Coinfecting Prion Strains Compete for a Limiting Cellular Resource

Prion strain interference can influence the emergence of a dominant strain from a mixture; however, the mechanisms underlying prion strain interference are poorly understood. In our model of strain interference, inoculation of the sciatic nerve with the drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent prior to superinfection with the hyper (HY) strain of TME can completely block HY TME from causing disease. We show here that the deposition of PrP^Sc, in the absence of neuronal loss or spongiform change, in the central nervous system corresponds with the ability of DY TME to block HY TME infection. This suggests that DY TME agent-induced damage is not responsible for strain interference but rather prions compete for a cellular resource. We show that protein misfolding cyclic amplification (PMCA) of DY and HY TME maintains the strain-specific properties of PrP^Sc and replicates infectious agent and that DY TME can interfere, or completely block, the emergence of HY TME. DY PrP^Sc does not convert all of the available PrP^C to PrP^Sc in PMCA, suggesting the mechanism of prion strain interference is due to the sequestering of PrP^C and/or other cellular components required for prion conversion. The emergence of HY TME in PMCA was controlled by the initial ratio of the TME agents. A higher ratio of DY to HY TME agent is required for complete blockage of HY TME in PMCA compared to several previous in vivo studies, suggesting that HY TME persists in animals coinfected with the two strains. This was confirmed by PMCA detection of HY PrP^Sc in animals where DY TME had completely blocked HY TME from causing disease.Prions are infectious agents of animals, including humans, which are comprised of PrP^Sc, a misfolded isoform of the noninfectious host encoded protein PrP^C (17, 24, 50, 63). Prion diseases of humans are unique neurodegenerative disorders in that they can have either a sporadic, familial, or infectious etiology. Prions cause disease in economically important domestic and wild animal species such as bovine spongiform encephalopathy in cattle and chronic wasting disease in wild and captive cervids (20, 62). Prion diseases can be zoonotic as illustrated by the transmission of bovine spongiform encephalopathy to humans that resulted in the emergence of variant Creutzfeldt-Jacob disease (14, 19, 22, 23, 46, 61, 68). Prion diseases are inevitably fatal and there are currently no effective treatments (21).Prion strains are defined by a characteristic set of features that breed true upon experimental passage (33, 34). Strain-specific differences have been identified in incubation period, clinical signs, agent distribution, overdominance, host range, neuropathology, and biochemical properties of PrP^Sc (5, 10, 11, 13, 28, 34, 42, 44). Strain-specific conformations of PrP^Sc are hypothesized to encode prion strain diversity; however, it is not understood how these differences result in the distinct strain properties (11, 19, 40, 47, 59, 66).Prion strain interference may be involved in the emergence of a dominant strain from a mixture as could occur during prion adaptation to a new host species or during prion evolution (4, 36, 43, 48, 56). In the natural prion diseases, there are examples where an individual host may be infected with more than one prion strain (15, 25, 55, 57, 58). Experimentally, coinfection or superinfection of prion strains can result in interference where a blocking, long incubation period strain extends the incubation period or completely blocks a superinfecting, short incubation period strain from causing disease (26, 27). Prion interference has been described in experimental studies of mice and hamsters infected with a wide variety of prion strains and routes of inoculation, suggesting it may be a common property of prion disease (3, 27, 52, 53, 60).It has been proposed that prion strains compete for a shared “replication site”; however, mechanistic details are not known, and it is unclear whether the blocking strain destroys or occupies the replication sites required for the superinfecting strain (28). The transport to and relative onset of replication of interfering strains in a common population of neurons is an important factor that can determine which strain will emerge (8). In the present study, we sought to determine whether the blocking strain disables transport and spread of the superinfecting strain or whether prion interference is due to competition for a cellular resource.     

Cellular Prion Protein Promotes Regeneration of Adult Muscle Tissue

It is now well established that the conversion of the cellular prion protein, PrP^C, into its anomalous conformer, PrP^Sc, is central to the onset of prion disease. However, both the mechanism of prion-related neurodegeneration and the physiologic role of PrP^C are still unknown. The use of animal and cell models has suggested a number of putative functions for the protein, including cell signaling, adhesion, proliferation, and differentiation. Given that skeletal muscles express significant amounts of PrP^C and have been related to PrP^C pathophysiology, in the present study, we used skeletal muscles to analyze whether the protein plays a role in adult morphogenesis. We employed an in vivo paradigm that allowed us to compare the regeneration of acutely damaged hind-limb tibialis anterior muscles of mice expressing, or not expressing, PrP^C. Using morphometric and biochemical parameters, we provide compelling evidence that the absence of PrP^C significantly slows the regeneration process compared to wild-type muscles by attenuating the stress-activated p38 pathway, and the consequent exit from the cell cycle, of myogenic precursor cells. Demonstrating the specificity of this finding, restoring PrP^C expression completely rescued the muscle phenotype evidenced in the absence of PrP^C.The cellular prion protein (PrP^C) is a glycoprotein, prominently expressed in the mammalian central nervous system (CNS) and lymphoreticular system, that is anchored to the cell external surface through a glycolipidic moiety. The bad reputation acquired by PrP^C originates from the notion that an aberrant conformer of it (PrP^Sc) is the major component of the prion, the unconventional infectious particle that causes fatal neurodegenerative disorders, i.e., transmissible spongiform encephalopathies (TSE) or prion diseases (56). A wealth of evidence has suggested that the function of PrP^C is beneficial to the cell, but currently, our detailed comprehension of its physiology remains poor. In this respect, the availability of knockout (KO) paradigms for PrP^C has provided less crucial information than expected. Subtle phenotypes, e.g., mild neuropathologic, cognitive, and behavioral deficits, have been described in PrP-KO mice (17, 50), but these animals generally live a normal life span without displaying obvious developmental defects (8, 42). Importantly, the same holds true when the expression of PrP^C is postnatally abrogated (40). The extensive search for PrP^C''s raison d''être has ascribed to the protein a plethora of functions (for updated reviews, see references 1 and 35); among these, roles in cell adhesion, migration, and differentiation have been proposed whereby PrP^C could act by modulating different cell-signaling pathways (63). In this framework, a variety of neuronal proteins have been hypothesized to interact with PrP^C (reviewed in references 1 and 11), for example, cell adhesion molecules or extracellular matrix proteins, which could explain the capacity of PrP^C to mediate the neuritogenesis and neuronal differentiation observed in several cell model systems (13, 22, 23, 27, 36, 59, 64).Although neurons are generally regarded as the model of choice for unraveling the function of PrP^C, the expression of the protein in several other organs suggests that PrP^C has a conserved role in different tissues. Thus, important insight into PrP^C function may also be provided by the analysis of extraneural tissues. One such tissue is skeletal muscle, which has been shown to express PrP^C at significant levels (43, 46) and has been found to upregulate PrP^C levels under stress conditions (71). On the other hand, ablation of the PrP gene has been shown to directly affect skeletal muscles, for example, by enhancing oxidative damage (30) or by diminishing tolerance for physical exercise (51). Skeletal muscles have also been associated with prion pathology, as evidenced by the accumulation of PrP^Sc (or PrP^Sc-like forms) in the muscles of TSE-affected humans and animals (2, 3, 6, 21, 53, 67) and by transgenic-mouse models of some inherited TSEs (16). In addition, overexpression of wild-type (WT) PrP^C (25, 68), or expression of TSE-associated mutants of the protein (16, 66), generates myopathic traits in transgenic mice.In light of these notions, and because intact muscle tissues are more amenable to in vivo manipulations than neural tissue, we set out to analyze the potential role of PrP^C in tissue morphogenesis (38, 41, 46) using an in vivo skeletal-muscle paradigm from two congenic mouse lines expressing (WT) or not expressing (PrP-KO) PrP^C. Importantly, to verify that the PrP-KO muscle phenotype was specifically dependent on the absence of PrP^C, we used PrP-KO mice reconstituted with a PrP transgene (PrP-Tg). The applied protocol consisted of first characterizing the degeneration of the hind-limb tibialis anterior (TA) muscle and then evaluating the myogenic process from the response to inflammation to the full recovery of the muscle. By combining acute insult with adult age, this strategy also had the potential to bypass possible compensatory mechanisms that might mask PrP-KO phenotypes during embryogenesis and/or in adulthood under normal conditions (65).In this study, we provide evidence that, compared to animals expressing PrP^C (WT and PrP-Tg), recovery from damage of adult skeletal muscles was significantly slower in PrP-KO mice. Analysis of the different stages of muscle regeneration allowed us to conclude that PrP^C is one of the factors that govern the early phases of this process, in which the proliferation and differentiation of myogenic precursor cells take place.     

