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Published online on May 29, 2007, 10.1073/pnas.0702662104
PNAS | June 5, 2007 | vol. 104 | no. 23 | 9741-9746


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From the Cover
BIOLOGICAL SCIENCES / GENETICS
Formation of native prions from minimal components in vitro

Nathan R. Deleault*, Brent T. Harris{dagger}, Judy R. Rees{ddagger}, and Surachai Supattapone*,§,

Departments of *Biochemistry, {dagger}Pathology, {ddagger}Community and Family Medicine (Biostatistics and Epidemiology), and §Medicine, Dartmouth Medical School, Hanover, NH 03755

Edited by Reed B. Wickner, National Institutes of Health, Bethesda, MD, and approved April 26, 2007 (received for review March 24, 2007)


   Abstract
Top
 Abstract
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The conformational change of a host protein, PrPC, into a disease-associated isoform, PrPSc, appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt–Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrPC and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrPSc molecules in a purified system requires accessory polyanion molecules. In addition, we found that PrPSc molecules could be formed de novo from these defined components in the absence of preexisting prions. Inoculation of samples containing either prion-seeded or spontaneously generated PrPSc molecules into hamsters caused scrapie, which was transmissible on second passage. These results show that prions able to infect wild-type hamsters can be formed from a minimal set of components including native PrPC molecules, copurified lipid molecules, and a synthetic polyanion.

polyanion | PrP | purified | spontaneous | de novo

A critical event in the pathogenesis of prion disease appears to be misfolding of the host-encoded prion protein (PrPC) into a pathogenic isoform (PrPSc). According to the "protein-only" hypothesis, infectious mammalian prions may be comprised exclusively of PrPSc molecules (1). Work using refolded, recombinant proteins in both mammalian and fungal systems has confirmed the remarkable concept that pure proteins can harbor infectious properties (2–4). However, the mechanism by which wild type PrPC molecules are converted into infectious PrPSc molecules is currently unknown.

Studies using model systems have also suggested that host-encoded factors other than PrPC may be required to propagate prions in vitro and in vivo (5–10). Furthermore, the restricted range of neuronal and nonneuronal cell types that are susceptible to infection by prions also suggests the existence of prion propagation cofactors (11–13). Although no specific cofactors have been identified to date, several studies have shown that various polyanionic compounds, such as host-encoded RNA and proteoglycan molecules, appear to stimulate prion-seeded conversion of PrPC into PrPSc molecules in vitro (10, 14–17).

A powerful approach to identify the requirements for prion formation is the use of in vitro PrPSc conversion systems, such as the cell-free conversion assay (18–20) and the protein misfolding cyclic amplification (PMCA) technique (21–24). Recently, Castilla et al. (22) serially propagated PrPSc molecules and infectious prions in vitro by subjecting brain homogenates to PMCA. Building on this advance, we investigated whether purified PrPC molecules could function as a substrate for the propagation of PrPSc molecules and infectious prions in vitro. To perform these studies, it was necessary to develop a protocol to purify PrPC from native brain tissue because recombinant PrP produced in Escherichia coli is an inefficient substrate for PMCA reactions, even when reconstituted with crude brain homogenates (25). During the course of these studies, we identified conditions under which purified substrates could propagate infectious prions in vitro and also unexpectedly discovered that infectious prions could be generated spontaneously from purified, noninfectious components.


   Results
Top
 Abstract
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Seeded PrPSc Propagation with Purified Substrates. We previously reported that partially purified hamster PrPC molecules efficiently convert into a protease-resistant conformation upon incubation with PrPSc seed and synthetic polyanions in vitro (10). To obtain a more homogeneous preparation of hamster PrPC substrate for the current studies, we developed a modified purification protocol using a combination of detergent solubilization, Protein A agarose, PrP immunoaffinity, and cation exchange chromatographic steps. This protocol yielded a preparation containing 30- to 33-kDa proteins, as determined by SDS/PAGE (Fig. 1, lane 3). We analyzed the chemical composition of the purified preparation using a variety of sensitive techniques and detected only hamster PrPC plus equimolar quantities of 20-carbon fatty acids [described in supporting information (SI) Materials and Methods].


Figure 1
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  		Fig. 1. Silver stain analysis of purified PrPC substrate. Twelve percent SDS/PAGE showing (from left to right): crude, detergent-solubilized brain supernatant; preparation mock-purified from Prnp0/0 mouse brains; preparation purified from normal hamster brains; and molecular weight markers.

