1-Methylthiodihydroceramide, a Novel Analog of Dihydroceramide, Stimulates Sphinganine Degradation Resulting in Decreased de Novo Sphingolipid Biosynthesis*

1-Methylthiodihydroceramide (10 μm) decreased de novo ceramide biosynthesis by about 90% in primary cultured cerebellar neurons. Accordingly, de novo formation of sphingomyelin and of glycosphingolipids, all of which contain ceramide in their backbone, was reduced in a time- and concentration-dependent manner by up to 80%. Complex sphingolipid synthesis was restored upon addition of dihydroceramide or ceramide, in micromolar concentrations, to the culture medium, suggesting that none of the glycosyltransferases involved in glycosphingolipid biosynthesis is inhibited by this analog. Assays of the enzymes catalyzing sphinganine biosynthesis, as well as its N-acylation to form dihydroceramide, revealed that they were also not affected. In contrast, there was a 2.5-fold increase in the activity of sphinganine kinase. Reduction of de novosphingolipid biosynthesis by 1-methylthiodihydroceramide is therefore due to its ability to deplete cells of newly formed free sphinganine. As a consequence of depletion of sphinganine levels, 1-methylthiodihydroceramide disrupted axonal growth in cultured hippocampal neurons in a manner similar to that reported for direct inhibitors of sphingolipid synthesis; thus, there was essentially no axon growth after incubation with 1-methylthiodihydroceramide between days 2 and 3, and co-incubation with short acyl chain analogs of ceramide (5 μm) antagonized the inhibition of growth. Interestingly, the d-erythro and thel-threo isomere were equally effective, but the corresponding free base as well as other structurally related compounds did not affect either sphingolipid biosynthesis or neuronal growth.

they are primarily components of the plasma membrane. SLs contain a ceramide backbone, which anchors them in the outer leaflet of the lipid bilayer. The ceramide backbone can be modified by attachment of phosphorylcholine, to form sphingomyelin (SM), or by attachment of one or more sugar residues, to form glycosphingolipids (GSLs). GSLs form cell-and speciesspecific profiles known to change characteristically during development, differentiation, and transformation, suggesting that they play a role in cell-cell interactions and in cell adhesion (1). Gangliosides, the sialic acid containing GSLs, are particularly abundant in neuronal cells, and their involvement in neuritogenesis and possibly synaptogenesis has been extensively studied (2,3). During the last few years studies have also been initiated to examine the role of endogenous SLs in neuronal growth (4 -7). In these studies inhibitors of SL biosynthesis, such as fumonisin B 1 (FB 1 ) and PDMP (DL-threo-1phenyl-2-decanoylamino-3-morpholino-1-propanol), as well as inhibitors of lysosomal GSL degradation, like conduritol Bepoxide (5), have been employed. In addition, evidence has emerged during the last few years that SL metabolites such as ceramide (Cer), sphingosine, and sphingosine 1-phosphate (SPP) play an important role as intracellular signaling molecules for a variety of different targets (8). Sphingosine and SPP, originally proposed as negative regulators of protein kinase C (9), were shown to play alternative signaling roles as mitogenic second messengers (8). Cer is involved in what has become known as the "sphingomyelin cycle" (10). For example, Cer serves as a mediator of cellular senescence (11), apoptosis, and differentiation in many cell types (12,13). The increasing amount of data concerning the role of SL metabolites in cellular signal transduction strongly suggests that SL metabolism is tightly regulated. The identification of factors interfering with SL metabolism and the examination of their mode of action is therefore of great importance.
