C-Terminal Truncated α-Synuclein Fibrils Contain Strongly Twisted β-Sheets

Abstract

C-terminal truncations of monomeric wild-type alpha-synuclein (henceforth WT-αS) have been shown to enhance the formation of amyloid aggregates both in vivo and in vitro and have been associated with accelerated progression of Parkinson's disease (PD). The correlation with PD may not solely be a result of faster aggregation, but also of which fibril polymorphs are preferentially formed when the C-terminal residues are deleted. Considering that different polymorphs are known to result in distinct pathologies, it is important to understand how these truncations affect the organization of αS into fibrils. Here we present high-resolution microscopy and advanced vibrational spectroscopy studies that indicate that the C-terminal truncation variant of αS, lacking residues 109-140 (henceforth referred to as 1-108-αS), forms amyloid fibrils with a distinct structure and morphology. The 1-108-αS fibrils have a unique negative circular dichroism band at ∼230 nm, a feature that differs from the canonical ∼218 nm band usually observed for amyloid fibrils. We show evidence that 1-108-αS fibrils consist of strongly twisted β-sheets with an increased inter-β-sheet distance and a higher solvent exposure than WT-αS fibrils, which is also indicated by the pronounced differences in the 1D-IR (FTIR), 2D-IR, and vibrational circular dichroism spectra. As a result of their distinct β-sheet structure, 1-108-αS fibrils resist incorporation of WT-αS monomers.

Figure 1

Seeding/cross-seeding profiles/rates of WT-αS (black circles) and 1–108-αS (blue diamonds). Representative aggregation profiles of monomeric WT-αS with WT-αS seeds (a), monomeric 1–108-αS with 1–108-αS seeds (b), monomeric 1–108-αS with WT-αS seeds (c), and monomeric WT-αS with 1–108-αS seeds (d), respectively. The monomer (circles for WT-αS and diamonds for 1–108-αS) concentration was 35 μM, and the seed (shown as bars) concentration was 1% (v/v) in PBS buffer at 37 °C. Shaded regions in panels a–d indicate SD from an aggregation experiment with at least 6 replicates. (e) Aggregation seeding rates obtained from a linear fit of the first 3000 s of the normalized seeded aggregation curves (Experimental Section) from three independent aggregation experiments. Red arrows in panels a–d refer to time-points at which samples were obtained for STEM images shown in Supporting Figure S2.

Figure 2

STEM micrographs (a, b), CD spectra (c), and FE fluorescence spectra (d) of WT-αS and 1–108-αS fibrils. Inset in panels a and b show suspensions of αS fibrils after 2 h. Settled aggregates of fibrils (densely white) are seen in the case of 1–108-αS fibrils only (b). Plotted data in c and d from WT-αS are depicted in black and that of 1–108-αS in blue. (c) CD spectra of WT-αS and 1–108-αS before (dotted lines) and after (solid lines) aggregation. Suspensions of fibrils were filtered to get rid of monomeric αS prior to measurement (Experimental Section). (d) Fluorescence emission spectra (λ_exc = 420 nm) of FE-dye interacting with αS fibrils showing that the short-emission N* band (∼510 nm) is red-shifted for 1–108-αS fibrils with a higher band ratio of fluorescence emission peaks compared to the WT-αS fibrils.

Figure 3

Structural characterization of WT-αS and 1–108-αS fibrils. All data from experiments with WT-αS are depicted in black and that of 1–108-αS in blue. (a) FTIR spectra of αS fibrils of the amide-I region. In gray and light blue are the calculated spectra as described in the text for an intersheet distance of 8.98 and 10.3 Å, respectively. (b, c) Perpendicular 2D-IR spectra of WT-αS (b), 1–108-αS fibrils (c), and their corresponding diagonal slices (d). (e) X-ray diffraction patterns of partially aligned αS fibrils depicting differences in the short-range reflections (arrows in gray background indicate peaks corresponding to intersheet distances) of WT-αS and 1–108-αS fibrils. (f, g) AFM amplitude images of αS fibrils on mica. Purified fibrils of WT-αS (f) show typical rod-like fibrillar morphology, while the 1–108-αS fibrils (g) show both higher-ordered fibrillar structures and sparse rod-like fibrils (insets). The scale bar is 1 μm. (h) VCD spectra of WT-αS and 1–108-αS fibrils.