Fig. 2: Amplification of pathogenic α-synuclein from PD and MSA patients by PMCA and characterisation of the resulting assemblies.

a PMCA was performed on PD (top panels, in red and corresponding controls, blue) and MSA (bottom panels, in green) and the corresponding controls (in blue). Patient brain homogenates (2% w:v) for the first cycle, in PMCA buffer (150 mM KCl, 50 mM Tris-HCl, pH 7.5) containing monomeric α-synuclein (100 µM). The following amplification cycles were seeded by 2 or 5% (v:v) of the preceding amplification cycles for PD and MSA, respectively. The amounts of brain homogenates and PMCA-amplified assemblies used in each amplification reaction were defined through an optimisation study aimed at maintaining high stringency by minimising the de novo aggregation of α-synuclein under the experimental conditions we used. The time at which an aliquot from a given amplification cycle was withdrawn for a subsequent amplification reaction (last timepoint for each amplification cycle) was also defined through an optimisation study aimed at avoiding the formation of de novo α-synuclein fibrillar assemblies. The curves represent an average of n = 4 replicates ± SD. b Electron micrographs of α-synuclein assemblies obtained after the third cycle of amplification by PMCA from each of the three PD, five MSA cases and de novo-generated α-synuclein fibrils and ribbons. For PD3, we examined the temporal gyrus (PD 3G) as well as the cingulate cortex (PD 3C). Scale bar: 200 nm. c Limited proteolytic patterns of α-synuclein assemblies obtained after the third cycle of amplification by PMCA from PD-, MSA- and de novo-generated α-synuclein fibrils and ribbons. Monomeric α-synuclein concentration is 100 µM. Proteinase K concentration is 3.8 µg/ml. Samples were withdrawn from the reaction, immediately after PK addition (lane most to the left, labelled 0) and at time 1, 5, 15, 30 and 60 min. PAGE analysis was performed and the gels were stained with Coomassie blue. The intensity of the proteolytic band generated at 15 min and indicated by the arrowhead was normalised to that of α-synuclein at time 0 for each PD (red) and MSA (green) patient sample (n = 4 PD and n = 5 MSA). PD patient-derived assemblies exhibited higher resistance to proteolysis than MSA patient-derived assemblies. In panel c, ***P < 0.001, two-sided unpaired Student’s t test. Source data for a, c are provided as a Source Data file.