To obtain RK13 cell lines stably expressing mouse and values were calculated using Anova

To obtain RK13 cell lines stably expressing mouse and values were calculated using Anova. new PrPSc synthesis and as a receptor for pathogenic signaling. Our data reveal PrPC downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains. Introduction Prions are transmissible brokers responsible for incurable neurodegenerative diseases in humans and animals. Contamination with prions is unique in that they may manifest after a lag phase that can exceed 5 decades from the time of exposure (1). The generation of infectivity from recombinant prion protein (PrP) (2) supports the protein-only hypothesis, whereby prion replication corresponds to a conformational transition from the host-encoded, cellular prion protein (PrPC) substrate into a misfolded infectivity-associated form called PrPSc. Initiation of prion disease has an obligatory dependence upon the introduction of a PrPSc seed, and susceptibility to disease is usually blocked by PrP gene null alleles (3). Prion disease pathogenesis is usually associated with a progressive accumulation of PrPSc levels, with the qualification that PrPSc may encompass subforms that are biophysically and perhaps toxicologically distinct (4, 5). Besides PrPC, the Shadoo (Sho) glycoprotein is also expressed in the central nervous system. Sho has a similarity to the N-terminal region of PrP (6, 7) and interacts with several proteins that also interact with PrPC (8). While the function of Sho remains to be determined, a notable discovery was that levels of the Sho protein are reduced (downregulated) in prion disease (7, 9). Since this effect is apparent in the preclinical phase of disease (10, 11), we speculated that this phenomenon might be of broader importance for pathogenesis. In particular, the status of PrPC protein levels during prion contamination has been investigated little, because of the unaltered steady-state levels of the corresponding mRNA (12, 13) and a co-occurrence of PrPSc that may be present in molar excess. Here, to investigate PrPC, while avoiding the confounding presence of PrPSc, velocity gradient centrifugation was used to separate monomeric PrPC from oligomeric and high molecular assemblies made up of PrPSc (14). We then applied the conformation-dependent immunoassay (CDI), which steps a hidden epitope of PrPSc subsequent to its exposure by use of a chemical denaturant. By means of this 2-step procedure, we were able to analyze levels of different PrP species, including PrPC, protease-resistant PrPSc MK-5108 (VX-689) (rPrPSc), and protease-sensitive PrPSc (sPrPSc) (5). Our studies exhibited both quantitative and qualitative alterations in PrPC, with a number of implications for our understanding of prion disease pathogenesis. Results PrPC downregulation at end stage in scrapie, Creutzfeldt-Jakob disease, and chronic wasting disease models. The procedure to couple velocity gradient centrifugation with CDI (as explained in the Introduction) has been validated previously for samples derived from hamsters (5) and humans (15). To extend this approach to the analysis of mouse PrP, we recalibrated conditions for the use of the monoclonal antibody 12B2 (mouse PrP epitope at residues 88C92) (ref. 16 and Physique ?Determine1,1, A and B). We then applied the recalibrated technique to the analysis of mouse brain homogenates; these homogenates were derived from WT mouse brains harvested at clinical end stage after contamination with the Rocky Mountain Laboratory (RML) isolate of mouse-adapted scrapie prions. Our analyses exhibited that 93% of the upper gradient fractions, fractions 9 and 10, are composed of PrPC, while rPrPSc and sPrPSc make up the remaining 7% (Physique ?(Figure1B).1B). Since PrPC is the predominant PrP isoform, we attempted to use the protein within these gradient fractions as a substrate in an in vitro assay to make PrPSc; this MBP assay procedure is called protein misfolding cyclic amplification (PMCA) and is conceptually similar to PCR amplification of nucleic acids. However, proteins within the upper fractions of the velocity gradients were incompatible with our PMCA conditions and showed no detectable amplification, even though the levels of PrPC and concentrations of PrPSc after seeding with infected brain homogenate were comparable to the PMCA control experiment with nonfractionated material (Supplemental Physique 1; see complete unedited blots in the supplemental material; supplemental material available online MK-5108 (VX-689) with this article; doi: 10.1172/JCI72241DS1). Nonetheless, the low infectivity of these fractions, as determined by the standard scrapie cell assay (SSCA; a cell MK-5108 (VX-689) monolayer assay to determine the infectious titer for prions), supports the notion that this major component of these upper fractions is indeed PrPC (Physique ?(Figure1D).1D). In comparing RML prion-infected and noninfected animals, we discovered that.