©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Regulatory Role for Sphingolipids in Neuronal Growth
INHIBITION OF SPHINGOLIPID SYNTHESIS AND DEGRADATION HAVE OPPOSITE EFFECTS ON AXONAL BRANCHING (*)

Andreas Schwarz (§) , Elizabeth Rapaport(§)(¶) , Koret Hirschberg , Anthony H. Futerman (**)

From the (1) Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (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.


INTRODUCTION

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)() 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.

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 (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.

In the current report, we demonstrate that both FB 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.


EXPERIMENTAL PROCEDURES

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 (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).

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.


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/CHOH/HO/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/CHOH/CaCl (0.22% (w/v)) (60:35:8 (v/v/v)) as the developing solvent. H-SLs were visualized by autoradiography using EnHance 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)).

The effect of CBE on GlcCer metabolism was analyzed using N-(1-[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).

Cells were observed using either a Plan Apochromat 40/1.3 or 63/1.4 oil objective of a Zeiss Axiovert 35 microscope with appropriate filters for fluorescein or rhodamine fluorescence.


RESULTS

We previously demonstrated that inhibition of ceramide (Cer) synthesis in hippocampal neurons by FB 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).

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 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.

We next examined the effect of addition of FB 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.

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 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.

We next examined the correlation between axonal branching and the extent of inhibition of SL metabolism. Incubation with 10 or 50 µM FB 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).

Incubation with 100 or 500 µM CBE caused accumulation of [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 and PDMP on Axonal Branching

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 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.




DISCUSSION

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.

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, 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.

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 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.

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.

  
Table: Time course of the effect of FB and PDMP on axonal growth between days 2 and 3 in culture

FB (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

10 µM FB 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.



FOOTNOTES

*
This work was supported by the Basic Research Foundation of the Israel Academy of Science and Humanities, by Grant 91-00278 from the United States-Israel Binational Science Foundation, Jerusalem, Israel, and by the Minerva Foundation, Munich/Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Both authors contributed equally to this study.

Permanent address: Dept. of Biochemistry, University of Bath, Bath, Avon BA2 7AY, United Kingdom.

**
Incumbent of the Recanati Career Development Chair in Cancer Research. To whom correspondence should be addressed. Tel.: 972-8-342704; Fax: 972-8-344112; E mail: BMFUTER@WEIZMANN.WEIZMANN.AC.IL.

The abbreviations used are: SLs, sphingolipids; CBE, conduritol B-epoxide; Cer, ceramide; FB, 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.