Role of the Lymphoreticular System in Prion Neuroinvasion from the Oral and Nasal Mucosa

Prion neuroinvasion from peripheral tissues involves agent replication in the lymphoreticular system (LRS) prior to entry into the nervous system. This study investigated the role of the LRS in prion neuroinvasion from the oral and nasal mucosa in wild-type and immunodeficient mice and in hamsters infected with the HY and DY strains of the transmissible mink encephalopathy (TME) agent. Following inoculation at neural sites, all hosts were susceptible to prion disease and had evidence of prion infection in the brain, but infection of the LRS was found only in scrapie-infected wild-type mice and HY TME-infected hamsters. In the LRS replication-deficient models, prion neuroinvasion was not observed following intraperitoneal or oral inoculation. However, immunodeficient mice, which have impaired follicular dendritic cells, were susceptible to scrapie following intratongue and intranasal inoculation despite the absence of PrP^Sc in the tongue or the nasal cavity. For DY TME, hamsters were susceptible following intratongue but not intranasal inoculation and PrP^Sc was limited to nerve fibers of the tongue. These findings indicate that neuroinvasion from the tongue and nasal cavity can be independent of LRS infection but neuroinvasion was partially dependent on the strain of the prion agent and/or the host species. The paucity of PrP^Sc deposition in the oral and nasal mucosa from LRS replication-deficient hosts following neuroinvasion from these tissues suggests an infection of nerve fibers that is below the threshold of PrP^Sc detection and/or the transport of the prion agent along cranial nerves without agent replication.In natural and experimental prion infections originating in the periphery, prion agent replication in the lymphoreticular system (LRS) precedes agent entry and spread in the peripheral nervous system. In the LRS, follicular dendritic cells (FDCs) are the major target of prion infection, and blocking or reversing FDC maturation can prevent scrapie agent replication in the LRS (25, 26, 28, 30, 32). Other migrating cell populations may also influence the progression of experimental prion disease (27, 36). From the LRS, centripetal spread of the prion agent to the spinal cord or brain occurs by spread along nerve fibers of the peripheral nervous system. In the central nervous system, prion agent replication can induce neurodegeneration and disease after an incubation period that can last from weeks to years. For example, in lambs from flocks with endemic scrapie, agent replication is initially detected in the gut-associated lymphoid tissues prior to proximal and distal spread in the LRS, infection of peripheral nerves that innervate the LRS, and subsequent spread to the spinal cord (19, 42). In addition, scrapie agent infection of the vagal nerve, which innervates many peripheral organs including the digestive tract, results in axonal transport directly to the dorsal motor nucleus of the vagus in the brain stem (29, 41). The role of scrapie infection in the LRS in the latter pathway of neuroinvasion is unknown. A similar pathway of prion neuroinvasion occurs in mule deer experimentally infected with the chronic wasting disease agent with the exception that early infection is also established in the lymph nodes of the upper gastrointestinal tract (37, 38). Recent studies indicate that a similar pathway of neuroinvasion occurs in natural and experimental bovine spongiform encephalopathy (BSE) following oral exposure except that agent replication in the LRS is greatly reduced and appears restricted to portions of the gut-associated lymphoid tissues (13, 20, 39).There are natural prion diseases in sheep and cattle that do not exhibit the typical distribution of the prion agent in the brain and LRS that are presumably acquired via oral prion exposure (5, 33). The absence of the abnormal isoform of the prion protein, PrP^Sc, in the LRS and dorsal motor nucleus of the vagus in atypical scrapie and the H type or L type of BSE raises the question as to whether these cases are due to infection by an alternate route(s) other than ingestion or whether these cases have an etiology that is distinct from that of acquired prion diseases. Direct prion infection of nerve fibers or terminals in highly innervated tissues, such as the mucosa in the head, has been suggested to represent potential sites of prion agent entry that would not require prior agent replication in the LRS (4, 12, 31). The presence of scrapie or BSE infection in the retina, sensory fibers of the tongue, and nasal mucosa of sheep, goat, and/or cattle suggests that the eye, tongue, or nasal cavity could be alternate sites of prion agent entry into hosts (8, 11, 15, 16, 40). Experimental prion inoculation at these mucosal sites can cause prion disease and in some cases rapid neuroinvasion (4, 9, 17, 18). Another explanation for this distribution of infection is that centrifugal spread of the prion agent away from the brain and along cranial nerves could serve as a pathway for prion infection and accumulation in these mucosal tissues (4, 10, 43).In this work, we investigated the role of the LRS in prion neuroinvasion from the oral and nasal cavities. In order to investigate neuroinvasion following neural and extraneural routes of inoculation in which prion replication is blocked in the LRS, we used two rodent models for prion infection. In muMT mice, which lack mature B cells, and in lymphotoxin-α (LTα) null mice, FDCs do not undergo maturation, and as a result, these mice do not develop clinical disease following intraperitoneal inoculation of the scrapie agent but are susceptible following direct inoculation into the brain (23, 30). In a second model, the HY and DY strains of the transmissible mink encephalopathy (TME) agent were used to investigate neuroinvasion in Syrian hamsters. The HY and DY TME agents can replicate in the nervous system, but the DY TME agent does not replicate in the LRS, and therefore, the DY TME agent is not pathogenic following intraperitoneal (i.p.) inoculation (2, 3). Following intratongue (i.t.) or intranasal (i.n.) inoculation, prion neuroinvasion was independent of scrapie agent replication in the LRS of immunodeficient mice, but evidence for scrapie infection of peripheral nerve fibers or olfactory neurons at these mucosa was lacking. In hamsters, i.t. inoculation of the HY or DY TME agent resulted in PrP^Sc deposition in nerve fibers and prion disease, but only the HY TME agent caused disease following i.n. inoculation. These findings suggest that neuroinvasion from the oral and nasal mucosa in LRS replication-deficient rodents can be independent of LRS infection, but the paucity of PrP^Sc at these mucosal sites of exposure in immunodeficient mice and DY TME-infected hamsters suggests that neuroinvasion is due to either a low-level prion infection of the nervous system at the site of inoculation or transport of the prion agent in axons in the absence of agent replication at the site of prion entry. These findings indicate that these mucosal tissues may not exhibit early evidence of infection and therefore will prove difficult to identify as a portal for agent entry.     

