoptosis were also demonstrated with primary neuron cultures. Accordingly, these results indicate that abnormal sulfatide metabolism/trafficking through direct internalization can induce neuronal cell apoptosis through endosome-generated ceramide accumulation [26–28] and/or lysosome swelling and sulfatide toxicity [29,30]. EXPERIMENTAL Materials
Semi-synthetic palmitoyl sulfatide (C16:0 -sulfatide), semisynthetic perdeuterated N-octadecanoyl galactosylceramide (d35 C18:0 -GalCer), pentadecanoyl galactosylceramide (C15:0 -GalCer), bovine brain sulfatides (C42 H81 NO11 S) and bovine brain GalCer (C48 H93 NO8 ) were purchased from Matreya (Pleasant Gap, PA, U.S.A.). Synthetic sphingolipids including heptadecanoyl (C17:0 ) sphingomyelin, heptadecanoyl (C17:0 ) ceramide and C16 sphingosine analogue as well as most of the synthetic phospholipids used as internal standards were obtained from Avanti Polar Lipids. The APO-BrdU TUNEL assay kit and fluorescence probes, including MitoProbe JC-1 assay kit, LysoTracker Red DND-99, BODIPY® (4,4-difluoro-4-bora3a,4a-diaza-s-indacene) FL C50 -ceramide and annexin V were purchased from Invitrogen. Antibodies, including anti-LAMP2b (lysosome-associated membrane protein 2b), anti-EEA1 (early endosome antigen 1) and anti-transferrin receptor, were purchased from Abcam. An nSMase (neutral sphingomyelinase) inhibitor (GW4869) and a β-galactosidase inhibitor [MTG (methyl βD-thiogalactopyranoside)] were purchased from Sigma–Aldrich Chemical Co. All other chemical reagents were of the best grade c The Authors Journal compilation c 2008 Biochemical Society
available and were obtained from either Thermo Fisher Scientific or Sigma–Aldrich Chemical Co., or as indicated. Delta TPG glassbottom dishes for live-cell confocal microscopy were purchased from Bioptechs. Cell culture
NB cells were obtained from A.T.C.C. (CRL-2268; A.T.C.C., Manassas, VA, U.S.A.) and were grown in Eagle’s modified MEM (minimum essential medium) supplemented with 2 % (v/v) fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate and 0.1 mM non-essential amino acids in a 5 % CO2 /95 % air atmosphere at 37 ◦C (Eagle’s MEM; A.T.C.C.). NB cells were incubated with the modified Eagle’s MEM containing various amounts of sulfatide or GalCer (either bovine brain GalCer or d35 C18:0 -GalCer) and 50 µM methyl-β-cyclodextrin for different time intervals as indicated. Sulfatides or GalCer were freshly added to the media from a concentrated stock solution (40 mM in DMSO) prior to exchanging the media. The final DMSO concentration was less than 0.2 % (v/v). Methyl-β-cyclodextrin (50 µM) was included in the media to enhance sulfatide or GalCer solubility. Control experiments were conducted with NB cells incubated with medium containing 50 µM of methyl-β-cyclodextrin and an appropriate concentration of DMSO. Apoptosis and staining determination using fluorescence microscopy and flow cytometry
NB cells were collected after treatment with various amounts of sulfatides or GalCer for different time intervals as indicated. NB cells were also incubated in the absence or presence of GW4869 (5 µM; an nSMase inhibitor) or MTG (3 mM; a βgalactosidase inhibitor) in a culture medium containing sulfatides or GalCer for up to 2 days. The cells were stained with the MitoProbe JC-1 assay kit for the determination of mitochondrial membrane depolarization [31]; with annexin V for measurement of phosphatidylserine translocation [32], with an Alexa Fluor® 488 dye-labelled anti-BrdU (bromodeoxyuridine) antibody for determination of DNA fragments [33], with LysoTracker Red DND-99 for the detection of lysosomes, or with BODIPY® FL C5:0 -ceramide for determination of ceramide distribution; each according to the manufacturer’s instructions. The stained cells were analysed by flow cytometry and/or confocal fluorescence microscopy as specified. Live-cell confocal microscopic analyses were conducted with Delta TPG glass-bottom dishes by using a Zeiss LSM-510 laser confocal fluorescence microscope. Negative control experiments were conducted in cell culture medium supplemented with all components (including DMSO and methylβ-cyclodextrin) except sulfatides. Isolation of cellular membrane/organelles from NB cells
Endosome- and lysosome-enriched fractions were isolated following a flotation-gradient fractionation method as described previously [34,35] with minor modifications. Briefly, NB cells were treated with or without sulfatides (60 µM) or GalCer (60 µM) for different time intervals as indicated. The cells were collected from the culture dish after treatment with a 0.25 % trypsin/EDTA solution for 2 min and washed twice by centrifugation at 200 g for 5 min at 4 ◦C with 50 ml of HB (homogenization buffer: 250 mM sucrose, 20 mM Hepes and 0.5 mM EGTA, pH 7.0). Each washed cell pellet was resuspended gently in HB and homogenized with a ground glass cell homogenizer (15 strokes) to lyse the cells. The homogenate was centrifuged at 800 g for 10 min at 4 ◦C to isolate the post-nuclear
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supernatant. The pellets containing cell debris and nuclei were collected and stored for lipid analysis. To separate mitochondria from the endosome/lysosome-enriched fraction, the post-nuclear supernatant was centrifuged at 50 000 g for 5 min at 4 ◦C. The supernatant was subsequently centrifuged at 34 000 rev./min for 15 min at 4 ◦C (SW 41Ti rotor; Beckman) to separate the microsomal fraction. The pellet containing lysosomes/endosomes was then diluted 1:1 (v/v) with a 62 % sucrose solution to produce a solution of 40.6 % sucrose. The diluted vesicular fraction (1 ml) was transferred to the bottom of a transparent Beckman SW41Ti centrifuge tube and was overlaid sequentially with 1.5 ml of a 35 % sucrose solution, 1.5 ml of a 30 % sucrose solution and then 2 ml of a 25 % sucrose solution. The tube was then filled with HB and centrifuged at 27 000 rev./min utilizing an SW41-Ti rotor (Beckman) at 4 ◦C for 2 h to yield different layers. Each fraction was collected separately using a syringe with a 22G × 4 inch needle and stored at –20 ◦C. Endosomal and lysosomal fractions were identified by Western blot analyses utilizing antibodies against specific endosomal (EEA1) and lysosomal (LAMP2b) markers. Briefly, equal amounts of protein (determined by protein assay) from the isolated subcellular fractions were separated by SDS/PAGE and transferred on to Immobilon-P membranes. Non-specific binding sites were blocked with milk powder (5 %, w/v) for 30 min in blocking buffer [Tris-buffered saline, pH 7.6, 0.05 % Tween 20 and 3 % (w/v) non-fat dried milk]. Then primary antibodies (1:500 dilution, 1 µg/ml final concentrations) were incubated for 16 h at 4 ◦C. Horseradish-peroxidase-linked secondary antibody was used in combination with an enhanced chemiluminescence detection system (Pierce SuperSignal) to visualize immunoreactive bands. Crude plasma membranes were prepared from sucrose-densitygradient ultracentrifugation as previously described [36,37] with minor modifications. NB cells were washed with isoosmotic HB as described above. After the final wash, the pellet was resuspended very gently in HB with protease inhibitors, homogenized with 20 strokes of a cell homogenizer and pushed through a 22G needle 20 times. After removing intact cells and nuclei by centrifugation at 1500 g, the post-nuclear supernatant was centrifuged at 12 000 g to remove mitochondria. The plasma membranes were pelleted at 15 000 g and resuspended in HB. All steps were performed at 4 ◦C. The isolated plasma membrane fractions were characterized by Western blot analysis of transferring receptor. Lipid sample preparation
