In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system.
Activation of caspase-3 requires proteolytic processing of the inactive zymogen into p18 and p12 subunits. We generated a rabbit polyclonal antiserum, CM1, which recognizes the p18 subunit of cleaved caspase-3 but not the zymogen. CM1 demonstrated an apparent specificity for activated caspase-3 by specifically immunolabelling only apoptotic but not necrotic cortical neurons in vitro. In the embryonic mouse nervous system, CM1 immunoreactivity was detected in neurons undergoing programmed cell death and was markedly increased in Bcl-xL-deficient embryos and decreased in Bax-deficient embryos. CM1 immunoreactivity was absent in the nervous system of caspase-3-deficient mouse embryos and in neurons cultured from caspase-3-deficient mice. Along with neuronal somata, extensive neuritic staining was seen in apoptotic neurons. These studies indicate that caspase-3 is activated during apoptosis in the developing nervous system in vivo and that CM1 is a useful reagent for its in situ detection.
Srinivasan
A
A
IDUN Pharmaceuticals, Inc., La Jolla, California 92037, USA. anu@idun.com
Roth
K A
KA
Sayers
R O
RO
Shindler
K S
KS
Wong
A M
AM
Fritz
L C
LC
Tomaselli
K J
KJ
D000818:Animals; D000918:Antibody Specificity; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000201:enzymology / Q000254:growth & development; D053148:Caspase 3; D020169:Caspases / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D002540:Cerebral Cortex / Q000166:cytology; D003374:Coumarins / Q000494:pharmacology; D003429:Cross Reactions; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003561:Cytarabine / Q000494:pharmacology; D004792:Enzyme Precursors / Q000032:analysis / Q000276:immunology; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D019169:Jurkat Cells; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D009842:Oligopeptides / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D011817:Rabbits; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C112287:Ac-aspartyl-glutamyl-valyl-aspartyl-aminomethylcoumarin; D000964:Antimetabolites, Antineoplastic; C494826:BAX protein, human; C494811:BCL2L1 protein, human; C494827:Bax protein, mouse; C494812:Bcl2l1 protein, mouse; D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D003561:Cytarabine; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases