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.     

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.     

Discovery of 2-Aminothiazoles as Potent Antiprion Compounds

Prion diseases are fatal, untreatable neurodegenerative diseases caused by the accumulation of the misfolded, infectious isoform of the prion protein (PrP), termed PrP^Sc. In an effort to identify novel inhibitors of prion formation, we utilized a high-throughput enzyme-linked immunosorbent assay (ELISA) to evaluate PrP^Sc reduction in prion-infected neuroblastoma cell lines (ScN2a). We screened a library of ∼10,000 diverse small molecules in 96-well format and identified 121 compounds that reduced PrP^Sc levels at a concentration of 5 μM. Four chemical scaffolds were identified as potential candidates for chemical optimization based on the presence of preliminary structure-activity relationships (SAR) derived from the primary screening data. A follow-up analysis of a group of commercially available 2-aminothiazoles showed this class as generally active in ScN2a cells. Our results establish 2-aminothiazoles as promising candidates for efficacy studies of animals and validate our drug discovery platform as a viable strategy for the identification of novel lead compounds with antiprion properties.Prion diseases belong to a class of neurodegenerative, protein-conformation disorders whose unifying pathological mechanism is the misprocessing and aggregation of normally benign soluble proteins. These “proteinopathies,” which include Alzheimer''s, Parkinson''s, and Huntington''s diseases, as well as the frontotemporal dementias—including Pick''s disease—and amyotrophic lateral sclerosis (ALS), are uniformly fatal after a period of neurodegeneration, characterized clinically by dementia and motor dysfunction (20). Prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy, are characterized by the formation of “spongiform” vacuolation of the brain (13). Disease onset is caused by the accumulation of a β-sheet-rich, infectious isoform of the prion protein, termed PrP^Sc (1, 16, 19, 20), which is formed from the α-helix-rich cellular prion protein, termed PrP^C. This conversion can occur spontaneously or be induced by the presence of a number of different autosomally dominant mutations of the PrP gene (8, 21). Alternatively, prion disease can result from exogenous exposure to PrP^Sc (20). The infectious nature of prions results from the ability of PrP^Sc to induce its own production by stimulating the alternative folding of PrP^C (18). Although PrP^Sc formation has been demonstrated to be the primary pathogenic event in prion disease, the precise mechanism that features in its formation and the ensuing neurodegeneration remains largely unknown.Despite the lack of a detailed understanding of the cellular mechanism of prion propagation, numerous studies have been directed toward development of therapeutics targeting prions. Screenings utilizing prion-infected cell lines have identified a number of compounds that reduce the level of PrP^Sc in culture. These include pentosan polysulfate (PPS), dextran sulfate (DS), HPA-23, Congo red, suramin, dendritic polyamines, and quinacrine, among others; for a comprehensive review, see reference 22. However, none of these have been shown to be effective against a broad range of prion strains in animal models when administered after clinical signs manifest, and none have been shown to modify the disease course in human clinical studies (22).The current array of antiprion compounds has been discovered mostly by ad hoc, low-throughput screening of small sets of known bioactive compounds. Most antiprion compounds that are active in prion-infected cell lines have failed in vivo, which highlights the need for a sustained, high-throughput drug discovery effort. Such studies need to rapidly screen large libraries of new chemical entities in vitro, analyze the pharmacokinetic and pharmacodynamic properties of the primary hits in vivo, and optimize the chemical properties of lead compounds. In two semi-high-throughput screenings for antiprion compounds, an analysis of ∼2,000-member compound libraries of known drugs and natural products identified sets of 17 and 8 active compounds (10). None of these have been reported to be effective in vivo.In the present study, we assessed the antiprion activity of ∼10,000 compounds encompassing a diverse set of chemical entities and identified a set of 121 compounds that induce the clearance of PrP^Sc. Subsequent analysis of a number of compounds with the 2-aminothiazole scaffold informed the structure-activity relationship (SAR) of this lead class.     