NB cells were detached after treatment with 0.25 % trypsin/EDTA solution for 2 min, pelleted by centrifugation at 200 g for 10 min, and washed with ice-cold PBS three times. Each cell pellet was separately homogenized in 200 µl of 0.1 × ice-cold PBS by using a disposable cell homogenizer (Thermo Fisher Scientific). Protein concentrations of individual cell homogenates were determined using a bicinchoninic acid protein assay kit (Pierce) with BSA as standard. Each homogenate (150 µl) was transferred on to a borosilicate glass culture tube (size 16 mm × 100 mm) to which 4 ml of chloroform/methanol (1:1, v/v) and 1.8 ml of LiCl solution (50 mM) were added to each individual test tube. A premixed solution of internal standards for global lipid analysis [typically, C17:0 sphingomyelin (2.25 nmol/mg of protein), C17:0 -ceramide (0.7 nmol/mg of protein), C16:0 -sulfatide (2.5 nmol/mg of protein) and C16 -sphingosine analogue (0.1 nmol/mg of protein), as well as necessary internal standards for quantification of individual molecular species of other lipid classes] was also added to each sample based on protein content prior to the extraction of lipids.
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Lipids were extracted using a modified Bligh and Dyer procedure as previously described [23]. Each lipid extract was reconstituted with a volume of 500 µl of chloroform/methanol (1:1, v/v)/mg of protein. The lipid extracts were flushed with nitrogen, capped, and stored at –20 ◦C for ESI–MS analyses (typically analysed within 1 week) as described in [38]. Lipid extracts from fetal bovine serum, cell culture medium and isolated membranes/organelles were also similarly prepared in the presence of proper internal standards. MS analyses of lipids
Shotgun lipidomics analyses were performed on a QqQ (triple quadrupole) mass spectrometer (Thermo Fisher TSQ Quantum Ultra) equipped with an electrospray ion source and operated with an Xcalibur software system. The diluted lipid extract solution was infused directly into the ESI source at a flow rate of 4 µl/min with a syringe pump and lipids were analysed as described in [38,39] on the basis of intrasource separation [40] and multidimensional MS [41]. The contents of individual lipid molecular species were quantified using a two-step procedure [23]. Quantification of sphingosine content was performed by comparison of the intensities of the sphingosine ion peaks with that of the selected internal standard (i.e. C16 -sphingosine analogue) in the tandem mass spectrum acquired by neutral loss scanning of 48 u as previously described [42]. All MS spectra and tandem MS spectra were automatically acquired by a customized sequence subroutine operated under Xcalibur software. Data processing of twodimensional MS analyses including ion peak selection, baseline correction, data transfer, peak intensity comparison, 13 C deisotoping and quantification were conducted using programmed Microsoft Excel macros as outlined previously [38]. Primary neuron cultures of embryonic mouse cortex and treatment with sulfatide
Mouse pure primary neuron cultures were prepared as described previously [43]. Briefly, pregnant Swiss Webster mice (15–16 days gestation; Taconic Farms) were anaesthetized with halothane by inhalation in accordance with institutional guidelines. The pregnant female mice were then killed by cervical decapitation and the brains of embryos were removed. Dissociated neocortices were plated on to 6- or 24-well culture plates (Falcon) at a density of 5–8 hemispheres per plate that had been coated with poly-D-lysine (100 µg/ml) and laminin (5 µg/ml) prior to plating. Cultures were maintained in a humidified incubator under a 5 % CO2 atmosphere at 37 ◦C. Cells were grown in MEM supplemented with 20 mM glucose (or as indicated), 26 mM sodium bicarbonate, 2 mM L-glutamine, 5 % fetal bovine serum and 5 % (v/v) horse serum (Hyclone), 1 × penicillin/streptomycin, and 1 × Fungizone for 7 and 15 days. Cytosine arabinoside (4 µM) was added 2–3 days after the plating to halt the growth of non-neuronal cells. Cultured primary neurons were incubated with the medium described above containing various amounts of sulfatide and 50 µM methyl-β-cyclodextrin for different time intervals as indicated, as described above for treatment of NB cells. RESULTS Sulfatides are directly internalized by NB cells in a time- and dose-dependent manner through a phagocytotic process
To initially investigate sulfatide metabolism and homoeostasis in the nervous system, we examined whether exogenous sulfatides could be directly taken up from the culture media by neuronal c The Authors Journal compilation c 2008 Biochemical Society
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Figure 1
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Representative ESI mass spectra of the lipid extracts of NB cells before and after supplementation with bovine brain sulfatides in the culture media
NB cells were grown in modified Eagle’s MEM supplemented with bovine brain sulfatides (60 µM) in the presence of 50 µM methyl-β-cyclodextrin for 0 h (A) and 5 h (B), detached with trypsin/EDTA solution for 2 min, pelleted, and washed three times at 4 ◦C. Lipids from each individual cell pellet were extracted after addition of internal standards by using a Bligh and Dyer procedure as described in the Experimental section. Mass spectra were acquired after direct infusion of the diluted lipid extract into an ESI mass spectrometer (Thermo Fisher TSQ Quantum Ultra) in the negative-ion mode. The assigned sulfatide molecular species were identified using precursor-ion scanning of 97 Th as previously described [39]. IS, internal standard; GPIns, phosphatidylinositol. In the insets, the mass spectra in the mass range m /z 875–925 are expanded. The mass spectrum in the inset of (B) clearly displays the additional ions corresponding to sulfatide molecular species in comparison with that in the inset of (A).
