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 e...

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Bibliographic Details
Published in:The Journal of neuroscience 1997-08, Vol.17 (16), p.6165-6178
Main Authors: Ellerby, H. Michael, Martin, Seamus J, Ellerby, Lisa M, Naiem, Shahrouz S, Rabizadeh, Shahrooz, Salvesen, Guy S, Casiano, Carlos A, Cashman, Neil R, Green, Douglas R, Bredesen, Dale E
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - pharmacology
Animals
Apoptosis - drug effects
Apoptosis - physiology
Atractyloside - pharmacology
Caspase 3
Caspases
Cell Extracts - pharmacology
Cell-Free System
Cells, Cultured
Cerebellum - cytology
Cysteine Endopeptidases - metabolism
Cytochrome c Group - pharmacology
Enzyme Inhibitors - pharmacology
Enzyme Precursors - metabolism
Estrogen Antagonists - pharmacology
Mitochondria - enzymology
Molecular Sequence Data
Neurons - cytology
Neurons - enzymology
Neurons - ultrastructure
Peptides
Proto-Oncogene Proteins c-bcl-2 - biosynthesis
Rats
Rats, Sprague-Dawley
Sequence Homology, Amino Acid
Staurosporine - pharmacology
Tamoxifen - pharmacology
Wasp Venoms - pharmacology
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Summary: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.
ISSN:0270-6474
1529-2401
DOI:10.1523/jneurosci.17-16-06165.1997