Increased accumulation of the amyloid-l_ peptide (A[_) or specific isoforms (AI342) is a major pathogenic event underlying neurodegeneration in all forms of Alzheimer's disease (AD). In the first four years of this project, we have focused on the role of presenilin (PS) FAD mutations in apoptosis, and how apoptosis influences AI_ production. However, evidence has mounted to suggest that while apoptosis-induced AI_ generation may occur following acute injury to the CNS, other pathogenic mechanisms are likely involved in A[3 production owing to familial AD mutations in APP and the PS genes. Cloning of the PS genes has led to the initial characterization of the protease called y-secretase that cleaves APP at the C-terminal end of AI3. y-secretase is a heteromeric complex of proteins, in which only two components have been identified to date, PS and nicastrin. FAD mutations in PSI increase the ratio of A[_az:Alg40 and are likely to involve a number of as of yet unidentified proteins in the y-secretase/PS1. Thus, in the coming funding period, we propose to expand upon specific aim 3 of the original application, by exploring how the y-secretase complex/PSI and FAD mutations in PS lead to alterations in the maturation and processing of APP and affect AI3 production. In our preliminary data, we show that nicastrin and thirteen unknown proteins co- immunoprecipitate with PS1 C- and N-terminal fragments from a sodium carbonate-washed lysate, thereby representing potentially novel membrane-associated components and/or substrates of the 7-secretase/PS1 complex. We have also identified a subcellular fraction in the Golgi/endosomes harboring the complex, together with its APP C-terminal substrates. We have tentatively already identified one of the unknown protein bands in the complex. To follow up on these findings and extend our studies of the effect of FAD presenilin mutations on y-secretase activity, we propose to identify and characterize novel components of the y-secretase_S 1 complex, especially those that modulate AI3 production and the A1342/A13tot,riatio. We will also test polymorphisms in genes that encode novel components of the y-secretase/PS1 complex, for family-based association with AD. We plan to determine the subcellular localization and elucidate the physiological functions of the y-secretase/PS1 complex. Finally, we are performing in vitro y-secretase assays and reconstituting the isolated complex into unilamellar liposomes to study its activity in vitro. The overall goal of these studies is to define the pathogenetic mechanism by which more than 100 FAD mutations in PS affect At3 generation, and to ultimately identify potential targets for reducing A[3 generation in AD. PLHEORMANCE SITE(S) (orgamzatton, ctty, state) > Massachusetts General Hospital Genetics and Aging Research Unit Building 114, 16th Street Charlestown, MA 02129 KEY PERSONNEL. See instructions. Use continuation pages as neededto provide _e required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name Organization Role on Project Rudolph E. Tanzi, Ph.D. Massachusetts General Hospital P.I. Dora M. Kovacs, Ph.D. Massachusetts General Hospital Co-P.I. Sangram S. Sisodia, Ph.D. University of Chicago Collaborator Michael Przybylski, Ph.D. University of Konstanz Collaborator Matthew Frosch, M.D., Ph.D. Harvard Medical School Collaborator Gregory J. Hannon, Ph.D. Cold Spring Harbor Laboratory Collaborator Weiming Xia, Ph.D. Harvard Medical School Collatorator PHS 398 (Rev. 05/01) Page 2 Form Page 2 Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. Principal Investigator/Program Director (Last, first, middle): Tanzi, Rudolph E. > The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. Type density and size must conform to limits and specifications provided in the PHS 398 Instructions. RESEARCH GRANT TABLE OF CONTENTS Page Numbe_ Face Page ....................................................................................................... Description,