Abstracts

Rationale: We have shown that generalized seizures trigger mitochondrial and lysosomal membrane permeabilization, allowing escape of death-promoting proteins/enzymes to cytosol and nuclei following 3-h seizures. Mitochondrial cytochrome c (cyt c) unexpectedly translocated not only to cytosol, but also to nuclei, along with apoptosis-inducing factor (AIF) and endonuclease G (endoG). In this study we investigated how soon after seizure onset that both translocation of cyt c, AIF and endoG, and lysosomal cathepsins B and D and DNase II, occur.Methods: Adult Wistar rats underwent 60-min and 3-h lithium-pilocarpine-induced status epilepticus (LPCSE), at the end of which they had either in situ transcardiac perfusion-fixation of their brains, with subsequent removal and processing for H & E staining, and cyt c, AIF, endoG, cathepsins B and D and DNase II immunoreactivity (n=3 in each group), or their brains were removed and the amygdala-piriform cortex regions were dissected in one block rapidly on ice, snap-frozen in liquid N2 and processed for subcellular fractionation and western blotting (n=4 for each western blot).Results: In the piriform cortex and amygdala of the 60-min seizure group, H & E staining showed scattered dark, basophilic neurons, some of normal size and some beginning to shrink, in the piriform cortex and amygdala, but no shrunken acidophilic neurons, a sign of irreversible neuronal necrosis. Following 3-h seizures, shrunken acidophilic neurons began to appear. In the piriform cortical and amygdalar neurons of control rats, cyt c, AIF, endoG, cathepsins B and D and DNase II appeared in a punctate distribution in the cytoplasm. However, 3 h and 60 min after seizure onset, all 6 proteins/enzymes had translocated to many neuronal nuclei. At 60 min, the translocation was seen in some of the neurons prior to cellular shrinkage and nuclear pyknosis. The western blots confirmed the nuclear translocation of the mitochondrial proteins, beginning 60 min after seizure onset (results for the lysosomal enzymes are pending).Conclusions: This is the first evidence of nuclear translocation of mitochondrial cyt c, AIF and endoG, as well as lysosomal cathepsins B and D and DNase II, before clear signs of irreversible neuronal death, and suggests that these proteins/enzymes could contribute to the chromatin condensation and DNA fragmentation that occur following 3-h LPCSE-induced neuronal necrosis.