(Received for publication, June 27, 1995; and in revised form, November 6, 1995)
From the
Plasmalopsychosine, a characteristic fatty aldehyde conjugate of
-galactosylsphingosine (psychosine) found in brain white matter,
enhances p140
(Trk A) phosphorylation and
mitogen-activated protein kinase (MAPK) activity and as a consequence
induces neurite outgrowth in PC12 cells. The effect of
plasmalopsychosine on neurite outgrowth and its prolonged activation of
MAPK was similar to that of nerve growth factor (NGF), and the effect
was specific to neuronal cells. Plasmalopsychosine was not capable of
competing with cold chase-stable, high affinity binding of NGF to Trk
A, indicating that plasmalopsychosine and NGF differ in terms of Trk
A-activating mechanism. Tyrosine kinase inhibitors K-252a and
staurosporine, known to inhibit the neurotrophic effect of NGF, also
inhibited these effects of plasmalopsychosine, suggesting that
plasmalopsychosine and NGF share a common signaling cascade.
Plasmalopsychosine prevents apoptosis of PC12 cells caused by serum
deprivation, indicating that it has ``neurotrophic
factor-like'' activity. Taken together, these findings indicate
that plasmalopsychosine may play an important role in development and
maintenance of the vertebrate nervous system.
A novel cationic glycosphingolipid (GSL), ()plasmalopsychosine (PLPS), was previously isolated from
white matter of human brain. The conjugates through 3,4- and 4,6-cyclic
plasmal linkage were identified and termed PLPS A and B,
respectively(1) , and these compounds were chemically
synthesized(2) . PLPS has a unique molecular shape in which two
aliphatic hydrophobic tails, Sph and fatty aldehyde, are oriented in
opposite directions. This is in striking contrast to all other GSLs,
which have two hydrophobic tails (Sph and fatty acid) oriented in the
same direction (Fig. 1).
Figure 1:
Structures of cerebroside
(-galactosylceramide), psychosine (
-galactosyl-Sph), and
4,6-PLPS.
Nerve growth factor (NGF) is
required for the survival and development of neurons in the sympathetic
and sensory nervous systems(3, 4) . NGF induces signal
transduction through activation of tyrosine kinase associated with its
receptor p140 (Trk A) (5, 6) and causes a phenotypic change of PC12 cells
involving neurite outgrowth, which has been regarded as a criterion of
neuronal cell differentiation(7, 8) . Various
gangliosides and sialic acid derivatives have been shown to promote
neuritogenesis of Neuro 2A cells in the presence or absence of
NGF(7) , but ganglioside-enhanced neuritogenesis of PC12 has so
far been observed only in the presence of NGF(9) .
Signal pathways from cell surface receptors to nuclear events are of central importance for understanding control of cell differentiation and proliferation. It has become obvious that the signal modulatory activity of GSLs as originally described (10, 11) resides not only in the entire complex structures but also in their backbone structures and metabolites, e.g. lyso-GSLs (12) and de-N-acetyl gangliosides(13) , Sph(14) , and its derivatives(15, 16, 17, 18) . Psychosine is known to inhibit kinase C (1, 14) and mitochondrial cytochrome c oxidase(19) . Based on this background, we studied the effect of PLPS on neuronal differentiation and associated transmembrane signal changes in PC12 cells.
Mouse NGF and
anti-MAPK (erk1-CT) antibody was purchased from Upstate Biotechnology
Inc. (Lake Placid, NY). Anti-p140 (Trk A) antibody was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). K-252a
was purchased from Kamiya Biochemical Co. (Thousand Oaks, CA). A stock
solution (2 mM) was prepared in dimethyl sulfoxide and stored
at -20 °C. All other reagents were purchased from Sigma (St.
Louis, MO).
In order to examine MAPK activation by PLPS in nonneuronal cell lines, murine fibroblast BALB/c 3T3 A31 and human fibroblast WI38 cells were treated with PLPS, and MAPK activity was assayed as described above.
Figure 2:
Neurite outgrowth of PC12 cells induced by
PLPS. 3 10
cells were seeded in 35-mm dishes,
incubated for 24 h, and then incubated for 72 h with experimental
reagent. Processes longer than two cell body diameters were counted as
neurites. Acetylcholine esterase staining was also performed according
to the procedure of Tago et al.(42) to confirm their
identity as true neurites (data not shown). A, untreated
controls; B, psychosine (5 µg/ml); C, EGF (30
nM); D, NGF (100 ng/ml); E, 4,6-PLPS (5
µg/ml); F, 3,4-PLPS (5 µg/ml). Magnification,
200
.
