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(Received for publication, November
1, 1994; and in revised form, November 29, 1994) From the
We have used serum-deprived cultures of wild type and
genetically modified PC12 cells to investigate the molecular mechanisms
by which monosialoganglioside (G Neuronal cell death is a prominent feature of normal nervous
system development (1, 2, 3) and is as well a
disastrous consequence of a number of neurodegenerative disorders (4) and of various nervous system insults or
injuries(5, 6) . In addition to neurotrophic growth
factors, a variety of agents have been described that can prevent
neuronal cell death and these have both experimental and clinical
potential(7, 8, 9, 10, 11, 12) .
Among these agents are gangliosides, a class of sialic acid-containing
glycosphingolipids mainly associated with the plasma membrane and
particularly abundant in the nervous system(13, 14) .
Both in vitro and in vivo experiments indicate that
exogenously supplied monosialoganglioside (G The molecular mechanism by which G The aim of the present studies has been to define
the molecular mechanism by which G
For cell survival
experiments, washed cells were resuspended in RPMI 1640 medium and
plated in 0.5 ml at a density of 8-10
Figure 1:
Temporal aspects of the
effect of G
In past work, we showed that protein kinase C does not appear to
play a role in the G
Figure 2:
Effect of K-252a (200 nM) on
serum-free cell survival induced by G
To
further test the role of Trk in mediating the survival-promoting
effects of G
Figure 3:
The effects of G
To determine whether the difference in G
Figure 4:
The effects of G
Figure 5:
Effect
of G
Figure 6:
Effect of NGF and G
Figure 7:
Effect of G
We next tested whether G
Figure 8:
Effect of G
The present study explores the mechanism by which
gangliosides exert trophic actions on neurons. In particular, we
studied the capacity of G Both our
past (38) and present findings are consistent with a similarity
in mechanism between NGF and G A previous study has shown that G Our observations with PC12 cells expressing kinase inactive Trk
provide an additional valuable clue regarding mechanism. Experiments
with fibroblasts co-expressing catalytically competent and incompetent
Trks show, as in the case of other receptor tyrosine kinases, that
NGF-induced Trk dimerization and autophosphorylation are required for
initiation of NGF-mediated signal transduction(68) . Our
findings corroborate this for PC12 cells and demonstrate that such
dimerization and autophosphorylation are necessary for promotion of
serum-free survival by NGF in this system. Moreover, suppression of the
G Our findings regarding ganglioside effects
on Trk activity raise several intriguing issues. For one, why is
G A
related issue concerns the types of responses mediated by G Mediation of G In summary, we have provided evidence that the
molecular mechanism by which G
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 3074-3080
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ganglioside
INVOLVEMENT OF Trk NEUROTROPHIN RECEPTORS (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) rescues neuronal cells
from apoptotic death elicited by withdrawal of trophic support. Our
findings indicate that G-promoted survival can be mediated
in part by the Trk NGF receptor as well as by TrkB, and potentially by
tyrosine kinase receptors for additional neurotrophic growth factors.
Experiments employing K-252a, an inhibitor of Trk kinases, and PC12
cells overexpressing a dominant inhibitory form of Trk both indicate
that a portion of the survival-promoting activity of G is
evoked by receptor dimerization and autophosphorylation. In consonance
with this we find that G stimulates Trk tyrosine
autophosphorylation and Trk-associated protein kinase activity. These
observations may provide a mechanism to account for the reported in
vitro and in vivo trophic actions of G.
) (
)inserts into membranes (15, 16) and can
potentiate neuronal cell responses to neurotrophic
factors(17, 18, 19, 20) . Moreover,
like neurotrophic factors, G can protect neurons against
diverse treatments that can otherwise induce their
death(21, 22, 23, 24, 25, 26, 27, 28, 29) .
Furthermore, gangliosides have been implicated in the modulation of
several protein kinase activities (30, 31, 32, 33, 34, 35) .
