From the
Sphingolipids, particularly gangliosides, are enriched in
neuronal membranes where they have been implicated as mediators of
various regulatory events. We recently provided evidence that
sphingolipid synthesis is necessary to maintain neuronal growth by
demonstrating that in hippocampal neurons, inhibition of ceramide
synthesis by Fumonisin B
A number of studies have examined the effects of disrupting the
metabolism or intracellular transport of the molecular components
presumed necessary to maintain neuronal growth. In cultures of rat
sympathetic ganglia, the formation of dendrites and axons has different
requirements for protein synthesis
(1) . In cultured hippocampal
neurons, both axonal and dendritic growth is disrupted by antisense
oligonucleotides to the molecular motor, kinesin
(2) , and
Brefeldin A, which blocks vesicle flow out of the Golgi apparatus,
inhibits axonal growth
(61) . The growth of rat cortical neurons
and PC12 cells is retarded by inhibition of SNAP-25 expression
(3) , an essential member of the cell fusion apparatus in adult
neurons. Thus, neuronal growth can be regulated by modifying either the
supply of molecular components or the molecular motors involved in
vesicular transport. Alternatively, growth could also be regulated by
modifying cytoskeletal components
(4) .
Little information is
available about the relationship between neuronal development and the
synthesis, degradation, and intracellular transport of lipids. Lipids
are the major structural components of intracellular vesicles and
membranes, and regulating their metabolism could provide an additional
mechanism of controlling neuronal development. We have therefore
initiated studies to examine the role of sphingolipids
(SLs)
Our approach differs in two respects from most studies that
have attempted to examine the role of SLs in neuronal development.
First, the use of well-characterized cultured hippocampal neurons, in
which axons and dendrites can be distinguished biochemically and
anatomically
(9, 10) , permits analysis of the role of
SLs in the growth of both types of processes and on the development of
neuronal polarity. Second, the use of inhibitors of SL metabolism
(11) permits examination of the role of endogenous SLs
in neuronal growth. We recently demonstrated
(5) that Fumonisin
B
In
the current report, we demonstrate that both FB
Hippocampal Cultures Hippocampal neurons were cultured at low density as described
(5, 9) . Briefly, the dissected hippocampi of embryonic
day 18 rats (Wistar), obtained from the Weizmann Institute Breeding
Center, were dissociated by trypsinization (0.25% (w/v), for 15 min at
37 °C). The tissue was washed in magnesium/calcium-free
Hank's balanced salt solution (Life Technologies, Inc.) and
dissociated by repeated passage through a constricted Pasteur pipette.
Cells were plated in minimal essential medium (MEM) with 10% horse
serum, at a density of 8,000-12,500 cells/13-mm glass coverslip
(Assistent, Germany) that had been precoated with
poly-L-lysine (1 mg/ml). After 3-4 h, coverslips were
transferred into 24-well Multidishes (Nunc) containing a monolayer of
astroglia. Neurons were placed with the cells facing downward and were
separated from the glia by paraffin ``feet.'' Cultures were
maintained in MEM which included N2 supplements
(9) , 0.1% (w/v)
ovalbumin, and 0.1 mM pyruvate.
For biochemical analysis,
neurons were cultured at high density (230,000 cells/24-mm glass
coverslip) and maintained in 100-mm plastic Petri dishes containing
glial cocultures, as above. Analysis of Neuronal Morphology Stock solutions of FB
Parameters of axonal length and branching were determined as
follows (see the control panel in Fig. 3for an example
of how parameters were measured). (i) The length of the total axon
plexus includes the length of the parent axon and all axonal branches.
(ii) The length of the longest axon indicates the distance between the
site of emergence of the parent axon from the cell body and the most
distal growth cone in cases where an axon has one or more branches.
This parameter equals the length of the total axon plexus (see (i)) in
cases where the parent axon did not branch. (iii). 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.
The effect of CBE on GlcCer metabolism was analyzed using
N-(1-[
Cells
were observed using either a Plan Apochromat 40
We previously demonstrated that inhibition of ceramide (Cer)
synthesis in hippocampal neurons by FB
Changes in the number of axonal branch points/cell
on day 3 provided the most striking indication of the effect on axonal
morphology induced by modulating Cer or GlcCer metabolism. The mean
number of axonal branch points was reduced by approximately 50% on day
3 compared to control cells after both FB
We next examined the effect of addition of FB
The early stages of development of
cultured hippocampal neurons are characterized by rapid axonal growth,
with the length of the total axon plexus increasing by approximately
150-200 µm between days 2-3 (Figs. 2 and 4). Each
neuron also has four to five minor processes (Fig. 3) which
acquire typical characteristics of dendrites after 5-6 days in
culture
(10) . Minor processes grow at a much slower rate.