Much of the current knowledge on SL metabolism has been derived from studies with compounds specifically inhibiting defined steps of SL biosynthesis (14). Taking into consideration that dihydroceramide (DH-Cer) does not mimic the effects of Cer in signaling pathways (15,16), we are studying the effects of DH-Cer analogs on SL biosynthesis and on neuronal growth. We now demonstrate that treatment of cerebellar neurons with 1-methylthiodihydroceramide (1-MSDH-Cer) strongly interferes with de novo Cer synthesis, and hence SL formation, by stimulating the catabolism of sphinganine, a vital precursor of * This work was supported by Deutsche Forschungsgemeinschaft SFB 284 and by the German-Israeli Foundation for Scientific Research and Development. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Materials-Six-day-old NMRI (Navy Marine Research Institute) mice were obtained from Dr. Brigitte Schmitz from the Institut fü r Anatomie und Physiologie der Haustiere of the University of Bonn, Germany. Embryonic day 18 Wistar rats were from the Weizmann Institute Breeding Center, Rehovot, Israel.
Cell Culture-Granule cells were cultured from cerebella of 6-day-old mice according to the method of Trenkner and Sidman (20). Cells were isolated by mild trypsinization (0.05%, w/v) and dissociated by repeated passage through a constricted Pasteur pipette in a DNase solution (0.1%, w/v). The cells were then suspended in DMEM containing 10% heat-inactivated horse serum and plated onto poly-L-lysine-coated 35-mm diameter Petri dishes (Costar) (6 ϫ 10 6 cells/dish). 24 h after plating, cytosine arabinoside was added to the medium (4 ϫ 10 Ϫ5 M) to arrest the division of non-neuronal cells (21).
Hippocampal neurons were cultured at low density as described (22) with some modifications (5). In brief, the dissected hippocampi of embryonic day 18 rats (Wistar) were dissociated by trypsinization (0.25% w/v, for 15 min at 37°C). The tissue was washed in Mg 2ϩ /Ca 2ϩ -free Hank's balanced salt solution (Life Technologies, Inc.) and dissociated by repeated passage through a constricted Pasteur pipette. Cells were plated in MEM with 10% horse serum, at a plating density of 6,000 cells per 13-mm glass coverslip that had been precoated with poly-L-lysine (1 mg/ml). After 3-4 h, coverslips were transferred into 100-mm Petri dishes (Nunc) containing a monolayer of astroglia. Cultures were maintained in serum-free medium (MEM) which included N2 supplements, ovalbumin (0.1%, w/v) and pyruvate (0.1 mM).
Analysis of Neuronal Morphology in Cultured Hippocampal Neurons-Stock solutions of the DH-Cer analogs in ethanol were added to cultures of hippocampal neurons to give final concentrations of 10 M; control cultures were incubated with ethanol alone (final concentration 1%). In some experiments, neurons were incubated with a short acyl chain analog of Cer, N-6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminohexanoyl-D-erythro-ceramide (C 6 -NBD-Cer) (5 M), dissolved in ethanol to give a final ethanol concentration in the medium of 1%. After various times, neurons were fixed in 1% glutaraldehyde in phosphate-buffered saline for 20 min at 37°C and mounted for microscopic examination. Neurons were examined by phase contrast microscopy using a Zeiss Axiovert 35 microscope (Achroplan 32 ϫ/0.4, Ph 2 objective). Axons were identified as long, thin processes of uniform diameter (22), and the parameters of branching were determined as described previously (5). In brief, an axon was considered to branch when the process that it gave rise to was more than 15 m long. Thin filipodia, which were occasionally observed along the entire length of the axon, were not considered as branches. Only those cells in which the whole axon plexus could be unambiguously delineated were measured. Cells with no axon (i.e. cells in which the longest process was less than 20 m longer than the next longest (minor) process; non-polarized cells) were excluded from length measurements as were cells in which more than one axon emerged from the cell body. Values were pooled from two separate cultures (in which 30 -50 cells were counted per coverslip on two individual coverslips per treatment) and statistical analysis performed using the Student's t test.
