Methods enabling prion replication ex vivo are important for advancing prion studies. However, few such technologies exist, and many prion strains are not amenable to them. Here we describe a prion organotypic slice culture assay (POSCA) that allows prion amplification and titration ex vivo under conditions that closely resemble intracerebral infection. Thirty-five days after contact with prions, mouse cerebellar slices had amplified the abnormal isoform of prion protein, PrP(Sc), >10(5)-fold. This is quantitatively similar to amplification in vivo, but fivefold faster. PrP(Sc) accumulated predominantly in the molecular layer, as in infected mice. The POSCA detected replication of prion strains from disparate sources, including bovines and ovines, with variable detection efficiency. Pharmacogenetic ablation of microglia from POSCA slices led to a 15-fold increase in prion titers and PrP(Sc) concentrations over those in microglia-containing slices, as well as an increase in susceptibility to infection. This suggests that the extensive microglial activation accompanying prion diseases represents an efficacious defensive reaction.

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Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrPC, efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrPC selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrPSc and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrPC, into a β-sheet-rich, aggregated isoform, PrPSc. We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the β2–α2 loop in the NMR structure at pH 4.5 and 20 °C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well-ordered β2–α2 loop at 20 °C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrPD167S developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the β2–α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.

Prior to invading the nervous system, prions frequently colonize lymphoid organs and sites of inflammatory lymphoneogenesis, where they colocalize with Mfge8+ follicular dendritic cells (FDCs). Here, we report that soft-tissue granulomas, a frequent feature of chronic inflammation, expressed the cellular prion protein (PrPC, encoded by Prnp) and the lymphotoxin receptor (LTbetaR), even though they lacked FDCs and did not display lymphoneogenesis. After intraperitoneal prion inoculation, granulomas of Prnp(+/+) mice, but not Prnp(-/-) granulomas or unaffected Prnp(+/+) skin, accumulated prion infectivity and disease-associated prion protein. Bone-marrow transfers between Prnp(+/+) and Prnp(-/-) mice and administration of lymphotoxin signaling antagonists indicated that prion replication required radioresistant PrPC-expressing cells and LTbetaR signaling. Granulomatous PrPC was mainly expressed by stromal LTbetaR+ mesenchymal cells that were absent from unaffected subcutis. Hence, granulomas can act as clinically silent reservoirs of prion infectivity. Furthermore, lymphotoxin-dependent prion replication can occur in inflammatory stromal cells that are distinct from FDCs.

Prions have been documented in extra-neuronal and extra-lymphatic tissues of humans and various ruminants affected by Transmissible Spongiform Encephalopathy (TSE). The presence of prion infectivity detected in cervid and ovine blood tempted us to reason that kidney, the organ filtrating blood derived proteins, may accumulate disease associated PrPSc. We collected and screened kidneys of experimentally, naturally scrapie-affected and control sheep for renal deposition of PrPSc from distinct, geographically separated flocks. By performing Western blot, PET blot analysis and immunohistochemistry we found intraepithelial (cortex, medulla and papilla) and occasional interstitial (papilla) deposition of PrPSc in kidneys of scrapie-affected sheep. Interestingly, glomerula lacked detectable signals indicative of PrPSc. PrPSc was also detected in kidneys of subclinical sheep, but to significantly lower degree. Depending on the stage of the disease the incidence of PrPSc in kidney varied from approximately 27% (subclinical) to 73.6% (clinical) in naturally scrapie-affected sheep. Kidneys from flocks without scrapie outbreak were devoid of PrPSc. Here we demonstrate unexpectedly frequent deposition of high levels of PrPSc in ovine kidneys of various flocks. Renal deposition of PrPSc is likely to be a pre-requisite enabling prionuria, a possible co-factor of horizontal prion-transmission in sheep.

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