cells. ESI–MS-based shotgun lipidomics analyses of cellular lipids demonstrated that sulfatides were rapidly imported into NB cells from the culture media (Figure 1). Specifically, MS analyses revealed that increased levels of sulfatides were present in the cell lipid extracts after incubation with sulfatide (60 µM)-containing medium for only 1 h (spectra not shown) and large amounts of sulfatides accumulated after 5 h (Figure 1B) relative to control (Figure 1A). Results from sulfatide dose–response experiments for different time intervals indicated that the total cellular uptake of sulfatides was dependent on their initial concentration in the media and upon the incubation duration, but was non-selective for different sulfatide molecular species with different chain length fatty acyl amides, degrees of unsaturation and the presence of a hydroxyl moiety at the α-position of the acyl amide (Figure 2). Next, to distinguish two potential mechanisms through which NB cells internalized sulfatides, i.e. through a phagocytotic process (internalized within the lumen of vesicles) compared with through insertion of sulfatides into the plasma membrane followed by internalization as membrane components of vesicles, we determined the sulfatide content in isolated plasma membrane fractions. We found that only residual amounts of sulfatides were present in isolated plasma membranes upon sulfatide supplementation for longer (e.g. 24 h), but not shorter (e.g. < 12 h), incubation intervals, which could be explained by cross-contamination from other subcellular membrane compartments. Therefore the present study indicates that a phagocytotic process is probably the primary mechanism responsible for the internalization of sulfatides by NB cells. By using shotgun lipidomics, we determined the changes in the content of supplemented sulfatides in the media following different incubation time intervals at two different initial concentrations of sulfatide (Figure 3). The decrease in media sulfatide content (normalized to NB cell protein content for comparison) should represent the amount of internalized sulfatide in the c The Authors Journal compilation c 2008 Biochemical Society
Figure 2 Accumulation of sulfatides in NB cells in a time- and dosedependent manner upon sulfatide supplementation NB cells were grown and collected after addition of bovine brain sulfatides at the indicated concentrations for 3 days (A, B) or after treatment with 60 µM of sulfatides for the indicated time intervals (C, D). Lipids of the treated cells were extracted by using a modified Bligh and Dyer method and the levels of individual sulfatide molecular species were quantified by using shotgun lipidomics as described in the Experimental section. The contents of the major sulfatide molecular species, i.e. C24:1 (♦) and C24:0 (䊐) sulfatide are shown. The results represent the means + − S.D. for three independent experiments.
treated NB cells under the experimental conditions employed. Comparisons of the net decreases in media sulfatide content (Figure 3) with the net accumulation of sulfatides within the
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0.004 s−1 for the internalization of sulfatide by NB cells was determined under the experimental conditions.
Sulfatide internalization facilitates NB cell apoptosis
Figure 3 Temporal loss of sulfatide content in the media at the different initially supplemented concentrations of sulfatides NB cells were incubated with the modified Eagle’s MEM containing 20 µM (䊏; equivalent to approx. 40 nmol/mg of cell protein) and 60 µM (䊉; equivalent to approx. 120 nmol/mg of cell protein) of bovine brain sulfatides and 50 µM of methyl-β-cyclodextrin for different time intervals as indicated. The culture media at the indicated time intervals were collected and the residual sulfatide contents in the media were determined by using shotgun lipidomics as described in the Experimental section. Decreases in sulfatide contents of the media (i.e. the internalized amounts of sulfatides by NB cells) were calculated by subtraction of the determined residual sulfatide contents from those initially supplemented and normalization to the protein contents of the corresponding cells. The curves represent the best fitting to the experimental data by using an irreversible first-order kinetic model through a least-squares regression analysis.
cell pellets (Figure 2C) revealed significant decreases in cellular sulfatide content after prolonged incubation in the presence of sulfatide (e.g. > 15 h). This difference represents the net hydrolysis of sulfatides after internalization, which is consistent with significant increases in ceramide and sphingosine contents in the cells after treatment (see below). Next, we hypothesized that the kinetics of the sulfatide internalization process obeyed an irreversible first-order reaction as follows: M = M0 [1 − exp(−kt)]
(1)
where M is the decrease in media sulfatide content in nmol/mg of cell protein; M 0 is the initial supplemented sulfatide content in the medium in nmol/mg of cell protein; k is an apparent internalization rate constant; and t is the time interval of sulfatide treatment. Our experimental results agreed well with this model through a least-squares regression analysis (see curves in Figure 3). Therefore an apparent rate constant of 0.051 + −
Figure 4
During the course of the above studies, we found that NB cell death became obvious after treatment with either high (i.e. 60 µM) concentrations of sulfatides for a short period (i.e. 12 h) or with lower doses of sulfatides for a longer duration. For example, substantial depolarization of mitochondrial membranes was detected by the shift in fluorescence of MitoProbe JC-1 by both confocal fluorescence microscopy (Figure 4) and flow cytometry (Figures 5A and 5B) since the fluorescence wavelength of this probe depends on its aggregation state. In healthy cells, the JC-1 probe is absorbed by the mitochondria (due to its membrane potential) and forms aggregates within the mitochondria. These aggregates show red fluorescence (Figure 4A). However, when cells undergo apoptosis, the mitochondrial electrochemical potential collapses and JC-1 diffuses out of the mitochondria and is present in a monomeric state, which exhibits a green fluorescence following excitation (Figure 4C). NB cell apoptosis under these experimental conditions was very apparent as substantiated by TUNEL staining (Figures 5C and 5D) and phosphatidylserine translocation using an annexin V probe (results not shown). Clearly, the occurrence of sulfatide-induced apoptosis in NB cells closely paralleled the concentration of sulfatides in the media and the duration of the treatment (comparison of Figures 2 and 5).
Contribution of the dramatic accumulation of ceramides and lysosomal swelling to NB cell apoptosis after treatment with sulfatides
To identify the biochemical mechanism(s) contributing to sulfatide-induced cell apoptosis, the contents of lipid molecular species from NB cells were first determined using shotgun lipidomics. It was demonstrated that a significant accumulation of ceramide occurred after treatment of the cells with sulfatides (Figure 6), whereas the contents of other lipid classes were not altered in the early stages of cell apoptosis. Moreover, the accumulation of ceramide molecular species was also dependent on the concentration of supplemented sulfatides in the media and the duration of the treatment (Figure 6). In addition, the profile of the accumulated ceramide molecular species closely matched
Mitochondrial membrane depolarization upon supplementation with sulfatides
NB cells were treated with bovine brain sulfatides at the concentration of 0 µM (A), 60 µM (B) and 120 µM (C) for 24 h at 37 ◦C. Mitochondrial membrane depolarization was determined by using a MitoProbe JC-1 assay kit in combination with a Zeiss LSM-510 laser confocal fluorescence microscope as described in the Experimental section. The JC-1 fluorescence probe exhibits a potential-dependent accumulation in functional mitochondrial membrane where it exhibits a red fluorescence. A decrease in the red/green fluorescence intensity ratio indicates mitochondrial depolarization. Scale bar, 10 µm. c The Authors Journal compilation c 2008 Biochemical Society
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Figure 5 Mitochondrial membrane depolarization and apoptosis of NB cells induced with increasing sulfatide concentrations and treatment intervals NB cells were treated at the indicated concentrations of bovine brain sulfatides for 24 h (A, C) or treated with 60 µM of sulfatides for the indicated time intervals (B, D). Mitochondrial membrane depolarization (A, B) was determined by using the MitoProbe JC-1 assay kit at a λex of 488 nm. The fluorescence intensities of the emission between 515 and 545 nm (green) or between 560 and 615 nm (red) were determined by using an FACSCaliburTM flow cytometer (Becton Dickinson, San Jose, CA, U.S.A.). Cell apoptosis (C, D) was determined by measurement of the DNA fragmentation of apoptotic cells by using the APO-BrdU TUNEL assay kit at a λex of 488 nm. The fluorescence intensities of the emission between 515 and 545 nm (green) were determined by flow cytometry. The results represent the means + − S.D. for three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 by using a Student’s t test in comparison with controls.
that of the sulfatide molecular species acquired by NB cells from the medium (Figure 6 compared with Figure 2). This indicates that most of the accumulated ceramide was generated from the direct hydrolysis of sulfatides (except C16:0 -ceramide, which is the most abundant endogenous species normally present in NB cells). This conclusion was supported by the results of additional experiments. First, shotgun lipidomics analysis did not show the presence of GalCer molecular species, a predicted hydrolysis product of sulfatide, within the limit of detection, indicating that sulfatide degradation did not occur through this intermediate reaction step. Secondly, quantitative comparisons indicated that the accumulated ceramide content accounted for most, but not all, of the sulfatide internalized by the cells as calculated from Figures 2, 3 and 6, suggesting that further degradation of ceramide was occurring. Determination of the altered sphingosine contents upon sulfatide supplementation supports this conclusion (Figure 6D). Previous studies have demonstrated that sulfatide-mediated lysosomal swelling and the resultant toxicity (due to the deficiency in sulfatidase and/or saposin B) contribute to the pathogenesis of metachromatic leukodystrophy [12]. Thus we next investigated whether these processes were responsible for NB cell apoptosis upon sulfatide supplementation. Lysosomes were stained with LysoTracker Red DND-99 and visualized by confocal fluorescence microscopy. Importantly, experiments demonstrated a substantial enlargement of lysosomes in NB cells upon sulfatide supplementation (Figures 7A–7D and 7G). To examine the cumulative toxic effects of both lysosomal swelling and ceramide accumulation on sulfatide-induced NB cell apoptosis, we next investigated the cellular distribution of ceramide, and specifically its presence in lysosomes, by employing a fluorescent ceramide analogue (i.e. ceramide BODIPY® ) followed by confocal fluorescence microscopic analysis. These experiments demonstrated that ceramide was widely distributed throughout the cell, but was excluded from the nucleus in control cells (i.e. performed in the absence of sulfatides for 12 h) (Figure 7E), whereas ceramide dramatically accumulated, predominantly in smaller vesicles, upon supplementation with 60 µM sulfatide for 12 h (Figure 7H). Merging of the fluorescent staining patterns of ceramide distribution and lysosomal location upon sulfatide treatment revealed overlapping, primarily within smaller vesicles surrounding the nucleus (Figures 7F and 7I), indicating the accumulation of ceramide, probably within the membrane bilayer of smaller lysosomes. However, the marked accumulation of ceramides in other locations including the nucleus indicates that lysosomal swelling and ceramide toxicity might represent distinct processes in sulfatide-induced NB cell apoptosis. It should be pointed out that the accumulation of ceramide in the nucleus and the role of such an accumulation in cell apoptosis remain to be explored. Endosomes and lysosomes play distinct roles in sulfatide-induced NB cell apoptosis
Figure 6 Accumulation of ceramide and sphingosine in NB cells in a timeand/or dose-dependent manner upon sulfatide supplementation NB cells were grown and collected after supplementation with bovine brain sulfatides at the indicated concentrations for 3 days (A, B, D) or the treatment of the cells with 60 µM of sulfatides for the indicated time intervals (C). Lipids of the treated cells were extracted by using a modified Bligh and Dyer method and the contents of individual ceramide molecular species and sphingosine were quantified by using shotgun lipidomics as described in the Experimental section. The contents of the major ceramide molecular species including C16:0 - (䊏), C24:1 (♦) and C24:0 - (䊐) ceramides are shown. The results represent the means + − S.D. for three independent experiments. c The Authors Journal compilation c 2008 Biochemical Society
To clarify further (i) whether sulfatide uptake occurs through an endocytotic pathway, (ii) if lysosomal enlargement is due to sulfatide accumulation, and (iii) whether ceramide accumulation and lysosomal swelling play differential roles in NB cell apoptosis, we isolated and characterized the endosome-enriched and lysosome-enriched fractions from both NB control cells and those treated with bovine brain sulfatides (60 µM) for 48 h (Figure 8). The endosome fraction included both early and late endosomes. Surprisingly, shotgun lipidomics analyses demonstrated that not only was a high concentration of sulfatides (440 + − 35 nmol/mg of
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Figure 7
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Detection of enlarged cellular lysosomes and localization of ceramide after the treatment of NB cells with sulfatides
NB cells were treated with 0 µM (A, D, E), 60 µM (B, G, H), or 120 µM (C) bovine brain sulfatides for 12 h at 37 ◦C. Lysosomes were detected with LysoTracker Red DND-99 (A–D, G) by using a Zeiss LSM-510 laser confocal fluorescence microscope. A λex of 543 nm and a λem range 560–615 nm were used. The location of ceramide was determined with an analogue of ceramide BODIPY® (E, H) by using a λex of 488 nm and a λem range 515–545 nm. The samples were mounted and viewed using a Zeiss LSM-510 laser confocal fluorescence microscope. The merged images (F) and (I) are overlays of (D) and (E), and (G) and (H) respectively. Scale bar, 10 µm.
Figure 8 Western blot analysis of isolated endosome- and lysosomeenriched fractions Proteins (20 µg), each from endosome- and lysosome-enriched fractions as indicated, were loaded on to the gel and subjected to SDS/PAGE. Proteins were transferred on to Immobilon-P membranes and blocked for 30 min in blocking buffer (Tris-buffered saline, pH 7.6, 0.05 % Tween 20 and 3 % non-fat dried milk). After a 16 h, 4 ◦C incubation with primary antibody (1 µg/ml anti-LAMP2b or anti-EEA1, 1:500 dilution) diluted in blocking buffer followed by washing, the blot was incubated for 30 min with appropriate secondary anti-IgG–horseradish peroxidase conjugate. The membrane was washed three times for 10 min each and developed with SuperSignal chemiluminescent substrate (Pierce).
protein following treatment compared with undetectable levels of sulfatides in controls) present in the isolated endosome fraction, but substantial amounts of ceramide were also present (51 + − 4 nmol of ceramide/mg of protein following sulfatide treatment compared with 18 + − 2 nmol of ceramide/mg of protein in controls) (Figures 9A and 9B). The total increased sulfatide and increased ceramide contents in the isolated endosome fraction (from ∼ 4 × 107 cells) were 19 + − 5 and 1.6 + − 0.5 nmol (Figures 9C and 9D) respectively. This finding not only established a predominant pathway of sulfatide internalization by NB cells, but also localized the site of ceramide production and identified the importance of endosomes in sulfatide metabolism. Considerably larger yields of lysosome-enriched fractions could be isolated from sulfatide-treated NB cells in comparison with those from control cells, which was consistent with the observed enlargement of lysosomes by confocal fluorescence microscopy. Shotgun lipidomics analysis again showed the c The Authors Journal compilation c 2008 Biochemical Society
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Dramatic accumulation of sulfatides and ceramides in endosomes and lysosomes
NB cells were supplemented with bovine brain sulfatides or GalCer at a concentration of 0 µM (white bars), 60 µM sulfatide (black bars) or 60 µM GalCer (hatched bars) for 48 h at 37 ◦C. The endosome- and lysosome-enriched fractions were isolated by sucrose-density-gradient ultracentrifugation as described in the Experimental section. The concentrations of sulfatides and ceramides in the lipid extracts of these isolated fractions were determined by shotgun lipidomics (A, B). The total sulfatide or ceramide contents were calculated from the determined lipid concentrations shown ∗∗ ∗∗∗ in (A, B) and the total proteins in the isolated fractions (C, D). The results represent the means + − S.D. for three independent experiments. P < 0.01 and P < 0.001 by using a Student’s t test in comparison with controls.
substantial accumulation of sulfatides, increasing from below detectable levels in control to 485 + − 40 nmol/mg of protein in the sulfatide-treated cells (Figure 9A). Thus the total amount of sulfatides in the isolated lysosomes from treated cells (approx. 4 × 107 ) was 250 + − 25 nmol (Figure 9C). Similarly, a significant increase in the ceramide concentration in the isolated lysosome fraction following sulfatide treatment relative to controls (elevated from 8 + − 1 to 13 + − 2 nmol of ceramide/mg of protein) was also identified (Figure 9B). The total net increase in lysosomal ceramide content was approx. 2.5 nmol (Figure 9D). These results indicate that most of the internalized sulfatides accumulated in lysosomes, whereas endosomes produced large amounts of ceramides upon sulfatide supplementation.
Mechanisms leading to increased ceramide content in endosomes upon sulfatide supplementation
To determine the biochemical mechanism(s) leading to increased ceramide content in endosomes upon sulfatide supplementation, several experiments were performed. First, we sought to exclude the possibility that the increased ceramide content was due to internalization of ceramides present in the fetal bovine serum in the cell culture media. We determined the profile (Figure 10A) and content of ceramide in the serum and calculated that serum ceramides would maximally contribute to ∼ 40 mol% of the observed increase in endosomal ceramides assuming that all available serum ceramides were taken up through a phagocytotic process and present in the endosomes. However, the distinct profile of the ceramide molecular species present in endosomes and that in serum support a minimal contribution of serum cera c The Authors Journal compilation c 2008 Biochemical Society
mides (i.e. < 10 mol%) to the endosomal ceramide pool (Figure 10). One potential cellular response to the presence of excess sulfatide is the up-regulation or activation of nSMase. It is well known that lipid rafts and/or caveolae are highly enriched in sphingomyelin and are involved in most endocytotic pathways [44,45]. The next experiment was performed to determine whether increased sphingomyelin hydrolysis contributed to the increased ceramide content in endosomes. It was found that the content of ceramide in endosomes was reduced by greater than 20 mol% after treatment of NB cells with 60 µM sulfatide in the presence of an nSMase inhibitor (5 µM GW4869) for 2 days in comparison with controls performed in the absence of the inhibitor. Intriguingly, over 50 mol% of the GW4869-mediated ceramide decrease was in the content of C16:0 -ceramide molecular species, indicating that a large amount of C16:0 -ceramide resulted from the sphingomyelin hydrolysis upon sulfatide supplementation. Moreover, analysis of the ceramide molecular species profile in isolated endosome-enriched fractions showed that alterations in the contents of cellular C24:1 - and C24:0 -ceramides were predominant (Figure 10B), indicating the degradation of the internalized sulfatides in endosomes. To further support this mechanism, we conducted an additional experiment utilizing the β-galactosidase inhibitor, MTG (3 mM), during the incubation of NB cells with 60 µM sulfatide. The presence of MTG led to a 47 mol% reduction of the sulfatide-induced ceramide increase in isolated endosomes, suggesting that sulfatide hydrolysis via β-galactosidase activity contributed to most of the increased ceramide content in endosomes. Molecular species analyses demonstrated that the resultant reduction of ceramide content
Sulfatide-induced apoptosis
Figure 10
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Distinct profiles of ceramide molecular species present in endosomes after treatment of NB cells with sulfatide and those in serum
Lipids from serum or endosomes were isolated by sucrose-density-gradient ultracentrifugation after NB cells, treated with sulfatide (60 µM) for 48 h, were extracted after addition of internal standards by using a Bligh and Dyer procedure as described in the Experimental section. Mass spectra were acquired after direct infusion of the diluted lipid extract into an ESI mass spectrometer (Thermo Fisher TSQ Quantum Ultra) in the negative-ion mode as described in the Experimental section. MS analyses were performed by neutral loss scanning of 240.2 u (corresponding to the loss of 2-trans -palmitoleyl alcohol from negatively charged ceramide molecular species).
in the presence of the β-galactosidase inhibitor occurred mainly from C24:1 - and C24:0 -ceramide molecular species. Finally, the contribution of GalCer to the increased ceramide content in endosomes was examined, since the β-galactosidase inhibitor employed is not specific to blocking the hydrolysis of sulfatides but may prevent the degradation of many βgalactosyl-linked sphingolipids including GalCer [46]. Identical incubations of NB cells with GalCer (instead of sulfatide) led to a similar pattern of cell apoptosis as with sulfatide treatment. It was also found that a substantial amount of GalCer accumulated in endosomes (from an undetectable level to 291 + − 35 nmol/mg of protein after treatment of NB cells with 60 µM GalCer for 2 days) (Figure 11A), which was comparable with the endosomal accumulation of sulfatide after exogenous sulfatide treatment (Figure 9A). Surprisingly, in contrast with the dramatic accumulation of ceramide content in both endosomal and lysosomal compartments after treatment of NB cells with sulfatide, no significant increases in ceramide content in both endosomes and lysosomes were detected after supplementation of the cells with GalCer (Figure 9B, comparison of the different profiles shown in Figures 10B and 11B). This observation was further supported by results from the treatment of NB cells with d35 -C18:0 -GalCer, in which no resultant d35 -C18:0 ceramide at m/z 599.6 was detected (Figure 11B). These results led to the following conclusions. First, no GalCer hydrolysis activity was present under the experimental conditions examined, which was consistent with our observation that no GalCer intermediate was present during sulfatide hydrolysis. This supports a pathway of direct hydrolysis of sulfatide to ceramide, but not through a prior desulfation step before the generation of ceramide. Secondly, the mechanism(s) leading to cell apoptosis after treatment with GalCer were different from those with sulfatide. Since our focus in the present study was not on the consequences of GalCer treatment, identification of the biochemical mechanism(s) responsible for the GalCer-induced NB
cell apoptosis was not further explored. Thirdly, this set of experiments further confirms that the contribution of serum ceramide (through an internalization process) to the increased ceramide content in the endosomal compartment was minimal. Sulfatide/ceramide accumulation and cell apoptosis in primary neurons upon sulfatide supplementation
To verify that the demonstrated apoptosis of NB cells induced by sulfatide treatment was not due to an artefact of mitotic tumour cell lines, we conducted identical experiments with primary neurons. A very similar pattern of the sulfatide-induced changes in primary neurons to NB cells was demonstrated, including dramatic accumulation of sulfatide and ceramide contents in primary neurons as determined by shotgun lipidomics and the induction of substantial cell apoptosis, as substantiated by the TUNEL assay (Figure 12). Moreover, the patterns of accumulated sulfatide and ceramide contents in primary neurons upon supplementation of sulfatide (60 µM) for 6 h were essentially comparable with those in NB cells (compare Figures 12A and 12B with Figures 2C and 6C respectively). However, it was intriguing that two marked differences between primary neurons and NB cells upon treatment with sulfatide were present. First, in contrast with NB cells, sulfatide levels in primary neurons were found to decrease after treatment with sulfatide (60 µM) for 6 h (Figure 12A). Secondly, significant sulfatide-induced cell apoptosis occurred at a significantly lower concentration of sulfatide or after a considerably shorter interval of treatment (comparison of Figures 12C and 12D with Figures 5C and 5D). DISCUSSION
