Two alternative pathways for generating transmissible prion disease de novo
© Makarava et al. 2015
Received: 8 September 2015
Accepted: 27 October 2015
Published: 10 November 2015
Previous studies established that prion disease with unique strain-specific phenotypes could be induced by in vitro-formed recombinant PrP (rPrP) fibrils with structures different from that of authentic prions, or PrPSc. To explain the etiology of prion diseases, new mechanism proposed that in animals the transition from rPrP fibrils to PrPSc consists of two main steps: the first involves fibril-induced formation of atypical PrPres, a self-replicating but clinically silent state, and the second consists of atypical PrPres-dependent formation of PrPSc via rare deformed templating events.
In the current study, atypical PrPres with characteristics similar to those of brain-derived atypical PrPres was generated in vitro. Upon inoculation into animals, in vitro-generated atypical PrPres gave rise to PrPSc and prion disease with a phenotype similar to those induced by rPrP fibrils. Significant differences in the sialylation pattern between atypical PrPres and PrPSc suggested that only a small sub-fraction of the PrPC that is acceptable as a substrate for PrPSc could be also recruited by atypical PrPres. This can explain why atypical PrPres replicates slower than PrPSc and why PrPSc outcompetes atypical PrPres.
This study illustrates that transmissible prion diseases with very similar disease phenotypes could be produced via two alternative procedures: direct inoculation of recombinant PrP amyloid fibrils or in vitro-produced atypical PrPres. Moreover, this work showed that preparations of atypical PrPres free of PrPSc can give rise to transmissible diseases in wild type animals and that atypical PrPres generated in vitro is an adequate model for brain-derived atypical PrPres.
Prions are proteinaceous infectious agents that are underlying causes of fatal neurodegenerative diseases known as prion diseases or transmissible spongiform encephalopathies (TSE) . Prions consist of misfolded, aggregated states of the normal, cellular form of the prion protein (PrPC). Prion diseases can arise spontaneously, be inherited or be acquired through transmission . Prions spread between organisms or from cell to cell by replicating their disease-specific misfolded structures via a template-assisted mechanism . This mechanism postulates that PrPSc template recruits and converts PrPC expressed by a host into PrPSc. According to this mechanism, the folding pattern of a newly formed PrPSc accurately replicates that of the PrPSc template, and prion replication exhibits high fidelity .
Recent studies demonstrated that in the presence of certain cellular components such as RNA and/or lipids, highly pure recombinant PrP converts into authentic PrPSc conformations in vitro that can effectively induce transmissible prion diseases in animals [3–6]. At the same time, transmissible prion disease could also be induced by recombinant PrP (rPrP) amyloid fibrils formed in vitro in the absence of any co-factors although at low efficiency [7–12]. Bearing in mind that the structures of rPrP fibrils produced in vitro were found to be fundamentally different from that of PrPSc [13, 14], these studies raised the possibility that an unexplored mechanism responsible for the etiology of prion diseases exists [15, 16]. According to this mechanism, referred to as deformed templating, a self-replicating state with one folding pattern can seed structurally different self-replicating states with alternative folding patterns when exposed to a new replication environment . The newly generated self-replicating states that fit well to the new environment outcompete the original template. A new replication environment might include, but is not limited to, new biochemical or cellular conditions, a new substrate with altered amino acid sequence or posttranslational modifications, or presence of prion inhibitors [17–22]. In direct support of the deformed templating mechanism, molecular imaging revealed that switching between alternative folding patterns can occur within individual PrP amyloid fibrils when amyloid seeds were exposed to heterologous substrate in vitro . In another illustration of the deformed templating mechanism, novel, structurally altered self-replicating PrPSc states that were absent in the original seeding material emerged upon changes to the biochemical environment, which involved a depletion of RNA from protein cyclic amplification reactions and subsequent addition of RNA to the reactions . In addition, upon change in pH, non-infectious fibrils of recombinant fungal prion protein HET-s with a stacked β-sheet architecture gave rise to the alternative, infectious state of HET fibrils with a β-solenoid structure .
The current work is inspired by recent findings that atypical PrPres with characteristics similar to those of brain-derived atypical PrPres can be generated in vitro by seeding Protein Misfolding Cyclic Amplification reactions with beads (PMCAb) using rPrP fibrils (Fig. 1). Three questions have been asked in the current work. The first is whether atypical PrPres generated in vitro gives a rise to PrPSc and prion disease in animals (Fig. 1). The second is whether the strain-specific disease phenotype associated with in vitro-generated atypical PrPres is similar to those of synthetic strains produced by direct inoculation of animals by rPrP fibrils. The third is whether evolution of authentic PrPSc and development of prion diseases can be accelerated if the first of the two main steps, i.e. formation of atypical PrPres, is accomplished in vitro.