Between days 2-3, minor processes grew approximately 30-40
µm, and addition of either FB
We next examined the
correlation between axonal branching and the extent of inhibition of SL
metabolism. Incubation with 10 or 50 µM FB
Incubation with 100 or 500 µM
CBE caused accumulation of [
The major
finding of our current work is that inhibition of SL synthesis and
degradation have opposite effects on the formation or stabilization of
collateral axonal branches. In contrast, no effect is observed on the
formation of the parent axon during its emergence from the cell body.
The fact that inhibition of SL synthesis and degradation effect growth
in hippocampal neurons is not entirely unexpected. SLs have been
implicated in a variety of cellular phenomena, including cell-cell
interaction, differentiation, and adhesion
(7, 34) .
Addition of exogenous SLs, particularly GMs, has dramatic effects on
neuronal growth in primary neuronal cultures and in neuroblastoma
(8) . It has also been demonstrated that accumulation of SLs in
animal models of lysosomal storage disorders is accompanied by abnormal
appearance of ectopic dendrites at the axonal hillock
(35) .
Manipulation of endogenous SLs in hippocampal neurons might therefore
be predicted to affect growth, as has been observed in cells and
tissues of non-neuronal origin
(16, 17, 18, 36, 37) and in
neuroblastoma
(38) . However, ours is the first study that
implicates SL metabolism in a particular facet of neuronal growth,
namely collateral branch formation or stabilization.
Little is known
about the mechanisms of formation of collateral axonal branches. The
formation of a branch must involve local disruption or inactivation of
microtubules
(39, 40) . In human neuroblastoma cells,
MAP-2 accumulates at the branch points of neurites
(41) , and
presumably in axons of hippocampal neurons, which lack MAP-2, another
microtubule-associated protein is involved in modifying the
cytoskeleton. However, in addition to structural alterations in the
cytoskeleton
(40) , distortion of the plasma membrane in the
region of formation of the new branch must occur to permit extension of
the fillipodia and lammelipodia that characterize growth cone
protrusions. In motile fibroblasts, in which the leading edge bears
many structural similarities to the neuronal growth cone, disruption of
the supply of vesicular material by Brefeldin A inhibits protrusional
activity at the leading edge
(42) resulting in inhibition of
cell motility; similar effects are observed after treatment with
FB
The reduction in the number of axonal branches induced by
inhibition of SL synthesis cannot be explained by a decrease in the
total mass of cellular SLs, since incubation with FB
Alternatively, SLs
per se could play an important role in the formation and
stabilization of axonal branches. Various GMs have been implicated as
mediators of neuronal adhesion
(7, 34) . In embryonic
chick retinal neural cells, different GMs have different adhesive
properties and the extent of GM-mediated adhesion varies with embryonic
age
(53) . In non-neuronal cells, adhesion is reduced after
incubation with PDMP
(19, 20, 36) . Altering SL
levels may also affect physical properties of the plasma membrane. For
instance, exogenous GMs have been shown to stimulate axonal growth by
promoting calcium influx into cells
(54, 55) , and
altering their levels may negate or enhance these effects. Altering the
property of the membrane may affect cytoskeletal structure and thus
disrupt growth
(56, 57) . Finally, the effects observed
may be explained by a role of SLs in a signal transduction pathway
(58) , either by the direct involvement of ceramide or a
derivative, or by the regulation of another pathway. In Madin-Darby
canine kidney cells, incubation with PDMP results in enhancement of
bradykinin-stimulated inositol trisphosphate formation
(59) ,
whereas incubation with CBE causes a reduction in inositol
trisphosphate formation
(22) .
Whichever of these
possibilities proves to be responsible for the effects observed on
axonal branching, the effects of the inhibitors suggest that ongoing SL
synthesis is necessary for axonal branching. Axonal branch formation
presumably requires ongoing axon elongation. SLs may directly affect
axon elongation or may effect a developmental sequence that precedes
axon branching. The ability of neurons to develop normal axons after
12-14 days in culture even though SL synthesis is significantly
reduced might suggest that SLs are not necessary for the development of
neuronal polarity. However, although axons appear to develop normally
after 12-14 days, no precise morphological analyses are possible
at this stage, and no studies have yet been performed to examine
whether other physiological parameters are altered.
In conclusion,
we have demonstrated that SLs, particularly glyco-SLs, are involved in
the formation or stabilization of axonal branches in cultured
hippocampal neurons. We are currently examining the levels and types of
SLs synthesized during development, and their targeting to axonal or
dendritic domains, in order to distinguish between the various possible
molecular mechanisms that could account for these effects.
