Spread Of Prions Research Articles

The immunobiology of prion diseases

Individuals infected with prions succumb to brain damage, and prion infections continue to be inexorably lethal. However, many crucial steps in prion pathogenesis occur in lymphatic organs and precede invasion of the central nervous system. In the past two decades, a great deal has been learnt concerning the cellular and molecular mechanisms of prion lymphoinvasion. These properties are diagnostically useful and have, for example, facilitated preclinical diagnosis of variant Creutzfeldt-Jakob disease in the tonsils. Moreover, the early colonization of lymphoid organs can be exploited for post-exposure prophylaxis of prion infections. As stromal cells of lymphoid organs are crucial for peripheral prion infection, the dedifferentiation of these cells offers a powerful means of hindering prion spread in infected individuals. In this Review, we discuss the current knowledge of the immunobiology of prions with an emphasis on how basic discoveries might enable translational strategies.

Lack of prion transmission by sexual or parental routes in experimentally infected hamsters

Prion diseases are a group of neurodegenerative disorders affecting humans as well as captive and wild animals. The mechanisms and routes governing the natural spread of prions are not completely understood and several hypotheses have been proposed. In this study, we analyzed the effect of gender in prion incubation period, as well as the possibility of prion transmission by sexual and parental contact using 263K infected hamsters as a model. Our results show that males have significantly longer incubation periods compared with females when exposed to the same quantity of infectious material. Importantly, no evidence of sexual or parental prion transmission was found, even 500 d after sexual contact or birth, respectively. Western blotting and PMCA were unable to detect sub-clinical levels of PrPSc in experimental subjects, suggesting a complete absence of prion transmission by these routes. Our results show that sexual and parental transmission of prions does not occur in this model. It remains to be studied whether this conclusion is valid also for other prion strains and species.

The spread of prions

The spread of prions

Targeted knock-down of cellular prion protein expression in myelinating Schwann cells does not alter mouse prion pathogenesis

In naturally acquired transmissible spongiform encephalopathies, the pathogenic agents or prions spread from the sites of initial peripheral uptake or replication to the brain where they cause progressive and fatal neurodegeneration. Routing via the peripheral nervous system is considered to be one of the main pathways to the central nervous system. Replication of prions in Schwann cells is viewed as a potentially important mechanism for efficient prion spread along nerves. Here we used a Cre-loxP mouse transgenetic approach to disrupt host-encoded prion protein (PrP(C)) specifically in myelinating Schwann cells. Despite the use of infection routes targeting highly myelinated nerves, there was no alteration in mouse prion pathogenesis, suggesting that conversion-dependent, centripetal spread of prions does not crucially rely on PrP(C) expressed by myelinating Schwann cells.

Infectivity versus Seeding in Neurodegenerative Diseases Sharing a Prion-Like Mechanism.

Prions are considered the best example to prove that the biological information can be transferred protein to protein through a conformational change. The term “prion-like” is used to describe molecular mechanisms that share similarities with the mammalian prion protein self-perpetuating aggregation and spreading characteristics. Since prions are presumably composed only of protein and are infectious, the more similar the mechanisms that occur in the different neurodegenerative diseases, the more these processes will resemble an infection. In vitro and in vivo experiments carried out during the last decade in different neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's diseases (PD), and amyotrophic lateral sclerosis (ALS) have shown a convergence toward a unique mechanism of misfolded protein propagation. In spite of the term “infection” that could be used to explain the mechanism governing the diversity of the pathological processes, other concepts as “seeding” or “de novo induction” are being used to describe the in vivo propagation and transmissibility of misfolded proteins. The current studies are demanding an extended definition of “disease-causing agents” to include those already accepted as well as other misfolded proteins. In this new scenario, “seeding” would be a type of mechanism by which an infectious agent can be transmitted but should not be used to define a whole “infection” process.

