Sterol-activated amyloid beta fibril formation

Abstract

The metabolic processes that link Alzheimer's disease (AD) to elevated cholesterol levels in the brain are not fully defined. Amyloid beta (Aβ) plaque accumulation is believed to begin decades prior to symptoms and to contribute significantly to the disease. Cholesterol and its metabolites accelerate plaque formation through as-yet-undefined mechanisms. Here, the mechanism of cholesterol (CH) and cholesterol 3-sulfate (CS) induced acceleration of Aβ₄₂ fibril formation is examined in quantitative ligand binding, Aβ₄₂ fibril polymerization, and molecular dynamics studies. Equilibrium and pre-steady-state binding studies reveal that monomeric Aβ₄₂•ligand complexes form and dissociate rapidly relative to oligomerization, that the ligand/peptide stoichiometry is 1-to-1, and that the peptide is likely saturated in vivo. Analysis of Aβ₄₂ polymerization progress curves demonstrates that ligands accelerate polymer synthesis by catalyzing the conversion of peptide monomers into dimers that nucleate the polymerization reaction. Nucleation is accelerated ∼49-fold by CH, and ∼13,000-fold by CS - a minor CH metabolite. Polymerization kinetic models predict that at presumed disease-relevant CS and CH concentrations, approximately half of the polymerization nuclei will contain CS, small oligomers of neurotoxic dimensions (∼12-mers) will contain substantial CS, and fibril-formation lag times will decrease 13-fold relative to unliganded Aβ₄₂. Molecular dynamics models, which quantitatively predict all experimental findings, indicate that the acceleration mechanism is rooted in ligand-induced stabilization of the peptide in non-helical conformations that readily form polymerization nuclei.

Keywords: Alzheimer’s disease; Aβ(42) peptide; amyloid beta plaque; cholesterol; cholesterol sulfate; dimer; fibril; mechanism; molecular dynamics; monomer; structure; sulfotransferase 2B1b.

Figure 1

Sterol binding to Aβ₄₂at equilibrium.A, DHE affinity for monomeric Aβ4₂. A solution containing DHE (10 nM, 0.83 × K_d) and K₂PO₄ (50 mM), pH 7.4, 25 °C ± 2 deg. C, was titrated with Aβ₄₂ (0–200 nM, 0–31 × K_d). Binding was detected via the binding-induced 4.4 (±0.2)-fold increase in DHE fluorescence (λ_ex = 325 nm, λ_em= 375 nm). Fluorescence intensity, I, is plotted relative to the intensity at zero titrant, I₀. Titrations were performed independently in triplicate and the line passing through the data is the least-squares fit to a single-binding-site model. B, Aβ₄₂:DHE stoichiometry. Aβ₄₂ was titrated into to a solution containing DHE (fixed at 1.0 μM, 62 × K_d) and K₂PO₄ (50 mM), pH 7.4, 25 °C ± 2 deg. C. DHE fluorescence (λ_ex = 325 nm, λ_em = 375 nm) is plotted versus [Aβ₄₂]/[DHE]. The line descending from the titration breakpoint indicates a stoichiometry of 1:1. C and D, CH and CS affinities. Affinities were determined by competitive binding versus DHE. A solution containing DHE (10 nM, 0.67 × K_d), Aβ₄₂ (20 nM, 1.3 × K_d), and K₂PO₄ (50 mM), pH 7.4, 25 °C ± 2 deg. C, was titrated with CH (5.0–500 nM, 0.05–5 × K_d) or CS (5.0–500 nM, 0.15–33 × K_d). Binding was monitored via the decrease in DHE fluorescence (λ_ex = 325, λ_em = 375) caused by DHE displacement from Aβ₄₂. Controls ensured that <2.0% of Aβ₄₂ oligomerized during the measurement. Titrations were performed in triplicate and averaged. The solid lines through the data represent least-squares fits to a competitive single-binding-site model. C and D, insets highlight the behavior of the titration over ligand concentrations that span their reported CMCs (indicated by arrows). K_d values are reported in Table 1.

Figure 2

Sterol binding to Aβ₄₂—pre-steady state studies.A, DHE binding. Reactions were monitored via binding induced changes in DHE fluorescence using a stopped-flow fluorimeter (λ_ex = 325, λ_em > 400 nm). Fluorescence intensity, I, is reported relative to the intensity in the absence of ligand, I_o. A solution containing Aβ₄₂ (2.0 μM, 74 × K_d), K₂PO₄ (50 mM), pH 7.4, 25 °C ± 2 deg. C was rapidly mixed (1:1, v/v) with a solution identical except that Aβ₄₂ was replaced by DHE (40 nM, 2.7 × K_d). The bindingreaction curve shown is the average of five independent progress curves. The averaged curve was a least-squares fit to a single-exponential equation and the resulting best fit (indicated by the red line) yielded k_obs. B, DHE k_on and k_off. k_obs values obtained as in Panel A were determined in triplicate at varying Aβ₄₂ concentrations and the averaged values are shown plotted versus [Aβ₄₂]. k_on and k_off are given by the slope and intercept of a linear least-squares fit of the k_obsversus [Aβ₄₂] plot. C and D, CH and CS displacement reactions. A solution containing DHE (10 μM, 630 × K_d), K₂PO₄ (50 mM), pH 7.5, 25 °C ± 2 deg. C was rapidly mixed (1:1, v/v) with a solution in which DHE was replaced with Aβ₄₂ (0.10 μM, 0.83 × K_d) and CH (2.0 μM, 17 × K_d), Panel C, or, Aβ₄₂ (0.10 μM, 2.9 × K_d) and CS (0.60 μM, 17 × K_d), Panel D. The final DHE concentration (5.0 μM, 330 × K_d) displaces ∼98 % of either CH (1.0 μM, 8.3 × K_d) or CS (0.30 μM, 8.3 × K_d); hence, the dissociation reactions are pseudo first order. k_off was obtained from a least-squares fit to a single-exponential equation (shown as red line passing through the data).

