Fig 1.
Generation and confirmation of in vitro generated α-syn oligomers and fibrils.
A) Oligomers generated by resuspending lyophilised recombinant α-syn at high concentration (10mg/mL) incubated on ice and subsequently isolated using gel-filtration. Oligomers (O) are collected between 18–22 min. and monomers (M) between 38–43 min. Depicted gel filtration chromatogram is representative of oligomer elution profile observed in more than 10 oligomer preparations B) Representative graph of hydrodynamic radius in nm of isolated particles determined using DLS. Graph shows a merged image of the hydrodynamic radius (x-axis) of oligomers (dark grey) and monomers (white). Intensity of signal is depicted on the y-axis. (n = 3). C) Representative image of antigenicity of α-syn monomers, oligomers, and preformed fibrils to BD, FILA5 and MJF14 was determined using dot blot. Dots consist of 100ng protein spotted in duplicates (n = 3). Representative image of negative stain EM shows ultrastructure of native α-syn oligomer D) and E) ultrastructure of in vitro formed α-syn fibrils (n = 4).
Fig 2.
Cross-linking of α-syn monomers and oligomers.
A) α-syn monomers and oligomers were cross-linked with FA at different concentrations for 30 min. Representative immunoblot of monomers (left) and oligomers (right) show ASY1 binding. Monomeric α-syn situates at ~17kDa. Depletion of the ~17kDa α-syn band and presence of ASY-1 reactivity in the stacking gel suggest efficient cross-linking upon FA treatment of oligomers. (n = 4) B) Representative dot blot of 100ng non-treated- or 1.6% FA cross-linked α-syn monomers and oligomers using aggregation-specific FILA5 and MJF14 antibody. Prior to dot-blot, one subset of native- and cross-linked oligomers were treated with 0.4% SDS for 1 h. to assess oligomer stability (n = 3).
Fig 3.
Optimization of α-syn oligomer cross-linking.
A-C) α-syn oligomers were cross-linked with FA in a concentration of 0–3.2% for 30- or 60 min and subjected to immunoblot using ASY1 antibody to detect α-syn species (A). Monomer ~17kDa ASY-1 positive bands were quantified to assess degree of cross-linking and results are shown in B and C. Bar graphs show means ± SD obtained in three individual experiments. D-G) Monomers (black bars) and oligomers (grey bars) were cross-linked using different FA concentrations (0%-3.2% FA) for 60 min. The antigenicity of cross-linked monomers and oligomers were assessed via dot blot using ASY1 (D), BD (E), FILA5 (F) and MJF14 (G) antibodies. Figures show means ± SD of signal intensity, minus subtracted background signal, relative to non-cross-linked monomer samples (n = 4).
Fig 4.
Oligomers cross-linked using FA maintain their size and structure and resist denaturation.
A) Hydrodynamic radius of oligomers was monitored during 1.6% FA cross-linking using DLS. Measurements were taken before FA treatment (0 min) and 15-, 30- and 60 min after initiation of cross-linking (n = 3). B) Negative stain EM image shows ultrastructure of α-syn oligomer cross-linked for 60 min using 1.6% FA (n = 3). C) Thioflavin T fluorescence signal of 1.5μg α-syn monomer, oligomer, 1.6% FA cross-linked oligomer and preformed fibril (excitation at 450 nm and emission at 486 nm). Figure show means ± SD of three independent experiments. *p<0.05, **p<0.01, n.s. = not significant. D) Native and 1.6% FA cross-linked α-syn oligomer were treated with 6M Urea for 6h at RT or left untreated. Serial dilutions were analyzed by ELISA as previously described by Lassen et al. [23], utilizing the aggregated specific antibody MJF14. The levels are measured as absorbance at 450 nm. Two-way ANOVA for repeated measures followed by Tukey's multiple comparison test was used for ELISA experiment. Figure show means ± SD of three independent experiments. ****p<0.0001.