Sphingolipid Labeling, Extraction, and Analysis-After 4 to 5 days in culture, cerebellar neurons were rinsed two times with serum-free MEM and incubated in the presence of the DH-Cer analogs added as complexes with bovine serum albumin to the culture medium (MEM) containing 0.3% heat-inactivated horse serum. Metabolic labeling of SLs was performed as described previously (23). The SLs were labeled by incubation with [ 14 C]serine (2 Ci/ml), 14 Clabeled Cer, or DH-Cer (0.125 Ci/ml). After the indicated times cells were harvested and lipids extracted from cell pellets with 6 ml of chloroform/methanol/water/pyridine (10:5:1:0.1, by volume) for 24 -48 h at 50°C. Phospholipids were degraded by mild alkaline hydrolysis with methanolic NaOH (50 mM) for 2 h at 37°C. The lipid extracts were desalted by reversed-phase chromatography on LiChroprep RP18, applied to TLC plates, and chromatographed with chloroform/methanol, 0.22% CaCl 2 (60:35:8, by volume); SLs were visualized by autoradiography and identified by their R F values and enzymatic digestion (23). To separate Cer from DH-Cer, radioactive bands comigrating with authentic Cer were scraped off the TLC plate, re-extracted, and re-chromatographed on borate plates developed in chloroform/methanol (90:10, by volume). Radioactive bands were evaluated by the bio-imaging analyzer Fujix Bas 1000 using software TINA 2.08 (Raytest, Straubenhardt, Germany) and additionally scraped from the TLC plate and measured by liquid scintillation counting.
Analysis of Sphingosine and Sphinganine Mass-After treatment of cells with DH-Cer analogs (see above), mass measurements of free sphingoid bases were conducted by HPLC (24) using C 20 -sphinganine as an internal standard. Briefly, lipids were extracted from cell pellets, and extracts were then hydrolyzed in 50 mM NaOH (2 h, 37°C) to remove phospholipids. Free sphingoid bases were subsequently derivatized with o-phthaldialdehyde and determined by HPLC. C 18 -sphinganine and C 18 -sphingosine were identified by comparison of their retention times with that of the respective standards. To determine total SL mass, the lipid extracts were acid-hydrolyzed according to Gaver and Sweeley (25) with 0.5 N argon-saturated, methanolic HCl for 16 h at 50°C to liberate sphingoid bases from GSLs, ceramides, and SM, prior to determination by HPLC as mentioned above.
Losses during extraction or hydrolysis were considered by addition of a defined amount of C 12 C 12 -ceramide to replicate samples. C 12 C 12ceramide was obtained by N-acylation of C 12 -sphingosine, as described (18); C 12 -sphingosine was synthesized according to published methods (26).
The values obtained for sphingosine and sphinganine were corrected for free sphingosine and free sphinganine, as determined above, to give the complex SL content (total minus free).
Enzyme Assays-Cell homogenates obtained by sonication of the cell pellet in the respective incubation buffer for 2 min on ice were used as an enzyme source in the enzyme assays described below, except for sphinganine kinase.
Serine palmitoyltransferase was measured (27) using [ 14 C]serine and palmitoyl-CoA as substrates. The assay mixture contained 0.1 M Hepes (pH 7.4), 5 mM dithiothreitol, 10 mM EDTA, 50 M pyridoxal phosphate, 1.2 mM [ 14 C]serine (1.6 Ci), 0.15 mM palmitoyl-CoA, and 120 g of cell protein, in a total volume of 100 l. After incubation for 10 min at 37°C, reactions were terminated by addition of chloroform/ methanol (5:3, by volume). The lipids were extracted by phase separation and applied to a TLC plate that was developed with chloroform/ methanol, 2 M NH 3 (40:10:1, by volume). The radiolabel in 3-dehydrosphinganine was measured by scanning or cutting out the regions of interest and scintillation counting.
3-Dehydrosphinganine reductase was determined using 3-dehydrosphinganine and NADPH as substrates. 3-Dehydrosphinganine was synthesized as described (26). The reaction mixture contained 0.1 M phosphate buffer (pH 7.0), 1 mM MgCl 2 , 0.5% (w/v) Nonidet P-40, 150 M NADPH, 120 M dehydrosphinganine, and 50 g of cell protein in a total volume of 100 l. After 15 min at 37°C the reaction was terminated by addition of 250 l of chloroform and 100 l of methanol. Lipid extraction was performed as described for serine palmitoyltransferase. Sphinganine was determined by HPLC as described above. Background values were determined in negative controls in which boiled protein was used as the enzyme source.
