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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 in culture. The neurons grown overnight and receiving no further treatment (A and C) or treated for 45 min with 20 μM LY294002 (B and D) are shown in phase-contrast optics (A and B) or stained with the fluorescent nuclear dye DAPI (C and D). (E). Bcl-xL staining in neurons injected 12 h earlier with an empty expression plasmid. (F). Bcl-xL staining in neurons injected 12 h earlier with a plasmid expressing antisense Bcl-xL RNA. Bar, 10 μM.
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fig6: Micrographs of adult SCG neurons in culture. The neurons grown overnight and receiving no further treatment (A and C) or treated for 45 min with 20 μM LY294002 (B and D) are shown in phase-contrast optics (A and B) or stained with the fluorescent nuclear dye DAPI (C and D). (E). Bcl-xL staining in neurons injected 12 h earlier with an empty expression plasmid. (F). Bcl-xL staining in neurons injected 12 h earlier with a plasmid expressing antisense Bcl-xL RNA. Bar, 10 μM.

Mentions: Adult sympathetic neurons surviving without neurotrophic factors had smooth cell bodies, a smooth spherical nucleus, and unbroken short neurites (Fig. 6 A). Neurons grown without neurotrophins retained this appearance throughout the period of the study (up to 5 d in vitro). However, neurite outgrowth could be stimulated from these neurons after this prolonged period of culture without neurotrophins by adding NGF to the culture medium (unpublished data). This indicates that although NGF is not required for survival, it is capable of promoting neurite outgrowth from these neurons. A similar phenomenon has been observed for neonatal Bax-deficient sympathetic neurons that survive in culture without NGF, but display very poor neurite outgrowth unless NGF is added to the medium (Deckwerth et al., 1996).


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 in culture. The neurons grown overnight and receiving no further treatment (A and C) or treated for 45 min with 20 μM LY294002 (B and D) are shown in phase-contrast optics (A and B) or stained with the fluorescent nuclear dye DAPI (C and D). (E). Bcl-xL staining in neurons injected 12 h earlier with an empty expression plasmid. (F). Bcl-xL staining in neurons injected 12 h earlier with a plasmid expressing antisense Bcl-xL RNA. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Micrographs of adult SCG neurons in culture. The neurons grown overnight and receiving no further treatment (A and C) or treated for 45 min with 20 μM LY294002 (B and D) are shown in phase-contrast optics (A and B) or stained with the fluorescent nuclear dye DAPI (C and D). (E). Bcl-xL staining in neurons injected 12 h earlier with an empty expression plasmid. (F). Bcl-xL staining in neurons injected 12 h earlier with a plasmid expressing antisense Bcl-xL RNA. Bar, 10 μM.
Mentions: Adult sympathetic neurons surviving without neurotrophic factors had smooth cell bodies, a smooth spherical nucleus, and unbroken short neurites (Fig. 6 A). Neurons grown without neurotrophins retained this appearance throughout the period of the study (up to 5 d in vitro). However, neurite outgrowth could be stimulated from these neurons after this prolonged period of culture without neurotrophins by adding NGF to the culture medium (unpublished data). This indicates that although NGF is not required for survival, it is capable of promoting neurite outgrowth from these neurons. A similar phenomenon has been observed for neonatal Bax-deficient sympathetic neurons that survive in culture without NGF, but display very poor neurite outgrowth unless NGF is added to the medium (Deckwerth et al., 1996).

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