Apoptosis is an important mechanism of cell death in embryonic and adult brain. The CASPASEs are a family of cysteine proteases (at least 12 have been identified) homologous to the gene product of ced-3 in the nematode c-elegans. These 12 members of the interleukin-1 converting enzyme (ICE) family can be classified into 3 subfamilies according to their amino acid sequence. CASPASE 1 (ICE) and CASPASE 3 (CPP32) are prototypical family members for ICE subfamily and CPP32 subfamily, respectively, which cleave endogenously expressed proteins in a cascade leading to apoptotic cell death. Histochemical (TUNEL staining) and biochemical (DNA laddering) changes consistent with apoptosis have been reported in cerebral ischemia. The investigators have obtained preliminary evidence showing that cysteine protease inhibitors decrease tissue injury and enhance functional recovery in brain models of reversible or permanent ischemia as well as in delayed onset ischemic cell death.
Four specific aims propose to expand these novel findings and examine the hypothesis that CASPASEs are major mediators of ischemic cell death and that deficiency or blockade of CASPASE activation decreases neurological dysfunction. First the investigators propose to characterize transcriptional and translational changes both spatially and temporally in ICE and CPP32 in each ischemic model. Then they will explore mechanisms of drug action by examining CASPASE activities, the appearance of cleavage and protein degradation products after treatment. It is proposed to combine molecular (in situ hybridization, Northern blot, Western immunoblots), biochemical (ELISA, fluorogenic assays) and histochemical tools (immunohistochemistry) with well characterized models of cerebral ischemia to determine the therapeutic potential of CASPASE inhibitors. Mutant mice deficient in gene expression of either ICE or CPP32 will be used to test the hypothesis that ICE and CPP32 deficiency reduce ischemic injury. The investigators will also administer cysteine protease inhibitors to these mutants to determine specificity of drug action as well as to define more precisely involvement of ICE and CPP32 in ischemic cell death. By so doing, these experiments are intended to explore mechanisms and treatment strategies related to cell death during cerebral ischemia.
