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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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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.
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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.

Mentions: On the basis of these data, we hypothesized that the synergistic survival seen with NGF and KCl might be mediated by a convergent stimulation of the Ras–PI3-kinase–Akt pathway. To test this hypothesis, NGF-selected neurons were induced for 15 min with 5 ng/ml NGF plus 10 mM KCl, a combination that mediated survival synergistically (Fig. 1 a), and were then analyzed biochemically. Western blot analysis with antiphospho-Akt, followed by quantitative densitometry, revealed that while 10 mM KCl had no detectable effect on Akt phosphorylation (Fig. 7, a and c), the addition of 10 mM KCl to either 2.5 or 5 ng/ml NGF led to levels of Akt phosphorylation that were higher than either of these concentrations of NGF alone (Fig. 7, a and c). Similar results were obtained when ERK phosphorylation was examined. 10 mM KCl did not detectably increase ERK tyrosine/threonine phosphorylation above controls, but the addition of 10 mM KCl to either 2.5 or 5 ng/ml NGF led to significantly higher levels of ERK phosphorylation than did treatment with either concentration of NGF alone (Fig. 7, a and b).


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)

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.
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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.
Mentions: On the basis of these data, we hypothesized that the synergistic survival seen with NGF and KCl might be mediated by a convergent stimulation of the Ras–PI3-kinase–Akt pathway. To test this hypothesis, NGF-selected neurons were induced for 15 min with 5 ng/ml NGF plus 10 mM KCl, a combination that mediated survival synergistically (Fig. 1 a), and were then analyzed biochemically. Western blot analysis with antiphospho-Akt, followed by quantitative densitometry, revealed that while 10 mM KCl had no detectable effect on Akt phosphorylation (Fig. 7, a and c), the addition of 10 mM KCl to either 2.5 or 5 ng/ml NGF led to levels of Akt phosphorylation that were higher than either of these concentrations of NGF alone (Fig. 7, a and c). Similar results were obtained when ERK phosphorylation was examined. 10 mM KCl did not detectably increase ERK tyrosine/threonine phosphorylation above controls, but the addition of 10 mM KCl to either 2.5 or 5 ng/ml NGF led to significantly higher levels of ERK phosphorylation than did treatment with either concentration of NGF alone (Fig. 7, a and b).

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