Experimental Verification of a Traceback Phenomenon in Prion Infection

The clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD) correlate with the allelotypes (M or V) of the polymorphic codon 129 of the human prion protein (PrP) gene and the electrophoretic mobility patterns of abnormal prion protein (PrP^Sc). Transmission of sCJD prions to mice expressing human PrP with a heterologous genotype (referred to as cross-sequence transmission) results in prolonged incubation periods. We previously reported that cross-sequence transmission can generate a new prion strain with unique transmissibility, designated a traceback phenomenon. To verify experimentally the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into mice expressing human PrP with the 129M/M genotype. These 129M/M mice showed altered neuropathology and a novel PrP^Sc type after a long incubation period. We then passaged the brain homogenate from the 129M/M mouse inoculated with sCJD-VV2 prions into other 129M/M or 129V/V mice. Despite cross-sequence transmission, 129V/V mice were highly susceptible to these prions compared to the 129M/M mice. The neuropathology and PrP^Sc type of the 129V/V mice inoculated with the 129M/M mouse-passaged sCJD-VV2 prions were identical to those of the 129V/V mice inoculated with sCJD-VV2 prions. Moreover, we generated for the first time a type 2 PrP^Sc-specific antibody in addition to type 1 PrP^Sc-specific antibody and discovered that drastic changes in the PrP^Sc subpopulation underlie the traceback phenomenon. Here, we report the first direct evidence of the traceback in prion infection.Creutzfeldt-Jakob disease (CJD) is a lethal transmissible neurodegenerative disease caused by an abnormal isoform of prion protein (PrP^Sc), which is converted from the normal cellular isoform (PrP^C) (1, 23). The genotype (M/M, M/V, or V/V, where M and V are allelotypes) at polymorphic codon 129 of the human prion protein (PrP) gene and the type (type 1 or type 2) of PrP^Sc in the brain are major determinants of the clinicopathological phenotypes of sporadic CJD (sCJD) (15-18). Type 1 and type 2 PrP^Sc are distinguishable according to the size of the proteinase K-resistant core of PrP^Sc (PrP^res) (21 and 19 kDa, respectively), reflecting differences in the proteinase K cleavage site (at residues 82 and 97, respectively) (15, 18). According to this molecular typing system, sCJD can be classified into six subgroups (MM1, MM2, MV1, MV2, VV1, or VV2).The homology of the PrP genes between inoculated animals and the inoculum determines the susceptibility to prion infection. Transmission of sCJD prions to mice expressing human PrP with a nonhomologous genotype (referred to as cross-sequence transmission) results in a relatively long incubation period (10, 12). Meanwhile, the cross-sequence transmission can generate a new prion strain. Transmission of sCJD-VV2 prions to mice expressing human PrP with the 129M/M genotype generates unusual PrP^res intermediate in size between type 1 and type 2 (10). We have designated this unusual PrP^res with an upward size shift (Sh+) from the inoculated type 2 template MM[VV2]2^Sh+ PrP^res, where the notation is of the following form: host genotype [type of inoculated prion] type of generated PrP^res.Similar to the MM[VV2]2^Sh+ PrP^res, the intermediate-sized PrP^res has been observed in the plaque-type of dura mater graft-associated CJD (p-dCJD) (10, 13). Furthermore, a transmission study using p-dCJD prions revealed that PrP-humanized mice with the 129V/V genotype were highly susceptible to p-dCJD prions despite cross-sequence transmission (10). In addition, these 129V/V mice inoculated with p-dCJD prions produced type 2 PrP^res (10). These findings suggest that p-dCJD could be caused by cross-sequence transmission of sCJD-VV2 prions to individuals with the 129M/M genotype. We have designated this phenomenon “traceback.” The traceback phenomenon was discovered for the first time by a transmission study using variant CJD (vCJD) prions (2). Mice expressing bovine PrP were highly susceptible to vCJD prions because vCJD was caused by cross-sequence transmission of bovine spongiform encephalopathy prions to human. These findings suggest that a traceback study can be a powerful tool to identify the origin of prions (2, 10, 11). However, the traceback phenomenon has not been verified experimentally despite the abundant circumstantial evidence described above.To verify the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into PrP-humanized mice with the 129M/M genotype as an experimental model of p-dCJD. Thereafter, we inoculated these MM[VV2]2^Sh+ prions into PrP-humanized mice with the 129M/M or 129V/V genotype and compared the incubation period, neuropathology, and the type of PrP^res in the brain. Here, we report the first direct evidence of the traceback in prion infection.     

Infection of Cell Lines with Experimental and Natural Ovine Scrapie Agents

Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.Prion diseases are a group of neurodegenerative diseases affecting humans and animals, including scrapie in sheep and goats and bovine spongiform encephalopathy (BSE) in cattle. A feature of prion diseases and, in particular, of scrapie, is the existence of different strains (6) which influence pathology and is most probably related to the conformation of the pathogenic form of the prion protein (PrP^Sc). The susceptibility of sheep to scrapie is determined by the PrP genotype; codons 136, 154, and 171 determine relative resistance and susceptibility, with amino acids valine (V), arginine (R), and glutamine (Q) at these positions (known as VRQ) being considered the sheep PrP allele most susceptible to classical scrapie (3).An array of diagnostic tests exist for prion diseases, aimed at the detection of the disease-associated protease-resistant form of the naturally occurring PrP^C protein, termed PrP^Sc or PrP^res after partial protease digestion. However, the level of detectable PrP^Sc does not quantitatively correlate with prion infectivity (2) and the current biochemical analysis of PrP^Sc cannot always determine the strain (6, 7).Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimate in ruminant transmissible spongiform encephalopathy (TSE) research. Conventional mouse bioassays using wild-type mice are generally slow (>150 days, and considerably longer, >600, days for obtaining infectious titer information) and require multiple mice to be dosed (typically 6 or more) at each dilution of infectious material. Therefore, the development of an approach using ex vivo cell-based assays remains an ethically and economically desirable alternative. Using cell lines permissive to mouse-passaged scrapie strains, Klöhn et al. have developed a cell-based assay for measuring de novo infection and the titer of mouse-passaged scrapie (18).The main limitation of adopting a cell-based approach is the scarcity of cell lines permissive to infection with natural TSE strains (for a review, see references 31 and 34), as the majority of permissive cell lines can only be infected with rodent-adapted strains of scrapie and BSE (4, 9, 16, 20, 23, 24, 29, 33, 36). While there are currently no cell lines reported to be permissive to bovine BSE or human TSE diseases, there are cell lines which express ovine PrP that have been shown to be permissive to natural scrapie infection (1, 35). There is also one fibroblast-like deer cell line that is able to propagate chronic wasting disease (27).Two of the sheep scrapie-susceptible cell lines are the MovS6 cell line (1), a Schwann cell line derived from the tg301 transgenic mouse, and the Rov9 cell line (35), based on a stably transfected rabbit kidney epithelial cell line (RK13) that does not express endogenous PrP. Both express the VRQ allele of ovine PrP, the latter upon induction with doxycycline (35). These cell lines were found to be permissive to infection with a PrP genotype-matched VRQ homozygous scrapie field case, and de novo PrP^Sc maintained its phenotype when used as an inoculum in mouse bioassays (1, 35). Using fluorescence-activated cell sorting, Falanga et al. isolated Rov9 subclones that produce higher levels of PrP^C and PrP^Sc than the parental cell line when infected (11).The primary objective of this study was to assess the permissiveness of the Rov9 and MovS6 cell lines to a panel of scrapie isolates from a range of sheep breeds with a range of PrP genotypes. Second, subcloning of the Rov9 cell line was undertaken in an attempt to identify subclones with greater sensitivity and more diverse permissibility to ovine scrapie isolates.     

Residues Surrounding the Glycosylphosphatidylinositol Anchor Attachment Site of PrP Modulate Prion Infection: Insight from the Resistance of Rabbits to Prion Disease

