From the Kekulé-Institut für Organische Chemie und
Biochemie, Universität Bonn, 53121 Bonn, Federal Republic of
Germany and the § Department of Membrane Research and
Biophysics, Weizmann Institute of Science,
Rehovot 76100, Israel
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INTRODUCTION |
Sphingolipids (SLs)1 are
found in all eukaryotic cells, where 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 species-specific
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 B1
(FB1) and PDMP
(DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), as well as inhibitors of lysosomal GSL degradation, like conduritol B-epoxide (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 SL biosynthesis. Furthermore, this analog significantly
reduces the rate of axonal growth in cultured hippocampal neurons, in a
manner similar to that reported for compounds that directly inhibit Cer
synthesis (4, 5, 13, 17).
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EXPERIMENTAL PROCEDURES |
Materials--
Six-day-old NMRI (Navy Marine Research Institute)
mice were obtained from Dr. Brigitte Schmitz from the Institut
für 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.
The DH-Cer analogs, 1-MSDH-Cer (the D-erythro
and the L-threo stereoisomer), as well as the
corresponding free base, 1-deoxy-DH-Cer and pyrrolidine-DH-Cer (see
Fig. 1), were synthesized in our laboratory as described (18).
L-threo-C12-Sphinganine was
synthesized in our laboratory according to Clasen (19). Semi-truncated
14C-labeled Cer and DH-Cer were obtained by
N-acylation of sphingosine and sphinganine, respectively,
with 1-[14C]octanoic acid (162.8 GBq/mol) as described
(18). L-[3-14C]Serine (2.0 GBq/mmol) was
purchased from Amersham-Buchler (Braunschweig, Germany). Fumonisin
B1, trypsin, L-serine, and palmitoyl-CoA were from Sigma (Deisenhofen, Germany). Culture media (Dulbecco's modified Eagle's medium (DMEM) and minimum essential medium (MEM)) were obtained from Life Technologies, Inc. (Karlsruhe, Germany). Horse serum
(heat-inactivated before use) was from Cytogen (Berlin, Germany). DNase
was from Boehringer Mannheim (Mannheim, Germany). LiChroprep RP-18 and
Silica Gel 60 were purchased from Merck (Darmstadt, Germany). Ultima
Gold was from Packard (Groningen, Netherlands). All other chemicals
were of analytical grade and obtained from Sigma (Deisenhofen, Germany)
or Merck (Darmstadt, Germany).
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 × 106 cells/dish). 24 h after plating,
cytosine arabinoside was added to the medium (4 × 10
5 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
Mg2+/Ca2+-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 µM; 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 (C6-NBD-Cer) (5 µM), 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 ×/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 µm 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 µm 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 [14C]serine (2 µCi/ml), 14C-labeled Cer, or DH-Cer (0.125 µCi/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%
CaCl2 (60:35:8, by volume); SLs were visualized by
autoradiography and identified by their RF 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
C20-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.
C18-sphinganine and C18-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 C12C12-ceramide to replicate
samples. C12C12-ceramide was obtained by
N-acylation of C12-sphingosine, as described
(18); C12-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
[14C]serine and palmitoyl-CoA as substrates. The assay
mixture contained 0.1 M Hepes (pH 7.4), 5 mM
dithiothreitol, 10 mM EDTA, 50 µM pyridoxal phosphate, 1.2 mM [14C]serine (1.6 µCi),
0.15 mM palmitoyl-CoA, and 120 µg of cell protein, in a
total volume of 100 µl. 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
NH3 (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
MgCl2, 0.5% (w/v) Nonidet P-40, 150 µM
NADPH, 120 µM dehydrosphinganine, and 50 µg of cell protein in a total volume of 100 µl. After 15 min at 37 °C the reaction was terminated by addition of 250 µl of chloroform and 100 µl 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-[4,5-3H]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 µM stearoyl-CoA, 50 µM labeled sphinganine (0.5 µCi), which was previously
sonicated for 2 min on ice in the buffer solution, and 120 µg of cell
protein mixture, in a total volume of 80 µl. 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 µg/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 × g for 90 min, and the
supernatants stored at
70 °C. The protein concentration of supernatants was about 1 mg/ml.
100-150 µg of cytosol were used in the in vitro
sphinganine kinase assay. Sphinganine (50 µM) 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 µg/ml leupeptin and
aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM 4-deoxypyridoxine, in a final volume of 200 µl. The
reaction was started by addition of 10 µl of
[
-32P]ATP (1-2 µCi, 20 mM) and
MgCl2 (100 mM) and incubated for 30 min at
37 °C. The reaction was terminated by addition of 20 µl of 1 N HCl followed by 0.8 ml of chloroform/methanol/HCl
(100:200:1, by volume). After vigorous vortexing, 240 µl 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.
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RESULTS |
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-[3-14C]serine into
cellular SLs. After 48 h preincubation with 10 µM each analog and an additional 24 h of labeling, a drastic
reduction of Cer formation (>80%) 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 µM 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.

