Gamma-Secretase is the final enzymatic step generating Abeta via intramembranous cleavage of APP. The same enzymatic activity appears to be responsible for cleaving other substrates, including Notch. Presenilin, initially identified as a gene in which mutations account for the vast majority of early onset autosomal dominant AD, is a major component of gamma-secretase. Enzymatic activity also depends on nicastrin, Aph- 1, and pen-2. We propose a model in which gamma-secretase components assemble, interact with substrates at a docking site, then cleave and release substrates. To test this model, we have developed a novel morphological technique based on advanced fluorescent microscopy methods, Fluorescence Lifetime Imaging (FLIM), as well as molecular and biochemical assays of APP and Notch cleavage. FLIM allows us to examine protein-protein proximity in living cells. Already we have identified APP-PS1 and Notch-PS1 interactions that are present even when gamma-secretase inhibitors or dominant negative PS-1 mutations are used to block gamma-secretase activity, supporting a hypothesis that there is a non-catalytic docking site closely associated with gamma-secretase. Aim 1 focuses on understanding where in the cell interactions between gamma-secretase and APP and Notch occur, and examining in depth the molecular characterization of the putative docking site. Because FLIM provides pixel level resolution, we will test the hypothesis that Notch interacts with PS 1 after activation by ligand at or near the cell surface. In aim 2, we will test the hypothesis that substrates compete with one another, and that PS-1 mutations alter substrate interactions with gamma-secretase. Finally, in collaboration with Core C and Project 1, aim 3 outlines experiments targeted at additional gamma-secretase components (Nicastrin, Aph 1a and b, and pen-2) using RNAi, dominant negative, and over-expression approaches, to determine if genetic or pharmacologic manipulations alter gamma-secretase -substrate interactions. Taken together, our studies address 4 fundamental questions: the spatial paradox of where in the cell gamma-secretase and its substrates come together, whether there are distinct and separable docking and catalytic sites, where they are in the gamma-secretase complex, and whether substrates that give rise to novel signal transduction cascades compete with one another. Taken together, we will perform a detailed, integrated study of gamma-secretase-substrate interactions that may lead to new avenues for therapeutic interventions.