A. Sofer and A. H. Futerman, J. Biol. Chem., in press.

I. Meivar-Levy, A. Bershadsky, and A. Futerman, unpublished observation.

A. Schwarz and A. H. Futerman, unpublished observations.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Lein, P. J., and Higgins, D. (1991) Brain Res. Dev. Brain Res. 60, 187-196 [Medline] [Order article via Infotrieve]
  2. Ferreira, A., Niclas, J., Vale, R. D., Banker, G., and Kosik, K. S. (1992) J. Cell Biol. 117, 595-606 [Abstract]
  3. Osen-Sand, A., Catsicas, M., Staple, J. K., Jones, K. A., Ayala, G., Knowles, J., Grenninigloh, G., and Catsicas, S. (1993) Nature 364, 445-448 [CrossRef][Medline] [Order article via Infotrieve]
  4. Joshi, H. C., and Baas, P. W. (1993) J. Cell Biol. 121, 1191-1196 [CrossRef][Medline] [Order article via Infotrieve]
  5. Harel, R., and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481 [Abstract/Free Full Text]
  6. Yu, R. K., and Saito, M. (1989) in Neurobiology of Glycoconjugates (Margolis, R. U., and Margolis, R. K., eds) pp. 1-42, Plenum Press, New York
  7. Zeller, C. B., and Marchase, R. B. (1992) Am. J. Physiol. 262, C1341-C1355
  8. Ledeen, R. W. (1989) in Neurobiology of Glycoconjugates (Margolis, R. J., and Margolis, R. K., eds) pp. 43-83, Plenum Press, New York
  9. Goslin, K., and Banker, G. (1991) Culturing Nerve Cells, pp. 251-281, MIT Press, Cambridge, MA
  10. Dotti, C. G., Sullivan, C. A., and Banker, G. A. (1988) J. Neurosci. 8, 1454-1468 [Abstract]
  11. Futerman, A. H. (1994) Trends. Glycosci. Glycotechnol. 6, 143-153
  12. Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., and Merrill, A. H. (1991) J. Biol. Chem. 266, 14486-14490 [Abstract/Free Full Text]
  13. Hanada, K., Nishijima, M., Kiso, M., Hasegawa, A., Fujita, S., Ogawa, T., and Akamatsu, Y. (1992) J. Biol. Chem. 267, 23527-23533 [Abstract/Free Full Text]
  14. Radin, N. S., and Vunnam, R. R. (1981) Methods. Enzymol. 72, 673-684 [Medline] [Order article via Infotrieve]
  15. Radin, N. S., Shayman, J. A., and Inokuchi, J. (1993) Adv. Lipid Res. 26, 183-213 [Medline] [Order article via Infotrieve]
  16. Uemura, K., Sugiyama, E., Tamai, C., Hara, A., Taketomi, T., and Radin, N. S. (1990) J. Biochem. ( Tokyo) 108, 525-530 [Abstract]
  17. Shayman, J. A., Deshmukh, G. D., Mahdiyoun, S., Thomas, T. P., Wu, D., Barcelon, F. S., and Radin, N. S. (1991) J. Biol. Chem. 266, 22968-22974 [Abstract/Free Full Text]
  18. Felding-Habermann, B., Igarashi, Y., Fenderson, B. A., Park, L. S., Radin, N. S., Inokuchi, J., Strassmann, G., Hanada, K., and Hakamori, S. (1990) Biochemistry 29, 6314-6322 [Medline] [Order article via Infotrieve]
  19. Inokuchi, J., Momosaki, K., Shimeno, H., Nagamatsu, A., and Radin, N. S. (1989) J. Cell. Physiol. 141, 573-583 [Medline] [Order article via Infotrieve]
  20. Kan, C. C., and Kolesnick, R. N. (1992) J. Biol. Chem. 267, 9663-9667 [Abstract/Free Full Text]
  21. Legler, G. (1977) Methods. Enzymol. 46, 368-381 [Medline] [Order article via Infotrieve]
  22. Mahdiyoun, S., Deshmukh, G. D., Abe, A., Radin, N. S., and Shayman, J. A. (1992) Arch. Biochem. Biophys. 292, 506-511 [Medline] [Order article via Infotrieve]
  23. Caceres, A., Banker, G. A., and Binder, L. (1986) J. Neurosci. 6, 714-722 [Abstract]
  24. Hirschberg, K., Rodger, J., and Futerman, A. H. (1993) Biochem. J. 290, 751-757 [Medline] [Order article via Infotrieve]
  25. van Echten, G., Iber, H., Stotz, H., Takatsuki, A., and Sandhoff, K. (1990) Eur. J. Cell Biol. 51, 135-139 [Medline] [Order article via Infotrieve]
  26. Williams, M. A., and McCluer, R. H. (1980) Int. Soc. Neurochem. 35, 266-269
  27. Futerman, A. H., and Pagano, R. E. (1991) Biochem. J. 280, 295-302 [Medline] [Order article via Infotrieve]
  28. Meivar-Levy, I., Horowitz, M., and Futerman, A. H. (1994) Biochem. J. 303, 377-382 [Medline] [Order article via Infotrieve]