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.     

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.     

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.     

Tunnelling nanotubes: A highway for prion spreading?

The discovery of tunnelling nanotubes (TNTs) and their proposed role in long intercellular transport of organelles, bacteria and viruses have led us to examine their potential role during prion spreading. We have recently shown that these membrane bridges can form between neuronal cells, as well as between dendritic cells and primary neurons and that both endogenous and exogenous PrP^Sc appear to traffic through these structures between infected and non-infected cells. Furthermore, prion infection can be efficiently transmitted from infected dendritic cells to primary neurons only in co-culture conditions permissive for TNT formation. Therefore, we propose a role for TNTs during prion spreading from the periphery to the central nervous system (CNS). Here, we discuss some of the key steps where TNTs might play a role during prion neuroinvasion.Key words: tunnelling nanotubes, TNTs, prion, PrP^Sc, prion spreading, dendritic cells, neuroinvasionPrion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that have been found in a number of species, including scrapie in sheep, bovine spongiform encephalopathy in cattle (BSE), Chronic wasting disease in deer and Creutzfeldt-Jacob, the Gerstmann-Straüssler-Scheinker syndrome, fatal familial insomnia and kuru in humans (reviewed in ref. ¹). Human TSEs can be sporadic, genetic or acquired by infection. A new variant of Creutzfeldt-Jakob disease (termed vCJD) was reported from the UK in 1996.² The majority of vCJD cases diagnosed to date resulted from a peripheral exposure via the consumption of BSE-contaminated food. Pathological features of TSE diseases can include gliosis, neuronal cell loss and spongiform changes, but the common feature of all members of this group of diseases is the build-up of an aberrant form of the host cellular protein PrP^C, named PrP^Sc (from scrapie). The normal cellular isoform, PrP^C, is an endogenous glycosylphosphatidyl inositol (GPI)-anchored protein present in numerous tissues in mammals, including neurons and lymphoid cells. While the exact function of PrP^C remains unclear, evidence suggest putative roles in neuroprotection, cell adhesion and signal transduction (reviewed in refs. ³ and ⁴). According to the ‘protein-only hypothesis,’ the causative agents of prion diseases are proteinaceous infectious particles (‘prions’), which are composed essentially of misfolded PrP^C, or PrP^Sc.^5,6 Prions replicate through a molecular mechanism in which abnormally folded PrP^Sc acts as a catalyst and serves as a template to convert normal PrP^C molecules into PrP^Sc.^5,6 PrP^Sc differs from PrP^C in the conformation of its polypeptide chain, which is enriched in β-sheets and is protease resistant. Although the conversion process is believed to have a predominant role in the pathogenesis of prion diseases, the cellular and molecular basis for the pathogenic conversion of PrP are still unknown.Another important question is how PrP^Sc spreads to and within the brain. After oral exposure, PrP^Sc accumulates into lymphoid tissues, such as the spleen, lymph nodes or Peyer''s patches, prior to neuroinvasion.^7–9 The exact mechanisms and specific cells involved in the spreading from the gastrointestinal track to the lymphoid system and to the peripheral nervous system (PNS), leading to neuroinvasion of the CNS remain to be elucidated. However, a range of evidence suggests that the accumulation of PrP^Sc within lymphoid tissues is necessary for efficient neuroinvasion.^9–11 In particular it has been shown that PrP^Sc accumulates first within follicular dendritic cells (FDCs)¹² and macrophages.¹³ FDCs are stromal-differentiated cells in the germinal centres of activated lymphoid follicles. A number of studies have demonstrated that FDCs play a critical role during spreading of infection since their absence greatly impaires the neuroinvasion process.