The present study has quantitatively investigated the mechanism of sulfatide uptake and sulfatide-induced apoptosis in neuronal c The Authors Journal compilation c 2008 Biochemical Society
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Accumulation of d35 -C18:0 -GalCer, but not d35 -C18:0 -ceramide in endosomes after treatment of NB cells with d35 -C18:0 -GalCer
NB cells were grown and collected after addition of d35 -C18:0 -GalCer at a concentration of 60 µM for 48 h. Endosomes from NB cells after treatment with d35 -C18:0 -GalCer were isolated by sucrose-density-gradient ultracentrifugation. Lipids of the isolated endosomes were extracted in the presence of internal standards (i.e. C15:0 -GalCer and C17:0 -ceramide for quantification of GalCer and Cer, respectively) and analysed by using shotgun lipidomics as described in the Experimental section. (A) Mass spectrum acquired from the neutral loss of 162 u (corresponding to the loss of hexose from lithiated GalCer molecular species) in the positive-ion mode. The abundant ion at m /z 769.7 corresponding to lithiated d35 -C18:0 -GalCer indicates the accumulation of GalCer in NB cells after treatment. (B) Negative-ion ESI tandem mass spectrum acquired from the neutral loss of 240.2 u (corresponding to the loss of 2-trans -palmitoleyl alcohol from negatively charged ceramide molecular species). The absent ion at m /z 599.5, corresponding to d35 -C18:0 -ceramide, indicates the absence of GalCer hydrolysis in the endosomes upon d35 -C18:0 -GalCer supplementation.
cells, which represents a cellular model of metachromatic leukodystrophy. By employing shotgun lipidomics, confocal fluorescence microscopy, flow cytometry and other techniques, we have demonstrated that: (i) NB cells readily internalize the sulfatide–methyl-β-cyclodextrin complexes from the medium through a phagocytotic process; (ii) a large portion of the internalized sulfatides were degraded directly to ceramide in the endosomal compartment; (iii) supplementation with sulfatides dramatically induced NB cell apoptosis; (iv) ceramide accumulation, lysosomal swelling and the resultant cytotoxicity probably contributed to NB cell apoptosis after sulfatide treatment; and (v) most importantly, accumulation of sulfatide and ceramide as well as cell apoptosis upon sulfatide supplementation were also demonstrated in primary neurons. To the best of our knowledge, direct degradation of sulfatides to ceramides has not been previously shown to occur in endosomes. These accumulated ceramides are clearly sufficient to initiate and/or participate in the programmed apoptotic cascade in NB cells and primary neuron cultures. Whether such a pathway is present in neurons in vivo remains to be investigated. However, we have previously demonstrated that sulfatide-containing ApoEassociated lipoprotein particles are present in the cerebrospinal fluid [16,21] and that these particles can be absorbed by neurons through an endocytotic pathway involving low-density lipoprotein receptors or related family members [21,25]. Alternatively, a deficiency in any of the enzymes involved in the degradation of the internalized sulfatide (e.g. sulfatidase and saposin B) results in its accumulation in lysosomes, leading to metachromatic leukodystrophy [12]. The lysosomal accumulation of sulfatides in this genetic disease strongly supports the role of endocytotic processes in sulfatide trafficking, metabolism and homoeostasis in the nervous system. Our current sulfatide-induced neuronal c The Authors Journal compilation c 2008 Biochemical Society
cell apoptotic model is closely analogous to that of the pathophysiological phenotype of metachromatic leukodystrophy. Similarities between the sulfatide-induced apoptosis of NB cells and primary neurons explored in the present study and that which occurs in metachromatic leukodystrophy may provide insights into the pathogenesis of this disease. For example, in addition to the toxic accumulation of sulfatides in the lysosomes of patients with metachromatic leukodystrophy, excessive production of ceramides by endosomal enzymes may also play an important role in the disease since ceramide accumulation is sufficient to induce cell apoptosis [27]. From the present study, it is also conceivable that neuronal cells that do not express the low-density lipoprotein receptor or its related family members (to absorb ApoE particles) may internalize these particles directly through a phagocytotic process. The significance of this apparently slower process in comparison with a receptor-mediated endocytotic process is unknown and remains to be explored. Recently, we have demonstrated a substantial depletion of sulfatides (accompanied by a dramatic elevation in ceramide content) in post-mortem brain tissues from subjects at the earliest clinically recognizable stages of AD relative to age-matched, cognitively normal controls [13,15]. To date, it is not clear whether the increased brain ceramide content of AD patients is generated directly from the accelerated sulfatide degradation that occurs during the pathogenesis of AD. However, our present study suggests that direct degradations of sulfatides to ceramides in both endosomes and lysosomes represent plausible mechanisms, although an activated nSMase activity may partially contribute to the increased ceramide content. Intriguingly, the profiles of the accumulated sulfatide and ceramide molecular species display some distinct differences. Since C24:1 - and C24:0 -sulfatide molecular species are predominant
Sulfatide-induced apoptosis
Figure 12 Substantial apoptosis occurred in parallel with the accumulation of sulfatides and ceramides in primary neurons Primary neurons of embryonic mouse cortex were prepared as described in the Experimental section. The primary neurons were grown and collected after treatment with bovine brain sulfatides at a concentration of 60 µM for the indicated time intervals (A, B, D), or at the indicated concentrations (C) for 24 h. The levels of sulfatide and ceramide (A, B) in the lipid extracts of the sulfatide-treated primary neurons were determined by using shotgun lipidomics as described in the Experimental section. Cell apoptosis (C, D) was determined through measurement of the DNA fragmentation of apoptotic cells using the APO-BrdU TUNEL assay kit at a λex of 488 nm. The fluorescence intensities of the emission between 515 and 545 nm (green) were determined by flow cytometry. The results represent the means + − S.D. for three independent experiments. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 by using a Student’s t test in comparison with controls.
