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Prion diseases are rapidly progressive, incurable neurodegenerative disorders caused by misfolded, aggregated proteins known as prions, which are uniquely infectious. Remarkably, these infectious proteins have been responsible for widespread disease epidemics, including kuru in humans, bovine spongiform encephalopathy in cattle, and chronic wasting disease in cervids, the latter of which has spread across North America and recently appeared in Norway and Finland. The hallmark histopathologic features include widespread spongiform encephalopathy, neuronal loss, gliosis, and deposits of variably-sized aggregated prion protein ranging from small, soluble oligomers to long, thin, unbranched fibrils, depending on the disease. Here we explore recent advances, from the function of the cellular prion protein to the dysfunction triggering neurotoxicity, as well as mechanisms underlying prion spread between cells, and the effect of prion conformation on spreading pathways. We also highlight key findings that have revealed new therapeutic targets and consider unanswered questions for future research.

International audience; Transmissible spongiform encephalopathies (TSEs) are a group of progressive, invariably fatal diseases that affect the nervous system of many mammals including humans. The key molecular event in the pathogenesis of TSEs is the conversion of the cellular prion protein PrPC into a disease-associated isoform PrPSc. The "protein-only hypothesis" argues that PrPSc itself is the infectious agent. In effect, PrPSc can adopt several structures that represent different prion strains. The interspecies transmission of TSEs is difficult because of differences between the host and donor primary PrP sequence. However, transmission is not impossible as this occurred when bovine spongiform encephalopathy spread to humans causing variant Creutzfeldt-Jakob disease (vCJD). This event determined a need for a thorough understanding of prion replication and transmission so that we could be one step ahead of further threats for human health. This chapter focuses on these concepts and on new insights gained into prion propagation mechanisms.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are neurological diseases that can be transmitted through a number of different routes. A wide range of mammalian species are affected by the disease. After peripheral exposure, some TSE agents accumulate in lymphoid tissues at an early stage of disease prior to spreading to the nerves and the brain. Much research has focused on identifying the cells and molecules involved in the transmission of TSE agents from the site of exposure to the brain and several crucial cell types have been associated with this process. The identification of the key cells that influence the different stages of disease transmission might identify targets for therapeutic intervention. This review highlights the involvement of mononuclear phagocytes in TSE disease. Current data suggest these cells may exhibit a diverse range of roles in TSE disease from the transport or destruction of TSE agents in lymphoid tissues, to mediators or protectors of neuropathology in the brain.

Prion diseases are subacute neurodegenerative diseases that affect humans and a range of domestic and free-ranging animal species. These diseases are characterized by the accumulation of PrP (Sc), an abnormally folded isoform of the cellular prion protein (PrP (C)), in affected tissues. The pathology during prion disease appears to occur almost exclusively within the central nervous system. The extensive neurodegeneration which occurs ultimately leads to the death of the host. An intriguing feature of the prion diseases, when compared with other protein-misfolding diseases, is their transmissibility. Following peripheral exposure, some prion diseases accumulate to high levels within lymphoid tissues. The replication of prions within lymphoid tissue has been shown to be important for the efficient spread of disease to the brain. This article describes recent progress in our understanding of the cellular mechanisms that influence the propagation of prions from peripheral sites of exposure (such as the lumen of the intestine) to the brain. A thorough understanding of these events will lead to the identification of important targets for therapeutic intervention, or alternatively, reveal additional processes that influence disease susceptibility to peripherally-acquired prion diseases.

The fungal prion-forming domain HET-s(218-289) forms infectious amyloid fibrils at physiological pH that were shown by solid-state NMR to be assemblies of a two-rung β-solenoid structure. Under acidic conditions, HET-s(218-289) has been shown to form amyloid fibrils that have very low infectivity in vivo, but structural information about these fibrils has been very limited. We show by x-ray fiber diffraction that the HET-s(218-289) fibrils formed under acidic conditions have a stacked β-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked β-sheet fibrils nucleate the formation of the infectious β-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious β-solenoid fibrils) fibrillization. Several serial passages of stacked β-sheet-seeded solutions lead to fibrillization kinetics similar to homogeneously seeded solutions. Our results directly show that structural mutation can occur between substantially different amyloid architectures, lending credence to the suggestion that the processes of strain adaptation and crossing species barriers are facilitated by structural mutation. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