Sphinganine N-acyltransferase was assayed using D-erythro-[4,5-3 H]sphinganine (obtained as described before (3)) and stearoyl-CoA as substrates. The reaction contained 0.1 M Tris buffer (pH 7.4), 0.5 mM dithiothreitol, 100 M stearoyl-CoA, 50 M labeled sphinganine (0.5 Ci), which was previously sonicated for 2 min on ice in the buffer solution, and 120 g of cell protein mixture, in a total volume of 80 l. After incubation for 15 min at 37°C, the lipids were extracted, separated by TLC (chloroform/methanol/water, 80:10:1, by volume), and radiolabeled Cer was determined by scanning or cutting out the regions of interest and scintillation counting.
Sphinganine kinase was determined as described by Olivera et al. (28). Briefly, cells were washed with cold phosphate-buffered saline and scraped in 0.1 M phosphate buffer (pH 7.4) containing 20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, 1 mM sodium orthovanadate, 15 mM NaF, 10 g/ml leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM 4-deoxypyridoxine. Cells were then disrupted by freeze-thawing, centrifuged at 105,000 ϫ g for 90 min, and the supernatants stored at Ϫ70°C. The protein concentration of supernatants was about 1 mg/ml. 100 -150 g of cytosol were used in the in vitro sphinganine kinase assay. Sphinganine (50 M) was added as a complex with bovine serum albumin. The reaction mixtures contained 20 mM Tris buffer (pH 7.4), 5% glycerol, 1 mM mercaptoethanol, 0.25 mM EDTA, 1 mM sodium orthovanadate, 15 mM NaF, 10 g/ml leupeptin and aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM 4-deoxypyridoxine, in a final volume of 200 l. The reaction was started by addition of 10 l of [␥-32 P]ATP (1-2 Ci, 20 mM) and MgCl 2 (100 mM) and incubated for 30 min at 37°C. The reaction was terminated by addition of 20 l of 1 N HCl followed by 0.8 ml of chloroform/methanol/HCl (100:200:1, by volume). After vigorous vortexing, 240 l of 2 N KCl were added and phases separated by centrifugation. The labeled lipids in the organic phase were resolved by TLC on Silica Gel 60 with 1-butanol/methanol/acetic acid/water (80:20:10:2, by volume), and visualized by autoradiography.

1-MSDH-Cer Decreases de Novo Ceramide and More Complex Sphingolipid Formation in Primary Cultured Cerebellar
Neurons-The effect of four different short chain DH-Cer analogs ( Fig. 1) on de novo Cer biosynthesis in primary cultured cerebellar neurons was studied by examining the incorporation of L-[3-14 C]serine into cellular SLs. After 48 h preincubation with 10 M each analog and an additional 24 h of labeling, a drastic reduction of Cer formation (Ͼ80%) was observed only in the presence of 1-MSDH-Cer (Fig. 1). As illustrated in Fig. 1 the inhibitory effect of 1-MSDH-Cer on Cer formation was not stereospecific since both stereoisomers were equally effective. The D-erythro-and the L-threo isomer caused an 82% and 87% decrease of Cer labeling, respectively (compounds and lanes 1 and 2 in Fig. 1). In contrast, Cer formation was almost unchanged (about 15% decrease) in the presence of pyrrolidine-DH-Cer and of 1-deoxy-DH-Cer (compounds and lanes 3 and 4 in Fig. 1). We have also studied the effect of 10 M free base of 1-MSDH-Cer (compound 5 in Fig. 1). This compound caused a much less decrease of Cer labeling (by 28%), suggesting that the presence of an N-acyl-group is essential for the observed biological effect.