^8,11,14,15 However, because FDCs are immobile cells, it is not clear how they acquire PrP^Sc and how it spreads from the FDCs to the PNS. FDCs and nerve synapses occupy different anatomical sites^16,17 and therefore the lack of physical contact between the gut and FDCs and between FDCs and the nerve periphery imply the presence of intermediate mechanisms for the transport of PrP^Sc. Dendritic cells (DCs) have been proposed to play a critical role in the transport of PrP^Sc from the gut to FDCs.¹⁸ DCs function as sentinels for incoming pathogens. Bone-marrow dendritic cells (BMDCs) are migratory cells that are able to transport proteins within Peyer''s patches and into mesenteric lymph nodes.¹⁹ Interestingly, mucosal dendritic cells which play a role in the transport of intestinal antigen for presentation to Peyer''s patches and to mesenteric lymph nodes, can also extend trans-epithelial dendrites to directly sample bacteria in the gut.^20,21 However, the transport of PrP^Sc from FDCs to the PNS remains controversial and evidence for a direct role of DCs during this process has been debated.^22,23 Several mechanisms have been proposed for the intercellular transfer of PrP^Sc, including cell-cell contact, transfer via exosomes or by GPI-painting.^24–26 For example, similar to other types of pathogens such as HIV-1, which was proposed to follow the “exosomal” pathway to be released from the cells,²⁷ it has been shown that the supernatant of prion infected cells contain large amount of PrP^Sc in membranous vesicles known as exosomes.^25,28 Thus, it was suggested that exosomes might be a way to spread prion infection in vivo.^25,28 Recently, a different type of vesicles known as plasma membrane-derived microvesicles, were also described as a potential spreading mechanism during neuroinvasion.²⁹In 2004, Rustom and colleagues discovered a new mechanism of long distance intercellular communication in mammalian cells, called tunnelling nanotubes (TNTs).³⁰ TNTs are transient, long, actin-rich projections that allow for long-distance intercellular communication (reviewed in refs. ^31–33). TNT-like structures have been described to form in vitro between numerous cell types, including neuronal and immune cells.^30,34,35 These studies demonstrated that TNT-like structures formed bridges or channels between distant cells that can be used to transfer material between cells, including Lysotracker positive or endosomal vesicles, calcium fluxes, bacteria or viruses through their cytoplasms or along the surface of the nanotubes.^31–33 Interestingly, a model GPI-anchored protein, GFP-GPI, was found to move at the surface of these tubes³⁴ and while studying the neuritic transport of prions in neuronal cells, Magalhães and colleagues noticed a strong correlation between internalized PrP-res and Lysotracker positive vesicles in neurites,³⁶ suggesting that PrP-res might also be able to transfer through TNTs during prion cell-cell spreading.The results from the studies mentioned above and random observations of TNT-like structures in neuronal model cell cultures first led us to study whether these structures could in fact provide an efficient mechanism for prion cell-cell spreading.³⁷ We initially characterized TNT-like structures in the mouse catecholaminergic neuronal cell line, Cath.a-Differentiated cells (CAD cells) a well-recognized neuronal cell model for prion infection.³⁸ Under our culturing conditions, over 40% of the CAD cells could efficiently form actin-rich TNT-like structures between differentially labelled cell populations. In CAD cells, these nanotubes were very heterogeneous, both in length and in diameters. Indeed, TNT-like structures had lengths ranging from 10 to 80 µm and while over 70% of the nanotubes had diameters smaller than 200 nm, the remaining TNT-like structures had larger diameters (200 to 800 nm). We demonstrated that vesicles of lysosomal origins, a fluorescent form of PrP (GFP-PrP), infectious Alexa-PrP^Sc, as well as both endogenous and exogenous PrP^Sc could traffic within TNTs between neuronal cells (Fig. 1). The lysosomal and GFP-PrP vesicles observed to move through TNTs had a directed movement with a speed in the range of actin-mediated motors,³⁷ consistent with previous studies suggesting the involvement of an actomyosin-dependent transport.