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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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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.
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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.

Mentions: One possible explanation for this synergy is that KCl causes enhanced synthesis and/or secretion of a nonneurotrophin growth factor, thereby conditioning the media and causing enhanced survival. If this hypothesis were true, the synergistic survival should be density-dependent. To test this possibility, sympathetic neurons were plated in 50 ng/ml NGF at densities of 350 and 1,500 cells/well for five days (Fig. 2). Neurons were then switched to varying concentrations of NGF and/or KCl for an additional two days. The survival of these cultures was measured by MTT assay. This analysis (Fig. 2) demonstrated that the cell density had no effect on synergistic survival mediated by NGF and KCl.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: One possible explanation for this synergy is that KCl causes enhanced synthesis and/or secretion of a nonneurotrophin growth factor, thereby conditioning the media and causing enhanced survival. If this hypothesis were true, the synergistic survival should be density-dependent. To test this possibility, sympathetic neurons were plated in 50 ng/ml NGF at densities of 350 and 1,500 cells/well for five days (Fig. 2). Neurons were then switched to varying concentrations of NGF and/or KCl for an additional two days. The survival of these cultures was measured by MTT assay. This analysis (Fig. 2) demonstrated that the cell density had no effect on synergistic survival mediated by NGF and KCl.

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