Fig. 1: EVs cleave α-syn Preformed Fibrils (PFFs) in vitro.
From: Proteolytic activities of extracellular vesicles attenuate A-synuclein aggregation
A Mouse PFFs (mPFFs) were incubated in vitro for 24 h at 37oC with and without EVs (Exo), and in the presence or absence of EDTA or EDTA with protease inhibitors cocktail (PIs) at a PFF:EV ratio 1:2. Immunoblotting with the Syn-1 antibody identified a truncated α-syn 10 kDa band upon incubation of mPFFs with EVs that was inhibited in the presence of protease inhibitors. B Western blotting of samples from (A) for α-syn with the C-20 antibody revealed that EVs degraded monomeric and oligomeric α-syn. The use of protease inhibitor cocktail inhibited the proteolytic effect of EVs on mPFFs and aided the formation of oligomeric α-syn species. C Human PFFs (two different preparations H1-H2 PFFs) were incubated as in (A) and (B) with EVs in the presence or absence of protease inhibitors and analyzed with Western blotting using the Syn1. As with the mPFFs, hPFFs were readily cleaved by the EVs in the same manner. D Western blotting of samples from (C) for α-syn with the C-20 antibody showed that inhibition of EV proteolytic activities resulted in increased formation of HMW α-syn species as with mPFFs. E Investigation of EV proteolysis in a time-dependent manner. hPFFs incubated with EVs displayed effective proteolysis at 6 h in a ratio of PFFs: EVs equal to 1:2. Staining for flotillin was used as a loading control for the EVs. Representative blots shown, n = 3.
