Untwisted α-Synuclein Filaments Formed in the Presence of Lipid Vesicles

Abstract

Accumulation of filamentous aggregates of α-synuclein is a pathological hallmark of several neurodegenerative diseases, including Parkinson's disease (PD). The interaction between α-synuclein and phospholipids has been shown to play a critical role in the aggregation of α-synuclein. Most structural studies have, however, been focused on α-synuclein filaments formed in the absence of lipids. Here, we report the structural investigation of α-synuclein filaments assembled under the quiescent condition in the presence of anionic lipid vesicles using electron microscopy (EM), including cryogenic electron microscopy (cryo-EM). Our transmission electron microscopy (TEM) analyses reveal that α-synuclein forms curly protofilaments at an early stage of aggregation. The flexible protofilaments were then converted to long filaments after a longer incubation of 30 days. More detailed structural analyses using cryo-EM reveal that the long filaments adopt untwisted structures with different diameters, which have not been observed in previous α-synuclein fibrils formed in vitro. The untwisted filaments are rather similar to straight filaments with no observable twist that are extracted from patients with dementia with Lewy bodies. Our structural studies highlight the conformational diversity of α-synuclein filaments, requiring additional structural investigation of not only more ex vivo α-synuclein filaments but also in vitro α-synuclein filaments formed in the presence of diverse cofactors to better understand the molecular basis of diverse molecular conformations of α-synuclein filaments.

Figure 1:

Aggregation kinetics of α-synuclein (60 μM) in the absence and presence of the DMPS SUVs (100 μM) at pH 6.5 (a) and pH 7.4 (b) under the quiescent condition. ThT fluorescence of all of the α-synuclein samples in the absence of the DMPS vesicles was nearly zero at the incubation periods.

Figure 2:

TEM images of G51D α-synuclein at pH 6.5 and 30 °C (a), WT α-synuclein at pH 6.5 and 30 °C (b), G51D α-synuclein at pH 6.5 and 37 °C (c), and WT α-synuclein at pH 6.5 and 37 °C (d). Protein samples (60 μM) were incubated for 7 days in the presence of DMPS (100 μM) lipid vesicles of an average diameter of 70 nm. No filaments were observed in the absence of the lipid vesicles.

Figure 3:

Representative cryo-EM micrographs of G51D α-synuclein (60 μM) incubated for 30 days in the presence of DMPS (100 μM) vesicles with an average diameter of 70 nm.

Figure 4:

(a–f) Representative reference-free 2D class averages of G51D α-synuclein filaments formed in the presence of DMPS SUVs were obtained with a box size of 56 nm.

Figure 5:

(a) CD spectra of α-synuclein filaments (solid lines) and fibrils (dotted lines) formed in the presence and absence of DMPS lipids, respectively. CD spectra of the DMPS-derived WT and G51D α-synuclein (10 μM) were measured using a 1 mm pathlength quartz cuvette on a Jasco 815 spectrometer. (b) Aggregation kinetics of WT and G51D α-synuclein (60 μM) seeded with the DMPS-derived α-synuclein filaments (0.6 μM) in the presence of ThT at 37 °C under constant agitation at 250 rpm. Aggregation kinetics were monitored by measuring ThT fluorescence with excitation and emission wavelengths of 440 and 482 nm, respectively. (c) Proteinase K digestion analyses of DMPS-derived WT (red) and G51D (yellow) α-synuclein filaments. α-Synuclein filaments (14 μM) were mixed with proteinase K (0.5 ng/μL) and incubated for 1 min (bands 1 and 2), 10 min (bands 3 and 4), and 20 min (bands 5 and 6). The core bands after the digestion are marked in red (WT) and yellow (G51D). Bands 7 and 8 are the α-synuclein monomer and the protein ladder, respectively. Protein samples were run on 4–12% SDS-PAGE gel. (d) MTT cell viability assay for the DMPS-derived WT and G51D α-synuclein filaments at 10, 20, and 40 μM monomeric concentrations.