FB
10 µM FB
We thank Rivi Zisling for expert help in preparing
and maintaining the hippocampal cultures and members of the laboratory
for critical reading of the manuscript and helpful comments.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(FB
) disrupted axonal
outgrowth (Harel, R. and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481). We now analyze further the relationship
between neuronal growth and sphingolipid metabolism by examining the
effect of an inhibitor of glucosylceramide synthesis,
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol
(PDMP) and by examining the effects of both FB
and PDMP at
various stages of neuronal development. No effects of FB
or
PDMP were observed during the first 2 days in culture, but by day 3
axonal morphology was significantly altered, irrespective of the time
of addition of the inhibitors to the cultures. Cells incubated with
FB
or PDMP had a shorter axon plexus and less axonal
branches. FB
appeared to cause a retraction of axonal
branches between days 2 and 3, although long term incubation had no
apparent effect on neuronal morphology or on the segregation of axonal
or dendritic proteins. In contrast, incubation of neurons with
conduritol B-epoxide, an inhibitor of glucosylceramide degradation,
caused an increase in the number of axonal branches and a corresponding
increase in the length of the axon plexus. A direct correlation was
observed between the number of axonal branch points per cell and the
extent of inhibition of either sphingolipid synthesis or degradation.
These results suggest that sphingolipids play an important role in the
formation or stabilization of axonal branches.
(
)
in neuronal growth
(5) . SLs are
strong candidates as regulators of growth since they are enriched in
neurons, and their levels and types, particularly gangliosides (GMs),
change significantly during development and differentiation
(6, 7) . The effect of adding exogenous SLs or GMs to
primary neuronal cultures and neuroblastoma cell lines has been
extensively studied
(8) , but the variety of effects observed
has complicated analysis of the mechanisms by which SLs regulate
growth.
(FB
), an inhibitor of dihydroceramide
synthesis
(12) (Fig. 1), disrupts axonal growth. These
results are consistent with experiments which suggest that SLs are
essential for cell growth in Chinese hamster ovary cells
(13) .
Figure 1:
The metabolism of
sphingolipids. The inhibitors used in this study are shown in
bold, and the intracellular location of the enzymatic
reactions (reviewed in Ref. 60) in
italics.
In order to further elucidate the role of SLs in neuronal
development, we now compare the effect of FB with that of
1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor
of glucosylceramide (GlcCer) synthesis
(14, 15) (Fig. 1). PDMP affects growth in a number of cells,
including murine neuroblastoma
(16) and Madin-Darby canine
kidney cells
(17) , inhibits the T cell proliferative response
(18) , and affects adherence in both melanoma
(19) and
leukemia
(20) cells. All of these phenomena have been
attributed to depletion of intracellular SLs. In addition, we compare
the effects of PDMP and FB
with those of conduritol
B-epoxide (CBE), an inhibitor of lysosomal glucocerebrosidase
(21) , the enzyme that cleaves GlcCer (Fig. 1). CBE causes
accumulation of GlcCer in a variety of cell types (see for example,
Ref. 22) although no effects on cell growth have been reported.
and PDMP
reduce axonal growth in cultured hippocampal neurons by altering the
ability of neurons to either form or stabilize axonal branches. CBE has
an opposite effect, causing an increase in axonal growth and branching.
These data suggest that SLs play an important role in axonal
development, and imply that modulation of SL metabolism may provide a
mechanism for regulating neuronal growth.
(Division of Food Science and
Technology, CSIR, Pretoria, South Africa), PDMP, and CBE (Matreya Inc.,
Pleasant Gap, PA) were dissolved in 20 mM HEPES buffer, pH 7.4
and added to cultures to give final concentrations as indicated under
``Results''; control cultures were incubated with HEPES
buffer alone. 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), and neurite lengths quantified by use
of a measuring reticle in one of the eyepieces. The investigator did
not know the source of the coverslip being counted. The accuracy of the
counting method was confirmed by comparing measurements obtained by
different investigators. Axons were identified as long, thin processes
of uniform diameter
(9) , and dendrites were identified by
immunofluorescence using an anti-MAP 2 antibody
(23) (see
below).
Figure 3:
Morphological characteristics of
hippocampal neurons. Camera lucid drawings of representative cells on
day 3 in culture after addition of inhibitors on day 0. Cells incubated
with either FB or PDMP have a shorter axon plexus and less
axonal branch points, whereas cells treated with CBE have more axonal
branch points/cell. Note the heterogeneity of the minor processes at
this stage of development. The bottom right cell in the
control panel indicates how parameters of axonal length and
branching were measured. Dendrites are marked by arrowheads,
the longest axon by an asterisk, and axonal branch points by
arrows. The bar corresponds to 100
µm.