Figure 3: Dose dependence of 4,6-PLPS, and additive effect of NGF on PC12 neuritogenesis. PC12 cells were incubated with 20 ng/ml NGF and/or 4,6-PLPS at the indicated concentrations for 72 h. Each data point represents mean ± S.D. of three separate experiments performed in triplicate. Neurites were counted as described under ``Experimental Procedures.'' Open bars, 4,6-PLPS alone; shaded bars, 4,6-PLPS plus 20 ng/ml NGF. The additive effect was statistically significant (*, p < 0.001;**, p < 0.05) by Student's t test.
Figure 4: Time course of MAPK activation in PC12 cells by PLPS. PC12 cells were treated for the indicated times with NGF plus 4,6-PLPS, NGF, 4,6-PLPS, EGF, or 3,4-PLPS. Panel A, time course of MAPK activation. Data points as in Fig. 3. Panel B, phosphorylated myelin basic protein.
Figure 5:
PLPS induces phosphorylation of
p140 (Trk A) in PC12 cells, and both K-252a and
staurosporine block PLPS-dependent tyrosine phosphorylation of
p140
. After preincubation for 10 min with or without 100
nM K-252a (lanes 3, 6, and 9) or
staurosporine (lanes 4, 7, and 10), cells
were treated for 5 min with 100 ng/ml NGF or 5 µg/ml 4,6- or
3,4-PLPS (lanes 2-4, 5-7, and 8-10, respectively). Lysates (containing 30 µg of
protein) from PC12 cells were immunoprecipitated with
anti-p140
antibody. Immunoprecipitates were
subjected to SDS-polyacrylamide gel electrophoresis followed by
immunoblotting with anti-phosphotyrosine antibody PY20 or rabbit
anti-mouse IgG, and visualized by autoradiography with ECL kit for 24
h.
Figure 6: Effect of K-252a and staurosporine on MAPK activation and neurite outgrowth by PLPS. Cells were incubated in the presence or absence of K-252a (100-200 nM) or staurosporine (2.5-200 nM) in culture medium, and were further incubated with or without NGF or PLPS for 5 min. MAPK activation (panel A) and neurite outgrowth (panel B) were examined. Data points were as in Fig. 3, except that each experiment was performed in duplicate. Panel B, a, 4,6-PLPS (5 µg/ml); b, 4,6-PLPS (5 µg/ml) plus K-252a (100 nM); c, 4,6-PLPS (5 µg/ml) plus staurosporine (5 nM); d, untreated (control). PC12 cells were treated with these agents for 48 h, and neurite outgrowth was examined.
Figure 7:
Cold chase-stable NGF binding to PC12
cells. Serial dilutions of NGF, 4,6-PLPS, 3,4-PLPS, and G were assayed for their ability to inhibit stable binding of
I-NGF to Trk A. Data points were as in Fig. 3.
Figure 8:
Effect of PLPS on long-term survival of
PC12 cells in serum-free medium. A, cells were washed and
plated in 0.5 ml of RPMI 1640 medium containing either no additive or
the indicated agents. Viable cells were counted on the indicated days.
The number of surviving cells is expressed as a percentage relative to
the number initially plated (8-10 10
; defined
as 100%). Data points were as in Fig. 3. B, agarose gel
electrophoresis of DNA from cells treated with 4,6- or 3,4-PLPS or NGF
in serum-free medium for 3 days. 5 µg of DNA was analyzed by 1.5%
agarose gel electrophoresis. The marker is HindIII digest of
DNA. Lane 1, control; lane 2, 72 h, serum-free
medium; lane 3, 100 ng/ml NGF in serum-free medium; lane
4, 5 µM 4,6-PLPS in serum-free medium; lane
5, 5 µM 3,4-PLPS in serum-free medium. C,
DNA fluorescence histogram (log scale) of propidium iodine-stained
cells. a-e, same as lanes 1-5,
respectively, of B. Bar 1, apoptotic cells; bar
2, G
/G
cells; bar 3, S and
G
-M cells; bar 4, total
cells.
Neuritogenesis of neuronal cell lines such as neuroblastoma,
glioma, and pheochromocytoma has been used often as a model of neuronal
differentiation and its biochemical mechanism. Mouse neuroblastoma cell
line Neuro2a shows strong neuritogenic response to exogenous addition
of various gangliosides and synthetic sialosyl compounds in the absence
of NGF. Gangliosides and NGF function synergistically to affect
neuritogenesis in Neuro2a cells(7, 26) . Various
gangliosides, including G, are mitogens for astroglial
cells, and induce astroglial differentiation. These compounds have been
claimed to mimic glial maturation factor(27, 28) .
