Disease phenotype of classical sheep scrapie is changed upon experimental passage through white-tailed deer.

Prion agents occur in strains that are encoded by the structure of the misfolded prion protein (PrPSc). Prion strains can influence disease phenotype and the potential for interspecies transmission. Little is known about the potential transmission of prions between sheep and deer. Previously, the classical US scrapie isolate (No.13-7) had a 100% attack rate in white-tailed deer after oronasal challenge. The purpose of this study was to test the susceptibility of sheep to challenge with the scrapie agent after passage through white-tailed deer (WTD scrapie). Lambs of various prion protein genotypes were oronasally challenged with WTD scrapie. Sheep were euthanized and necropsied upon development of clinical signs or at the end of the experiment (72 months post-inoculation). Enzyme immunoassay, western blot, and immunohistochemistry demonstrated PrPSc in 4 of 10 sheep with the fastest incubation occurring in VRQ/VRQ sheep, which contrasts the original No.13-7 inoculum with a faster incubation in ARQ/ARQ sheep. Shorter incubation periods in VRQ/VRQ sheep than ARQ/ARQ sheep after passage through deer was suggestive of a phenotype change, so comparisons were made in ovinized mice and with sheep with known strains of classical sheep scrapie: No. 13-7 and x-124 (that has a more rapid incubation in VRQ/VRQ sheep). After mouse bioassay, the WTD scrapie and x-124 isolates have similar incubation periods and PrPSc conformational stability that are markedly different than the original No. 13-7 inoculum. Furthermore, brain tissues of sheep with WTD scrapie and x-124 scrapie have similar patterns of immunoreactivity that are distinct from sheep with No. 13-7 scrapie. Multiple lines of evidence suggest a phenotype switch when No. 13-7 scrapie prions are passaged through deer. This represents one example of interspecies transmission of prions resulting in the emergence or selection of new strain properties that could confound disease eradication and control efforts.

Characterization of Classical Sheep Scrapie in White-tailed Deer after Experimental Oronasal Exposure.

BACKGROUND: Classic scrapie is a prion disease of sheep and goats that is associated with accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the prion disease of cervids. This study was conducted to determine the susceptibility of white-tailed deer (WTD) to the classic scrapie agent. METHODS: We inoculated WTD (n = 5) by means of a concurrent oral/intranasal exposure with the classic scrapie agent from sheep or oronasally with the classic scrapie agent from goats (n = 6). RESULTS: All deer exposed to the agent of classic scrapie from sheep accumulated PrPSc. PrPSc was detected in lymphoid tissues at preclinical time points, and necropsies in deer 28 months after inoculation showed clinical signs, spongiform lesions, and widespread PrPSc in neural and lymphoid tissues. Western blots on samples from the brainstem, cerebellum, and lymph nodes of scrapie-infected WTD have a molecular profile similar to CWD and distinct from samples from the cerebral cortex, retina, or the original classic scrapie inoculum. There was no evidence of PrPSc in any of the WTD inoculated with classic scrapie prions from goats. CONCLUSIONS: WTD are susceptible to the agent of classic scrapie from sheep, and differentiation from CWD may be difficult.

Bovine adapted transmissible mink encephalopathy is similar to L-BSE after passage through sheep with the VRQ/VRQ genotype but not VRQ/ARQ.

BACKGROUND: Transmissible mink encephalopathy (TME) is a fatal neurologic disease of farmed mink. Evidence indicates that TME and L-BSE are similar and may be linked in some outbreaks of TME. We previously transmitted bovine adapted TME (bTME) to sheep. The present study compared ovine passaged bTME (o-bTME) to C-BSE and L-BSE in transgenic mice expressing wild type bovine prion protein (TgBovXV). To directly compare the transmission efficiency of all prion strains in this study, we considered the attack rates and mean incubation periods. Additional methods for strain comparison were utilized including lesion profiles, fibril stability, and western blotting. RESULTS: Sheep donor genotype elicited variable disease phenotypes in bovinized mice. Inoculum derived from a sheep with the VRQ/VRQ genotype (o-bTMEVV) resulted in an attack rate, incubation period, western blot profile, and neuropathology most similar to bTME and L-BSE. Conversely, donor material from a sheep with the VRQ/ARQ genotype (o-bTMEAV) elicited a phenotype distinct from o-bTMEVV, bTME and L-BSE. The TSE with the highest transmission efficiency in bovinized mice was L-BSE. The tendency to efficiently transmit to TgBovXV mice decreased in the order bTME, C-BSE, o-bTMEVV, and o-bTMEAV. The transmission efficiency of L-BSE was approximately 1.3 times higher than o-bTMEVV and 3.2 times higher than o-bTMEAV. CONCLUSIONS: Our findings provide insight on how sheep host genotype modulates strain genesis and influences interspecies transmission characteristics. Given that the transmission efficiencies of L-BSE and bTME are higher than C-BSE, coupled with previous reports of L-BSE transmission to mice expressing the human prion protein, continued monitoring for atypical BSE is advisable in order to prevent occurrences of interspecies transmission that may affect humans or other species.