U posljednjih 10 godina Hrvatskom zavodu za javno zdravstvo prijavljeno je 15 bolesnika oboljelih od Creutzfeldt-Jakobove bolesti (CJB). Tijekom lipnja 2012. godine u Općoj bolnici Varaždin (OBV) preminuo je 58-godišnji bolesnik premješten s Klinike za neurologiju KBC Zagreb pod sumnjom na sporadični oblik CJB. Nakon obdukcije bolesnika, koja je provedena u našoj ustanovi, dijagnoza CJB potvrđena je patohistološkom analizom mozga u Kliničkom institutu za neurologiju Medicinskog fakulteta u Beču. Povjerenstvo za sprječavanje i suzbijanje bolničkih infekcija OBV izradilo je upute o mjerama i načinu postupanja s prostorima, opremom i instrumentima koji su bili potencijalno kontaminirani prionima. Obzirom da se radilo o bolesniku koji je 2,5 mjeseca prije postavljene dijagnoze bio neurokirurški operiran u našoj ustanovi, a upotrijebljeni neurokirurški instrumenti nisu bili tretirani po principu dekontaminacije priona, postavljeno je pitanje potencijalnog prijenosa priona kontaminiranim instrumentima na 29 bolesnika koji su naknadno neurokirurški operirani. Analizom neurokirurškog zahvata utvrdili smo da su neurokirurški instrumenti bili u kontaktu s kožom, potkožjem, fascijama mišića, kostima i intervertebralnom hrskavicom bolesnika, ali ne i s tvrdom moždanom ovojnicom, niti je za vrijeme operacije bilo likvoreje. Zaključili smo da tijekom neurokirurškog zahvata nije došlo do kontakta instrumenata s tkivima visokog i srednjeg rizika za prijenos priona te je rutinski postupak s neurokirurškim instrumentima (rutinsko čišćenje i sterilizacija) bio dostatan za sprječavanje prijenosa priona, sukladno smjernicama britanske i kanadske grupe za kontrolu CJB. Naknadnim praćenjem nije registriran niti jedan slučaj sekundarne CJB u 29 bolesnika operiranih nakon ovog bolesnika u našoj bolnici., In the last 10 years there have been 15 cases of Creutzfeldt-Jakob disease (CJD) reported to the Croatian Institute of Public Health/Department of Epidemiology. In June 2012, a 58-year-old patient that had been relocated from the Department of Neurology of the University Hospital Centre Zagreb to General Hospital Varaždin, died from a suspected sporadic form of Creutzfeldt-Jakob disease. After autopsy was performed in our hospital, CJD was confirmed by histopathological analysis of the brain in the Clinical Institute of Neurology/Medical University of Vienna. The Hospital Infection Control Committee drafted guidelines for infection control measures regarding work areas, equipment and instruments that were potentially contaminated with prions. Since the patient had undergone a neurosurgical operation in our institution 2.5 months prior to CJD diagnosis, and the neurosurgical instruments used during this procedure were not processed according to the prion decontamination principles, a question was raised regarding a potential prion transmission via contaminated instruments to the 29 patients who subsequently underwent neurosurgical operation. By analyzing the neurosurgical procedure that the patient underwent, we established that neurosurgical instruments were in contact with the patient's skin, subcutaneous tissue, fascia muscle, bones and intervertebral cartilage and not with the dura mater and that there was no cerebrospinal fluid drainage during surgery either. Therefore, we concluded that during neurosurgery the instruments did not come into contact with tissues of higher or medium risk for transmission of prions and that routine procedure for cleaning and sterilization of neurosurgical instruments was sufficient for preventing prion transmission, which is in accordance with the British and Canadian CJD infection control guidelines. Subsequent follow-up recorded not a single case of secondary CJD among the 29 patients who underwent surgery after the infected patient in our hospital.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases that affect several species, such as scrapie in sheep or goats and CJD in humans. In several species, neurological disease is preceded by TSE agent accumulation in lymphoid tissues prior to neuroinvasion. While oral transmission is considered the most common route for scrapie, transmission can also occur through lesions to the skin or mucosa, for example in the mouth or gastrointestinal tract due to rough feed, or birth associated skin damage. Scrapie has also been experimentally transmitted through skin scarification in mice. Following scrapie infection via skin scarification, PrPSc accumulates in the draining lymph node (LN) before spreading to other organs in the lymphoreticular system. It is not yet known by what means the scrapie agent is transported from the skin to the draining LN. Dendritic cells (DCs) in the skin have been found to transport viruses, such as HIV or Dengue, from the skin, thereby raising the question whether DCs or Langerhans cells (LCs), located within the epidermis, play a role in the uptake and transport of the TSE agent from the skin to the draining LN. CD11c is a cell surface marker traditionally used to identify or isolate DCs from other cell types. Mice and rats are naturally resistant to Diphtheria toxin (DTX). A transgenic mouse line was created where the Diphtheria toxin receptor (DTR) was expressed on CD11c+ cells. The presence of this receptor on CD11c+ cells allowed for the temporary conditional depletion of CD11c+ cells following a single injection of DTX. The cells repopulate the tissues within a time frame specific to the tissues the cells are located in. These mice were used to determine whether the absence of CD11c+ cells at the time of scrapie infection via the skin had an effect on the early accumulation of PrPSc within the lymphoid tissues and on disease progression. Immunohistochemical analysis demonstrated that early PrPSc accumulation in the draining LNs was delayed following depletion of CD11c+ cells, indicating that their potential role in the transport of the scrapie agent from the skin. Scrapie incubation period was not affected by the absence of the CD11c+ cells at the time of infection. Recent findings show that CD11c is not exclusive to DCs and is also expressed on macrophage populations. Following DTX-mediated depletion, DCs repopulate the tissues much faster than CD11c+ macrophages. Scrapie infection was carried out in the skin in DTX treated mice after DCs had repopulated the tissues but before macrophage numbers had returned, to determine whether macrophages rather than DCs played a role in the early accumulation of PrPSc in the draining LNs. No differences in PrPSc accumulation were observed in mice depleted of macrophages compared to controls and there was no effect on disease incubation period. Another transgenic mouse line was used, where DTX expression on langerin+ cells (LCs and langerin+ DCs in the dermis), allowed for their temporary depletion through DTX treatment. Following langerin+ cell depletion, increased PrPSc accumulation was observed in the draining LNs 7 weeks post infection, but did not affect the incubation period of disease. These results indicate that the absence of LCs somehow accelerated PrPSc accumulation, and that LCs might play a preventative role in early stages after infection.Histopathological analysis was used to complement microarray studies aimed to determine what immune responses were associated with scarification and DTXmediated depletion of cells within the skin and whether these responses might be linked to disease transmission. DCs and LCs in the skin appear to play different roles in the early stages following scrapie infection via the skin, but the lack of effect on incubation period does not rule out the involvement of other cell types or cell-free mechanisms of scrapie agent spread from the skin.

