biostudies-literatureVekrellis KMichael J. Fox Foundation for Parkinson's ResearchNational Recovery and Resilience plan, Next Generation EUEuropean Regional Development Fund277https://www.ebi.ac.uk/biostudies/studies/S-EPMC12480265biostudies-literatureUnknown11(1)Extracellular vesicles (EVs) are nano-sized lipid vesicles released into the extracellular space. We investigated the role of mouse brain-derived EVs in α-synuclein (α-syn) degradation and pathology transmission. Using sucrose gradient isolation and biochemical characterization, we found that EVs harbor active proteases that cleave both monomeric α-syn and pre-formed fibrils (PFFs). Protease activity and inhibitor profiling identified cathepsins B and S as key enzymes mediating this cleavage. EV-mediated proteolysis reduced the seeding capacity of α-syn PFFs in vitro and in vivo, whereas protease inhibition enhanced aggregation. Proteomic analysis revealed a restricted protease repertoire within EV cargo. Our findings suggest that EVs regulate extracellular α-syn levels via proteolysis, thNPJ Parkinson's diseaseProteolytic activities of extracellular vesicles attenuate A-synuclein aggregation.PMC12480265022676TAEDR-0535850Τ2ΕDK- 02813Kolianou ASkarveli MKokoli MVekrellis KMelachroinou KSotiropoulou GSamiotaki MLamprokostopoulou AZingkou EfalseProteolytic activities of extracellular vesicles attenuate A-synuclein aggregation.Extracellular vesicles (EVs) are nano-sized lipid vesicles released into the extracellular space. We investigated the role of mouse brain-derived EVs in α-synuclein (α-syn) degradation and pathology transmission. Using sucrose gradient isolation and biochemical characterization, we found that EVs harbor active proteases that cleave both monomeric α-syn and pre-formed fibrils (PFFs). Protease activity and inhibitor profiling identified cathepsins B and S as key enzymes mediating this cleavage. EV-mediated proteolysis reduced the seeding capacity of α-syn PFFs in vitro and in vivo, whereas protease inhibition enhanced aggregation. Proteomic analysis revealed a restricted protease repertoire within EV cargo. Our findings suggest that EVs regulate extracellular α-syn levels via proteolysis, th2025-01-01T00:00:00Z2025 Sep2026-06-03T23:01:32.506Z2026-05-02T03:12:12.366ZS-EPMC124802654102273610.1038/s41531-025-01122-9