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

Vaillant AR, Mazzoni I, Tudan C, Boudreau M, Kaplan DR, Miller FD - J. Cell Biol. (1999)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4.

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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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).
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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).

Mentions: Previous work with cortical neurons indicates that depolarization-induced survival requires an autocrine neurotrophin loop (Ghosh et al. 1994). To determine if a similar mechanism could explain the functional synergy observed here, we asked whether KCl-mediated survival requires Trk receptor activation. Lysates of neurons maintained in varying concentrations of NGF or KCl were immunoprecipitated with anti-panTrk, and the level of Trk receptor activation monitored by Western blot analysis with antiphosphotyrosine (Fig. 4 a). Both 5 and 10 ng/ml NGF led to a robust increase in Trk autophosphorylation, but Trk autophosphorylation was undetectable in the presence of KCl, as previously reported by ourselves and others (Franklin et al. 1995; Bamji et al. 1998).


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

Vaillant AR, Mazzoni I, Tudan C, Boudreau M, Kaplan DR, Miller FD - J. Cell Biol. (1999)

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).
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Related In: Results  -  Collection

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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).
Mentions: Previous work with cortical neurons indicates that depolarization-induced survival requires an autocrine neurotrophin loop (Ghosh et al. 1994). To determine if a similar mechanism could explain the functional synergy observed here, we asked whether KCl-mediated survival requires Trk receptor activation. Lysates of neurons maintained in varying concentrations of NGF or KCl were immunoprecipitated with anti-panTrk, and the level of Trk receptor activation monitored by Western blot analysis with antiphosphotyrosine (Fig. 4 a). Both 5 and 10 ng/ml NGF led to a robust increase in Trk autophosphorylation, but Trk autophosphorylation was undetectable in the presence of KCl, as previously reported by ourselves and others (Franklin et al. 1995; Bamji et al. 1998).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4.

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.

Show MeSH
Related in: MedlinePlus