Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial, and postmitochondrial phases.
Apoptosis is a fundamental process required for normal development of the nervous system and is triggered during neurodegenerative disease. To dissect the molecular events leading to neuronal cell death, we have developed a cell-free model of neuronal apoptosis. The model faithfully reproduces key elements of apoptosis, including chromatin condensation, DNA fragmentation, caspase activation/processing, and selective substrate cleavage. We report that cell-free apoptosis is activated in premitochondrial, mitochondrial, and postmitochondrial phases by tamoxifen, mastoparan, and cytochrome c, respectively, allowing a functional ordering of these proapoptotic modulators. Furthermore, this is the first report of mitochondrial-mediated activation of cell-free apoptosis in a cell extract. Although Bcl-2 blocks activation at the premitochondrial and mitochondrial levels, it does not affect the postmitochondrial level. The cell-free system described here provides a valuable tool to elucidate the molecular events leading to neuronal cell death.
Ellerby
H M
HM
The Burnham Institute, La Jolla Cancer Research Center, La Jolla, California 92037, USA.
Martin
S J
SJ
Ellerby
L M
LM
Naiem
S S
SS
Rabizadeh
S
S
Salvesen
G S
GS
Casiano
C A
CA
Cashman
N R
NR
Green
D R
DR
Bredesen
D E
DE
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001278:Atractyloside / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases; D002457:Cell Extracts / Q000494:pharmacology; D002474:Cell-Free System; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003574:Cytochrome c Group / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism; D004965:Estrogen Antagonists / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D019311:Staurosporine / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology; D014862:Wasp Venoms / Q000494:pharmacology
D002457:Cell Extracts; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D004965:Estrogen Antagonists; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D014862:Wasp Venoms; D013629:Tamoxifen; D001278:Atractyloside; C037500:mastoparan; D000255:Adenosine Triphosphate; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D019311:Staurosporine