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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 SCG neurons in culture. The neurons were grown in 96-well plates either as single cells in isolation (black bars) or single cells cocultured with one to four nonneuronal cells per well (hatched bars). The means and standard errors are shown for three experiments, with a total of 36 single neurons and 42 single neurons cocultured with nonneuronal cells.
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fig1: Survival of adult SCG neurons in culture. The neurons were grown in 96-well plates either as single cells in isolation (black bars) or single cells cocultured with one to four nonneuronal cells per well (hatched bars). The means and standard errors are shown for three experiments, with a total of 36 single neurons and 42 single neurons cocultured with nonneuronal cells.

Mentions: Although it has been reported that mature SCG neurons survive without added neurotrophic factors in dissociated cultures enriched for neurons (Orike et al., 2000), it is possible that the small number of nonneuronal cells present in these cultures might produce sufficient neurotrophic factors to sustain neuronal survival. To demonstrate conclusively that mature sympathetic neurons are capable of surviving independently of factors produced by other cells, 12-wk adult rat SCG neurons were plated at single cell density in 96-well plates. The great majority of these neurons (>75%) survived for at least 5 d in defined, serum-free medium without added neurotrophic factors, and their survival was not enhanced by coculturing them with nonneuronal cells in these wells (Fig. 1) . Having established that mature sympathetic neurons can survive autonomously and independently of trophic support from other cells, we embarked upon a series of experiments to elucidate the molecular basis of their survival independence.


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 in culture. The neurons were grown in 96-well plates either as single cells in isolation (black bars) or single cells cocultured with one to four nonneuronal cells per well (hatched bars). The means and standard errors are shown for three experiments, with a total of 36 single neurons and 42 single neurons cocultured with nonneuronal cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Survival of adult SCG neurons in culture. The neurons were grown in 96-well plates either as single cells in isolation (black bars) or single cells cocultured with one to four nonneuronal cells per well (hatched bars). The means and standard errors are shown for three experiments, with a total of 36 single neurons and 42 single neurons cocultured with nonneuronal cells.
Mentions: Although it has been reported that mature SCG neurons survive without added neurotrophic factors in dissociated cultures enriched for neurons (Orike et al., 2000), it is possible that the small number of nonneuronal cells present in these cultures might produce sufficient neurotrophic factors to sustain neuronal survival. To demonstrate conclusively that mature sympathetic neurons are capable of surviving independently of factors produced by other cells, 12-wk adult rat SCG neurons were plated at single cell density in 96-well plates. The great majority of these neurons (>75%) survived for at least 5 d in defined, serum-free medium without added neurotrophic factors, and their survival was not enhanced by coculturing them with nonneuronal cells in these wells (Fig. 1) . Having established that mature sympathetic neurons can survive autonomously and independently of trophic support from other cells, we embarked upon a series of experiments to elucidate the molecular basis of their survival independence.

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