Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169479&req=5

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.

Mentions: We then determined the functional necessity of this convergent activation for sympathetic neuron survival. We first examined Ras: NGF-dependent neurons were infected with the dnRas adenovirus, were switched to 5 ng/ml NGF plus 10 mM KCl, and survival was determined two days later by MTT assays. This analysis (Fig. 8 a) revealed that inhibition of Ras decreased the synergistic survival ∼50%, a result similar to that seen with NGF or KCl alone (Fig. 6 a). Coincident with the decrease in neuronal survival observed with dnRas, we also observed a partial decrease in the downstream activation of Akt and the ERKs, as assessed by Western blots with phosphorylation-specific antibodies (Fig. 8 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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169479&req=5

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.
Mentions: We then determined the functional necessity of this convergent activation for sympathetic neuron survival. We first examined Ras: NGF-dependent neurons were infected with the dnRas adenovirus, were switched to 5 ng/ml NGF plus 10 mM KCl, and survival was determined two days later by MTT assays. This analysis (Fig. 8 a) revealed that inhibition of Ras decreased the synergistic survival ∼50%, a result similar to that seen with NGF or KCl alone (Fig. 6 a). Coincident with the decrease in neuronal survival observed with dnRas, we also observed a partial decrease in the downstream activation of Akt and the ERKs, as assessed by Western blots with phosphorylation-specific antibodies (Fig. 8 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