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The Ras/Phosphatidylinositol 3-Kinase and Ras/ERK Pathways Function as Independent Survival Modules Each of Which Inhibits a Distinct Apoptotic Signaling Pathway in Sympathetic Neurons

2000; Elsevier BV; Volume: 275; Issue: 12 Linguagem: Inglês

10.1074/jbc.275.12.8817

ISSN

1083-351X

Autores

Luzheng Xue, James H. Murray, Aviva M. Tolkovsky,

Tópico(s)

Melanoma and MAPK Pathways

Resumo

Ras promotes robust survival of many cell systems by activating the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, but little is understood about the survival functions of the Ras/ERK pathway. We have used three different effector-loop mutant forms of Ras, each of which activates a single downstream effector pathway, to dissect their individual contributions to survival of nerve growth factor (NGF)-dependent sympathetic neurons. The PI3-kinase pathway-selective protein RasVal-12Y40C was as powerful as oncogenic RasVal-12 in preventing apoptosis induced by NGF deprivation but conferred no protection against apoptosis induced by cytosine arabinoside. Identical results were obtained with transfected Akt. In contrast, the ERK pathway-selective protein RasVal-12T35S had no protective effects on NGF-deprived neurons but was almost as strongly protective as RasVal-12 against cytosine arabinoside-induced apoptosis. The protective effects of RasVal-12T35S against cytosine arabinoside were completely abolished by the ERK pathway inhibitor PD98059. RasVal-12E37G, an activator of RalGDS, had no survival effect on either death pathway, similar to RasS17N, the full survival antagonist. Thus, Ras provides two independent survival pathways each of which inhibits a distinct apoptotic mechanism. Our study presents one of the few clear-cut cases where only the Ras/ERK, but not the Ras/PI3K/Akt pathway, plays a dominant survival signaling role. Ras promotes robust survival of many cell systems by activating the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, but little is understood about the survival functions of the Ras/ERK pathway. We have used three different effector-loop mutant forms of Ras, each of which activates a single downstream effector pathway, to dissect their individual contributions to survival of nerve growth factor (NGF)-dependent sympathetic neurons. The PI3-kinase pathway-selective protein RasVal-12Y40C was as powerful as oncogenic RasVal-12 in preventing apoptosis induced by NGF deprivation but conferred no protection against apoptosis induced by cytosine arabinoside. Identical results were obtained with transfected Akt. In contrast, the ERK pathway-selective protein RasVal-12T35S had no protective effects on NGF-deprived neurons but was almost as strongly protective as RasVal-12 against cytosine arabinoside-induced apoptosis. The protective effects of RasVal-12T35S against cytosine arabinoside were completely abolished by the ERK pathway inhibitor PD98059. RasVal-12E37G, an activator of RalGDS, had no survival effect on either death pathway, similar to RasS17N, the full survival antagonist. Thus, Ras provides two independent survival pathways each of which inhibits a distinct apoptotic mechanism. Our study presents one of the few clear-cut cases where only the Ras/ERK, but not the Ras/PI3K/Akt pathway, plays a dominant survival signaling role. nerve growth factor cytosine arabinoside extracellular signal-regulated kinase hemagglutinin phosphatidylinositol 3-kinase superior cervical ganglion mitogen-activated protein kinase/ERK kinase cytomegalovirus phosphate-buffered saline guanine nucleotide dissociation stimulator myristoylation/palmitoylation One of the central problems posed by degenerative disorders involving postmitotic cells is how to prevent cell death. We have demonstrated previously that p21Ras (Ras) protein plays a pivotal role in mediating survival by nerve growth factor (NGF)1 and other cytokines in rat sympathetic (SCG) neurons (1.Nobes C.D. Buckmaster A.E. Tolkovsky A.M. J. Auton. Nerv. Syst. 1991; 33: 213-214Abstract Full Text PDF Scopus (8) Google Scholar, 2.Nobes C.D. Tolkovsky A.M. Eur. J. Neurosci. 