Fig. 4
Different CSF fractions differently affect α-syn aggregation. A Scheme of the CSF fractionation procedure. From a starting aliquot of 4.5 mL of CSF in PBS 1x, we collected 6 aliquots containing compounds of different molecular weight and froze them in liquid nitrogen. After every filtration with centrifugal filters, the flow-through of the filtered fraction was passed to a filter with smaller cut-off. B The addition of CSF fractions, whole NC-CSF pool, and PBS (40 μL) was analysed by ThT protein aggregation assay to evaluate effects on α-syn spontaneous aggregation. Background signal was corrected by subtracting the average fluorescence of three replicates containing PBS, whole CSF and CSF fractions without α-syn. All ThT fluorescence traces are represented as average intensity over 3 replicates with error bars representing the SEM. C Mean fitted A2 parameters (fitting was not possible for samples with whole CSF and the > 100 kDa fraction) and maximum fluorescence values (Fmax) estimated from individual ThT traces. Two scales of fluorescence intensity were used to better compare the results. Represented values correspond to the average of three replicates with error bars reflecting the SEM. D Relative concentration (emPAI score multiplied by protein molecular weight) of the most abundant protein constituents measured by nLC-nESI HRMS/MS. Apolipoproteins scores were summed together, with ApoA1 and ApoE being the most abundant (~ 85% of the total). Scores for fractions 10–3 and < 3 kDa are not shown since the protein content of these fractions was negligible with respect to the others