Prion diseases are a group of transmissible, invariably fatal neurodegenerative diseases that affect both humans and animals. According to the protein-only hypothesis, the infectious agent is a prion (proteinaceous infectious particle) that is composed primarily of PrP^Sc, the disease-associated isoform of the cellular prion protein, PrP. PrP^Sc arises from the conformational change of the normal, glycosylphosphatidylinositol (GPI)-anchored protein, PrP^C. The mechanism by which this process occurs, however, remains enigmatic. Rabbits are one of a small number of mammalian species reported to be resistant to prion infection. Sequence analysis of rabbit PrP revealed that its C-terminal amino acids differ from those of PrP from other mammals and may affect the anchoring of rabbit PrP through its GPI anchor. Using a cell culture model, this study investigated the effect of the rabbit PrP-specific C-terminal amino acids on the addition of the GPI anchor to PrP^C, PrP^C localization, and PrP^Sc formation. The incorporation of rabbit-specific C-terminal PrP residues into mouse PrP did not affect the addition of a GPI anchor or the localization of PrP. However, these residues did inhibit PrP^Sc formation, suggesting that these rabbit-specific residues interfere with a C-terminal PrP^Sc interaction site.Prion diseases, traditionally known as transmissible spongiform encephalopathies (TSE), are a group of invariably fatal neurodegenerative diseases that affect both humans and animals. According to the protein-only hypothesis, an abnormal isoform of the host-encoded prion protein (PrP^C), referred to as PrP^Sc, is the sole or major component of the infectious agent causing these diseases (33). These disorders affect a wide range of mammals and include diseases such as Creutzfeldt-Jakob disease (CJD), variant CJD, Gerstmann-Straüssler-Scheinker (GSS) syndrome, kuru, and fatal familial insomnia (FFI) in humans, scrapie in sheep and goats, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE) in cattle. The term “prion” was first used to describe the unique infectious agent and was derived from “proteinaceous infectious particle” to distinguish it from conventional pathogens such as bacteria and viruses (33).To date, rabbits are one of the few mammalian species reported to be resistant to prion infection. Rabbits do not develop clinical disease after inoculation with brain tissue from individuals affected by the human prion diseases CJD and kuru, or by a number of animal forms of the disease, including scrapie and transmissible mink encephalopathy (TME) (12). In addition, mouse neuroblastoma (MNB) cells overexpressing rabbit PrP are also resistant to prion infection (45). Evidence that rabbit cells per se have the correct cellular machinery to support prion propagation has come from studies using the rabbit kidney epithelial cell line RK13. Upon transfection with appropriate PrP-expressing transgenes, these cells are a highly efficient and robust model of prion infection (6, 25, 41, 43). RK13 cells do not have detectable levels of endogenous rabbit PrP^C and are therefore ideal for studying exogenous PrP^C and the propagation of prions from different species (6). Originally, it was shown that RK13 cells overexpressing ovine PrP became susceptible to infection with scrapie (43), and more recently, RK13 cells expressing rodent PrP^C, from either the mouse or the bank vole, were readily infected by prions adapted to and propagated in these two species (6, 41). RK13 cells expressing human PrP^C, however, were resistant to infection with human prions derived directly from a patient with sporadic CJD (25). Since RK13 cells overexpressing PrP are a well-established model of prion propagation, we can therefore conclude that while these cells apparently have the appropriate cellular machinery to support prion propagation, it may be a characteristic of the rabbit prion protein itself that results in the resistance of this species to prion infection. However, the loss of a cellular cofactor may also be a contributing factor.Analysis of the rabbit PrP amino acid sequence shows that it has all the features previously described for members of the PrP protein family, including an N-terminal signal peptide, an octapeptide repeat region, and a C-terminal signal sequence (26). While amino acid sequence comparison of both mouse and rabbit PrP species reveals 87% sequence homology, there are 22 amino acid differences between the two, and several of these reside in regions of PrP known to be important in PrP^Sc formation. In scrapie-infected MNB cells, the residues Gly99 and Met108 within the N terminus, Ser173 within the central region, and Ile214 within the C terminus of rabbit PrP were shown to inhibit PrP^Sc generation when incorporated into mouse PrP, suggesting that multiple amino acid residues in rabbit PrP inhibit PrP^Sc formation (45). Approximately one-third (9/33 residues in the immature sequence) of the amino acid difference between mouse and rabbit PrPs was shown to occur at the glycosylphosphatidylinositol (GPI) anchor attachment site (see Fig. S1 in the supplemental material). As yet, studies involving this region of rabbit PrP have not been performed. Therefore, this region of rabbit PrP may provide further insight into the resistance of rabbits to prion infection.GPI anchor addition occurs via a transamination reaction in the endoplasmic reticulum (ER) following cleavage of the C-terminal signal sequence (39). There is no consensus sequence with which to identify the C-terminal cleavage site, but there are three key C-terminal elements: (i) the cleavage site, or ω site, where the GPI anchor attaches to the COOH group of the ω amino acid; (ii) a hydrophilic spacer region of 8 to 12 amino acids (ω + 1 up to ω + 10); and (iii) a hydrophobic region of 10 to 20 amino acids (ω + 11 onwards) (9). Analysis of known GPI-anchored proteins has given rise to sequence motifs in the C-terminal signal peptide allowing the prediction of the ω site of proteins. Due to the complexity of experimentally determining the ω site of GPI-anchored proteins, relatively few of the many known GPI-anchored proteins have had their ω sites determined (36 of 340 proteins in 2008) (32) The ω site of hamster PrP was determined experimentally to be at amino acid 231 (34) and is predicted to be at the same site for PrPs from all mammals, based on amino acid sequence comparison. Amino acid substitutions near the ω site of mouse PrP revealed that mouse PrP has an ω site at residue 230 (17). It was also shown that single amino acid substitutions at and near the ω site of mouse PrP affect the anchoring and conversion efficiency of PrP (17). It is therefore possible that the amino acids at the C terminus and within the GPI anchor signal sequence of rabbit PrP lead to the resistance to prion infection.To date, no protein structures containing a GPI anchor have been determined by X-ray crystallography, and although the nuclear magnetic resonance (NMR) structures of mouse and rabbit PrP have been solved, they do not contain any structural information for the residues immediately preceding the GPI anchor. We therefore created a mutant mouse PrP model containing rabbit PrP-specific amino acids at the ω site to investigate whether these residues are involved in rabbit resistance to prion infection. Here we demonstrate that the GPI anchor attachment site is an important site that controls the ability of PrP to be converted into PrP^Sc and that residues ω and ω + 1 of PrP are important modulators of this pathogenic process.     

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP^Sc) are largely unknown. PrP^Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP^C). One manifestation of PrP^Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP^Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP^Sc. Here, we compare the structures of PrP^Sc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrP^Sc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP^Sc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP^Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.Transmissible spongiform encephalopathies (TSEs),² or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy (“mad cow” disease) (1–7). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrP^C, to a pathological isoform, PrP^Sc (1–5). According to the “protein-only” model, PrP^Sc itself represents the infectious prion agent (1, 8); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrP^C and templating the conversion of the latter protein to the PrP^Sc state (9, 10). Although molecular details of such a mechanism of disease propagation remain largely unknown, the general principle of protein-based infectivity is supported by a wealth of experimental data (1–7).PrP^C is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrP^C, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11–13). Conversely, the PrP^Sc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1–5, 14, 15). Little specific structural information is available, however, for this isoform beyond low resolution biochemical and spectroscopic characterization. Thus, the structure of PrP^Sc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases.Many efforts have been made over the years to recapitulate PrP^Sc formation and prion propagation in vitro. Early studies have shown that PrP^C can be converted with remarkable species and strain specificities to a PrP^Sc-like conformation (as judged by PK resistance) simply by incubation with PrP^Sc from prion-infected animals (16, 17). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18). An important more recent study showed that both PrP^Sc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19), a procedure called protein misfolding cyclic amplification (PMCA) (20). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrP^C as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21). Unfortunately, the quantities of infectious PrP^Sc generated by PMCA using purified brain-derived PrP^C are very small, precluding most structural studies.In contrast to brain-derived PrP^C, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22–28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP^C variant encompassing residues 89–231, the infectivity titer of these “synthetic prions” was extremely low (29) or absent altogether (4). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrP^Sc (26, 30), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrP^Sc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrP^PMCA or rPrP-res^(Sc)) (31). These aggregates display a PK digestion pattern that is much more closely related to PrP^Sc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrP^PMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures.     

Glycosylation of PrPC Determines Timing of Neuroinvasion and Targeting in the Brain following Transmissible Spongiform Encephalopathy Infection by a Peripheral Route

