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Journal of Lipid Research, Vol. 42, 1197-1202, August 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

Apoptosis of Neuro2a cells induced by lysosphingolipids with naturally occurring stereochemical configurations

Correspondence to: Makoto Ito, To whom correspondence should be addressed., makotoi{at}agr.kyushu-u.ac.jp (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Lysosphingolipids, which lack the fatty acid moiety of sphingolipids, are known to be accumulated in some variants of sphingolipid storage diseases. Here, we report that lysosphingolipids with naturally occurring stereochemical configurations induce apoptosis in mouse neuroblastoma Neuro2a cells. The intracellular dehydrogenase activity and [³H]thymidine incorporation of Neuro2a cells were strongly suppressed by the addition of lysosphingolipids in a dose-dependent manner, whereas the parental sphingolipids had no effect. Intranucleosomal DNA fragmentation, chromatin condensation, and phosphatidylserine externalization, which are typical features of apoptosis, were observed when the cells were cultured with 40;–80 µM of lysosphingolipids for 24;–48 h in the presence of 5% fetal calf serum. Activation of caspase-3-like enzyme occurred after addition of lysosphingolipids followed by incubation at 37°C for 24 h. The addition of an inhibitor of caspases, ZVAD-fmk, to the Neuro2a cell culture completely inhibited the elevation of caspase-3 activity but not the DNA fragmentation.

These results may indicate that a caspase-3 independent signaling pathway is involved in the lysosphingolipid-induced apoptosis and suggest that accumulation of lysosphingolipids, but not parental sphingolipids, triggers the apoptotic cascade in neuronal cells of patients with sphingolipidoses. — Sueyoshi, N., T. Maehara, and M. Ito. Apoptosis of Neuro2a cells induced by lysosphingolipids with naturally occurring stereochemical configurations. J. Lipid Res. 2001. 42: 1197;–1202.

Supplementary key words: apoptosis, sphingolipidosis, lysosphingolipid, sphingolipid, glycosphingolipid, sphingolipid ceramide N-deacylase

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Sphingolipids may function as endogenous and exogenous modulators for various cell functions (1) (2) (3). Lysosphingolipids, which lack the fatty acid moiety of sphingolipids, have been detected in normal tissues at very low levels (4) and are accumulated in some cases of inherited sphingolipid storage disease (5) (6) (7). Several reports indicate the possible involvement of lysosphingolipids in the pathogenesis of sphingolipidosis. Miyatake and Suzuki (8) suggested that the galactosylpsychosine (galactosylsphingosine), which accumulated in patients with Krabbe disease, a ß-galactosylceramidase-deficient lysosomal disease, caused widespread demyelination. It was suggested that the accumulation of galactosylpsychosine could account for the formation of multinuclear globoid cells in the brains of patients with globoid cell leukodystrophy due to inhibition of cytokinesis and actin reorganization (9). Accumulation of sphingosylphosphorylcholine (SPC), a lyso-derivative of sphingomyelin (SM), may contribute to the pathophysiology of Niemann-Pick disease type A by deleteriously affecting mitochondrial function and Ca²+ uptake (10). Apoptotic cells have actually been observed in the brains of patients with sphingolipidoses (11). However, little is known about the cause of apoptosis in neural cells of sphingolipidoses.

We found a novel sphingolipid-degrading enzyme [sphingolipid ceramide N-deacylase (SCDase)] in the culture supernatants of Pseudomonas sp. TK4 (12) and Shewanella alga NS589 (13). The enzyme hydrolyzes the N-acyl linkage of various glycosphingolipids and SM to produce their lyso-forms. Using SCDase or SCDase-producing bacteria as a microbial catalyst, various lysosphingolipids were successfully prepared in good yield and high purity (13) (14). SPC thus prepared showed the naturally occurring D-erythro-(2S, 3R)-configuration, whereas the chemically prepared SPC is a mixture of D-erythro/L-threo;–stereoisomers (13).