Materials and methods
Expression and purification of rPrP and formation of rPrP fibrils
Syrian hamster full-length rPrP encompassing residues 23–231 was expressed and purified according to a previously described procedure  with minor modifications . Lyophilized rPrP was dissolved in 5 mM HEPES, pH 7.0, immediately before use. To form fibrils, a mixture of 0.5 mg/ml rPrP with 50 mM MES, pH 6.0, and 2.0 M or 0.5 M guanidine hydrochloride (GdnHCl) was incubated at 37 °C under continuous agitation. Amyloid formation was confirmed by thioflavin T fluorescence assay as described previously . Fibrils were dialyzed against 10 mM sodium acetate, pH 5.0 for storage. If required, fibrils were heat-treated in PBS, pH 7.4 in the presence of 5 mg/ml BSA (Sigma, Cat. #A3294) or 5 % brain homogenate prepared from healthy hamsters (see below) as described before [10, 29].
Preparation of 10 % brain homogenate (BH)
10 % (wt/vol) brain homogenates were prepared in PBS, pH 7.4, using glass/Teflon homogenizers attached to a cordless 12 V compact drill (Ryobi) as previously described .
Protein misfolding cyclic amplification with beads (PMCAb)
10 % normal brain homogenate (NBH) from healthy hamsters was prepared as described previously  and used as a substrate for PMCAb . For the first round, 10 μl of water-diluted rPrP fibrils were added to 90 μl of NBH resulting in the 5 μg/ml final rPrP concentration in the reaction. For the reactions seeded with brain-derived PrPSc, 10 μl of scrapie BH from inoculated animals were diluted in PBS to achieve the desired final concentration and added to 90 μl of NBH. The standard sonication program consisted of 20 s sonication pulses at ~150 W applied every 20 min during a 24 h period. For each subsequent round, 10 μl of the reaction from the previous round were added to 90 μl of fresh substrate. Each PMCAb reaction was carried out in the presence of two 3/32” Teflon beads (amazonsupply.com). To analyze production of PK-resistant PrP material in PMCAb, 10 μl of sample were supplemented with 5 μl of SDS and 5 μl of PK to a final concentration of SDS and PK of 0.25 % and 50 μg/ml, respectively, followed by incubation at 37 °C for 1 h. The digestion was terminated by addition of SDS-sample buffer and heating the samples for 10 min in a boiling water bath.
Protein misfolding cyclic amplification with partially deglycosylated substrate (dgPMCAb) and generation of atypical PrPres in vitro
Each hamster received 50 μl of inoculum intracerebrally, under 2 % O2/4 minimum alveolar concentration (MAC) isoflurane anesthesia. After inoculation, animals were observed daily for disease using a ‘blind’ scoring protocol. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore (Assurance Number A32000-01; Permit Number: 0215002).
Proteinase K digestion of brain homogenates
For the PK digestion in sarcosyl, an aliquot of 10 % BH was prepared as described previously . Briefly, 10 % BH was mixed with an equal volume of 4 % sarcosyl in PBS, supplemented with 50 mM Tris, pH 7.5, and digested with 20 μg/ml PK (New England BioLabs) for 30 min at 37 °C with 1000 rpm shaking using a DELFIA plate shaker (Wallac) placed in 37 °C incubator. PK digestion was stopped by adding SDS sample buffer and heating the samples for 10 min in a boiling water bath. Samples were loaded onto NuPAGE 12 % Bis-Tris gels, transferred to PVDF membrane, and probed with 3F4 or SAF-84 antibodies.
Analysis of conformational stability and Proteinase K resistance
10 % brain homogenate was diluted 10 times into PMCAb conversion buffer, then supplemented with an equal volume of GdnHCl solution in PBS to a final concentration of GdnHCl ranging from 0.4 to 4 M, and incubated at room temperature for 1 h. Next, nine volumes of 2 % sarkosyl in PBS were added to all samples followed by 1 h incubation at room temperature, and then the samples were treated with 20 μg/mL PK for 1 h at 37 °C with shaking. The digestion was stopped with 2 mM PMSF, and the proteins were precipitated in four volumes of ice-cold acetone, incubated overnight at −20 °C, and subsequently centrifuged for 30 min at 16000 x g. Pellets were dried for 30 min, resuspended in 1 × SDS-sample buffer, loaded into NuPAGE 12 % bisTris gels, then transferred to PVDF membrane, and stained with 3F4 or SAF-84 antibody. To analyze PK resistance, 10 % BH was diluted 10-fold into PMCAb conversion buffer, then supplemented with 0.2 % SDS and digested with serially diluted glycerol-free PK (Sigma, Cat. # P6556) for 1 h at 37 °C. The digestion was terminated by addition of SDS-sample buffer and heating the samples for 10 min in a boiling water bath.
PNGase F treatment of PrPSc
10 % brain homogenate was diluted with an equal volume of 4 % sarcosyl in PBS, pH 7.4, digested with 20 μg/ml PK as described above, deglycosylated following the procedure described previously , and assayed by Western blot with 3 F4 antibody.
