Fig.2.

Impact of storage conditions on alpha-synuclein pre-formed fibril (aSyn PFF) structure and pathogenicity. A-C) Representative transmission electron microscopy (TEM) images of aSyn PFF samples stored at varying temperatures before sonication. A) Samples stored at room temperature for 3–4 weeks display long fibrillar structures characteristic of unsonicated aSyn PFFs. B) Samples stored at –80°C for 4–5 months display long fibril structures as well as fractured, smaller aggregates. C) Samples stored in liquid nitrogen for 4–5 months display a distinct morphology characterized by fractured fibrils and non-specific aggregates (arrowhead). D–F) Analysis of the impact of storage conditions on aSyn PFF pathogenicity in primary hippocampal neuron cultures. Primary hippocampal neurons were exposed to fibrils at 0.2 μg/mL and fixed 6 days later. D–E) Immunofluorescence was performed using an antibody to pS129 aSyn to visualize inclusions (green) or to neurofilament heavy chain (blue) to visualize axons. Scale bars = 50 μm. Fibrils were either (D) generated immediately before adding to primary neurons or (E) stored at –80°C for 6 months before use. F) Quantitation of the percent area occupied by pS129 aSyn immunoreactivity reveals a highly significant (p < 0.001) difference between fresh and frozen aSyn PFFs, with much greater levels of pS129 aSyn induced by fresh aSyn PFFs as compared to frozen aSyn PFFs. Graph depicts mean values with error bars denoting standard deviation. aSyn, alpha-synuclein; PFF, pre-formed fibril; RT, room temperature; liquid N₂, liquid nitrogen; p-α-syn, pS129 aSyn.