Lichens: Unexpected anti-prion agents?

The prion diseases sheep scrapie and cervid chronic wasting disease are transmitted, in part, via an environmental reservoir of infectivity; prions released from infected animals persist in the environment and can cause disease years later. Central to controlling disease transmission is the identification of methods capable of inactivating these agents on the landscape. We have found that certain lichens, common, ubiquitous, symbiotic organisms, possess a serine protease capable of degrading prion protein (PrP) from prion-infected animals. The protease functions against a range of prion strains from various hosts and reduces levels of abnormal PrP by at least two logs. We have now tested more than twenty lichen species from several geographical locations and from various taxa and found that approximately half of these species degrade PrP. Critical next steps include examining the effect of lichens on prion infectivity and cloning the protease responsible for PrP degradation. The impact of lichens on prions in the environment remains unknown. We speculate that lichens could have the potential to degrade prions when they are shed from infected animals onto lichens or into environments where lichens are abundant. In addition, lichens are frequently consumed by cervids and many other animals and the effect of dietary lichens on prion disease transmission should also be considered.Key words: prion, lichen, degradation, environment, Chronic Wasting disease, protease, transmissible spongiform encephalopathy     

Degradation of the disease-associated prion protein by a serine protease from lichens

The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.     

Generation of a new form of human PrP(Sc) in vitro by interspecies transmission from cervid prions

Prion diseases are infectious neurodegenerative disorders that affect humans and animals and that result from the conversion of normal prion protein (PrP(C)) into the misfolded prion protein (PrP(Sc)). Chronic wasting disease (CWD) is a prion disorder of increasing prevalence within the United States that affects a large population of wild and captive deer and elk. Determining the risk of transmission of CWD to humans is of utmost importance, considering that people can be infected by animal prions, resulting in new fatal diseases. To study the possibility that human PrP(C) can be converted into the misfolded form by CWD PrP(Sc), we performed experiments using the protein misfolding cyclic amplification technique, which mimics in vitro the process of prion replication. Our results show that cervid PrP(Sc) can induce the conversion of human PrP(C) but only after the CWD prion strain has been stabilized by successive passages in vitro or in vivo. Interestingly, the newly generated human PrP(Sc) exhibits a distinct biochemical pattern that differs from that of any of the currently known forms of human PrP(Sc). Our results also have profound implications for understanding the mechanisms of the prion species barrier and indicate that the transmission barrier is a dynamic process that depends on the strain and moreover the degree of adaptation of the strain. If our findings are corroborated by infectivity assays, they will imply that CWD prions have the potential to infect humans and that this ability progressively increases with CWD spreading.     

Subclinical prion disease induced by oral inoculation

Natural transmission of prion disease is believed to occur by peripheral infection such as oral inoculation. Following this route of inoculation, both the peripheral nervous system and the lymphoreticular system may be involved in the subsequent neuroinvasion of the central nervous system by prions, which may not necessarily result in clinical signs of terminal disease. Subclinical prion disease, characterized by the presence of infectivity and PrP(Sc) in the absence of overt clinical signs, may occur. It is not known which host factors contribute to whether infection with prions culminates in a terminal or subclinical disease state. We have investigated whether the level of host PrP(c) protein expression is a factor in the development of subclinical prion disease. When RML prion inoculum was inoculated by either the i.c. or intraperitoneal route, wild-type and tga20 mice both succumbed to terminal prion disease. In contrast, orally inoculated tga20 mice succumbed to terminal prion disease, whereas wild-type mice showed no clinical signs. However, wild-type mice sacrificed 375 or 525 days after oral inoculation harbored significant levels of brain PrP(Sc) and infectivity. These data show that same-species transmission of prions by the oral route in animals that express normal levels of PrP(c) can result in subclinical prion disease. This indicates that the level of host PrP(c) protein expression is a contributing factor to the regulation of development of terminal prion disease. Events that increase PrP(c) expression may predispose a prion-infected animal to the more deleterious effects of prion pathology.     

Prions adhere to soil minerals and remain infectious

An unidentified environmental reservoir of infectivity contributes to the natural transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) in sheep, deer, and elk. Prion infectivity may enter soil environments via shedding from diseased animals and decomposition of infected carcasses. Burial of TSE-infected cattle, sheep, and deer as a means of disposal has resulted in unintentional introduction of prions into subsurface environments. We examined the potential for soil to serve as a TSE reservoir by studying the interaction of the disease-associated prion protein (PrP(Sc)) with common soil minerals. In this study, we demonstrated substantial PrP(Sc) adsorption to two clay minerals, quartz, and four whole soil samples. We quantified the PrP(Sc)-binding capacities of each mineral. Furthermore, we observed that PrP(Sc) desorbed from montmorillonite clay was cleaved at an N-terminal site and the interaction between PrP(Sc) and Mte was strong, making desorption of the protein difficult. Despite cleavage and avid binding, PrP(Sc) bound to Mte remained infectious. Results from our study suggest that PrP(Sc) released into soil environments may be preserved in a bioavailable form, perpetuating prion disease epizootics and exposing other species to the infectious agent.     

