Detection of caspase-9 activation in the cell death of the Bcl-x-deficientmouse embryo nervous system by cleavage sites-directed antisera

Caspases, which play crucial roles during apoptosis, are activated from the ir inactive proforms in a sequential cascade of cleavage by other members o f 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 ant iapoptotic member of the Bcl-2 family, prevents activation of caspases in v itro. Little is known, however, about the relation between caspase-9 and Bc l-xL during development of the mammalian nervous system. We used antisera a gainst two cleavage sites in mouse caspase-9 that recognize only the activa ted form of mouse caspase-9, and we examined immunohistochemically the acti vation of mouse caspase-9 in the nervous system of Bcl-x-deficient mouse em bryos. Mouse caspase-9 is processed at both D-353 D-368, but if is processe d preferentially at D-368 during apoptosis of cultured cells induced by var ious 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 ap optosis in the caudal portion of the ventral hindbrain, anterior horn cells , and dorsal root ganglia neurons of the normal mouse embryos and against c aspase-9/caspase-3-independent apoptosis in the dorsal region of the nervou s system including the dorsal spinal cord. Furthermore, we demonstrate that Bcl-xL blocks cytochrome c release from mitochondria, causing activation o f caspase-9 in anterior horn cells and dorsal root ganglia neurons in mouse embryos at embryonic day 11.5. (C) 2000 Elsevier Science BN. All rights re served.