1995; 7: 344-350Crossref PubMed Scopus (71) Google Scholar, 3.Nobes C.D. Reppas J.B. Markus A. Tolkovsky A.M. Neuroscience. 1996; 70: 1067-1079Crossref PubMed Scopus (41) Google Scholar). However, the mechanisms used by Ras to protect the neurons from apoptosis are not yet fully understood. Active Ras associates with multiple downstream targets to exert its biological effects (4.White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 5.White M.A. Nicolette C. Minden A. Polverino A. Aelst L.V. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (628) Google Scholar) including Raf-1 kinase, the catalytic subunit of a phosphatidylinositol 3-kinase (PI3K), and the Ral guanine nucleotide dissociation stimulator (RalGDS) (6.Joneson T. White M.A. Wigler M.H. Bar-Sagi D. Science. 1996; 271: 810-812Crossref PubMed Scopus (358) Google Scholar). PI3K, which is directly stimulated by Ras (7.RodriguezViciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar) and promotes survival through PKB/Akt in numerous cell systems (8.Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1190) Google Scholar), is persistently activated by NGF in SCG neurons (9.Virdee K. Xue L. Hemmings B.A. Goemans C. Heumann R. Tolkosvky A.M. Brain Res. 1999; 137: 129-142Google Scholar) and has been shown to mediate survival in sympathetic (9.Virdee K. Xue L. Hemmings B.A. Goemans C. Heumann R. Tolkosvky A.M. Brain Res. 1999; 137: 129-142Google Scholar) and sensory neurons (10.Klesse L.J. Parada L.F. J. Neurosci. 1998; 18: 10420-10428Crossref PubMed Google Scholar) as well as robust neurite outgrowth (9.Virdee K. Xue L. Hemmings B.A. Goemans C. Heumann R. Tolkosvky A.M. Brain Res. 1999; 137: 129-142Google Scholar). Moreover, expression of active PI3K (11.Philpott K.L. McCarthy M.J. Klippel A. Rubin L.L. J. Cell Biol. 1997; 139: 809-815Crossref PubMed Scopus (220) Google Scholar) or PKB/Akt (9.Virdee K. Xue L. Hemmings B.A. Goemans C. Heumann R. Tolkosvky A.M. Brain Res. 1999; 137: 129-142Google Scholar, 12.Crowder R.J. Freeman R.S. J. Neurosci. 1998; 18: 2933-2943Crossref PubMed Google Scholar) is sufficient to protect SCG neurons from apoptosis induced by NGF withdrawal. In contrast, whereas p42 and p44 mitogen-activated protein kinases (ERKs), which mediate the Ras/Raf-1 pathway, are strongly and persistently activated by NGF (13.Virdee K. Tolkovsky A.M. Eur. J. Neurosci. 1995; 7: 2159-2169Crossref PubMed Scopus (64) Google Scholar, 14.Creedon D.J. Johnson Jr., E.M. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 20713-20718Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), ERK activity is not required for survival support by NGF or other cytokines (14.Creedon D.J. Johnson Jr., E.M. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 20713-20718Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 15.Virdee K. Tolkovsky A.M. J. Neurochem. 1996; 67: 1801-1805Crossref PubMed Scopus (95) Google Scholar). A third Ras-interacting protein, RalGDS, contributes to cell transformation (4.White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), but its functions in neurons are still unclear. In PC12 cells its overexpression inhibited neurite outgrowth induced by NGF suggesting a dominant-negative effect (16.Goi T. Rusanescu G. Urano T. Feig L.A. Mol. Cell. Biol. 1999; 19: 1731-1741Crossref PubMed Scopus (81) Google Scholar). Recently, we uncovered a putative role for ERK activity in SCG neuron survival by demonstrating that the MEK inhibitor PD98059, which abolishes ERK activity (14.Creedon D.J. Johnson Jr., E.M. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 20713-20718Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 15.Virdee K. Tolkovsky A.M. J. Neurochem. 1996; 67: 1801-1805Crossref PubMed Scopus (95) Google Scholar), dramatically increased apoptosis induced by araC treatment in the presence of NGF (17.Anderson C.N.G. Tolkovsky A.M. J. Neurosci. 