Recent clinical trials support the possible use of G for
treatment of neuronal disorders and injury(36, 37) . may protect neurons
from degeneration has remained elusive. We recently reported that
gangliosides can rescue cultured neuronal cells from death induced by
withdrawal of trophic support(38) . In particular, ganglioside
G was able to maintain the long term survival of
NGF-deprived sympathetic neurons and to prevent the apoptotic death of
PC12 cells caused by withdrawal of serum or of NGF in the absence of
serum. The ability of G to promote serum-free survival of
PC12 cells provides a potentially powerful model system with which to
study the molecular basis for the neurotrophic-like actions of
gangliosides. Serum-deprived PC12 cells undergo rapid death that can be
prevented by NGF and other trophic
agents(12, 39, 40) . Analysis of DNA from
PC12 cells deprived of trophic support for as little as 2-3-h
reveals a pattern of internucleosomal DNA fragmentation characteristic
of apoptotic cell death(12) . This permits acute experiments to
be performed under conditions that would themselves prove lethal over
longer term exposure(41) . The existence of a growing number of
genetically modified PC12 cell variants further enhances the utility of
this model system for studying the survival-promoting actions of
G. exerts trophic actions
on neurons. We provide evidence based on experiments with PC12 cells
and their variants that G-induced prevention of apoptotic
neuronal death is in part mediated by Trk neurotrophin receptors.
Materials
Mouse NGF was prepared from adult male
submaxillary glands as described previously (42) and was used
at a concentration of 100 ng/ml. Recombinant human BDNF was obtained
from Regeneron Pharmaceuticals Inc. (Tarrytown, NY). Ganglioside
G was purified according to methods described by
Tettamanti et al.(43) and was provided by FIDIA
Research Laboratories (Abano Terme, Italy). The purity of the compound
was over 99% as determined by highperformance thin layer
chromatography. G was dissolved in PBS at a concentration
of 10
M, autoclaved and diluted to the
desired concentration directly in the culture medium. K-252a was
purchased from Kamiya Biomedical Company (Thousand Oaks, CA). A stock
solution (2 mM) was prepared in dimethyl sulfoxide and stored
at -20 °C. The stock solution was diluted with culture medium
just before use. All other reagents were purchased from Sigma.Cell Culture
PC12 cells and various PC12
cell-derived lines were cultured as described previously on
collagen-coated dishes in RPMI 1640 medium supplemented with 10%
heat-inactivated horse serum and 5% fetal bovine serum(44) .
For the experiments in serum-free medium, cells were extensively washed
in serum-free RPMI 1640 as described
previously(12, 38, 40) .
10
/well
in 24-well plastic culture dishes coated with rat tail collagen. To
feed, but to avoid loss of floating cells, fresh medium (0.2 ml) was
added to the cultures on days 1, 5, and 10.Cell Counts
To avoid loss of floating cells, the
culture medium was removed, centrifuged (500 g, 5
min), and replaced with 0.2 ml of a solution that lyses the cell
membrane but leaves the nuclei intact(45) . The nuclei were
counted in a hemacytometer. Counts were performed on triplicate wells
and are presented as means ± S.E. The results are presented
relative to the cell number initially plated per well (designated as
100).Transfection and Selection of Permanently Transfected
Clones
The derivation and properties of PC12nnr5, PC12nnr5-T1,
and PC12nnr5-T14 cells have been previously
described(46, 47) . To construct PC12nnr5 cells stably
expressing TrkB, transfection and selection were performed as described
previously (48) with a rat TrkB cDNA (49) subcloned
into the pVCOSneo expression vector (kindly provided by Drs. T. Stitt,
D. Glass and G. Yancopoulos, Regeneron Inc. Tarrytown, NY). PC12 cells
stably expressing a kinase inactive Trk were derived by transfection
with a cDNA encoding human Trk mutated at amino acid 538 to exchange
lysine for asparagine (50) and sublconed into the pCMV-IRV
expression vector(51) . The presence and overexpression of the
kinase-inactive Trk was verified by both Western (52) and
Northern blotting(53) .DNA Fragmentation
Experiments were performed as
described by Edwards et al.(54) and Batistatou and
Greene(41) . In brief, PC12 cells were washed and plated
(6-8 10
/100-mm dish) in RPMI 1640 medium with
or without indicated additives. After incubation for the indicated
times at 37 °C, the cells were triturated off the substrate,
centrifuged at 800 g for 5 min, and the pellet was
washed twice with ice-cold phosphate-buffered saline
(Ca
Mg-free). Soluble DNA was
extracted and resuspended in TE buffer (10 mM Tris, pH 8; 1
mM EDTA). Subsequently the samples were incubated with 50
mg/ml DNase-free RNase (Boehringer Mannheim) at 37 °C for 30 min.
