Figure 5.

Differential seeding by fibrillar α-Syn polymorphs in primary neuronal cultures. (A) A schematic representation of the protocol used to assess the seeding of endogenous α-Syn by exogenous fibrillar α-Syn polymorphs fibrils, ribbons, and fibrils-91 is shown. Primary mature hippocampal cultured neurons (prepared from WT C57BL6J mice) were exposed to fibrillar α-Syn polymorphs (250 nM, 15 min in fresh culture medium) at DIV 14. After extensive washing, the cells were transferred back to the original culture medium. Neurons were fixed at DIV 21 and immunolabeled for pS129-α-Syn. (B) Seeded endogenous α-Syn aggregation after exposure of primary neuronal cultures to the fibrillar α-Syn polymorphs fibrils, ribbons, and fibrils-91 is shown, imaged using the monoclonal anti-pS129-α-Syn antibody 81A. (C) Quantification of the area occupied by aggregated pS129-α-Syn after exposure to the different fibrillar α-Syn polymorphs is shown. The box plot shows median, interquartile range, and 10–90% distribution. Number of images (n): fibrils, 30; ribbons, 34; fibrils-91, 34, from three independent experiments. A Mann-Whitney test was performed, ^∗∗∗p < 0.001. The dot plot shows the averaged value per experiment. (D) Seeded endogenous pS129-α-Syn (green) within axons (red, labeled with anti-τ antibody) is shown. (E) pS129-α-Syn bundles (green) are stained by the autophagy marker p62 (red) in the cell body, but not in the processes (arrows). (F) pS129-Syn bundles (green) are stained by ubiquitin (red). (G) 1% sarkosyl extract from unseeded or polymorph-seeded neurons is shown. Western blots after migration of extracts on SDS-PAGE gels without stacking layer and probing for pS129-α-Syn (left), endogenous mouse α-Syn (middle), and tubulin (right) are shown. ^∗ represents the correct detected bands. Aggregated pS129-α-Syn can be detected in ribbons- and fibrils-91-seeded neurons (left and middle). Soluble endogenous mouse α-Syn is detected in all conditions (middle).