Molecular biology and pathology of prion strains in sporadic human prion diseases

Abstract

Prion diseases are believed to propagate by the mechanism involving self-perpetuating conformational conversion of the normal form of the prion protein, PrP(C), to the misfolded, pathogenic state, PrP(Sc). One of the most intriguing aspects of these disorders is the phenomenon of prion strains. It is believed that strain properties are fully encoded in distinct conformations of PrP(Sc). Strains are of practical relevance to human prion diseases as their diversity may explain the unusual heterogeneity of these disorders. The first insight into the molecular mechanisms underlying heterogeneity of human prion diseases was provided by the observation that two distinct disease phenotypes and their associated PrP(Sc) conformers co-distribute with distinct PrP genotypes as determined by the methionine/valine polymorphism at codon 129 of the PrP gene. Subsequent studies identified six possible combinations of the three genotypes (determined by the polymorphic codon 129) and two common PrP(Sc) conformers (named types 1 and 2) as the major determinants of the phenotype in sporadic human prion diseases. This scenario implies that each 129 genotype-PrP(Sc) type combination would be associated with a distinct disease phenotype and prion strain. However, notable exceptions have been found. For example, two genotype-PrP(Sc) type combinations are linked to the same phenotype, and conversely, the same combination was found to be associated with two distinct phenotypes. Furthermore, in some cases, PrP(Sc) conformers naturally associated with distinct phenotypes appear, upon transmission, to lose their phenotype-determining strain characteristics. Currently it seems safe to assume that typical sporadic prion diseases are associated with at least six distinct prion strains. However, the intrinsic characteristics that distinguish at least four of these strains remain to be identified.

Figure 1. Representation of the original identification of two PrP^Sc conformers as distinct strains subsequently named types 1 and 2

The two PrP^Sc conformers, referred to as types 2 and 1, were respectively associated with fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (fCJD^D178N) (A), two familial prion diseases that share the same D178N mutation in the PrP gene but have distinct phenotypes as demonstrated by the opposite topography of the lesions in thalamus and cerebral cortex (B). PrP^Sc types 2 and 1 also show distinct electrophoretic profiles (C), which can be appreciated by the distinct mobilities to kDa 19 and kDa 21 of the lowest electrophoretic bands corresponding to the unglycosylated fragments of PrP resistant to protease digestion. The different electrophoretic mobilities of the two PrP^Sc conformers support the notion that they have different conformations represented as different geometric forms (C). PrP^Sc types 2 and 1 associated with FFI and fCJD^E200K another form of familial CJD was replicated upon inoculation to receptive mice (D), even in the absence of the original PrP gene mutation. Familial CJD^D178N could not be transmitted to the same transgenic mice probably because 129M/129V mismatch. : The light blue triangle represents the conformation of the PrP^Sc conformer from FFI^D178N; : The green hexagon represents the conformation of PrP^Sc from fCJD^D178N; : The red square represents the conformation of PrP^Sc from fCJD^E200K which although being type one as fCJD^D178N- associated PrP^Sc is presumably different based on the fCJD^E200K distinct phenotype.

Figure 2. Western blots of PrP^Sc types 1 and 2, and of PrP^Dis associated with VPSPr-129MM

The different electrophoretic mobilities of the PrP^Sc types 1 (T1A) and 2 (T2A-C) are evident. The capital letters identify the glycoform ratios that may occur in PrP^Sc types 1 and 2. The overrepresentation of the middle band containing the monoglycosylated isoform is indicated with A; B indicates the overrepresentation of the upper band (diglycosylated isoform) and C that of the lowest band (unglycosylated isoform). The ladder-like profile formed by the PrP^Dis indicated as VPSPr is from VPSPr-129MM but this profile is shared by the three VPSPr 129 genotypes. All preparations have been treated with proteinase K. The sporadic Creutzfeldt-Jakob disease (sCJD) and variant (v) CJD preparations have been probed with 3F4, while the sporadic fatal insomnia (sFI) and the variably protease-sensitive prionopathy (VPSPr) preparations have been probed with IE4, both monoclonal antibodies to the prion protein.

Figure 3. Diagram of the cleavage pattern of PrP^Sc types 1 and 2 associated with the three 129 genotypes

The large arrowheads indicate the major sites of cleavage by proteinase K, which are further identified with the residue numbers of the prion protein (PrP) (lowest bar). The smaller arrowheads indicate the minor cleavage sites. Minor cleavage sites extend more towards the C-terminus in PrP^Sc type 1, while they extend more toward the N-terminus in at least 2 of the PrP^Sc type 2 preparations (MV2 and VV2). This phenomenon is more prominent when PrP^Sc types 1 and 2 are associated with the 129MV genotype indicating that in this association, PrP^Sc 1 and 2 N-termini are more ragged.

Figure 4. Conformation stability immunoassay (CSI) of the unglycosylated isoform of PrP^Sc from sCJDMM1, sCJDMM2 and sCJDMM1-2

To measure the stability of PrP^Sc, increasing concentrations of GdnHCl are used to unfold PrP^Sc, which in turn becomes progressively more sensitive to digestion with PK. The concentration of guanidine applied to achieve the digestion of 50% of the PrP^Sc with proteinase K (PK) is indicated by [GdnHCl]_1/2 (shown by the vertical color coded broken lines). The CSI curve shows that the stability of the PrP^Sc type 1 when it is present alone as in sCJDMM1 (red hollow diamond) is significantly different from the stability that it shows when it co-occurs with PrP^Sc type 2 as in sCJDMM1-2 (green solid triangle) ([GdnHCl]_1/2 values 2.96M vs. 1.96M, respectively; p<10^-5). Furthermore, the CSI curve of the PrP^Sc type 1 from sCJDMM1-2 becomes indistinguishable from that of PrP^Sc type 2 from sCJDMM2 (black square with hollow circle) and sCJDMM1-2 (blue solid circle), which are similar indicating no change in CSI characteristics between PrP^Sc 2 when alone or co-occurring with type 1. CSI profiles are smooth curves best matching data (mean ± SD) obtained at different concentrations of guanidine.

Figure 5. Proposed relationships between the 129 genotypes, PrP^Sc types and subtypes, phenotypes and strains in sporadic Creutzfeldt-Jakob (sCJD) and sporadic fatal insomnia (sFI)

The three 129 genotypes (and possibly other polymorphisms) are believed to influence the formation of PrP^Sc type 1 or 2 as the 129MM genotype is preferentially associated with type 1 while 129MV and 129VV are associated with type 2. The six possible combinations of the three 129 genotypes with PrP^Sc types 1 and 2 are associated with six distinct disease phenotypes, five belonging to sCJD and one to sFI. The pairing of 129 genotype-PrP^Sc type combinations with the phenotypes is not stringent. Notable exceptions are the association of the distinct MM1 and MV1 combinations with one phenotype (MM1/MV1) and the association of the single MM2 combination with two phenotypes (MM2 sCJD and MM2 sFI). It is proposed that the six phenotypes are paired with six distinct prion strains, three of which are relatively prevalent, while three are rare and might result from variations in the strain selection process. The distinct strain proposed to be associated with sCJDMV2 is questionable because of the inability to maintain its distinctive characteristics after transmission [5]. Coexistence of types 1 and 2 associated with the three 129 genotypes and the strains possibly associated with the novel disease variably protease-sensitive prionopathy (VPSPr), are not shown for simplicity, and because the presence of true strains in VPSPr is currently unclear.