Analysis of the band comigrating with authentic Cer revealed that DH-Cer accounts for only 5-10% of the total amount of radioactively labeled Cer in untreated control cells after 24 h of [ 14 C]serine labeling (not shown), suggesting that in these cells most of the de novo formed DH-Cer is desaturated to Cer.
The effect of the respective compounds (see Fig. 1) on the incorporation of radiolabeled serine into cellular SLs was examined. Fig. 2 depicts the results obtained with both stereoisomers of 1-MSDH-Cer and with the free base (the deacylated 1-MSDH-Cer). Both L-threo-and D-erythro-1-MSDH-Cer strongly reduced incorporation of [ 14 C]serine into cellular SLs (by 78 and 73%, respectively), whereas the free base was much less effective (32% reduction of overall SL labeling). The other two DH-Cer analogs (1-deoxy-DH-Cer and pyrrolidine-DH-Cer) were even less, if at all, effective (not shown). Taken together our results indicate that the various effects of the respective compounds on Cer biosynthesis (see Fig. 1) were paralleled by analogous effects of these compounds on ongoing SL formation. This is not surprising since Cer is the direct biosynthetic precursor of cellular SLs.  (Table I). In the presence of 10 M 1-MSDH-Cer, levels of radiolabeled SM were reduced by ϳ90%, but labeling of GSL was less affected, by ϳ80% for GlcCer and ϳ50% for ganglioside G Q1b . The radiolabeled lipid, SX (see Fig. 2), was affected even less (by ϳ40%, Table I revealed that there was only a small increase in the efficacy of the D-erythro stereoisomer after 48 and 72 h compared to 24 h (66 and 71% versus 55%, respectively, compared with un-treated controls. Results not shown).

1-MSDH-Cer Decreases [ 14 C]Serine Incorporation into Cellular Sphingolipids in a Dose-and Time-dependent Manner-
1-MSDH-Cer Does Not Inhibit Biosynthetic Enzymes-The de novo biosynthetic pathway of SLs begins with the condensation of serine and palmitoyl-CoA by serine palmitoyltransferase to form 3-dehydrosphinganine. Reduction of 3-dehydrosphinganine by a NADPH-dependent reductase leads to formation of sphinganine, which is N-acylated to form DH-Cer. Preincubation of cells with 1-MSDH-Cer for 72 h had no effect on any of these enzyme activities when measured in vitro compared with untreated controls (Table II). Furthermore, direct addition of 1-MSDH-Cer to the in vitro enzyme assay, in concentrations up to 100 M, also had no effect on any of the enzyme activities (not shown).
As shown above, addition of 10 M 1-MSDH-Cer reduced de novo biosynthesis of all SLs in cerebellar neurons, but to a different extent, using [ 14 C]serine to label SLs. To determine whether these results were due to alteration of Cer metabolism, neurons were labeled with 28 M 14 C-labeled semi-truncated DH-Cer or Cer, in the presence or absence of 1-MSDH-Cer. Both DH-Cer and Cer were glycosylated and processed to the characteristic profile of GSLs (Fig. 3), demonstrating that 1-MSDH-Cer does not interfere with any of the glycosyltransferases involved in GSL biosynthesis. Furthermore, the formation of labeled SM and dihydrosphingomyelin was not altered when Cer or DH-Cer was added to the culture medium. As illustrated in Fig. 3, SM migrated as a double band only when labeled DH-Cer was used as a biosynthetic precursor of cellular SLs, indicating that, as previously suggested (29), some desaturation might also occur at the level of SM.

1-MSDH-Cer Depletes Cells of Free Sphinganine by Stimulating Its
Catabolism-There are two potential mechanisms to decrease SL biosynthesis as follows: (i) by inhibition or downregulation of biosynthetic enzymes, and (ii) by stimulation of the degradation of a vital precursor. As shown above, the first possibility can be excluded as a means to explain the mechanism of action of 1-MSDH-Cer. We therefore examined whether 1-MSDH-Cer enhances the rate of SL degradation. The precursor for (DH)Cer formation in the de novo biosynthetic pathway is sphinganine, and the rate-limiting enzyme for sphinganine degradation is sphinganine kinase. We measured sphinganine kinase activity in vitro in the presence and absence of 1-MSDH-Cer, as well as in the cytosol of cells cultured in the presence of the analog for various periods. In the in vitro assay, 100 M  Table I.

