Abstract
Using a well documented ex vivo system consisting of rodent cerebellar granule cells (CGCs) the activation of caspases 3 and 6 during apoptosis induced by withdrawal of trophic support was analyzed. At the time of deprivation, the addition of the irreversible, broad-spectrum caspase inhibitor zVADfmk or the cell permeable, caspase 6 inhibitor CP-VEID-cho can transiently suppress the appearance of apoptosis, including the early appearance of DNA fragmentation. Using immunoblotting and fluorogenic peptide assays we observe deprivation-induced activation of caspases 3 and 6, but not caspase 9. Furthermore, active caspase 6 is capable of processing and activating procaspase 3 in cellular extracts prepared from non-apoptotic CGCs, whereas caspase 3 failed to activate caspase 6. In consonant with this, the cell permeable caspase 6 inhibitor prevented deprivation-induced caspase 3 activation whereas a cell permeable caspase 3 inhibitor, CP-DEVD-cho, had no effect on caspase 6 activation. This would indicate that caspase 6 is a significant inducer of the early caspase 3 activity in apoptotic CGCs.
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