Only those cells in which the whole axon plexus could be
unambiguously delineated were measured. The values obtained are
therefore probably underestimates of the mean length of the axon plexus
in the entire cell population as cells with longer axons (and more
branch points) would have a higher probability of either crossing other
axons or fasciculating. 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 to five separate cultures (in
which 50 cells were counted per coverslip on two individual
coverslips/treatment) and statistical analysis performed using the
Student's t test. Analysis of SL Metabolism in Hippocampal Neurons The effect of FB and PDMP on SL synthesis was determined by
incubating neurons with [4,5-
H]dihydrosphingosine
(24) . High density cultures were incubated with FB
or PDMP immediately after transferring the coverslips to Petri
dishes containing glial cocultures. After 66 h, neurons were
transferred to 100-mm Petri dishes containing 5 ml of MEM and the
appropriate inhibitors, and 5
10
counts/min of
[4,5-
H]-dihydrosphingosine (10 Ci/mmol) for a
further 6 h. Coverslips were then washed with Hank's balanced
salt solution, cells removed by scraping with a rubber policeman, and
coverslips washed four times with water. The suspended cells were
lyophilized, and the dry material extracted using
CHCl
/CH
OH/H
O/pyridine (60:30:6:1
(v/v/v/v)) for 48 h at 48 °C
(25) . Phospholipids were
degraded by mild alkaline hydrolysis with 100 mM methanolic
NaOH for 2 h at 37 °C. The lipid extracts were desalted by
reversed-phase chromatography using RP18 (Merck)
(26) .
H-SLs were separated by thin layer chromatography using
CHCl
/CH
OH/CaCl
(0.22% (w/v))
(60:35:8 (v/v/v)) as the developing solvent.
H-SLs were
visualized by autoradiography using En
Hance
spray (DuPont NEN) and identified using authentic SL standards.
Plates were washed three times using diethylether and
H-SLs
recovered from the plates by scraping. Radioactivity was determined by
liquid scintillation counting in a Packard Tri-Carb 1500 scintillation
counter using Ultima gold scintillation fluid (Packard)/water (8:1
(v/v)).
C]hexanoyl)-D- erythro-glucosylsphingosine
([
C]hexanoyl GlcCer) as substrate
(27) .
Neurons were incubated as above with a defatted bovine serum
albumin
[
C]hexanoyl GlcCer complex for 6 h,
and after lyophilization, extracted and analyzed by thin layer
chromatography as described
(28) .Immunofluorescence
Thy-1
Coverslips were washed in 2.5% fetal calf serum in
Hank's balanced salt solution and incubated with an anti-Thy-1.1
(Serotec, Oxford, United Kingdom) monoclonal antibody diluted 1:100 in
MEM containing 10% bovine serum albumin. After 30 min at 37 °C,
coverslips were washed in 2.5% fetal calf serum and fixed in 4%
formaldehyde for 20 min at 37 °C, prior to incubation with
biotin-conjugated goat anti-mouse IgG followed by
indocarbocyanine-conjugated streptavidin (Jackson).
Gap-43, MAP 2, and Synaptophysin
Neurons were
fixed in 4% paraformaldehyde in phosphate-buffered saline containing 4%
sucrose for 20 min at 37 °C. For MAP 2 and GAP-43, cells were
permeabilized in methanol at -20 °C for 10 min; for
synaptophysin, cells were permeabilized with 0.25% Triton X-100 for 5
min at 37 °C. The anti-GAP 43 (Boehringer Manheim, Germany),
anti-MAP 2, and anti-synaptophysin (Biomakor, Israel) antibodies were
diluted 1:1000, 1:300, and 1:100, respectively, in phosphate-buffered
salin containing 10% bovine serum albumin. A rhodamine-conjugated goat
anti-mouse second antibody was used for detection (Jackson).
/1.3 or
63
/1.4 oil objective of a Zeiss Axiovert 35 microscope with
appropriate filters for fluorescein or rhodamine fluorescence.
reduced the length
of the longest axon between days 2 and 3 in culture and that addition
of Cer together with FB
reversed this effect
(5) .
To analyze further the role of SL metabolism in neuronal development,
we have now compared the effects of FB
with those of PDMP
and CBE, inhibitors of GlcCer synthesis and degradation, respectively
(Fig. 1). We have also examined the effects of these inhibitors
on additional parameters of axonal growth and on the growth of the
minor processes which eventually mature into dendrites
(10) .
Modulation of SL Metabolism Affects Axonal Branching
In
control cells, the length of the axon plexus increased by approximately
150-200 µm/day during the first 3 days in culture
(Fig. 2). Addition of either 50 µM PDMP
(Fig. 2) or 10 or 50 µM FB (not shown)
on day 0 had no significant effect on the rate of axonal growth during
the first 2 days in culture, but significantly reduced axonal growth
between days 2 and 3, consistent with our previous report using 10
µM FB
(5) . In control cells, 88% of
cells had axons
250 µm long on day 3 compared to 55% on day 2,
whereas after PDMP treatment, only 52% of cells had axons
250
µm compared to 57% on day 2 (Fig. 2).
Figure 2:
Axonal growth in cultured hippocampal
neurons. Neurons were incubated with 50 µM PDMP on day 0
immediately after cells were placed in multiwell dishes containing
cocultures of glial cells. After various times, coverslips were removed
from the dishes, fixed, and mounted prior to measurement of the length
of the axon plexus. The normalized distributions of the length of the
axon plexus for control and PDMP-treated cells are shown for days 1
(), 2 (
), and 3 (
) in culture. Measurements are
taken from three separate cultures in which approximately 50
cells/coverslip were counted on two individual
coverslips/day.
