Pathologic and biochemical characterization of PrPSc from elk with PRNP polymorphisms at codon 132 after experimental infection with the chronic wasting disease agent

Abstract

Background: The Rocky Mountain elk (Cervus elaphus nelsoni) prion protein gene (PRNP) is polymorphic at codon 132, with leucine (L132) and methionine (M132) allelic variants present in the population. In elk experimentally inoculated with the chronic wasting disease (CWD) agent, different incubation periods are associated with PRNP genotype: LL132 elk survive the longest, LM132 elk are intermediate, and MM132 elk the shortest. The purpose of this study was to investigate potential mechanisms underlying variations in incubation period in elk of different prion protein genotypes. Elk calves of three PRNP genotypes (n = 2 MM132, n = 2 LM132, n = 4 LL132) were orally inoculated with brain homogenate from elk clinically affected with CWD.

Results: Elk with longer incubation periods accumulated relatively less PrP^Sc in the brain than elk with shorter incubation periods. PrP^Sc accumulation in LM132 and MM132 elk was primarily neuropil-associated while glial-associated immunoreactivity was prominent in LL132 elk. The fibril stability of PrP^Sc from MM132 and LM132 elk were similar to each other and less stable than that from LL132 elk. Real-time quaking induced conversion assays (RT-QuIC) revealed differences in the ability of PrP^Sc seed from elk of different genotypes to convert recombinant 132 M or 132 L substrate.

Conclusions: This study provides further evidence of the importance of PRNP genotype in the pathogenesis of CWD of elk. The longer incubation periods observed in LL132 elk are associated with PrP^Sc that is more stable and relatively less abundant at the time of clinical disease. The biochemical properties of PrP^Sc from MM132 and LM132 elk are similar to each other and different to PrP^Sc from LL132 elk. The shorter incubation periods in MM132 compared to LM132 elk may be the result of genotype-dependent differences in the efficiency of propagation of PrP^Sc moieties present in the inoculum. A better understanding of the mechanisms by which the polymorphisms at codon 132 in elk PRNP influence disease pathogenesis will help to improve control of CWD in captive and free-ranging elk populations.

Keywords: Chronic wasting disease; Conformational stability; Elk; Prion protein; RT-QuIC.

Fig. 1

Spongiform change observed in elk inoculated with the CWD agent. a Spongiform change in the dorsal motor nucleus of the vagus nerve in elk #2 (MM132). (Hematoxylin and eosin, original magnification 20×). b White matter vacuolation in the corpus callosum in elk #8 (LL132). (Hematoxylin and eosin, original magnification 10×)

Fig. 2

Spongiform change and patterns of PrP^Sc immunoreactivity observed in elk inoculated with the CWD agent. a Intraneuronal immunoreactivity in the hypoglossal nucleus in elk #5 (LL132) elk. b Glial associated immunoreactivity in the cerebellar white matter of elk #5 (LL132). c Glial associated immunoreactivity in the lateral geniculate nucleus of elk #5 (LL132). a-c immunostained using monoclonal anti-PrP antibody F99/96.7.1, original magnification 40×

Fig. 3

The fibril stability of PrP^Sc from MM132 and LM132 elk was lower than the fibril stability of PrP^Sc from LL132 elk. Homogenates of infected elk brain were incubated in GdnHCl at the indicated concentration as described in Methods, with remaining PrP^Sc, as detected by EIA, expressed as a fraction of the signal after treatment with 0.25 M GdnHCl. a Comparison of individual animals of MM132, LM132 and LL132 genotype elk, as indicated. Data were averaged across 4–6 technical replicates to generate each curve. b Average curves for LL132 elk (closed symbols) as compared to MM132 and LM132 elk (open symbols) from (a); error bars depict +/− the standard error of the mean (SEM) of the biological replicates

Fig. 4

The relative amount of PrP^Sc in the brainstem (obex) is strongly associated with incubation period. The amount of PrP^Sc in the brain was calculated using optical density readings from an antigen-capture enzyme immunoassay (EIA). The relative amount of PrP^Sc in the brain of the elk with the lowest EIA result in the linear range was designated a baseline value of 1.0. Results for other elk are expressed as a ratio relative to the baseline elk. Frozen obex was not available for elk #2 (MM132) so this animal is not included in the figure

Fig. 5

RT-QuIC detection of seeding activity in elk brain samples using mature length recombinant elk prion protein 132 L (a) and 132 M (b) substrates. RT-QuIC assays seeded with MM132 (green), LM132 (red), LL132 (black) or negative seed (yellow) are shown. RT-QuIC reaction mixtures were seeded with 10^− 2 dilutions of normalized brain homogenate. A final 400 mM NaCl was used with each substrate. Data are presented as mean ThT fluorescence of quadruplicate reactions