1999; 19: 664-673Crossref PubMed Google Scholar). These experiments raised the possibility that besides the Ras/PI3K pathway, the Ras/ERK pathway might also protect against apoptosis of NGF-deprived neurons. There is great interest in understanding the role of the Ras/ERKs pathway in survival since it is becoming increasingly clear that there exist PI3K/Akt-independent survival signaling pathways and that PI3K/Akt activity induced by some cytokines is not being utilized for survival (18.Kulik G. Weber M.J. Mol. Cell. Biol. 1998; 18: 6711-6718Crossref PubMed Scopus (230) Google Scholar, 19.Carson J.P. Kulik G. Webe M.J. Cancer Res. 1999; 59: 1449-1453PubMed Google Scholar, 20.Scheid M.P. Duronio V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7439-7444Crossref PubMed Scopus (276) Google Scholar), although the identity of the alternative survival signals was not determined. However one study (20.Scheid M.P. Duronio V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7439-7444Crossref PubMed Scopus (276) Google Scholar) excluded ERK activity from being the alternative pathway mediating Akt-independent survival. In PC12 cells it was suggested that ERKs promoted survival by inhibiting c-Jun N-terminal kinase/p38 stress kinases (21.Xia Z.G. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5045) Google Scholar), but in SCG neurons we found no obligate relationship between the two processes (13.Virdee K. Tolkovsky A.M. Eur. J. Neurosci. 1995; 7: 2159-2169Crossref PubMed Scopus (64) Google Scholar). In cardiomyocytes ERK activity was induced by oxidative stress and was shown to limit damage by inducing cyclooxygenase-2 expression and production of prostacyclin (22.Adderley S.B. Fitzgerald D.J. J. Biol. Chem. 1999; 274: 5038-5046Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar), but the relationship to apoptosis was not clear. Moreover, ERK activity is not induced by oxidative signals in SCG neurons. The role of the Ras/ERK signaling pathway in suppression of apoptosis thus remains largely unresolved. In this study we investigate the relative importance of each of the signal pathways downstream of p21Ras to determine whether these pathways are synergistic in protection against apoptosis, whether there are limits to protection by the PI3K pathway, and whether there are circumstances in which the ERK pathway might play a predominant role in survival. To this end, we have studied the effects of three different effector-loop domain point mutants of Ha-Ras(G12V) (RasVal-12) which interact with different single effectors in mammalian cells (4.White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 5.White M.A. Nicolette C. Minden A. Polverino A. Aelst L.V. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (628) Google Scholar, 6.Joneson T. White M.A. Wigler M.H. Bar-Sagi D. Science. 1996; 271: 810-812Crossref PubMed Scopus (358) Google Scholar, 7.RodriguezViciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 23.KauffmanZeh A. RodriguezViciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1075) Google Scholar). Ras(G12V,T35S) (RasVal-1235S) binds to Raf-1 and activates the mitogen-activated protein kinase pathway with about 20% efficiency of RasVal-12 (7.RodriguezViciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar). It shows no interaction with PI3-kinase p110α subunit but interacts with RalGDS very slightly; Ras(G12V,E37G) (RasVal-12E37G) binds to RalGDS but not to Raf-1 or p110α; Ras(G12V,Y40C) (RasVal-1240C) binds and activates PI3-kinase p110α but not Raf-1 or RalGDS. Previously it was suggested that RasVal-1240C but not RasVal-1235S suppresses c-Myc-induced apoptosis in fibroblasts (23.KauffmanZeh A. RodriguezViciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1075) Google Scholar). By using these constructs, we demonstrate that RasVal-1240C is as efficient as RasVal-12 in protecting against the death of NGF-deprived neurons. In contrast, only RasVal-1235S could protect against apoptosis induced by araC, the Ras/PI3K pathway playing no role in this protection. These results were corroborated using active Akt and the ERK pathway inhibitor PD98059. The RalGDS-selective proteins RasVal-12E37G had no protective effects. Thus unlike cycling cells where the three Ras pathways are synergistic for transformation (5.White M.A. Nicolette C. Minden A. Polverino A. Aelst L.V. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (628) Google Scholar), in sympathetic neurons the two Ras downstream pathways that are implicated in survival are neither additive nor synergistic. Rather, they function as independent mechanisms to support neuron survival by suppressing two different mechanisms of apoptotic induction. Plasmids pDCR-Ha-Ras (G12V,T35S), pDCR-Ha-Ras (G12V,E37G), and pDCR-Ha-Ras (G12V,Y40C) in which HA-tagged proteins were expressed under the CMV promoter were generous gifts from Dr. Michael H. Wigler (Cold Spring Harbor Laboratory). pRK5-Myc-Ha-Ras (G12V) and pEXV-Myc-Ha-Ras (S17N) cDNA and the rat monoclonal anti-Ras antibody Y13-259 were kindly provided by Professor Alan Hall (MRC-LMCB, UCL, UK). Ras(S17N) cDNA was subcloned into the pRK5-Myc expression vector that contains a CMV promoter (Ras17N). pCMV-m/p-HA-PKBα (a membrane-targeted form of PKB/Akt containing sites for myristoylation/palmitoylation (m/p-Akt)) was provided by Dr. Brian Hemmings (Friedrich Miescher-Institut, Basel, Switzerland). After amplification, all constructs were fully sequenced to confirm fidelity of mutations. LipofectAMINE was purchased from Life Technologies, Inc. Monoclonal antibodies to hemagglutinin (HA) and c-Myc were from Babco. Antibody to active ERK was from Promega and antibody to phospho-Akt was from New England Biolabs. Anti-mouse IgG conjugated with Cy3 was from Jackson ImmunoResearch Laboratories. Cytosine arabinoside (araC) and Hoechst 33342 were from Sigma. PD98059 was purchased from Calbiochem. Superior cervical ganglia (SCG) were dissected from 1-day-old rat pups, and sympathetic neurons were extracted as described previously (13.Virdee K. Tolkovsky A.M. Eur. J. Neurosci. 1995; 7: 2159-2169Crossref PubMed Scopus (64) Google Scholar). Briefly, SCG were digested for 40 min at 37 °C in 0.1% trypsin, and a single-cell suspension was obtained by triturating the digested ganglia through a narrow-bore flame-polished Pasteur pipette. To purify the neurons, cells were preplated twice for about 1 h each onto collagen-coated culture dishes in L15-CO2 medium containing 5% rat serum under 5% CO2, 37 °C, and nonadhering neurons were collected by centrifugation. HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1 mml-glutamine, penicillin (100,100 IU/ml), and streptomycin (100,100 UG/ml) in 5% CO2 at 37 °C. Transfection was carried out following the protocol from Life Technologies, Inc., with some modifications. Briefly, newly isolated neurons were plated onto poly-l-lysine/laminin-coated glass coverslips in 24-well plates and cultured for 3 h in L15-CO2 medium containing 3% rat serum and 20 ng/ml NGF. After one wash with serum-free medium without antibiotics, the cells were incubated for 4 h in the same medium containing 3 μl of LipofectAMINE and 0.15 pmol of plasmid DNA per well. Transfections were terminated by replacing the transfection mixture with the culture medium. After 36 h of transfection, cells were washed twice with medium lacking NGF and incubated in the same medium for 20 h in the presence or absence of NGF and/or 1 mm araC. In some cases, 80 μm PD98059 was added. HeLa cells were passaged 1 day before transfection and transfected using the same protocol used for neurons. After 1 day of expression, the cells were washed with Dulbecco's modified Eagle's medium, serum-starved for 20 h to remove endogenous signaling through Ras, and then lysed for Western blot analysis. SCG neurons were fixed in 3% paraformaldehyde for 20 min at room temperature. After three washes with PBS, the cells were permeabilized in PBS containing 1% BSA and 0.1% saponin followed by 1 h incubation with primary mouse antibodies to the appropriate tag (anti-c-Myc or -HA). Cells were then stained with Cy3-conjugated anti-mouse IgG for 1 h and washed twice in PBS/BSA/saponin and once in the same buffer containing Hoechst 33342. The coverslips were mounted in Vectashield (Vector). Expression of mutant Ras and apoptosis in the transfected population were determined by direct visual counting under a fluorescence microscope. The scores from the entire cohort of transfected neurons (4–6 coverslips) were pooled to represent