Pericellular Proteases Modulate Cellular Infection by Extracellular Prion Deposits

Introduction: Secreted proteases are critical for maintaining extracellular matrix (ECM) homeostasis and actively contribute to ECM remodeling following brain injury and parenchymal damage. Extracellular deposits of Proteinase K-resistant PrP (PrPSc) are a pathological hallmark of prion diseases, but how host-pathogen interactions lead to prion dissemination and progressive degeneration are not well characterized. Objectives: Using a gene loss-of-function approach, we aimed to identify pericellular proteases that modulate prion infection induced by extracellular prion aggregates. Methods: To model extracellular prion deposits, we made use of fibrillar prion protein (PrP) aggregates that accumulate at the cell surface of persistently prion-infected neuroblastoma cells and penetrate the ECM. Upon trituration of cells, stable, highly infectious scaffolds are retained on the substrate. To identify key pericellular proteases, we transcriptionally silenced genes encoding secreted or membrane-bound proteases in uninfected reporter cells, which were then seeded onto infectious scaffolds. Results: Among the 31 expressed pericellular proteases screened, gene silencing of serine protease 2 (Prss2) and serine protease 36 (Prss36) resulted in the most pronounced reduction of prion infection, by 70% and 69%, respectively. Coagulation factor 7 (F7) and cathepsin L (Ctsl) also significantly reduced infection levels, by 45% and 44%. In contrast, loss of function of plasminogen activator, tissue type (Plat), matrix metalloproteinase 15 (Mmp15) and Mmp17 increased prion infection by 260%, 160% and 138%, respectively. Conclusion: Our findings provide a quantitative framework to classify pericellular proteases as either aggregation-enhancing or protective, offering mechanistic insight into their distinct modes of proteolytic processing.