High-resolution NMR characterization of low abundance oligomers of amyloid-β without purification

Abstract

Alzheimer's disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-β (Aβ). The aggregation of Aβ leads to diverse oligomeric states, each of which may be potential targets for intervention. Obtaining insight into Aβ oligomers at the atomic level has been a major challenge to most techniques. Here, we use magic angle spinning recoupling (1)H-(1)H NMR experiments to overcome many of these limitations. Using (1)H-(1)H dipolar couplings as a NMR spectral filter to remove both high and low molecular weight species, we provide atomic-level characterization of a non-fibrillar aggregation product of the Aβ1-40 peptide using non-frozen samples without isotopic labeling. Importantly, this spectral filter allows the detection of the specific oligomer signal without a separate purification procedure. In comparison to other solid-state NMR techniques, the experiment is extraordinarily selective and sensitive. A resolved 2D spectra could be acquired of a small population of oligomers (6 micrograms, 7% of the total) amongst a much larger population of monomers and fibers (93% of the total). By coupling real-time (1)H-(1)H NMR experiments with other biophysical measurements, we show that a stable, primarily disordered Aβ1-40 oligomer 5-15 nm in diameter can form and coexist in parallel with the well-known cross-β-sheet fibrils.

Figure 1. Biophysical characterization of Aβ_1-40 disordered oligomers.

(a) CD spectra, (b) ThT fluorescence, and (c) bis-ANS fluorescence of fibrillar (black), the spin-X-isolated oligomer (blue), and freshly dissolved (red) samples of Aβ_1-40. In panel c, the emission spectrum of bis-ANS in solution is shown in grey. (d) Distributions of the hydrodynamic radii of the freshly dissolved Aβ_1-40 (red) and the spin-X-isolated Aβ_1-40 oligomer (blue) determined by DLS. Representative AFM and TEM images of the Aβ_1-40 oligomers (e) and fibrils (f). Scale bars in both images are 100 nm. All experiments were performed in 10 mM sodium phosphate buffer, pH 7.4 at 25  °C.

Figure 2. RFDR-based 2D ¹H/¹H chemical shift correlation spectra of freshly dissolved (red) and aggregated (blue) forms of Aβ_1-40.

(a) Side-chain to Hα, (b) side-chain, and (c) Hβ-Hα and Hα -Hα regions of the overlaid 2D spectra were recorded under 2.7 kHz MAS. The dotted circle highlights the Ser and Gly fingerprints of the aggregated Aβ_1-40 sample. Peak assignments are given for the mixed Aβ_1-40 sample. The spectra were acquired with a 50 ms mixing time at 600 MHz in 100% D₂O, 10 mM sodium phosphate buffer, pH 7.4, and 37 °C. Total Aβ_1-40 concentrations for both samples were 462 μM; the estimated oligomer concentration in the aggregated sample is 35 ± 12 μM. The acquisition time was 4 days.

Figure 3. MAS spectra of the filtered disordered Aβ_1-40 oligomer.

An overlay of the assigned Hα regions from RFDR-based 2D ¹H/¹H (blue) and 2D ¹H/¹H TOCSY (red) spectra acquired at 298 K with 10 kHz MAS. The filtered oligomer sample was lyophilized and re-hydrated to double its initial concentration, making for a total Aβ_1-40 of ~35 μM. The RFDR and TOCSY based 2D ¹H/¹H spectra were acquired with mixing times of 50 and 70 ms, respectively, at 600 MHz in 100% D₂O, 10 mM sodium phosphate buffer, pH 7.4. RFDR spectra were acquired at 25 °C under 10 kHz MAS. Assignments are given for the RFDR-based 2D ¹H/¹H spectra. The non-uniform sampling based data acquisition time was 4 hours.

Figure 4. The disordered Aβ_1-40 oligomer grows in size while maintaining its morphology.

(a) DLS experiments at 19 days demonstrate that the disordered oligomer not only remains disordered, but also grows in size as well with a distribution of hydrodynamic radii at 8.6 nm and 65.3 nm and polydispersity of 14.9% and 37.8%, respectively. (b) The strong minimum at ~200 nm in the CD spectrum of the disordered oligomer after 19 days reveals that the oligomer does not progress to a fibrillar state. Two-dimensional spectra of the fingerprint region of (c) TOCSY (70 ms mixing) and (d) NOESY (250 ms mixing) of the disordered Aβ_1–40 oligomers recorded at 4 days (blue) and 19 days (red). The filtered oligomer sample was lyophilized and re-hydrated to double its initial concentration, making for a total Aβ_1-40 of ~35 μM. After 19 days (red), almost all peaks are broadened beyond detection in both TOCSY and NOESY spectra.

Figure 5. Comparison of ¹H/¹⁵N HSQC spectra of the freshly dissolved (red) and the spin-X-isolated disordered oligomer (blue) of Aβ_1-40 (after 4 days) recorded from a 900 MHz spectrometer.

Both experiments were performed in 10 mM phosphate buffer, pH 7.4, and 10% D₂O at 25 °C.

Figure 6. Simultaneously occurring aggregation pathways of Aβ_1-40.

Early aggregates maintain structural similarity to the stable, disordered Aβ_1-40 oligomers observed at late aggregation stages. The early aggregates either (i) solely nucleate the disordered oligomers or (ii) act as a single nucleating seed from which the two distinct aggregation pathways bifurcate.