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.