Structural Studies of Alzheimer's beta-Amyloid Fibrils

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Progress in FY2022 has been in the following areas: (1) AMYLOID-BETA FIBRIL STRUCTURES FROM CRYO-ELECTRON MICROSCOPY: We have obtained high-quality cryo-EM images of 42-residue amyloid-beta (Ab42) fibrils obtained by seeding with amyloid-enriched extract from human brain tissue. The images show two coexisting polymorphs with distinct molecular structures. "Type 1" fibrils have an approximately U-shaped Ab42 conformation, two cross-beta subunits with 2-fold screw symmetry, inter-subunit salt bridge interactions between Lys28 sidechains and the C-terminal carboxylate groups, and polar interactions within each subunit between Asp23 and Asn27 sidechains. These structural features have not been observed in previous structural studies of Ab42 fibrils, either grown in vitro or isolated from brain tissue. Thus, this is a new "fold" for Ab42 fibrils, and is especially relevant to Alzheimer's disease (AD) because our fibrils were derived from cortical tissue of AD patients. "Type 2" fibrils have the more common S-shaped Ab42 conformation and two-fold symmetry between cross-beta subunits, but with unusual inter-subunit Lys16-Ala42 salt bridge interactions, as well as unusual Asp1-Lys28 salt bridges. A paper describing these results is in preparation. (2) INITIAL STEPS IN AMYLOID-BETA OLIGOMERIZATION REVEALED BY TIME-RESOLVED SOLID STATE NMR: In FY2022, we initiated a project to investigate the initial steps in oligomerization by the 40-residue amyloid-beta peptide (Ab40), using the time-resolved solid state NMR methods that have been developed in our lab over the past several years. In these experiments, an Ab40 solution at pH 12 (where Ab40 is soluble as a monomer) is neutralized within 1 ms by rapid mixing with a pH 7 buffer. This triggers the oligomerization process. After variable time intervals, the solution is rapidly frozen, then examined by DNP-enhanced low-temperature solid state NMR. Surprisingly, the solid state NMR spectra show the development of beta-strand secondary structure and intermolecular contacts immediately (i.e., within 1 ms). The spectra then do not change significantly for at least 400 ms. Time-resolved static light scattering measurements, carried out with a novel stopped-flow method, show that Ab40 molecules are primarily in a dimeric state after 1 ms, but then assemble into oligomers consisting of approximately eight Ab40 molecules after 400 ms. Thus, our data show that beta-strand secondary structure develops at the very earliest stage of oligomerization, and oligomers subsequently grow without detectable changes in Ab40 conformation or structural order. These studies are the first characterization of structural properties of the earliest amyloid-beta oligomers. A paper describing these results will be prepared in the next several months.