25 μL samples were heated for 10 min in a boiling water bath in the presence of gel loading buffer, solubilized for 1 h at room temperature with 200 μL solubilization buffer (8 M Urea, 2 % (wt/vol) CHAPS, 5 mM TBP, 20 mM TrisHCl pH 8.0), then alkylated by adding 7 μL of 0.5 M iodoacetamide and incubated for 1 h at room temperature in the dark. Then, 1150 μL of ice-cold methanol was added and samples were incubated for 2 h at −20 °C. After centrifugation at 16,000 g at 4 °C, supernatant was discarded and the pellet was re-solubilized in 160 μL rehydration buffer (7 M urea, 2 M thiourea, 1 % (wt/vol) DTT, 1 % (wt/vol) CHAPS, 1 % (wt/vol) Triton X-100, 1 % (vol/vol) ampholyte, trace amount of Bromophenol Blue). Fixed immobilized pre-cast IPG strips (cat. # ZM0011, Life Technologies, Carlsbad, CA) with a linear pH gradient 3–10 were rehydrated in 155 μL of resulting mixture overnight at room temperature inside IPG Runner cassettes (cat. # ZM0008, Life Technologies). Isoelectrofocusing (first dimension separation) was performed at room temperature with rising voltage (175 V for 15 min, then 175–2,000 V linear gradient for 45 min, then 2,000 V for 30 min) on Life Technologies Zoom Dual Power Supply using the XCell SureLock Mini-Cell Electrophoresis System (cat. # EI0001, Life Technologies). The IPG strips were then equilibrated for 15 min consecutively in (i) 6 M Urea, 20 % (vol/vol) glycerol, 2 % SDS, 375 mM Tris-HCl pH 8.8, 130 mM DTT, and (ii) 6 M Urea, 20 % (vol/vol) glycerol, 2 % SDS, 375 mM Tris-HCl pH 8.8, 135 mM iodoacetamide, and loaded on 4–12 % Bis-Tris ZOOM SDS-PAGE pre-cast gels (cat. # NP0330BOX, Life Technologies). For the second dimension, SDS-PAGE was performed for 1 h at 170 V. Immunoblotting was performed as described elsewhere, blots were stained using 3F4 or SAF-84 antibody.
The brains of two animals from the second passage of F0.5-derived atypical PrPres were evaluated for the presence of spongiform changes and intensity of PrP immunostaining. Formalin-fixed brain halves divided at the midline (left hemisphere) were processed for hematoxylin-eosin (H&E) stain and immunohistochemistry staining using the mouse monoclonal anti-PrP antibody 3F4 (1:1000, Covance). Blocks were treated in formic acid (96 %) before being embedded in paraffin. For detection of disease-associated PrP, blocks were pretreated by 15 min hydrated autoclaving at 121 °C in trisodium citrate buffer, pH 6.0 with 0.05 % Tween 20, followed by 5 min in 88 % formic acid.
Triggering atypical PrPres in vitro is a generic property of rPrP fibrils
In previous studies, upon inoculations into animals, rPrP fibrils were shown to induce misfolding of PrPC into atypical PrPres [11, 12]. To test whether this process could be recapitulated in vitro, rPrP fibrils were prepared according to the protocols used in previous studies for making fibrils that induced transmissible prion diseases [10–12, 29]. Regardless of the experimental protocol, all fibril preparations triggered atypical PrPres in a PMCAb format that used partially deglycosylated PrPC substrate, referred to as dgPMCAb (Fig. 2a) . Notably, dgPMCAb was previously found to favor selective replication of brain-derived atypical PrPres over PrPSc . When serial PMCAb reactions, instead of dgPMCAb, were seeded with the same fibril preparations, no PK-resistant products were found (Fig. 2a). This result is in agreement with the previous studies that rPrP fibrils do not seed PrPSc nor contain small amounts of PrPSc amplifiable by PMCAb [11, 12, 33]. rPrP fibril-induced formation of atypical PrPres in dgPMCAb was robust and highly reproducible (Fig. 1b). No PK-resistant products were found in non-seeded dgPMCAb control reactions (Fig. 1b). For further experiments, we choose rPrP fibrils prepared in 0.5 M GdnHCl (abbreviated as F0.5), as they were the most effective in inducing prion disease in animals .
In vitro preparation of atypical PrPres materials for animal bioassay
To prepare atypical PrPres for inoculation, dgPMCAb reactions were seeded with F0.5 fibrils and subjected to ten serial rounds. The products of the 10th rounds, abbreviated as F0.5-induced atypical PrPres, were inoculated into Syrian hamsters. In parallel to the F0.5-induced atypical PrPres, brain-derived atypical PrPres material free of PrPSc was also produced for inoculation using the following strategy. When subjected to a serial dgPMCAb, atypical PrPres readily amplifies, whereas PrPSc does not. To ensure the absence of PrPSc the following procedures were followed. First, S05 brain material of an asymptomatic animal containing predominantly atypical PrPres was used to seed dgPMCAb . Second, for seeding, this material was diluted 109-fold that corresponds to the limiting dilution of atypical PrPres (Fig. 1c) (dgPMCAb titration of atypical PrPres material is in Additional file 1: Figure S1). Third, the substrate for dgPMCAb was treated with RNase, a procedure that selectively amplifies atypical PrPres but not PrPSc . Fourth, 18 serial dgPMCAb rounds with 10-fold dilutions between rounds were conducted to completely dilute out any initial PrPSc material, if such would still present after the initial 109-fold dilution (Fig. 2c). Moreover, to confirm absence of PrPSc in the preparation of brain-derived atypical PrPres amplified in dgPMCAb, the products of the 18th dgPMCAb round were subjected to six serial PMCAb rounds (Fig. 2d). Previously, we showed that six PMCAb rounds were sufficient for amplifying a single PrPSc particle to the amounts detectible by Western blot . As expected, no PrPSc was detected by Western blot after six PMCAb rounds in the preparation of atypical PrPres (Fig. 2d).