³⁹ Interestingly, active transfer of endogenous PrP^Sc, lysosomal or GFP-PrP vesicles occurred through TNTs with larger diameters, suggesting distinct roles for the different TNT-like structures observed.³⁷ These results do not seem to be specific to CAD cells since the transfer of GFP-PrP throught TNTs was observed in different types of transfected cells, including HEK293 cells (unpublished data). Furthermore, these results were in agreement with previous observations by Onfelt and colleagues showing the presence of a fluorescent GPI model protein (GFP-GPI) in TNTs formed between EBV-transformed human B cells³⁴ suggesting that different GPI-anchored proteins can be transferred along the surface and inside vesicles within TNTs. In order to determine the relevance of this type of intercellular communication in the case of prion diseases, it was necessary to evaluate the trafficking of the pathological form of PrP (PrP^Sc) within TNTs, by analyzing the transfer of endogenous PrP^Sc between chronically infected ScCAD cells and non-infected CAD cells. By immunofluorescence after guanidium treatment, endogenous PrP^Sc was found inside TNTs and in the cytoplasm of recipient non-infected CAD cells. Similar to exogenous PrP^Sc, endogenous PrP^Sc particles were not present in non-infected CAD cells not in contact with ScCAD cells after overnight co-cultures, thus excluding exosomal transfer or protein shedding.³⁷ Similarly, no transfer was observed between cells in direct contact with one another or upon treatment with latrunculin, which inhibits TNT formation. Strikingly, the transfer of endogenous PrP^Sc was visible only when TNTs were present, demonstrating that in vitro, PrP^Sc can efficiently exploit TNTs to spread between cells of neuronal origin. These data suggested that TNTs could be a mechanism for prion spreading within the cells of the CNS.Open in a separate windowFigure 1Endogenous PrP^Sc transfer from ScCAD cells to CAD cells via TNTs. Endogenous PrP^Sc is found in punctate structures inside TNTs and in the cytoplasms of recipient cells. CAD cells were transfected with Cherry-PLAP (red) and co-cultured with ScCAD for 24 h. Cells were fixed, treated with Gnd and immunostained for PrP using SAF32 Ab (green). (A) Merge projection of Z-stacks obtained with a confocal Andor spinning-disk microscope. (B) Three-dimensional reconstruction of (A) using OsiriX software. (C) Zoom in on TNT-like structures. PrP^Sc is found in vesicular structures inside TNTs and in the cytoplasm of the recipient non-infected CAD cells (see blue arrow heads). Scale bar represents 10 µm.Interestingly, DCs were shown to form networks of TNTs both in vitro⁴⁰ and in vivo.⁴¹ In an elegant study, Watkins and Salter demonstrated that DCs could propagate calcium flux upon cell stimulation to other cells hundreds of microns away through TNTs, both between DCs and between DCs and THP-1 monocytes.⁴⁰ These data suggested the possibility that DCs could form tubular connections with neuronal cells in order to transport PrP^Sc to the PNS via TNTs. Using BMDCs in co-cultures with both cerebellar granular neurons (CGNs) and primary hippocampal neurons, we showed that BMDCs could form networks of TNTs with both types of neurons. Furthermore, these TNTs appeared to be functional, allowing for the transport of Lysotracker positive vesicles and infectious Alexa-PrP^Sc between loaded BMDCs and primary neurons, suggesting that DCs could transfer the infectious prion agent to primary neuronal cultures through TNTs. By using filters and conditions unfavorable for other mechanisms of transport, we found that moRK13 cells,²⁸ as well as CGNs (unpublished data), could be infected by co-cultures with BMDCs loaded with infectious brain homogenate.