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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 after manipulating expression of Bcl-xL and Bcl-2. The neurons were injected with expression plasmids containing Bcl-xL and Bcl-2 cDNAs in the sense or antisense orientations, Bcl-xS cDNA in the sense orientation 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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fig8: Survival of adult sympathetic neurons after manipulating expression of Bcl-xL and Bcl-2. The neurons were injected with expression plasmids containing Bcl-xL and Bcl-2 cDNAs in the sense or antisense orientations, Bcl-xS cDNA in the sense orientation 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 members of the Bcl-2 family of proteins play key roles in controlling caspase activation, we carried out additional experiments to assess the importance of different members of this family in regulating adult sympathetic neuron survival. To ascertain the importance of antiapoptotic members of the Bcl-2 family in sustaining the survival of adult SCG neurons, we injected plasmids expressing specific antisense RNAs to interfere with the synthesis of these proteins. We also injected separate sets of neurons with an empty expression plasmid to control for the injection procedure and studied the effect of overexpressing antiapoptotic proteins. There was no significant difference in the survival of control-injected neurons compared with uninjected neurons (unpublished data), indicating that the microinjection procedure did not affect neuronal survival. Overexpression of the antiapoptotic proteins Bcl-xL or Bcl-2 had little effect on survival. Whereas neurons overexpressing Bcl-xL survived slightly better than control-injected neurons, the survival of Bcl-2–overexpressing neurons was not significantly different from control-injected neurons (Fig. 8) . In contrast, injection of plasmids containing Bcl-xL or Bcl-2 in the reverse orientation caused a marked reduction in neuronal survival. Expression of antisense Bcl-xL had the most dramatic effect, with over three quarters of the neurons dying within the first 24 h after injection, and by 5 d, <10% of the neurons were surviving. Expression of antisense Bcl-2 reduced the number of surviving neurons to ∼25% after 5 d (Fig. 8). To confirm that adult SCG neurons normally express Bcl-xL protein and that expression of this protein is reduced by antisense Bcl-xL RNA, we used immunocytochemistry to study Bcl-xL expression after injection of the antisense Bcl-xL plasmid. Whereas control-injected neurons exhibited intense Bcl-xL immunofluorescence (Fig. 6 E), neurons injected with the antisense Bcl-xL plasmid were either unlabeled by the Bcl-xL antiserum or exhibited negligible Bcl-xL immunofluorescence 12 h after injection (Fig. 6 F). Taken together, these results suggest that the endogenous expression of Bcl-xL and, to a lesser, extent Bcl-2 is required for the survival of adult SCG neurons.


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 after manipulating expression of Bcl-xL and Bcl-2. The neurons were injected with expression plasmids containing Bcl-xL and Bcl-2 cDNAs in the sense or antisense orientations, Bcl-xS cDNA in the sense orientation 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

fig8: Survival of adult sympathetic neurons after manipulating expression of Bcl-xL and Bcl-2. The neurons were injected with expression plasmids containing Bcl-xL and Bcl-2 cDNAs in the sense or antisense orientations, Bcl-xS cDNA in the sense orientation 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 members of the Bcl-2 family of proteins play key roles in controlling caspase activation, we carried out additional experiments to assess the importance of different members of this family in regulating adult sympathetic neuron survival. To ascertain the importance of antiapoptotic members of the Bcl-2 family in sustaining the survival of adult SCG neurons, we injected plasmids expressing specific antisense RNAs to interfere with the synthesis of these proteins. We also injected separate sets of neurons with an empty expression plasmid to control for the injection procedure and studied the effect of overexpressing antiapoptotic proteins. There was no significant difference in the survival of control-injected neurons compared with uninjected neurons (unpublished data), indicating that the microinjection procedure did not affect neuronal survival. Overexpression of the antiapoptotic proteins Bcl-xL or Bcl-2 had little effect on survival. Whereas neurons overexpressing Bcl-xL survived slightly better than control-injected neurons, the survival of Bcl-2–overexpressing neurons was not significantly different from control-injected neurons (Fig. 8) . In contrast, injection of plasmids containing Bcl-xL or Bcl-2 in the reverse orientation caused a marked reduction in neuronal survival. Expression of antisense Bcl-xL had the most dramatic effect, with over three quarters of the neurons dying within the first 24 h after injection, and by 5 d, <10% of the neurons were surviving. Expression of antisense Bcl-2 reduced the number of surviving neurons to ∼25% after 5 d (Fig. 8). To confirm that adult SCG neurons normally express Bcl-xL protein and that expression of this protein is reduced by antisense Bcl-xL RNA, we used immunocytochemistry to study Bcl-xL expression after injection of the antisense Bcl-xL plasmid. Whereas control-injected neurons exhibited intense Bcl-xL immunofluorescence (Fig. 6 E), neurons injected with the antisense Bcl-xL plasmid were either unlabeled by the Bcl-xL antiserum or exhibited negligible Bcl-xL immunofluorescence 12 h after injection (Fig. 6 F). Taken together, these results suggest that the endogenous expression of Bcl-xL and, to a lesser, extent Bcl-2 is required for the survival of adult SCG neurons.

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