The decrease in the
rate of axonal growth was reflected in altered axonal morphologies of
the neurons on day 3 (Fig. 3). Not only was the length of the
total axon plexus reduced (see Fig. 2), but the number of axonal
branch points/cell decreased, as did the length of the longest axon. In
contrast, incubation with CBE resulted in an increase in the length of
the axon plexus and the number of axonal branch points/cell
(Fig. 3).
and PDMP
treatment (Fig. 4 C), the length of the total axon plexus
by 30-40% (Fig. 4 A), and the length of the longest
axon by 25-30% (Fig. 4 B). In CBE-treated cells,
the mean number of axonal branch points was increased by approximately
40% (Fig. 5 C) compared to control cells on day 3, the
length of the total axon plexus by 20% (Fig. 5 A), but
the length of the longest axon increased by less than 10%
(Fig. 5 B). The number of axonal branch points was also
increased significantly on day 2 (Fig. 5 C). In contrast,
neither FB
, PDMP, or CBE had any effect on the formation of
the initial (parent) axon. The percent of polarized neurons ( i.e. cells in which the longest process was at least 20 µm longer
than the next longest (minor) process) was 50-60, 70-80,
and 85-90% on days 1, 2, and 3, respectively, for control and
inhibitor-treated cells. In addition, the mean length of the axon
plexus in the population of cells with no axonal branches was similar
in control and cells treated with inhibitors during the first 3 days in
culture.
Figure 4:
Effect of addition of FB and
PDMP on day 0 on axonal development. Neurons were incubated with 50
µM PDMP (
) or 10 µM FB
(
) on day 0 and the length of the total axon plexus
( A), the length of the longest axon ( B), and the
number of axonal branch points/cell ( C) measured on days 1, 2,
and 3. Each bar represents the mean of measurements ±
S.E. from three separate cultures, in which approximately 50
cells/coverslip were counted on two individual coverslips/treatment.
Statistical differences ( p < 0.001) with control cells
(
) are indicated by
asterisks.
Figure 5:
Effect of addition of CBE on day 0 on
axonal development. Neurons were incubated with 100 µM CBE
(&cjs2098;) on day 0 and parameters of axonal development measured on
days 1, 2, and 3. Each bar represents the mean of measurements
± S.E. from three to four separate cultures, in which
approximately 50 cells/coverslip were counted on two individual
coverslips/treatment. Statistical differences ( p < 0.001)
with control cells () are indicated by
asterisks.
The effect of CBE was abolished when cells were incubated
with FB and CBE together on day 0. The mean number of
axonal branch points/cell was 1.6 ± 0.1 ( n =
210) in control cells, 0.83 ± 0.1 ( n = 215) in
FB
-treated cells, and 1.0 ± 0.1 ( n =
204) in cells treated with FB
and CBE together. The ability
of FB
to antagonize the effects of CBE indicate that SL
synthesis is required for CBE to mediate its effects on axonal
morphology.
and PDMP on day 1 on axonal development. No difference was
observed between control and FB
- or PDMP-treated cells on
day 2 (0.66 ± 0.06, 0.67 ± 0.07, and 0.60 ± 0.09
axonal branch points/cell, respectively), but similar to experiments
when inhibitors were added on day 0, a decrease in axon length and
branching was observed on day 3 (1.33 ± 0.10 (control), 0.67
± 0.06 (FB
), 0.66 ± 0.06 (PDMP) axonal branch
points/cell). Thus, irrespective of whether FB
or PDMP were
added on day 0 (Figs. 2 and 4), 1, or 2
(5) , axonal outgrowth
was always affected on day 3.
or PDMP on days 0 and 2
had no significant effect on either the length or the number of minor
processes on day 3.
Relationship between Inhibition of SL Metabolism and
Axonal Branching
Previous biochemical analysis
(5) demonstrated that FB inhibited (dihydro)ceramide
and ganglioside synthesis during the first 4 days in culture.
Ganglioside GD
was virtually undetectable by
immunofluorescence or thin layer chromatography immunostaining on day 3
after incubation with FB
on day 0 or 2
(5) ;
further, levels of cell surface cholera toxin, which binds to
ganglioside GM
, were also reduced after incubation with
FB
(62) .
(
)
Similarly, we now
observed that GD
levels were significantly reduced at the
cell surface when analyzed by immunofluorescence on days 1, 2, or 3
after addition of either PDMP or FB
on day 0 (not shown).
