A novel mechanism of phenotypic heterogeneity in Creutzfeldt-Jakob disease.

One of remarkable features of sporadic Creutzfeldt-Jakob disease (sCJD) is the great phenotypic variability. Understanding the molecular basis of this variability has important implications for the development of therapeutic approaches. It is well established that, in many cases, phenotypic heterogeneity of sCJD is under control of two determinants: the genotype at the methionine (M)/valine (V) polymorphic codon 129 of the human prion protein gene and the type, 1 or 2, of the pathogenic and disease-related form of the prion protein, PrPD. However, this scenario fails to explain the existence of distinct heterozygous sCJDMV2 subtypes, where heterogeneity occurs without any variation of the 129 allotype and PrPD type. One of these subtypes, denoted sCJDMV2C, associated with PrPD type 2, is characterized by widespread spongiform degeneration of the cerebral cortex (C). The second variant, denoted sCJDMV2K, features prominent deposition of PrPD amyloid forming kuru type (K) plaques. Here we used a mass spectrometry based approach to test the hypothesis that phenotypic variability within the sCJDMV2 subtype is at least partly determined by the abundance of 129 M and 129 V polymorphic forms of proteinase K-resistant PrPD (resPrPD). Consistent with this hypothesis, our data demonstrated a strong correlation of the MV2C and MV2K phenotypes with the relative populations of protease-resistant forms of the pathogenic prion proteins, resPrPD-129 M and resPrPD-129 V, where resPrPD-129 M dominated in the sCJDMV2C variant and resPrPD-129 V in the sCJDMV2K variant. This finding suggests an important, previously unrecognized mechanism for phenotypic determination in human prion diseases.

Gerstmann-Sträussler-Scheinker disease revisited: accumulation of covalently-linked multimers of internal prion protein fragments.

Despite their phenotypic heterogeneity, most human prion diseases belong to two broadly defined groups: Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). While the structural characteristics of the disease-related proteinase K-resistant prion protein (resPrPD) associated with the CJD group are fairly well established, many features of GSS-associated resPrPD are unclear. Electrophoretic profiles of resPrPD associated with GSS variants typically show 6-8 kDa bands corresponding to the internal PrP fragments as well as a variable number of higher molecular weight bands, the molecular nature of which has not been investigated. Here we have performed systematic studies of purified resPrPD species extracted from GSS cases with the A117V (GSSA117V) and F198S (GSSF198S) PrP gene mutations. The combined analysis based on epitope mapping, deglycosylation treatment and direct amino acid sequencing by mass spectrometry provided a conclusive evidence that high molecular weight resPrPD species seen in electrophoretic profiles represent covalently-linked multimers of the internal ~ 7 and ~ 8 kDa fragments. This finding reveals a mechanism of resPrPD aggregate formation that has not been previously established in prion diseases.

PMCA-replicated PrPD in urine of vCJD patients maintains infectivity and strain characteristics of brain PrPD: Transmission study.

The presence of abnormal, disease-related prion protein (PrPD) has recently been demonstrated by protein misfolding cyclic amplification (PMCA) in urine of patients affected with variant Creutzfeldt-Jakob disease (vCJD), a prion disease typically acquired from consumption of prion contaminated bovine meat. The complexity and multistage process of urine excretion along with the obligatory use of PMCA raise the issue of whether strain characteristics of the PrPD present in vCJD brains, such as infectivity and phenotype determination, are maintained in urine excreted PrPD and following amplification by PMCA. We inoculated transgenic mice expressing normal human PrP with amplified urine and brain homogenate achieving the same 100% attack rate, similar incubation periods (in both cases extremely long) and histopathological features as for type and severity of the lesions. Furthermore, PrPD characteristics analyzed by immunoblot and conformational stability immunoassay were indistinguishable. Inoculation of raw vCJD urine caused no disease, confirming the extremely low concentration of PrPD in vCJD urine. These findings show that strain characteristics of vCJD brain PrPD, including infectivity, are preserved in PrPD present in urine and are faithfully amplified by means of PMCA; moreover, they suggest that the PrPD urine test might allow for the diagnosis and identification of disease subtype also in sporadic CJD.

Variable Protease-Sensitive Prionopathy Transmission to Bank Voles.

Variably protease-sensitive prionopathy (VPSPr), a recently described human sporadic prion disease, features a protease-resistant, disease-related prion protein (resPrPD) displaying 5 fragments reminiscent of Gerstmann-Sträussler-Scheinker disease. Experimental VPSPr transmission to human PrP-expressing transgenic mice, although replication of the VPSPr resPrPD profile succeeded, has been incomplete because of second passage failure. We bioassayed VPSPr in bank voles, which are susceptible to human prion strains. Transmission was complete; first-passage attack rates were 5%-35%, and second-passage rates reached 100% and survival times were 50% shorter. We observed 3 distinct phenotypes and resPrPD profiles; 2 imitated sporadic Creutzfeldt-Jakob disease resPrPD, and 1 resembled Gerstmann-Sträussler-Scheinker disease resPrPD. The first 2 phenotypes may be related to the presence of minor PrPD components in VPSPr. Full VPSPr transmission confirms permissiveness of bank voles to human prions and suggests that bank vole PrP may efficiently reveal an underrepresented native strain but does not replicate the complex VPSPr PrPD profile.