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Fig. 1.
Effect of different dihydroceramide analogs
on [14C]serine incorporation into cellular ceramide.
Primary cultured cerebellar neurons were incubated for 72 h in the
absence (C) or presence of 10 µM the
respective analog (the lane numbers correspond to the
structure numbers). Medium was renewed every 24 h.
[14C]Serine was added for the last 24 h. Then cells
were harvested and lipids extracted and isolated as described under
"Experimental Procedures." Ceramides were separated by TLC using
chloroform/methanol/water (80:10:1, by volume) as solvent system.
Radioactively labeled lipids were visualized by autoradiography and
quantitatively evaluated as described under "Experimental
Procedures." The mobilities of authentic ceramide (Cer)
and glucosylceramide (GlcCer) are indicated. OR,
origin.
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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
[14C]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 [14C]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.

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Fig. 2.
Effect of stereoisomers and deacylation of
1-MSDH-Cer on incorporation of [14C]serine into cellular
sphingolipids. Cultured cerebellar neurons were incubated in the
absence (lane 2) or presence of 10 µM either
the L-threo-isomer (lane 1), the
D-erythro-isomer (lane 3), or the
free base 1-methylthiodihydrosphingosine (lane 4) for
72 h. Medium was renewed every 24 h.
[14C]Serine was added to the medium for the last 24 h. Then cells were harvested and cellular SLs extracted and isolated as
described under "Experimental Procedures." The TLC plate was
developed in chloroform/methanol, 0.22% CaCl2 (60:35:8, by
volume). The mobilities of authentic SLs are given. The nature of the
band marked SX is not known. HPLC measurements demonstrated
that this band was neither sphingosine nor sphinganine. For
abbreviations see Table I.
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1-MSDH-Cer Decreases [14C]Serine Incorporation into
Cellular Sphingolipids in a Dose- and Time-dependent
Manner--
Newly synthesized SLs were labeled for 24 h with
L-[3-14C]serine in the presence of increasing
concentrations (up to 50 µM) of 1-MSDH-Cer, after 48 h preincubation with the DH-Cer analog. A drastic decrease of
radioactive labeling of SLs (~60%) was observed with concentrations
as low as 5 µM 1-MSDH-Cer (Table
I). In the presence of 10 µM 1-MSDH-Cer, levels of radiolabeled SM were reduced by
~90%, but labeling of GSL was less affected, by ~80% for GlcCer and ~50% for ganglioside GQ1b. The radiolabeled lipid,
SX (see Fig. 2), was affected even less (by ~40%, Table
I). A concentration of 50 µM 1-MSDH-Cer was cytotoxic
after 72 h of treatment, with fragmentation of neurites and of the
plasma membrane.
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Table I
Dose dependence of the effect of 1-MSDH-Cer on [14C]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-[3-14C]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 ±20% of the respective data given here.
The terminology for gangliosides (GQ1b, GT1b,
GD1b, GD1a, GM1) is according to Svennerholm
(43). Other abbreviations are as follows: SM, sphingomyelin; SX, unknown lipid; LacCer, lactosylceramide; GlcCer,
glucosylceramide.
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Studies of the time course of the effect of 10 µM
1-MSDH-Cer on the incorporation of radiolabeled serine into cellular
SLs 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 untreated controls. Results not shown).
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 µM, also had no effect on any of the enzyme
activities (not shown).
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Table II
The effect of 1-MSDH-Cer on the activity of enzymes involved in
DH-Cer biosynthesis
Primary cultured cerebellar neurons were incubated in the absence
(control) or presence of 1-MSDH-Cer (10 µM). Medium was renewed every 24 h. After 72 h cells were harvested and
enzyme activities determined in the cell homogenate as described under "Experimental Procedures." Results are means of three different experiments with at least double determinations.
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As shown above, addition of 10 µM 1-MSDH-Cer reduced
de novo biosynthesis of all SLs in cerebellar neurons, but
to a different extent, using [14C]serine to label SLs. To
determine whether these results were due to alteration of Cer
metabolism, neurons were labeled with 28 µM
14C-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.