³⁷ Overall, these data indicate that TNTs could be an efficient mechanism of prion transmission between immune cells and neuronal cells, as well as between neuronal cultures. Since DCs can interact with peripheral neurons,⁴² we propose that TNTs could be involved in the process of neuroinvasion at multiple stages, from the peripheral site of entry to the PNS by neuroimmune interactions with DCs, allowing neurons to retrogradely transport prions to the CNS, and within the CNS (Fig. 2).Open in a separate windowFigure 2Transport of PrP^Sc via TNTs, an alternative spreading mechanism during neuroinvasion. Studies in our laboratory suggest that TNTs allow for the intracellular transport of PrP^Sc between dendritic cells and neurons and between neurons (see inset). The exact mechanism of transport remains to be determined. For instance, it is still not clear, whether PrP^Sc is strictly transported within endocytic vesicles, or whether it can slide along the surface or be transported as aggregosomes within the tubes. Similarly, the types of motors used, as well as the possible gated mechanisms to enter the recipient cells are not known. Because of the high propensity of DCs to form TNTs with different cell types, we propose that TNTs could play important roles in delivering PrP^Sc to the proper cell types along the neuroinvasion route. For instance, DCs could deliver PrP^Sc from the peripheral entry sites to FDCs in the secondary lymphoid tissues (2) or in a less efficient manner, they might occasionally directly transport PrP^Sc to the PNS (1). They could also bridge the immobile FDC networks and the PNS (3), since we have shown that DCs can form TNTs with nerve cells. Finally, once PrP^Sc has reached its final destination within the CNS, TNTs might play a final role in the spreading of PrP^Sc within the brain between neurons and possibly between neuronal cells and astrocytes (4).Recently, it was demonstrated that the distance between FDCs and the neighbouring PNS was critical for prion neuroinvasion.⁴³ Indeed, in the spleen of CD19^−/− mice, FDC networks were found to be 50% closer to the nerve fibers compared to wild-type mice.⁴³ The authors suggested that the increase in prion spreading efficiency in these mice was directly dependent on the reduction in the distance between the FDC networks and the PNS in these mice. These results would be consistent with a mechanism of transfer such as exosomes release. However, shortening the distance between FDCs and the PNS would also reduce the route of transport that mobile cells would have to travel and increase the chances for transfer of prions to the PNS, resulting in an increase in prion spreading efficiency. While the importance of FDCs in prion replication during the spreading to the CNS seems to be clear,^11,14,15 their specific role in the transfer of prions and their possible interactions with other mobile cells are much more debated.^22,23 In order to bridge the gap between FDCs and the PNS, a role for DCs as possible carriers of PrP^Sc has been postulated. Aucouturier and colleagues have previously shown, using RAG-1^−/− mice, which are deficient in FDCs, that CD11c⁺ DCs infected with 139A were able to carry prion infection to the CNS, without accumulation and replication in lymphoid organs,²² thus suggesting that DCs are able to transport prions from the periphery to nerve cells. Recently, however, another study using TNFR1^−/− mice, deficient for FDCs, suggested that DCs were unlikely candidates in the transport of prion to the PNS.