Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation

Abstract

We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.

Keywords: amyloid aggregation; cryoelectron microscopy; neurodegeneration; protein misfolding; toxic oligomer.

Fig. 1.

Determination of the size distribution of the purified αS oligomeric samples. (A) HPLC-SEC analysis of the composition of the oligomeric samples; the oligomeric fraction eluted at 14.5 mL and the monomeric fraction at 22 mL (Top). The elution volumes for each protein standard used for the calibration are shown above (see also SI Appendix, SI Experimental Procedures). The fractions corresponding to the oligomeric peak were collected and separated in three groups: large oligomer HPLC fraction (area depicted in blue in the zoomed-in image of the oligomeric peak), medium-size oligomer HPLC fraction (green area), and small oligomer HPLC fraction (orange area). The three oligomeric solutions were then concentrated and reinjected into the HPLC-SEC column at the same mass concentration for their analysis (Bottom). (B) Native PAGE gel showing the different mobility of fibrillar (F), oligomeric (O), and monomeric (M) αS in comparison with protein markers (first lane). (C) Sedimentation velocity analysis of the αS oligomeric sample. The solid line represents the size distribution of sedimenting species obtained by c(s) analysis (Experimental Procedures). In Inset, the experimental data (symbols), their fit to the model (solid lines), and their residuals (below) are shown. For clarity, only one in every nine scans is represented. (D) Representative DLS-derived size distribution of 45 μM monomeric (red bars), 5 µM oligomeric (blue bars), and 3 µM fibrillar (black bars) αS solutions.

Fig. 2.

Morphological and structural characterization of the oligomeric αS species. Examples of AFM images of monomeric (A), oligomeric (B), and fibrillar (C) αS species. The color-coding represents the surface topography (height), and the scale bar is shown at the bottom of each image. (D) Representative TEM image of the αS oligomeric samples. Far-UV CD (E), FTIR (F), ThT fluorescence (G), and ANS fluorescence spectra (H) of monomeric (red line), oligomeric (blue line), and fibrillar αS solutions (black line) at the same mass concentration. The spectrum of the buffer is also shown as thin dashed black lines. In F, the position of the aborbance band characteristic of antiparallel β-sheet structure present in the oligomeric species but absent in the fibrillar forms is highlighted in blue.

Fig. 3.

Detailed characterization of the individual structural properties of the different sized subgroups of oligomers. (A) AU sedimentation velocity experiments with the oligomeric samples in the presence of different urea concentrations. A zoomed-in view of the size profile of the oligomeric fraction is shown in Inset. (B) Fractions of monomeric (in red) and oligomeric species (in blue) in the oligomeric samples, as a function of urea concentration, estimated by AU. The fraction of the two main oligomeric size subgroups is also represented: the 15S oligomer subgroup in green and the 10S oligomer subgroup in orange. The average sedimentation coefficient of the oligomeric fraction at different urea concentrations is also shown (black circles). The error bars represent experimental errors. Correlation of the secondary structure content (C) and the degree of hydrophobic surface area exposed to the solvent (represented as the wavelength of the maximum fluorescence emission of ANS) (D) with the size of the oligomers (blue symbols represent the experimental data and the line, the correlation function; Sl Appendix, SI Experimental Procedures). Estimates of the β-sheet content by FTIR of two independent oligomeric samples, corresponding to one prepared with freshly purified protein and another prepared with reused flow-through solutions (orange symbols; Sl Appendix, Fig. S4), overlie with the overall trend obtained by far-UV CD analysis (blue symbols). The orange and green arrows indicate the estimated averaged β-sheet content for the 10S and 15S oligomeric subgroup, respectively.

Fig. 4.

Three-dimensional reconstructions of the two main size subgroups of oligomers of purified oligomeric samples. (A) Example of cryoEM image of an oligomeric sample. Representatives of the two main particle subgroups are highlighted: with arrows, for the 15S oligomeric species; arrowheads, for the 10S oligomers. (B) Typical side view (Top) and end-on view (Bottom) of the small oligomeric subgroup (corresponding to the 10S oligomer subgroup) according to cryoEM. (C) The same views for the large structural group (corresponding to the 15S oligomer subgroup). (D) Two orthogonal views, side (Left) and end-on (Right), of the 3D reconstruction of the average structure for the 10S oligomer subgroup. (E) The same views for the 3D reconstruction of the structure representing the 15S oligomer subgroup.

Fig. 5.

Comparison of the purified αS oligomers with toxic oligomers previously found during αS fibril formation. (A) The single-molecule FRET efficiency distribution of a purified αS oligomeric sample (shown in gray bars) compared with the FRET distributions of the two main oligomeric species (type A and type B) previously found during αS fibril formation. (B) Proteinase K degradation curves of the different protein species: monomers in red, oligomers in blue, and fibrils in black. The data represented correspond to the average and SE of three different experiments. The degradation profile of type B oligomers is shown by green circles. (C) Time-resolved cytoplasmatic ROS production (HEt: dihydroethidium) in rat midbrain neuronal cultures after exposure to the different αS species: monomers in red, oligomers in blue, and fibrils in black. (D) Dose–response effect of monomers (M), oligomers (O), and fibrils (F) on the rate of cytoplasmatic ROS production. The basal rate of ROS production was taken as 100%. ***P < 0.0001. (E) Calcein release from LUVs of different POPS:POPC ratios after being incubated with monomeric (red bars), oligomeric (blue bars), and fibrillar (black bars) αS, at two different protein:lipid ratios (1:10 and 1:100). The pore-forming peptide, melittin, was used as positive control (green bars). The signal obtained when the detergent Triton X-100 was added to the vesicles was taken as 100%. Data points are averaged triplicates, and the error bars represent the SDs. *P < 0.5; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.