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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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Micrographs of adult SCG neurons stained for Akt and phospho-Akt. The neurons were stained for Akt (A–C) or phospho-Akt (E–G) after being grown overnight and treated for 45 min with 10 ng/ml NGF (A and E), received no treatment (B and F) or were treated for 45 min with 20 μM LY294002 (C and G) before fixation. D and H show phase-contrast and epifluorescence micrographs of a neuron in a preparation from which the primary antiserum was omitted from the staining protocol, showing no detectable immunofluorescence. Bar, 10 μM.
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fig5: Micrographs of adult SCG neurons stained for Akt and phospho-Akt. The neurons were stained for Akt (A–C) or phospho-Akt (E–G) after being grown overnight and treated for 45 min with 10 ng/ml NGF (A and E), received no treatment (B and F) or were treated for 45 min with 20 μM LY294002 (C and G) before fixation. D and H show phase-contrast and epifluorescence micrographs of a neuron in a preparation from which the primary antiserum was omitted from the staining protocol, showing no detectable immunofluorescence. Bar, 10 μM.

Mentions: To ascertain if the active, phosphorylated form of Akt is present in adult SCG neurons surviving without neurotrophic factors and whether this is dephosphorylated after inhibition of PI 3-kinase, we used immunocytochemistry to visualize Akt protein and phospho-Akt protein in SCG neurons grown with and without the PI 3-kinase inhibitor LY294002. Fig. 5 A shows that SCG neurons were clearly immunoreactive for phospho-Akt protein when grown at low density in defined medium alone. Although phospho-Akt immunoreactivity was clearly reduced by treatment with LY294002 for 45 min (Fig. 5G), expression of total Akt protein was little affected as revealed by the similar level of Akt immunofluorescence in the presence and absence of LY294002 (Fig. 5, B and C). There was no obvious change in the expression of either phospho-Akt immunoreactivity or total Akt protein in adult SCG neurons treated for 45 min with NGF (Fig. 5, A and E). No staining was observed when the primary antibodies were omitted from the staining protocol (Fig. 5 H), indicating that there was no detectable nonspecific staining. Taken together, these results suggest that Akt is constitutively active in adult SCG neurons surviving without neurotrophic factors and that PI 3-kinase is required for sustaining Akt activation.


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)

Micrographs of adult SCG neurons stained for Akt and phospho-Akt. The neurons were stained for Akt (A–C) or phospho-Akt (E–G) after being grown overnight and treated for 45 min with 10 ng/ml NGF (A and E), received no treatment (B and F) or were treated for 45 min with 20 μM LY294002 (C and G) before fixation. D and H show phase-contrast and epifluorescence micrographs of a neuron in a preparation from which the primary antiserum was omitted from the staining protocol, showing no detectable immunofluorescence. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Micrographs of adult SCG neurons stained for Akt and phospho-Akt. The neurons were stained for Akt (A–C) or phospho-Akt (E–G) after being grown overnight and treated for 45 min with 10 ng/ml NGF (A and E), received no treatment (B and F) or were treated for 45 min with 20 μM LY294002 (C and G) before fixation. D and H show phase-contrast and epifluorescence micrographs of a neuron in a preparation from which the primary antiserum was omitted from the staining protocol, showing no detectable immunofluorescence. Bar, 10 μM.
Mentions: To ascertain if the active, phosphorylated form of Akt is present in adult SCG neurons surviving without neurotrophic factors and whether this is dephosphorylated after inhibition of PI 3-kinase, we used immunocytochemistry to visualize Akt protein and phospho-Akt protein in SCG neurons grown with and without the PI 3-kinase inhibitor LY294002. Fig. 5 A shows that SCG neurons were clearly immunoreactive for phospho-Akt protein when grown at low density in defined medium alone. Although phospho-Akt immunoreactivity was clearly reduced by treatment with LY294002 for 45 min (Fig. 5G), expression of total Akt protein was little affected as revealed by the similar level of Akt immunofluorescence in the presence and absence of LY294002 (Fig. 5, B and C). There was no obvious change in the expression of either phospho-Akt immunoreactivity or total Akt protein in adult SCG neurons treated for 45 min with NGF (Fig. 5, A and E). No staining was observed when the primary antibodies were omitted from the staining protocol (Fig. 5 H), indicating that there was no detectable nonspecific staining. Taken together, these results suggest that Akt is constitutively active in adult SCG neurons surviving without neurotrophic factors and that PI 3-kinase is required for sustaining Akt activation.

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