²³ In this study, the authors showed that in TNFR1^−/− mice, ME7 or 139A infected DCs were inefficient in transferring infection to the PNS. The authors suggested that the differences between the results obtained with RAG-1^−/− mice and TNFR1^−/− mice could be due to the differences in the levels of innervation of the spleen in RAG-1^−/−mice compared to TNFR1^−/− mice. They suggested that in RAG-1^−/− mice, DCs could spread prion infection because their spleens are highly innervated, compared to TNFR1^−/− or wild-type mice, therefore increasing the propensity of DCs to encounter nerve cells and tranfer the prion agents. Because of the reduction in the levels of innervation in the spleens of wild-type mice, the authors concluded that DCs are unlikely candidate for the transport of prions directly to the PNS [see (1) in Fig. 2]. However, since these studies are using mice deficient for FDCs, it remains unclear what type of interactions might occur between FDCs and DCs, and how DCs might be able to transport prions from FDCs to the PNS in wild-type mice [see (2) in Fig. 2]. Indeed, both studies show that under the right circumstances, DCs can interact with nerve cells, similar to what was recently shown in infected mice⁴² and in agreement with our findings that DCs can form TNTs with neurons.³⁷ Within this scenario, it is clear that to determine the specific role of DCs during the spreading of prions from the gut to the PNS, the transfer mechanisms between DCs and other cell types, especially FDCs and peripheral neurons, need to be better characterized.Overall, these in vitro data strongly point toward TNTs as one possible mechanism of prion spreading. The next step will be to identify these structures in vivo and to determine whether prion spreading in vivo is the result of passive mechanisms, such as exosome release, active intercellular transport along and within TNTs or whether prions will use any means available to reach their targets. Recently, TNT-like structures were imaged in a mouse cornea,⁴¹ suggesting that while challenging, the visualization of in vivo trafficking of prions in lymphoid tissues such as in lymph nodes or in the spleen as well as in brain organotypic cultures might be possible and could be used to reveal the presence of TNTs.The discovery of the existence of nanotubular membrane bridges in vitro has opened-up a new field of research. Channels, called plasmodesmata,⁴⁴ connecting plant cells have long been known to play crucial roles in the transport of nutrients, molecules and signals during development and some of their functions were recently compared with some of the recently proposed functions of TNTs.⁴⁵ Furthermore, in vivo long, actin-rich filopodia like structures were found to be crucial during development.^46–49 For example, these structures exist in developing sea urchin embryos and were proposed to play a role in signalling and patterning during gastrulation.⁴⁷ Similar roles were proposed for thin filopodia-like structures observed during dorsal closure in drosophila.⁴⁹ In addition, TNT-like structures were observed in the mouse cornea between DCs and were shown to increase under inflammatory conditions.⁴¹ The authors postulated that these TNT-like structures could play a role in Ag-specific signalling, especially as a response to eye inflammation. Therefore, the possibility that TNTs might play numerous roles during cell development, in the immune system and as conduits for the spreading of pathogens could lead to major changes in the way we view animal cell interactions. Specifically, understanding how pathogens usurp these cellular connections to spread could allow for the screening and the identification of new therapeutic inhibitors. To this aim, characterizing the basic mechanism of TNT formation within cell model systems will be necessary to improve the knowledge of TNTs in general, to analyze the transfer of pathogens more specifically, and to identify key molecules during this process. In the case of prions, whether they hijack nanotubes to spread between cells or whether prions increase the formation of filopodia and TNT-like structures similar to some viruses^33,50 and/or the efficiency of transfer remain to be determined. Overall, in this specific field, the constant improvement of cell imaging techniques and the emergence of imaging tools to study prion spreading^{36,37,51–53} could lead to exciting new insights both in the physiology of these intercellular connections and in the pathology of these devastating diseases.     

