Prion Strain Characterization of a Novel Subtype of Creutzfeldt-Jakob Disease

Abstract

In 2007, we reported a patient with an atypical form of Creutzfeldt-Jakob disease (CJD) heterozygous for methionine-valine (MV) at codon 129 who showed a novel pathological prion protein (PrP^TSE) conformation with an atypical glycoform (AG) profile and intraneuronal PrP deposition. In the present study, we further characterize the conformational properties of this pathological prion protein (PrP^TSE MV^AG), showing that PrP^TSE MV^AG is composed of multiple conformers with biochemical properties distinct from those of PrP^TSE type 1 and type 2 of MV sporadic CJD (sCJD). Experimental transmission of CJD-MV^AG to bank voles and gene-targeted transgenic mice carrying the human prion protein gene (TgHu mice) showed unique transmission rates, survival times, neuropathological changes, PrP^TSE deposition patterns, and PrP^TSE glycotypes that are distinct from those of sCJD-MV1 and sCJD-MV2. These biochemical and experimental data suggest the presence of a novel prion strain in CJD-MV^AGIMPORTANCE Sporadic Creutzfeldt-Jakob disease is caused by the misfolding of the cellular prion protein, which assumes two different major conformations (type 1 and type 2) and, together with the methionine/valine polymorphic codon 129 of the prion protein gene, contribute to the occurrence of distinct clinical-pathological phenotypes. Inoculation in laboratory rodents of brain tissues from the six possible combinations of pathological prion protein types with codon 129 genotypes results in the identification of 3 or 4 strains of prions. We report on the identification of a novel strain of Creutzfeldt-Jakob disease isolated from a patient who carried an abnormally glycosylated pathological prion protein. This novel strain has unique biochemical characteristics, does not transmit to humanized transgenic mice, and shows exclusive transmission properties in bank voles. The identification of a novel human prion strain improves our understanding of the pathogenesis of the disease and of possible mechanisms of prion transmission.

Keywords: Creutzfeldt-Jakob disease; humanized mice; prion strain; prions.

FIG 1

Biochemical and conformational properties of PrP^TSE in MV^AG, MV1, and MV2 brain samples. (A) Characterization of PrP^TSE soluble and insoluble in nondenaturing detergents. Shown are detergent-soluble (S1) and detergent-insoluble (P3) fractions obtained from MV^AG, MV1, and MV2 frontal cortices following ultracentrifugation and proteinase K digestion. In MV^AG cortices, the S1 fraction PrP^TSE is represented by a monoglycosylated and an unglycosylated band, while in the P3 fraction, the unglycosylated band (arrow) shows an upper band of 23 and a lower band of 19 kDa. In contrast, PrP^TSE in MV1 and MV2 cortices shows three bands representing the differently glycosylated isoforms of PrP^TSE, and the unglycosylated band migrates at 19 and 21 kDa, respectively. The membranes were probed with 3F4. (B) Conformational stability assay in MV^AG, MV1, and MV2 brain homogenates. Western blot analyses of total brain homogenate following denaturation with various concentrations of GdnHCl from 0 to 2.5 M. In MV^AG homogenates, PrP^TSE was digested at 2.0 M GdnHCl, and the unglycosylated band separated as two bands (arrows) with distinct conformational stabilities. The conformational stability of PrP^TSE was analyzed by plotting the fraction of PrP^TSE as a function of the GdnHCl concentration. The GdnHCl_1/2 value for MV^AG was 0.8 M; for MV1, 2.0 M; and for MV2, 2.5 M. The membranes were probed with 3F4. (C) Fractionation of PrP^TSE aggregates in MV^AG, MV1, and MV2 brain homogenates. The brain homogenates were sedimented in a 10 to 60% sucrose gradient. (Right) After sedimentation, half samples were PK treated (MV^AG, MV1, and MV2). PrP^TSE MV^AG was distributed across all the fractions, albeit concentrated in the top (lanes 1 to 4) and bottom (lanes 9 to 11) fractions. A similar separation pattern was observed for PrP^TSE MV1. In contrast, PrP^TSE MV2 sedimented in the lower fractions. Following PK treatment, PrP^TSE MV^AG was composed of large aggregates, while PrP^TSE MV1 was mainly concentrated in the upper and lower fractions and PrP^TSE MV2 at the bottom (the PrP synthetic peptides in the first lane were used as PrP size markers). The representations of PrP^TSE size aggregates were produced by plotting the amount of PrP as a function of the sucrose concentration. The membranes were probed with 3F4.