Thus, the inability of PDMP and FB
to affect axonal
development on days 1 and 2 was not due to differences in the efficacy
of the inhibitors on different days.
inhibited the synthesis of
[4,5-
H]dihydroceramide from
[4,5-
H]dihydrosphingosine by 63 and 80%,
respectively, resulting in a corresponding decrease in
[
H]sphingomyelin (SM),
[
H]GlcCer, and
[
H]ganglioside synthesis
(Fig. 6 D). The total incorporation of
[4,5-
H]dihydrosphingosine into
H-SLs
was reduced by 54% with 10 µM FB
and by 90% by
50 µM FB
(Fig. 6 D). However,
both concentrations caused a significant decrease in the number of
axonal branch points/cell (Fig. 6 A). Lower
concentrations of FB
(0.1 and 1 µM) had no
significant effect on
H-SL synthesis
(Fig. 6 D) or on the number of axonal branch points
(Fig. 6 A).
Figure 6:
Inhibition of SL metabolism and effect on
axonal branching. Various concentrations of FB ( A and D), PDMP ( B and E), or CBE ( C and F) were added to either low ( A-C) or high
density ( D-F) cultures on day 0. The number of axonal branch
points/cell was measured on day 3 ( A-C). Results are means
± S.E. from three separate cultures in which approximately 50
cells/coverslip were counted on two individual coverslips/treatment.
Statistical differences ( p < 0.05) with control cells are
indicated by asterisks. Inhibition of SL metabolism was
quantified as described under ``Experimental Procedures.''
Radioactive precursors were added at 66 h and neurons extracted at 72
h. In panels D and E, results are shown as
incorporation of [4,5-
H]dihydrosphingosine into
[
H]SM (), [
H]GlcCer (), and
[
H]gangliosides (). The incorporation into
[
H]gangliosides represents the sum of the
incorporation into gangliosides GM
, GM
,
GD
, GD
, GT
, and GQ
.
In panel F, [
C]hexanoyl GlcCer levels
remaining after incubation are shown as a percent of total
[
C]hexanoyl SLs; the only other lipid obtained
after incubation with [
C]hexanoyl GlcCer was
[
C]hexanoyl Cer. Panels D-F show means
of data from three to four independent
experiments.
Likewise, a correlation was observed
between the extent of inhibition of [H]GlcCer
synthesis and axonal branching. Incubation with 50 and 250
µM PDMP resulted in inhibition of
[
H]GlcCer synthesis by 41 and 61%, respectively,
and of [
H]ganglioside synthesis by 44 and 79%
(Fig. 6 E). The total incorporation of
[4,5-
H]dihydrosphingosine into
H-SLs
was only inhibited by 5% using 50 µM PDMP, since at this
concentration [
H]SM synthesis was increased by
80% compared to control cells (Fig. 6 E). At 250
µM PDMP, the total incorporation of
[4,5-
H]dihydrosphingosine into
H-SLs
was inhibited by 51% and [
H]SM synthesis was
similar to control cells (Fig. 6 E). Thus, at 50
µM PDMP, the synthesis of GlcCer and gangliosides is
specifically inhibited, and SM synthesis is enhanced. At higher
concentrations (250 µM), SM synthesis is also inhibited by
PDMP, but to a far lesser extent than the inhibition of GlcCer
synthesis (Fig. 6 E). Similar effects on SL synthesis
using PDMP have been reported in other cells
(29) . However,
axonal branching was reduced by a similar amount using both 50 and 250
µM PDMP (Fig. 6 B), whereas lower
concentrations (0.5, 5 µM) had no effect on axonal
branching (Fig. 6 B), or
H-SL synthesis
(Fig. 6 E).
C]hexanoyl GlcCer
(Fig. 6 F) and reduction in levels of
[
C]hexanoyl Cer. Both concentration of CBE had a
similar effect on axonal branching (Fig. 6 C) whereas
lower concentrations (1 and 10 µM) had no effect on either
[
C]hexanoyl GlcCer degradation
(Fig. 6 F) or axonal branching (Fig. 6 C).
Together, these biochemical data demonstrate a correlation between SL
metabolism and axonal branching. Inhibition of SL synthesis results in
a decrease in the number of axonal branch point/cell, whereas
inhibition of SL degradation results in a increase in the number of
axonal branch point/cell.
Time Course of the Effect of FB
To characterize further the role of SLs in
axonal branching, the number of axonal branch points was measured every
6 h between days 2 and 3. In control cells, the mean number of axonal
branches increased linearly from 48 to 72 h (). This is
reflected in the normalized distribution of the number of axonal branch
points/cell (Fig. 7), in which the percent of cells with no
branches decreased from 48 to 72 h, and the number of cells with one,
two, or more branches increased in control cells. No effect was
observed after addition of either 10 µM FB and PDMP on
Axonal Branching
or
50 µM PDMP on day 0 on the mean number of axonal branches
during the first 54-60 h in culture, but for the next 12-18
h the mean number of branch points was significantly reduced
(). For FB
-treated cells, the percent of cells
with no branches decreased for the first 60 h similar to control cells,
but increased at 66 and 72 h; the percent of cells with one or two
branch points decreased during this period, suggesting that inhibition
of SL synthesis enhances regression of axonal branches between
60-72 h (Fig. 7). Similar results were obtained using 50
µM FB
.
