PRNP mutations initially generate an alternatively misfolded PrP species that dissuades prion replication

Abstract

Mutations in the PRNP gene, which encodes the prion protein (PrP), cause genetic prion disease. However, how PRNP mutations lead to spontaneous prion formation remains poorly understood. Building off the observation that expression of mutant bank vole PrP (BVPrP) in mice causes spontaneous prion disease, we sought to identify early events in prion formation by expressing mutant BVPrP in cultured cells lacking endogenous PrP. Expression of D178N- and E200K-mutant, but not wild-type BVPrP, in CAD5-PrP-/- and N2a-PrP-/- cells generates an alternatively misfolded PrP species, which we term PrPAM, that is detergent-insoluble and resistant to digestion with the protease thermolysin. PrPAM is also present in the brains of young, asymptomatic knock-in mice expressing mutant BVPrP. However, PrPAM does not appear to be a direct precursor of prions since 1) the prion disease-protective G127V substitution enhances PrPAM levels when coupled with the D178N or E200K mutations; 2) small molecule anti-prion drugs fail to reduce PrPAM accumulation in cells; 3) unlike cells expressing wild-type BVPrP, cells expressing D178N- or E200K-mutant BVPrP are largely resistant to infection with several prion strains; and 4) PrPAM-containing cell homogenate does not contain seeds that induce misfolding of wild-type BVPrP in either cells or the RT-QuIC assay. These results raise the possibility that prion disease-causing PRNP mutations initially generate a protective misfolded PrP species that is refractory to prion formation, which provides a potential explanation for why genetic prion diseases manifest later in life despite the presence of a mutation from birth.