Rho GTPases Differentially Modulate Prion Infection in Neuronal Cells

Introduction: The plasma membrane is a critical site for pathogen entry and constitutes a platform for prion protein conversion. Targeting essential regulators of endocytosis may thus inform on critical molecular pathways that drive prion propagation. Rho GTPases (Cdc42, Rac1, and RhoA), anchored to the cytoplasmic leaflet of the plasma membrane, are among the key regulators of clathrin-independent endocytosis.

Objectives:  Using a gene loss-of-function approach followed by de novo prion infection, we aimed to explore the role of Rho GTPase-mediated endocytic pathways in prion internalisation.

Methods: We transcriptionally silenced genes encoding upstream mediators and downstream effectors responsible for initiating macropinocytosis (Rac1, Cyfip1, Wasf2), RhoA-mediated endocytosis (Rhoa, Rock2, Diaph1/3), and the CLIC/GEEC pathway (Cdc42, Wasl). Following the transfection with target-specific and control siRNA pools, prion-susceptible Neuro2a (S7) cells were infected with exosomes harvested from RML-infected cells. Prion levels were quantified using the Scrapie Cell Assay, and effect levels were analysed using a linear mixed model.

Results/Discussion: Transcriptional silencing of genes involved in the CLIC/GEEC pathway reduced de novo prion infection by up to 74%. Strikingly, Cdc42 knockdown was nearly as effective as Prnp knockdown in blocking prion accumulation. In comparison, targeting pathways associated with macropinocytosis and RhoA-mediated endocytosis reduced prion infection by up to 20% and 36%, respectively.

Conclusion: Our results highlight the role of Cdc42 GTPase in de novo prion infection with exosomes, suggesting a potential route for establishing a prion infection in previously healthy cells via the CLIC/GEEC pathway.

Funding Information: Medical Research Council PhD Studentship (grant number: MC_ST_00037).