Structural basis for increased toxicity of pathological aβ42:aβ40 ratios in Alzheimer disease

Abstract

The β-amyloid peptide (Aβ) is directly related to neurotoxicity in Alzheimer disease (AD). The two most abundant alloforms of the peptide co-exist under normal physiological conditions in the brain in an Aβ(42):Aβ(40) ratio of ∼1:9. This ratio is often shifted to a higher percentage of Aβ(42) in brains of patients with familial AD and this has recently been shown to lead to increased synaptotoxicity. The molecular basis for this phenomenon is unclear. Although the aggregation characteristics of Aβ(40) and Aβ(42) individually are well established, little is known about the properties of mixtures. We have explored the biophysical and structural properties of physiologically relevant Aβ(42):Aβ(40) ratios by several techniques. We show that Aβ(40) and Aβ(42) directly interact as well as modify the behavior of the other. The structures of monomeric and fibrillar assemblies formed from Aβ(40) and Aβ(42) mixtures do not differ from those formed from either of these peptides alone. Instead, the co-assembly of Aβ(40) and Aβ(42) influences the aggregation kinetics by altering the pattern of oligomer formation as evidenced by a unique combination of solution nuclear magnetic resonance spectroscopy, high molecular weight mass spectrometry, and cross-seeding experiments. We relate these observations to the observed enhanced toxicity of relevant ratios of Aβ(42):Aβ(40) in synaptotoxicity assays and in AD patients.

FIGURE 1.

Aβ₄₀ and Aβ₄₂ interact directly. 10 μm covalently tethered biotinylated Aβ₄₀ or Aβ₄₂ to streptavidin-coated SA chips show binding between 10 μm Aβ variants. The sensorgram presents the interaction between Aβ₄₀-Aβ₄₂ (orange), Aβ₄₂-Aβ₄₂ (black), Aβ₄₀-Aβ₄₀ (blue), and the negative control nonspecific binding between Aβ₄₂-KAAEAAAKKFFE (gray).

FIGURE 2.

The monomeric structures of Aβ₄₂:Aβ₄₀ ratios are identical at atomic level. Shown is the overlay of the ¹H-¹⁵N HSQC spectra immediately after sample preparation of a, ¹⁵N-labeled Aβ₄₀ in pure Aβ₄₀ sample (black), ratio 1:9_{(15 N)} (green) and ratio 3:7_{(15 N)} (red). b, ¹⁵N-labeled Aβ₄₂ in pure Aβ₄₂ sample (black), ratio 1_{(15 N)}:9 (red), and ratio 3_{(15 N)}:7 (green). For representation purposes and clarity, we have artificially introduced a systematic shift of the spectra of the 1:9 and 3:7 Aβ₄₂:Aβ₄₀ ratios.

FIGURE 3.

Morphology and detailed structure analysis of fibrils of Aβ₄₂:Aβ₄₀ ratios reveals similar structural characteristics. a, TEM images of Aβ ratios obtained upon aggregation for 2 weeks at 25 °C without agitation. Top left panel, pure Aβ₄₀; top right panel, ratio 1:9; bottom left panel, ratio 3:7; bottom right panel, pure Aβ₄₂. Bar, 200 nm. b, fiber diffraction patterns of Aβ ratios obtained upon aggregation for 4 weeks at 25 °C without agitation. Top left panel, pure Aβ₄₀; top right panel, ratio 1:9; bottom left panel, ratio 3:7; bottom right panel, pure Aβ₄₂. c, overlay showing the normalized x-ray scattering intensity function of D-spacing plotted from b.

FIGURE 4.

