Research ArticleBIOPHYSICS

Secondary nucleation and elongation occur at different sites on Alzheimer’s amyloid-β aggregates

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Science Advances  17 Apr 2019:
Vol. 5, no. 4, eaau3112
DOI: 10.1126/sciadv.aau3112
  • Fig. 1 Analysis of the effects of clusterin on the aggregation kinetics of Aβ(M1-42).

    (A to C) Kinetic reaction profiles for the aggregation of 4 μM Aβ(M1-42) solutions are shown in each panel from left (blue) to right (green) in the absence and presence of 7.5, 37, 75, and 135 nM clusterin, with each color representing repetitions at the same concentration. The integrated rate law for Aβ(M1-42) aggregation in the absence of clusterin using the rate constants, previously determined by a least-squares error function, is shown as a dark blue line in each case (10). Predicted profiles of the specific inhibition processes of (A) primary nucleation, (B) fibril elongation, and (C) secondary nucleation generated by clusterin are shown as continuous lines. Note the characteristic differences in the changes in the shape of the reaction profiles in each case. The prediction for the case where the molecular chaperone suppresses only elongation events matches closely the experimental data in the presence of different concentrations of clusterin. (D) Kinetic reaction profiles for the aggregation reaction of a 2 μM Aβ(M1-42) solution seeded with 100 nM preformed fibrils in the absence and presence of 7.5, 37, 75, and 135 nM clusterin. The lines represent the integrated rate laws for Aβ(M1-42) aggregation, where the elongation rate has been selectively reduced. The apparent elongation reaction rates as a function of the molecular chaperone concentration evaluated from the fitting in (B) and (D) are reported in (E) for both unseeded and seeded reactions. The continuous line in (E) represents a simplified correlation between the elongation rate and the binding affinity constant (see Materials and Methods), from which KD,37°C = 8 nM is determined. (F) Comparison between the experimental data reported in (B) and theoretical predictions of the reaction profiles calculated from a kinetic model, which considers the association and dissociation rate constants in the reaction scheme with KD,37°C = 2.5 nM. (G and H) Schematic diagrams showing the molecular pathways involved in Aβ(M1-42) aggregation (G) and the mechanism by which clusterin perturbs the aggregation process (H).

  • Fig. 2 Analysis of clusterin interactions with Aβ(M1-42) fibrils using immunogold TEM.

    Aβ(M1-42) fibrils formed under quiescent condition imaged as is (A and B) and after sonication (C and D) were incubated with BSA and clusterin and stringently washed. Incubation with an anti-mouse secondary antibody conjugated to a gold particle showed no nonspecific labeling (A and C), whereas incubation with an anti-clusterin monoclonal antibody followed by an anti-mouse secondary antibody conjugated to a gold particle shows the presence of clusterin interacting with the Aβ(M1-42) fibrils (black dots). Scale bars, 100 nm.

  • Fig. 3 Microfluidic analysis of clusterin binding to Aβ(M1-42) fibrils.

    (A) Schematic diagram of the microfluidic diffusional sizing device used in this work indicating its most relevant components (33). (B) The bar charts show the average size and fraction of the species in the large-size range in the absence and presence of Aβ(M1-42) fibrils. The average sizes and the fraction of species in the large-size range reported are the means and SDs of at least three independent repetitions. (C) Diffusion profiles acquired at 12 different positions along the microfluidic channel for a 0.8 μM clusterin solution in the absence (red curves) and presence (black dashed curves) of 17.5 μM preformed Aβ(M1-42) fibrils in 20 mM sodium phosphate buffer at pH 8.0. (D) The size distributions in the absence (blue) and presence (green) of 2 μM Aβ(M1-42) fibrils were evaluated by fitting model simulations on the basis of advection-diffusion equations to the experimental diffusion profiles reported in (C) (see the Supplementary Materials). (E) Binding curve of clusterin to 17.5 μM Aβ(M1-42) fibrils in 20 mM sodium phosphate at pH 8.0 and 21°C measured by the microfluidic diffusion technique. In a first set of experiments (squares), different concentrations of clusterin were incubated with previously generated Aβ(M1-42) fibrils and size distributions were measured after 48 hours of incubation to ensure equilibrium conditions. In a second set of experiments (circles), different concentrations of clusterin were incubated with 17.5 μM Aβ(M1-42) fibrils and BSA at equimolar concentrations to clusterin. Each point represents the mean and SD of at least two independent repetitions. The regression line represents the best fit to the nonlinear Langmuir binding isotherm with KD = 0.67 ± 0.19 μM and M = 0.80 ± 0.08 μM [corresponding to one clusterin molecule per 21 Aβ(M1-42) monomers], with R2 = 0.97.

  • Fig. 4 Brichos and clusterin exhibit modular and additive behavior of their specific inhibition processes.

    (A) Kinetic profiles of 3 μM Aβ(M1-42) solutions in 20 mM sodium phosphate buffer at pH 8.0 in the absence and presence of 18 nM clusterin and 2 μM proSP-C Brichos added either individually or together as indicated at 37°C. (B) The additivity of the inhibition effects reveals that the sites associated with the two different microscopic steps of elongation and secondary nucleation are distinct. Continuous lines represent model simulations where either the elongation rate constant (green line), secondary nucleation rate constant (red line), or both (blue line) have been selectively inhibited.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaau3112/DC1

    Fig. S1. Analysis of the effects of clusterin on the aggregation kinetics of Aβ(M1-42) at 37°C.

    Fig. S2. Analysis of the effects of clusterin on the aggregation kinetics of Aβ(M1-42) at 21°C.

    Fig. S3. Seeding experiments of Aβ(M1-42) in the presence and absence of clusterin.

    Fig. S4. Diffusion profiles of specific samples at 12 different positions in a microfluidic device.

    Fig. S5. Analysis of interfering effects of transient proteins on clusterin inhibition activity.

    Fig. S6. Effects of the fluorescent label of clusterin on the inhibition process on Aβ(M1-42) aggregation.

    Fig. S7. Kinetic analysis on the aggregation kinetics of Aβ(M1-42) in the presence of Brichos and clusterin separately and combined.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Analysis of the effects of clusterin on the aggregation kinetics of Aβ(M1-42) at 37°C.
    • Fig. S2. Analysis of the effects of clusterin on the aggregation kinetics of Aβ(M1-42) at 21°C.
    • Fig. S3. Seeding experiments of Aβ(M1-42) in the presence and absence of clusterin.
    • Fig. S4. Diffusion profiles of specific samples at 12 different positions in a microfluidic device.
    • Fig. S5. Analysis of interfering effects of transient proteins on clusterin inhibition activity.
    • Fig. S6. Effects of the fluorescent label of clusterin on the inhibition process on Aβ(M1-42) aggregation.
    • Fig. S7. Kinetic analysis on the aggregation kinetics of Aβ(M1-42) in the presence of Brichos and clusterin separately and combined.

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