Seeded growth of beta-amyloid fibrils from Alzheimer's brain-derived fibrils produces a distinct fibril structure

Abstract

Studies by solid-state nuclear magnetic resonance (NMR) of amyloid fibrils prepared in vitro from synthetic 40-residue beta-amyloid (Abeta(1-40)) peptides have shown that the molecular structure of Abeta(1-40) fibrils is not uniquely determined by amino acid sequence. Instead, the fibril structure depends on the precise details of growth conditions. The molecular structures of beta-amyloid fibrils that develop in Alzheimer's disease (AD) are therefore uncertain. We demonstrate through thioflavin T fluorescence and electron microscopy that fibrils extracted from brain tissue of deceased AD patients can be used to seed the growth of synthetic Abeta(1-40) fibrils, allowing preparation of fibrils with isotopic labeling and in sufficient quantities for solid-state NMR and other measurements. Because amyloid structures propagate themselves in seeded growth, as shown in previous studies, the molecular structures of brain-seeded synthetic Abeta(1-40) fibrils most likely reflect structures that are present in AD brain. Solid-state (13)C NMR spectra of fibril samples seeded with brain material from two AD patients were found to be nearly identical, indicating the same molecular structures. Spectra of an unseeded control sample indicate greater structural heterogeneity. (13)C chemical shifts and other NMR data indicate that the predominant molecular structure in brain-seeded fibrils differs from the structures of purely synthetic Abeta(1-40) fibrils that have been characterized in detail previously. These results demonstrate a new approach to detailed structural characterization of amyloid fibrils that develop in human tissue, and to investigations of possible correlations between fibril structure and the degree of cognitive impairment and neurodegeneration in AD.

Fig. 1.

Negatively stained TEM images of β-amyloid fibrils. (A-C) Fibrils extracted from AD brain tissue. Yellow arrows indicate carbon lace of the sample grid. (D-F) Aβ_1–40 fibrils observed 4 hours after seeding of a monomeric Aβ_1–40 solution with AD brain amyloid. (G-I) Aβ_1–40 fibrils in the brain 1/generation 3, brain 2/generation 3, and control/generation 3 samples, respectively. Red arrows indicate the predominant fibril morphology in brain-seeded samples. Blue arrows indicate other morphologies. Green scale bars, 400 nm.

Fig. 2.

Amyloid fibril formation kinetics monitored by ThT fluorescence. (A) Unseeded Aβ_1–40 solution (open square and dotted line) and solutions seeded with sonicated synthetic Aβ_1–40 fibrils (open circles and dashed line) or sonicated amyloid from brain 2 (open triangles and solid line). Brain amyloid data are scaled down by a factor of 2. (B) Unseeded Aβ_1–40 solution (open squares and dotted line) and solutions seeded with brain material from a non-demented, 25-year-old man (open circles and dashed line) or a non-demented, 56-year-old woman (open triangles and solid line). Lines are empirical fits with a stretched exponential function (dotted line in A, all lines in B), a monoexponential function (dashed line in A), and the difference between two monoexponential functions (solid line in A).

Fig. 3.

2D solid-state ¹³C NMR spectra of Aβ_1–40 fibrils. (A-C) Aliphatic regions of spectra for brain 1/generation 3, brain 2/generation 3, and control/generation 3. Fibrils were uniformly ¹⁵N,¹³C-labeled at F19, V24, G25, A30, I31, L34, and M35. Chemical shift assignment paths are shown, with solid and dashed lines in (A) and (B) indicating majority and minority signals, respectively. The distinction between majority and minority signals is unclear in (C). (D) Comparison of secondary shifts (i.e., deviations from random coil values) for brain-seeded fibril majority signals (brain 1/generation 3, green triangles) with previously reported secondary shifts for purely synthetic fibrils with twisted (red circles) and striated ribbon (black squares) morphologies.

Fig. 4.

Aromatic and aliphatic regions of 2D solid-state ¹³C NMR spectra with 500-millisecond RAD mixing periods. (A-C) brain 1/generation 3, brain 2/generation 3, and control/generation 3 fibrils, respectively. Relevant chemical shift assignment paths are shown. Vertical dashed lines with arrowheads indicate spin polarization transfers from aliphatic carbons of I31 (A-C) and V24 (C only) to F19 aromatic carbons that produce inter-residue crosspeaks and reflect sidechain-sidechain contacts. One-dimensional slices below each two-dimensional spectrum are taken at positions indicated by the color-coded horizontal dashed lines. Green slice, I31 C_α; red and blue slices, F19 aromatic; magenta, V24 C_γ.

Fig. 5.

Internuclear distances in brain-seeded Aβ_1–40 fibrils. (Upper) Measurements of ¹⁵N-¹³C dipole-dipole couplings between sidechains of K28 and D23 in brain 1/generation 3 (open squares) and brain 2/generation 3 (open circles) fibrils, using the frequency-selective rotational echo double resonance technique with detection of D23 carboxylate ¹³C NMR signals and dephasing by K28 sidechain ¹⁵N nuclei. Comparison with simulated curves for ¹⁵N-¹³C pairs indicates a distance of 3.4 ± 0.2 Å. (Lower) Measurements of intermolecular ¹³C-¹³C couplings for ¹³C labels at the V12 carbonyl (open circles) and A21 methyl (open triangles) sites in brain 1/generation 3 fibrils, using the PITHIRDS-CT technique. Comparison with simulated curves indicates 4.8 ± 0.2 Å intermolecular distances for both sites. Simulations are for linear chains of three ¹³C nuclei with initial spin polarization on the central nucleus, and include a 75-millisecond transverse spin relaxation time. Experimental signals are corrected for natural-abundance contributions by subtraction of a 20% (open circles) or 10% (open triangles) constant baseline.