Neuritogenesis in primary culture of normal rat astroglial cells, rat
astroglioma cell line GA-1, and rat Schwannian glioma line 354-A is
strongly promoted by various synthetic sialosyl derivatives, including
- and
-sialosylcholesterol(29) . Neuritogenesis of
two human neuroblastoma lines, GOTO and NB-1, was strongly promoted
when nM order concentrations of the specific ganglioside GQ1b
were added to culture medium(30) . This effect was ascribed to
activation of a GQ1b-sensitive cell surface protein kinase, and claimed
to be present on the neuroblastoma cells(31) . Neurite
outgrowth and cholinergic differentiation in Neuro2a cells were
recently shown to be induced by transfection and expression of
sialyltransferase gene for synthesis of b-series gangliosides, i.e. CMP-sialic acid: NeuAc
2
3Gal
2
8-sialyltransferase(32) .
However, none of the above studies included attempts to clarify the mechanism of transmembrane signaling and its possible modulation by exogenous addition of gangliosides or their analogs, or endogenous stimulation of b-series ganglioside synthesis. Transmembrane signaling in neuronal cells is currently thought to be initiated by activation of tyrosine kinase associated with neurotrophin receptors (Trk family) (33) .
Neuritogenesis of rat pheochromocytoma PC12 cells has
been employed as a model of neuronal differentiation induced by NGF.
NGF causes PC12 cells to undergo neuritogenesis through activation of
its receptor-associated tyrosine kinase, Trk A. Although various
gangliosides enhance the neuritogenic effect of NGF in PC12 cells,
gangliosides or other GSLs alone in the absence of NGF do not induce
neuritogenesis(9) . The present study indicates that one novel
GSL, PLPS, a peculiar fatty aldehyde conjugate of -galactosyl-Sph
(psychosine)(1) , is capable of inducing not only
neuritogenesis but also prolonged survival of PC12 cells in the absence
of serum and NGF, through prevention of apoptosis. PLPS is present in
brain white matter, but not gray matter, in humans and some animals. Of
two types of PLPS, i.e. 4,6- and 3,4-cyclic acetal
derivatives, 4,6-PLPS shows strong NGF-like activity, and 3,4-PLPS has
weaker activity. This is the first case in which a GSL species induces
NGF-like activity in PC12 cells in the absence of NGF. The effect of
PLPS in inducing differentiation of PC12 cells provides an opportunity
to study PLPS-dependent transmembrane signaling mechanism in these
cells.
Mixing experiments with NGF and PLPS revealed an additive effect on neuritogenesis when half-saturating doses of both agents were used. When saturating doses were used, there was no clear additive effect. These findings suggest that PLPS may interact with cell surface membranes in a different way than NGF, and indirectly activate NGF receptor. Consequently, PLPS shares the common pathway induced by NGF, leading to activation of specific genes required for the differentiation process.
The established signal transduction pathway
associated with NGF-dependent differentiation is initiated by
activation of NGF receptor kinase (p140, Trk A) (5, 6) and leads to prolonged activation of
MAPK(34, 35) . We studied these two processes in
relation to differentiation induction of PC12 cells by NGF and/or PLPS.
Similar degrees of p140
(Trk A) phosphorylation were
induced when PC12 cells were incubated with NGF alone or PLPS alone. No
phosphorylated band was detected in cells stimulated in the presence of
100 nM K-252a or staurosporine, both of which are known
tyrosine kinase inhibitors and neuritogenesis
blockers(23, 24, 25) . The degree of Trk A
activation was much stronger in the presence of 4,6- than 3,4-PLPS,
consistent with the relative degree of neuritogenesis induction. Prompt
and prolonged MAPK activation was also observed when PC12 cells were
treated with 4,6-PLPS, NGF, or a combination. The degree of MAPK
activation by 3,4-PLPS was considerably smaller, again comparable with
the degree of neuritogenesis induction.
Neuritogenic effect and prolonged MAPK activation by PLPS were also observed in glioma cell line Neuro2A. PLPS did not activate MAPK in fibroblast line BALB/c 3T3 A31 or human foreskin fibroblast line WI38 and did not initiate EGF receptor phosphorylation in PC12 cells (data not shown). Therefore, its effect is specific to NGF-susceptible neuronal cells and may require the presence of NGF receptor. Whether PLPS affects activation of other types of neuronal growth factor receptors (Trk B, Trk C, etc.) remains to be studied.
Data accumulated so far indicate that PLPS mimics the activity of NGF through activation of NGF receptor kinase to the same extent as NGF, triggering a common signal transduction cascade that leads to activation of MAPK. MAPK switches on various transcription factors necessary for activation of genes required for induction of neuronal differentiation (including neuritogenesis, cholinergic activity, and cell survival through prevention of apoptosis).
