Host Determinants of Prion Strain Diversity Independent of Prion Protein Genotype

Abstract

Phenotypic diversity in prion diseases can be specified by prion strains in which biological traits are propagated through an epigenetic mechanism mediated by distinct PrP(Sc) conformations. We investigated the role of host-dependent factors on phenotypic diversity of chronic wasting disease (CWD) in different host species that express the same prion protein gene (Prnp). Two CWD strains that have distinct biological, biochemical, and pathological features were identified in transgenic mice that express the Syrian golden hamster (SGH) Prnp. The CKY strain of CWD had a shorter incubation period than the WST strain of CWD, but after transmission to SGH, the incubation period of CKY CWD was ∼150 days longer than WST CWD. Limited proteinase K digestion revealed strain-specific PrP(Sc) polypeptide patterns that were maintained in both hosts, but the solubility and conformational stability of PrP(Sc) differed for the CWD strains in a host-dependent manner. WST CWD produced PrP(Sc) amyloid plaques in the brain of the SGH that were partially insoluble and stable at a high concentration of protein denaturant. However, in transgenic mice, PrP(Sc) from WST CWD did not assemble into plaques, was highly soluble, and had low conformational stability. Similar studies using the HY and DY strains of transmissible mink encephalopathy resulted in minor differences in prion biological and PrP(Sc) properties between transgenic mice and SGH. These findings indicate that host-specific pathways that are independent of Prnp can alter the PrP(Sc) conformation of certain prion strains, leading to changes in the biophysical properties of PrP(Sc), neuropathology, and clinical prion disease.

Importance: Prions are misfolded pathogenic proteins that cause neurodegeneration in humans and animals. Transmissible prion diseases exhibit a spectrum of disease phenotypes and the basis of this diversity is encoded in the structure of the pathogenic prion protein and propagated by an epigenetic mechanism. In the present study, we investigated prion diversity in two hosts species that express the same prion protein gene. While prior reports have demonstrated that prion strain properties are stable upon infection of the same host species and prion protein genotype, our findings indicate that certain prion strains can undergo dramatic changes in biological properties that are not dependent on the prion protein. Therefore, host factors independent of the prion protein can affect prion diversity. Understanding how host pathways can modify prion disease phenotypes may provide clues on how to alter prion formation and lead to treatments for prion, and other, human neurodegenerative diseases of protein misfolding.

FIG 1

Body weight of SGH during prion infection with chronic wasting disease. Mock-infected (○) and CWD-infected hamsters (□ and △) (see Table 1, recipient groups H1733 for WST CWD and H1727 for CKY CWD) were monitored weekly over the course of infection for mean body weight (A) and the percent change in body weight (BW) from the prior week or BW replacement value (B). The green dashed line in panel B indicates no change in the BW replacement value, whereas values above or below the line indicate a net gain or loss, respectively, in body weight. A solid blue box at 32 weeks postinfection is indicated for WST CWD. The error bars indicate the standard errors of the mean and values changed when hamsters were sacrificed due to illness and for collection of age-matched controls (e.g., at 50 to 60 weeks and at 70 to 80 weeks postinfection). For illustration purposes, data points are plotted for every other week.

FIG 2

PrP^Sc deposition pattern in the brains of Syrian golden hamsters (SGH) infected with WST and CKY CWD. Brain sections from CKY CWD (A, D, G, B, E, H, and K), WST CWD (C, F, I, J, and L), and mock (M, N, and O) infections in SGH were stained for PrP^Sc by IHC (brown deposit) or stained with hematoxylin and eosin (A, D, G, J, M, N, and O). Brain tissue was from the second (CKY CWD) and third (WST CWD) serial passages in SGH. VC, visual cortex; M, motor cortex; Cg, cingulate cortex; S1, somatosensory cortex; Pir, piriform cortex; HC, hippocampus; SC, superior colliculus; SNR, substania nigra; RN, red nucleus; MG, medial geniculate nucleus; VPM, ventral posterior thalamic nucleus; IPR, interpedunclar nucleus; PN, pretectal nucleus; ZI, zona incerta; H, hypothalamic nuclei; AM, amygdala; Rt, reticular formation; CPu, caudate-putamen; GP, globus pallidum; LS, lateral septal nucleus; MS, medial septal nucleus; ic, internal capsule; cc, corpus callous; LV, lateral ventricle; 3, third ventricle.

FIG 3

PrP^Sc deposition associated with brain ventricles and vasculature. PrP^Sc IHC (B, D, and F) and hematoxylin and eosin stain (A, C, and E) in adjacent tissue sections of CKY CWD (A and B)- and WST CWD (C to F)-infected SGH. (A and B) Heavy PrP^Sc deposition associated with the ependymal cell layer (e) of the third ventricle (3). PrP^Sc plaques (white asterisks and black arrowheads) were located below the ependymal cells, PrP^Sc extended through this cell layer, and PrP^Sc projected into the third ventricle. (C and D) A large PrP^Sc plaque (white asterisk) at the brain surface was found adjacent to PrP^Sc deposition surrounding an arteriole (a) in the pia. (E and F) Several PrP^Sc plaques were observed along the midline of the cerebral cortex. Large unicentric and multicentric hematoxylin-positive plaques (white arrowhead and asterisk, respectively) were often observed in WST CWD but were less common in CKY CWD infection of SGH. Brain tissue was from the second (CKY CWD) and third (WST CWD) serial passages in SGH. Scale bar, 20 μm.

