Cofactor molecules: Essential partners for infectious prions

Abstract

The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrP^Sc, a misfolded conformer of the normal prion protein, PrP^C. However, to date, all wild type protein-only PrP^Sc preparations lack significant levels of prion infectivity. Using a systemic biochemical approach, our laboratory isolated and identified two different endogenous cofactor molecules, RNA (Deleault et al., 2003 [50]; Deleault et al., 2007 [59]) and phosphatidylethanolamine (Deleault et al., 2012 [61]; Deleault et al., 2012 [18]), which facilitate the formation of prions with high levels of specific infectivity, leading us to propose to the alternative hypothesis that cofactor molecules are required to form wild type infectious prions (Deleault et al., 2007 [59]; Deleault et al., 2012 [18]; Geoghegan et al., 2007 [57]). In addition, we found that purified cofactor molecules restrict the strain properties of chemically defined infectious prions (Deleault et al., 2012 [18]), suggesting a "cofactor selection" model in which natural variation in the distribution of strain-specific cofactor molecules in different parts of the brain may be responsible for strain-dependent patterns of neurotropism (Deleault et al., 2012 [18]; Geoghegan et al., 2007 [57]).

Keywords: Cofactor; Cofactor selection; Mammalian; Phosphatidylethanolamine; Phospholipid; Polyanion; Prion; RNA; Specific infectivity.

Fig. 1

Cofactor molecules are required to form infectious prions. Western blots of reconstituted sPMCA reactions show adaptation of autocatalytic PrP^Sc molecules in the presence and absence of PE cofactor, as indicated. (−PK) = samples not subjected to proteinase K digestion; all other samples were proteolyzed. All reactions were initially seeded with cofactor PrP^Sc molecules. Reactions containing PE continued to propagate an ~18 kDa PK-resistant core (top blot). Approximately 40% of reactions lacking PE failed to propagate (middle blot), while ~60% adapted into a protein-only PrP^Sc conformer with an ~16 kDa PK-resistant core (bottom blot). End-point bioassays were performed in normal C57BL mice to determine the specific infectivity of in vitro-generated recombinant PrP^Sc molecules.

Fig. 2

Propagation with a single cofactor restricts prion strain diversity. (A) Regional neuropathology of infected mice. Profiles of vacuolation scores of animals inoculated with samples containing either input prions or cofactor PrP^Sc molecules, as indicated. Prion strains: OSU, red squares; Me7, orange circles; 301C, green triangles. Brain regions: CC, cerebral cortex (all layers); H, hippocampus; T, thalamus; HT, hypothalamus; Mid, midbrain; BS, brain stem; Cb, cerebellum. The mean values (n = 5–8) are shown±SEM. (B) Cofactor restriction in vitro. Schematic diagram illustrating the principle of “cofactor restriction.” In vitro propagation with a single cofactor forces multiple prion strains to adapt into a single PrP^Sc conformer with identical strain properties.

Fig. 3

Infectivity and strain information can be fully restored from protein-only PrP^Sc. End-point bioassays were performed in normal bank voles to determine the specific infectivity of in vitro-generated samples. Cofactor PrP^Sc was used as a seed to produce protein-only PrP^Sc by sPMCA propagation in the absence of cofactors. Protein-only PrP^Sc was subsequently used as a seed to produce [protein-only ➔ BH PrP^Sc] by sPMCA propagation in crude bank vole brain homogenate in a cofactor-dependent reaction. Bioassay results show restoration of both high specific infectivity and original strain properties.

Fig. 4

Restored PrPSc preserves original strain information. (A) Regional neuropathology bank voles with cofactor recPrP^Sc or [protein-only ➔ BH PrP^Sc]. Profiles of vacuolation scores of animals inoculated with either cofactor recPrP^Sc (black squares) or [protein-only ➔ BH PrP^Sc] (blue circles) show nearly identical patterns of neurotropism. Mean values±SEM are shown. N = 6 for all measurements except for [protein-only ➔ BH PrP^Sc] cerebellum and pons, where N = 3. (B) Cofactor selection in vitro. Schematic diagram illustrating the principle of “cofactor selection.” In vitro propagation of single prion strain in a diverse mixture of potential cofactor molecules (e.g., crude brain homogenate) faithfully maintains the original PrP^Sc conformation and strain properties of the seed.

Fig. 5

Cofactor selection model explains neurotropism. Schematic diagram shows hypothetical model to explain why strain-specific PrP^Sc conformers have characteristic patterns of neurotropism. The model proposes that each prion strain has a PrP^Sc conformation that can only be formed by a specific set of endogenous cofactors and that different endogenous cofactors are proposed to be naturally enriched in different regions of the brain (top of diagram). Therefore, each strain-specific PrP^Sc conformer would be expected replicate and accumulate preferentially in the brain regions that have higher levels of the specific cofactor molecules that are able to facilitate its formation (bottom of diagram).