F0.5-induced atypical PrPres propagates in vivo and gives rise to PrPSc
Bioassay of rPrP amyloid fibrils
Euthanized, days post inocul.
F0.5 rPrP fibrils (1st passage)
F0.5 fibril-induced atypical PrPres (1st passage)
2 at 441, 582, 2 at 630, 5 at 636
Brain-derived atypical PrPres amplified in dgPMCA
atypical PrPres from S05 brainc
BH from animal inoculated with F0.5 fibril-induced atypical PrPres (2nd passage)
Transition from atypical PrPres to PrPSc and a competition between atypical PrPres and PrPSc
To elucidate relationship between atypical PrPres and PrPSc, the relative amounts of two states were compared by Western blot in four groups: animals inoculated with F0.5 fibrils, F0.5-induced atypical PrPres, S05 brain-derived atypical PrPres amplified in dgPMCAb, and with diluted S05 brain material not subjected to dgPMCA (Fig. 3b, d). Several observations could be made. First, atypical PrPres were found in all animals from all four groups, whereas PrPSc were observed only in a fraction of animals and in smaller amounts than atypical PrPres (Fig. 3d). Such dynamics support the hypothesis that atypical PrPres is the first product of PrPC misfolding, that it faithfully propagates in vivo independently of PrPSc and that it precedes PrPSc. Second, small amounts of PrPSc detectible by Western blot were found in the brains of animals infected with the preparations of brain-derived atypical PrPres amplified in dgPMCAb that lacked PrPSc. This result suggests that atypical PrPres not only precedes, but also gives rise to PrPSc. Not surprisingly, the highest amounts of PrPSc were found in the group inoculated with S05 brain material, since in addition to atypical PrPres it contained small amount of PrPSc. Brain-derived atypical PrPres enriched through dgPMCA lacked PrPSc and thus resulted in formation of very low amounts of PrPSc upon inoculation, similar to the amount of PrPSc found in the brains inoculated with F0.5-induced atypical PrPres. Interestingly, without the interference of PrPSc, atypical PrPres accumulated to a greater degree. Unexpectedly, PrPSc amounts accumulated in the brains of animals inoculated with F0.5 fibril-induced or brain-derived atypical PrPres did not exceed the amount of PrPSc found in some animals inoculated with F0.5 fibrils. This observation, however, is consistent with the two-step mechanism, according to which (i) atypical PrPres gives rise to PrPSc in a stochastic manner and (ii) PrPSc competes with atypical PrPres (Fig. 1). As judged from PK-resistance and conformational stability assays, both PrPSc and atypical PrPres were extremely resistant to PK degradation and GdnHCl-induced denaturation (Additional file 2: Figure S2). Such high conformational stability and stability to proteolytic degradation explains why both states can co-exist and compete with each other for a long time. Upon inoculation of high amounts of preformed atypical PrPres, PrPSc arises, but has a low chance of outcompeting atypical PrPres in the 1st passage.
Atypical PrPres and PrPSc are structurally different
Two alternative biochemical pathways produce synthetic strains with similar disease phenotype
Next, we tested whether in vitro-generated atypical PrPres results in a disease phenotype similar to those previously described for the synthetic strains triggered by inoculation of rPrP fibrils directly. Brain material from the animal inoculated with F0.5-induced atypical PrPres and euthanized at 441 days post inoculation was used for the second passage. Using animal euthanized at the early time point allowed initiating the second passage approximately 200 days before the remaining animals from this group were euthanized. In the second passage, clinical disease was observed in 4 out of 6 animals after 500 dpi (Table 1). In agreement with the slow disease progression of synthetic hamster strains described previously [11, 12], the clinical disease here progressed very slowly. Animals approached terminal stages approximately 120 days after the first clinical signs and were euthanized at 630 days due to the old age. Animals displayed the same set of clinical symptoms including rough coat, dry skin and obesity as was previously observed for the synthetic strains [10–12].
PNGase treatment revealed that the sizes of the PK-resistant cores were the same for PrPSc from the second passages of F0.5-induced atypical PrPres and the second passage of S05, both of which were ~0.5-1 kDa shorter than that of the 263K strain (Fig. 7c). In summary, the disease phenotype observed in a second passage of F0.5-induced atypical PrPres was very similar, if not identical to, that described in previous studies for S05 .
