Structural Characterization of Plaque Seeded Amyloid-β Fibrils by Magic Angle Spinning NMR

Abstract

Accumulation of plaques consisting of amyloid fibrils of the peptide amyloid-β (Aβ) in the brain is one of the hallmarks of Alzheimer’s disease (AD). Aβ is prone to polymorphism and the structure of fibrils is sensitive to the conditions under which they are formed. The two dominant forms of Aβ are Aβ₁₋₄₀ and Aβ₁₋₄₂. Aβ₁₋₄₂ is more neurotoxic and aggregates faster. While studies of Aβ₁₋₄₀ have not found any consensus on a single structure, in the case of Aβ₁₋₄₂ three solid state nuclear magnetic resonance (NMR) studies conducted by different research groups have found essentially the same structure. However, since Aβ is prone to polymorphism it is not clear if this consensus structure reflects what is present in the brain of an AD patient. Unfortunately, NMR studies require isotopic labelling which is not possible in vivo, but amyloid fibrils display a seeding behavior where mature fibrils catalyze the formation of additional fibrils from peptide monomers. The work presented in this thesis focuses on preparing and characterizing fibrils by using plaques isolated from an AD patient’s brain as seeds for isotopically labelled Aβ₁₋₄₂ monomers. The goal of this process was to prepare isotopically labelled fibrils that reflect the structures found in the brain. We have demonstrated that we can reproducibly prepare such seeded samples, which display a single set of NMR peaks indicating a single molecular fold of Aβ. Interestingly, the NMR spectra of the plaque seeded samples do not match the previously identified structure of Aβ₁₋₄₂ found by three groups. I applied cutting edge solid state NMR techniques to obtain site specific spectral assignments and distance constraints. I have calculated a structural model for the plaque seeded fibrils based on those NMR derived constraints that converged in the region from D23-A42. We have also collected cryogenic electron microscopy (cryo-EM) images of the seeded fibrils. Even though the NMR spectra show a single set of peaks, we have been unable to reconstruct high resolution electron density maps due to heterogeneity in the width and twist of the fibrils.