one independent experiment, which was repeated between 2 and 6 times. To determine the amount of apoptosis in the total population of neurons for each treatment (labeled Total in Figs. 4 and Figure 6, Figure 7, Figure 8), random fields of neurons from each well (1000–1500 cells per well) were scored for apoptosis using Hoechst, and the results were pooled to give a single value per experiment. Figs. 4 and 6 show the total percentage of survival obtained after each treatment and also the difference in percentage survival between the total population of NGF-deprived or araC-treated neurons (set to 0) and each of the other treatments or transfections. Statistical comparisons were performed using two-tailed Student's t test.Figure 8m/p-Akt fails to support the survival of araC-treated SCG neurons. A, HeLa cells were transfected with m/p Akt for 1 day and then serum-starved for 20 h, and extracts were prepared to examine gene expression and kinase activities by Western blotting using antibodies to HA tag, phospho-Akt, and active ERKs. A strong band around 62 kDa is recognized by the anti-HA antibody (left panel). The overexpressed m/p-Akt strongly enhanced Akt phosphorylation but had no effect on Erk activity (right panel). B, SCG neurons were transfected with m/p-Akt. After 36 h, the neurons were washed and treated with normal growth medium, or NGF-deprived, or exposed to 1 mmaraC in the absence of NGF for 20 h as indicated. 200–2000 transfected neurons were counted per treatment. m/p-Akt significantly reduced the death of NGF-deprived neurons but showed no protection against araC-induced apoptosis. Data correspond to the mean of three independent experiments; the error bars are standard deviations (p < 0.01 +NGF/total versus−NGF/total not significant, +NGF/Akt versus −NGF/Akt;p > 0.05 −NGF+araC/total versus−NGF+araC/Akt).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Quantitative analysis of survival or apoptosis of neurons expressing various Ras mutant proteins treated with araC in the presence or absence of NGF. SCG neurons were transfected and treated according to the same time course as described in Figs. 4 and 5. A, neurons exposed to 1 mmaraC in the presence of NGF (+NGF+ araC); B,neurons exposed to 1 mm araC in the absence of NGF (−NGF+ araC). Between 200 and 800 transfected neurons were counted for each treatment depending on the expression or death level. The left-hand y axis shows percentages of survival of neurons, and the right-hand y axis shows the difference compared with the value obtained for the mean percentage survival in the total population of araC-treated neurons in the presence (A) or absence (B) of NGF. Data presented correspond to the mean of 2–4 independent experiments; theerror bars are standard deviations or range of values (n = 2, 37G). A, compared with the neuronal survival in the normal medium (+ NGF,left-hand side), araC (+NGF+ araC,right-hand side) caused about 25–35% reduction of neuronal survival in the total, RasVal-12-, or RasVal-1240C-expressing neurons but not in RasVal-1235S-expressing neurons (p < 0.01 +NGF versus +NGF+araC/total/12V/40C/35S; not significant, +NGF+araC/total versus +NGF+araC/12V/40C/35S). B, compared with neuron protection by RasVal-12 or RasVal-1240C but not RasVal-1235S or RasVal-1237G during NGF withdrawal (− NGF , left-hand side), RasVal-12 or RasVal-1235S did, but RasVal-1240C or RasVal-1237G did not, block araC-triggered neuronal death (−NGF+ araC, right-hand side) (p< 0.001, −NGF+araC/total versus −NGF+araC/12V/35S; not significant, −NGF+araC/total versus −NGF+araC/40C /37G).View Large Image Figure ViewerDownload Hi-res image Download (PPT) After three washes with ice-cold PBS, HeLa cells were solubilized in lysis buffer (20 mm Tris-HCl, pH 7.4, 250 mm sucrose, 1 mm EDTA, 1 mm EGTA, 1 mm sodium orthovanadate, 10 mm sodium glycerophosphate, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.1% 2-mercaptoethanol, 1 mm benzamidine, 5 mg/ml leupeptin, 0.2 mm phenylmethanesulfonyl fluoride, and 1% Triton X-100). The lysates were fractionated by SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to a nitrocellulose