The results in the current study are important for the following reasons. First, this work illustrates that transmissible prion disease with very similar if not identical disease phenotypes can be generated via two experimental procedures that utilize alternative biochemical pathways. The initial step of both pathways involves an in vitro conversion of rPrP into an amyloid fibrilar state. However, the first procedure involves direct inoculation of rPrP fibrils into animals, whereas the second protocol relies on production of atypical PrPres in vitro in reactions seeded with rPrP fibrils. Second, this study provides the first direct illustration that preparations of atypical PrPres free of PrPSc produced transmissible diseases when inoculated into wild type animals. Atypical PrPres is a self-replicating, transmissible, but clinically silent state. Third, the current work provides strong support for the mechanism established in previous studies and presented in Fig. 1 that atypical PrPres is an alternative transmissible state, which is also a precursor of PrPSc [11, 12]. Fourth, the current study demonstrates that atypical PrPres generated in vitro using dgPMCAb is an adequate model for atypical PrPres formed in brain. Indeed, inoculation of in vitro-generated F0.5-induced atypical PrPres led to the de novo formation of atypical PrPres in animals. Moreover, both F0.5-induced atypical PrPres and brain-derived atypical PrPres gave rise to PrPSc upon serial transmission. In both atypical PrPres states, F0.5-induced and brain-derived, monoglycosylated PrP was the predominant glycoform and hypersialylated diglycosylated isoforms were missing. This is in contrast to the glycosylation pattern of all currently known hamster strains of synthetic or natural origin, in which diglycosylated PrP is the predominant glycoform . In support of previous studies, this work argues that atypical PrPres can be faithfully and selectively propagated in the dgPMCAb .
The mechanism in Fig. 1 postulates that evolution of synthetic prions involves two main steps. Completing the first step, which involved a transition from rPrP fibrils to atypical PrPres, in vitro instead of in vivo did not accelerate the disease development. This result suggests that the major barrier on a pathway toward PrPSc is associated with the second step, i.e. generation of PrPSc via deformed templating events, which are believed to be rare and stochastic. The data on the dynamics between the two forms suggest that atypical PrPres and PrPSc compete with each other (Figs. 3 and 7). Atypical PrPres gives rise to PrPSc in stochastic events that might occur earlier or later in the course of infection. In experiments where the first PrPSc arises from atypical PrPres before a critical amount of atypical PrPres is accumulated, PrPSc is able to propagate efficiently and rapidly replaces atypical PrPres (Fig. 3d). If PrPSc appears later in the course of the infection when the amount of atypical is above a certain threshold, PrPSc replication is slowed down. Inoculation of atypical PrPres material, which was selectively amplified in dgPMCA and free of PrPSc, proved its ability to produce PrPSc in animals. However, it also provided a selective advantage to the propagation of atypical PrPres conformations. Under such inoculation conditions, PrPSc accumulation during the first passage was very weak. Several passages might be needed for the PrPSc to rich a level, at which it exceed and effectively outcompete atypical PrPres as it was observed with S05 . With the atypical PrPres inhibiting propagation of the disease-related forms we have an interesting case of a “protective misfolding”, where PrPC recruitment into an alternative misfolded, PK-resistant, non-toxic, self-replicating state delays development of a deadly disease.
Atypical PrPres is remarkable with respect to its biochemical and biological properties. While fully transmissible in animal studies, this is a clinically silent state, which in the absence of PrPSc does not cause prion diseases or clinical symptoms [11, 12, 25]. Atypical PrPres forms large plaques and diffuse synaptic oligomers, however, neither is associated with neurotoxic or pathogenic effects . Atypical PrPres is structurally different from PrPSc, as evident from the differences in length of PK-resistant fragments, glycoform ratios and RNA-dependency of their amplification in vitro [11, 12, 26, 27]. Moreover, the current study established that atypical PrPres and PrPSc exhibit significantly different sialylation patterns. In accordance with previous work , PrPSc was found to contain PrP molecules with a wide range of sialylation levels encompassing molecules with both highly and moderately sialylated glycans. In contrast to PrPSc, atypical PrPres does not recruit PrPC with high levels of sialylation. Negatively charged sialic acid residues create electrostatic repulsion between neighboring N-linked glycans. Differences in sialylation status suggest that atypical PrPres has much stronger structural constraints for recruiting diglycosylated or heavily sialylated molecules than PrPSc. As a result, only a fraction of PrPC that is acceptable as a substrate for PrPSc is also acceptable to atypical PrPres. Differences in sialylation offer one of the possible explanations why atypical PrPres replicates slower than PrPSc and why PrPSc can eventually outcompete atypical PrPres. Differences in sialylation pattern could also be responsible for the lack of toxicity by atypical PrPres. This hypothesis remains to be tested. Alternatively, the lack of neurotoxicity could be due to the absence of structural determinants associated with toxic effects. Another hypothesis that would be interesting to consider in future studies is whether sialylation deficiency observed in certain genetic or metabolic disorders can facilitate conversion of PrPC into alternative self-replicating states similar to atypical PrPres or CTF12/13 associated with sCJD.