Mutant PrPSc conformers induced by a synthetic peptide and several prion strains

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP^Sc(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4°C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP^Sc(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a β-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP^Sc(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP^Sc in the brains of Tg196 mice. Our results argue that MoPrP^Sc(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP^C(P101L) as well as in Tg196 mice expressing low levels of MoPrP^C(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a β-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.The discovery that brain fractions enriched for prion infectivity contain a protein (rPrP^Sc) that is resistant to limited proteolytic digestion advanced prion research (8, 37). N-terminal truncation of rPrP^Sc produced a protease-resistant fragment, denoted PrP 27-30, that is readily measured by Western blotting, enzyme-linked immunosorbent assay, or immunohistochemistry. The measurement of PrP^Sc was dramatically changed with the development of the conformation-dependent immunoassay (CDI), which permitted detection of full-length rPrP^Sc as well as previously unrecognized protease-sensitive forms of PrP^Sc (39).The CDI depends on using anti-PrP antibodies that react with an epitope exposed in native PrP^C but that do not bind to native PrP^Sc. Upon denaturation, the buried epitope in PrP^Sc becomes exposed and readily reacts with anti-PrP antibodies. Using the CDI, we discovered that most PrP^Sc is protease sensitive, which we designate sPrP^Sc. Whether sPrP^Sc is an intermediate in the formation of rPrP^Sc remains to be determined. In Syrian hamsters inoculated with eight different strains of prions, the ratio of rPrP^Sc to sPrP^Sc was different for each strain and the concentration of sPrP^Sc was proportional to the length of the incubation time (39).In earlier studies, transgenic (Tg) mice, denoted Tg2866, expressing high levels of PrP(P101L) were used to model Gerstmann-Sträussler-Scheinker (GSS) disease caused by the P102L point mutation. In the brains of several lines of mice expressing high levels of PrP(P101L), no rPrP^Sc(P101L) was detectable (26, 27, 47). This was particularly perplexing since these Tg mice expressing high levels of PrP(P101L) developed all facets of prion-induced neurodegeneration, including multicentric PrP amyloid plaques. Moreover, brain extracts from ill Tg2866 mice transmitted disease to Tg196 mice expressing low levels of PrP(P101L) that infrequently developed spontaneous neurodegeneration (29).In humans with GSS, several different mutations of the PrP gene (PRNP) resulting in nonconservative amino acid substitutions have been identified (23). In these patients, the clinical presentation, disease course, and amounts of rPrP^Sc in the brain are variable. Brain extracts from humans who died of GSS were inoculated into apes and monkeys, but the transmission rates were not correlated with the levels of PrP^Sc in the inoculum (1, 2, 9, 32). In a limited study, GSS(P102L) was transmitted to Tg mice expressing a chimeric mouse-human (MHu2 M) PrP transgene carrying the P102L mutation but not to Tg mice expressing MHu2M PrP without the mutation (47). In another study, GSS(P102L) human prions were transmitted to Tg mice expressing MoPrP(P101L) in which the transgene was incorporated through gene replacement (31). The use of gene replacement permits all of the regulatory elements that control the wild-type (wt) MoPrP gene to modulate the expression of MoPrP(P101L). In these mice, the expression level of MoPrP(P101L) in brain is likely to be similar to that in Tg196 mice.When we synthesized a 55-mer MoPrP peptide composed of residues 89 to 143 containing the P101L mutation and folded it under conditions favoring a β-structure, it induced neurodegeneration in Tg196 mice (29). When the peptide was not folded into a β-structure, it did not produce disease in Tg196 mice. We report here that the peptide-initiated disease in Tg196 mice could be serially transmitted to other Tg196 mice using brain extracts from the peptide-inoculated Tg196 mice. Using procedures derived from the CDI, brain extracts from inoculated Tg196 mice were found to contain sPrP^Sc(P101L), from which a 22- to 24-kDa PrP fragment was generated by limited digestion with proteinase K (PK) at 4°C and selective precipitation with phosphotungstate (PTA) (25, 39). In the interest of clarity, we have designated digestion at 4°C as “cold PK” and simply refer to standard digestion at 37°C as “PK.” To aid in distinguishing rPrP^Sc(P101L) from sPrP^Sc(P101L), their properties based on the work reported here and in other previously published papers are listed in Table ​Table11 (39, 40).

TABLE 1.

Characteristics of PrP(P101L) isoforms

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.     

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.     

Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.For human immunodeficiency virus type I (HIV-1) to enter a target cell, the gp120 subunit of the viral envelope glycoprotein (Env) must engage CD4 and a coreceptor on the cell surface. Although numerous coreceptors have been identified in vitro, the two most important coreceptors in vivo are the CCR5 (3, 11, 19, 22, 24) and CXCR4 (27) chemokine receptors. HIV-1 variants that can use only CCR5 (R5 viruses) are critical for HIV-1 transmission and predominate during the early stages of infection (86, 90). The importance of CCR5 for HIV-1 transmission is underscored by the fact that individuals bearing a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (15, 49, 84). Although R5 viruses typically persist into late disease stages, viruses that can use CXCR4, either alone (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of individuals infected with subtype B or D viruses (12, 39, 44). Although not required for disease progression, the appearance of X4 and/or R5X4 viruses is associated with a more rapid depletion of CD4⁺ cells in peripheral blood and faster progression to AIDS (12, 44, 77, 86). However, it remains unclear whether these viruses are a cause or a consequence of accelerated CD4⁺ T cell decline (57). The emergence of CXCR4-using viruses has also complicated the use of small-molecule CCR5 antagonists as anti-HIV-therapeutics as these compounds can select for the outgrowth of X4 or R5X4 escape variants (93).Following triggering by CD4, gp120 binds to a coreceptor via two principal interactions: (i) the bridging sheet, a four-stranded antiparallel beta sheet that connects the inner and outer domains of gp120, together with the base of the V3 loop, engages the coreceptor N terminus (NT); and (ii) more distal regions of V3 interact with the coreceptor extracellular loops (ECLs) (13, 14, 36-38, 43, 59, 60, 78, 79, 88). Although both the NT and ECL interactions are important for coreceptor binding and entry, their relative contributions vary among different HIV-1 strains (23). For example, V3 interactions with the ECLs, particularly ECL2, serve a dominant role in CXCR4 utilization (7, 21, 50, 63, 72), while R5 viruses exhibit a more variable use of CCR5 domains, with the NT interaction being particularly important (4, 6, 20, 67, 83). Although V3 is the primary determinant of coreceptor preference (34), it is unclear how specificity for CCR5 and/or CXCR4 is determined, and, in particular, it is unknown how X4 tropism is acquired. Several reports have shown that the emergence of X4 tropism correlates with the acquisition of positively charged residues in the V3 stem (17, 29, 87), particularly at positions 11, 24, and 25 (8, 17, 28, 29, 42, 75), raising the possibility that these mutations directly or indirectly mediate interactions with negatively charged residues in the CXCR4 ECLs. However, Env domains outside V3, including V1/V2 (9, 32, 45, 46, 61, 64, 65, 80, 95) and even gp41 (40), can also contribute to coreceptor switching, and it is unclear mechanistically or structurally how X4 tropism is determined.We previously derived a replication-competent variant of the R5X4 HIV-1 clone R3A that contained a markedly truncated V3 loop (47). This Env was generated by introducing a mutation termed ΔV3(9,9), which deleted the distal 15 amino acids of V3. The ΔV3(9,9) mutation selectively ablated X4 tropism but left R5 tropism intact, consistent with the view that an interaction between the distal half of V3 and the ECLs is critical for CXCR4 usage (7, 21, 43, 50, 59, 60, 63, 72). This V3-truncated virus provided a unique opportunity to address whether CXCR4 utilization could be regained on a background in which this critical V3-ECL interaction had been ablated and, if so, by what mechanism. Here, we characterize two novel X4 variants of R3A ΔV3(9,9) derived by adapting this virus to replicate in CXCR4⁺ CCR5⁻ SupT1 cells. We show that R3A ΔV3(9,9) could indeed reacquire X4 tropism but through two markedly different mechanisms. One X4 variant, designated ΔV3-X4A, acquired changes in the V3 remnant that reestablished an interaction with the CXCR4 ECLs; the other, ΔV3-X4B, acquired changes outside V3 that engendered interactions with the CXCR4 NT. These divergent evolutionary pathways led to profound differences in sensitivity to the CXCR4 antagonist AMD3100, with ΔV3-X4A showing increased sensitivity relative to R3A and with ΔV3-X4B becoming completely resistant. These findings demonstrate the contributions that interactions with distinct coreceptor regions have in mediating tropism and drug sensitivity and illustrate how HIV''s remarkable evolutionary plasticity in adapting to selection pressures can be exploited to better understand its biological potential.     

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.     

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.     

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.