TABLE I Dose dependence of the effect of 1-MSDH-Cer on [ 14 C]serine
incorporation into cellular sphingolipids Cerebellar neurons were incubated with the indicated concentrations of 1-MSDH-Cer for 72 h. Medium was renewed every 24 h. L-[3-14 C]Serine was added to the medium for the last 24 h. Cells were then harvested, and SLs were isolated, separated, and quantified as described under "Experimental Procedures." Data are expressed as percentages of the control values that were obtained by treating cells exactly as described but in the absence of 1-MSDH-Cer. The values presented have been taken from one representative experiment; the results of two additional experiments were all within the range of Ϯ20% of the respective data given here. The terminology for gangliosides (G Q1b , G T1b , G D1b , G D1a , G M1 ) is according to Svennerholm (43). Other abbreviations are as follows: SM, sphingomyelin; SX, unknown lipid; LacCer, lactosylceramide; GlcCer, glucosylceramide.   Fig. 2 lane 3).
b These values are from one experiment with triple determinations. manner (Fig. 4). Maximal stimulation was reached after 24 h, although results from individual cell cultures showed considerable statistical scatter after longer incubation times (Fig. 4).
To determine if this stimulation of sphinganine kinase reduced the level of cellular sphinganine, we determined the concentration of free sphinganine in primary cultured neurons under different culture conditions. To increase the levels of free sphinganine, cells were preincubated for 24 h with 25 M FB 1 in MEM (serine-free), to mimic conditions as in the SL labeling experiments described above. Cells were then chased in DMEM (42 mg serine/liter) for 24 h in the absence or presence of 1-MSDH-Cer (10 M), FB 1 , or L-threo-C 12 -sphinganine, a competitive inhibitor of sphingosine kinase (30). 1-MSDH-Cer caused a drastic reduction of sphinganine, irrespective of the incubation conditions used (Fig. 5). 1-MSDH-Cer reduced the level of free sphinganine by more than 90% (so that in some of the samples the long chain base was hardly detectable) in neurons chased in control medium, as well as in FB 1 containing medium. L-threo-C 12 -sphinganine alone increased the intracellular level of free sphinganine about 2.5-fold compared with controls, demonstrating that it strongly interfered with its degradation by inhibiting its phosphorylation. Simultaneous addition of 1-MSDH-Cer antagonized this effect causing a 90% reduction of the free sphinganine concentration when compared with cells treated with the kinase inhibitor alone. Thus 1-MSDH-Cer almost completely depletes cells of sphinganine, a vital precursor of Cer formation, by stimulating its phosphorylation.