Figure 7:
Time course of the effect of
FB on axonal branching between 48-78 h. FB
(
) was added on day 0 and the percent distribution of axonal
branch points/cell compared to control neurons (
) at the
indicated times. Cells with more than six branches (less than 2% of the
cell population) were not included in the graph. Data are taken from
the same cells as in Table I and are represented as means ± S.E.
from three to five separate cultures in which 50 cells were counted on
two individual coverslips. Errors cannot be seen in cases where error
bars are smaller than the size of the symbol. Statistical differences
( p < 0.05) with control cells are shown by
asterisks.
To determine whether inhibition of
SL synthesis had an acute effect on axonal branching between days 2 and
3, FB was added to cultures at various times and axonal
morphology analyzed 6 h later. Addition of FB
at 48 or 54 h
had no effect on the distribution of axonal branches at 54 and 60 h,
respectively (not shown). However, addition of FB
at 60 h
caused an increase in the percent of cells with no branches when
measured at 66 and 72 h () and a decrease in the percent
of cells with one, two, or three branches (not shown). Similarly,
addition of FB
at 66 h increased the percent of cells with
no branches at 72 h (not shown). The similarity between the acute
effects of FB
and the effects when FB
was added
immediately after cells were plated indicates that SL synthesis plays
an important role in the formation or stabilization of axonal branches
during a particular period of development.
Long Term Inhibition of SL Synthesis Has No Effect on
Neuronal Polarity
Cultured hippocampal neurons develop a dense
axonal network after 3-4 days in culture rendering it difficult
to delineate the entire axon plexus of any one cell. Thus, rates of
axonal growth cannot be determined in older cells. The long term effect
of FB on neuronal development was therefore examined by
analyzing the distribution of various proteins that are localized in
either axons or dendrites. 10 µM FB
was added
to cultures every 2 days for a period of 12-14 days. No GD
was detected at the cell surface by immunofluorescence after
FB
treatment although in control cultures, only
20-30% of cells were GD
-positive after 14 days in
culture. FB
had no discernible effect on the polarized
distribution of any of the proteins tested, including the axonal
markers GAP-43 (Fig. 8, A and B)
(30) and synaptophysin (Fig. 8, C and D)
(31) , the dendritic marker MAP-2 (Fig. 8, E and
F)
(23) , and Thy-1 (Fig. 8, G and
H)
(32) ; similar results were obtained using 50
µM FB
. Determining the precise distribution of
Thy-1 was problematic
(33) due to the presence of
Thy-1-negative cells in culture (see arrow in
Fig. 8H). These results demonstrate that long term
inhibition of SL synthesis had no deleterious effect on the segregation
of axonal or dendritic markers, although it was not possible to examine
whether inhibiting SL synthesis affected the rate of axonal growth or
branching in older cells.
Figure 8:
The polarity of axonal and dendritic
markers is unaffected by long term incubation with FB.
Neurons were incubated with 10 µM FB
on day 0,
and additional doses of the drug were added to the medium every 2 days.
After 12-14 days, the distribution of GAP-43 ( A and
B), synaptophysin ( C and D), MAP-2 ( E and F), and Thy-1 ( G and H) were
analyzed by immunofluorescence as described under ``Experimental
Procedures.'' The left-hand panels show cells observed by
phase contrast microscopy and the right-hand panels cells by
immunofluorescence. A Thy 1-negative cell is indicated by an arrow in panel H. The bar corresponds to 10
µm.
The Formation of Axonal Branches
Examining the
role of SLs in modulating neuronal growth is particularly attractive in
hippocampal neurons cultured at low density. These neurons are well
characterized and have provided novel information about the sequence of
events leading to the formation of axons and dendrites during the
development of neuronal polarity
(9, 10) . In the
initial stages of growth (stages 1-2; see Ref. 10), each neuron
develops a number of short processes that cannot be distinguished by
morphological or biochemical criteria. After some hours (with the exact
time varying considerably between individual cells), one of the
processes starts to grow rapidly (10-15 µm/h). Growth is
irregular and consists of periods of active elongation followed by
intervals of inactivity. The rapidly growing process develops axonal
characteristics (stage 3) and can be further distinguished from the
minor processes by the presence of proteins such as GAP-43
(30) and synaptophysin
(31) . Axonal branches arise as
collaterals, and as each new branch emerges, the growth cone of the
original axon loses its typical lamellipodial appearance and elongation
stops
(10) . During branch formation, some branches retract
while others continue to elongate, with some branches reaching
distances further from the cell body than the parent axon. The final
stages of neuronal growth (stages 4 and 5) occur when the minor
processes elongate, albeit at much slower rates than the axon, and
eventually acquire the characteristics of dendrites.
