Figure 1

DMSO increases α-syn particle-size, aggregation-dependent antibody recognition and stimulates fibrillation (A and B) Dynamic light scattering analysis of α-syn (0.5 mg/mL, 35 µM) and carbonic anhydrase (0.5 mg/mL, 17 µM) incubated for 1 h with increasing concentrations of DMSO (0%, 2%, 5% and 10%). The figure demonstrates a mass% distribution based on the scattering intensity, with log scaled hydrodynamic radius depicted on the X-axis. Representative figure of three independent replicates (n = 3). (C) Dot blot of 100 ng of α-syn protein. Prior to loading, recombinant α-syn was incubated for 30 min at room temperature (RT) under non-agitating conditions in a concentration of 10 µg/mL (0.69 µM) in PBS alone or in combination with indicated concentrations of DMSO. α-syn was visualized using total α-syn (SYN-1) or MJFR-14–6-4–2 antibodies. (D) Quantification of MJFR-14–6-4–2/SYN-1 α-syn signal ratio normalized to 0% DMSO (n = 3, *p < 0.05, ***p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparison test). (E) Lyophilized recombinant α-syn was re-suspended in sterile PBS alone (0% DMSO) or in combination with increasing amounts of DMSO (1%, 5% or 10%). A sample with 10% DMSO alone was included as negative control. The samples were incubated at 37 °C and 1050 rpm in a final α-syn concentration of 0.5 mg/mL (35 µM). ThT measurements were performed on 10 µl samples added to 100 μl of 40 μM ThT (final concentration) and signal measured (excitation at 450 nm and emission at 486 nm). Values were normalized to day 0 measurements for each sample and fitted as a sigmoidal growth curve. Representative figure of three independent replicates (n = 3). (F) Sedimentation assay of samples depicted in E) after 7 days of incubation. Equal volumes of each sample (0%, 1%, 5% and 10% DMSO) were pelleted by 25.000×g centrifugation for 30 min at 20 °C. The pellets were re-suspended and resolved on SDS-PAGE and stained using Coommassie Brilliant Blue. Depicted supernatant and pellet gel stainings were developed on two individual SDS-PAGE gels (n = 3). (G) Lyophilized recombinant β-syn was re-suspended in sterile PBS alone (0% DMSO) or in combination with 10% DMSO. The samples were incubated at 37 °C and 1050 rpm in a final β-syn concentration of 0.5 mg/mL (35 µM). ThT measurements were performed on 10 µl samples (excitation at 450 nm and emission at 486 nm), and normalized to day 0 measurements. Representative figure of three independent replicates (n = 3).