The
major question of how PLPS activates Trk A remains to be elucidated.
PLPS, in contrast to unlabeled NGF, does not inhibit binding of I-labeled NGF to the NGF receptor (Trk A). This suggests
that PLPS does not bind to the NGF binding site on Trk A.
The
co-presence of G ganglioside potentiates the effect of NGF
in terms of neuritogenesis in PC12 cells. Unlike PLPS, G
in the absence of NGF has no neuritogenic effect(9) . It
was reported recently that G
at very high concentration
(0.5-2 mM) incubated with PC12 cells had only a weak
enhancing effect on Trk A phosphorylation(36) . This weak
stimulatory effect by G
alone, even at mM order
concentration, was not sufficient to induce neuritogenic
differentiation. G
at mM order concentration may
induce membrane perturbation, which somehow indirectly induces weak Trk
A phosphorylation. In contrast, PLPS at much lower concentration
(1-20 µM) strongly induces Trk A phosphorylation and
consequent differentiation of PC12 cells.
Our results indicate that
PLPS does not compete with binding of NGF to Trk A (Fig. 7). It
is possible that PLPS does not bind to the same binding site as NGF on
Trk A within the receptor. Although the exact mechanism for
phosphorylation of Trk A by PLPS remains to be elucidated, it was
reported recently that G phosphorylates Trk A and binds to
Trk A in SDS-resistant fashion(37) . However, these authors did
not perform a binding assay, and they state that the exact site for
binding of G
to Trk protein is not known at present. They
also report that treatment with tunicamycin, a potent N-glycosylation inhibitor, results in loss of association of
G
and Trk A protein, and disappearance of tyrosine kinase
activity of fully glycosylated 140-kDa Trk A. This suggests that
different mechanisms are involved in binding of G
to Trk A
and phosphorylation of Trk A by G
. We found that G
did not affect binding of
I-NGF to Trk A (Fig. 7). Based on all of these results, we conclude that the
mechanism of Trk A phosphorylation by PLPS or G
differs
from that by NGF and that PLPS and NGF share a common signaling cascade
from NGF receptor Trk A to MAPK. Further study is necessary to
determine the precise mechanism by which PLPS activates Trk A.
Possible explanations for the observed effects of PLPS are as follows. (a) PLPS has a novel structure with two aliphatic chains oriented in opposite directions, and this morphology may cause membrane reorganization and local perturbation, inducing NGF receptor activation via some yet unknown mechanism. (b) The novel structure of PLPS may bind one NGF receptor to another, accelerating receptor-receptor interaction without binding of NGF, although the binding site of PLPS on NGF receptor may well be different from that NGF. This could enhance Trk A dimerization and consequent phosphorylation. (c) PLPS may affect modulators of Trk A such as P75(33, 38) . In order to distinguish between these possibilities, we are in the process of synthesizing radioactive PLPS.
Apoptosis of PC12 cells results within a few hours from serum
withdrawal in culture and can be prevented by the addition of
NGF(39) . We demonstrated clearly that PLPS has an
apoptosis-preventing (neurotrophic) effect similar to that of NGF in
serum-deprived cells for a prolonged period (7 days). In other words,
PLPS has ``neurotrophic factor-like activity.'' Psychosine is
absent in normal neuronal tissue, but it is known to accumulate in
association with a genetic deficiency of ceramide -galactosidase
in brain white matter (Krabbe's
disease)(1, 40) . Psychosine is strongly cytotoxic,
and its accumulation is regarded as the cause of neuronal dysfunction
in Krabbe's disease. As shown in this study, psychosine inhibits
endogenous MAPK activity in PC12 cells (Table 1). Most
psychosine-treated cells were swollen and detached. Psychosine inhibits
neurite outgrowth and MAPK activation by NGF (data not shown). Plasmal
conjugation of psychosine results in a product (PLPS) showing a
completely opposite physiologic effect from psychosine in the sense
that it shows strong neutrotrophic factor-like activity and enhanced
Trk A phosphorylation leading to prolonged MAPK activation. The process
of cyclic aldehyde formation is biochemically unknown, yet it is
crucial for conversion of neurotoxic psychosine into neurotrophic PLPS
in normal neuronal cells. This process is presumably highly active in
normal human brain, since PLPS is present in significant quantity in
this tissue, whereas psychosine is completely absent. It is possible
that plasmal conjugation reaction is defective in Krabbe's
disease, along with enhanced ceramidase activity that converts
cerebroside to psychosine(40, 41) .
The presence of a novel lipid factor in normal brain that mimics NGF activity through activation of Trk A and MAPK may be important in development and maintenance of the vertebrate nervous system.