Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production.

Alzheimer's disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds - identified here using cells and tissues expressing wt human APP - in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects.

The small co-chaperone p23 overexpressing transgenic mouse.

Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we described the role of p23 a small co-chaperone protein in preventing ER stress-induced cell death. p23 is cleaved by caspases at D142 to yield p19 (a 19 kDa product) during ER stress-induced cell death. Mutation of the caspase cleavage site not only blocks formation of the 19 kDa product but also attenuates the cell death process triggered by various ER stressors. Thus, uncleavable p23 (p23D142N) emerges as a reasonable candidate to test for potential inhibition of neurodegenerative disease phenotype that features misfolded proteins and ER stress. In the present work we report the generation of transgenic mouse lines that overexpress wild-type p23 or uncleavable p23 under the control of a ROSA promoter. These mice should prove useful for studying the role of p23 and/or uncleavable p23 in cellular stress-induced cell death.