The BH3-only protein Puma is both necessary and sufficient for neuronal apoptosis induced by DNA damage in sympathetic neurons.
DNA damage activates apoptosis in several neuronal populations and is an important component of neuropathological conditions. While it is well established that neuronal apoptosis, induced by DNA damage, is dependent on the key cell death regulators p53 and Bax, it is unknown which proteins link the p53 signal to Bax. Using rat sympathetic neurons as an in vitro model of neuronal apoptosis, we show that cytosine arabinoside is a DNA damaging drug that induces the expression of the BH3-only pro-apoptotic genes Noxa, Puma and Bim. Increased expression occurred after p53 activation, measured by its phosphorylation at serine 15, but prior to the conformational change of Bax at the mitochondria, cytochrome c (cyt c) release and apoptosis. Hence Noxa, Puma and Bim could potentially link p53 to Bax. We directly tested this hypothesis by the use of nullizygous mice. We show that Puma, but not Bim or Noxa, is a crucial mediator of DNA damage-induced neuronal apoptosis. Despite the powerful pro-apoptotic effects of overexpressed Puma in Bax-expressing neurons, Bax nullizygous neurons were resistant to Puma-induced death. Therefore, Puma provides the critical link between p53 and Bax, and is both necessary and sufficient to mediate DNA damage-induced apoptosis of sympathetic neurons.
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
Andreas
A
Wyttenbach
Aviva M
AM
Tolkovsky
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D003561:Cytarabine / Q000494:pharmacology; D004249:DNA Damage / Q000502:physiology*; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012694:Serine / Q000378:metabolism; D017783:Superior Cervical Ganglion / Q000166:cytology*; D013997:Time Factors; D014162:Transfection / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000172:deficiency / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C533952:Bbc3 protein, rat; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D003561:Cytarabine; D049452:Green Fluorescent Proteins; D012694:Serine; D020932:Nerve Growth Factor; C497208:Mdm2 protein, rat; D051736:Proto-Oncogene Proteins c-mdm2; D048052:Mitogen-Activated Protein Kinase 3