To test the ability of purified PrPC to propagate prions in vitro, we used the serial dilution/propagation protocol described by Castilla et al. (22) (Fig. 2A). We first tested the ability of several purified substrate mixtures to facilitate the PMCA propagation of PrPSc molecules in reactions originally seeded with Sc237 PrP27-30 molecules. The results showed that a substrate mixture containing both purified PrPC plus synthetic poly(A) RNA molecules successfully propagated PrPSc molecules for 16 consecutive rounds of PMCA (Fig. 2B Bottom), whereas poly(A) RNA or PrPC molecules alone failed to propagate PrPSc (Fig. 2B Top and Middle). These results indicate that the propagation of Sc237 PrPSc in vitro requires both PMCA sonication and an accessory polyanion. No propagation was obtained without sonication (SI Fig. 5).


Figure 2
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  		Fig. 2. Seeded PrPSc propagation by using purified substrates. (A) Schematic diagram of serial dilution and propagation paradigm used in subsequent experiments, adapted from Castilla et al. (22). (B and C) Western blots showing samples subjected to 16 rounds of PMCA, serial dilution, and propagation as depicted in A. (B) Samples originally seeded with Sc237 PrP27-30 were propagated in substrate containing either poly(A) RNA alone (top gel), purified PrPC alone (middle gel), or PrPC plus poly(A) RNA (bottom gel). (C) Samples originally seeded with 139H PrP27-30 were propagated in substrate containing either purified PrPC alone (top gel), or poly(A) PrPC plus poly(A) RNA (bottom gel). In all gels, a sample containing PrPC not subjected to proteinase K digestion is shown in the first lane as a reference for comparison of electrophoretic mobility (PrPC-PK). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K (+PK).

To determine whether the propagation of other prion strains might also require an accessory polyanion, we performed similar serial PMCA-propagation experiments using 139H PrP27-30 molecules as the initial seed. Again, PrPC alone failed to propagate PrPSc, whereas PrPC plus poly(A) RNA successfully propagated PrPSc during all 16 days tested (Fig. 2C). These results show that in vitro propagation of 139H PrPSc also depends on the presence of an accessory polyanion, and therefore the phenomenon of polyanion dependence is not limited to the propagation of one specific prion strain.

We tested other charged polymeric compounds for their ability to support purified PrPSc propagation and found that only single-stranded polyanions sufficed for this process (SI Fig. 6). To determine whether accessory polyanions are also required for the maintenance of purified PrPSc propagation, we used nuclease-treated, PMCA-generated PrPSc molecules to seed subsequent propagation reactions containing either purified PrPC substrate alone or PrPC plus poly(A) RNA. These experiments confirmed that polyanions are required to maintain PMCA propagation of PrPSc molecules (SI Fig. 7).

We next measured the sensitivity of PMCA using purified substrates. The results of this experiment showed that the minimum dilution of the PMCA product from a typical PrPSc propagation experiment required to seed PrPSc formation in the next round is between 1:5,000 and 1:10,000, and the minimum dilution of the PMCA product required to seed PrPSc formation after three successive propagation rounds is {approx}10–11 (equivalent to 1 fl of undiluted PMCA product) (SI Fig. 8). The seven orders of magnitude increase in sensitivity gained by performing three rounds of PMCA propagation resembles the previously reported increased sensitivity of multiple-round PMCA reactions using brain homogenate substrate (24, 26). We estimate that the concentration of PrPSc molecules present in a sample of undiluted PMCA product to be {approx}400 ng/ml as determined by comparison with reference amounts of recombinant PrP. Using this value, we calculate that the minimum number of PrPSc molecules required to seed PrPSc formation in a three-round PMCA propagation experiment is {approx}7 monomers (no. of monomers = reaction volume 100 µl x limiting dilution 10–11x estimated PrPSc concentration 400 ng/ml x monomeric PrP molecular weight 35,000 ÷ Avogadro's number). Interestingly, this calculated value is close to the measured minimum size of brain-derived infectious scrapie particles (2–6 PrPSc monomers) (27–29) as well as the minimum number of PrPSc monomers (equal to 26) required to initiate serial PMCA propagation reactions in crude homogenates (26). It should be noted that approximately half of the PrPC preparations generated were less sensitive, i.e., required seed more concentrated than a 10–11 dilution of PMCA product to initiate a three-round PMCA propagation experiment; such preparations were not used for subsequent experiments.

Spontaneous Formation of PrPSc Molecules. During the studies described above, we performed a serial PMCA-propagation experiment using PrPC plus poly(A) RNA substrate that was not originally seeded with infectious prions. To our surprise, we observed that PrPSc molecules spontaneously appeared during the "mock" propagation of these unseeded substrates (Fig. 3A). The spontaneously appearing (de novo) PrPSc molecules migrated on SDS/PAGE with an apparent molecular mass of {approx}28 kDa after proteinase K treatment, similar to the apparent molecular mass of brain-derived Sc237 PrP27-30. Once formed, de novo PrPSc molecules serially propagated the formation of PrPSc molecules for the remainder of the experiment.