DNA samples were subjected to electrophoresis on a 1.2% agarose gel,
blotted onto Gene Screen Plus Membrane (DuPont NEN) and analyzed with
radioactive probes from digested total PC12 cell genomic DNA. Probe
labeling was performed according to Sambrook et al.(55) and hybridization according to the
manufacturer's protocol (DuPont-NEN).In Vitro Trk-Kinase Assay
Experiments were
performed by modification of the method described by Berg et
al.(56) . In brief, subconfluent cultures of PC12 cells
grown on 150-mm tissue culture dishes were incubated overnight with
RPMI 1640 medium containing 1% HS and incubated for the indicated time
with G, NGF, or without additive. The cells were rinsed
twice with phosphate-buffered saline and solubilized in 1 ml of lysis
buffer (10 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.4%
deoxycholate, 66 mM EDTA, 1 mM vanadate, 0.1 mM molybdate, 1 mM phenylmethylsulfonyl fluoride, 5
µg/ml aprotinin, and 5 µg/ml leupeptin). Following a 10-min
incubation on ice, the samples were centrifuged at 60,000 rpm for 15
min at 4 °C to remove nuclei and cellular debris. The clarified
lysates were normalized for protein concentration and brought to 1-ml
total volume in lysis buffer. The lysates were incubated for 2 h with
anti-Trk antibody (1:200 dilution), immunoprecipitated with protein
A-Sepharose beads, and washed as follows: one wash in buffer A (20
mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1%
Triton X-100, 0.2% bovine serum albumin), three washes with buffer B
(20 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 0.1%
SDS, and 0.2% bovine serum albumin), and finally one wash with 50
mM Tris-HCl at pH 8. The beads were resuspended in 60 µl
of kinase buffer containing 50 mM HEPES, pH 7.4, 20 mM MnCl
, 5 mM MgCl, 1 mM dithiothreitol, 20 µM ATP, and 5 µCi of
[
-
P]ATP, incubated for 10 min at 30 °C,
and the reaction was stopped by adding 20 µl of 4 sample
buffer. P-Labeled proteins were analyzed by SDS-PAGE on
7.5% polyacrylamide gels and visualized by autoradiography.Trk Tyrosine Autophosphorylation
Trk tyrosine
autophosphorylation was determined as described previously by Kaplan et al.(57) . All treatments were in culture medium
containing 1% horse serum.
Temporal Aspects of the Actions of G
Detection of internucleosomal DNA
fragmentation provides a sensitive means to monitor early events of
apoptotic death. In PC12 cultures, such DNA fragmentation is evident by
3 h of serum withdrawal and NGF can prevent this if it is added within
the first 1-1.5 h of this time(41) . Previously, we
showed that application of G on
Apoptosis of PC12 Cells at the time of serum
deprivation blocks early apoptotic DNA fragmentation as well as cell
death(38) . To determine the time within which G can prevent apoptosis, PC12 cell cultures were deprived of serum
and the ganglioside was added after delays of 0-3 h. After a
total of 3 h of serum deprivation, soluble DNA was isolated and
analyzed. As shown in Fig. 1, DNA fragmentation was prevented
when G was added to the cultures up to a delay of
1-1.5 h, and thus was present for only the last 1.5-2 h of
incubation. These results are strikingly similar to those achieved with
NGF and indicate that G prevents apoptosis by means of a
relatively rapid mechanism.