Transmissible spongiform encephalopathy (TSE) infectivity naturally spreads from site of entry in the periphery to the central nervous system where pathological lesions are formed. Several routes and cells within the host have been identified as important for facilitating the infectious process. Expression of the glycoprotein cellular PrP (PrP^C) is considered a key factor for replication of infectivity in the central nervous system (CNS) and its transport to the brain, and it has been suggested that the infectious agent propagates from cell to cell via a domino-like effect. However, precisely how this is achieved and what involvement the different glycoforms of PrP have in these processes remain to be determined. To address this issue, we have used our unique models of gene-targeted transgenic mice expressing different glycosylated forms of PrP. Two TSE strains were inoculated intraperitoneally into these mice to assess the contribution of diglycosylated, monoglycosylated, and unglycosylated PrP in spreading of infectivity to the brain. This study demonstrates that glycosylation of host PrP has a profound effect in determining the outcome of disease. Lack of diglycosylated PrP slowed or prevented disease onset after peripheral challenge, suggesting an important role for fully glycosylated PrP in either the replication of the infectious agent in the periphery or its transport to the CNS. Moreover, mice expressing unglycosylated PrP did not develop clinical disease, and mice expressing monoglycosylated PrP showed strikingly different neuropathologic features compared to those expressing diglycosylated PrP. This demonstrates that targeting in the brain following peripheral inoculation is profoundly influenced by the glycosylation status of host PrP.Transmissible spongiform encephalopathies (TSE) or prion diseases are a group of fatal neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, bovine spongiform encephalopathies (BSE) in cattle, and chronic wasting disease (CWD) in deer and elk (30). These diseases can be sporadic, familial, or acquired by infection, and the common hallmark is a distinct pathology in the central nervous system (CNS) characterized by neuronal loss, spongiform degeneration, and gliosis (38, 46).Expression of the host-encoded cellular PrP (PrP^C) is fundamental for the onset of disease since PrP-deficient mice are refractory to TSE infection (11, 31). PrP^C is a glycoprotein with two consensus sites for attachment of N-linked glycans (at codons 180 and 196 in the mouse) which are variably occupied, producing di-, mono-, and unglycosylated PrP (43). The diversity in glycosylation, combined with the complexity of added sugars, results in a large number of glycosylated forms of PrP (41). A central event associated with TSE infection is the conformational conversion of PrP^C into an abnormal protease-resistant form, PrP^Sc (39). PrP^Sc is deposited in brain and, in some but not all cases, in peripheral organs of individuals affected by TSE (21).Although the pathology associated with TSE is found in the brain, the periphery is the most natural route of acquiring infection. Evidence suggests that oral transmission via contaminated food is linked with transmission of BSE to humans, resulting in variant CJD (vCJD) (10, 47), and blood transfusion has been identified as a probable route of human-to-human transmission of vCJD (23, 27, 36). Moreover, parenteral administration of contaminated human tissue-derived therapeutics has been shown to facilitate iatrogenic spread of these diseases (8, 46). It is therefore important to understand the mechanisms that allow the infectious agent to propagate in the periphery and be transported to the CNS prior to the onset of neurodegeneration in the brain.Many studies have been conducted to understand routes of transmission (for a review see references 1 and 29). Lymphoid tissues such as the spleen have been shown to play a fundamental role in agent replication and propagation in the very early stages of disease. Indeed, studies of splenectomized and asplenic mice have shown the lymphoreticular system (LRS) to be an important site for TSE agent replication (14, 26). The periphery also appears to have a role in processing the infectious agent following intracerebral (i.c.) inoculation as PrP^Sc accumulates in the spleen shortly after inoculation and before accumulation of the abnormal protein in the brain (15, 17). Within the LRS, follicular dendritic cells (FDC) have been shown to be important for the uptake of infectivity and subsequent spreading toward the CNS (7, 28, 33, 35). Several studies have also suggested the peripheral nervous systems (PNS) as a potential route of infectivity to the brain, implicating the vagus and sciatic nerves in this process (5, 20, 25, 34).Expression of PrP^C in the peripheral tissues appears to be an important prerequisite for the transport of infectivity to the CNS following peripheral routes of inoculation. Indeed, it has been proposed that a continuous chain of cells expressing PrP^C is fundamental for TSE neuroinvasion (6, 40), with overexpression of endogenous PrP in the PNS greatly facilitating the spread of infectivity (19). Thus, host PrP appears to have a fundamental role in the uptake, transport, and replication of the infectious agent (6). Moreover, it has been suggested that the different PrP^C glycoforms may influence the timing of neuroinvasion by directly influencing the interaction with the infectious agent (19). However, the mechanism by which the different glycoforms are involved in these processes remains to be determined.In order to investigate the role of PrP^C glycosylation in TSE disease after peripheral infection with different TSE strains, we have used our inbred gene-targeted transgenic mice expressing different glycosylated forms of PrP. These mice expressed PrP with no sugars at the first (designated G1/G1 in homozygous mice) or the second glycosylation site (G2/G2) or both (G3/G3) under the control of the endogenous PrP promoter (13). We have previously shown that following intracerebral inoculation, all glycotypes are susceptible to infection with at least one TSE strain and that the type of PrP glycosylation in the host influenced the incubation period but not the distribution of pathological lesions in the brain (45). Here, we examine the influence of host PrP glycosylation on the peripheral acquisition of infection and demonstrate that, unlike the intracerebral route, mice without PrP glycosylation were resistant to disease and that the different glycoforms had a profound influence on not only the timing of disease but also the type and distribution of the PrP^Sc deposits in the brain.     

Detection of PrPsc in Blood from Sheep Infected with the Scrapie and Bovine Spongiform Encephalopathy Agents

The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrP^sc, a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrP^sc was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrP^sc was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrP^sc is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification.Transmission of variant Creutzfeldt-Jakob disease (vCJD) has been linked with blood transfusion in four reported cases in Great Britain (19, 24, 26, 32), indicating that this is likely to be an efficient route of transmission. Such findings highlight a significant risk to recipients of vCJD-contaminated blood components, and blood services in the United Kingdom have responded by putting in place precautionary measures, including leucodepletion. However, it remains uncertain whether such a procedure is able to remove all prion infectivity. For example, in two studies by Gregori et al. (13, 14) only 42 and 72% of infectivity was removed by leucodepletion from blood from hamsters with scrapie. Therefore, a rapid blood test for vCJD that is able to screen for likely infected blood is critical given that the presymptomatic stages of vCJD are long and that the prevalence of infection in the human population is unknown (6, 9). This knowledge has given rise to concerns that a large-scale vCJD epidemic could occur by human-to-human transmission (16, 21).Infectivity in human blood is consistent with the demonstration of transmission of disease by blood transfusion in sheep incubating both scrapie and experimental BSE infection (17, 18, 20). Transmission was demonstrated from both whole blood and buffy coat fractions from sheep blood, indicating a cellular source of prions although, from studies done in rodent models, it is likely that the plasma fraction also contains infectivity (4, 13, 14). Furthermore, transmission was possible from sheep showing clinical signs and from sheep that were infected but still in the preclinical phase. However, identification of the abnormal prion protein (PrP^sc) in blood as a surrogate marker for infection has proved more elusive (3). Recently, PrP^sc has been amplified from the blood of experimentally infected rodents (5, 25, 28) and from sheep naturally infected with scrapie agent (29) using protein misfolded cyclic amplification (PMCA), but often these studies take days or weeks to complete. Here, we demonstrate, using a ligand-based immunoassay, that PrP^sc is associated with blood leukocytes from sheep with terminal scrapie or bovine spongiform encephalopathy (BSE) and in sheep incubating scrapie prior to the onset of clinical signs. This assay is a modification of a test that has been validated for use as a postmortem test for BSE, scrapie, and chronic wasting disease (CWD) in Europe and the United States (7).     

Conformational Properties of ��-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP^C) to a disease-specific isomer (PrP^Sc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP^Sc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP^C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrP^C structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)² (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrP^Sc, differs from the normal cellular form (PrP^C) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrP^C, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrP^C to PrP^Sc. The most coherent and general model proposed thus far is that PrP^C fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrP^Sc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrP^Sc-dependent conversion reactions (18–20) or through conversion of PrP^C in the absence of a PrP^Sc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrP^Sc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrP^C and PrP^Sc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrP^Sc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrP^Sc (but not PrP^C) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrP^Sc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP.     

Antiprion Drugs as Chemical Tools to Uncover Mechanisms of Prion Propagation

A number of drugs active against prions either in vitro, in cellular systems or in vivo in animal models have been isolated in various screening assays. In this minireview, we would like to suggest, that in addition to their direct interest as potential therapeutic agents, these molecules could be used as original research tools to understand prion propagation. The use of antiprion compounds as tool to understand fundamentals of prion propagation relies on reverse screening approaches. These global genetic and/or biochemical approaches aim to identify the intracellular target(s) and mechanism of action of the drugs. Once those are known, the biological activity of the compounds can be optimized on a rational basis, their potential side effects understood and minimized. In vitro enzyme-based screening assays can then be designed to allow discovery of new, more potent and selective molecules. Here we describe the main comprehensive biochemical and genetical approaches to realize reverse screening approaches based on antiprion drugs. We will finish by discussing the interest of using drug inactivation of specific targets as a substitute to genetic inactivation.Key Words: prion, amyloid fibers, protein folding, protein chaperone, antiprion drugs, reverse screeningA number of drugs have been isolated as active against mammalian prion (reviewed in ref. ¹). For most of these molecules, the mode of action and targets remain largely unknown. In principle, two main modes of action for antiprion drugs can be envisioned: either in cis directly on PrP^C/PrP^Sc, or in trans by interfering with the activity of cellular factors required for prion propagation. Some compounds are thought to bind directly to PrP^C or PrP^Sc (action in cis). Among these compounds are Congo Red (CR), Pentosan Polysulfate (PPS) or Glycosaminoglycans (GAGs). Other compounds are thought to act in trans by affecting PrP^C or PrP^Sc indirectly. Among these molecules are various lysosomotropic factors including the antimalarial drugs Quinacrine (QC) and Chloroquine. Indeed, the lysosome is a potential site of conversion of PrP^C to PrP^Sc.² In addition, a recent report,³ proposes that QC''s antiprion activity is related to its ability to redistribute cholesterol from the plasma membrane to intracellular compartments, thereby destabilizing membrane domains. This conclusion was drawn from correlation experiments indicating that drugs known to display cholesterol-redistributing activity (but structurally unrelated to QC) also have antiprion potency. Finally, QC was also shown to interact directly with PrP.^4,5 The example of QC, with these conflicting results, thus illustrates the need for unbiaised and global approaches not driven by a preconcieved hypothesis on the drug mode of action. To our knowledge, no such approaches were applied for any of the known antiprion drugs, with the noticeable exception of chlorpromazine for which a haploinsufficiency profiling screen (HIP, see in the later section) has been published.⁶ In this minireview, we discuss the various advantages of defining extensively intracellular targets of antiprion drugs. We then would like to present some global approaches that can be applied to uncover, in an unbiaised manner, cellular mechanism(s) of action of compounds active against prions. To finish we propose that, once intracellular targets have been clearly identified, the drugs can be used to do “chemical genetics” to inactivate cellular target(s) which could be especially useful in situations where classical inactivation by mutagenesis is unpractical (for instance if redundant multigenic families are involved).     