FIG 2

Western blot comparison of PrP^TSE patterns in the brains of the three TgHu mouse genotypes and in the corresponding CJD inocula. Mice inoculated with sCJD-MV1 (lanes 2 to 4) produced a PrP^TSE type identical to that of the original inoculum (lane 1) regardless of the genotype. In the MV2 group (lanes 5 to 8), only the HuVV genotype (lane 8) reproduced the original type 2 PrP^TSE (lane 5). Atypical CJD PrP^TSE MV^AG (lane 9) with features identical to those of the original inoculum was observed only in HuVV mice (lane 12). No PrP^TSE was detected in HuMM and HuMV mice injected with CJD-MV^AG (lanes 10 and 11). Approximate molecular masses (kilodaltons) are on the right. The membranes were probed with 3F4.

FIG 3

Biochemical and histopathological findings observed in bank voles inoculated with CJD-MV^AG. (A) Western blot analysis of proteinase K-resistant PrP^TSE in brains of individual voles infected with CJD-MV^AG in comparison with representative voles infected with sCJD-MV1 (type A in voles) (40) or sCJD-MV2 (type C in voles) (40). Samples were loaded as indicated above the blots either before or after treatment with PNGase to remove N-linked oligosaccharides. Replica blots were revealed with MAb Sha31 (top) (the Sha31 epitope is in the core of the PrP fragments), which was expected to bind all protease-resistant C-terminal PrP fragments, or with SAF32 (bottom) (the SAF32 epitope is in the octarepeat region of PrP at the extreme N-terminal end of PK-resistant PrP fragments), which was expected to bind only PrP fragments cleaved N terminally to the last octarepeat. Three individual voles from the CJD-MV^AG group were selected because they were representative of the electrophoretic variability of PK-resistant PrP fragments observed in the whole group, encompassing cases displaying the preferentially high (H), preferentially low (L), or intermediate (I) pattern (see the text). Note that with Sha31, upon deglycosylation, the unglycosylated PK-resistant PrP fragments from MV^AG appeared as less defined bands than MV1 and MV2, suggesting the presence of PrP fragments of variable size. Among these, the H pattern included PrP fragments similar to or higher than MV1, the I pattern encompassed apparent MWs between those of MV1 and MV2, and the L pattern seemed similar to that of MV2. With SAF32, which binds to MV1 but not MV2 PK-resistant PrP, high-MW PrP fragments were evident in H-type patterns and with decreasing intensity also in types I and L, suggesting that in all samples from MV^AG there was copresence of PrP fragments with different N-terminal cleavages. (B) Histopathological analysis of the brains of two representative voles with low (a and c) or high (b and d) cortical involvement. The occipital cortex was relatively spared (a) or severely affected (b), while the thalamus was similarly affected in both voles (c and d). Bars, 40 μm. (C) Immunohistochemical examination of voles' infected brains revealed different PrP^TSE deposition patterns, which included synaptic/punctate (a), intraneuronal (b and c), intra-astrocytic and intramicroglial (d), perivascular (e), and perivacuolar (f). Bars, 20 μm. (D) Magnification of peculiar intraneuronal engulfment of PrP^TSE observed in colliculus (a), external capsule (b and d), and vestibular nucleus (c). Bars, 25 μm.

FIG 4

Representative immunohistochemistry of bank voles inoculated with sCJD-MV1 and sCJD-MV2. Shown are patterns of PrP^TSE observed by immunohistochemistry in the thalamus of voles inoculated with positive controls, sCJD-MV1 and sCJD-MV2. In the brains of sCJD-MV1-affected voles, there were extracellular punctate PrP^TSE deposits, while in sCJD-MV2-affected voles, there were remarkable granular intracellular depositions in glia and neurons in the same area.