on PC12 cell DNA fragmentation. PC12 cells
were washed and plated in serum-free RPMI 1640 medium. G (60 µM) was either not added (0 h) or was added to
the cultures immediately after plating (3 h) or for various
intermediate times (2.5, 2, 1.5, 1, or 0.5 h). Parallel cultures were
treated for 3 h with 100 ng/ml NGF. After 3 h of exposure to serum-free
media soluble DNA was isolated from all cultures and analyzed as
described under ``Materials and
Methods.''
Extracellular Calcium and Protein Kinase A Are Not
Involved in the Mechanism by Which G
Extracellular Ca Prevents
Apoptosis is a possible
regulator of neuronal survival/death that could be involved in rapid
ganglioside actions. For instance, influx of Ca mediates the neurotoxicity induced by excitatory amino acids or
glucose deprivation (for review, see (6) and (58) )
and pretreatment of neurons with G is reported to reduce
the protracted elevation of free cytosolic Ca and
neurotoxic effects brought about by such
conditions(59, 60) . To test the role of extracellular
Ca in the actions of G, PC12 cells were
deprived of serum and incubated for 3 h either with no additive or with
G, both in medium depleted of free Ca (i.e. RPMI 1640 medium supplemented with 1 mM EGTA or Ca-free Dulbecco's modified
Eagle's medium). Despite the absence of extracellular
Ca, G retained its ability to inhibit
internucleosomal DNA cleavage under these conditions (data not shown). -promoted survival of PC12
cells(38) . Activators of protein kinase A have been found to
promote survival of both serum-deprived PC12 cells and NGF-deprived
neurons (8, 40) and thus this kinase is a potential
rapidly activated mediator for such actions of G. To
assess this, we employed the A126-1B2 variant line of PC12 cells that
is defective in protein kinase A activity and that is unresponsive to
permeant cAMP derivatives(40, 61) . When serum was
withdrawn, G, like NGF, promoted survival of A126-1B2
cells (data not shown). These findings indicate that protein kinase A
neither mediates nor is required for the survival-promoting actions of
this ganglioside.Role of Trk in Mediating the Survival-promoting Actions
of G
The rapid actions of G in
preventing apoptosis of serum-deprived PC12 cells suggested the
possible role of a transmembrane signaling pathway. One such potential
pathway involves that for NGF. NGF-promoted survival of PC12 cells
requires the expression of Trk, a receptor tyrosine kinase that
undergoes rapid activation and autophosphorylation upon NGF binding (47, 51, 57, 62) . To assess the
potential role of Trk-associated tyrosine kinase activity in the
survival-promoting actions of G, we employed the bacterial
product K-252a. This drug selectively blocks a variety of the actions
of NGF on PC12 cells(63, 64) , including its ability
to maintain survival in serum-free medium (40) . Such
inhibition appears to be a consequence of the selective suppression by
K-252a of the tyrosine kinase activity of the Trk family of
neurotrophin receptors(56) . PC12 cultures were pretreated with
200 nM K-252a for 1 h in serum-free medium, exposed to either
no additive, G, NGF, or insulin and then tested for long
term cell survival. Fig. 2shows that K-252a not only blocks the
survival effect of NGF by 90%, but also partially (by approximately
50%) inhibits the effect of G. In contrast, the drug does
not block the survival- or proliferation-promoting actions of insulin,
indicating both specificity and nontoxicity. These findings raise the
possibility that Trk tyrosine kinase activity may play at least a
partial role in G-mediated neuronal survival.
and NGF (Panel
A) or insulin (Panel B). PC12 cells were washed and
plated for 1 h with RPMI 1640 medium and, where indicated, with K-252a
before the addition of the indicated agents. Cell numbers were
determined at 8 days. The number of surviving cells is presented
relative to the number initially plated (designated 100). Error
bars represent S.E. (n = 3). Comparable results
were achieved in three independent
experiments.
