Progress in FY2013 has been in the following areas: AMYLOID FIBRIL STRUCTURES DERIVED FROM BRAIN TISSUE: We have previously developed a protocol for partial purification of amyloid from brain tissue obtained at autopsy, and for using this material as a "seed" for growing fibrils from synthetic, isotopically-labeled peptide. With this protocol, 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 Alzheimer's disease patient, we found that there is a single fibril structure in this tissue, a surprising result. We have performed numerous solid state NMR measurements and electron microscopy measurements on these brain-seeded fibrils, with isotopic labeling at specific sites and with uniform isotopic labeling. From these data, we have developed a full structural model for brain-derived fibrils, the first of its kind. The brain-derived fibrils are structurally similar to fibrils that we have created synthetically and characterized in previous years, but have some unique features (e.g., the entire 40-residue peptide sequence is structurally ordered). Solid state NMR and electron microscopy studies of a second patient showed a single fibril structure in frontal, occipital, and temporal lobe tissue (same structure in all three brain regions), but this structure is different from the structure in the first patient. Interestingly, the two patients had different clinical histories and neuropathology, suggesting a link between fibril structure and disease development. These results will be published in the September 12, 2013 issue of Cell. (Collaboration with Prof. S.C. Meredith, University of Chicago). We are now screening tissue samples from a series of Alzheimer's disease patients with several distinct clinical history categories, using solid state NMR spectra and electron microscope images as "fingerprints" of fibril structure. Preliminary results indicate that most AD patients have the same predominant fibril structure, but that patients with rapidly progressing AD have distinct fibril structures. These experiments will be completed and published in FY14. (Collaboration with Prof. J.C. Collinge, University College London) KINETICS AND THERMODYNAMICS OF AMYLOID FIBRIL GROWTH: We have used atomic force microscopy to measure the intrinsic beta-amyloid fibril shrinkage rates and monomer-concentration-dependent fibril extension rates of two different 40-residue beta-amyloid fibril polymorphs. The ratio of shrinkage rate to growth rate equals the polymorph-specific thermodynamic quasi-equilibrium solubility, which we corroborate with direct monomer concentration measurements by UV absorption and direct measurements of polymorph interconversion by solid state NMR. We find that "striated ribbon" fibrils are slightly more stable than "twisted" fibrils at 24 C, with greater extension rates. At 37 C, the two polymorphs have indistinguishable stabilities, with markedly lower solubilities and greater extension rates than at 24 C. These results help explain the prevalence and persistence of polymorphism in amyloid fibrils, and the dependence of structure on growth conditions.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$599,499
Indirect Cost
City
State
Country
Zip Code
Tycko, Robert (2016) Molecular Structure of Aggregated Amyloid-β: Insights from Solid-State Nuclear Magnetic Resonance. Cold Spring Harb Perspect Med 6:
Tycko, Robert (2015) Amyloid polymorphism: structural basis and neurobiological relevance. Neuron 86:632-45
Potapov, Alexey; Yau, Wai-Ming; Ghirlando, Rodolfo et al. (2015) Successive Stages of Amyloid-β Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization. J Am Chem Soc 137:8294-307
Tycko, Robert (2014) Physical and Structural Basis for Polymorphism in Amyloid Fibrils. Protein Sci :
Lu, Jun-Xia; Qiang, Wei; Yau, Wai-Ming et al. (2013) Molecular structure of β-amyloid fibrils in Alzheimer's disease brain tissue. Cell 154:1257-68
Qiang, Wei; Kelley, Kevin; Tycko, Robert (2013) Polymorph-specific kinetics and thermodynamics of β-amyloid fibril growth. J Am Chem Soc 135:6860-71
Qiang, Wei; Yau, Wai-Ming; Luo, Yongquan et al. (2012) Antiparallel β-sheet architecture in Iowa-mutant β-amyloid fibrils. Proc Natl Acad Sci U S A 109:4443-8
McDonald, Michele; Box, Hayden; Bian, Wen et al. (2012) Fiber diffraction data indicate a hollow core for the Alzheimer's aβ 3-fold symmetric fibril. J Mol Biol 423:454-61
Cloe, Adam L; Orgel, Joseph P R O; Sachleben, Joseph R et al. (2011) The Japanese mutant Aβ (ΔE22-Aβ(1-39)) forms fibrils instantaneously, with low-thioflavin T fluorescence: seeding of wild-type Aβ(1-40) into atypical fibrils by ΔE22-Aβ(1-39). Biochemistry 50:2026-39
Lu, Jun-Xia; Yau, Wai-Ming; Tycko, Robert (2011) Evidence from solid-state NMR for nonhelical conformations in the transmembrane domain of the amyloid precursor protein. Biophys J 100:711-9

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