A number of atypical or short PK-resistant fragments has been identified in human and animal prion diseases. PK-resistant fragments referred to as CTF12/13 that encompass residues 154/156-231 and 162/167-231 were found in the majority of patients with sCJD . C-terminal PK-resistant species of similar size were also found in iatrogenic CJD  and in mice infected with mouse-passaged hamster scrapie . Furthermore, similar C-terminal PK-resistant fragments were found in atypical bovine spongiform encephalopathy, which is believed to be sporadic in origin , and in certain types of ovine scrapie . While the size and position of PK-resistant fragments of atypical PrPres described in the current study are very similar to those found in human and animal diseases of natural origin, it is not clear whether they are structurally related or play similar role in disease etiology. Moreover, the relationship of the C-terminal fragments found in sCJD, iatrogenic CJD or in atypical bovine spongiform encephalopathy to PrPSc is also uncertain. Taking into account results presented here, one can speculate that CTF12/13 species are precursors of PrPSc and important for the etiology of sporadic CJD. Testing this hypothesis would involve selective amplification of sCJD-derived CTF12/13 species in dgPMCAb to generate CTF12/13 material free of PrPSc and then testing whether this material induce prion diseases with sCJD phenotype in humanized mice. Comparison of the sialylation status of atypical PK resistant fragments in prion diseases of natural and synthetic origin would help to establish a relationship between them as well as their relationship with PrPSc.
This work highlights the role of deformed templating in transformation and evolution of protein self-replicating states. The deformed templating model predicts emergence of new, structurally altered self-replicating states upon changes in replication environment . In agreement with this prediction, treatments with anti-prion drugs or prion inhibitors were found to result in transformation of strain-specific properties and emergence of drug-resistant prions [20, 43–45].
In summary, the current work illustrate for the first time that transmissible prion disease with similar disease phenotype can be produced via two alternative procedures: direct inoculation of recombinant PrP amyloid fibrils or in vitro-produced atypical PrPres. Moreover, this work showed that preparations of atypical PrPres free of PrPSc can give rise to transmissible diseases in wild type animals and that atypical PrPres generated in vitro is an adequate model for brain-derived atypical PrPres. Finally, the current study illustrates a remarkable case of competition between two transmissible states formed by the same protein, where one state being clinically silent, whereas another being neurotoxic and responsible for progression of disease. Differences in sialylation patterns between PrPSc and atypical PrPres revealed that only a fraction of PrPC that is acceptable as a substrate for PrPSc is also acceptable to atypical PrPres. This explains why atypical PrPres replicates slower than PrPSc and why PrPSc can eventually outcompete atypical PrPres. Other amyloidogenic proteins that are responsible for a range of neurodegenerative disease are known to spread via prion-like mechanisms [46, 47]. It remains to be determined whether competition between clinically silent and toxic states is a general phenomenon or specific to the prion protein.
bovine serum albumin
sporadic Creutzfeldt Jakob Disease
Protein misfolding cyclic amplification with partially deglycosylated substrate
days post inoculation
Normal brain homogenate
Phosphate buffered saline buffer
Protein misfolding cyclic amplification with beads
- PNGase F:
- PrPC :
normal, cellular form of the prion protein
- PrPSc :
disease-associate, infectious form of the prion protein
recombinant prion protein
Transmissible Spongiform Encephalopathies
We thank Pamela Wright for editing the manuscript. Financial support for this study was provided by National Institute of Health Grants R01 NS045585 and R01 NS074998.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Prusiner SB. Prion diseases and the BSE crisis. Science. 1997;278:245–51.View ArticlePubMedGoogle Scholar
- Cohen FE, Prusiner SB. Pathologic conformations of prion proteins. Annu Rev Biochem. 1998;67:793–819.View ArticlePubMedGoogle Scholar
- Wang F, Wang X, Yuan CG, Ma J. Generating a Prion Bacterially Expressed Recombinant Prion Protein. Science. 2010;327:1132–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Z, Zhang Y, Wang F, Wang X, Xu Y, Yang H, et al. De novo generation of infectious prions with bacterially expressed recombinant prion protein. Faseb J. 2013;27:4768–75.PubMed CentralView ArticlePubMedGoogle Scholar
