Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNAfragmentation in transient cerebral ischemia and in neuronal cultures

Nuclear changes, including internucleosomal DNA fragmentation, are characte ristic features of neuronal apoptosis resulting from transient cerebral isc hemia and related brain insults for which the molecular mechanism has not b een elucidated. Recent studies suggest that a caspase-3-mediated mechanism may be involved in the process of nuclear degradation in ischemic neurons. In this study, we cloned from rat brain a homolog cDNA encoding caspase-act ivated deoxyribonuclease (CAD)/DNA fragmentation factor 40 (DFF40), a 40 kD a nuclear enzyme that is activated by caspase-3 and promotes apoptotic DNA degradation. Subsequently, we investigated the role of CAD/DFF40 in the ind uction of internucleosomal DNA fragmentation in the hippocampus in a rat mo del of transient global ischemia and in primary neuronal cultures under isc hemia-like conditions. At 8-72 hr after ischemia, CAD/DFF40 mRNA and protei n were induced in the degenerating hippocampal CA1 neurons. CAD/DFF40 forme d a heterodimeric complex in the nucleus with its natural inhibitor CAD (IC AD) and was activated after ischemia in a delayed manner (>24 hr) by caspas e-3, which translocated into the nucleus and cleaved ICAD. Furthermore, an induced CAD/DFF40 activity was detected in nuclear extracts in both in vivo and in vitro models, and the DNA degradation activity of CAD/DFF40 was inh ibited by purified ICAD protein. These results strongly suggest that CAD/DF F40 is the endogenous endonuclease that mediates caspase-3-dependent intern ucleosomal DNA degradation and related nuclear alterations in ischemic neur ons.