FIGURE 4.

Exosome-bound preformed fibrils (PFFs) induce minor pathology following injection in wild-type (wt) mouse brain. (A) Exosomes from α-syn KO brains were loaded with recombinant α-syn PFFs. Successful binding of PFFs on exosomes was verified by the shift of the purified exosome–PFF complex to fractions of lower sucrose density following ultracentrifugation on sucrose gradient as assessed with the Syn-1 antibody. (B) The amount of PFFs loaded to exosomes was determined by western blot analysis using the C20 antibody and comparing different dilutions of the exosome–PFF fraction with known concentrations of PFFs. Flotillin was used as a vesicular marker. Coronal midbrain sections of free and exosome-bound PFF-injected animals at 2 (C) and 7 (D) months post injection were stained with antibodies to phosphorylated α-syn and TH. At 2 months post injection, robust pathology was detected following free PFF injection as seen by accumulation of phosphorylated α-syn. Western blot analysis verified the presence of sodium dodecyl sulfate (SDS)-soluble phosphorylated α-syn species in the midbrain of PFF-injected animals. (D) At 7 months post injection, phosphorylated α-syn accumulations were significantly more in PFFs compared with exosome-bound PFF-injected animals. Graph shows the% phosphorylated α-syn accumulations within tyrosine hydroxylase (TH) neurons. (E) Stereological assessment of SNpc neurons in PFFs and exosome-bound PFF-injected animals at 7 months post injection. Significant TH-neuronal loss occurs following free PFF injections. Graph depicts the TH neurons as a % ratio of ipsilateral to contralateral side. Statistical analysis by Student’s t-test, n = 4 per group. ****p < 0.0001, **p < 0.01.