1-MSDH-Cer Reduces the Mass of Total Sphingolipids-As demonstrated above, 1-MSDH-Cer stimulates the catabolism of sphinganine in cultured neurons and thus strongly interferes with cellular SL biosynthesis. However, the analog had little or no effect on total SL levels after 24 h incubation. There was, however, a small but significant reduction (15-20%) of sphingosine at longer incubation times (Table III). Based on mor-  1, 4, and 6). Medium was renewed every 24 h. [ 14 C]Serine (lanes 1 and 2), 28 M [ 14 C]semi-truncated ceramide (lanes 3 and 4), or [ 14 C]semi-truncated dihydroceramide (lanes 5 and 6) were subsequently added to the culture medium. After an additional 24 h, cells were harvested and lipids analyzed as described under "Experimental Procedures." The R F values of authentic SLs and semi-truncated (st) SLs are given. Note that due to lower hydrophobicity, the R F values of semi-truncated SLs are lower than those of their physiologic counterparts, and the R F values of the desaturated semi-truncated SLs biosynthesized from exogenous semi-truncated Cer (lanes 3 and 4) are slightly lower than those of the respective saturated SL species formed from semi-truncated DH-Cer (lanes 5 and 6) as well. Cer, ceramide; for other abbreviations see Table I 1-MSDH-Cer Blocks Axonal Growth-Although cerebellar neurons cultured according to the methods described above are useful for biochemical analysis (23, 31), they are less useful for accurate determination of parameters of neuronal growth, for which cultured hippocampal neurons (22) have proved an invaluable tool (4,5,13,17). It has been previously demonstrated that the synthesis of GlcCer from Cer is required to sustain both normal axonal growth (4,5,13) and also axonal growth stimulated by growth factors (17) in hippocampal neurons; these studies were performed using FB 1 , an inhibitor of (DH)-Cer synthesis (32), and PDMP, an inhibitor of GlcCer synthesis (33,34). Incubation with either inhibitor at 48 h in culture resulted in a decrease in the length of the axonal plexus and a reduction in the number of axonal branch points compared with untreated cells at 72 h in culture. Addition of C 6 -NBD-Cer together with the inhibitors at 48 h reversed the inhibitory effect of FB 1 on axonal growth (4, 13, 17) but not of PDMP (13,17).
We have now examined the effect of four different DH-Cer analogs (see Fig. 1) on axonal growth (Table IV). Hippocampal neurons were incubated with 10 M each analog between 48 and 72 h in culture. The number of axonal branch points was measured at 72 h. Both stereoisomers of 1-MSDH-Cer completely blocked axonal growth between 48 and 72 h, as indicated by the 50% reduction of the number of axonal branch points per cell at 72 h compared to control cells; however, the other two analogs exhibited no effect (Table IV and Fig. 6). Similar to results obtained with FB 1 (4,13,17), the effect of 1-MSDH-Cer could be antagonized by the simultaneous addition of C 6 -NBD-Cer to the medium, confirming that both stereoisomers of 1-MSDH-Cer decrease levels of DH-Cer and Cer biosynthesis ( Fig. 6 and Table IV). DISCUSSION In this study, we have described an SL analog that stimulated the activity of sphinganine kinase in vivo, resulting in increased rates of sphinganine degradation and, as a consequence, significantly reduced rates of SL synthesis. This novel analog, 1-MSDH-Cer, may provide an important new means of manipulating levels of SL synthesis without accumulation of toxic SL intermediates, such as sphinganine and sphingosine that accumulate upon incubation with FB 1 .
Previous studies from our laboratory have shown that exogenous sphingosine homologs of different chain length as well as the biosynthetically stable azidosphingosine (23, 27) and the cis-configured 4-methylsphingosine (35) cause a decrease of de novo SL biosynthesis in primary cultured neurons by specifically interfering with serine palmitoyltransferase activity. Our results revealed a striking correlation of the relative order and magnitude of the sphingosine analogs in terms of the percentage of the phosphorylated metabolites in cultured cells and the inhibitory effect of the respective compound on serine palmitoyltransferase activity, supporting the idea that 1-phosphates are the link between sphingosine metabolites and serine palmitoyltransferase regulation (35).
In the present study we examined the effects of truncated DH-Cer analogs (with a chain length corresponding to 12 car-  In some experiments C 6 -NBD-D-erythro-Cer was simultaneously added. The number of branch points per axon at 48 h was 0.062 Ϯ 0.08. For comparison, the reduction in the number of branch points per axon in the presence of FB 1 (10 M) was 39%, compared to 50% for L-threo-and 40% for D-erythro-1-MSDH-Cer. Each value represents the mean Ϯ S.E. of measurements from two separate cultures, in which 50 cells per coverslip were counted on two individual coverslips per treatment. bon atoms in the sphingoid and in the fatty acid moiety, respectively) with changed polar head groups on SL metabolism as well as on neuronal growth using primary cultured neuronal cells. All of the analogs tested lack the 1-hydroxyl group and are thus resistant to glycosylation as well as to phosphorylation at this position. However, only 1-MSDH-Cer was found to exhibit a pronounced effect on both de novo SL biosynthesis and consequently on neuronal growth. The effect of 1-MSDH-Cer was not stereospecific. Both, the D-erythro and the L-threo isomer exerted similar effects on de novo SL biosynthesis and on axonal growth in cerebellar and hippocampal neurons. The corresponding free base (1-methylthiodihydrosphingosine) had almost no effect. Together, these results suggest that several structural requirements are essential for the observed effect of 1-MSDH-Cer.
