Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival - PubMed (original) (raw)

Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival

A R Vaillant et al. J Cell Biol. 1999.

Abstract

In this report, we have examined the mechanisms whereby neurotrophins and neural activity coordinately regulate neuronal survival, focussing on sympathetic neurons, which require target-derived NGF and neural activity for survival during development. When sympathetic neurons were maintained in suboptimal concentrations of NGF, coincident depolarization with concentrations of KCl that on their own had no survival effect, synergistically enhanced survival. Biochemical analysis revealed that depolarization was sufficient to activate a Ras-phosphatidylinositol 3-kinase-Akt pathway (Ras-PI3-kinase-Akt), and function-blocking experiments using recombinant adenovirus indicated that this pathway was essential for approximately 50% of depolarization-mediated neuronal survival. At concentrations of NGF and KCl that promoted synergistic survival, these two stimuli converged to promote increased PI3-kinase-dependent Akt phosphorylation. This convergent PI3-kinase-Akt pathway was essential for synergistic survival. In contrast, inhibition of calcium/calmodulin-dependent protein kinase II revealed that, while this molecule was essential for depolarization-induced survival, it had no role in KCl- induced Akt phosphorylation, nor was it important for synergistic survival by NGF and KCl. Thus, NGF and depolarization together mediate survival of sympathetic neurons via intracellular convergence on a Ras-PI3-kinase-Akt pathway. This convergent regulation of Akt may provide a general mechanism for coordinating the effects of growth factors and neural activity on neuronal survival throughout the nervous system.

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Figures

Figure 1

Figure 1

NGF and KCL synergistically regulate sympathetic neuron survival. a, Sympathetic neuron survival in response to various combinations of NGF and KCl, as monitored using MTT assays. Neonatal sympathetic neurons were cultured in 50 ng/ml NGF for five days, washed free of neurotrophin-containing medium, and then incubated for two days in various concentrations of NGF (ng/ml) and/or KCl (mM), as indicated on the x-axis. Data is derived from one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. In these assays, absolute values are normalized so that the value obtained with 20 ng/ml NGF is considered 100% survival. Error bars represent SD. *Indicate those NGF plus KCl values that are significantly different (P < 0.01, t test) from 2.5 (*) or 5 ng/ml NGF (**). b, Quantitative analysis of sympathetic neuron apoptosis in response to various combinations of NGF and KCl, as monitored by TUNEL. NGF-selected neurons were incubated for two days in different concentrations of NGF (ng/ml) and/or KCl (mM), as indicated on the x-axis. Each point represents combined data from three separate experiments, each of which was performed in triplicate. The data are expressed as percentage of TUNEL-negative cells/total Hoechst-labeled nuclei. Error bars and significance are as in a.

Figure 2

Figure 2

Sympathetic neuron survival in response to NGF and KCl is similar at different cell densities. Sympathetic neuron survival at different densities, as monitored by MTT assays. Neonatal sympathetic neurons were cultured in 50 ng/ml NGF for five days at 350 or 1,500 cells/well, washed free of neurotrophin-containing medium, and then incubated for two days in various concentrations of NGF (ng/ml) or KCl (mM), as indicated on the x-axis. Data is derived from one representative experiment performed in triplicate. In these assays, absolute values are normalized so that the value obtained with 10 ng/ml NGF is considered 100% survival. Error bars represent SD. *Indicate the NGF plus KCl values that are significantly different (P < 0.01, t test) from 2.5 (*) or 5 ng/ml NGF (**). Insets are phase-contrast photographs of representative fields of neurons at the different cell densities.

Figure 3

Figure 3

Quantitation of sympathetic neuron apoptosis in response to NGF and KCl. Immunofluorescence microscopy showing Hoechst 33258 (left) and TUNEL (right) of the same field of sympathetic neurons treated with NGF and/or KCL. Neurons were cultured five days in 50 ng/ml NGF, and then switched into the following concentrations of NGF and/or KCl for an additional two days before analysis: 5 ng/ml NGF (a and b); 10 ng/ml NGF (c and d); 10 mM KCl (e and f); 50 mM KCl (g and h); and 5 ng/ml NGF + 10 mM KCl (i and j). TUNEL labeling is markedly reduced in j, compared with that seen in b or f. Bar, 100μm.