- Deleault NR, Piro JR, Walsh DJ, Wang F, Ma J, Geoghegan JC, et al. Isolation of phosphatidylethanolamine as a solitary cofactor for prion formation in the absence of nucleic acids. Proc Acad Natl Sci U S A. 2012;109:8546–51.View ArticleGoogle Scholar
- Deleault NR, Walsh DJ, Piro JR, Wang F, Wang X, Ma J, et al. Cofactor molecules maintain infectious conformation and restrict strain properties in purified prions. ProcAcadNatlSciUSA. 2012;109:E1938–46.View ArticleGoogle Scholar
- Legname G, Baskakov IV, Nguyen HOB, Riesner D, Cohen FE, DeArmond SJ, et al. Synthetic mammalian prions. Science. 2004;305:673–6.View ArticlePubMedGoogle Scholar
- Colby DW, Giles K, Legname G, Wille H, Baskakov IV, DeArmond SJ, et al. Design and construction of diverse mammalian prion strains. ProcAcadNatlSciUSA. 2009;106:20417–22.View ArticleGoogle Scholar
- Colby DW, Wain R, Baskakov IV, Legname G, Palmer CG, Nguyen HO, et al. Protease-sensitive synthetic prions. PLoS Pathogen. 2010;6:e1000736.View ArticleGoogle Scholar
- Makarava N, Kovacs GG, Bocharova OV, Savtchenko R, Alexeeva I, Budka H, et al. Recombinant prion protein induces a new transmissible prion disease in wild type animals. Acta Neuropathol. 2010;119:177–87.PubMed CentralView ArticlePubMedGoogle Scholar
- Makarava N, Kovacs GG, Savtchenko R, Alexeeva I, Ostapchenko VG, Budka H, et al. A New Mechanism for Transmissible Prion Diseases. J Neurosci. 2012;32:7345–55.PubMed CentralView ArticlePubMedGoogle Scholar
- Makarava N, Kovacs GG, Savtchenko R, Alexeeva I, Budka H, Rohwer RG, et al. Genesis of mammalian prions: from non-infectious amyloid fibrils to a transmissible prion disease. PLoS Pathogen. 2011;7:e1002419.View ArticleGoogle Scholar
- Wille H, Bian W, McDonald M, Kendall A, Colby DW, Bloch L, et al. Natural and synthetic prion structure from X-ray fiber diffraction. ProcAcadNatlSciUSA. 2009;106:16990–5.View ArticleGoogle Scholar
- Ostapchenko VG, Sawaya MR, Makarava N, Savtchenko R, Nilsson KP, Eisenberg D, et al. Two amyloid states of the prion protein display significantly different folding patterns. J Mol Biol. 2010;400:908–21.PubMed CentralView ArticlePubMedGoogle Scholar
- Makarava N, Baskakov IV. Genesis of transmissible protein states vie deformed templating. Prion. 2012;6:252–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Makarava N, Baskakov IV. The Evolution of Transmissible Prions: The Role of Deformed Templating. PLOS Pathog. 2013;9:e1003759.PubMed CentralView ArticlePubMedGoogle Scholar
- Gonzalez-Montalban N, Lee YJ, Makarava N, Savtchenko R, Baskakov IV. Changes in prion replication environemnt cause prion strain mutation. Faseb J. 2013;27:3702–10.PubMed CentralView ArticlePubMedGoogle Scholar
- Ghaemmaghami S, Watts JC, Nquyen HO, Hayashi S, DeArmond SJ, Prusiner SB. Conformational transformation and selection of synthetic prion strains. J Mol Biol. 2011;413:527–42.PubMed CentralView ArticlePubMedGoogle Scholar
- Ghaemmaghami S, Colby DW, Nquyen HO, Hayashi S, Oehler A, DeArmond S, et al. Convergent Replication of Mouse Synthetic Prion Strains. Am J Pathol. 2013;182:866–74.PubMed CentralView ArticlePubMedGoogle Scholar
- Berry DB, Lu D, Geva M, Watts JC, Bhardwaj S, Oehler A, et al. Drug resistance confounding prion therapeutics. Proc Acad Natl Sci USA. 2013;110:E4160–9.View ArticleGoogle Scholar
- Mahal SP, Jablonski J, Suponitsky-Kroyter I, Orlschlegel AM, Herva ME, Oldstone M, et al. Propagating of RML prions in mice expressing PrP devoid of GPI anchor leads to formation of a novel, stable prion strain. PLOS Pathog. 2012;8:e1002746.PubMed CentralView ArticlePubMedGoogle Scholar
- Cancellotti E, Mahal SP, Somerville R, Diack A, Brown D, Piccardo P, et al. Post-translational changes to PrP alter transmissible spongiform encephalopathy strain properties. EMBO J. 2013;32:756–69.PubMed CentralView ArticlePubMedGoogle Scholar
- Makarava N, Ostapchenko VG, Savtchenko R, Baskakov IV. Conformational switching within individual amyloid fibrils. J Biol Chem. 2009;284:14386–95.PubMed CentralView ArticlePubMedGoogle Scholar
- Wan W, Bian W, McDonald M, Kijac A, Wemmer DE, Stubbs G. Heterogeneous Seeding of a Prion Structure by a Generic Amyloid Form of the Fungal Prion-forming Domain HET-s(218–289). J Biol Chem. 2013;288:29604–12.PubMed CentralView ArticlePubMedGoogle Scholar
- Kovacs GG, Makarava N, Savtchenko R, Baskakov IV. Atypical and classical forms of the disease-associated state of the prion protein exhibit distinct neuronal tropism, deposition patterns, and lesion profiles. Am J Pathol. 2013;183:1539–47.PubMed CentralView ArticlePubMedGoogle Scholar
- Makarava N, Savtchenko R, Baskakov IV. Selective amplification of classical and atypical prions using modified protein misfolding cyclic amplification. J Biol Chem. 2013;288:33–41.PubMed CentralView ArticlePubMedGoogle Scholar
- Klimova N, Makarava N, Baskakov IV (2015) The diversity and relationship of prion protein self-replicating states. Virus Research in press.
