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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 expressing dominant negative Akt. The neurons were injected with an expression plasmid containing the kinase-inactive mutant of PKB/Akt 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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fig4: Survival of adult SCG neurons expressing dominant negative Akt. The neurons were injected with an expression plasmid containing the kinase-inactive mutant of PKB/Akt 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: Because the downstream effector of PI 3-kinase, Akt, plays a key role in mediating the survival response of neurons to neurotrophins (Crowder and Freeman, 1998; Vaillant et al., 1999), we investigated whether Akt activation is required for the survival of adult SCG neurons. Injection of an expression plasmid that directs the synthesis of a kinase-inactive mutant of Akt that acts as a dominant-negative protein (Virdee et al., 1999) caused the death of a substantial number of adult SCG neurons. Over 70% of these neurons had died 5 d after injection with the dominant-negative Akt expression plasmid compared with 20% of control-injected neurons (Fig. 4) . The magnitude of neuronal death effected by expression of dominant-negative Akt was similar to that caused by expression of Δp85 or overexpression of Rukl. These results suggest that Akt is the main downstream effector of PI 3-kinase in sustaining the survival of adult SCG neurons in the absence of added neurotrophic factors.


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 expressing dominant negative Akt. The neurons were injected with an expression plasmid containing the kinase-inactive mutant of PKB/Akt 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

fig4: Survival of adult SCG neurons expressing dominant negative Akt. The neurons were injected with an expression plasmid containing the kinase-inactive mutant of PKB/Akt 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: Because the downstream effector of PI 3-kinase, Akt, plays a key role in mediating the survival response of neurons to neurotrophins (Crowder and Freeman, 1998; Vaillant et al., 1999), we investigated whether Akt activation is required for the survival of adult SCG neurons. Injection of an expression plasmid that directs the synthesis of a kinase-inactive mutant of Akt that acts as a dominant-negative protein (Virdee et al., 1999) caused the death of a substantial number of adult SCG neurons. Over 70% of these neurons had died 5 d after injection with the dominant-negative Akt expression plasmid compared with 20% of control-injected neurons (Fig. 4) . The magnitude of neuronal death effected by expression of dominant-negative Akt was similar to that caused by expression of Δp85 or overexpression of Rukl. These results suggest that Akt is the main downstream effector of PI 3-kinase in sustaining the survival of adult SCG neurons in the absence of added neurotrophic factors.

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