Figure 4

Figure 4

KCl signals via a TrkA-independent mechanism. a, KCl does not cause Trk autophosphorylation. NGF-selected sympathetic neurons were washed free of neurotrophin, and then exposed to various concentrations of NGF (ng/ml) or KCl (mM) for 15 min. Cellular lysates were immunoprecipitated with anti-panTrk and probed with antiphosphotyrosine (p-tyr) to visualize Trk autophosphorylation. All samples were normalized for equal amounts of protein. b, Dominant-negative TrkA inhibits NGF-mediated, but not KCl-mediated sympathetic neuron survival. NGF-selected neurons were infected with recombinant adenovirus expressing dnTrkA at titers ranging from 10 to 150 MOI, or with 200 MOI of a control adenovirus expressing the TTA transactivator, and were then switched to 10 ng/ml NGF (10N) or 50 mM KCl (50K) one day later. After 48 h, cell survival was measured by MTT assays. As controls, uninfected sister cultures were either withdrawn from NGF (0N), or maintained in 10 ng/ml NGF or 50 mM KCl. Results are those obtained in one representative experiment performed in triplicate, and represent the mean ± SD. Similar results were obtained in three separate experiments. *Represents those values that are significantly different from 10 ng/ml NGF alone (P < 0.01). c, K252a blocks Trk activation in response to NGF, whereas nifedipine has no effect. NGF-selected neurons were treated with various concentrations of NGF (ng/ml) or KCl (mM) for 15 min in the presence of 200 nm K-252a or 1 μM nifedipine (NIF), and then probed for phosphotyrosine (p-tyr). All samples were normalized for equal amounts of protein. d and e, K252a selectively blocks NGF-mediated sympathetic neuron survival, whereas nifedipine selectively blocks KCl-mediated survival. d, NGF-selected neurons were treated for two days with 200 nm K-252a in the presence of 10 ng/ml NGF (10N) or 50 mM KCl (50K) and survival was measured by MTT assays. Results derive from one representative experiment performed in triplicate and each point is the mean ± SD. Similar results were obtained in three separate experiments. *Indicates those values that are significantly different from 10 ng/ml NGF (P < 0.01). e, Quantitative analysis of TUNEL-labeled sympathetic neurons treated with 200 nm K-252a or 1 μM nifedipine (NIF) in the presence of 10 ng/ml NGF (10N) or 50 mM KCl (50K) for two days. Data are expressed as the percentage of TUNEL-negative cells/total Hoechst-positive nuclei. Each point represents combined data from three separate experiments, each performed in triplicate, and is the mean ± SD. *Indicates those values that are significantly different from 10 ng/ml NGF (*P < 0.01) or 50 mM KCl (**P < 0.01).

Figure 5

Figure 5

Activation of ERK and Akt kinases by NGF and KCl. a and b, ERK activation in response to increasing concentrations of NGF or KCl. a, Western blot analysis of equal amounts of protein derived from sympathetic neurons treated for 15 min with increasing concentrations of NGF (ng/ml) or KCl (mM), as detected with antibodies to tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERKs), tyrosine-phosphorylated ERKs (P-tyr ERKs), or to total ERK protein (total ERKs). The upper band in the doublet is ERK1 and the lower is ERK2. Note that all three panels are reprobes of the same blot. b, In vitro ERK activation assay using myelin basic protein (MBP) as a substrate in lysates of sympathetic neurons treated for 15 min with increasing concentrations of NGF (ng/ml) or KCl (mM). All samples were normalized for equal amounts of protein. c–e, Akt activation in response to increasing concentrations of NGF or KCl. c, Western blot analysis of equal amounts of protein derived from sympathetic neurons treated for 15 min with increasing concentrations of NGF (ng/ml) or KCl (mM), as detected with antibodies to serine-phosphorylated Akt (P-ser Akt) or to total Akt protein (Total Akt). Note that both panels are reprobes of the same blot. d, In vitro Akt activation using histone H2B as a substrate in lysates of sympathetic neurons treated for 15 min with increasing concentrations of NGF (ng/ml) or KCl (mM). All samples were normalized for equal amounts of protein. e, Quantitative in vitro Akt activity assay using an Akt-specific substrate in lysates of sympathetic neurons treated for 15 min with increasing concentrations of NGF (ng/ml) or KCl (mM). In sister cultures, neurons were coincidently treated with 100 μM of the PI3-kinase inhibitor LY294002 (LY). Bars represent the mean ± SD. Note that inhibition of PI3-kinase with LY294002 abolishes Akt activity induced by NGF or KCl.

