Activation of apoptosis, specifically the activation of caspases, leading to neuronal cell death may account for neurodegeneration in Alzheimer's disease. Since caspase-2, an initiator caspase, can act as a trigger to send a cell into apoptosis, its activity is not only regulated at the protein level but also at the level of alternative pre-mRNA splicing. Specific protein binding to an intronic sequence element, In 100, is critical for proper splice site usage. In 100 acts as a decoy acceptor site that causes exon skipping. We propose to develop and validate reagents for a high throughput assay that will be used to screen a compound library for small molecules that will disrupt the specific protein binding to Ini 00. A reporter gene will be added to a caspase-2 minigene construct to allow a convenient and cost effective screening. The proper regulation of this caspase-2 reporter minigene will be determined by comparison to the endogenous gene assayed in the same cells. Cell lines expressing the caspase-2 minigenes constructed in this Phase I application will be used to screen a small molecule library in the Phase II study focused on the development of an anti-neurodegenerative therapeutic.
