Abstract
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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphate / pharmacology
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Animals
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Apoptosis / drug effects
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Apoptosis / physiology*
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Atractyloside / pharmacology
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Caspase 3
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Caspases*
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Cell Extracts / pharmacology
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Cell-Free System
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Cells, Cultured
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Cerebellum / cytology*
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Cysteine Endopeptidases / metabolism
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Cytochrome c Group / pharmacology
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Enzyme Inhibitors / pharmacology
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Enzyme Precursors / metabolism
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Estrogen Antagonists / pharmacology
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Intercellular Signaling Peptides and Proteins
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Mitochondria / enzymology*
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Molecular Sequence Data
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Neurons / cytology*
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Neurons / enzymology
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Neurons / ultrastructure
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Peptides
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Proto-Oncogene Proteins c-bcl-2 / biosynthesis
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Rats
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Rats, Sprague-Dawley
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Sequence Homology, Amino Acid
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Staurosporine / pharmacology
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Tamoxifen / pharmacology
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Wasp Venoms / pharmacology
Substances
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Cell Extracts
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Cytochrome c Group
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Enzyme Inhibitors
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Enzyme Precursors
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Estrogen Antagonists
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Intercellular Signaling Peptides and Proteins
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Peptides
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Proto-Oncogene Proteins c-bcl-2
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Wasp Venoms
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Tamoxifen
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Atractyloside
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mastoparan
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Adenosine Triphosphate
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Casp3 protein, rat
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Caspase 3
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Caspases
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Cysteine Endopeptidases
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Staurosporine