A systematic investigation of production of synthetic prions from recombinant prion protein

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic'' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.     

Mouse-adapted sporadic human Creutzfeldt-Jakob disease prions propagate in cell culture

Cell based models used for the study of prion diseases have traditionally employed mouse-adapted strains of sheep scrapie prions. To date, attempts to generate human prion propagation in cell culture have been unsuccessful. Rabbit kidney epithelial cells (RK13) are permissive to infection with prions from a variety of species upon expression of cognate PrP transgenes. We explored RK13 cells expressing human PrP for their utility as a cell line capable of sustaining infection with human prions. RK13 cells processed exogenously expressed human PrP similarly to exogenously expressed mouse PrP but were not permissive to infection when exposed to sporadic Creutzfeldt-Jakob disease prions. Transmission of the same sporadic Creutzfeldt Jakob disease prions to wild-type mice generated a strain of mouse-adapted human prions, which efficiently propagated in RK13 cells expressing mouse PrP, demonstrating these cells are permissive to infection by mouse-adapted human prions. Our observations underscore the likelihood that, in contrast to prions derived from non-human mammals, additional unidentified cofactors or subcellular environment are critical for the generation of human prions.     

Prions,prionoids and pathogenic proteins in Alzheimer disease

Like patients with prion disease, Alzheimer patients suffer from a fatal, progressive form of dementia. There is growing evidence that amyloid-β (Aβ) aggregates may be transmissible similar to prions, at least under extreme experimental conditions. However, unlike mice infected with prion protein (PrP) prions, those inoculated with Aβ do not die. The transmission of Aβ and PrP thus differs conspicuously in the neurological effects they induce in their hosts, the difference being no less than a matter of life and death. Far from being a mere academic nuance, this distinction between Aβ and PrP begs the crucial questions of what, exactly, controls prion toxicity and how prion toxicity relates to prion infectivity.     

Transmission Properties of Human PrP 102L Prions Challenge the Relevance of Mouse Models of GSS

Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene (PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Sträussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease.     

Mammalian prions: Tracking the infectious entities

Protein misfolding is central to the pathogenesis of several neurodegenerative disorders. Among these disorders, prion diseases are unique because they are transmissible. The conversion of the host-encoded GPI-anchored PrP protein into a structurally altered form is crucially associated with the infectious and neurotoxic properties of the resulting abnormal PrP. Many lines of evidence indicate that distinct aggregated forms with different size and protease resistance are produced during prion multiplication. The recent isolation of various subsets of abnormal PrP, along with the improved biochemical tools and infectivity detection assays have shed light on the diversity of abnormal PrP protein and may give insights into the features of the more infectious subsets of abnormal PrP.Key words: prions, aggregates, infectivity, EST, PrP protein     

Accumulation of pathological prion protein PrPSc in the skin of animals with experimental and natural scrapie

Prion infectivity and its molecular marker, the pathological prion protein PrP(Sc), accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrP(Sc) was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles). Here, we address the question of whether prions target the skin and show widespread PrP(Sc) deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrP(Sc) was detected before the onset of symptoms, but the bulk of skin-associated PrP(Sc) accumulated in the clinical phase. PrP(Sc) was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrP(Sc) in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrP(Sc) by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission.     

Detection of infectious prions in urine

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). The mechanism of prion transmission is unknown. In this study, we attempted to detect prions in urine of experimentally infected animals. PrP(Sc) was detected in approximately 80% of the animals studied, whereas no false positives were observed among the control animals. Semi-quantitative calculations suggest that PrP(Sc) concentration in urine is around 10-fold lower than in blood. Interestingly, PrP(Sc) present in urine maintains its infectious properties. Our data indicate that low quantities of infectious prions are excreted in the urine. These findings suggest that urine is a possible source of prion transmission.     

Vertical transmission of bovine spongiform encephalopathy prions evaluated in a transgenic mouse model

In this work we show evidence of mother-to-offspring transmission in a transgenic mouse line expressing bovine PrP (boTg) experimentally infected by intracerebral administration of bovine spongiform encephalopathy (BSE) prions. PrP(res) was detected in brains of newborns from infected mothers only when mating was allowed near to the clinical stage of disease, when brain PrP(res) deposition could be detected by Western blot analysis. Attempts to detect infectivity in milk after intracerebral inoculation in boTg mice were unsuccessful, suggesting the involvement of other tissues as carriers of prion dissemination. The results shown here prove the ability of BSE prions to spread centrifugally from the central nervous system to peripheral tissues and to offspring in a mouse model. Also, these results may complement previous epidemiological data supporting the occurrence of vertical BSE transmission in cattle.     

Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.

The 'protein only' hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the 'protein only' hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).     

Alteration of the chronic wasting disease species barrier by in vitro prion amplification

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids now detected in 19 states of the United States, three Canadian provinces, and South Korea. Whether noncervid species can be infected by CWD and thereby serve as reservoirs for the infection is not known. To investigate this issue, we previously used serial protein misfolding cyclic amplification (sPMCA) to demonstrate that CWD prions can amplify in brain homogenates from several species sympatric with cervids, including prairie voles (Microtus ochrogaster) and field mice (Peromyscus spp.). Here, we show that prairie voles are susceptible to mule deer CWD prions in vivo and that sPMCA amplification of CWD prions in vole brain enhances the infectivity of CWD for this species. Prairie voles inoculated with sPMCA products developed clinical signs of TSE disease approximately 300 days prior to, and more consistently than, those inoculated with CWD prions from deer brain. Moreover, the deposition patterns and biochemical properties of protease-resistant form of PrP (PrP(RES)) in the brains of affected voles differed from those in cervidized transgenic (CerPrP) mice infected with CWD. In addition, voles inoculated orally with sPMCA products developed clinical signs of TSE and were positive for PrP(RES) deposition, whereas those inoculated orally with deer-origin CWD prions did not. These results demonstrate that transspecies sPMCA of CWD prions can enhance the infectivity and adapt the host range of CWD prions and thereby may be useful to assess determinants of prion species barriers.     

Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin

Disease-related PrP(Sc) [pathogenic PrP (prion protein)] is classically distinguished from its normal cellular precursor, PrP(C)(cellular PrP) by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease-resistant fragments of PrP(Sc) using PK (proteinase K), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrP(Sc) in its full-length form. In the present study, we show that thermolysin can degrade PrP(C) while preserving both PK-sensitive and PK-resistant isoforms of disease-related PrP in both rodent and human prion strains. For mouse RML (Rocky Mountain Laboratory) prions, the majority of PK-sensitive disease-related PrP isoforms do not appear to contribute significantly to infectivity. In vCJD (variant Creutzfeldt-Jakob disease), the human counterpart of BSE (bovine spongiform encephalopathy), up to 90% of total PrP present in the brain resists degradation with thermolysin, whereas only approximately 15% of this material resists digestion by PK. Detection of PK-sensitive isoforms of disease-related PrP using thermolysin should be useful for improving diagnostic sensitivity in human prion diseases.     

Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.     

Prions on the move

Prions consist mainly, if not entirely, of PrP(Sc), an aggregated conformer of the host protein PrP(C). Prions come in different strains, all based on the same PrP(C) sequence, but differing in their conformations. The efficiency of prion transmission between species is usually low, but increases after serial transmission in the new host, suggesting a process involving mutation and selection. Even within the same species, the transfer of prions between cell types entails a selection of favoured 'substrains', and propagation of prions in the presence of an inhibitory drug can result in the appearance of drug-resistant prion populations. We propose that prion populations are comprised of a variety of conformers, constituting 'quasi-species', from which the one replicating most efficiently in a particular environment is selected.     

Inherited Prion Disease A117V Is Not Simply a Proteinopathy but Produces Prions Transmissible to Transgenic Mice Expressing Homologous Prion Protein

Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene (PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP^Sc), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP (^CtmPrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP^Sc was demonstrated in the brains of recipient transgenic mice. This PrP^Sc rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of ^CtmPrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.     

Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions

Prions are unconventional infectious agents composed exclusively of misfolded prion protein (PrP(Sc)), which transmits the disease by propagating its abnormal conformation to the cellular prion protein (PrP(C)). A key characteristic of prions is their species barrier, by which prions from one species can only infect a limited number of other species. Here, we report the generation of infectious prions by interspecies transmission of PrP(Sc) misfolding by in vitro PMCA amplification. Hamster PrP(C) misfolded by mixing with mouse PrP(Sc) generated unique prions that were infectious to wild-type hamsters, and similar results were obtained in the opposite direction. Successive rounds of PMCA amplification result in adaptation of the in vitro-produced prions, in a process reminiscent of strain stabilization observed upon serial passage in vivo. Our results indicate that PMCA is a valuable tool for the investigation of cross-species transmission and suggest that species barrier and strain generation are determined by the propagation of PrP misfolding.