- Bocharova OV, Breydo L, Parfenov AS, Salnikov VV, Baskakov IV. In vitro conversion of full length mammalian prion protein produces amyloid form with physical property of PrPSc. JMolBiol. 2005;346:645–59.Google Scholar
- Bocharova OV, Makarava N, Breydo L, Anderson M, Salnikov VV, Baskakov IV. Annealing PrP amyloid firbils at high temperature results in extension of a proteinase K resistant core. J Biol Chem. 2006;281:2373–9.View ArticlePubMedGoogle Scholar
- Makarava N, Kovacs GG, Savtchenko R, Alexeeva I, Budka H, Rohwer RG, et al. Stabilization of a prion strain of synthetic origin requires multiple serial passages. J Biol Chem. 2012;287:30205–14.PubMed CentralView ArticlePubMedGoogle Scholar
- Gonzalez-Montalban N, Makarava N, Ostapchenko VG, Savtchenko R, Alexeeva I, Rohwer RG, et al. Highly Efficient Protein Misfolding Cyclic Amplification. PLoS Pathogen. 2011;7:e1001277.View ArticleGoogle Scholar
- Gonzalez-Montalban N, Makarava N, Savtchenko R, Baskakov IV. Relationship between Conformational Stability and Amplification Efficiency of Prions. Biochemistry. 2011;50:7933–40.PubMed CentralView ArticlePubMedGoogle Scholar
- Piro JR, Wang F, Walsh DJ, Rees JR, Ma J, Supattapone S. Seeding Specificity and Ultrastructural Characteristics of Infectious Recombinant Prions. Biochemistry. 2011;50:7111–6.PubMed CentralView ArticlePubMedGoogle Scholar
- Makarava N, Savtchenko R, Alexeeva I, Rohwer RG, Baskakov IV. Fast and ultrasensitive method for quantitating prion infectivity titer. Nature Commun. 2012;3:741.View ArticleGoogle Scholar
- Katorcha E, Makarava N, Savtchenko R, D’Azzo A, Baskakov IV. Sialylation of prion protein controls the rate of prion amplification, the cross-species barrier, the ratio of PrPSc glycoform and prion infectivity. PLOS Pathog. 2014;10:e1004366.PubMed CentralView ArticlePubMedGoogle Scholar
- Stahl N, Baldwin MA, Hecker R, Pan KM, Burlingame AL, Prusiner SB. Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid. Biochemistry. 1992;31:5043–53.View ArticlePubMedGoogle Scholar
- Nishina K, Deleault NR, Mahal S, Baskakov I, Luhrs T, Riek R, et al. The Stoichiometry of Host PrPC Glycoforms Modulates the Efficiency of PrPSc formation in vitro. Biochemistry. 2006;45:14129–39.View ArticlePubMedGoogle Scholar
- Zou WQ, Capellari S, Parchi P, Sy MS, Gambetti P, Chen SG. Identification of Novel Proteinase K-resistant C-terminal Fragments of PrP in Creutzfeldt-Jakob Disease. J Biol Chem. 2003;278:40429–36.View ArticlePubMedGoogle Scholar
- Satoh K, Muramoto T, Tanaka T, Kitamoto N, Ironside JW, Nagashima K, et al. Association of an 11–12 kDa protease-resistant prion protein fragment with subtypes of dura graft-associated Creutzfeldt-Jakob disease and other prion diseases. J Gen Virol. 2003;84:2885–93.View ArticlePubMedGoogle Scholar
- Lawson VA, Priola SA, Meade-White K, Lawton M, Chesebro B. Flexible N-terminal region of prion protein influences conformation of protease resistant prion protein isoforms associated with cross-species scrapie infection in vivo and in vitro. J Biol Chem. 2004;279:13689–95.View ArticlePubMedGoogle Scholar
- Biacabe AG, Jacobs JG, Bencsik A, Langeveld JP, Baron TG. H-type bovine spongiform encephalopathy: complex molecular features and similarities with human prion diseases. Prion. 2007;1:61–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Baron T, Bencsik A, Vulin J, Biacabe AG, Morignat E, Verchere J, et al. A C-terminal protease-resistant prion fragment distinguishes ovine “CH1641-like” scrapie from bovine classical and L-Type BSE in ovine transgenic mice. PLOS Pathog. 2008;4:e1000137.PubMed CentralView ArticlePubMedGoogle Scholar
- Li J, Browning S, Mahal SP, Oelschlegel AM, Weissmann C. Darwinian evolution of prions in cell culture. Science. 2010;327:869–72.PubMed CentralView ArticlePubMedGoogle Scholar
- Giles K, Berry DB, Condello C, Hawley RC, Gallardo-Godoy A, Bryant C, et al. (2015) Different 2-aminothiazole therapeutics produce distinct patterns of scrapie prion neuropathology in mouse brains. J Pharmacol Exp Ther in press.
- Oelschlegel AM, Weissmann C. Acquisition of Drug Resistance and Dependence by Prions. PLOS Pathog. 2013;9:e1003158.PubMed CentralView ArticlePubMedGoogle Scholar
- Soto C, Estrada L, Castilla J. Amyloids, prions and the inherent infectious nature of misfolded protein aggregates. Trends BiochemSci. 2006;31:150–5.View ArticleGoogle Scholar
- Walker LC, Jucker M. Neurodegenerative Diseases: Expanding the Prion Concept. Annu Rev Neurosci. 2015;38:87–103.View ArticlePubMedGoogle Scholar