Dynamics of oligomer populations formed during the aggregation of Alzheimer's Aβ42 peptide

Abstract

Oligomeric species populated during the aggregation of the Aβ42 peptide have been identified as potent cytotoxins linked to Alzheimer's disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach that combines theory, experiment and simulation, we reveal, in molecular detail, the mechanisms of Aβ42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we found that most Aβ42 oligomers dissociate into their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar structures. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to those of aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases.

Figure 1. Experimental procedures for the quantitative measurement of Aβ42 oligomer populations during an ongoing amyloid fibril self-assembly reaction using tritium labelling or mass spectrometry.

(a) We incubated varying concentrations of Aβ42 or Aβ40 monomers and collected aliquots at desired time points during the aggregation reaction. For each time point ($\Delta t$), we used centrifugation to remove fibrils. We then isolated the oligomeric fraction, encompassing species in the range of trimers to ca. 22-mers, through size-exclusion chromatography (SEC). We used a Superdex 75 column for which the void volume is ca. 7 mL and the monomer elutes at 14-16 mL. After separation through SEC, we used (b) liquid scintillation counting or (c) mass spectrometry (MS) to measure oligomer concentrations. In (b), we used liquid scintillation counting to measure the absolute mass concentration of peptides eluting between 7 and 13 mL in the case of ³H-labelled Aβ42. In (c), we used MS of natural abundance peptides, in which case each fraction (1 mL) was lyophilised, redissolved in 20 μL H₂O, supplemented by a defined amount of ¹⁵N-Aβ42 (10 pmol) and AspN enzyme, digested overnight, and analysed by MALDI-TOF MS. The peptide concentration in each fraction was determined as the ratio r of the integrated area of the ¹⁴N peak at 1906 m/z and the ¹⁵N peak at 1928 m/z as $c=r\times 10$ nM. The total oligomer concentration at each time point $\Delta t$ was calculated as the sum over fractions 7-12. The relative Aβ concentration in each fraction was then calculated by dividing c by the summed concentrations over fractions 7-12. (d) Observed concentration of oligomers versus aggregation time $\Delta t$. This procedure, which requires 10-16 minutes for oligomer isolation (Materials and Methods in Supplementary Sec. 1), provides a rapid and quantitative readout of the time evolution of oligomeric populations.

Figure 2. Kinetic analysis of Ab42 oligomer populations elucidates the molecular pathways of their dynamics during amyloid aggregation.

(a)-(c) Experimental measurements of (i) fibril formation at varying initial concentrations of Aβ42 (from Ref. [16]) and (ii) time evolution of the concentration of oligomers recorded starting from 5 μM Aβ42, and best fits (solid lines) to the integrated rate laws corresponding to different mechanistic scenarios for Aβ42 oligomer dynamics (Supplementary Secs. 3-5): (a) one-step nucleation producing elongation-competent oligomers, (b) two-step nucleation via oligomer conversion to growth-competent fibrils, and (c) two-step nucleation via conversion of unstable oligomers. See Supplementary Sec. 6 and Supplementary Table 1 for a detailed description of the fitting procedure and a list of fitting parameters. (d)-(e) Experimental measurements of fibril and oligomer kinetics at 5 μM Aβ42 in the absence (d) and presence (e) of 5 μM of the Brichos chaperone domain from proSP-C to detect the presence of off-pathway oligomers, i.e. oligomers that do not appreciably contribute to the reactive flux towards fibrils on the time scale of the experiment. Fibril mass measurements were fitted to the analytical expression for the aggregation time course (Supplementary Eq. 25) to determine how the overall rates constants for primary and secondary nucleation are affected by Brichos (Supplementary Sec. 6.4). The rate parameters determined in this way were then used to predict successfully the effect of Brichos on the oligomer concentration, without introducing any additional fitting parameters (Supplementary Eq. 28); this shows that suppressing oligomer formation on fibril surfaces affects equally the reactive fluxes towards oligomers and fibrils, indicating that the majority of oligomers is on-pathway to fibrils.

Figure 3. Concentration dependence of the molecular pathways of Aβ42 oligomer dynamics.

(a)-(c) Experimental measurements of the time evolution of oligomeric populations at varying concentrations of Aβ42 reveal the concentration dependence of oligomer conversion. (a) Global fit of experimental oligomer concentration data for 2.5, 5 and 10 μM Aβ42 to the integrated rate law corresponding to the model shown in Fig. 2c. Shaded areas indicate 68% confidence bands. See Supplementary Sec. 6 and Supplementary Tables 1-2 for a list of fitting parameters. (b) Concentration dependence of the fractional contribution of unconverted oligomers towards the reactive flux to mature fibrils. Error bars indicate standard deviation. (c) Extracted reaction orders for oligomer formation, oligomer conversion, and overall two-step secondary nucleation. (d)-(h) Computer simulation model of Aβ42 aggregation probes concentration dependence of oligomer conversion. (d) Possible protein and aggregate states in the computer model. (e) Mechanism of secondary nucleation in the computer simulations: monomers adsorb onto the fibril surface, and detach as oligomers, which then convert into fibrils in solution at a later time. However, based on the analysis of our experimental data, we cannot exclude the possibility that structural conversion and dissociation of Aβ42 oligomers occur in contact with, or close, to the fibril surface. (f) Rate of conversion of detached oligomers at varying monomer concentrations. (g) The fraction of converted oligomers in the total oligomer population at 3 different monomer concentrations. (h) Reaction orders for oligomer formation, oligomer conversion and overall two-step secondary nucleation as measured in the simulations.

Figure 4. Schematic illustration of the reaction pathways of oligomers during amyloid aggregation and the associated reaction rates determined in this work for Aβ42.

Amyloid fibril proliferation occurs through a two-step nucleation mechanism involving oligomer formation followed by oligomer conversion into fibrillar structures. The heterogeneous ensemble of oligomers includes not only converting species but consists mainly of unstable oligomers that can dissociate back to monomers. Oligomers undergo repeated cycles of formation-dissociation before eventually converting into species that are able to grow into new fibrils. The reaction rates are shown here for Aβ42 at a concentration of 5 μM (rate constants in Supplementary Sec. 6.3) and are to be interpreted as averages over the heterogeneous ensemble of oligomers. The geometric mean of the rates of oligomer formation, oligomer conversion and fibril elongation (which constitute the autocatalytic cycle of fibril self-replication, Supplementary Sec. 5.3) yields the characteristic rate of amyloid fibril formation (Supplementary Sec. 6.5 and Supplementary Fig. 17).