Figure 3
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  		Fig. 3. Formation of PrPSc molecules de novo during serial PMCA propagation of unseeded purified substrates. (A) Western blot showing unseeded samples containing PrPC plus poly(A) RNA subjected to 16 rounds of PMCA, serial dilution, and propagation. A sample containing PrPC not subjected to proteinase K digestion is shown in the first lane as a reference for comparison of electrophoretic mobility (PrPC-PK). All other samples were subjected to limited proteolysis with 50 µg/ml proteinase K (+PK). (B) Representative slot blot showing the formation of protease-resistant PrP molecules de novo in multiple propagation experiments. Unseeded samples were propagated for 15 rounds in either the presence or absence of poly(A) RNA, as indicated by plus (+) and minus (–) symbols, respectively. In experiments designated by asterisks, propagation was not carried beyond the 12th round because a preliminary assay performed at that stage already detected PrPSc in several preceding rounds. The experiment designated by the # symbol was performed entirely in a prion-free laboratory. (C) Histogram showing the temporal distribution of the first detectable PrPSc signals during serial propagation of unseeded PrPC plus poly(A) RNA substrate mixtures in 10 separate propagation experiments, 7 of which were carried out simultaneously in the same sonicator.

We next conducted a control experiment to investigate the possibility that environmental contamination during sonication or sample processing could have been responsible for the apparently spontaneous production of PrPSc molecules described above. Specifically, we purified PrPC substrate and performed an unseeded propagation experiment entirely in a different laboratory that never been exposed to prions, using only uncontaminated reagents and equipment including: commercially purchased frozen brains obtained from hamsters raised in an off-site commercial facility, new chemical buffers, a new sonicator directly shipped from the manufacturer, a new tube rack, and new sample tubes taken from a previously unopened container. Daily volume transfers were performed on an open bench within this prion-free environment by using aerosol barrier pipette tips and disposable gloves. Under these conditions, we again observed the spontaneous formation of PrPSc, indicating that PrPSc molecules can form under prion-free conditions (Fig. 3B, experiment designated with # symbol).

We next performed a series of unseeded propagation experiments to characterize the polyanion dependence and kinetics of spontaneous PrPSc formation. The results show that, whereas unseeded PMCA reactions containing both PrPC and poly(A) RNA could produce PrPSc molecules spontaneously, reactions containing PrPC substrate alone failed to produce de novo PrPSc molecules. (Fig. 3B). Furthermore, the spontaneous generation of PrPSc in experiments containing PrPC plus poly(A) RNA appeared to be stochastic; PrPSc first became detectable during different propagation rounds in different experiments (Fig. 3 B and C). The stochastic nature of spontaneous PrPSc formation indicates that this process: (i) occurs relatively infrequently (< 1 conversion event per 6 x 1011 input PrPC molecules per PMCA round) and (ii) is unlikely to be caused by the amplification of preexisting PrPSc molecules.

We also compared the detergent solubility and glycosylation patterns of de novo PrPSc molecules with brain-derived PrP27-30 and prion-seeded, in vitro-generated PrPSc molecules. The results of these biochemical studies showed that, like both native and seeded in vitro-generated PrPSc molecules, de novo PrPSc molecules are relatively detergent-insoluble (SI Fig. 9) and contain N-linked glycans (SI Fig. 10).

In Vitro-Generated, Purified PrPSc Molecules Are Infectious. To test whether in vitro-generated, purified PrPSc molecules are infectious, we inoculated wild-type hamsters intracerebrally with various samples derived from our serial PMCA-propagation experiments. The results of these bioassays show that in vitro-propagated, serially diluted PrPSc molecules originally seeded with Sc237 or 139H prions as well as de novo PrPSc molecules formed and propagated without seeds (including a sample prepared in a prion-free environment by using new equipment), all caused scrapie in inoculated hamsters (Table 1). Neuropathological studies revealed typical spongiform degeneration, astrogliosis, and PrP deposition (Fig. 4), accompanied by the accumulation of PrPSc (SI Fig. 11) in the brains of hamsters inoculated with either seeded or unseeded PrPSc molecules.


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  		Table 1. Transmission of brain-derived and in vitro-generated prions to normal Syrian hamsters.

 


Figure 4
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  		Fig. 4. Representative histological fields of the CA2 hippocampus region in control animals and animals inoculated with in vitro-generated PrPSc molecules. Rows from top to bottom: (top row) uninoculated, normal 190 day old hamster; (second row) terminally ill hamster inoculated with Sc237-seeded, serially propagated PrPSc molecules; (third row) terminally ill hamster inoculated with 139H-seeded, serially propagated PrPSc molecules; (bottom row) terminally ill hamster inoculated with spontaneously generated, serially propagated PrPSc molecules. Hematoxylin and eosin (H&E) staining, as well as glial fibrillary acidic protein (GFAP) and PrP immunohistochemical staining results are shown for each group. (Scale bar, 100 µm.)

