Fig. 7: De novo aggregation analysis of α-syn in the presence and absence of SVD-1a.

A De novo ThT assay of 10 µM α-syn with and without 20 µM SVD-1a. ThT fluorescence progression was measured in a 96-well non-binding half-area plate (Corning, USA) with a FLUOStar plate reader (BMG labtech, GE) at λ_ex = 448 nm and λ_em = 482 nm with 300 rpm continuous orbital shaking between reads. For induction of aggregation, one borosilicate bead per well was added (d = 3 mm, Hilgenberg, GE). Data are shown as mean values with ±SD (n = 5). B CD secondary structure analysis of de novo aggregation samples. Samples were incubated as described in (A) without added ThT (n = 3) and subsequently pooled for CD analysis. Far-UV ellipticity of the samples was measured in a quartz cuvette (l = 10 mm) in a J-1100 CD-spectrometer (Jasco, GE). In addition to (A), a sample with 20 µM SVD-1a alone was incubated under identical conditions and later used as a reference for the sample with α-syn and SVD-1a. For this sample (α-syn + SVD-1a (after incubation)), the SVD-1a reference subtracted CD spectrum is shown. C Samples from (A) were isolated directly after incubation and diluted in PBS pH 7.4 to a final concentration of 1 µM α-syn monomer equivalent. 5 µl diluted sample was 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 in (C) are representative of the observed species identified on all surface sections. D and E 10 µM α-syn monomer was incubated—analogous to the samples in (A)—with or without 5, 10, or 20 µM SVD-1a for 7 days (n = 7). The replicates were then united and loaded onto a 100 kDa MWCO centrifugal concentrator. In addition, a 10 µM α-syn sample was prepared and subjected directly to size-based fractionation. D α-Syn in the flow through (MWCO < 100 kDa) was quantified using an α-syn specific ELISA (BioLegend, Human α-Synuclein (Colorimetric), 448607, n = 3). E Retentate and flow-through samples were analyzed using SDS–PAGE with silver staining. The whole gel is shown in Supplementary Fig. 20.