Spread of Classic BSE Prions from the Gut via the Peripheral Nervous System to the Brain

An experimental oral bovine spongiform encephalopathy (BSE) challenge study was performed to elucidate the route of infectious prions from the gut to the central nervous system in preclinical and clinical infected animals. Tissue samples collected from the gut and the central and autonomic nervous system from animals sacrificed between 16 and 44 months post infection (mpi) were examined for the presence of the pathological prion protein (PrP(Sc)) by IHC. Moreover, parts of these samples were also bioassayed using bovine cellular prion protein (PrP(C)) overexpressing transgenic mice (Tgbov XV) that lack the species barrier for bovine prions. A distinct accumulation of PrP(Sc) was observed in the distal ileum, confined to follicles and/or the enteric nervous system, in almost all animals. BSE prions were found in the sympathetic nervous system starting at 16 mpi, and in the parasympathetic nervous system from 20 mpi. A clear dissociation between prion infectivity and detectable PrP(Sc) deposition became obvious. The earliest presence of infectivity in the brain stem was detected at 24 mpi, whereas PrP(Sc) accumulation was first detected after 28 mpi. In summary, our results decipher the centripetal spread of BSE prions along the autonomic nervous system to the central nervous system, starting already halfway in the incubation time.

Plasmacytoid dendritic cells sequester high prion titres at early stages of prion infection.

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.

Co-Infection with the Friend Retrovirus and Mouse Scrapie Does Not Alter Prion Disease Pathogenesis in Susceptible Mice

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrPSc) of the normally soluble protease-sensitive host prion protein (PrPC) is the major component of the infectious prion. During the course of prion disease, PrPSc accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrPSc in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.

Mice deficient in p52/NF-κB as a model for studying prion disease

AbstractA tight functional link between the central nervous and peripheral immune system closely related to the activity of NF-κB transcription factor has been evidenced in numerous brain pathological conditions. One of the most instructive examples is a prion neuroinvasion and the development of transmissible spongioform encephalopathy (TSE) or prion diseases. The failure of peripherally administrated prions to elicit disease in immune-deficient mice indicates that preserved organ microarchitecture is crucial for TSE pathogenesis and it seems to go through topographic relationships between follicular dendritic cells and sympathetic endings in lymphoid organs. New insight into the molecular requirements for follicular dendritic cell (FDC) development in NF-κB p52 deficient mice highlights the role of NF-κB transcription factor in prion disease progression. However, adoptive transfer of wild type bone marrow cells (NF-κB +/+ background) into p52 deficient animals (NF-κB -/- background) does not correct the FDC defect. RT-PCR analysis of the expression level of mRNA for LTa, LTb, TNFRI and BLC of the splenic cells from p52/NF-κB mutant/wt BM chimera did not reveal major changes compared to wt/wt BM chimeras, indicating that their normal expression within splenic tissue does not assure for complete restoration of the FDC network in p52/NF-κB mutant. This implies that the distance between FDCs and splenic nerve terminals around the central arteriole is longer and could interfere with prion spreading in lymphoid organs prior to neuroinvasion. In light of the fact that prion neuroinvasion highly depends on the physical distance between sympathetic nerve endings and FDCs dendritic extensions, it is not hard to imagine that this could be the way to approach the problem of prion diseases development.

Multifaceted roles of tunneling nanotubes in intercellular communication.

Cell-to-cell communication and exchange of materials are vital processes in multicellular organisms during cell development, cell repair, and cell survival. In neuronal and immunological cells, intercellular transmission between neighboring cells occurs via different complex junctions or synapses. Recently, long distance intercellular connections in mammalian cells called tunneling nanotubes (TNTs) have been described. These structures have been found in numerous cell types and shown to transfer signals and cytosolic materials between distant cells, suggesting that they might play a prominent role in intercellular trafficking. However, these cellular connections are very heterogeneous in both structure and function, giving rise to more questions than answers as to their nature and role as intercellular conduits. To better understand and characterize the functions of TNTs, we have highlighted here the latest discoveries regarding the formation, structure, and role of TNTs in cell-to-cell spreading of various signals and materials. We first gathered information regarding their formation with an emphasis on the triggering mechanisms observed, such as stress and potentially important proteins and/or signaling pathways. We then describe the various types of transfer mechanisms, in relation to signals and cargoes that have been shown recently to take advantage of these structures for intercellular transfer. Because a number of pathogens were shown to use these membrane bridges to spread between cells we also draw attention to specific studies that point toward a role for TNTs in pathogen spreading. In particular we discuss the possible role that TNTs might play in prion spreading, and speculate on their role in neurological diseases in general.

Tauons and Prions: Infamous Cousins?

The paradigm of Alzheimer's disease (AD) is one subject to frequent change: what was thought to be a rare form of pre-senile dementia was revealed as a wide-spread malady; where amyloid-β was deemed the sole causative agent for the better part of 20 years, tau protein was shown to play a crucial role in AD genesis. With the discovery of possible prion-like phenomena in this disease supposedly driven by cell-autonomous processes, an evaluation of the similarities and differences between tau-driven neurodegeneration and prion disease becomes necessary. In this article, we provide a comparison of the template agent genesis, filament assembly, as well as intra- and inter-individual spread of prions and tauons.

Sheep with Scrapie and Mastitis Transmit Infectious Prions through the Milk

Prions are misfolded proteins that are infectious and naturally transmitted, causing a fatal neurological disease in humans and animals. Prion shedding routes have been shown to be modified by inflammation in excretory organs, such as the kidney. Here, we show that sheep with scrapie and lentiviral mastitis secrete prions into the milk and infect nearly 90% of naïve suckling lambs. Thus, lentiviruses may enhance prion transmission, conceivably sustaining prion infections in flocks for generations. This study also indicates a risk of prion spread to sheep and potentially to other animals through dietary exposure to pooled sheep milk or milk products.

Characterization of the role of dendritic cells in prion transfer to primary neurons

TSEs (transmissible spongiform encephalopathies) are neurodegenerative diseases caused by pathogenic isoforms (PrPSc) of the host-encoded PrPc (cellular prion protein). After consumption of contaminated food, PrPSc deposits rapidly accumulate in lymphoid tissues before invasion of the CNS (central nervous system). However, the mechanisms of prion spreading from the periphery to the nervous system are still unclear. In the present study, we investigated the role of DCs (dendritic cells) in the spreading of prion infection to neuronal cells. First, we determined that BMDCs (bone-marrow-derived DCs) rapidly uptake PrPSc after exposure to infected brain homogenate. Next, we observed a progressive catabolism of the internalized prion aggregates. Similar experiments performed with BMDCs isolated from KO (knockout) mice or mice overexpressing PrP (tga20) indicate that both PrPSc uptake and catabolism are independent of PrPc expression in these cells. Finally, using co-cultures of prion-loaded BMDCs and cerebellar neurons, we characterized the transfer of the prion protein and the resulting infection of the neuronal cultures. Interestingly, the transfer of PrPSc was triggered by direct cell-cell contact. As a consequence, BMDCs retained the prion protein when cultured alone, and no transfer to the recipient neurons was observed when a filter separated the two cultures or when neurons were exposed to the BMDC-conditioned medium. Additionally, fixed BMDCs also failed to transfer prion infectivity to neurons, suggesting an active transport of prion aggregates, in accordance with a role of TNTs (tunnelling nanotubes) observed in the co-cultures.

Spreading of prions from the immune to the peripheral nervous system: a potential implication of dendritic cells

The implication of dendritic cells (DCs) in the peripheral spreading of prions has increased in the last few years. It has been recently described that DCs can transmit prions to primary neurons from the central nervous system. In order to improve the understanding of the earliest steps of prion peripheral neuroinvasion, we studied, using an in vitro model, the effect of exposing primary peripheral neurons to scrapie-infected lymphoid cells. Thanks to this system, there is evidence that bone marrow dendritic cells (BMDCs) are in connection with neurites of peripheral neurons via cytoplasmic extensions. BMDCs are competent to internalize prions independently from the expression of cellular prion protein (PrP(C)) and have the capacity to transmit detergent-insoluble, relatively proteinase K-resistant prion protein (PrP(Sc)) to peripheral neurons after 96 h of coculture. Furthermore, we confirmed the special status of the peripheral nervous system in front of prion diseases. Contrary to central neurons, PrP(Sc) infection does not disturb survival and neurite outgrowth. Our model demonstrates that PrP(Sc)-loaded dendritic cells and peripheral nerve fibers that are included in neuroimmune interfaces can initiate and spread prion neuroinvasion.

Paracrine Diffusion of PrPC and Propagation of Prion Infectivity by Plasma Membrane-Derived Microvesicles

Cellular prion protein (PrPc) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1–1 µm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrPC diffusion and prion infectivity transmission. We first identified PrPC in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrPSc is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrPSc-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrPC diffusion and signaling as well as to the process of prion spread and neuroinvasion.

From mad cows to sensible blood transfusion: the risk of prion transmission by labile blood components in the United Kingdom and in France

Transfusion transmission of the prion, the agent of variant Creutzfeldt-Jakob disease (vCJD), is now established. Subjects infected through food may transmit the disease through blood donations. The two nations most affected to date by this threat are the United Kingdom (UK) and France. The first transfusion cases have been observed in the UK over the past 5 years. In France, a few individuals who developed vCJD had a history of blood donation, leading to a risk of transmission to recipients, some of whom could be incubating the disease. In the absence of a large-scale screening test, it is impossible to establish the prevalence of infection in the blood donor population and transfused patients. This lack of a test also prevents specific screening of blood donations. Thus, prevention of transfusion transmission essentially relies at present on deferral of "at-risk" individuals. Because prions are present in both white blood cells and plasma, leukoreduction is probably insufficient to totally eliminate the transfusion risk. In the absence of a screening test for blood donations, recently developed prion-specific filters could be a solution. Furthermore, while the dietary spread of vCJD seems efficiently controlled, uncertainty remains as to the extent of the spread of prions through blood transfusion and other secondary routes.

Faecal shedding, alimentary clearance and intestinal spread of prions in hamsters fed with scrapie

Shedding of prions via faeces may be involved in the transmission of contagious prion diseases. Here, we fed hamsters 10 mg of 263K scrapie brain homogenate and examined the faecal excretion of disease-associated prion protein (PrPTSE) during the course of infection. The intestinal fate of ingested PrPTSE was further investigated by monitoring the deposition of the protein in components of the gut wall using immunohistochemistry and paraffin-embedded tissue (PET) blotting. Western blotting of faecal extracts showed shedding of PrPTSE in the excrement at 24–72 h post infection (hpi), but not at 0–24 hpi or at later preclinical or clinical time points. About 5% of the ingested PrPTSE were excreted via the faeces. However, the bulk of PrPTSE was cleared from the alimentary canal, most probably by degradation, while an indiscernible proportion of the inoculum triggered intestinal infection. Components of the gut-associated lymphoid tissue (GALT) and the enteric nervous system (ENS) showed progressing accumulation of PrPTSE from 30 days post infection (dpi) and 60 dpi, respectively. At the clinical stage of disease, substantial deposits of PrPTSE were found in the GALT in close vicinity to the intestinal lumen. Despite an apparent possibility of shedding from Peyer’s patches that may involve the follicle-associated epithelium (FAE), only small amounts of PrPTSE were detected in faeces from clinically infected animals by serial protein misfolding cyclic amplification (sPMCA). Although excrement may thus provide a vehicle for the release of endogenously formed PrPTSE, intestinal clearance mechanisms seem to partially counteract such a mode of prion dissemination.

Immunolocalisation of PrP Sc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy

The causative agent of transmissible spongiform encephalopathies (TSE) is PrP Sc, an infectious, misfolded isoform of the cellular prion protein (PrP C). The localisation and trafficking of PrP Sc and sites of conversion from PrP C to PrP Sc are under debate, particularly since most published work did not discriminate between PrP C and PrP Sc. Here we describe the localisation of PrP C and PrP Sc in a scrapie-infected neuroblastoma cell line, ScN2a, by light and electron microscopic immunolocalisation. After eliminating PrP C with proteinase K, PrP Sc was detected at the plasma membrane, endocytosed via clathrin-coated pits and delivered to early endosomes. Finally, PrP Sc was detected in late endosomes/lysosomes. As we detected PrP Sc at the cell surface, in early endosomes and in late endosomes/lysosomes, i.e. locations where PrP C is also present, our data imply that the conversion process could take place at the plasma membrane and/or along the endocytic pathway. Finally, we observed the release of PrP C/PrP Sc via exocytotic pathways, i.e. via exosomes and as an opaque electron-dense mass which may represent a mechanism of intercellular spreading of infectious prions.

Rodent models for prion diseases

Until today most prion strains can only be propagated and the infectivity content assayed by experimentally challenging conventional or transgenic animals. Robust cell culture systems are not available for any of the natural and only for a few of the experimental prion strains. Moreover, the pathogenesis of different transmissible spongiform encephalopathies (TSE) can be analysed systematically by using experimentally infected animals. While, in the beginning, animals belonging to the natural host species were used, more and more rodent model species have been established, mostly due to practical reasons. Nowadays, most of these experiments are performed using highly susceptible transgenic mouse lines expressing cellular prion proteins, PrP, from a variety of species like cattle, sheep, goat, cervidae, elk, hamster, mouse, mink, pig, and man. In addition, transgenic mice carrying specific mutations or polymorphisms have helped to understand the molecular pathomechanisms of prion diseases. Transgenic mouse models have been utilised to investigate the physiological role of PrP(C), molecular aspects of species barrier effects, the cell specificity of the prion propagation, the role of the PrP glycosylation, the mechanisms of the prion spread, the neuropathological roles of PrP(C) and of its abnormal isoform PrP(D) (D for disease) as well as the function of PrP Doppel. Transgenic mouse models have also been used for mapping of PrP regions involved in or required for the PrP conversion and prion replication as well as for modelling of familial forms of human prion diseases.

Altered lymphocyte homeostasis after oral prion infection in mouse

Transmissible spongiform encephalopaties (TSEs) or prion diseases develop as central nervous system (CNS) disorders characterized by extremely long incubation periods. Although TSEs do not go along with inflammatory infiltrates and/or antibody production against the prion protein (PrP Sc), the immune system plays an important role in pathogenesis as long as different lymphoid organs (Peyer's patches, lymph nodes and spleen) may facilitate the accumulation and further spread of prions after peripheral exposure. In this work we investigated the changes in lymphoid and dendritic cell (DC) populations as well as the implications of different cytokines during disease progression after experimental oral inoculation of prions in a transgenic mouse model. At different days post-inoculation (dpi), T and B lymphocytes and DC populations from lymphoid organs, blood and brain were analyzed by flow cytometry and immunohistochemistry. Besides time related variations in lymphoid cell numbers due to the aging of the animals significant changes related with the infection were found in mesenteric lymph nodes, peripheral blood leukocytes (PBLs) as well as in spleen, affecting the CD4/CD8 ratio. In contrast, little or no variation was detected in Peyer's Patches or in thymus either associated with aging or the infection status. At individual time points significant differences between infected and control mice were seen in the CD8, CD4 and DC populations, with less evidence of differences in the B cell compartment. Finally, a pro-inflammatory phenotype occurred at early times in the spleen, where the levels of lymphotoxin-β mRNA were found augmented with respect to controls. Altogether, these results suggest that normal regulation of lymphocyte populations becomes altered along the progression of a prion infection.