Several reports have indicated that lysosphingolipids are accumulated with their parental sphingolipids in sphingolipidosis (5) (6) (7), and apoptosis has actually been observed in the brain of some gangliosidosis patients (11). Nevertheless, there is no report that describes the relationship between lysosphingolipid accumulation and apoptosis. This report describes for the first time that lysosphingolipids, but not sphingolipids, with naturally occurring stereochemical configurations induce apoptosis in mouse neuroblastoma Neuro2a cells via a caspase-3 independent pathway. This result strongly suggests that accumulation of lysosphingolipids, but not sphingolipids, triggers the apoptotic cascade in neuronal cells of patients with sphingolipidoses.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Materials
A crude ganglioside mixture was prepared from bovine brain by the method described in (15). GM1a was prepared using sialidase-producing Pseudomonas sp. YF-2 as described previously (16). GM2, prepared from the brain of a patient with Tay-Sachs disease, was kindly donated by Dr. Y. Hirabayashi (RIKEN, Japan). DMEM and PBS were obtained from Nissui Seiyaku Co., Ltd. (Japan). SM (from egg yolk) and RNase A were purchased from Sigma. [Methyl-1', 2'-³H]thymidine (119 Ci/mmol) was purchased from Amersham Pharmacia Biotech and proteinase K from Wako (Japan). The cell counting kit was from Dojindo (Japan). All other chemical reagents were of analytical grade. Bacterial SCDase was prepared as described in (14) or purchased from Takara Shuzo Co. (Japan).

Preparation of various lysosphingolipids
Naturally occurring D-erythro-SPC was prepared from SM using SCDase-producing Shewanella alga NS589 as a microbial catalyst as described in (13). Lyso-GM1a and lyso-GM2 were prepared from GM1a and GM2, respectively, by using SCDase from Pseudomonas sp. TK4 as described in (14). Fast atom bombardment mass spectrometry and NMR confirmed the structure of the lysosphingolipids prepared (13).

Cell culture
Mouse neuroblastoma Neuro2a cells were obtained from Dr. H. Higashi (Mitsubishi Kasei Institute of Life Sciences, Japan). Neuro2a cells were cultured in DMEM supplemented with 2 mM glutamine, 60 mg/l kanamycin, and 10% calf serum at 37°C in an atmosphere of 95% air and 5% CO₂.

Assay of cell viability by the modified MTT assay
The viability of the cells was evaluated by the modified MTT assay using a cell counting kit (Dojindo, Japan). The principle of MTT and its modified assay is the same but the cell counting kit utilizes WST-1 [2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, sodium salt] as a substrate instead of MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]. WST-1 is reduced by intracellular dehydrogenase to generate WST-1-formazan, which shows the maximal absorbance at 450 nm. Thus, a decrease in the absorbance at 450 nm represents the reduction of the viability of cells.

Assay for DNA synthesis
Neuro2a cells were seeded at the concentration of 4 x 10⁴ cells/well in a 96-well plate containing 200 µl of DMEM supplemented with 2 mM glutamine, 60 mg/l kanamycin, and 5% FCS. After incubation at 37°C for 4 h, the cells were treated with SM, GM1a, GM2, or their lyso-derivatives at specific concentrations. After 18 h, 0.1 µCi of [³H]thymidine was added and the cultures were incubated at 37°C for 4 h. The [³H]thymidine incorporation into trichloroacetic acid-insoluble materials was measured by a LS6500 liquid scintillation counter (Beckman).

Flowcytometric analysis
Neuro2a cells were incubated at 37°C for 48 h in DMEM supplemented with 5% FCS containing GM1a (80 µM), lyso-GM1a (80 µM), or stearic acid (40 µM). Then, an aliquot of the cells (1.5 x 10⁶) was washed, fixed with 50% ethanol, and then stained with propidium iodide (PI). Flowcytometric analyses were performed by FACStar plus (Becton-Dickinson).

Measurement of phosphatidylserine externalization by annexin V binding
Neuro2a cells (1 x 10⁶ cells) were pelleted and resuspended in binding buffer (10 mM HEPES-NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂), and then incubated at room temperature for 20 min with 1 µg/ml FITC-labeled annexin V (Dako Japan Co., Ltd., Japan) and 2 µg/ml PI. The extent of annexin V staining in PI-negative populations of Neuro2a cells was determined by flowcytometric analysis with a FACSCalibur (Becton-Dickinson).

Apoptotic morphology
Neuro2a cells were treated with several concentrations of lyso-GM1a in DMEM supplemented with 2 mM glutamine, 60 mg/l kanamycin, and 5% FCS at 37°C for 48 h. The cells (5 x 10⁵) were fixed with a 1% glutaraldehyde solution for 1 h, collected by centrifugation at 2,000 rpm for 5 min, and then resuspended in PBS. The cell nuclei were stained with Hoechst 33258 (Wako, Japan) and examined under a fluorescent microscope with a UV combination filter.