Figure 3

Formation of CH•, CS•, and unliganded Aβ₄₂fibrils.A, fibril formation. Fibril formation was monitored via the fluorescence increase (λ_ex = 450 nm, λ_em = 482 nm) associated with the binding of ThT, which binds Aβ oligomers ≥5-mers. Reaction conditions: Aβ₄₂ (1.0 μM), ThT (30 μM), ligand (CH (3.0 μM, 17 × K_d) or CS (1.6 μM, 17 × K_d) or no ligand), DMSO (0.5 % v/v), K₂PO₄ (50 mM), pH 7.4, 25 °C ± 2 deg. C. Progress curves were performed in triplicate, averaged, and fit numerically using the NEF polymerization model (see Results and discussion). Lines passing through the data represent the best fits of the NEF model and the associated rate constants are listed in Table 2.

Figure 4

Nucleation reaction order. Reactions were monitored via binding-induced changes in ThT fluorescence (λ_ex = 450 nm, λ_em = 482 nm). All reactions were performed under the following conditions: ThT (30 μM), DMSO (0.5 % v/v), and K₂PO₄ (50 mM), pH 7.4, 25 °C ± 2 deg. C. Ligand concentrations were adjusted to maintain free [ligand] at 17 × K_d. A, unliganded Aβ₄₂ nucleation. Titrant concentrations: Aβ₄₂ (1.0, 2.0, 3.0, or 4.0 μM). B, CH•Aβ₄₂ nucleation. Titrant concentrations: Aβ₄₂ (0.5, 1.0, 1.5, or 2.0 μM) and CH ([Aβ₄₂] + 17 × K_d; 2.5, 3.0, 3.5, and 4.0 μM). C, CS•Aβ₄₂ nucleation. Titrant concentrations: Aβ₄₂ (0.25, 0.50, 0.75, 1.0 μM) and CS ([Aβ₄₂] + 17 × K_d; 0.85, 1.1, 1.35, and 1.60 μM). Reactions were run in triplicate and plotted versus [Aβ₄₂]². Nucleation rate constants are given by the slope of linear least-squares fits shown as lines passing through the data.

Figure 5

MD-predicted Aβ₄₂monomer structures.A, the CH•Aβ₄₂ monomer. B, the CS•Aβ₄₂ monomer. All residues in direct contact with the ligand are shown in “stick” and labeled. Small red spheres mark the Aβ₄₂ peptide C-terminal residue, A42.

Figure 6

Dimerization studies.A and B, dimerization is monomer dependent. Eight monomer forms (unliganded peptide, CH•peptide Forms 1–6, and CS•peptide) were tested in MD simulations for their tendency to form dimers over a 5.0 μs time interval. Panel A presents progress curves for the species that formed dimers — i.e., CS•peptide (CS), Form 3 CH•peptide (CH), and unliganded peptide. Panel B shows the progress curves for CH Forms that did not yield dimers. The curves are numbered according to the CH Forms given in Fig. S2. C–F, dimer structures. Panels C–E present the time average of the structures in the plateaus of the CS•, CH•, and unliganded-peptide progress curves, respectively. Panel F presents the structure of the unliganded-peptide intermediate that rapidly forms and slowly rearranges to the structure seen in Panel E.

Figure 7

CS•Aβ₄₂oligomerization studies.A, CS•Aβ₄₂ octamer assembly. Eight CS•Aβ₄₂ monomers are seen spontaneously assembling into an octamer. Black and red lines trace the oligomerization status of the two peptides that initiate oligomerization. The simulation was initiated with eight monomers randomly positioned in a 10 × 10 × 10 nm cube of water, PO₄ (50 mM), KCl (0.10 mM), pH 7.4, 25 °C. B, interface formation. Each progress curve shows the transition of two interface-forming species from an early-contact stage to a fully formed interface. The seven transitions associated with octamer assembly are included in the figure. The curves are separated based on reaction rate into two classes — dimer interface formation, and oligomer interface formation (which includes monomer/oligo and oligo/oligo interfaces). The progress curves in each class were least-squares fit to a single exponential equation, the best-fit k_obs values within each class were averaged and the average value was used to generate the solid lines seen passing through the datasets. C, predicted structure of the CS octamer.

Figure 8

Aβ₄₂fibril formation versus CS percentage. Fibril formation was detected via binding-induced changes in ThT fluorescence (λ_ex = 450 nm, λ_em = 482 nm). Reaction conditions: Aβ₄₂ (1.0 μM), CS percent (as indicated), [CS] + [CH] (20 μM), ThT (30 μM), DMSO (0.5 % v/v), K₂PO₄ (50 mM), pH 7.4, 25 °C ± 2 deg. C. Progress curves were performed in triplicate and averaged data are shown. Lines passing through the data represent the behavior predicted by the NEF model using the Table 2 rate constants.