Oxidative stress, defined as an abnormal accumulation of reactive oxygen species (ROS), disrupts normal cell signaling pathways often resulting in cell death by apoptosis or necrosis. Calcium signaling is involved in pathways leading to oxidant induced cell death. In preliminary experiments, the investigator and his associates show that oxidant induced apoptosis results in a marked elevation of cyclic adenosine diphosphoribose (cADPR), which is a metabolite of NAD that elicits calcium mobilization. The elevation of cADPR is due to activation of a membrane cADPR synthase, linked to the activation of the mitochondrial permeability transition (PT), and causally related to increases in levels of intracellular free calcium. These data have led to the investigator's hypothesis, that cADPR functions in a signaling pathway that is activated by oxidant stress and leads to disruption of calcium homeostasis and to oxidant induced cell death. He will test this hypothesis in PC12 cells, which are widely used as a model system of neuronal cell death relating to oxidant toxicity. Studies will be conducted in PC12 cells overexpressing Bcl-2, a potent inhibitor of the mitochondrial PT and apoptosis, and in a control cell line containing only vector, to allow segregation of events upstream and downstream from the effector phase of apoptosis. The first specific aim is to identify the cADPR synthase(s) activated by oxidant stress. A molecular genetic approach will be used to determine if the target enzyme is one of two known cADPR synthases (CD38 or BST-l) or a novel enzyme. According to the investigator, identification of the synthase is a prerequisite to studies of the mechanism of oxidant induced activation of cADPR and evaluation of the therapeutic potential of this metabolism. The second specific aim will establish whether cADPR formation is obligatory in oxidant induced apoptosis and will identify specific functions of cADPR in cell death. PC12 cells will be stably transfected with inducible sense and antisense cDNA constructs encoding the cADPR synthase activated by oxidant stress. Enzyme depletion and overexpression will then be used to assess the role of cADPR synthase in the cell death pathway, as monitored by examination of specific biochemical markers of apoptosis. The third specific aim will determine if cADPR synthase activation is involved in initiating pathways of apoptosis specific to ROS, or whether it results from ROS generated in the effector phase of apoptosis. Initiators will include tumor necrosis factor alpha (TNF-(), anti-Fas antibodies, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Staurosporine, and serum withdrawal. The studies are designed to advance our understanding of mechanisms of cell death and provide potentially new targets for reduction of oxidant induced cell death that results from re-perfusion injury following vascular or cerebral ischemia.
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