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 hav e developed a cell-free model of neuronal apoptosis. The model faithfu lly reproduces key elements of apoptosis, including chromatin condensa tion, DNA fragmentation, caspase activation/processing, and selective substrate cleavage. We report that cell-free apoptosis is activated in premitochondrial, mitochondrial, and postmitochondrial phases by tamo xifen, mastoparan, and cytochrome c, respectively, allowing a function al ordering of these proapoptotic modulators. Furthermore, this is the first report of mitochondrial-mediated activation of cell-free apopto sis in a cell extract. Although Bcl-2 blocks activation at the premito chondrial and mitochondrial levels, it does not affect the postmitocho ndrial level. The cell-free system described here provides a valuable tool to elucidate the molecular events leading to neuronal cell death.