in the supplemented bovine brain sulfatides (Figure 1), it is not surprising that these molecular species dramatically accumulated in NB cells upon their inclusion in the medium (Figure 2). C16:0 ceramide is the predominant molecular species under normal cell culture conditions (i.e. treatment for 0 h), which represents a major component of de novo biosynthesis and/or sphingomyelin hydrolysis (Figures 6B and 11B). In parallel with the profile of intracellular sulfatides that accumulated following treatment with exogenous sulfatides, C24:1 - and C24:0 -ceramide molecular species were significantly increased in a dose-dependent manner (Figure 6B). Decreases in the contents of C24:1 - and C24:0 ceramides upon inhibition of β-galactosidase activity support this pathway of ceramide generation. The significant accumulation of C16:0 -ceramide primarily resulted from sphingomyelin hydrolysis due to activated nSMase activity which occurred upon sulfatide supplementation, as evidenced by the addition of the nSMase inhibitor GW4869. On the basis of the depletion of sulfatides from the medium (Figure 3 and eqn 1), the corresponding internalization and accumulation of sulfatides in NB cells (Figure 2), and the increase in cellular ceramide and sphingosine contents (Figure 6), a quantitative comparison of sulfatide uptake and degradation was performed. It was found that sulfatide degradation was not severe after sulfatide supplementation for < 15 h, since the internalized amounts of sulfatide were essentially comparable with the accumulated amounts of sulfatides (compare Figure 2 with Figure 3) and the levels of sulfatide degradation products (i.e. ceramides and sphingosine) were relatively low. In contrast, after treatment of NB cells with sulfatides for > 15 h, the accumulated amounts of sulfatides were no longer equivalent to the internalized
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amounts, even when considering the generated amounts of ceramides and sphingosine, indicating that further degradation of sphingosine occurred. To verify that the demonstrated accumulation of toxic lipids and the resultant apoptosis of NB cells induced by sulfatide treatment also occurred in primary neurons, we performed essentially identical experiments with cultured mouse primary neurons. Interestingly, we not only demonstrated the rapid accumulation of sulfatide and ceramide in primary neurons as well as substantial apoptosis of the primary cells, but also found that the effects of abnormal sulfatide metabolism and trafficking on primary neurons were more severe than those occurring in NB cells under similar conditions. The results suggest that NB cells are capable of tolerating considerably higher levels of toxic lipids than primary neurons, probably due to a reduced sensitization to ceramide toxicity. The lower level of sulfatide accumulation manifest in primary neurons upon sulfatide supplementation (Figure 12A) strongly supports this possibility. However, we cannot exclude other potential explanations such as a more rapid generation of ceramides through other pathways in primary neurons in comparison with that in NB cells. During the present study, we investigated the potential biochemical mechanisms contributing to apoptotic sequelae of NB cells following sulfatide supplementation. By using shotgun lipidomics, confocal fluorescence microscopy and/or flow cytometry, we found the dramatic and specific accumulation of sulfatides and ceramides in NB cells in a time- and dose-dependent manner. Fractionation of subcellular organelles followed by shotgun lipidomics analyses showed that ceramides were primarily generated in endosomes, whereas sulfatides predominantly accumulated in lysosomes. We can reasonably assume that the protein concentrations present in endosomes and lysosomes are similar since these organelles are closely interrelated and their contents are interchangeable. Accordingly, the determined sulfatide and ceramide contents in the isolated endosome and lysosome fractions were directly compared. Notably, the sulfatide concentrations present in endosomes and lysosomes are not significantly different on sulfatide treatment (Figure 9A), indicating that inefficient and/or limited sulfatide degradation occurs in lysosomes, which probably serve as a storage depot for this sulfated lipid. In contrast, the substantially low concentration of ceramide in lysosomes in comparison with that in endosomes upon sulfatide treatment (Figure 9B) suggests that further degradation of ceramide or ceramide leakage to the cytosol or both occurs in lysosomes. The increased sphingosine content (Figure 6D) and the broad distribution of ceramide in the cells (Figure 7) support both possibilities. Therefore these results indicate that abnormal sulfatide metabolism induces neuronal cell apoptosis in which endosomes and lysosomes probably play distinct roles, i.e. the generation of ceramide and the accumulation of toxic lipids respectively. Collectively, the present study provides insights into the abnormal sulfatide trafficking and metabolism associated with AD and metachromatic leukodystrophy. This work was supported by the National Institute on Aging of the National Institutes of Health (grants R01 AG23168 and R01 AG31675) and by the Neurosciences Education and Research Foundation.
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