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Fig. 3.
Effect of 1-MSDH-Cer on the metabolism of
exogenous (dihydro)ceramide in primary cultured neurons.
Cerebellar neurons were incubated for 48 h in the absence
(lanes 2, 3, and 5) or presence of 10 µM 1-MSDH-Cer (lanes 1, 4, and 6).
Medium was renewed every 24 h. [14C]Serine
(lanes 1 and 2), 28 µM
[14C]semi-truncated ceramide (lanes 3 and
4), or [14C]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
RF values of authentic SLs and semi-truncated (st) SLs are given. Note that due to lower hydrophobicity,
the RF values of semi-truncated SLs are lower than
those of their physiologic counterparts, and the RF
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.
The nature of the band marked st-SX (just above st-SM) is not known, and we cannot explain at present the
heavy labeling of this band as a result of DH-Cer metabolism.
[14C]st-Cer,
N-[14C]octanoyl-D-erythro-sphingosine;
[14C]st-dihydroceramide,
N-[14C]octanoyl-D-erythro-sphinganine.
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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 down-regulation 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 µM (10-fold the concentration used
in the cell culture experiments) 1-MSDH-Cer caused only a slight
stimulation of enzyme activity to 138 ± 16% (data not shown).
However, pretreatment of cells with 10 µM 1-MSDH-Cer from
5 min up to 72 h clearly stimulated sphinganine kinase activity in
a time-dependent 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).

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Fig. 4.
Effect of 1-MSDH-Cer on sphinganine kinase
activity from primary cultured neurons. Primary cultured
cerebellar neurons were incubated for the indicated times in the
absence (C, control) or presence of 10 µM
1-MSDH-Cer. Medium was renewed every 24 h. Cells were harvested,
and sphinganine kinase was measured as described under "Experimental
Procedures." Data are means ± S.E. from three different
experiments. 100% sphinganine kinase activity of untreated controls
corresponds to ~20 pmol/min/mg.
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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 µM FB1 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 µM), FB1, or L-threo-C12-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 FB1
containing medium.
L-threo-C12-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.

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Fig. 5.
Effect of 1-MSDH-Cer, FB1, and
L-threo-C12-sphinganine on the
level of free sphinganine in cultured neurons. Primary cultured
cerebellar neurons were incubated for 24 h in MEM containing 25 µM FB1. Then the medium was discarded, and
the cells were rinsed three times and chased in DMEM containing no
further addition (C, controls), 25 µM
FB1, or 20 µM
L-threo-C12-sphinganine
(L-t-Sa) in the absence (white bars)
or presence (black bars) 10 µM 1-MSDH-Cer, as
indicated. After an additional 24 h, cells were harvested and free
sphinganine determined in the cellular lipid extracts as described
under "Experimental Procedures." The retention times of the
o-phthalaldehyde derivative of
L-threo-C12-sphinganine (with 12 carbon atoms), sphinganine, and C20-sphinganine used as an
internal standard were 2.4, 8.3, and 13.9 min, respectively. Data are
means ± S.E. from two different experiments with double determinations. 100% free sphinganine equals 1.2 nmol/mg
protein.
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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 morphology
and trypan blue exclusion, cells were still viable after these long times of treatment with 1-MSDH-Cer (10 µM).
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Table III
Effect of 1-MSDH-Cer on the mass of complex sphingolipids
Cerebellar neurons were cultured in the absence (control) or presence
of 10 µM of 1-MSDH-Cer. Medium was renewed every 24 h. After the indicated periods cells were harvested and lipids extracted. Sphingosine and sphinganine released after acid hydrolysis were determined as described under "Experimental Procedures."
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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 FB1, 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 C6-NBD-Cer together with the inhibitors at 48 h
reversed the inhibitory effect of FB1 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 µM 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
FB1 (4, 13, 17), the effect of 1-MSDH-Cer could be
antagonized by the simultaneous addition of C6-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).
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Table IV
The effect of different dihydroceramide analogs on neuronal development
between culture day 2 and 3
DH-Cer analogs were added to the culture medium of hippocampal neurons
at 48 h in culture. The number of axonal branch points was
determined at 72 h as described under "Experimental
Procedures." In some experiments
C6-NBD-D-erythro-Cer was simultaneously
added. The number of branch points per axon at 48 h was 0.062 ± 0.08. For comparison, the reduction in the number of branch points per axon
in the presence of FB1 (10 µM) was 39%, compared
to 50% for L-threo- and 40% for
D-erythro-1-MSDH-Cer. Each value represents the
mean ± S.E. of measurements from two separate cultures, in which
50 cells per coverslip were counted on two individual coverslips per
treatment.
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Fig. 6.
Morphological characteristics of hippocampal
neurons. Camera lucid drawings of representative cells at 72 h in culture after addition at 48 h of either 10 µM
L-threo-1-MSDH-Cer or 10 µM
L-threo-1-MSDH-Cer together with 5 µM
C6-NBD-D-erythro-Cer. The
bar corresponds to 50 µm.
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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
FB1.
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 carbon 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 rate-limiting 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 FB1, 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 GM1 (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 µM) is quite similar to the results
obtained in cerebellar neurons with FB1 (25 µM), known to specifically inhibit sphinganine
(sphingosine) N-acyltransferase (41). In contrast to
FB1 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, FB1 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 FB1 (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 C6-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.
We thank Andrea Raths and Martina Feldhoff
for excellent technical assistance and Judith Weisgerber for help in
preparing the figures.