Presence of DNA fragmentation and lack of neuroprotective effect in DFP45 knockout mice subjected to traumatic brain injury

Background: Apoptosis plays an important pathophysiologic role in neuronal cell loss and associated neurologic deficits following traumatic brain inju ry (TBI). DNA fragmentation represents one of the characteristic biochemica l features of neuronal apoptosis and is observed after experimental TBI. DF F45 and DFF40 are essential for DNA fragmentation in various models of apop tosis. Materials and Methods: We used mice deficient in DFF45 and wild-type contro ls. Oligonucleosomal DNA fragmentation induced by TBI was analyzed using in vivo and in vitro assays. Expression and integrity of DFF45 and DFF40 prot eins was assessed by Western analysis. Other outcome measurements included neurologic scoring, learning/memory tests, lesion volume measurements (MRI) , and assessment of cell viability in vitro among others. Results: We compared the effects of controlled cortical impact (CCI) trauma in DFF45 knockout mice and wild-type controls. Analysis of TBI-induced DNA fragmentation in brain cortex from wild-type and DFF45 knockout mice indic ates that, although somewhat delayed, oligonucleosomal cleavage of DNA occu rs after TBI in DFF45 knockout mice. DFF45 knockouts showed no significant differences in behavioral outcomes or lesion volumes after TBI as compared to wild-type controls. Using an in vitro reconstitution system, we also dem onstrated that cleavage of DFF45 by caspase-3 is not sufficient for DNA fra gmentation induced by protein extracts from rat brain cortex. We found that endonuclease activity induced in rat brain cortex following TBI depends on the presence of Mg2+ and Ca2+, but is not inhibited by Zn2+. Primary neuro nal cultures from DFF45 knockouts failed to show DNA laddering in response to staurosporine, but did show prominent, albeit delayed, DNA fragmentation following treatment with etoposide. In contrast, primary neurons from wild type animals demonstrated marked DNA fragmentation following treatment with staurosporine or etoposide. Conclusions: The results of this study suggest that, in addition to DFF45/4 0, other endonucleases may be essential for chromatin degradation during ne uronal apoptosis in adult brain after TBI.