pH Dependence of Amyloid-β Fibril Assembly Kinetics: Unravelling the Microscopic Molecular Processes

Abstract

Central to Alzheimer's disease (AD) is the assembly of the amyloid-beta peptide (Aβ) into fibrils. A reduction in pH accompanying inflammation or subcellular compartments, may accelerate fibril formation as the pH approaches Aβ's isoelectric point (pI). Using global fitting of fibril formation kinetics over a range of pHs, we identify the impact net charge has on individual fibril assembly microscopic rate constants. We show that the primary nucleation has a strong pH dependence. The titration behaviour exhibits a mid-point or pK_a of 7.0, close to the pK_a of Aβ histidine imidazoles. Surprisingly, both the secondary nucleation and elongation rate constants are pH independent. This indicates the charge of Aβ, in particular histidine protonation, has little impact on this stage of Aβ assembly. These fundamental processes are key to understanding the forces that drive the assembly of Aβ into toxic oligomers and fibrils.

Keywords: Alzheimer's Disease; Amyloid; Isoelectric Point; Kinetics; Molecular Mechanism.

Figure 1

pH‐dependent fibril formation kinetics of Aβ40 and Aβ42. ThT kinetic traces (n=4) at pH 6.0, 7.0 and 8.0 for Aβ40 (A) and Aβ42 (B), see also supplemental S2 and S3. Single representative traces for Aβ40 (C) and Aβ42 (D) between pH 6.0–8.0, from left (black, pH 6.0) to right (purple, pH 8.0). E) Plots of t _1/2 versus pH, with pK _a fitted. F) Plots of growth‐time versus pH; error bars are standard error of the mean (SEM) from four replicates.

Figure 2

pH effects primary nucleation processes of Aβ42 aggregation. A–C) Kinetics profiles of Aβ42 (5 μM) at pH 6.0–8.0, from left (black, pH 6.0) to right (purple, pH 8.0). The solid lines represent global fits of the kinetic traces when only primary nucleation (A), secondary nucleation (B) and fibril elongation (C) rate constants are altered to globally fit pH dependent traces. (D) Change in primary nucleation rate constants (k_n ) versus pH, derived from global fits in Figure 2A, error bars are SEM from four replicates. E) Schemes of the microscopic steps for primary nucleation, secondary nucleation, and fibril elongation.

Figure 3

Seeded fibril formation is pH independent. This seeded kinetics indicates secondary nucleation (k ₂) and elongation (k ₊) rates are independent of pH. A) ThT kinetic traces (n=4) at pH 6.0, 7.0 and 8.0 for Aβ40 (5 μM) with 10 % fibril seed, see also Figure S7. B) Single representative traces for Aβ40 between pH 6.0–8.0. C) t _1/2 versus pH, error bars are SEM from 4 replicates. D) Growth‐time versus pH.

Figure 4

pH has negligible effect on the morphology of Aβ40 and Aβ42 fibrils. Negatively stained TEM fibril images produced at pH 6.0, 7.0 and 8.0 for Aβ40 and Aβ42 (A, B). Further examples are shown Figure S8 and S9. Scale bars: 500 nm; inset 50 nm. Node‐to‐node distance of Aβ40 (C) and Aβ42 (D) fibril twists at pH 6.0, 7.0 and 8.0. N=50 individual fibrils are measured per condition. The median value is shown as a line, boxes are 25–75 % of median values, error bars are 1.5 interquartile range.