The deposition of the amyloid-beta (Abeta) protein in neuritic plaques has been regarded as the pathological hallmark of Alzheimer's disease (AD). Abeta is generated through the proteolysis of amyloid precursor protein (APP) by sequential actions of beta- and gamma-secretases. Causative mutations linked to APP or presenilins (PS1 and PS2) found in familial AD patients all result in increased production of the more amyloidogenic 42-residue Abeta isoform (Abeta42) versus the soluble 40-residue isoform (Abeta40). Both beta- and gamma-secretases have thus become important therapeutic targets. The objectives of this proposed study are to identify genetic modifiers that can modulate the selective cleavage of APP by gamma-secretase, providing basis for the invention of novel therapeutics to Abeta-centered pathogenesis of AD. gamma-Secretase is of particular interest, because it catalyzes the final proteolytic cleavage of APP within its transmembrane domain, and its activity could define the ratio of Abeta40 to Abeta42. Recent evidence has suggested that PSs are aspartyl proteases with gamma-secretase activity and that the co-expression of PS along with nicastrin, Aph-1, and Pen-2 in yeast is sufficient to reconstitute gamma-secretase activity, unambiguously providing the direct evidence of the molecular constituents of gamma-secretase. Most importantly, nicastrin has been demonstrated as a receptor of APP and Notch to recruit these substrates into the active gamma-secretase complexes. We thus reason that the protein-protein interactions between gamma-secretase and its substrates could be subject to endogenous regulation mediated by intracellular signaling mediators. Thus, it is plausible that the identification of genetic modifiers of gamma-secretase would not only advance our understanding of the normal and pathological roles of this protease, but also lead to the discovery of new drug prototypes and targets. Our previous efforts, supported by a recent ADDF-Elan grant (#271214 Elan) have allowed us to establish a number of cell-based assays based on the principle of bioluminescence resonance energy transfer (BRET) technology. By screening a kinase/phosphatase shRNA subset library with our novel BRET assays, we have identified a number of effective genes that exhibit differential effects on PS1-C99 and PS1-N∆E interactions. Using independent C99- and N∆E-based γ-secretase assays, we further confirmed that, among these newly identified genetic modifiers of gamma-secretase, epidermal growth factor receptors (ErbBs) consistently exhibit the differential regulation in modulating gamma-cleavage of C99 and N∆E. We thus hypothesize that ErbB-elicited signaling could play a crucial role in the regulation of substrate selectivity of gamma-secretase and be superior pharmacological targets for anti-AD therapy. Our study will likely expedite the development of more selective therapeutics against AD with minimal side effects. Ultimately, our studies will establish the pivotal role of ErbB-mediated signaling in controling the substrate selectivity of gamma-secretase and pave the way for the development of selective chemical modifiers that can selectively block gamma-secretase-mediated cleavage of APP without tampering the cleavage of Notch and its other physiological substrates.