DESCRIPTION (From the applicant's abstract): Research proposed in this application seeks to continue the applicant's genetic and molecular studies on Presenilin function and its role in the intracellular trafficking and proteolytic processing of its substrate proteins. Mutant human Presenilins influence the proteolysis of amyloid precursor protein (APP), resulting in an accelerated accumulation of the neurotoxic amyloid peptides during Alzheimer's disease. In the model organisms Caenorhabditis and Drosophila, Presenilins are required for Notch/Lin-12 developmental signaling. Presenilins have recently been shown to regulate proteolytic processing events during Notch receptor maturation and signaling that may be analogous to the Presenilin-dependent cleavages of APP in Alzheimer's disease. Finally, Presenilins have also been implicated in the cellular response to apoptotic stimuli in both mammalian cells and Drosophila. Mosaic tissue studies will be performed in vivo using newly isolated Presenilin gene mutants. Preliminary experiments have revealed integrin-like phenotypes in the mutant tissue clones, prompting the applicants to analyze the role of Presenilin in integrin cleavage using the genetic and biochemical approaches that have been used previously to demonstrate the effects of Presenilin on Notch processing. These studies may reveal shared feature of Presenilin substrates and lead to a better understanding of the specific pathway of protein processing controlled by Presenilin. A central goal of this proposal is to develop an extensive collection of new molecular probes to dissect Notch processing at much higher resolution than is currently possible. These reagents, including new antibodies and epitope-tagged constructs that can discriminate among Notch cleavage products, will be combined with mutational and proteolysis inhibition studies to identify the biochemical steps of Notch processing that involve Presenilin. Genetic and molecular screens for Presenilin-interacting factors will also be performed, taking advantage of the applicant's recent finding that the conserved C-terminus of Presenilin is a crucial functional domain. Finally, detailed parallel studies on the trafficking of Notch and other proteins will be undertaken in tissues lacking either Presenilin or another protein with known effect of subcellular trafficking, the SERCA-type Calcium-ATPase. These experiments are made possible by the applicant's recent isolation of Calcium-ATPase mutants, and they will address the unresolved issue of whether Presenilin is required for protein trafficking or only proteolysis. The studies proposed here will clarify the biochemical activity of Presenilin in the processing of Notch, APP and other proteins, and may ultimately increase our understanding of the molecular causes of Alzheimer's disease.