Figure 6

Figure 6

Ras, PI3-kinase, and Akt activity are important in KCl-mediated sympathetic neuron survival. a, Ras is involved in KCl-mediated survival. NGF-selected sympathetic neurons were infected with 200 MOI of adenovirus expressing either dnRas or GFP, and one day later were switched to 10 ng/ml NGF (10N) or 50 mM KCl (50K). 48 h later, survival was monitored by MTT assay. Results derive from one representative assay performed in triplicate; values represent the mean ± SD. Similar results were obtained in three separate experiments. *Indicate values that are significantly different (P < 0.01) from 10 ng/ml NGF (*) or 50 mM KCl (**). b and c, PI3-kinase, but not MEK activity, is involved in KCl-mediated survival. NGF-selected neurons were maintained for two days in 10 ng/ml NGF (10N) or 50 mM KCl (50K), plus or minus 100 μM of the PI3-kinase inhibitor LY294002 (LY) and/or 75 μM of the MEK inhibitor PD98059 (PD). Survival was then monitored by MTT assay (b) or TUNEL (c). In b, results represent data from one representative experiment performed in triplicate, and each point is the mean ± SD. In c, results represent the mean percentage of TUNEL-negative cells/total Hoechst-positive nuclei from three separate experiments, each performed in triplicate. *Asterisks indicate values that are significantly different (P < 0.01) from 10 ng/ml NGF (*) or 50 mM KCl (**). d, Akt is essential for a portion of KCl-mediated survival. NGF-selected sympathetic neurons were infected with 10–100 MOI of an adenovirus expressing dnAkt and one day later were switched into 10 ng/ml NGF (10N) or 50 mM KCl (50K). All neurons were also coinfected with 50 MOI of an adenovirus expressing the transactivator TTA. As controls, neurons were infected with 200 MOI of the TTA adenovirus alone. Neuronal survival was then measured two days later by MTT assay. Results represent data from one representative experiment performed in triplicate, and each point is the mean ± SD. Similar results were obtained in three separate experiments. *Indicate values that are significantly different (P < 0.01) from 10 ng/ml NGF (*) or 50 mM KCl (**). e, Western blot analysis for ERKs and Akt kinase in sympathetic neurons treated for 15 min with NGF (ng/ml) or KCl (mM), as analyzed using antibodies specific to tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERKs), tyrosine phosphorylated ERKs (P-tyr ERKs), serine-phosphorylated Akt (P-ser Akt), or total ERK or Akt protein. Some cultures were coincidently treated with 100 μM LY294002 (LY) or 75 μM PD98059 (PD). All samples were normalized for equal amounts of protein. The three ERK panels are reprobes of the same blot, as are the two Akt panels. f, Ras activity is necessary for KCl-mediated Akt phosphorylation. Western blot analysis for ERKs and Akt kinase in sympathetic neurons infected with 200 MOI dnRas, and then induced for 15 min with KCl (mM), using the same antibodies as in e. All ERK panels derive from reprobes of one blot, and the Akt panels derive from reprobes of another.

Figure 7

Figure 7

Suboptimal concentrations of NGF and KCl synergistically increase ERK and Akt kinase phosphorylation. a, Western blot analysis of sympathetic neurons induced for 15 min with various concentrations of NGF (ng/ml) and/or KCl (mM), using antibodies specific to tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERK), serine-phosphorylated Akt (P-ser Akt) or total ERK or Akt protein. All samples were normalized for equal amounts of protein. All ERK panels derive from reprobes of one blot, and the Akt panels reprobes of another. b and c, Quantitative analysis of ERK (b) or Akt (c) phosphorylation in response to various concentrations of NGF and/or KCl, obtained by scanning densitometry of Western blots similar to those shown in a. Note that 10 mM KCl (10K) does not produce signals that are detectably increased over control (0N) neurons, but treatment with 2.5 ng/ml NGF (2.5N) and 5 ng/ml NGF (5N) plus 10 mM KCl results in levels of ERK and Akt phosphorylation that are higher than either 2.5 or 5 ng/ml NGF alone, respectively.

