ADAM10-Mediated Proteolytic Remodelling of Signalling and Adhesion Proteins on Brain Cell-Derived Small Extracellular Vesicles

Proteases are common components of extracellular vesicles (EVs), yet the extent and functional relevance of ongoing proteolytic activity on EV surfaces remain largely unexplored. Such activity could significantly influence EV function and identity, with likely implications for EV-mediated signalling, recipient cell targeting, cargo delivery, and even translational applications ranging from biomarker discovery to therapeutic approaches. Here, we investigated the impact of sustained proteolysis on the composition of brain cell-derived EVs, focusing on A Disintegrin And Metalloprotease 10 (ADAM10), a key sheddase for signalling and adhesion proteins involved in neuronal and synaptic processes. Using primary rat cortical cultures, we found that numerous known ADAM10 substrates are part of small EVs (sEVs), and that their associated functions overlap with major sEV-mediated roles such as nervous system development, cell adhesion, and neurite outgrowth. Applying N-terminal proteomics to monitor sEV-derived cleavage fragments over time, we identified novel substrate candidates and demonstrated that sEV-associated ADAM10 activity remodels surface proteins involved in EV-cell interactions while generating soluble factors implicated in neuronal development. These findings suggest a previously unrecognised role for ADAM10 as a modulator of sEV composition and potentially cell-targeting specificity in the brain and position EVs as dynamic platforms for proteolytic processing 'on the move'.