Quantitative analysis of the time course of Aβ oligomerization and subsequent growth steps using tetramethylrhodamine-labeled Aβ

Abstract

Although amyloid β (Aβ) is a critical player in the pathology of Alzheimer's disease, there is currently little Information on the rate and extent of formation of oligomers that lead to the presence of Aβ fibrils observed in amyloid plaques. Here we describe a unique method to monitor the full time course of Aβ aggregation. In this method, Aβ is labeled with tetramethylrhodamine at a lysine residue on the N-terminal end. During aggregation, the fluorescence is quenched in a time-dependent manner in three distinct phases: an early oligomerization phase, an intermediate phase, and a growth phase. The oligomerization phase can be characterized as a monomer-dimer-trimer process for which we have determined the rate and equilibrium constants. The rate constants differ markedly between Aβ(1-42) and Aβ(1-40), with Aβ(1-42) showing a greater oligomerization propensity. The intermediate phase reflects slow clustering and reorganization of the oligomers, whereas the growth phase ultimately results in the formation of fibrillar material. The data are consistent with a conformational change being an important rate-limiting step in the overall aggregation process. The rates of all phases are highly sensitive to temperature and pH, with the pH-dependent data indicating important roles for lysine and histidine residues. From the temperature-dependent data, activation energies of oligomerization and fibrillization are estimated to be 5.5 and 12.1 kCal/mol, respectively. The methodologies presented here are simple and can be applied to other amyloidogenic peptides or proteins.

Fig. 1.

Normalized time course of changes in TMR fluorescence (□) and in CD (○). TMR-Aβ_1–42 (2 µM) was prepared in 20 mM phosphate buffer at pH 7.5 containing 15 mM NaCl, 0.1 mM EDTA, and 0.5 mM βMe. TMR fluorescence was monitored at 600 nm with excitation at 520 nm. The CD signal was monitored at 216 nm. Both measurements were performed on the same sample. Solid lines are sigmoidal fits to the data. The data were normalized by setting the fluorescence at zero time to 1 and that at long times to zero.

Fig. 2.

Time courses using TMR (red) or ThT fluorescence (blue) under various conditions. A–C use unlabeled or TMR-labeled Aβ_1–42 at (A) 25 °C, pH 7.5; (B) 50 °C, pH 7.5; and (C) 25 °C, pH 6.5. (D) Time course of unlabeled and TMR-labeled Aβ_1–40 at 25 °C, pH 7.5. All buffers contained 150 mM NaCl, 1 mM EDTA, and 5 mM βMe. The unlabeled Aβ samples contained 2 µM ThT and were monitored at an emission wavelength 470 nm with excitation at 438 nm. The data were normalized for comparative purposes.

Fig. 3.

Negative stain electron microscopy images of Aβ fibrils collected from the endpoints of the experiments shown in Fig. 2 prepared in pH 7.5 and incubated at 25 °C. (A) unlabeled Aβ_1–42, (B) unlabeled Aβ_1–40, (C) TMR-Aβ_1–42, and (D) TMR-Aβ_1–40.

Fig. 4.

Oligomerization of TMR-labeled Aβ. Time course of fluorescence change following dilution of a 100 µM stock solution containing monomeric (A) TMR-Aβ_1–42 or (B) TMR-Aβ_1–40, prepared in 4 M GdnCl, to final concentrations from 0.5 to 4.0 µM in 20 mM phosphate buffer at pH 7.5 containing 1 mM EDTA and 5 mM βMe. The black dots represent data, and the red lines are global fit of the data using a monomer-dimer-trimer model as described in Scheme 1. The rate constants obtained are summarized in Table 1. In all of the samples, the final concentration of GdnCl is 0.16 M. All experiments were performed at 25 °C without stirring.

Scheme 1.

Monomer-dimer-trimer process of Aβ oligomerization.

Fig. 5.

pH dependence of oligomerization and fibrillization. (A) Extent of loss of TMR fluorescence in 20 min following dilution of a 100 µM stock solution of monomeric TMR-Aβ_1–42 to 2.0 µM and (B) the inverse of half-time (t_1/2) of the growth phase of TMR-labeled Aβ_1–42 at different pH values. Empty squares represent data, and the solid lines represent sigmoidal fit of the data. The final buffer concentration was 20 mM phosphate at all pH values.

Fig. 6.

Temperature dependence of oligomerization and fibrillization. Arrhenius plots of (A) the extent of loss of TMR fluorescence in 20 min following dilution of a 100 µM stock solution of monomeric TMR-Aβ_1–42 to 2.0 µM and (B) the half-time of the growth phase of TMR-labeled Aβ_1–42 into 20 mM phosphate, pH 7.5 buffer at different temperatures. Empty squares represent data, and the solid lines represent fit of the data to Arrhenius equation as described in Eq. 1a.

Fig. 7.

Simulation of time course of oligomerization by Aβ_1–40 and Aβ_1–42. Time course of dimer and trimer formation for Aβ_1–42 (solid lines) and Aβ_1–40 (dotted lines) starting from 2 µM monomer. The curves were simulated by Kintek explorer (28) using the rate constants listed in Table 1.

Fig. 8.

Schematic view of aggregation based on time course of TMR fluorescence. The red curve is a typical fluorescence time course of TMR-Aβ. (A) Monomeric ensemble at t = 0, (B) small oligomers predominantly dimers and trimers formed during the oligomerization phase, (C) small oligomers cluster to larger oligomers during the intermediate or lag phase, and (D) β-structured fibrillar aggregates are formed and the solution is monomer depleted.