The amyloid peptide (Abeta) is the major constituent of senile plaques, a lesion characteristic of Alzheimer's disease (AD). Abeta is derived from the amyloid precursor protein (APP) by posttranslational proteolysis. BACE cleaves APP near its transmembrane region, generating the N-terminus of the Abeta peptide. Subsequently, gamma-secretase cleaves APP within its transmembrane domain, producing the C-terminus of Abeta which can be secreted from the cell or accumulate intracellularly. Alterations in the amount or type of Abeta produced are thought to be an important factor in the development of AD. The overall goal of Project 1 is to characterize cellular factors that influence both the amount and type of Abeta that is produced, concentrating on beta-site APP cleaving enzyme (BACE) and several newly discovered proteins that form part of the gamma-secretase complex. Two major N-terminal species of Abeta are generated by cleavage of APP by BACE, resulting in peptides that begin at positions Asp1 or Glu11. While Abeta11 species aggregate readily in vitro and are found in the brains of AD patients, their contributions to AD pathogenesis are not well understood. Project 1 will address the mechanisms that regulate N-terminal heterogeniety of Abeta in vitro, while Project 2 will investigate the ability of Abeta11/40-42 to from senile plaques in animal models. Abeta also exhibits heterogeniety at its C-terminus, with the most common Abeta species ending at either position 40 or 42. Abetax-42 aggregates more readily than Abetax-40, and increased Abeta42 production relative to Abeta40 has been linked to specific mutations in either APP or presenilin (PS) that result in familial Alzheimer's disease. Thus, factors that influence not just the efficiency of C-terminal cleavage, but also the precise location of cleavage could have profound effects on Abeta production and disease progression. At present, 3 proteins have been identified that, with PS, form the gamma-secretase complex, including two proteins that have been identified in the last several months: APH-1, a protein that probably spans the membrane multiple times, Pen-2, a protein that has two putative transmembrane domains, and Nicastrin, a type 1 membrane protein shown to interact with PS1 several years ago. We will study the cell biology of nicastrin, Pen-2, and APH-1, characterizing their membrane topology, posttranslational processing, the mechanisms by which they associate with each other or with PS, and their impact on the amount and type of Abeta produced at different subcellular locations.