  29. Okada, Y., Radin, N. S., and Hakomori, S. (1988) FEBS Lett. 235, 25-29 [CrossRef][Medline] [Order article via Infotrieve]
  30. Goslin, K., Schreyer, D. J., Skene, J. H. P., and Banker, G. (1988) Nature 336, 672-674 [CrossRef][Medline] [Order article via Infotrieve]
  31. Fletcher, T. L., Cameron, P., de Camilli, P., and Banker, G. (1991) J. Neurosci. 11, 1617-1626 [Abstract]
  32. Dotti, C. G., Parton, R. G., and Simons, K. (1991) Nature 349, 158-161 [CrossRef][Medline] [Order article via Infotrieve]
  33. Morris, R. J. (1985) Neuroscience 7, 133-160
  34. Hakamori, S. I. (1990) J. Biol. Chem. 265, 18713-18716 [Abstract/Free Full Text]
  35. Siegel, D. A., and Walkley, S. U. (1994) J. Neurochem. 62, 1852-1862 [Medline] [Order article via Infotrieve]
  36. Inokuchi, J., Jimbo, M., Momosaki, K., Shimeno, H., Nagamatsu, A., and Radin, N. S. (1990) Cancer Res. 50, 6731-6737 [Abstract]
  37. Shukla, G. S., Shukla, A., Inokuchi, J.-i., and Radin, N. (1991) Biochim. Biophys. Acta 1083, 101-108 [Medline] [Order article via Infotrieve]
  38. Uemura, K., Sugiyama, E., and Taketomi, T. (1991) J. Biochem. ( Tokyo) 110, 96-102 [Abstract]
  39. Bray, D., Thomas, C., and Shaw, G. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 5226-5229 [Abstract]
  40. Yu, W. Q., Ahmad, F. J., and Baas, P. W. (1994) J. Neurosci. 14, 5872-5884 [Abstract]
  41. Kirsch, J., Zutra, A., and Littauer, U. Z. (1990) J. Neurochem. 55, 1031-1041 [Medline] [Order article via Infotrieve]
  42. Bershadsky, A., and Futerman, A. H. (1994) Proc. Natl. Acad. Sci.U. S. A. 91, 5686-5689 [Abstract]
  43. Merrill, A. H., Van Echten, G., Wang, E., and Sandhoff, K. (1993) J. Biol. Chem. 268, 27299-27306 [Abstract/Free Full Text]
  44. Rosenwald, A. G., Machamer, C. E., and Pagano, R. E. (1992) Biochemistry 31, 3581-3590 [Medline] [Order article via Infotrieve]
  45. Rosenwald, A. G., and Pagano, R. E. (1993) J. Biol. Chem. 268, 4577-4579 [Abstract/Free Full Text]
  46. Walsh, F. S., and Doherty, P. (1992) in GPI Membrane Anchors (Cardoso de Almeida, M. L., ed) pp. 294-309, Academic Press, New York
  47. Simons, K., and Wandinger-Ness, A. (1990) Cell 62, 207-210 [Medline] [Order article via Infotrieve]
  48. Lisanti, M. P., Caras, I. W., Gilbert, T., Hanzel, D., and Rodriguez-Boulan, E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7419-7423 [Abstract]
  49. Brown, D. A., and Rose, J. K. (1992) Cell 68, 533-544 [Medline] [Order article via Infotrieve]
  50. Horvath, A., Sutterlin, C., Manning-Krieg, U., Movva, N. R., and Riezman, H. (1994) EMBO J. 13, 3687-3695 [Abstract]
  51. Lisanti, M. P., Tang, Z. L., and Sargiacomo, M. (1993) J. Cell Biol. 123, 595-604 [Abstract]
  52. Parton, R. G. (1994) J. Histochem. Cytochem. 42, 155-166 [Abstract/Free Full Text]
  53. Blackburn, C. C., Swank-Hill, P., and Schnaar, R. L. (1986) J. Biol. Chem. 261, 2873-2881 [Abstract/Free Full Text]
  54. Doherty, P., Ashton, S. V., Skaper, S. D., Leon, A., and Walsh, F. S. (1992) J. Cell Biol. 117, 1093-1099 [Abstract]
  55. Wu, G., Vaswani, K. K., Lu, Z.-H., and Ledeen, R. W. (1990) J. Neurochem. 55, 484-491 [Medline] [Order article via Infotrieve]
  56. Fentie, I. H., and Roisen, F. J. (1993) J. Neurocytol. 22, 498-506 [Medline] [Order article via Infotrieve]
  57. Spiegel, S., Yamada, K. M., Hom, B. E., Moss, J., and Fishman, P. H. (1986) J. Cell Biol. 102, 1898-1906 [Abstract]
  58. Hannun, Y. A. (1994) J. Biol. Chem. 269, 3125-3128 [Free Full Text]
  59. Shayman, J. A., Mahdiyoun, S., Deshmukh, G., Barcelon, F., Inokuchi, J., and Radin, N. S. (1990) J. Biol. Chem. 265, 12135-12138 [Abstract/Free Full Text]
  60. Futerman, A. H. (1994) in Current Topic in Membranes (Hoekstra, D., ed) pp. 93-110, Academic Press, Orlando, FL
  61. Jareb, M., and Banker, G. A. (1991) Soc. Neurosci.Abs. 17, 739
  62. Sofer, A., and Futerman, A. H. (1995) J. Biol. Chem. 270, in press

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.