Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue

Abstract

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-2₁ helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.

Fig. 1:. Schematic illustrating extraction and amplification of LBD Asyn fibrils derived from postmortem tissue samples.

a, Fibrils were isolated in an insoluble fraction by sequential extraction and centrifugation of tissue samples. The insoluble fraction seeds were combined with recombinant Asyn monomer and subjected to multiple rounds of sonication followed by quiescent incubation at 37 °C to generate amplified fibrils. b-d, Representative negative stain TEM images of LBD amplified fibrils.

Fig. 2:. Seeding properties of tissue-derived and amplified fibrils in HEK 293 biosensor cells expressing A53T Asyn-CFP/YFP.

Fluorescence images show cells seed with: a, LBD-Insoluble fraction. b, MSA-insoluble fraction. c, LBD-amplified fibrils. d, MSA-amplified fibrils. e, LBD-tissue soluble fraction. f, MSA- tissue soluble fraction. g, LBD-amplified fibrils soluble fraction. h, MSA-amplified fibril soluble fraction. Similar results were observed in more than three independent experiments examining amplified fibrils derived from caudate samples for three cases of LBD and three cases of MSA.

Fig. 3:. SSNMR assignments, long range contacts, and MPL measurements showing 2 protofilaments are the dominant population of fibrils.

a, ¹³C-¹³C correlation of ¹³C-¹⁵N labeled LBD fibril showing carbonyl (left panel) and aliphatic (right panel) with unambiguous assignments labeled on the spectrum. Data was acquired at 750 MHz ¹H frequency with 16.667 kHz magic-angle spinning and sample temperature 10±5 °C with 75 ms DARR mixing. b, 2D projections of a (left) CO-N-H 3D and (right) CA-N-H experiment with backbone assignments of a ¹³C, ²H, ¹⁵N labeled sample. Data in (b) were acquired at 750 MHz ¹H frequency with 33.333 kHz magic-angle spinning, using a 6 ms ¹⁵N-¹³C cross polarization. 3Ds were collected using non-uniform sampling followed by reconstruction using SMILE prior to Fourier transformation. c, Peak heights are plotted for unambiguous assignments from a uniformly sampled CANH 3D spectrum collected on the uniformly ¹³C,¹⁵N-labeled sample. Bars are normalized to the peak height for residue A76. Predicted secondary structure from TALOS-N is plotted below the peak height plot for the LBD tissue-seeded sample. d, TALOS-N derived dihedral Phi and Psi angles reveal a beta-sheet structure. Predicted RCI chemical shift ordered parameter is plotted in blue showing highly ordered (R² >0.9) regions between G36 and G41, T64 and T81, and A85 to V95.

Fig. 4:. Long-range correlations determined by SSNMR.

a, Long-range unambiguous correlations identified using 12 ms of PAR mixing between L38 V70, G73, S87, I88 found uniquely resolved within a ¹³C-¹³C 2D correlation experiment. b, 2D strip plots from a 3D ¹³C-¹³C-¹³C correlation spectrum showing correlations from three threonine (T72, T75, T92) residues that are disambiguated in F1 and F2 relative to the 2D spectrum. c, Long-range contacts identified within a hCAhhNH experiment showing close contact between L38 with A76 and V77 and G41 with A69. d, Mass per unit length histogram of the LBD fibril measured with dark-field, unstained TB-TEM micrograph.

Fig. 5:. Structure of LBD fibrils.

a, Cartoon representation of the LBD fibrils determined via SSNMR studies. b, 10 lowest energy structures of LBD fibril determined. Blue represent well-ordered regions with large amount of intermolecular contacts; red represent relatively disordered regions with few intermolecular contacts detectable using CP based polarization techniques. c, Inter-residue contacts identified from SSNMR CP based experiments. Magenta lines are intramolecular contacts manually identified within spectra, blue lines are correlations identified utilizing the PASD algorithm for probabilistic assignments.

Fig. 6:. Fibril growth rates for in vitro assembled fibrils and amplified fibrils in the presence of mutant Asyn monomer.

a, WT-normalized growth rates in the presence of Asyn monomer with familial PD mutations A53T, E46K and H50Q. b, WT-normalized growth rates in the presence of Asyn monomer with S87K, S87Q, G68Q and A76T mutations. The results show that the A53T mutation distinguishes in-vitro (Tris buffer) fibrils from the LBD and MSA amplified fibril forms and that the E46K-mutation distinguishes MSA amplified fibrils from LBD amplified fibrils. The S87Q and A76T mutations distinguish LBD amplified fibrils from in-vitro and MSA amplified fibril forms.

Fig. 7:. ssNMR structure of amplified Asyn resembles cryo-EM structure of extracted filaments.

a, Primary sequence of human Asyn, with beta-strands annotated with arrows. The amphipathic N-terminal region, hydrophobic non-amyloid beta-component of plaque (NAC) region, and acidic C-terminal region are colored blue, yellow, and red, respectively. The purple, green, and orange rectangles denote the residue ranges used for subsequent comparison studies. b, Atomic model of SSNMR LBD Asyn core structure with labeled N- and C- termini (PDB 8FPT). Areas of low confidence are depicted with transparent cartoons with grey highlights. Residue ranges modeled with high confidence are depicted as cartoons with sidechains as sticks and are highlighted in purple, green, and orange. c, Atomic model of cryo-EM LBD Asyn core structure (PDB 8A9L), colored as in b. The unidentified proteinaceous islands are in grey. High-confident residue ranges of the SSNMR structure are overlayed in purple, green, and orange, and root-mean-square deviations (r.m.s.d) of the main-chain atoms were calculated. d, Number of satisfied NMR-derived interatomic distances mapped onto the lowest-energy structure using Xplor-NIH calculations on a per-residue basis using initial assignments from PASD. The color scale extends from white, indicating few or no NMR-derived distances were observed for the residue, to red, indicating many NMR-derived distances were observed for the residue. e, Number of satisfied NMR-derived distances mapped onto the cryo-EM Asyn core structure (PDB 8A9L).