, we assessed the actions of this drug on
PC12nnr5 cells. PC12nnr5 cells are a mutant PC12-cell derived line that
express p75 NGF receptors, but not Trk(46, 51) . These
cells lack a variety of responses to NGF, including the capacity to be
maintained by this factor (but not by cAMP derivatives, insulin, or
fibroblast growth factor) in serum-free
medium(40, 46, 47) . The data in Fig. 3show that in contrast to its effect on PC12 cells,
G maintains only a fraction (about 50%) of PC12nnr5 cells
in serum-free medium. Moreover, this support is not blocked by K-252a.
, NGF and
insulin on serum-free survival of NGF-unresponsive PC12 cells
(PC12-nnr5 cells) and their sensitivities to K-252a. Cell treatments
were as described in the legend of Fig. 2. Cell numbers were
counted at 1 day. Error bars represent S.E. (n = 3). Comparable results were obtained in three different
experiments.
responsiveness
between PC12 and PC12nnr5 cells is due to the contrasting expression of
Trk, we next tested the actions of G on PC12nnr5 cells
permanently transfected with a cDNA encoding Trk. Such transfection
restores the ability of the mutant cells to respond to
NGF(47, 51) . As shown in Fig. 4A,
introduction of Trk also restores full responsiveness to G in terms of serum-free survival. In addition, as for PC12 cells,
G-supported serum-free survival of the transfectants
(PC12nnr5-T14 cells) is partially inhibited by K-252a. Interestingly,
the proportion of survival blocked by K-252a (40-60%) is similar
to the increase in survival that occurs after reintroduction of Trk.
Control experiments were also performed with a line of PC12nnr5 cells
(PC12nnr5-T1) transfected with the same cDNA, but lacking expression of
Trk(47) . In this case (Fig. 4B),
responsiveness to G and K-252a were similar to that in
non-transfected PC12nnr5 cells. Altogether, these observations support
the possibility that G works in part via a mechanism
dependent on the expression of Trk.
, NGF, and
insulin (all ± K-252a) on serum-free survival of Trk-transfected
PC12nnr5 (PC12-nnr5-T14 cells) (Panel A) and PC12nnr5
cells transfected with the same DNA, but lacking expression of Trk (PC12 nnr5-T1 cells) (Panel B). Cell treatment with
K-252a was as described in Fig. 2. Cell counts were determined
after 1 day and are expressed relative to those initially plated. Error bars represent S.E. (n =
9).
TrkB Can Mediate Survival-promoting Actions of
G
To test the specificity of the Trk requirement
for mediating survival-promoting actions of gangliosides, we also
assessed PC12nnr5 cells that were permanently transfected with TrkB, a
receptor for BDNF and neurotrophin 4/5(65) . Fig. 5shows that 30 and 60 µM G (as
well as BDNF) fully rescues these cells from serum-free death. In
addition, as in the case of PC12 cells and PC12nnr5 cells expressing
Trk, K-252a substantially inhibits this action of G (data
not shown). These observations indicate that at least one other member
of the Trk family can play a role in mediating the ability of
gangliosides to maintain survival.
and BDNF on serum-free survival of TrkB-transfected
PC12-nnr5 cells (TrkB-PC12nnr5 cells). Cells were washed free of serum
and incubated either without additive (NONE) or in the
presence of 100 ng/ml BDNF or with the indicated concentrations of
G. Cell numbers were determined at 7 days as described
under ``Materials and Methods'' and are expressed relative to
the number initially plated. Error bars represent S.E. (n = 6).
