Extracellular vesicles as novel mediators of pathology in Anderson–Fabry disease

Anderson-Fabry disease (AFD) is an X-linked lysosomal storage disorder caused by GLA mutations that reduce α-galactosidase A activity, leading to systemic accumulation of globotriaosylceramide (Gb3). Increasing evidence indicates that extracellular vesicles (EVs) contribute to pathogenic signal dissemination in lysosomal storage disorders. We hypothesized that EVs released from AFD-affected cells transfer toxic metabolites and molecular cues to recipient cells, thereby amplifying disease mechanisms. As a proof-of-concept study, urinary EVs were isolated from adults with AFD and healthy controls by ultracentrifugation and characterized by flow cytometry. Renal immortalized cells (HEK293) were treated with either healthy or AFD EVs to assess Gb3 accumulation, α-galactosidase A activity, and autophagic markers (Beclin-1, LC3 II/LC3-I ratio, and p62) at multiple time points. HEK293 cells exposed for 24 h to urinary EVs from 10 AFD patients showed Gb3 accumulation comparable to that observed in urinary epithelial cells from AFD subjects, along with increased α-galactosidase A activity relative to untreated and control-EV-treated cells. This was accompanied by impairment of autophagic flux as indicated by elevated LC3B-II/LC3-I ratios and accumulation of the autophagy substrate p62 after 72 h of exposure to AFD EVs. Autophagic inhibition was further supported by a significant reduction of Beclin-1 at 24-72 h in cells exposed to AFD EVs and increased Bcl-2 expression, a negative regulator of Beclin-1. These findings provide new insight into the potential involvement of EVs in AFD pathogenesis, suggesting that systemic manifestations may arise, at least in part, from EV-mediated transfer of pathogenic lipids and signaling molecules to recipient cells.