UNIVERSITY OF CHICAGO, THE
The ultimate goal of this project is to understand the structure of intermediates of fibrillogenesis, focusing on ¿-amyloid (A¿) of Alzheimer's Disease (AD). The specific aims of this proposal are as follows: 1. To examine, by solid-state NMR, A¿1-40 fibrils seeded by brain amyloid from patients who died with Alzheimer's Disease, i.e., fibrils seeded by ex mortuo brain amyloid. A¿1-40 can adopt more than one stable conformation in fibrils, and these conformations can be passed on to progeny fibrils. But which of the known conformations, if any, is adopted by A¿1-40 in the brains of patients with AD? To address this question, we purified brain amyloid from AD patients, and used this material to seed fibril formation by labeled A¿1-40. Exciting new data, resulting in a paper, indicate that brain-seeded fibrils have a novel structure, distinct from either of the known A¿1-40 fibril structures. To address the question of fibril heterogeneity and disease phenotype, we will also compare ex mortuo seeded fibrils formed using material from different anatomic locations of the brain, and from different patients. 2. To examine mutations of the A¿ peptide occurring adjacent to the "bend region". Of the point mutations (associated with familial AD) within the A¿ sequence itself, most are at positions 21-23, adjacent to the bend region that is essential for association of the two ¿-sheets of wild-type A¿ in fibrils. New solid-state NMR and other data on the Iowa mutant, D23N-A¿, reported in a paper, indicate that fibrils of this peptide have an unprecedented structure for full length A¿ fibrils, antiparallel ¿-sheets. In addition, new data on the Japanese mutation, ?E22-A¿ indicate that, contrary to published reports, it readily and abundantly makes ¿-sheet fibrils, and studies on these are in progress. 3. To examine A¿1-40 peptides modified by lipidation and other covalent additions by solid-state NMR; to examine these, and wild type- and point mutant A¿ by solution NMR of encapsulated peptides. This aim complements aims 1 and 2. Subaim 3A is to study modified A¿1-40 to model peptides arising in oxidative stress of AD, using solid-state NMR techniques. Much of this sub-aim examines progeny fibrils formed from seeding solutions of A¿1-40 by lipid-modified A¿1-40. Subaim 3B is to study both unmodified A¿1-40 and A¿1-40 modified by lipids, as well as point mutant A¿1-40, using encapsulation methods. In general, modified and unmodified peptides are to be studied in parallel. This approach will allow us to observe A¿ oligomers and monomers by high resolution NMR methods. PUBLIC HEALTH RELEVANCE: Alzheimer's Disease is associated with deposits of ¿-amyloid into neuritic plaques, blood vessels and other parts of the brain. Through the use of solid-state NMR and other techniques, structural models have been developed for ¿-amyloid fibrils, which show a great deal of polymorphism. In this project, we will investigate, by solid-state and solution NMR (complemented with other techniques), brain-seeded ¿-amyloid fibrils, which differ structurally from fibrils made from purely synthetic ¿-amyloid; point mutations (associated with familial Alzheimer's Disease) in a critical region of the ¿-amyloid peptide that lead to structural alterations of soluble oligomers and fibrils; and lipidated ¿-amyloid peptides which model modifications that occur during the pathogenesis of Alzheimer's Disease through oxidative stress.