Accelerated accumulation of retinal α-synuclein (pSer129) and tau, neuroinflammation, and autophagic dysregulation in a seeded mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by accumulation of misfolded α-synuclein within the central nervous system (CNS). Visual problems in PD patients are common, although retinal pathology associated with PD is not well understood. The purpose of this study was to investigate retinal pathology in a transgenic mouse model (TgM83) expressing the human A53T α-synuclein mutation and assess the effect of α-synuclein "seeding" on the development of retinal pathology. Two-month-old TgM83 mice were intracerebrally inoculated with brain homogenate from old (12-18 months) TgM83 mice. Retinas were then analyzed at 5 months of age. We analyzed retinas from 5-month-old and 8-month-old uninoculated healthy TgM83 mice, and old (12-18 months) mice that were euthanized following the development of clinical signs. Retinas of B6C3H mice (genetic background of the TgM83 mouse) served as control. We used immunohistochemistry and western blot analysis to detect accumulation of α-synuclein, pTauThr231, inflammation, changes in macroautophagy, and cell death. Raman spectroscopy was used to test the potential to differentiate between retinal tissues of healthy mice and diseased mice. This work demonstrates retinal changes associated with the A53T mutation. Retinas of non-inoculated TgM83 mice had accumulation of α-synuclein, "pre-tangle" tau, activation of retinal glial cells, and photoreceptor cell loss by 8 months of age. The development of these changes is accelerated by inoculation with brain homogenate from clinically ill TgM83 mice. Compared to non-inoculated 5-month-old TgM83 mice, retinas of inoculated 5-month-old mice had increased accumulation of α-synuclein (pSer129) and pTauThr231 proteins, upregulated microglial activation, and dysregulated macroautophagy. Raman spectroscopic analysis was able to discriminate between healthy and diseased mice. This study describes retinal pathology resulting from the A53T mutation. We show that seeding with brain homogenates from old TgM83 mice accelerates retinal pathology. We demonstrate that Raman spectroscopy can be used to accurately identify a diseased retina based on its biochemical profile, and that α-synuclein accumulation may contribute to accumulation of pTauThr231 proteins, neuroinflammation, metabolic dysregulation, and photoreceptor cell death. Our work provides insight into retinal changes associated with Parkinson's disease, and may contribute to a better understanding of visual symptoms experienced by patients.

Temporal Resolution of Misfolded Prion Protein Transport, Accumulation, Glial Activation, and Neuronal Death in the Retinas of Mice Inoculated with Scrapie.

Currently, there is a lack of pathological landmarks to describe the progression of prion disease in vivo. Our goal was to use an experimental model to determine the temporal relationship between the transport of misfolded prion protein (PrP(Sc)) from the brain to the retina, the accumulation of PrP(Sc) in the retina, the response of the surrounding retinal tissue, and loss of neurons. Retinal samples from mice inoculated with RML scrapie were collected at 30, 60, 90, 105, and 120 days post inoculation (dpi) or at the onset of clinical signs of disease (153 dpi). Retinal homogenates were tested for prion seeding activity. Antibody staining was used to assess accumulation of PrP(Sc) and the resulting response of retinal tissue. Loss of photoreceptors was used as a measure of neuronal death. PrP(Sc) seeding activity was first detected in all samples at 60 dpi. Accumulation of PrP(Sc) and coincident activation of retinal glia were first detected at 90 dpi. Activation of microglia was first detected at 105 dpi, but neuronal death was not detectable until 120 dpi. Our results demonstrate that by using the retina we can resolve the temporal separation between several key events in the pathogenesis of prion disease.

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

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.

Prion protein facilitates retinal iron uptake and is cleaved at the ß-site: Implications for retinal iron homeostasis in prion disorders.

Prion disease-associated retinal degeneration is attributed to PrP-scrapie (PrPSc), a misfolded isoform of prion protein (PrPC) that accumulates in the neuroretina. However, a lack of temporal and spatial correlation between PrPSc and cytotoxicity suggests the contribution of host factors. We report retinal iron dyshomeostasis as one such factor. PrPC is expressed on the basolateral membrane of retinal-pigment-epithelial (RPE) cells, where it mediates uptake of iron by the neuroretina. Accordingly, the neuroretina of PrP-knock-out mice is iron-deficient. In RPE19 cells, silencing of PrPC decreases ferritin while over-expression upregulates ferritin and divalent-metal-transporter-1 (DMT-1), indicating PrPC-mediated iron uptake through DMT-1. Polarization of RPE19 cells results in upregulation of ferritin by ~10-fold and ß-cleavage of PrPC, the latter likely to block further uptake of iron due to cleavage of the ferrireductase domain. A similar ß-cleavage of PrPC is observed in mouse retinal lysates. Scrapie infection causes PrPSc accumulation and microglial activation, and surprisingly, upregulation of transferrin despite increased levels of ferritin. Notably, detergent-insoluble ferritin accumulates in RPE cells and correlates temporally with microglial activation, not PrPSc accumulation, suggesting that impaired uptake of iron by PrPSc combined with inflammation results in retinal iron-dyshomeostasis, a potentially toxic host response contributing to prion disease-associated pathology.