Detection of caspase-9 activation in the cell death of the Bcl-x-deficient mouse embryo nervous system by cleavage sites-directed antisera.
Caspases, which play crucial roles during apoptosis, are activated from their inactive proforms in a sequential cascade of cleavage by other members of the caspase family. Caspase-9 is autoprocessed by the Apaf-1/cytochrome c pathway and acts at an early point in this cascade, whereas Bcl-xL, an antiapoptotic member of the Bcl-2 family, prevents activation of caspases in vitro. Little is known, however, about the relation between caspase-9 and Bcl-xL during development of the mammalian nervous system. We used antisera against two cleavage sites in mouse caspase-9 that recognize only the activated form of mouse caspase-9, and we examined immunohistochemically the activation of mouse caspase-9 in the nervous system of Bcl-x-deficient mouse embryos. Mouse caspase-9 is processed at both D(353) and D(368), but it is processed preferentially at D(368) during apoptosis of cultured cells induced by various stimuli and in the nervous system of Bcl-x-deficient mouse embryos. We show that Bcl-xL protects against caspase-9- and/or caspase-3-dependent apoptosis in the caudal portion of the ventral hindbrain, anterior horn cells, and dorsal root ganglia neurons of the normal mouse embryos and against caspase-9/caspase-3-independent apoptosis in the dorsal region of the nervous system including the dorsal spinal cord. Furthermore, we demonstrate that Bcl-xL blocks cytochrome c release from mitochondria, causing activation of caspase-9 in anterior horn cells and dorsal root ganglia neurons in mouse embryos at embryonic day 11.5.
Fujita
E
E
Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan.
Urase
K
K
Egashira
J
J
Miho
Y
Y
Isahara
K
K
Uchiyama
Y
Y
Isoai
A
A
Kumagai
H
H
Kuida
K
K
Motoyama
N
N
Momoi
T
T
D000818:Animals; D019556:COS Cells; D053453:Caspase 9; D020169:Caspases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002417:Cattle / Q000097:blood; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D004622:Embryo, Mammalian / Q000201:enzymology; D004789:Enzyme Activation / Q000502:physiology; D004847:Epithelial Cells / Q000201:enzymology; D005312:Fetal Blood; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D005727:Ganglia, Spinal / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D020287:In Situ Nick-End Labeling; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics; D008817:Mice, Mutant Strains / Q000235:genetics; D009420:Nervous System / Q000166:cytology / Q000196:embryology*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000172:deficiency* / Q000235:genetics; D012249:Rhombencephalon / Q000378:metabolism; D051020:bcl-X Protein
C494812:Bcl2l1 protein, mouse; D003574:Cytochrome c Group; D007106:Immune Sera; D010446:Peptide Fragments; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D016222:Fibroblast Growth Factor 2; C506434:Casp9 protein, mouse; D053453:Caspase 9; D020169:Caspases