Role of Trk Dimerization in Mediating
G
Following binding of ligand,
receptor dimerization and autophosphorylation are thought to be the
primary mechanisms underlying receptor tyrosine kinase-mediated
signaling(66, 67, 68) . These concepts have
been verified by experiments showing that co-expression of wild-type
and kinase defective mutant receptors results in the formation of
heterodimer and suppression of signaling(68, 69) . To
test the role of Trk dimerization on the survival-promoting actions of
G-induced Survival, we used PC12 transfectants (PC12 538-1-2 cells) that,
in addition to their normal complement of endogenous Trk, overexpress
(by 5-10-fold) human Trk mutated at lysine 538 so as to render it
kinase inactive. ()The transfected mutant Trk functions as a
``dominant-inhibitory'' receptor by forming inactive
heterodimers with normal Trk in the presence of NGF(68) . As
shown in Fig. 6, in contrast to a similarly transfected control
line that does not express the mutated Trk, the cells expressing
dominant-inhibitory receptors are not rescued by NGF from death in
serum-free medium. In contrast, full survival is promoted by insulin
(data not shown). The data in Fig. 6also reveal that, like
PC12nnr5 cells and in contrast to control PC12 cells, PC12 538-1-2
cells are only partially rescued from serum-free death by
G. Similar observations were obtained with an additional
independently-derived PC12 line that also overexpresses kinase-inactive
Trk (data not shown). These findings support the notion that Trk
dimerization and autophosphorylation play a role in the
survival-promoting actions of G.
on
serum-free survival of cells expressing dominant inhibitory Trk. The
PC12-538-1-2 line overexpressing kinase inactive Trk and a control line
similarly transfected, but not expressing the mutant Trk, were washed
free of serum and incubated either without additive (NONE), or
with 100 ng/ml NGF (NGF) or 40 µM G (G
). Numbers of surviving cells
were determined after 7 days and are expressed relative to the number
initially plated. Error bars represent S.E. (n = 3).
Effect of G
The partial sensitivity of
G on Trk-associated Protein Kinase
Activity and Trk Autophosphorylation actions to K-252a suggests that survival promoting
actions of ganglioside depend in part on activation of Trk-associated
protein kinase activity. As one test of this, we employed an in
vitro assay in which PC12 cells were treated in the presence of 1%
HS with either no additive, NGF (5 min) or G (30 min) and
then lysed and subjected to immunoprecipitation of Trk. The Trk
immunoprecipitates were then incubated in the presence of
[P]ATP and autophosphorylation activity was
detected by SDS-PAGE and autoradiography(56) . As shown in Fig. 7A, material from both NGF- and
G-treated cells showed an increase in phosphorylation of a
band corresponding to Trk (as corroborated by probing the
nitrocellulose with anti-Trk antibodies). The G concentration used in this experiment (500 µM)
reflects the presence of serum, which is known to affect the free
ganglioside concentration(70) , as well as the amount of
G required to support neuronal survival(38) .
However, a comparable result was achieve in serum-free medium with 60
µM G (data not shown). Quantification of
several independent experiments indicates that although the effect of
G is smaller than that of NGF, it is highly significant p = 0.05 (Fig. 7B). The relatively low
levels of radioactivity incorporated by Trk in the experiments with
G precluded direct analysis of the amino acid into which
it was incorporated.
on Trk-kinase
activity assessed in vitro. PC12 cells were incubated in RPMI
1640 medium containing 1% horse serum and either no additive (NONE), 100 ng/ml NGF (NGF) for 5 min or 500
µM G (G) for
30 min. The cells were extracted and equal amounts of protein
(6-9 mg) were subjected to immunoprecipitation with anti-Trk
antiserum. The immunoprecipitates were then assayed for in vitro kinase activity as described under ``Materials and
Methods.'' Panel A shows an autoradiogram of proteins
labeled during the in vitro phosphorylation. Other than the
band at 55 kDa (IgG), the bands in addition to Trk represent other
kinase substrates that co-immunoprecipitate with Trk(56) . Arrowhead shows position of Trk. Panel B shows the
quantification of radioactivity incorporated into Trk in three
independent experiments. Comparison of the values obtained in untreated (NONE) and G-treated cultures by a paired t test reveals that the difference is significant (p = 0.05).
