Prion disease tempo determined by host-dependent substrate reduction

Abstract

The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrP Sc ), which is derived from its cellular precursor (PrP C ), as well as downregulation of the PrP-like Shadoo (Sho) glycoprotein. Given the overlapping cellular environments for PrP C and Sho, we inferred that PrP C levels might also be altered as part of a host response during prion infection. Using rodent models, we found that, in addition to changes in PrP C glycosylation and proteolytic processing, net reductions in PrP C occur in a wide range of prion diseases, including sheep scrapie, human Creutzfeldt-Jakob disease, and cervid chronic wasting disease. The reduction in PrP C results in decreased prion replication, as measured by the protein misfolding cyclic amplification technique for generating PrP Sc in vitro. While PrP C downregulation is not discernible in animals with unusually short incubation periods and high PrP C expression, slowly evolving prion infections exhibit downregulation of the PrP C substrate required for new PrP Sc synthesis and as a receptor for pathogenic signaling. Our data reveal PrP C downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains.

Prion Infectivity Plateaus and Conversion to Symptomatic Disease Originate from Falling Precursor Levels and Increased Levels of Oligomeric PrP Sc Species

“…Velocity gradient centrifugation was used to separate monomeric, oligomeric, and high molecular assemblies of PrP as previously described (5). Briefly, the 400-l aliquots of 10% brain homogenate containing 2% Sarkosyl were clarified and applied to a 10 to 45% sucrose gradient prepared in PBS (pH 7.4) containing 1% Sarkosyl.…”

“…The validation of CDI has been reported by the authors and other laboratories (9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Here, we utilized the recently modified protocol that adapts CDI to murine samples (5). First, Lumitrac 600 High-Binding 96-well plates (E&K Scientific) were coated with monoclonal antibody (MAb) 8H4 (epitope 175-185) in 200 mM NaH 2 PO 4 containing 0.03% (wt/vol) NaN 3 (pH 7.5) (19).…”

“…In other studies, based on falling levels of the PrP-like Shadoo (Sho) protein, we recently demonstrated that the extended asymptomatic stage encompasses a different chemical parameter, namely, the posttranslational downregulation of PrP C levels (5). We surmised that a functional relationship might exist between the PrP C downregulation seen at disease endpoint ("⌬PrP C ," as measured against mock-infected control mice) in different prion diseases and the formation of PrP Sc measured by conformationdependent immunoassay (CDI) (5).…”

“…We surmised that a functional relationship might exist between the PrP C downregulation seen at disease endpoint ("⌬PrP C ," as measured against mock-infected control mice) in different prion diseases and the formation of PrP Sc measured by conformationdependent immunoassay (CDI) (5). We explored this relationship for RML, 139A, Sc237, and CJD prion isolates using an in vitro protein misfolding cyclic amplification system (6,7).…”

“…We explored this relationship for RML, 139A, Sc237, and CJD prion isolates using an in vitro protein misfolding cyclic amplification system (6,7). In titrated amplification reactions using low rPrP Sc concentrations (to model an early stage in disease), a 30% decrease in PrP C substrate produced a 54% drop in replication rate (from 118-to 54-fold) of protease-resistant PrP Sc (5). However, these earlier studies neither addressed the full time course of disease nor the formation of infectious prion particles.…”