FIG 4

Antigenic mapping of PrP^Sc from TME and CWD infection of transgenic mice and Syrian golden hamsters. (A) Prion-infected brain homogenates from HPrP7752KO mice and Syrian golden hamsters were digested with or without proteinase K (PK) and analyzed by SDS-PAGE and Western blotting with the anti-PrP antibodies 12B2, D13, and D18. The hamster amino acid (aa) sequence that is recognized (12B2) or used as a peptide antigen (D13 and D18) is indicated below each antibody. Brain tissues were from the second (CKY CWD) and third (WST CWD) serial passages in SGH. The three short horizontal bars between the PK-treated samples from HPrP7752KO mice and Syrian golden hamsters correspond to molecular masses of 20, 30, and 40 kDa. The bracket indicates the doublet PrP^Sc polypeptide found in CKY CWD. (B) Schematic of the linear amino acid map of the mature prion protein, including the octapeptide repeat (OR) region, two N-linked glycosylation sites, and carbohydrate structures (the Y-like branches). Limited PK digestion of PrP^Sc results in the degradation of the N-terminal region. This results in a ragged N terminus with new N termini around amino acid 90. The locations of the peptides used to generate the anti-PrP antibodies 12B2, D13, and D18 are indicated with horizontal bars below the prion protein map. Enzymatic deglycosylation of PK-treated PrP^Sc removes the two N-linked carbohydrate structures and results in an ∼25% reduction in molecular weight and a single major PrP^Sc polypeptide (see Fig. 5B).

FIG 5

Enrichment and deglycosylation of PrP^Sc from TME and CWD strains. Brain homogenates from SGH infected with HY TME, DY TME, WST CWD, and CKY CWD were enriched for PrP^Sc by detergent extraction, ultracentrifugation, and proteinase K digestion (A), and N-linked carbohydrates were removed after enzymatic deglycosylation with PNGase F (B). Brain tissue was from second (CKY CWD) and third (WST CWD) serial passages in SGH. Samples were analyzed by SDS-PAGE and Western blotting with anti-PrP D18 antibody. Short white horizontal lines are located below the aglycosylated polypeptide band of HY TME and placed between each lane for reference purposes.

FIG 6

Western blot of prion protein from the TME and CWD strains in Syrian golden hamsters after a conformation solubility and stability assay (CSSA). SGH brain homogenates from HY TME (A), DY TME (B), WST CWD (C), and CKY CWD (D) were extracted in Sarkosyl, incubated with GdnHCl from 0 to 4.0 M, and subjected to ultracentrifugation as described for the CSSA. For each GdnHCl treatment, the supernatant (S) and pellet (P) fraction after ultracentrifugation were analyzed in adjacent lanes by SDS-PAGE and Western blotting with anti-PrP D18 antibody. Brain tissue was from the second (CKY CWD) and third (WST CWD) serial passages in SGH. For each panel, two separate Western blots prepared for strain analysis, and for illustrative purposes, they were merged between the 2.0 and 2.5 M GdnHCl values.

FIG 7

Conformational stability and solubility of prion protein in transgenic mice and Syrian golden hamsters infected with TME and CWD strains. The CSSA was performed using three individual brains from the TME and CWD strains in transgenic mice and SGH. The percentage of insoluble prion protein (PrP) indicates the percentage in the pellet fraction when 100% is equal to the sum of the soluble and pellet fraction for a given [GdnHCl]. The CSSA results for the prion strains in SGH (A) and HPrP7752KO mice (B) are plotted on the same graph. The CSSA results for each individual prion strain in both host species are also plotted on a single panel (E through H). The CSSA results for uninfected SGH and HPrP7752KO mice (C), and the solubility of PrP in N-laurylsarcosine and 0 M GdnHCl from mock-infected and HY TME-infected HrP7752KO brain (D) are also illustrated. Brain tissue was obtained from the second (CKY CWD) and third (WST CWD) serial passages in SGH and from the fourth (CKY CWD) and third (WST CWD) serial passages in HPrP7752KO mice.

FIG 8

Western blot analysis of PrP^Sc following backpassage of CWD strains from Syrian golden hamsters (SGH) into transgenic mice. SGH brain samples from the second (CKY CWD) and third (WST CWD) serial passages were subsequently passaged back into transgenic mice (Mo) two times (e.g., lanes “1st” and “2nd”) as presented in Table 1. Brain homogenates were digested with proteinase K and analyzed by SDS-PAGE and Western blotting with anti-PrP D18 antibody. The two short horizontal bars at both ends of the membrane correspond to molecular masses of 20 and 30 kDa. The bracket indicates the doublet PrP^Sc polypeptide found in CKY CWD.