membrane, and probed with antibodies as indicated under "Results." The intensity of protein bands was quantified using a Leica Q500 Quantimet image analysis system. Newly isolated rat sympathetic neurons were transfected with five different mutant forms of human Ha-Ras as follows: RasVal-12, which is constitutively active but nonselective; RasVal-1235S, which couples almost exclusively to the Raf/ERK pathway; RasVal-1240C, which couples exclusively to the PI3K pathway; RasVal-1237G, which couples to RalGDS; and Ras17N, which is dominant-negative. Since the number of transfected sympathetic neurons was not high enough for a biochemical analysis by Western blotting, we first examined the fidelity of gene expression and pathway coupling of our constructs by using transfected HeLa cells (Fig. 1). Antibodies detecting either the c-Myc or HA tag epitopes fused to the N termini of the various Ras mutant proteins recognized two major bands of around 25 kDa in the transfected cells but not in control cells (Fig. 1, A and B). The lower bandsrepresent the mature post-translationally modified form of the transfected Ras proteins, and the upper bands consist of the unmodified forms of transfected Ras that can accumulate during overexpression. 2A. Hall, personal communication. To confirm the specificity of the various mutated forms of Ras for the signaling pathways, we probed the HeLa cell samples with antibodies to active phospho-ERKs, which are downstream of Raf-1, and phospho-Akt, which is downstream of PI3K. Fig. 1 C shows that RasVal-12strongly enhanced phosphorylation of both Akt and ERKs in the transfected HeLa cells; RasVal-1235S only increased ERK phosphorylation whereas RasVal-1240C only increased Akt phosphorylation thus confirming the lack of cross-reactivity between the signals induced by the two loop effector mutants. Neither Ras17N nor RasVal-1237G had any activatory effect on either kinase, confirming that these mutants do not activate either pathway. To assess the contribution of the various Ras effector pathways to neuronal survival, we scored the number of living and apoptotic neurons expressing different forms of Ras by fluorescence microscopy after staining with the DNA dye Hoechst 33342. Expression of the transfected Ras proteins was revealed by immunocytochemical staining of the c-Myc or HA tags. The extent of survival of Ras-overexpressing neurons was compared with that of the total population of cells in the same samples. As shown in Fig. 2 (a, c, e, g, and i), all the different forms of tagged Ras proteins were localized primarily to the plasma membrane, as expected of the mature protein, although there was also staining in the Golgi region, where the pre-processed form of Ras is accumulated. Staining was distributed in both neurosoma and neurites. Since these neurons were transfected prior to neurite outgrowth, Ras proteins must have been exported into the growing neurites, presumably by being included within membrane vesicles that provide neurite precursor building blocks. During the initial stages of apoptosis, neurites were collapsed, and Ras staining in the fragmented neurites was usually seen as small dots around the neurosoma (Fig. 2 c, lower neuron). Occasionally, overexpression of Ras proteins caused nuclear deformation that showed a crescentic or lobulated pattern of nuclei containing uncondensed DNA masses (Fig. 2 d, upper neuron). Deformation seemed to be due to the abundance of protein accumulated in the Golgi prior to export. When these neurons had healthy neurites they were scored as surviving cells. Expression of RasVal-12 and Ras17N confirmed our previous results using trituration of proteins that demonstrated the critical function of Ras signaling in SCG neuron survival (1.Nobes C.D. Buckmaster A.E. Tolkovsky A.M. J. Auton. Nerv. Syst. 1991; 33: 213-214Abstract Full Text PDF Scopus (8) Google Scholar, 2.Nobes C.D. Tolkovsky A.M. Eur. J. Neurosci. 1995; 7: 344-350Crossref PubMed Scopus (71) Google Scholar, 3.Nobes C.D. Reppas J.B. Markus A. Tolkovsky A.M. Neuroscience. 