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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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Survival of adult sympathetic neurons overexpressing Bad or Bax. The neurons were injected with Bad or Bax expression plasmids or with an empty plasmid (control plasmid). The neurons were initially grown for 12 h before injection, and the number of neurons surviving at intervals after injection is expressed as a percentage of the initial number of neurons injected. The means and standard errors for the combined results of three separate experiments are shown.
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fig9: Survival of adult sympathetic neurons overexpressing Bad or Bax. The neurons were injected with Bad or Bax expression plasmids or with an empty plasmid (control plasmid). The neurons were initially grown for 12 h before injection, and the number of neurons surviving at intervals after injection is expressed as a percentage of the initial number of neurons injected. The means and standard errors for the combined results of three separate experiments are shown.

Mentions: In addition to interfering with the synthesis of antiapoptotic members of the Bcl-2 family to ascertain the importance of these proteins in sustaining the survival of adult SCG neurons, we also overexpressed proapoptotic members of this family to see if this would compromise the survival of these neurons. The most marked effect on survival was observed by overexpressing Bad. Injection of a Bad expression plasmid caused the rapid death of the majority of adult SCG neurons, leaving <20% surviving 5 d after injection (Fig. 9) . Injection of a Bax expression plasmid caused much less neuronal death, leaving 40% surviving 5 d after injection (Fig. 9).


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 sympathetic neurons overexpressing Bad or Bax. The neurons were injected with Bad or Bax expression plasmids or with an empty plasmid (control plasmid). The neurons were initially grown for 12 h before injection, and the number of neurons surviving at intervals after injection is expressed as a percentage of the initial number of neurons injected. 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

fig9: Survival of adult sympathetic neurons overexpressing Bad or Bax. The neurons were injected with Bad or Bax expression plasmids or with an empty plasmid (control plasmid). The neurons were initially grown for 12 h before injection, and the number of neurons surviving at intervals after injection is expressed as a percentage of the initial number of neurons injected. The means and standard errors for the combined results of three separate experiments are shown.
Mentions: In addition to interfering with the synthesis of antiapoptotic members of the Bcl-2 family to ascertain the importance of these proteins in sustaining the survival of adult SCG neurons, we also overexpressed proapoptotic members of this family to see if this would compromise the survival of these neurons. The most marked effect on survival was observed by overexpressing Bad. Injection of a Bad expression plasmid caused the rapid death of the majority of adult SCG neurons, leaving <20% surviving 5 d after injection (Fig. 9) . Injection of a Bax expression plasmid caused much less neuronal death, leaving 40% surviving 5 d after injection (Fig. 9).

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