Nuclear translocation of mitochondrial cytochrome c, lysosomal cathepsins B and D, and three other death-promoting proteins within the first 60 minutes of generalized seizures.
We have shown that generalized seizures produce necrotic neurons with caspase-independent nuclear pyknosis and DNA fragmentation. In this study, we determined the time course of translocation of mitochondrial cytochrome c, apoptosis-inducing factor, endonuclease G, lysosomal cathepsins B and D, and DNase II with respect to signs of irreversible neuronal damage. Adult male Wistar rats underwent lithium-pilocarpine-induced seizures lasting for 60 min, 3 hr, and 3 hr with 6- or 24-hr survival periods, after which the brains were prepared for immunofluorescence microscopic examination of piriform cortex. Contrary to expectation, cytochrome c and cathepsins B and D translocated to neuronal nuclei with DNase II, endonuclease G, and apoptosis-inducing factor within 60 min of seizure onset and persisted for 24 hr after 3-hr seizures. After 60-min seizures, some neurons showed translocation of the death-promoting proteins in normal-appearing neurons, prior to their appearance in irreversibly damaged neurons. Western blots of subcellular fractions of cytochrome c and cathepsins B and D confirmed their nuclear translocation. This is the first evidence of nuclear translocation of cathepsins B and D and the first in vivo evidence of nuclear translocation of cytochrome c. The appearance of these mitochondrial proteins and lysosomal enzymes before signs of irreversible neuronal death suggests that they could contribute to seizure-induced nuclear pyknosis and DNA fragmentation.
Zhao
Shuangping
S
Experimental Neurology Laboratory, VA Greater Los Angeles Healthcare System, North Hills, CA 91343, USA.
Aviles
Ernesto R
ER
Fujikawa
Denson G
DG
D000704:Analysis of Variance; D000818:Animals; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D004195:Disease Models, Animal; D004569:Electroencephalography / Q000379:methods; D004829:Epilepsy, Generalized / Q000139:chemically induced / Q000473:pathology*; D004852:Epoxy Compounds / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D008094:Lithium; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure*; D010862:Pilocarpine; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D051033:Apoptosis Inducing Factor; D051017:Apoptosis Regulatory Proteins; D004852:Epoxy Compounds; C092940:cathestatin B; D010862:Pilocarpine; D014443:Tyrosine; D045304:Cytochromes c; D008094:Lithium; D003851:Deoxyribonucleases; D006026:Glycoside Hydrolases; C090534:endo-alpha-sialidase; D002402:Cathepsin D