An Ex Vivo Brain Slice Culture Model of Chronic Wasting Disease: Implications for Disease Pathogenesis and Therapeutic Development

Abstract

Chronic wasting disease (CWD) is a rapidly spreading prion disease of cervids, yet antemortem diagnosis, treatment, and control remain elusive. We recently developed an organotypic slice culture assay for sensitive detection of scrapie prions using ultrasensitive prion seeding. However, this model was not established for CWD prions due to their strong transmission barrier from deer (Odocoileus spp) to standard laboratory mice (Mus musculus). Therefore, we developed and characterized the ex vivo brain slice culture model for CWD, using a transgenic mouse model (Tg12) that expresses the elk (Cervus canadensis) prion protein gene (PRNP). We tested for CWD infectivity in cultured slices using sensitive seeding assays such as real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA). Slice cultures from Tg12, but not from prnp^-/- mice, tested positive for CWD. Slice-generated CWD prions transmitted efficiently to Tg12 mice. Furthermore, we determined the activity of anti-prion compounds and optimized a screening protocol for the infectivity of biological samples in this CWD slice culture model. Our results demonstrate that this integrated brain slice model of CWD enables the study of pathogenic mechanisms with translational implications for controlling CWD.

Figure 1

In vitro seeded amplification of CWD prions from organotypic slice cultures using the RT-QuIC assay and PMCA. (A) RT-QuIC seeding curves showing that slice cultures from Tg12 mice (prnp^+/−) efficiently seed the RT-QuIC assay producing strong positive peaks for the reactions seeded with 5 ng of CWD-infected slice culture homogenates at 42 dpi (red trace, labeled as Tg12 Slices CWD) or brain homogenates (BH) from a CWD-infected deer (blue trace, labeled as CWD BH). Slices cultured with NBH (grey trace) and prnp^−/− slices cultured with CWD BH (black trace) or NBH (green trace) did not show seeding activity during the reaction period. Each trace is a combined average of 3 technical replicates and 2 biological replicates from a pool of 7–10 slices. (B) A representative full-length Western blot from the third of three serial rounds of PMCA performed on BH from the CWD slice culture model. All samples were treated with PK, except NBH from Tg5037 mice (NBH PK (−) in the first lane) used as a migration (positive) control. Only the positive control and Tg12 slices inoculated with CWD deer BH show immunoreactivity. (C) Representative PrP^CWD immunohistochemistry (6H4 antibody) images showing a cross-section of 35-dpi slices. Slices from Tg12 mice inoculated with CWD deer BH showed immunoreactivity (top image), whereas NBH did not (bottom image). Scale bar, 10 µm. (D) Representative full-length Western blot showing the presence of PK-resistant CWD prions in Tg12, but not prnp^−/−, slice culture homogenates, all probed with the anti-prnp mouse monoclonal antibody POM1. (E) DHE conversion assay showing the significantly higher generation of reactive oxygen species (ROS) for CWD BH-infected cerebellar slices when compared to NBH-treated or prnp^−/− slice cultures. Data were analyzed using one-way ANOVA; n = 4 biological replicates. Data are mean ± SEM (*p ≤ 0.05, **p < 0.01, ***P < 0.001).

Figure 2

Seeding profile of CWD prions from organotypic slice cultures. (A–C) Representative traces from RT-QuIC assay of slices exposed to brain homogenates from normal (NBH) or CWD-infected deer and cultured up to 52 dpi. Each trace comprises 0.5-h time points represented as an average ThT fluorescence from n = 3–4 replicate wells. (A) Seeding activity in CWD-infected slices accelerated rapidly after 21 dpi (n = 3). (B) Lysates prepared from 52-dpi slices were serially diluted 10-fold, ranging from 50 fg to 50 ng. The reactions (n = 4), consisting of 5 µL of these dilutions as seed, revealed optimal seeding doses ranging between 50 pg to 50 ng. (C) Endpoint 10-fold serial dilution analysis (n = 4) of the CWD deer BH used as inoculum. (D) Quantification of seeding activity from different time points in the CWD slice culture model. The median seeding dose (SD₅₀) is defined as the amount giving sufficiently enhanced ThT fluorescence in half of the replicate wells. The Spearman-Kärber estimate of the SD₅₀, plotted against days post-inoculation of the slice culture, shows prion titers progressively increase to a maximum at 52 dpi.

Figure 3

The anti-prion activity of compounds and the capture of CWD prions from biological samples using slice cultures. (A) The anti-prion activity of the compounds astemizole, Congo red, and quinacrine was evaluated against a CWD-infected control and 5 ng, 0.5 ng, 50 pg serial log dilutions. Inhibitory activity depended on the seed dilution, except for astemizole, which had no apparent effect on the seeding activity of CWD prion-infected slice cultures. (B) RT-QuIC assay showing transmission of CWD prions from infected recto-anal mucosa-associated lymphoid tissue (RAMALT) samples to the slices cultured with RAMALT homogenates for 21 days when tested at log-fold dilutions of 50 ng to 50 pg. A CWD-infected RAMALT homogenate was used as a positive control, while a healthy RAMALT control homogenate served as a negative control. Each RT-QuIC trace represents the average fluorescence reading from 3 technical replicates of slice cultures.

Figure 4

Mouse bioassay of CWD prions from slice cultures. (A) Survival analysis of Tg12 mice intracranially inoculated with 5-µL lysates from homogenized CWD-inoculated brain slices during passage 1 (Pass 1) showing an incubation period of 208 days. Mice inoculated with NBH slices tested negative for PrP^CWD at the end of the experiment. When Tg12 mice (Pass 2) were similarly injected with 5-µL of inoculum from Pass 1 mouse brains, the mean incubation period was reduced to 139.5 dpi. (B) RT-QuIC assay reaction traces (n = 4 replicates) showing efficient CWD prion transmission to the brains of mice in passage 1. (C) Immunohistochemistry of paraffin-embedded tissue sections showing neuropathology and CWD prion plaques in multiple brain regions of mice from Pass 2. No immunoreactivity is present in a representative section of the thalamus from a negative control Tg12 mouse. Plaques were more abundant in the granular layer of the cerebellum and hippocampus. A diffuse pattern was observed in the hypothalamus and medulla while a mixed pattern manifested in the midbrain and thalamus. Scale bars: 50 μm.