Figure 8

Figure 8

The Ras–PI3-kinase–Akt pathway is required for the synergistic survival observed with NGF and KCl together. a, Ras activity is necessary for ∼50% of synergistic survival. NGF-selected sympathetic neurons were infected with 200 MOI of dnRas or GFP adenovirus, and one day later were switched into 5 ng/ml NGF (5N) plus or minus 10 mM KCl (10K). Neuronal survival was monitored two days later by MTT assay. Results represent the mean ± SD of one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. *Indicates those results that are significantly different (P < 0.01) from 5 ng/ml NGF plus 10 mM KCl. b, Dominant-negative Ras partially inhibits Akt and ERK phosphorylation induced by NGF plus KCl. Western blot analysis for Akt and ERK phosphorylation in sympathetic neurons infected with 200 MOI dnRas adenovirus and then maintained in 5 ng/ml NGF (5N) and/or 10 mM KCl (10K). Lysates were analyzed with antibodies specific for serine-phosphorylated Akt (P-ser Akt), tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERKs), or total Akt or ERK protein. All samples were normalized for equal amounts of protein. All ERK panels are reprobes of one blot, and Akt panels reprobes of another. c, PI3-kinase is necessary for all of the survival seen with NGF plus KCl. MTT assays of sympathetic neurons maintained in various concentrations of NGF (ng/ml) and/or KCl (mM) treated coincidently with 100 μM LY294002 (LY) and/or 75 μM PD98059 (PD). Results represent data obtained from one representative experiment performed in triplicate, and each point is the mean ± SD. Similar results were obtained in three separate experiments. *Indicates those values that are significantly different from 5 ng/ml NGF plus 10 mM KCl (*) or 10 ng/ml NGF plus 10 mM KCl (**). d, Akt activity is necessary for all of the survival seen with NGF plus KCl. NGF-selected sympathetic neurons were infected with 10–100 MOI dnAkt adenovirus and, one day later, were switched to 5 ng/ml NGF plus 10 mM KCl. All neurons infected with dnAkt adenovirus were also infected with 50 MOI of an adenovirus expressing the TTA transactivator. Control neurons were infected with 100 MOI of the TTA adenovirus alone. Results represent the mean ± SD of one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. *Indicates those values that are significantly different (P < 0.01) from 5 ng/ml NGF plus 10 mM KCl.

Figure 9

Figure 9

The role of CaMKII in survival mediated by NGF and/or KCl. a and b, CaMKII is necessary for KCl-mediated survival, but plays no role in the synergistic survival seen with NGF plus KCl. NGF-selected sympathetic neurons maintained in NGF and/or KCl for two days with or without 10 μM KN-62 and survival was then monitored by MTT assay (a) or TUNEL (b). In a, results represent the mean ± SD of one representative experiment performed in triplicate. Similar results were obtained in three separate experiments. In c, results represent the mean percentage of TUNEL-negative cells/total Hoechst-positive nuclei as determined in three separate experiments, each of which was performed in triplicate. In both panels, *indicates values that are significantly different (P < 0.01) from 50 mM KCl. c, CaMKII is necessary for ERK, but not Akt phosphorylation in response to KCl. Western blot analysis of equal amounts of protein derived from sympathetic neurons stimulated for 15 min with NGF (ng/ml) and/or KCl (mM) with or without 10 μM KN-62. Blots were probed with antibodies specific to tyrosine/threonine-phosphorylated ERKs (P-tyr/thr ERK), serine-phosphorylated Akt (P-ser Akt), or to total ERK or Akt protein. The ERK panels derive from reprobes of the same blot, and the Akt panels from reprobes of another.

Figure 10

Figure 10

Proposed models for signal-transduction pathways mediating survival in NGF or KCl alone (a) or synergistically in the presence of both NGF and KCl (b). a, Illustrates the proposed pathways that support sympathetic neuron survival when either NGF or KCl are alone. b, Illustrates the proposed convergence onto the Ras–PI3-kinase–Akt pathway that is essential for the synergistic survival seen at concentrations of NGF and KCl, which on their own are not sufficient for maximal survival.

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