Agarose gel analysis of DNA fragmentation
DNA fragmentation was analyzed by agarose gel electrophoresis as described by Selins and Cohen (17). In brief, fragmented genomic DNA was extracted by incubating cells in a 10 mM Tris-HCl buffer, pH 7.5, containing 10 mM EDTA, and 0.5% Triton X-100 at 4°C for 10 min. Under these conditions, intact genomic DNA was not extracted. The cell suspension was centrifuged at 15,000 rpm for 20 min, and the resultant supernatants were incubated at 37°C for 1 h in the presence of RNase A (400 µg/ml) and then for an additional 1 h in the presence of proteinase K (400 µg/ml). The fragmented DNA was precipitated with isopropyl alcohol and then analyzed by 2% agarose gel electrophoresis.

Preparation of the cytosolic extracts
The cytosolic extracts of apoptotic cells were prepared essentially as described by Lazebnik et al. (18). Briefly, cells were treated with an extraction buffer containing 50 mM PIPES-NaOH, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl₂, 1 mM dithiothreitol (DTT), 20 mM cytochalasin B, and a mixture of protease inhibitors (1 mM PMSF, 1 µg/ml pepstatin, 50 µg/ml antipain, and 10 µg/ml chymopapain) and then collected in a microtube. Cells were disrupted by five cycles of freezing and thawing. Crude extracts were obtained by centrifugation at 10,000 g for 15 min at 4°C and the supernatants were used as the cytosolic fraction.

Assay for caspase-3-like enzyme activity
Cell lysates (5 µg protein) were diluted to 150 µl with ICE standard buffer consisting of 100 mM HEPES-KOH, pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mM DTT, and 0.1 mg/ml ovalbumin, and incubated at 30°C for 30 min with 2 µM fluorescent substrate Ac-DEVD-MCA (Peptide Institute Inc., Japan). The fluorescence of the cleaved substrates was determined using a microplate reader (SHIMADZU CS-9300PC, Japan) set at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The viability of Neuro2a cells, which was defined as a reduction in intracellular dehydrogenase activity, decreased with the addition of lyso-GM1a and lyso-GM2 to the cell culture at 40 µM in the presence of 5% FCS, whereas GM1a and GM2 had no effect ( Fig 1A). The effect of lyso-GM1a on the cell viability was dose dependent (Fig 1B). [³H]thymidine incorporation was also strongly suppressed by the addition of SPC but not by SM (Fig 1C). Interestingly, after treatment with 80 µM lyso-GM1a, the genomic DNA of Neuro2a cells showed an apparent hypodiploid DNA peak that was clearly distinguished from the diploid DNA peak on flowcytometric analysis ( Fig 2A). The hypodiploid DNA peak was also found in apoptosis of mouse cytotoxic T-cell line CTLL-2 induced by ISP-1 that inhibits serine palmitoyltransferase (19). However, GM1a and stearic acid did not affect the genomic DNA (Fig 2A). After treatment with 50;–100 µM SPC or lyso-GM1a, the genomic DNA of Neuro2a cells showed a ladder pattern of fragmentation on agarose gel electrophoresis, which is a typical feature of apoptosis (Fig 2B). The DNA ladder was also observed after treatment with lyso-GM2 and glucosylpsychosine (glucosylsphingosine) at the same concentrations (data not shown). In addition, lyso-GM1a-treated cells showed chromatin condensation and nucleus fragmentation after staining with Hoechst 33258 (Fig 2C). To examine whether the lysosphingolipids induce cell death by apoptosis or by necrosis, flowcytometric analysis of Neuro2a cells was performed after double-staining with PI and annexin V, which could determine the membrane permeability (necrosis) and phosphatidylserine externalization (apoptosis), respectively. PI should be excluded from living cells. Phosphatidylserine externalization was found to increase in PI-negative populations of Neuro2a cells when treated with either SPC or lyso-GM1a (Fig 2D). These results clearly indicate that lysosphingolipids, but not parental sphingolipids, induce apoptosis in Neuro2a cells.