, like
NGF, affects Trk tyrosine phosphorylation in intact cells. Although
preliminary experiments with wild-type PC12 cells indicated an effect
of G, the signals were barely detectable. To enhance
sensitivity we therefore used PC12 cells overexpressing Trk (clone
6-24)(52) . These cells were exposed to either no additive,
G (15 min) or NGF (5 min) in the presence of 1% HS, lysed
and subjected to immunoprecipitation with anti-Trk antiserum. The
immunoprecipitates were then analyzed by Western immunoblotting using
anti-phosphotyrosine as probe. Fig. 8shows that although the
effect of G is less pronounced than that achieved with
short-term NGF exposure, it significantly enhances Trk tyrosine
phosphorylation. Reprobing of the same nitrocellulose membrane with
anti-Trk antibodies confirmed that an equal amount of Trk protein was
immunoprecipitated under each condition (data not shown).
on Trk tyrosine
autophosphorylation in PC12 cells overexpressing Trk (clone 6-24).
Cells were incubated in DMEM medium containing 1% horse serum and the
indicated additives for 5 min (NGF) or 15 min (G). The cells were extracted, and equal
amounts of protein were immunoprecipitated using anti-Trk antiserum.
The immunoprecipitates were subjected to SDS-PAGE. Proteins were then
electroblotted onto nitrocellulose and probed with anti-phosphotyrosine
antibody.
to rescue cultured PC12 cells
from apoptotic death induced by exposure to serum-free medium.
Serum-deprived PC12 cells share with sympathetic neurons survival
responsiveness to both NGF and
G(38, 39) . These and additional
observations (48) support the utility and appropriateness of
the PC12 cell system for our cell survival experiments.. As in the case of
NGF(12, 40, 41) , prevention of PC12 cell
apoptosis by G does not require transcription or
translation, does not appear to be mediated by or require activation of
either protein kinase C or A, and does not require the presence of
extracellular Ca. In addition, our results indicate
that G, as reported for NGF(41) , works within a
rapid time frame and prevents DNA fragmentation detected at 3 h of
serum withdrawal only when added to cultures within a delay of no more
than 1-1.5 h. antagonizes certain inhibitory actions of K-252a on PC12 cell
responses to NGF(35) . These findings raised the possibility
that G may share with K-252a the ability to interact with
the NGF signaling mechanism. The observation that K-252a is a potent
and specific inhibitor of Trk NGF receptor protein tyrosine kinase
activity in PC12 cells (56) further suggested that Trk could be
a target for ganglioside actions. This in turn led us to consider that
as NGF(51) , G acts to promote survival in part
via a mechanism dependent on expression and activation of Trk. A number
of our observations support this supposition. First, K-252a
significantly inhibits the survival-promoting actions of G on PC12 cells. Second, G supports only a fraction of
serum-deprived PC12nnr5 cell mutants that lack expression of Trk.
Third, transfection of PC12nnr5 cells with Trk restores full capacity
for ganglioside-mediated survival. Fourth, K-252a does not block
survival of the sub population of PC12nnr5 cells maintained by
G. However, K-252a does block full survival promotion by
G for PC12nnr5 cells transfected with Trk. Fifth, PC12
cells which have been transfected to express an excess of
kinase-inactive Trk in addition to their endogenous Trk and which
consequently do not respond to NGF, are supported by G only to the extent seen with Trk
PC12nnr5
cells. Lastly, we find enhancement of protein kinase activity in Trk
immunoprecipitates from G-treated cells and directly
detect ganglioside-mediated increases in Trk tyrosine phosphorylation. survival response by overexpression of kinase-inactive
Trk in PC12 cells strongly suggests that the ganglioside mechanism also
requires Trk dimerization and autophosphorylation. Past studies have
demonstrated that exogenously supplied G is rapidly
incorporated into cell membranes(15) . Thus, we hypothesize
that G, once integrated into neuronal membranes, causes
local changes in membrane properties that facilitate Trk dimerization
and consequent activation. Consistent with this are observations that
gangliosides synergize the efficacy of suboptimal levels of NGF, both in vitro and in vivo(20) . If this hypothesis
is correct, then it raises the further possibility that the role of
endogenous gangliosides may be to regulate interactions between
transmembrane proteins. able to maintain survival if it appears to be
considerably less effective than NGF in activating Trk? Several
different possibilities may be considered. For instance, the degree of
Trk tyrosine phosphorylation required to promote survival is likely to
be well below that observed upon initial NGF treatment. In this regard,
it is relevant to note that although short-term exposure of PC12 cells
to NGF elicits a very large and easily detectable increase in Trk
tyrosine phosphorylation, this rapidly falls and beyond several hours
of treatment, is only slightly above basal levels(57) .
