Structural heterogeneity of α-synuclein fibrils amplified from patient brain extracts

Abstract

Parkinson's disease (PD) and Multiple System Atrophy (MSA) are clinically distinctive diseases that feature a common neuropathological hallmark of aggregated α-synuclein. Little is known about how differences in α-synuclein aggregate structure affect disease phenotype. Here, we amplified α-synuclein aggregates from PD and MSA brain extracts and analyzed the conformational properties using fluorescent probes, NMR spectroscopy and electron paramagnetic resonance. We also generated and analyzed several in vitro α-synuclein polymorphs. We found that brain-derived α-synuclein fibrils were structurally different to all of the in vitro polymorphs analyzed. Importantly, there was a greater structural heterogeneity among α-synuclein fibrils from the PD brain compared to those from the MSA brain, possibly reflecting on the greater variability of disease phenotypes evident in PD. Our findings have significant ramifications for the use of non-brain-derived α-synuclein fibrils in PD and MSA studies, and raise important questions regarding the one disease-one strain hypothesis in the study of α-synucleinopathies.

Fig. 1. Amplification of aSyn aggregates from brain extracts.

a Immunoblotting of PMCA products with proteinase-K (PK) digestion. Similar PK-resistant signals were detected in PD samples (PD1-PD3), whereas nothing remained in the control sample (CT; brain extract from an individual, in which an α-synucleinopathy was excluded). An uncropped image of the blot is shown in Supplementary Fig. 1. b ThT-binding kinetics of recombinant aSyn with or without PMCA products as seeds. ThT-intensities for three PD samples (PD1-PD3) were averaged and used to derive error bars. c Relative ThT-fluorescence after seeding with different PMCA products. Error bars indicate standard deviation over three fluorescence measurements for each sample.

Fig. 2. Electron micrographs and circular dichroism spectra of aSyn fibrils.

a Electron micrographs of the two in vitro polymorphs hsAsyn (blue) and lsAsyn (red), as well aSyn fibrils amplified from brain extracts of a PD (green) and a MSA patient (purple; Table 1). b Circular dichroism spectra of aSyn fibrils.

Fig. 3. Fluorescent dyes distinguish aSyn aggregate structures.

a–c Normalized fluorescence spectra of curcumin (a), HS-68 (b) and FSB (c) in the presence of amyloid fibrils amplified from brain extracts of different patients (Table 1; PD/green, MSA/purple), as well as two in vitro aSyn polymorphs (hsAsyn/blue, lsAsyn/red)^,. d, e PCA of fluorescence spectra of the amyloid-binding dyes curcumin and HS-68 (d), and curcumin and FSB (e), in presence of aSyn fibrils. aSyn fibrils amplified from patient brain-extracts are identified according to Table 1. In addition to brain-extract amplified fibrils, the fluorescence spectra of various in vitro polymorphs were analyzed: hsAsyn (blue), lsAsyn (red), and amyloid fibrils formed through de novo aggregation under conditions of PMCA (termed de novo PMCA; yellow). For each sample, fluorescence spectra of the three dyes were measured independently twice, and the resulting values were joined combinatorially resulting in four data points per sample in order to represent the intrinsic variability in the fluorescence measurements.

Fig. 4. H/D exchange properties of aSyn in vitro polymorphs.

a–d Residue-specific HD-exchange profiles of different in vitro polymorphs (see text for further details). e Mapping of residue-specific protonation levels of hsAsyn on the structure of the hsAsyn filament (PDB code: 6A6B). The interface to the second filament, which involves A53, is marked. The scale bar indicates protonation levels relative to the minimum protonation level observed for V37-Q99. Residues with signal overlap were excluded from the analysis (shown in white). f Side view of (e). g, h Residue-specific differences in the protonation levels of hsAsyn and lsAsyn in the region from V37 to Q99 (g), mapped in (h) onto the core structure of hsAsyn fibrils (PDB code: 6A6B). Difference values were calculated on the basis of the average protonation levels observed for two independently seeded samples (hsAsyn I/II and lsAsyn I/II, respectively). Error bars represent std. NACore is the most hydrophobic part of the aSyn sequence. The interface between two aSyn filaments as seen in the structure of hsAsyn (PDB code: 6A6B) is marked.

Fig. 5. Molecular level insights into the structure of aSyn fibrils amplified from patient brain extracts.

a HD-exchange profiles of aSyn fibrils amplified from brain extracts of PD patients (left, green) and MSA patients (middle, purple). HD-exchange profiles of four de novo PMCA aSyn fibrils, which are not based on seeding/propagation, are shown in the right column (yellow to orange) together with the residue-specific average values for six de novo PMCA fibril samples. Error bars represent std. b PCA of protonation levels in aSyn fibrils (see text for further details). Independent repetition of HD exchange measurements for fibrils obtained from patient MSA5 are included (indicated by black arrows).

Fig. 6. Structural diversity in aSyn aggregates amplified from patient brain extracts.

a, b Differences between protonation levels in aSyn fibrils amplified from PD1/PD2- and MSA1/2-brain extracts, mapped in (b) on the core structure of hsAsyn fibrils. Difference values were calculated on the basis of the average protonation levels observed for PD1 and PD2 (and MSA1 and MSA2, respectively). Errors were calculated from the differences in protonation levels between the two patient profiles of either PD or MSA. Residues that experience faster solvent exchange in MSA1/2 fibrils (when compared to PD1/PD2 fibrils) are shown in orange, those with slower solvent exchange in green. Residues with signal overlap were excluded from the analysis (shown in gray in (b)).

Fig. 7. EPR spectroscopy of aSyn fibrils.

a Schematic representation of the location of MTSL spin labels (green balls) attached to residues 54 and 90. b Continuous wave EPR spectra of aSyn fibrils. Fits derived from a two-spin simulation are shown as colored lines. c Dynamic parameters (weight: fast/slow) of MTSL spins derived from fitting continuous wave EPR spectra. For both spin species, g ≈ [2.008, 2.006, 2.002] and A/h ≈ [17.8–19 18.5–19 93–110] MHz. The peak-to-peak isotropic linewidths (lwpp) for the two species were lwpp_(slow) = 0.52–0.67 mT and lwpp_(fast) = 0.3–0.4 mT, respectively. d Dipolar modulation (without background correction) of aSyn fibrils from 34 GHz four-pulse PELDOR experiments. e Dipolar modulation (corrected for background) and normalized distance distribution determined by 34 GHz four-pulse PELDOR experiments for hsAsyn (blue) and lsAsyn (red).