View larger version (27K): [in this window] [in a new window]   Figure 1. Growth inhibition of Neuro2a cells induced by lysosphingolipids. A: Cell viability of Neuro2a cells after treatment with gangliosides and their lyso-derivatives. Cells (4 x 104) were seeded into wells of a 96-well plate and cultured at 37°C overnight in DMEM supplemented with 5% FCS. Then, lyso-derivatives or parental gangliosides were added to the cell culture at a concentration of 40 µM. After 15 h, the viability of the cells was evaluated by the modified MTT assay using a cell counting kit (Dojindo, Japan) as described in Materials and Methods. B: Dose-dependent inhibition of cell viability by lyso-GM1a. Neuro2a cells were treated with several concentrations of lyso-GM1a at 37°C for 15 h and the cell viability was measured by the method described in A. C: DNA synthesis of Neuro2a cells after treatment with SM and its lyso-derivative (SPC). Cells (4 x 104) were seeded into wells of a 96-well plate. After 4 h, the cells were treated with SPC or SM for 18 h at the concentrations indicated. Then, 0.1 µCi [3H]thymidine was added and the cells incubated for 4 h. The incorporation of [3H]thymidine into trichloroacetic acid-insoluble materials was measured by a liquid scintillation counter. O.D., optical density.

View larger version (34K): [in this window] [in a new window]   Figure 2. Apoptosis of Neuro2a cells induced by lysosphingolipids. A: Flowcytometric analysis of Neuro2a cells. Cells were incubated at 37°C for 48 h with GM1a (80 µM), lyso-GM1a (80 µM), or stearic acid (40 µM) in DMEM supplemented with 5% FCS. Then, an aliquot of the cells (1.5 x 106) was washed, fixed with 50% ethanol, and stained by PI. Flowcytometric analyses were performed by FACStar plus (Becton-Dickinson). B: DNA ladder fragmentation. Neuro2a cells (5 x 105) in 2 ml were incubated at 37°C for 48 h with various concentrations of SPC or lyso-GM1a. Fragmented genomic DNA was then extracted and analyzed by 2% agarose gel electrophoresis as described in Materials and Methods. M; DNA marker X174/HincII digest. C: Chromatin condensation and nucleus fragmentation. An aliquot of Neuro2a cells was fixed with 1% glutaraldehyde for 1 h. Then, the nuclei were stained with Hoechst 33258 and observed under a fluorescence microscope as described in Materials and Methods. Arrowheads indicate the cells with condensation of chromatin. D: Phosphatidylserine externalization. Neuro2a cells were incubated at 37°C for 36 h in the presence or absence (control) of 60 µM lysosphingolipids. Cells were then double-stained with FITC-labeled annexin V and PI without fixation, and then subjected to flowcytometric analysis described in Materials and Methods. The extent of annexin V staining in PI-negative populations of Neuro2a cells was compared between lysosphingolipid-treated and control cells.

We examined whether caspase-3-like enzymes, which are proteases involved in most apoptotic processes, participate in lysosphingolipid-induced apoptosis using a specific substrate, Ac-DEVD-MCA. This chromogenic tetrapeptide is specifically hydrolyzed by caspase-3 (CPP32), but not caspase-1 (ICE) (20). In Neuro2a cells, an elevation of caspase-3-like activity was observed after addition of lyso-GM1a or SPC at 80 µM followed by incubation at 37°C for 24;–36 h ( Fig 3A). Simultaneously, a DNA ladder was detected on agarose gel electrophoresis (Fig 3B). The addition of an inhibitor for caspases, ZVAD-fmk, to the Neuro2a-cell culture inhibited the elevation in the protease activity (Fig 3C) but not the DNA fragmentation (Fig 3D). This result may indicate that a caspase-3-independent signaling pathway is involved in the apoptosis induced by lysosphingolipids. An apoptosis pathway that is not inhibited by ZVAD-fmk has also been observed in adenovirus death factor-induced apoptosis (21).

View larger version (59K): [in this window] [in a new window]   Figure 3. Activation of caspase-3-like enzyme by lysosphingolipids. A: Time course for caspase-3-like activity. Neuro2a cells were incubated at 37°C in the absence (control) or presence of lysosphingolipids (80 µM) for the indicated times. Cytosolic extracts were prepared from cells and the caspase-3-like activity in the lysates was measured using the fluorescent substrate Ac-DEVD-MCA. B: Time course for genomic DNA fragmentation. The genomic DNA was extracted from the cells and then analyzed by electrophoresis. C: Inhibition of caspases by specific inhibitor. Neuro2a cells were incubated at 37°C for 24 or 48 h with 80 µM of lyso-GM1a in the presence or absence of 50 µM ZVAD-fmk and the enzyme activity of cell lysates was determined after washing out the inhibitor from the cells. For 48 h incubation in the presence of inhibitor, the medium was changed at 24 h to a fresh one containing the inhibitor at the same concentration. D: Effects of an inhibitor for caspases on genomic DNA fragmentation. Details are described in Materials and Methods.