Anonymous screening of lymphoreticular tissues removed during routine surgery has been applied to estimate the UK population prevalence of asymptomatic vCJD prion infection. The retrospective study of Hilton et al (J Pathol 2004; 203: 733-739) found accumulation of abnormal prion protein in three formalin-fixed appendix specimens. This led to an estimated UK prevalence of vCJD infection of similar to 1 in 4000, which remains the key evidence supporting current risk reduction measures to reduce iatrogenic transmission of vCJD prions in the UK. Confirmatory testing of these positives has been hampered by the inability to perform immunoblotting of formalin-fixed tissue. Animal transmission studies offer the potential for `gold standard' confirmatory testing but are limited by both transmission barrier effects and known effects of fixation on scrapie prion titre in experimental models. Here we report the effects of fixation on brain and lymphoreticular human vCJD prions and comparative bioassay of two of the three prevalence study formalin-fixed, paraffin-embedded (FFPE) appendix specimens using transgenic mice expressing human prion protein (PrP). While transgenic mice expressing human PrP 129M readily reported vCJD prion infection after inoculation with frozen vCJD brain or appendix, and also FFPE vCJD brain, no infectivity was detected in FFPE vCJD spleen. No prion transmission was observed from either of the FFPE appendix specimens. The absence of detectable infectivity in fixed, known positive vCJD lymphoreticular tissue precludes interpreting negative transmissions from vCJD prevalence study appendix specimens. In this context, the Hilton et al study should continue to inform risk assessment pending the outcome of larger-scale studies on discarded surgical tissues and autopsy samples.

TOC Summary: An epidemic agent could have originated from such a cattle prion., Bovine spongiform encephalopathy (BSE) and BSE-related disorders have been associated with a single major prion strain. Recently, 2 atypical, presumably sporadic forms of BSE have been associated with 2 distinct prion strains that are characterized mainly by distinct Western blot profiles of abnormal protease-resistant prion protein (PrPres), named high-type (BSE-H) and low-type (BSE-L), that also differed from classical BSE. We characterized 5 atypical BSE-H isolates by analyzing their molecular and neuropathologic properties during transmission in transgenic mice expressing homologous bovine prion protein. Unexpectedly, in several inoculated animals, strain features emerged that were highly similar to those of classical BSE agent. These findings demonstrate the capability of an atypical bovine prion to acquire classical BSE–like properties during propagation in a homologous bovine prion protein context and support the view that the epidemic BSE agent could have originated from such a cattle prion.