Fig. 4: SVD-1a disassembles small α-syn PFF seeds into α-syn monomers.

Small α-syn PFF seeds were prepared as described previously. 100 or 200 nM monomer equivalent small α-syn PFF seeds were incubated with or without 400, 500, 1600 nM SVD-1a for 3 days at 37 °C in PBS pH 7.4. A and B Time-dependent DLS measurements with 200 nM small α-syn PFF seeds in the absence (A) and presence (B) of 500 nM SVD-1a. 1 ml sample was continuously measured in a sealed quartz cuvette at 37 °C under quiescent conditions every 60 s for 72 h in a SpectroSize 300 instrument (XtalConcepts, GE). Data are shown as a radius plot where the signal amplitude of each particle size is represented by the data point diameter. C One-phase exponential decay fit of “200 nM PFF + 500 nM SVD-1a” shown in (B). Fit was performed for particles in the range of 1–100 nm with an amplitude > 0.2. Outliers were excluded from fitting using a Q value of 1%. Fitting results: Span = 23.38 nm, rate constant = 0.132 1/s; half-life = 5.2 h. Fitting was performed using the “One phase exponential decay” fitting function from GraphPad Prism 10 (GraphPad Software Inc., USA). SVD-1a does not precipitate small α-syn PFF seeds as demonstrated by protein quantification after centrifugation, shown in Supplementary Fig. 12. D For AFM analysis, small α-syn PFF seeds pretreated with or without 400 and 1600 nM SVD-1a were incubated and dried on a freshly cleaved mica surface, followed by washing with ddH₂O and drying using a gentle stream of N₂. Analysis was performed using the NanoWizard 3 system (J-1100, JPK BioAFM, USA), recording multiple surface sections. The sections shown are representative of the observed species and particle density identified on all surface sections. E Exemplary Western blot of small α-syn PFF samples incubated with SVD-1a. 200 nM small α-syn PFFs (monomer equivalent) were incubated with increasing concentrations of SVD-1a for 72 h at 37 °C. PFF and monomer samples that were incubated without SVD-1a served as controls. α-Syn was detected with the antibody Syn211. A prominent α-syn monomer band was present at the expected molecular weight. Below the monomer bands, no further bands were visible. In the upper part of the blot, above the marker range, PFFs were detected, using a longer exposure time due to a weaker signal (see Supplementary Fig. 11 for the whole blot image with different exposure times). F Size-based fractionation by centrifugal concentration followed by ELISA quantification. 200 nM small α-syn PFF (monomer equivalent) were incubated with or without SVD-1a together with a 200 nM monomer control for 72 h at 37 °C. After incubation, the samples were fractioned using a 100 kDa MWCO centrifugal concentrator (Merck, Microcon DNA Fast Flow 100 MWCO). α-Syn content of flow through (red) and retentate (blue) was quantified using an α-syn specific ELISA (BioLegend, Human α-Synuclein (Colorimetric), 448607). Shown are the fractions of the retentate and flow through as a percentage of the total concentration identified for each sample. A linear regression line (blue or red dotted lines) was included for the SVD-1a-treated samples (left) to underline the concentration dependency of the SVD-1a treatment effect. The fractions identified for the α-syn monomer control sample are shown as a bar graph (right). Notably, the sample treated with 1000 nM SVD-1a shows the same distribution as the monomer control. Data are shown as mean values with ±SD (n = 3).