Sti1 Regulation of Hsp70 and Hsp90 Is Critical for Curing of Saccharomyces cerevisiae [PSI+] Prions by Hsp104

Although propagation of Saccharomyces cerevisiae prions requires Hsp104 protein disaggregating activity, overproducing Hsp104 “cures” cells of [PSI⁺] prions. Earlier evidence suggests that the Hsp70 mutant Ssa1-21 impairs [PSI⁺] by a related mechanism. Here, we confirm this link by finding that deletion of STI1 both suppresses Ssa1-21 impairment of [PSI⁺] and blocks Hsp104 curing of [PSI⁺]. Hsp104''s tetratricopeptide repeat (TPR) interaction motif was dispensable for curing; however, cells expressing Sti1 defective in Hsp70 or Hsp90 interaction cured less efficiently, and the Hsp90 inhibitor radicicol abolished curing, implying that Sti1 acts in curing through Hsp70 and Hsp90 interactions. Accordingly, strains lacking constitutive or inducible Hsp90 isoforms cured at reduced rates. We confirm an earlier finding that elevating free ubiquitin levels enhances curing, but it did not overcome inhibition of curing caused by Hsp90 defects, suggesting that Hsp90 machinery is important for the contribution of ubiquitin to curing. We also find curing associated with cell division. Our findings point to crucial roles of Hsp70, Sti1, and Hsp90 for efficient curing by overexpressed Hsp104 and provide evidence supporting the earlier suggestion that destruction of prions by protein disaggregation does not adequately explain the curing.Saccharomyces cerevisiae prions are self-replicating misfolded forms of normal cellular proteins. They are believed to propagate as amyloid, which is a highly ordered fibrous aggregate. What triggers prion formation is uncertain, but in order to be maintained in an expanding yeast population, prions must grow, replicate, and be transmitted to daughter cells during cell division. Growth occurs when soluble protein joins the fiber ends and is converted into the prion form (30, 52, 58). Replication is associated with fragmentation of prion polymers, which generates new prions from preexisting material (37, 50). Transmission is believed to occur by passive diffusion of prions with cytoplasm (57).Although it is uncertain to what extent cellular factors influence growth or transmission of prions, it is clear that the Hsp104 disaggregation machinery is necessary for prion replication (10, 17, 55, 70). Hsp104 is a hexameric AAA+ chaperone that protects cells from a variety of stresses by resolubilizing proteins from aggregates (24, 25, 53). With help from Hsp70 and Hsp40, it extracts monomers from aggregates and extrudes them through its central pore (24, 41, 68). This machinery could act in prion replication by extracting monomers from amyloid fibers (29, 68), which would destabilize the fibers, causing them to break into more numerous pieces that each can continue to propagate the prion.Paradoxically, overexpressing Hsp104 very efficiently “cures” cells of the [PSI⁺] prion, which is composed of the translation termination factor Sup35 (10). A widely held view of this curing is that elevating the cellular protein disaggregation activity causes complete destruction of prions. However, elevating Hsp104 has little or no effect on most other amyloidogenic prions (15, 16, 38, 47, 54, 66), although it can be inferred to cure [MCA] prions in cells also propagating a prion of an Mca1-Sup35 fusion (49). Together, these results suggest that prions of Sup35, and perhaps those of Mca1, are particularly sensitive to Hsp104 disaggregation activity. Alternatively, something in addition to or other than a simple increase in protein disaggregation is involved in the curing.Although protein disaggregation activity of Hsp104 is required for both thermotolerance and prion propagation, we and others have identified mutations in Hsp104 that affect these processes separately (27, 32, 39, 60). The ability of Hsp104 to thread proteins through its central pore, however, is required for both processes (29, 41, 68), so this distinction in Hsp104 function could be due to differences in how Hsp104 interacts with amorphous aggregates of thermally denatured proteins and highly ordered prion aggregates or with cofactors that interact with the different prions as substrates. In any scenario, efficiency and specificity of Hsp104 function are affected by interactions with other components of the disaggregation machinery, in particular the Hsp70s and Hsp40s, which are believed to interact first with substrates to facilitate action of Hsp100 family disaggregases (2, 71, 72).Increasing expression of either ubiquitin (Ub) or Ssb, an Hsp70 that has roles in protein translation and proteasome degradation, enhances Hsp104 curing of [PSI⁺] (3, 11, 12). Predictably, reducing expression of either of them reduces curing efficiency. The mechanisms underlying these effects are unknown, but the combined effects of Ssb and Ub are additive, suggesting that they act in different pathways. The role of Ub is indirect, as Sup35 is neither ubiquitylated nor degraded during curing. Whether other chaperones are involved in the effects of Ub on curing has not been investigated.Earlier we isolated a mutant of the Hsp70 Ssa1, designated Ssa1-21, that weakens and destabilizes [PSI⁺] propagation (33). We later isolated several Hsp104 mutants that suppress this antiprion effect (29). The Hsp104 mutants retain normal functions in thermotolerance, protein disaggregation, and prion propagation, but when overexpressed, they are unable to cure [PSI⁺], even in wild-type cells. These findings argue against a specific hypersensitivity of [PSI⁺] to disaggregation and support the notion that something distinct from or in addition to complete destruction of prions is involved in the curing. They also imply that Ssa1-21 and elevated Hsp104 inhibit [PSI⁺] prions by similar mechanisms. A prediction from this conclusion is that other suppressors of Ssa1-21 will also inhibit curing of [PSI⁺] by overexpressed Hsp104. Indeed, we find here that alterations that suppress Ssa1-21 inhibition of [PSI⁺] do interfere with curing of [PSI⁺] by overexpressed Hsp104. We also provide evidence that Hsp90 has a critical role in this curing and that the ability of Ub to enhance curing depends on proper function of Hsp90 machinery.     

