Cytochrome c release and caspase-3 activation during colchicine-induced apoptosis of cerebellar granule cells

The microtubule-disrupting agent colchicine is known to be neurotoxic towar d certain neuronal populations including cerebellar granule cells (CGCs). I n this study we investigated the involvement of cytochrome c release and ca spase-3 activation during colchicine-induced CGC apoptosis. Treatment of ra t CGCs with 1 mu M colchicine (for up to 24 h) caused high molecular weight DNA fragmentation and nuclear condensation. An involvement of group II cas pases (which includes caspase-3) was demonstrated by the proteolytic degrad ation of poly(ADP-ribose) polymerase (PARP) after 18 h exposure to colchici ne. Colchicine induced a time-dependent increase in Ac-Asp-Glu-Val-Asp-alph a-(4-methyl-coumaryl-7-amide) (DEVD-MCA) cleavage activity in CGCs, which w as blocked with a specific, peptide-based, aldehyde inhibitor of group II c aspases, i.e. DEVD-CHO. We also observed a time-dependent proteolysis of ca spase-3 as judged by the appearance of p17 which is one of the subunits of active caspase-3. Activation of caspase-3 during colchicine-induced apoptos is may be mediated by cytochrome c since there was a close correlation betw een the time courses of cytochrome c release from the mitochondria and of c aspase-3 activation. Furthermore, colchicine-induced apoptosis, as assessed by propidium iodide visualization of the nuclei, could be blocked by the c aspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (O-methyl) fluoromethyl keto ne.