Aβ₄₀ and Aβ₄₂ show different aggregation behavior in different Aβ₄₂:Aβ₄₀ ratios. a, pure Aβ₄₀ at a concentration of 180 μm does not aggregate within the timeframe of data collection. b, pure Aβ₄₂ at a concentration of 20 μm displays a lag phase and a sigmoidal transition from monomeric species into NMR invisible aggregates. c, 3:7_{(15 N)} ratio, whereby the Aβ sample is composed of 70% ¹⁵N-labeled Aβ₄₀, which is monitored via HSQC (140 μm Aβ₄₀ monomer concentration) and 30% unlabeled Aβ₄₂ (at a monomer concentration of 60 μm), which is simultaneously monitored via the amide region ¹⁵N-filtered one-dimensional NMR spectrum. d, the 3_{(15 N)}:7 ratio whereby the Aβ sample is composed of 30% ¹⁵N-labeled Aβ₄₂ and 70% unlabeled Aβ₄₀. e, the 1:9_{(15 N)} ratio with 10% unlabeled Aβ₄₂ (20 μm) and 90% ¹⁵N-labeled Aβ₄₀ (180 μm). f, the 1_{(15 N)}:9 ratio with 10% ¹⁵N-labeled Aβ₄₂ and 90% unlabeled Aβ₄₀. g, the 5_{(15 N)}:5 ratio whereby the ¹⁵N-labeled Aβ₄₂ and unlabeled Aβ₄₀ are present in equimolar amounts (60 μm of each alloform). h, the 5:5_{(15 N)} ratio whereby equimolar amounts (60 μm) of unlabeled Aβ₄₂ and ¹⁵N-labeled Aβ₄₀ are present. The blue symbols represent Aβ₄₀, and the black symbols correspond to Aβ₄₂.

FIGURE 5.

Oligomer formation by Aβ₄₂:Aβ₄₀ ratios shows a monomer addition process and a dynamic distribution of oligomeric species. Mass spectra of the different ratios with the high molecular weight detection spectra as insets whereby the blue trace is t = 1 h, the black trace is t = 3 h, and the red trace is t = 6 h. a, pure Aβ₄₀; b, 1:9 ratio; c, 3:7 ratio; d, pure Aβ₄₂. These aggregation patterns for the different ratios are also presented in supplemental Table S1.

FIGURE 6.

Cross-seeding reveals that Aβ₄₂ oligomers show plasticity, whereas Aβ₄₀ oligomers display a higher selectivity. a, TEM of seed preparations. Seeds were prepared by incubation of 50 μm Aβ ratios for 24 h followed by sonication at maximum power for 10 min. From left to right: pure Aβ₄₀, ratio 1:9; ratio 3:7, pure Aβ₄₂. Bar, 0.2 μm. Freshly prepared seeds were added to monomeric solutions of Aβ₄₂:Aβ₄₀ ratios at final concentrations of 0.5 μm and 25 μm, respectively. b, ThT of Aβ₄₀ monomers seeded with pure Aβ₄₀ seeds (blue dotted trace); with seeds from ratio 1:9 (green dotted trace), with seeds from ratio 3:7 (red dotted trace), and with seeds from pure Aβ₄₂ (black dotted trace). The blue dashed line represents the unseeded Aβ₄₀ control. c, ThT of ratio 1:9 monomers seeded with Aβ ratios as compared with the non-seeded aggregation curve. The colors are as described in b. d, ThT of ratio 3:7 monomers seeded with Aβ ratios as compared with the non-seeded aggregation curve (red dashed line). Colors are as described in b. e, ThT of Aβ₄₂ monomers seeded with Aβ ratios in comparison with the non-seeded sample (black dashed line). Colors are as described in b.

FIGURE 7.

Cross-seeding was monitored by NMR by recording one-dimensional proton spectra as a function of time with unlabeled pure Aβ₄₀ (a and b) and pure Aβ₄₂ (c and d) monomers using preformed Aβ₄₀ seeds (b and d) and Aβ₄₂ seeds (a and c). Pure Aβ₄₀ samples (blue) were prepared at a concentration of 180 μm, whereas pure Aβ₄₂ samples (black) were at a concentration of 20 μm. The addition of 10% (v/v) of a 50 μm (monomeric equivalent) seed preparation was added at the time points indicated by the arrow.

FIGURE 8.

Cross-seeding was monitored by ¹⁵N-filtered and ¹⁵N-edited NMR experiments with Aβ samples composed of the Aβ₄₂:Aβ₄₀ ratio 1_{(15 N)}:9 (a and b), whereby 20 μm¹⁵N-labeled Aβ₄₂ is present with 180 μm unlabeled Aβ₄₀ and the Aβ₄₂:Aβ₄₀ ratio 3_{(15 N)}:7 (c and d) with 60 μm¹⁵N-labeled Aβ₄₂ and 140 μm unlabeled Aβ₄₀. The addition of 10% (v/v) of a 50 μm (monomeric equivalent) preparation of preformed Aβ₄₀ seeds (b and d) and Aβ₄₂ seeds (a and c) is indicated by the arrows.