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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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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.
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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.

Mentions: To determine potential intracellular convergence points for KCl and NGF-mediated survival, we examined two downstream substrates; the ERKs (MAP kinases), which are activated in sympathetic neurons by depolarization (Franklin et al. 1995) and by NGF (Creedon et al. 1996), and the serine–threonine kinase Akt, which is activated by TrkA (Andjelkovic et al. 1998), and is required for NGF-mediated sympathetic neuron survival (Crowder and Freeman 1998). To perform these experiments, NGF-selected sympathetic neurons were acutely stimulated with varying concentrations of NGF or KCl for 15 min, and then analyzed biochemically. Western blot analysis of neuronal lysates with phospho-specific ERK antibodies revealed that both NGF and KCl caused a dose-dependent increase in ERK phosphorylation, as monitored by phospho-specific antibodies for tyrosine and threonine (tyr/thr) or for tyrosine alone (Fig. 5 a). We also performed in vitro kinase assays using myelin basic protein as a phospho-acceptor substrate to monitor ERK activity. This analysis revealed that while both NGF and KCl induced increased phosphotransferase activity of ERK in a concentration-dependent fashion, higher levels of activation were observed with NGF than with KCl (Fig. 5 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)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169479&req=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.
Mentions: To determine potential intracellular convergence points for KCl and NGF-mediated survival, we examined two downstream substrates; the ERKs (MAP kinases), which are activated in sympathetic neurons by depolarization (Franklin et al. 1995) and by NGF (Creedon et al. 1996), and the serine–threonine kinase Akt, which is activated by TrkA (Andjelkovic et al. 1998), and is required for NGF-mediated sympathetic neuron survival (Crowder and Freeman 1998). To perform these experiments, NGF-selected sympathetic neurons were acutely stimulated with varying concentrations of NGF or KCl for 15 min, and then analyzed biochemically. Western blot analysis of neuronal lysates with phospho-specific ERK antibodies revealed that both NGF and KCl caused a dose-dependent increase in ERK phosphorylation, as monitored by phospho-specific antibodies for tyrosine and threonine (tyr/thr) or for tyrosine alone (Fig. 5 a). We also performed in vitro kinase assays using myelin basic protein as a phospho-acceptor substrate to monitor ERK activity. This analysis revealed that while both NGF and KCl induced increased phosphotransferase activity of ERK in a concentration-dependent fashion, higher levels of activation were observed with NGF than with KCl (Fig. 5 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