Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons.
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.
Orike
N
N
Department of Anatomy and Developmental Biology, Royal Free Hospital School of Medicine, London NW3 2PF, United Kingdom.
Middleton
G
G
Borthwick
E
E
Buchman
V
V
Cowen
T
T
Davies
A M
AM
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D061945:Caspase Inhibitors; D002470:Cell Survival; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008845:Microinjections; D008856:Microscopy, Fluorescence; D009025:Morpholines / Q000494:pharmacology; D009363:Neoplasm Proteins; D009414:Nerve Growth Factors / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D017783:Superior Cervical Ganglion / Q000166:cytology*; D000077191:Wortmannin
D000590:Amino Acid Chloromethyl Ketones; D000730:Androstadienes; D061945:Caspase Inhibitors; D002867:Chromones; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D009025:Morpholines; D009363:Neoplasm Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D000077191:Wortmannin; C411889:Sh3kbp1 protein, rat; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494920:Akt1 protein, rat