Structural Studies of Alzheimer's beta-Amyloid Fibrils

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Progress in FY2010 has been in the following areas: 1. AMYLOID FIBRIL STRUCTURES DERIVED FROM BRAIN TISSUE: We have developed a new protocol for partial purification of amyloid from brain tissue obtained at autopsy, and a new protocol for using this material as a "seed" for growing fibrils from synthetic, isotopically-labeled peptide. With the new protocols, we can create 1 mg fibril samples suitable for solid state NMR and electron microscopy studies, starting with 1 g of brain tissue, in a single fibril growth step. Applying this protocol to fronto-temporal lobe and occipital lobe tissue from a diseased Alzheimer's disease patient, we find that there is a single fibril structure in this tissue, a surprising result. Moreover, based on NMR chemical shifts, this structure differs from any structures we have examined previously. Thus, the possibility exists that this is a particularly neurotoxic structure, which may be a key to understanding the pathogenesis of AD. We are in the process of developing a molecular structural model for this brain-derived beta-amyloid fibril. 2. SURPRISING ANTIPARALLEL BETA-SHEET STRUCTURE IN MUTANT BETA-AMYLOID FIBRILS: In collaboration with S.C. Meredith, we have recently shown that the Asp23-to-Asn mutant of human beta-amyloid (D23N mutant, or Iowa mutant) is capable of forming amyloid fibrils that contain antiparallel beta-sheets. This is the first demonstration that a full-length peptide or protein could form fibrils that contain antiparallel (rather than parallel) beta-sheets. We have now explored fibril formation conditions that produce homogeneous D23N beta-amyloid fibrils. Extensive exploration of growth and seeding conditions indicates that the antiparallel D23N structure is metastable relative to parallel beta-sheet structures. Nonetheless, using a novel filtration purification protocol, we have isolated relatively pure preparations of antiparallel D23N fibrils and have performed measurements that will allow us to propose a specific molecular structural model. 3. KINETICS AND THERMODYNAMICS OF FIBRIL GROWTH: We have used atomic force microscopy to monitor the extension rates and shrinkage rates of both wild-type and D23N mutant beta-amyloid fibrils, in an effort to assess the relative thermodynamic stabilities of various polymorphs. Data analysis is in progress.