In general, induction of apoptosis is somewhat difficult for neuronal cells. Actually, we confirmed that apoptosis-inducing reagents such as camptothecin, actinomycin D, and cycloheximide had no effect in Neuro2a cells under the present conditions ( Fig 4A), although these reagents efficiently induced apoptosis in human promyelocytic leukemia HL60 cells (Fig 4B). Lysosphingolipids induced apoptosis in Neuro2a cells (Fig 4A) and thus will prove a useful reagent for inducing apoptosis experimentally in neuronal cells in vitro as well as in vivo.

View larger version (40K): [in this window] [in a new window]   Figure 4. Sensitivity of Neuro2a cells to various apoptosis inducers. A: DNA ladder fragmentation of Neuro2a cells. Cells were treated at 37°C for 24 h with actinomycin D (2 µg/ml), camptothecin (10 µM), cycloheximide (200 µg/ml), valinomycin (20 µM), H2O2 (400 µM), or lysosphingolipids (100 µM). The genomic DNA was then extracted and analyzed by electrophoresis. B: DNA ladder fragmentation of human promyelocytic leukemia HL60 cells. The same experiment as described in A was performed using HL60 cells instead of Neuro2a cells.

Sphingolipidoses are inherited storage diseases resulting from an inability to catabolize sphingolipids due to a dysfunction of sphingolipid-degrading enzymes. Several lines of evidence indicate that lyso-forms of sphingolipids are accumulated with their parental sphingolipids in some cases of sphingolipidosis. For example, SPC is accumulated in the brains of patients with Niemann-Pick type A (5), lyso-GM1a and lyso-GM2 in those with GM1- and GM2-gangliosidosis, respectively (6) (7), and galactosylpsychosine in those with globoid cell leukodystrophy (22). The level of galactosylpsychosine in brain tissue of the twitcher mouse, which has a deficiency of lysosomal galactosylceramidase, increased up to 15 nmol/g wet weight (23). Similarly, the amount of lyso-GM2 in the brain of a patient with GM2-gangliosidosis was calculated to be 15 nmol/g wet weight, corresponding to an overall concentration of 15 µM (7). These data suggest that neuronal cells in some cases of sphingolipidosis would be exposed topically to lysosphingolipids at the concentrations described in the present study (40;–80 µM). It should be noted that the minimum concentration of lysosphingolipids to induce cytotoxic effects in Neuro2a cells decreased to 10;–30 µM if FCS was removed from the cell culture (data not shown).

Apoptosis has actually been observed in the brain in mouse models of GM2 gangliosidosis and in autopsy samples of brain and spinal cord from patients with Tay-Sachs and Sandhoff diseases (11). Recently, an apoptotic depletion of oligodendrocytes has also been reported in the twitcher mouse (24). Our study strongly suggests that lysosphingolipids, but not sphingolipids, are a potent inducer for apoptosis of neuronal cells in sphingolipidosis.

Lysosphingolipids are present in normal cells/tissues in very small quantities and accumulated in sphingolipidosis patients. However, the mechanism for the generation of lysosphingolipids in vivo has not yet been clarified in either the normal or pathogenic state. It has been reported that UDP-galactose:ceramide galactosyltransferase is involved in the formation of galactosylpsychosine, utilizing sphingosine as an acceptor instead of ceramide (25). An alternative pathway has also been reported; i.e., an acidic ceramidase hydrolyzed the N-acyl linkage of not only free ceramide but also ceramide in glucosylceramide to generate glucosylpsychosine (26). It is interesting that a nonmicrobial SCDase-like enzyme, which cleaves the N-acyl linkage of ceramides in SM as well as glucosylceramide to produce respective lyso-forms, was found in the skin tissue of patients with atopic dermatitis (27). Further study is necessary to reveal the molecular mechanisms for the generation of lysosphingolipid in the sphingolipidoses.

	FOOTNOTES

Abbreviations: FCS, fetal calf serum; GM1a, Galß1-3GalNAcß1-4(NeuAc2-3)Galß1-4Glcß1-1'ceramide; GM2, GalNAcß1-4(NeuAc2-3)Galß1-4Glcß1-1'ceramide; PI, propidium iodide; SCDase, sphingolipid ceramide N-deacylase; SM, sphingomyelin; SPC, sphingosylphosphorylcholine.

	ACKNOWLEDGMENTS

We wish to thank Dr. Takashi Nakamura, Kyushu University, for the valuable discussions and encouragement throughout the course of this work. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (B) (12140204) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Manuscript received October 31, 2000; and in revised form March 6, 2001; and in revised form April 16, 2001

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