Abstracts

Seizure-induced neuronal death is morphologically necrotic and caspase-independent, despite internucleosomal DNA cleavage (DNA laddering), a programmed process. We studied whether seizure-induced neuronal necrosis involves translocation of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (endoG) to the nucleus and cytochrome c (cyt c) to the cytoplasm, where AIF and endoG could be involved in large-scale (50 kb) and internucleosomal DNA fragmentation respectively. In caspase-independent, excitotoxic neuronal death, cyt c translocation from mitochondria to cytoplasm may occur because of non-specific outer membrane rupture from mitochondrial swelling., Adult Wistar rats were subjected to 3-h lithium-pilocarpine-induced status epilepticus (LPCSE), after which seizures were stopped with diazepam and phenobarbital. Six and 24 h later rats underwent in situ transcardiac perfusion-fixation of their brains, with subsequent removal and processing for H & E staining, TUNEL and AIF, endoG and cyt c immunoreactivity (n=3 in each group). Twenty-six brain regions were assessed for the degree of neuronal death, using H & E and TUNEL, and for translocation of AIF, endoG and cyt c, by brightfield immunohistochemical and immunofluorescent light-microscopic examination of the piriform cortex, which is one of the most damaged regions in the brain., The extent of neuronal necrosis and TUNEL positivity was similar to what we have described previously. Twenty-four h after SE, 19 of 26 brain regions had significant numbers of acidophilic neurons by H & E staining, and 10 of 26 regions had TUNEL-positive necrotic neurons. In the piriform cortical neurons of control rats AIF, endoG and cyt c appeared in a punctate distribution in the cytoplasm. Unexpectedly, 6 and 24 h after SE, cyt c, as well as AIF and endoG, had translocated to the pyknotic nuclei of necrotic neurons, and there was diffuse staining in the cytoplasm. Thus, in addition to the nuclear translocation of lysosomal cathepsin B and DNase II, which we reported last year, nuclear translocation of mitochondrial AIF, endoG and cyt c also occurred., Nuclear AIF and endoG could contribute to DNA fragmentation. Nuclear translocation of cyt c was unexpected, and has only been reported previously in HeLa cells in culture exposed to apoptotic stimuli, where it was associated with release of acetylated histone H2A from nucleus to cytoplasm and chromatin condensation. We are currently investigating earlier time points to determine when nuclear translocation first appears. Our results suggest that caspase-independent programmed mechanisms contribute to seizure-induced neuronal necrosis., (Supported by Department of Veterans Affairs.)