©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Prevention of Apoptotic Neuronal Death by G Ganglioside
INVOLVEMENT OF Trk NEUROTROPHIN RECEPTORS (*)

(Received for publication, November 1, 1994; and in revised form, November 29, 1994)

Giovanna Ferrari (1) Blake L. Anderson (1) Robert M. Stephens (2) David R. Kaplan (2) Lloyd A. Greene (1)(§)

From the  (1)Department of Pathology and Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, New York 10032 and (2)ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have used serum-deprived cultures of wild type and genetically modified PC12 cells to investigate the molecular mechanisms by which monosialoganglioside (G) 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.


INTRODUCTION

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) (^1)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) .

The molecular mechanism by which G 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.

The aim of the present studies has been to define the molecular mechanism by which 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.


EXPERIMENTAL PROCEDURES

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 10M, 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) .

For cell survival experiments, washed cells were resuspended in RPMI 1640 medium and plated in 0.5 ml at a density of 8-10 times 10^4/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 times 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 times 10^6/100-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 times g for 5 min, and the pellet was washed twice with ice-cold phosphate-buffered saline (CaMg-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(2), 5 mM MgCl(2), 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 times 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.


RESULTS

Temporal Aspects of the Actions of G on Apoptosis of PC12 Cells

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 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.


Figure 1: Temporal aspects of the effect of G 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 Prevents Apoptosis

Extracellular Ca 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).

In past work, we showed that protein kinase C does not appear to play a role in the G-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.


Figure 2: Effect of K-252a (200 nM) on serum-free cell survival induced by G 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.



To further test the role of Trk in mediating the survival-promoting effects of G, 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.


Figure 3: The effects of G, 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.



To determine whether the difference in G 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.


Figure 4: The effects of G, 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.


Figure 5: Effect of G 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-induced Survival

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, 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. (^2)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.


Figure 6: Effect of NGF and 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 on Trk-associated Protein Kinase Activity and Trk Autophosphorylation

The partial sensitivity of G 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.


Figure 7: Effect of G 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).



We next tested whether G, 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).


Figure 8: Effect of G 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.




DISCUSSION

The present study explores the mechanism by which gangliosides exert trophic actions on neurons. In particular, we studied the capacity of G 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.

Both our past (38) and present findings are consistent with a similarity in mechanism between NGF and G. 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.

A previous study has shown that G 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.

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 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.

Our findings regarding ganglioside effects on Trk activity raise several intriguing issues. For one, why is G 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.

A related issue concerns the types of responses mediated by G 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.

Mediation of G-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.

In summary, we have provided evidence that the molecular mechanism by which G 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.


FOOTNOTES

*
This work was partially supported by FIDIA Research Laboratories (to G. F.), by grants from the United States Public Health Service, National Institutes of Health (NINDS), and March of Dimes Birth Defects Foundation (to L. A. G.) and by National Cancer Institute (DHHS) contract N0I-CO-7410I with the ABL (to D. R. K.). 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.

§
To whom correspondence should be addressed: Dept. of Pathology and Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, 630 W. 168th St., New York, NY 10032. Tel.: 212-305-6369; Fax: 212-305-5498; lag3{at}columbia.edu.

(^1)
The abbreviations used are: G, NGF, nerve growth factor; PAGE, polyacrylamide gel electrophoresis; BDNF, brain-derived neurotrophic factor; HS, horse serum.

(^2)
M. E. Cunningham, D. M. Loeb, L. A. Greene, R. M. Stephens, and D. R. Kaplan, unpublished results.


ACKNOWLEDGEMENTS

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.


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