Compositional Determinants of Prion Formation in Yeast

Numerous prions (infectious proteins) have been identified in yeast that result from the conversion of soluble proteins into β-sheet-rich amyloid-like protein aggregates. Yeast prion formation is driven primarily by amino acid composition. However, yeast prion domains are generally lacking in the bulky hydrophobic residues most strongly associated with amyloid formation and are instead enriched in glutamines and asparagines. Glutamine/asparagine-rich domains are thought to be involved in both disease-related and beneficial amyloid formation. These domains are overrepresented in eukaryotic genomes, but predictive methods have not yet been developed to efficiently distinguish between prion and nonprion glutamine/asparagine-rich domains. We have developed a novel in vivo assay to quantitatively assess how composition affects prion formation. Using our results, we have defined the compositional features that promote prion formation, allowing us to accurately distinguish between glutamine/asparagine-rich domains that can form prion-like aggregates and those that cannot. Additionally, our results explain why traditional amyloid prediction algorithms fail to accurately predict amyloid formation by the glutamine/asparagine-rich yeast prion domains.Amyloid fibers are associated with a large number of neurodegenerative diseases and systemic amyloidoses. Amyloid fibrils are rich in a cross-beta quaternary structure in which β-strands are perpendicular to the long axis of the fibril (8).[URE3] and [PSI⁺] are the prion (infectious protein) forms of the Saccharomyces cerevisiae proteins Ure2 and Sup35, respectively (61). Formation of both prions involves conversion of the native proteins into an infectious, amyloid form. Ure2 and Sup35 have served as powerful model systems for examining the basis for amyloid formation and propagation. Both proteins possess a well-ordered functional domain responsible for the normal function of the protein, while a functionally and structurally separate glutamine/asparagine (Q/N)-rich intrinsically disordered domain is necessary and sufficient for prion aggregation and propagation (4, 26, 27, 52, 53). Both proteins can form multiple prion variants, which are distinguished by the efficiency of prion propagation and by the precise structure of the amyloid core (14, 54).Five other prion proteins have also been identified in yeast: Rnq1 (13, 46), Swi1 (15), Cyc8 (33), Mca1 (30), and Mot3 (1). Numerous other proteins, including New1, contain domains that show prion activity when inserted in place of the Sup35 prion-forming domain (PFD) (1, 42). Each of these prion proteins contains a Q/N-rich PFD. Similar Q/N-rich domains are overrepresented in eukaryotic genomes (28), raising the intriguing possibility that prion-like structural conversions by Q/N-rich domains may be common in other eukaryotes. However, we currently have little ability to predict whether a given Q/N-rich domain can form prions.A variety of algorithms have been developed to predict a peptide''s propensity to form amyloid fibrils based on its amino acid sequence, including BETASCAN (6), TANGO (17), Zyggregator (51), SALSA (62), and PASTA (55). These algorithms have been successful at identifying regions prone to amyloid aggregation and predicting the effects of mutations on aggregation propensity for many amyloid-forming proteins. However, they have generally been quite ineffective for Q/N-rich amyloid domains such as the yeast PFDs. For example, using the statistical mechanics-based algorithm TANGO (17), which predicts aggregation propensity based on a peptide''s physicochemical properties, Linding et al. found that the Sup35 and Ure2 PFDs both completely lack predicted β-aggregation nuclei (24). Similarly, yeast PFDs are generally lacking in the hydrophobic residues predicted by algorithms such as Zyggregator to nucleate amyloid formation.Why are these algorithms so effective for many amyloid-forming proteins but not for yeast PFDs? For most amyloid proteins, amyloid formation is driven by short hydrophobic protein stretches, and increased hydrophobicity is correlated with an increased amyloid aggregation propensity (34). In contrast, the yeast PFDs are all highly polar domains, due largely to the high concentration of Q/N residues and the lack of hydrophobic residues. High Q/N content is clearly not a requirement for a domain to act as a prion in yeast, since neither the mammalian prion protein PrP nor the Podospora anserina prion protein HET-s is Q/N rich, yet fragments from both proteins can act as prions in yeast (49, 50). However, the significant compositional differences between the yeast PFDs and most other amyloid/prion proteins suggest that there may be two distinct classes of amyloid-forming proteins driven by different types of interactions. Specifically, Q/N residues, which are predicted to have a relatively low amyloid propensity in the context of hydrophobic amyloid domains (34), may promote amyloid formation when present at sufficiently high density. Stacking of Q/N residues to form polar zippers has been proposed to stabilize amyloid fibrils (35). Consistent with this hypothesis, mutational studies of Sup35 indicate that Q/N residues are critical for driving [PSI⁺] formation (12), and expanded poly-Q or poly-N tracts are sufficient to drive amyloid aggregation (36, 63). Therefore, this paper examines the sequence features that allow the polar, Q/N-rich yeast PFDs to form prions.Mutational studies of the PFDs of Ure2 and Sup35 have shown that amino acid composition is the predominant feature driving prion formation (40, 41). Due to the unique compositional biases observed in the yeast PFDs, algorithms have been developed to identify potential PFDs based solely on amino acid composition (19, 28, 42). These algorithms are designed to produce a list of potential prion proteins that meet a specific set of criteria (such as high Q/N content) but are not able to predict the prion propensity of each member of the list or to predict the effects of mutations on prion formation. A recent study by Alberti et al. was the first to systematically test whether compositional similarity to known PFDs is sufficient to distinguish between Q/N-rich proteins that form prions and those that do not. They developed a hidden Markov model to identify domains that are compositionally similar to known PFDs and then analyzed the 100 highest-scoring Q/N-rich domains in a series of in vivo and in vitro assays (1). Remarkably, they discovered 18 proteins with prion-like activity in all assays. However, an equal number, including some of the domains with greatest compositional similarity to known PFDs, showed no prion-like activity.This inability to distinguish between Q/N-rich proteins that form prions and those that do not might seem to suggest that amino acid composition is not an accurate predictor of prion propensity. However, an alternative explanation is that known yeast PFDs are not an ideal training set for a composition-based prediction algorithm, since yeast prions are likely not optimized for maximal prion propensity. It is unclear whether yeast prion formation is a beneficial phenomenon providing a mechanism to regulate protein activity or a detrimental phenomenon analogous to human amyloid disease. [PSI⁺] can increase resistance to certain stress conditions (56), but the failure to observe [PSI⁺] in wild yeast strains (29) argues that beneficial [PSI⁺] formation is at most a rare event. If yeast prions are diseases, the PFDs certainly would not be optimized for maximum prion potential. If prion formation is a beneficial event allowing for rapid conversion between active and inactive states, the prion potential of the PFD would be optimized such that the frequencies of prion formation and loss would yield the optimal balance of prion and nonprion cells (25). Thus, specific residues might be excluded from yeast PFDs either because they inhibit prion formation or because they too strongly promote prion formation; bioinformatic analysis can reveal which residues are excluded from yeast PFDs but not why they are excluded. Accurate prediction of prion propensity requires understanding which deviations from known prion-forming compositions will promote prion formation and which will inhibit.We have therefore developed the first in vivo method to quantitatively determine the prion propensity for each amino acid in the context of a Q/N-rich PFD. As expected, we found proline and charged residues to be strongly inhibitory to prion formation; but surprisingly, despite being largely underrepresented in yeast PFDs, hydrophobic residues strongly promoted prion formation. Furthermore, although Q/N residues dominate yeast PFDs, prion propensity appears relatively insensitive to the exact number of Q/N residues. Using these data, we were able to distinguish with approximately 90% accuracy between Q/N-rich domains that can form prion-like aggregates and those that cannot. These experiments provide the first detailed insight into the compositional requirements for yeast prion formation and illuminate the different methods by which Q/N- and non-Q/N-rich amyloidogenic proteins aggregate.     

Role of ADAMs in the Ectodomain Shedding and Conformational Conversion of the Prion Protein