1996; 70: 1067-1079Crossref PubMed Scopus (41) Google Scholar). Overexpression of RasVal-12 had no deleterious effects on neurons maintained in the presence of NGF (Fig. 3,a and b; Fig. 4) (survival of RasVal-12-positive cells 91 ± 2.6% compared with 95.4 ± 0.6% in the total population) and completely blocked apoptosis induced by 20 h of NGF deprivation (survival of RasVal-12-positive cells 87 ± 4% compared with 69 ± 3.5% in the total population of neurons,p < 0.001 −NGF/total versus−NGF/RasVal-12). Most RasVal-12-expressing cells showed a healthy profile with robust neurites despite the absence of NGF (compare Figs. 2 a and 3 a). In contrast, Ras17N caused massive neuronal apoptosis in the presence of NGF (survival being reduced from 96 ± 0.6 to 41 ± 7.6%), there being a significant increase (by 28%; p < 0.005) in the percentage of cells with apoptotic nuclei compared with the value measured in the total population of NGF-deprived neurons (Fig. 2,c and d; Fig. 4). Little further apoptosis was observed in the Ras17N-expressing neurons that were also NGF-deprived (35 ± 3.3% survival) thus supporting the idea that Ras17N is only dominant-negative in the context of a counter-signal induced by NGF. Thus, most of the apoptosis that occurred in Ras17N-expressing neurons occurred prior to NGF deprivation. Furthermore, no proper neurite outgrowth could be observed in the Ras17N-positive neurons maintained in the presence of NGF (Fig. 2 c). Thus, constitutively active transfected RasVal-12 is sufficient to support neuron survival and rescue neurons from NGF deprivation, whereas dominant inactive Ras17N kills neurons by completely blocking survival signaling from NGF, implying that Ras activity is necessary for NGF-mediated survival. To examine which of the downstream pathways of Ras contribute to rescue from NGF withdrawal, SCG neurons were deprived of NGF after transfection with specific effector-loop domain mutant Ras constructs. In the absence of NGF, expression of RasVal-1240C (which activates the PI3K pathway) completely inhibited neuronal death induced by 20 h of NGF withdrawal (91.3 ± 1.1% survival, Fig. 4; p < 0.001 compared with −NGF/total) and did not reduce survival in the presence of NGF (93 ± 0.9%). Furthermore, RasVal-1240C (like RasVal-12) promoted effusive outgrowth of healthy and long neurites in both presence or absence of NGF (Figs. 2 g and 3 e). Thus, the activity of the Ras-linked PI3K pathway fully blocks the apoptotic signal and is sufficient for supporting neuronal survival in the absence of a neurotrophic factor. In contrast to RasVal-1240C, RasVal-1235S expression (which activates the Raf-1/ERK pathway) did not block neuronal death during NGF deprivation, the rate of survival of RasVal-1235S-positive cells (60 ± 5%) being similar to that of NGF-deprived cells in the total population (69 ± 3.5%) (Figs. 3, c and d and Fig. 4). In the presence of NGF, RasVal-1235S-overexpressing neurons underwent as much apoptosis (69 ± 2.4% survival) as the NGF-deprived neurons, suggesting that it may have some dominant-negative effects when the NGF survival signaling is active. Compared with RasVal-12-positive neurons, surviving RasVal-1235S-positive cells exhibited a thinner and less dense profile of neurites (Fig. 2, a and e, and Fig. 3, a and c). Thus the Ras/ERK pathway does not appear to deliver a protective signal against NGF withdrawal-induced neuronal death. To address the function of RalGDS pathway, RasVal-1237G was expressed in SCG neurons. As shown in Fig. 2 i,RasVal-1237G seemed to suppress neurite outgrowth in the presence of NGF, and many expressing cells did not display HA-stained neurites. In addition, a low proportion of survival (64 ± 3%) was scored in RasVal-1237G-positive neurons in the presence of NGF (Fig. 4) suggesting a slight dominant-negative effect of RasVal-1237G on neuronal survival signals. This interpretation was supported by the lack of further reduction in survival (57 ± 7%) when neurons were deprived of NGF for 20 h. Treatment with 1 mm araC can induce sympathetic neuron apoptosis in the presence of NGF as efficiently as NGF deprivation (24.Wallace T.L. Johnson E.M. J. Neur

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