The overall objective of this research proposal is the elucidation of the roles of the ICE family in the development of the nervous system, neuronal degenerative diseases and cerebral ischemia. In the nematode C. elegans the gene ced -3 is essential for cells to undergo programmed cell death. Interleukin-1b converting enzyme (ICE) has been identified as a vertebrate homologue of Ced-3. Expression of crmA, a specific inhibitor of ICE encoded by cowpox virus, inhibits neuronal cell death. Expression of a mutant ICE, in which the cysteine residue in the active site has been altered into a glycine residue, can also inhibit neuronal cell death. Expression of crmA and the mutant ICE prevents not only NGF-deprivation induced but also CNTF-deprivation induced neuronal cell death, while the latter is not inhibited by expression of bc1-2. The hypothesis is that activation of the ICE family is critical for neuronal cell death during development and under pathological conditions. To test this hypothesis, transgenic mice have been generated that express the mutant ICE under the control of neuronal specific enolase (NSE) promoter. A mouse model of stroke, middle cerebral artery (MCA) occlusion, was used to examine the resistance of ischemia injury offered by the mutant ICE. After 24 hrs MCA occlusion, the neuronal damages as measured by infarct sizes in the brains of mutant ICE transgenic mice are 47% smaller than that of wild type control. These experiments suggest that the ICE family plays a very important role in neuronal cell death induced by ischemia injury. The experiments proposed here are to determine the role and mechanism of the ICE family in neuronal cell death. The first Specific Aim is to continue the characterization of the mutant ICE transgenic mice by determining the expression patterns and levels of the mutant ICE, developmental neuronal cell death and resistance to ischemia and axotomy induced cell death in transient ischemia model and axotomy model. The second Specific Aim is to determine how the mutant ICE acts to inhibit apoptosis. The hypothesis is that the mutant ICE acts by inhibiting a member(s) of the ICE family or an activator of the ICE family. Immunoprecipitation and yeast two hybrid system will be used to characterize the proteins interacting with the mutant ICE. There are indications that multiple proteases are activated simultaneously during apoptosis and, thus, the mutant ICE may interact with a number of proteins. Additional previously unknown proteins that interact with the mutant ICE will be identified and purified to determine their sequence. The mechanism of inhibition will be determined using cotransfection and enzymatic assays. The third Specific Aim is to use cerebellum as a model system to determine the role and pathway of the ICE family in cerebella granular and Purkinje cell death and to examine whether inhibition of ICE may prevent developmental neuronal death and neuronal degeneration induced by the weaver and pcd mutation. The goal is to identify a genetic pathway of apoptosis in cerebellum, much like what has been described in C. elegans.