The cellular prion protein (PrP^C) is essential for the pathogenesis and transmission of prion diseases. PrP^C is bound to the plasma membrane via a glycosylphosphatidylinositol anchor, although a secreted, soluble form has also been identified. Previously we reported that PrP^C is subject to ectodomain shedding from the membrane by zinc metalloproteinases with a similar inhibition profile to those involved in shedding the amyloid precursor protein. Here we have used gain-of-function (overexpression) and loss-of-function (small interfering RNA knockdown) experiments in cells to identify the ADAMs (a disintegrin and metalloproteinases) involved in the ectodomain shedding of PrP^C. These experiments revealed that ADAM9 and ADAM10, but not ADAM17, are involved in the shedding of PrP^C and that ADAM9 exerts its effect on PrP^C shedding via ADAM10. Using dominant negative, catalytically inactive mutants, we show that the catalytic activity of ADAM9 is required for its effect on ADAM10. Mass spectrometric analysis revealed that ADAM10, but not ADAM9, cleaved PrP between Gly²²⁸ and Arg²²⁹, three residues away from the site of glycosylphosphatidylinositol anchor attachment. The shedding of another membrane protein, the amyloid precursor protein β-secretase BACE1, by ADAM9 is also mediated via ADAM10. Furthermore, we show that pharmacological inhibition of PrP^C shedding or activation of both PrP^C and PrP^Sc shedding by ADAM10 overexpression in scrapie-infected neuroblastoma N2a cells does not alter the formation of proteinase K-resistant PrP^Sc. Collectively, these data indicate that although PrP^C can be shed through the action of ADAM family members, modulation of PrP^C or PrP^Sc ectodomain shedding does not regulate prion conversion.The prion protein (PrP)³ is the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer and elk (1). In these diseases the cellular form of PrP (PrP^C) undergoes a conformational conversion to the infectious form PrP^Sc that is characterized biochemically by its resistance to digestion with proteinase K (PK) (2). Mature PrP^C is anchored to the extracellular surface of the cell membrane through a glycosylphosphatidylinositol (GPI) anchor and, like most GPI-anchored proteins, is clustered into cholesterol-rich, detergent-resistant membrane rafts (reviewed in Ref. 3). Although the precise subcellular site of conversion remains undefined, conformational conversion of PrP^C to PrP^Sc is believed to occur either at the cell surface or within the endocytic pathway (4–6).A number of studies indicate that modulation of PrP^C levels at the cell surface may represent a possible future disease intervention strategy. For example, the retention of PrP^C at the cell surface and concomitant prevention of its endocytosis through the use of PrP antibodies inhibited PrP^Sc formation (7). Furthermore, the sterol-binding polyene antibiotic filipin reduced endocytosis, and induced cellular release, of PrP^C with a concomitant reduction in PrP^Sc accumulation (8). More recently, it has been shown that modulation of cell surface PrP^C levels by the novel sorting nexin SNX33 can interfere with PrP^Sc formation in cultured cells (9). Nonetheless, the natural processes regulating PrP^C levels at the cell surface remain poorly defined. One such mechanism of regulation is via shedding of the bulk of the ectodomain of PrP^C either through cleavage of the polypeptide close to the GPI anchor or within the GPI anchor itself. Indeed, it has long been established that PrP^C can be shed into the medium of cultured cells and is present as a soluble form in vivo in human cerebrospinal fluid (10, 11).Numerous cell surface proteins can be proteolytically shed by the action of a group of zinc metalloproteinases known collectively as secretases or sheddases (reviewed in Refs. 12, 13). Whereas most proteolytically shed proteins are derived from transmembrane polypeptide-anchored substrates, several GPI-anchored proteins, including the folate receptor (14), the ecto-ADP-ribosyltransferase ART2.2 (15), and a GPI-anchored construct of angiotensin-converting enzyme (16) are shed by the action of metalloproteinases. We have previously shown that PrP^C can also be proteolytically shed from the cell surface through the action of one or more zinc metalloproteinases with similar properties to those of the α-secretases responsible for the shedding of the amyloid precursor protein (APP) of Alzheimer disease (17). This α-secretase-mediated ectodomain shedding of APP from the cell surface is carried out by at least three members of the a disintegrin and metalloproteinase (ADAM) family, namely ADAM9, -10, and -17 (reviewed in Ref. 18). In addition to cleavage by ADAMs, APP is also cleaved by the β-secretase, BACE1 (β-site APP-cleaving enzyme) and the γ-secretase complex to release the neurotoxic amyloid-β peptide (19). BACE1 itself is also subject to ectodomain shedding by as yet unidentified members of the ADAM family (20).The similarities between the ectodomain shedding of APP and PrP^C, in particular the similar profile of inhibition by a range of hydroxamate-based zinc metalloproteinase inhibitors (17), led us to investigate whether the same members of the ADAM family were also involved in the shedding of PrP^C. It should be noted that this ectodomain shedding of PrP^C by cleavage of the polypeptide chain near to the site (Ser²³¹) of GPI anchor addition in the C terminus of the protein is distinct from the so-called α-cleavage between residues 111 and 112 in the middle of the protein (21, 22). This latter “endoproteolytic” cleavage of PrP^C is reported to be carried out by members of the ADAM family (23, 24).To investigate the role of ADAMs in the ectodomain shedding of PrP^C, we used loss-of-function and gain-of-function experiments in cultured cells in which candidate PrP sheddases were either knocked down by siRNA or overexpressed. We have also further characterized the shedding of BACE1 by comparison to the shedding of APP and PrP^C. In addition, we have explored whether proteolytic shedding of PrP^C is of importance in regulating its conversion into PrP^Sc.     

Transcytosis of Murine-Adapted Bovine Spongiform Encephalopathy Agents in an In Vitro Bovine M Cell Model

Transmissible spongiform encephalopathies (TSE), including bovine spongiform encephalopathy (BSE), are fatal neurodegenerative disorders in humans and animals. BSE appears to have spread to cattle through the consumption of feed contaminated with BSE/scrapie agents. In the case of an oral infection, the agents have to cross the gut-epithelial barrier. We recently established a bovine intestinal epithelial cell line (BIE cells) that can differentiate into the M cell type in vitro after lymphocytic stimulation (K. Miyazawa, T. Hondo, T. Kanaya, S. Tanaka, I. Takakura, W. Itani, M. T. Rose, H. Kitazawa, T. Yamaguchi, and H. Aso, Histochem. Cell Biol. 133:125-134, 2010). In this study, we evaluated the role of M cells in the intestinal invasion of the murine-adapted BSE (mBSE) agent using our in vitro bovine intestinal epithelial model. We demonstrate here that M cell-differentiated BIE cells are able to transport the mBSE agent without inactivation at least 30-fold more efficiently than undifferentiated BIE cells in our in vitro model. As M cells in the follicle-associated epithelium are known to have a high ability to transport a variety of macromolecules, viruses, and bacteria from gut lumen to mucosal immune cells, our results indicate the possibility that bovine M cells are able to deliver agents of TSE, not just the mBSE agent.Transmissible spongiform encephalopathies (TSE) or prion diseases, including human Creutzfeldt-Jakob disease (CJD) and endemic sheep scrapie, are fatal neurodegenerative diseases. The host cellular prion protein (PrP^C), which is thought to have neuroprotective function, is expressed in both humans and a range of other animal species (36), and PrP^C expression is essential for TSE disease susceptibility (7). The prion hypothesis suggests that infectious abnormally folded prion protein (PrP^Sc) is the primary or sole composition of the infectious agent of TSE (known as the prion). However, the molecular composition of PrP^Sc remains speculative and unclear. It is well known that the detergent-insoluble and relatively proteinase K (PK)-resistant prion protein (PrP-res) is detectable in many kinds of TSE-infected tissues, including the brain. Although some studies have revealed that PrP-res does not correlate with infectivity levels in animal tissues as well as in subcellular fractions (37, 40), PrP-res is a useful surrogate marker for TSE infection.Bovine spongiform encephalopathy (BSE) is a TSE of cattle. The first case of BSE in the world was found in the United Kingdom in 1986 (41), and it spread to continental Europe, North America, and Japan. At present, BSE is a threat to human health because of the appearance of BSE-linked variant Creutzfeldt-Jakob disease (vCJD). The cattle BSE agent appears to spread to the cattle population through the consumption of rendered meat and bone meal contaminated with BSE-infected brain or spinal cord (32). Likewise, the transmission of vCJD to humans is likely to have occurred following the consumption of BSE-contaminated food (6, 13, 45). In cases of oral transmissions such as BSE and vCJD, TSE agents first have to cross the gut epithelium, but the exact mechanisms for intestinal invasion still are unknown.Intestinal epithelial cells are bound to each other by tight junctions. This close-packed structure forms a highly selective barrier for macromolecules and limits the access of pathogenic bacteria to the underlying host tissues (43). Gut epithelia are composed of two different epithelial types. One is the villous epithelium, and the other is the follicle-associated epithelium (FAE), which overlies gut-associated lymphoid tissues (GALTs) such as Peyer''s patches. The FAE is considerably different from the surrounding villous epithelium, in that it contains membranous (M) cells. Because M cells have a high capacity for the transcytosis of a wide range of macromolecules, viruses, and microorganisms, they are specialized epithelial cells and act as an antigen sampling system from the gut lumen (28). M cells are, however, exploited by some pathogenic microorganisms and viruses as the entry site to invade the body (20, 29). In fact, some experiments have proposed that M cells transport TSE agents (12) and that Peyer''s patches including the FAE are associated with TSE disease susceptibility (35). In contrast, some authors have suggested the M cell-independent pathway as the main transport route of TSE agents across the intestinal epithelium (16, 23, 27). The intestinal cell types involved in the transport of TSE agents therefore are still a matter of controversy at this stage.Recently, we succeeded in the establishment of a bovine intestinal epithelial cell line (BIE cells) and the development of an in vitro bovine M cell model by coculture with murine intestinal lymphocytes or the supernatant of bovine peripheral blood mononuclear cells (PBMC) stimulated by interleukin 2 (IL-2) (25). In this study, we investigate whether M cells can transport the murine-adapted BSE (mBSE) agent using BIE cells. We demonstrate here that M cell-differentiated BIE cells are able to deliver mBSE agents at least 30-fold more efficiently than undifferentiated BIE cells, although a small number of the mBSE agents pass through undifferentiated BIE cells. Our findings thus provide an insight into the uptake mechanisms of TSE agents, including the cattle BSE agent from the gut lumen.