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Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons.

Orike N, Middleton G, Borthwick E, Buchman V, Cowen T, Davies AM - J. Cell Biol. (2001)

Bottom Line: To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival.Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase).These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, Royal Free Hospital School of Medicine, London NW3 2PF, United Kingdom.

ABSTRACT
By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.

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Related in: MedlinePlus

Survival of adult SCG neurons cultured with inhibitors PI 3-kinase or MCK. The neurons were grown at low density in defined, serum-free medium alone (untreated) or containing 20 μM PD98059, 20 μM LY294002, or 20 μM Wortmannin. These reagents were added 3 h after plating when the neurons had become attached to the substratum. The number of neurons surviving at intervals after plating is expressed as a percentage of the initial number of neurons plated. The means and standard errors for the combined results of three separate experiments are shown.
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fig2: Survival of adult SCG neurons cultured with inhibitors PI 3-kinase or MCK. The neurons were grown at low density in defined, serum-free medium alone (untreated) or containing 20 μM PD98059, 20 μM LY294002, or 20 μM Wortmannin. These reagents were added 3 h after plating when the neurons had become attached to the substratum. The number of neurons surviving at intervals after plating is expressed as a percentage of the initial number of neurons plated. The means and standard errors for the combined results of three separate experiments are shown.

Mentions: We began our investigations of the survival independence of adult SCG neurons using pharmacological agents to inhibit signaling pathways that have been implicated in mediating the survival effects of growth factors. These and all subsequent experiments were carried out in low density–dissociated cultures of SCG neurons (<1,000 neurons per 60-mm petri dish) from which most nonneuronal cells were removed by differential sedimentation (>90% neurons). Addition of Wortmannin or LY294002 at concentrations shown previously to preferentially block PI 3-kinase activation (Okada et al., 1994, Vlahos et al., 1994) caused the rapid death of the majority of the neurons (Fig. 2) . Addition of PD98059 to the culture medium at concentrations shown previously to inhibit activation of MEK (Alessi et al., 1995) had little effect on neuronal survival, even when added repeatedly to the medium at daily intervals (Fig. 2). These results suggest that activation of PI 3-kinase is required for the neurotrophic factor–independent survival of adult SCG neurons, whereas MEK/MAP kinase signaling has no apparent role.


Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons.

Orike N, Middleton G, Borthwick E, Buchman V, Cowen T, Davies AM - J. Cell Biol. (2001)

Survival of adult SCG neurons cultured with inhibitors PI 3-kinase or MCK. The neurons were grown at low density in defined, serum-free medium alone (untreated) or containing 20 μM PD98059, 20 μM LY294002, or 20 μM Wortmannin. These reagents were added 3 h after plating when the neurons had become attached to the substratum. The number of neurons surviving at intervals after plating is expressed as a percentage of the initial number of neurons plated. The means and standard errors for the combined results of three separate experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196191&req=5

fig2: Survival of adult SCG neurons cultured with inhibitors PI 3-kinase or MCK. The neurons were grown at low density in defined, serum-free medium alone (untreated) or containing 20 μM PD98059, 20 μM LY294002, or 20 μM Wortmannin. These reagents were added 3 h after plating when the neurons had become attached to the substratum. The number of neurons surviving at intervals after plating is expressed as a percentage of the initial number of neurons plated. The means and standard errors for the combined results of three separate experiments are shown.
Mentions: We began our investigations of the survival independence of adult SCG neurons using pharmacological agents to inhibit signaling pathways that have been implicated in mediating the survival effects of growth factors. These and all subsequent experiments were carried out in low density–dissociated cultures of SCG neurons (<1,000 neurons per 60-mm petri dish) from which most nonneuronal cells were removed by differential sedimentation (>90% neurons). Addition of Wortmannin or LY294002 at concentrations shown previously to preferentially block PI 3-kinase activation (Okada et al., 1994, Vlahos et al., 1994) caused the rapid death of the majority of the neurons (Fig. 2) . Addition of PD98059 to the culture medium at concentrations shown previously to inhibit activation of MEK (Alessi et al., 1995) had little effect on neuronal survival, even when added repeatedly to the medium at daily intervals (Fig. 2). These results suggest that activation of PI 3-kinase is required for the neurotrophic factor–independent survival of adult SCG neurons, whereas MEK/MAP kinase signaling has no apparent role.

Bottom Line: To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival.Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase).These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, Royal Free Hospital School of Medicine, London NW3 2PF, United Kingdom.

ABSTRACT
By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.

Show MeSH
Related in: MedlinePlus