Progress in FY2019 has been in the following areas: (1) AMYLOID-BETA FIBRIL STRUCTURES FROM CRYO-ELECTRON MICROSCOPY: We have obtained high-quality cryo-EM images of 40-residue amyloid-beta (Ab40) fibrils obtained by seeding with amyloid-enriched extract from human brain tissue. We have modified standard image-processing software to include orientational correlations among fibril segments (particles) that come from the same fibrils in a given micrograph. With this modification, we have obtained high-resolution density maps (3.0 Angstrom reported resolution) that allow unambiguous molecular structure determination. The cryo-EM density reveals a surprising structure, consisting of two central cross-beta layers in which Ab40 molecules are fully extended, rather than adopting the U-shaped or S-shaped conformations seen in earlier studies of different fibril polymorphs. In addition to the two central layers, two additional layers show density attributable to Ab40 half-molecules. We tentatively interpret these half-molecule layers to cross-beta sheets formed by molecules with beta-hairpin conformations. Confirmation of beta-hairpin conformations will depend on solid state NMR measurements, which are currently in progress. We expect to publish the results of these cryo-EM investigations in early FY2020. (2) CHARACTERIZATION OF AMYLOID-BETA FIBRIL POLYMORPHS BRAIN TISSUE OF NON-DEMENTED INDIVIDUALS: In earlier work from our lab (Qiang et al., Nature 2017), we used solid state NMR spectroscopy to characterize amyloid-beta fibril polymorphisms in brain tissue of Alzheimer's disease (AD) patients. 2D solid state NMR spectra of brain-derived fibrils showed a single predominant polymorph for the 40-residue peptide (Ab40) and two predominant polymorphs for the 42-residue peptide (Ab42). We are now applying the same methods to brain tissue of individuals who were cognitively normal at the time of death, but who were found to have high levels of amyloid deposition in their cerebral cortex. Tissue for these experiments was obtained from the Religious Orders Study of the Rush Alzheimer's Disease Center. So far, we have examined Ab40 fibrils derived by seeded growth from amyloid-enriched extract of frontal cortex samples of eight individuals. We have found that the 2D solid state NMR spectra of these fibrils are not significantly different from spectra of Ab40 fibrils that were derived from cortical tissue of AD patients in our earlier work. Thus, cognitive impairment is not uniquely correlated with Ab40 fibril polymorphisms. Solid state NMR measurements on 42-residue peptide fibrils (Ab42) will be performed in early FY2020.
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