Figure 2.

Differential fibrillar α-Syn polymorphs binding and clustering on primary neuronal cultures. (A) Cultured hippocampal neurons (DIV 21–24) exposed for 5 or 60 min to the fibrillar α-Syn polymorphs fibrils, ribbons, fibrils-91, fibrils-65, and fibrils-110 (50 nM) labeled with ATTO-550, followed by fixation and immunolabeling of Homer (excitatory postsynapse marker), are shown (top two row: full field view; bottom two rows: boxed region). Full-field view of ATTO-550 fluorescence is shown in grayscale (top row) for better visualization of neuronal morphology. The images reveal striking differences in binding. Fibrils-91 bound much more efficiently than the ribbons and fibrils polymorphs. Fibrils-65 and fibrils-110 exhibited weak binding. All three polymorphs exhibited significant colocalization with Homer (bottom row). (B–D) Quantification of size (B), fluorescence intensity of fibrillar ATTO-550-α-Syn polymorphs clusters, density (C), number of fibrillar ATTO-550-α-Syn polymorphs clusters per μm², and synaptic colocalization (D) of fibrillar ATTO-550-α-Syn polymorph clusters obtained after thresholding are shown (see Material and Methods). (D) The proportion of synaptic fibrillar α-Syn polymorph clusters were similar for all polymorphs except fibrils-65 and fibrils-110. The box plot shows median, interquartile range, and 10–90% distribution. A Mann-Whitney test was performed to compare the distribution between 5 and 60 min; 60–75 images from four to five independent experiments. ^∗∗∗p < 0.001; ns, not significant. Dot plot shows the averaged value per experiment.