Although Trk autophosphorylation is barely detectable in cells
continuously exposed to NGF, this appears to be both necessary and
sufficient for promotion of survival. Thus, even a low level of Trk
activation mediated by G may be adequate to prevent
neuronal cell death. An alternative possibility is that
ganglioside-enhanced phosphorylation is restricted to a subset of the
various tyrosine residues that become autophosphorylated in response to
NGF, and that this subset includes sites involved in initiating
signaling pathways that promote survival. In this case, overall Trk
autophosphorylation evoked by G would be low, but would be
sufficiently elevated on those sites required to prevent death. in our system. If G actions on PC12 cells are
mediated in part by Trk, then why does it only elicit a subset of NGF
responses (e.g. survival) and not the entire set of responses
including stimulation of neurite outgrowth? Again, there are several
possible explanations. It may be that the overall level of signaling
required for promotion of survival and of neuronal differentiation are
different and that the signal generated by G is sufficient
only for the former. If this is the case it could explain the reported
effects of G in enhancing neurotrophin-induced neurite
outgrowth(17, 20, 29) . Alternatively, if
G treatment leads to autophosphorylation of only a subset
of Trk tyrosine residues affected by NGF, and, as appears to be the
case for Trk (50, 71, 72) and other receptor
tyrosine kinases(66, 67) , if different
phosphotyrosines on the intracellular domain of the receptor are
responsible for activating different signaling pathways, then G would be expected to mimic only a subset of NGF actions. -promoted survival responses does not
appear to be limited to Trk. We also observed enhanced rescue from
serum-free death with PC12nnr5 cells expressing a second member of the
Trk family, TrkB. Although we have not tested cells expressing TrkC, it
would seem highly likely that this receptor would also confer
sensitivity to G. In addition, because other tyrosine
receptor kinases can be activated by dimerization (including those that
bind growth factors with neurotrophic actions such as fibroblast growth
factor, insulin-like growth factors, and epidermal growth factor), it
may be further speculated that G, as appears to be the
case (18, 73) would have trophic actions on a wide
variety of neuronal cell types that do not necessarily express
neurotrophin receptors. The ability of G to affect
receptor tyrosine kinases in addition to Trk would also account for the
40-50% of K-252a-insensitive survival promoted by G in PC12 cell cultures and for the similar proportions of
ganglioside-promoted and K-252a-insensitive survival observed in
PC12nnr5 cultures. prevents neuronal death
depends at least in part on the presence and activation of neurotrophic
factor receptor tyrosine kinases. We propose that G achieves its trophic activity by favoring the dimerization of
such receptors and thereby at least partially mimicking the action of
their corresponding ligands. Understanding the mechanism underlying
G-induced survival of PC12 cells should be relevant to
comprehending the basis for the ameliorative actions of Gin vivo.
), NGF, nerve growth factor; PAGE,
polyacrylamide gel electrophoresis; BDNF, brain-derived neurotrophic
factor; HS, horse serum.)
We thank Drs. A. Batistatou and A. Rukenstein for
discussions and Drs. G. Yancopoulos, D. Glass, and T. Stitt for
providing the TrkB expression plasmid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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