In contrast to the sphingoid bases previously studied, reduction of de novo SL biosynthesis by 1-MSDH-Cer was not due to a decrease of serine palmitoyltransferase activity, the ratelimiting enzyme of SL biosynthesis, but rather to the stimulation of sphinganine kinase, the rate-limiting enzyme of long chain base degradation. Both, DH-Cer and sphingosine are intermediates of SL metabolism, but the former is primarily a biosynthetic metabolite, whereas the latter is exclusively a degradation product (29,36). It therefore appears that there might exist two different mechanisms for regulation of SL biosynthesis. First, accumulation of an SL degradation product (SPP) leads to down-regulation of serine palmitoyltransferase (27,35); alternatively, accumulation of a biosynthetic intermediate (DH-Cer) stimulates the catabolism of its biosynthetic precursor (Fig. 7). An unusual degradation of sphinganine also occurs in the presence of FB 1 , which causes an accumulation of sphinganine, a biosynthetic intermediate, by inhibiting sphinganine N-acyltransferase (37) (see also Fig. 7). In that study, however, sphinganine degradation was indirectly evaluated by its utilization for phosphatidylethanolamine synthesis and not by direct measurements of sphinganine kinase activity. The fact that 1-MSDH-Cer does not directly stimulate sphinganine kinase upon addition to the in vitro enzyme assay but only after preincubation of the cultured neurons suggests that it does not directly interact with the enzyme on molecular level. More likely, up-regulation of sphinganine kinase activity by 1-MSDH-Cer seems to be a complex process which requires cell integrity and longer incubation. Stimulation of sphingosine kinase of human erythroleukemia cells by phorbol 12-myristate 13-acetate also required preincubation of cells for at least 18 h with the phorbol ester (38). Unlike growth factors (39) or G M1 (40) which rapidly and transiently increase sphingosine kinase activity, stimulation of sphingosine kinase by phorbol ester appeared to be dependent on transcriptional as well as on translational events (38).
Reduction of SL biosynthesis by about 80% as well as some reduction of total SL mass after long incubation times (72-96 h) caused by 1-MSDH-Cer (10 M) is quite similar to the results obtained in cerebellar neurons with FB 1 (25 M), known to specifically inhibit sphinganine (sphingosine) N-acyltransferase (41). In contrast to FB 1 which caused a 20-fold increase of the amount of free sphinganine, 1-MSDH-Cer almost completely depleted the cells of sphinganine within 24 h. Moreover, when both compounds, FB 1 and 1-MSDH-Cer, were simultaneously supplied, the latter completely prevented sphinganine accumulation caused by the former.
It is intriguing to note that 1-MSDH-Cer has a similar effect on axonal growth of hippocampal neurons as that reported for FB 1 (4,5). Both reduce axonal length and the number of axonal branch points. In addition, the effect of both compounds on axonal growth can be fully reversed by the addition of C 6 -NBD-Cer to the culture medium, supporting the idea that endogenous SLs in general (4) or Cer (6) and GlcCer (5,13) in particular are involved in neuronal growth. These results therefore confirm the usefulness of 1-MSDH-Cer as an investigative tool for the manipulation of endogenous SL pathways. The carcinogenicity of fumonisins which most probably is due to the mitogenic effect of the sphingoid bases (42), known to accumulate as a result of the inhibited sphinganine (sphingosine) N-acyltransferase (32), could be overcome with 1-MSDH-Cer. Moreover, this compound might be clinically useful in the therapy of SL storage diseases.