, although with a different time course.
(
)
By analogy, the formation of collateral branches could be
regulated by modifying the supply or contents of membrane material to
the region of the axon where the branch is formed. Determination of the
mechanisms by which inhibition of SL metabolism affects this process
necessitates characterization of the molecular events in which SLs are
involved.
The Molecular Mechanisms by Which SLs Affect Axonal
Branching
Clues concerning the nature of the SL species that may
be responsible for the effects on collateral branch formation are
provided by the use of three inhibitors
(11) of SL metabolism
(Fig. 6). FB inhibits acylation of the sphingoid base
of SLs, resulting in accumulation of sphinganine and sphingosine
(12, 43) and a reduction of dihydroceramide,
sphingomyelin, and glycosphingolipids distal to the block, including
GlcCer (Figs. 1 and 6). PDMP inhibits glucosylation of Cer
(15) , resulting in accumulation of dihydroceramide, ceramide,
and sphingomyelin (Figs. 1 and 6), and reduction in SLs distal to the
block (see Ref. 16). CBE inhibits the removal of the glucose moiety
from GlcCer
(21) , resulting in accumulation of GlcCer
(22) , and preliminary data indicate that levels of gangliosides
also increase upon CBE treatment.
(
)
Common to all
three inhibitors is a change in intracellular levels of GlcCer and
glycosphingolipids, suggesting that a glycosphingolipid(s) may be
involved in mediating axonal branching. Moreover, the possible
involvement of SM can be excluded since at 50 µM PDMP,
[
H]SM levels were increased by 80%
(Fig. 6 E) compared to control cells even though the
number of axonal branch points was significantly reduced
(Fig. 6 B). We are currently attempting to determine
whether a particular glycosphingolipid species is involved in
regulating axonal branching, but preliminary data indicate that there
is no major change in the incorporation of
[4,5-
H]dihydrosphingosine into
[
H]GlcCer or
[
H]gangliosides during the first 3 days in
culture.
for
only short periods ( i.e. 6 h, see ) had identical
effects on axonal morphology as long term incubations ( i.e. 72
h, see Fig. 7). Thus, the effect on axonal branching appears to
be directly related to SL synthesis and presumably delivery of newly
synthesized molecules to the axonal growth cone. Inhibition of SL
synthesis and delivery could affect a number of cellular processes that
may be involved in the formation and stabilization of collateral axonal
branches. For instance, SL synthesis may be necessary for the delivery
to the axon of a protein involved in regulating branching. Incubation
of Chinese hamster ovary cells with PDMP slows down delivery of an
infected viral protein to the cell surface
(44) , although
similar studies using a short acyl chain analog of ceramide
(45) indicate that an alternative explanation for the effect of
PDMP may be warranted. In neurons, a number of proteins involved in
axonal outgrowth are anchored to the membrane via a
glycosylphosphatidylinositol (GPI) anchor
(46) . GPI-anchored
proteins are believed to aggregate with glyco-SLs in the trans-Golgi
network of polarized epithelial cells
(47, 48) and are
consequently cotransported
(49) to the apical plasma membrane.
If a similar process occurs in neurons
(32) , inhibition of
glyco-SL synthesis, such as occurs with FB
and PDMP, could
affect the delivery to the cell surface of a developmentally regulated
GPI-anchored protein. Indeed, it has recently been shown in yeast that
inhibition of Cer synthesis leads to a rapid and specific reduction in
the rate of transport of GPI-anchored proteins to the Golgi apparatus
without affecting the transport of soluble or transmembrane proteins
(50) . Likewise, inhibition of SL degradation with CBE could
affect the rate of internalization of a GPI-anchored protein from the
cell surface. Glyco-SLs and GPI-anchored proteins are colocalized in
non-clathrin-coated membrane invaginations
(51, 52) and
may be internalized along the same endocytic pathway. Inhibition of
GlcCer degradation could slow down the internalization of a
GPI-anchored protein whose presence at the cell surface mediates the
formation or stability of axonal branches.
Table:
Time
course of the effect of FB and PDMP on axonal growth
between days 2 and 3 in culture
(10 µM)
and PDMP (50 µM) were added to cultures on day 0 and the
number of axonal branch points compared to control cells at the
indicated times. Numbers represent the means of measurements ±
S.E. from three to five separate cultures in which 50 cells were
counted on two individual cover slips.
Table:
Acute effects of FB on axonal
development
was added to neurons
after 60 h in culture, and the percent of cells with no axonal branch
points was measured 6 or 12 h later. Results are means ± S.E.
taken from data for four to five separate cultures.
, Fumonisin B
; GlcCer,
glucosylceramide; GMs, gangliosides; GPI, glycosylphosphatidylinositol;
[
C]hexanoyl GlcCer,
N-(1-[
C]hexanoyl)-D- erythro-glucosylsphingosine;
PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; SM,
sphingomyelin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.