End-point titration data indicate that, during the course of the Sc237-seeded propagation experiment, the level of prion infectivity associated with the final, day-16 PMCA product ({approx}5 x 104 LD50 per ml) was {approx}4-fold lower than the level of infectivity associated with the input Sc237 PrP27-30 seed ({approx}2 x 105 LD50 per ml) (Table 1). Therefore, during the course of the 16-round, serial dilution experiment, the level of prion infectivity was relatively maintained, whereas the initial PrPSc seed was diluted >1015-fold. In contrast, Sc237-seeded samples propagated in either PrPC or poly(A) RNA substrate alone contained no prion infectivity detectable by bioassay (Table 1). Based on comparisons with known quantities of recombinant PrP on semiquantitative slot-blot assays, we estimate that the concentration of PrPSc molecules is {approx}400 ng/ml in the PMCA product and {approx}40 ng/ml in the PrP27-30 seed. Therefore, we calculate that there are {approx}1.4 x 107 in vitro-propagated PrPSc monomers (based on molecular mass of 35,000 Da) and {approx}4.3 x 105 PrP27-30 monomers (based on molecular mass of 28,000 Da) per LD50 unit. It should be noted that there is currently no agreed definition of "PrPSc," and therefore our method for measuring protease-resistant PrPSc molecules should be considered operational.

The infectious titer of the inoculum containing de novo PrPSc molecules derived from serial PMCA propagation of PrPC plus poly(A) RNA substrate mixture for 16 rounds was {approx}5 x 103 LD50 per ml. In contrast, no prion infectivity could be detected in the PrPC plus poly(A) RNA substrate mixture not subjected to PMCA (Table 1). Taken together, these results confirm that prion infectivity was generated de novo during the course of the serial PMCA propagation experiment, i.e., in the absence of infectious seed material.

To test whether scrapie caused by in vitro-generated PrPSc molecules is transmissible, we serially passaged brain homogenates prepared from animals originally inoculated with samples containing Sc237-seeded, 139H-seeded, or de novo PrPSc molecules. The results confirm that scrapie caused by any of the three original inocula could be efficiently transmitted to normal hamsters upon serial passage (Table 1). The brains of terminally ill animals that received the serial passage inocula displayed accumulation of PrPSc (SI Fig. 11A) and severe spongiform degeneration (data not shown).

Strain Properties of in Vitro-Generated Prions. Originally, we intended to analyze whether inocula derived from Sc237 and 139H could maintain strain differences upon in vitro propagation with purified substrates. However, we were unable to perform this analysis because the incubation time, biochemical, and neuropathological phenotypes of hamsters inoculated with PrP27-30 molecules derived from these two strains showed no statistically significant differences (Table 1; SI Fig. 12A, Fig. 4C, and SI Fig. 11B Upper). The reason for this apparent strain convergence is currently unknown but may be related to the digestion of strain-specific, protease-sensitive sPrPSc conformers during the PrP27-30 purification procedure (30, 31) or the removal of the N-terminal octarepeat region from protease-resistant rPrPSc molecules.

We next sought to compare the strain characteristics of animals inoculated with brain-derived PrP27-30 molecules to those of animals inoculated with in vitro-generated PrPSc molecules. Interestingly, animals inoculated with samples containing PrPSc molecules generated by in vitro propagation of Sc237 prions exhibited an altered relationship between incubation time and infectious titer compared with animals inoculated with samples containing the brain-derived Sc237 molecules used as input seed for the propagation experiment (Table 1). At infectious titers between 103 and 105 LD50 units/ml, animals inoculated with Sc237-seeded, in vitro-generated PrPSc molecules had incubation times {approx}50 days longer that animals inoculated with brain-derived Sc237 molecules. Animals inoculated with high concentrations of 139H-seeded in vitro-generated or de novo PrPSc molecules also displayed long scrapie incubation times (Table 1). All in vitro-generated PrPSc inocula displayed shortened incubation times upon second passage (Table 1).

Clinically, hamsters inoculated with Sc237-seeded, 139H-seeded, or de novo PrPSc molecules were indistinguishable from each other. All three groups of animals showed typical signs of scrapie in the terminal phase, including ataxia, trembling, circling, broad gait, and inability to maintain or regain upright posture. However, nearly all of the animals in these three groups also displayed atypical clinical signs during the early symptomatic phase, namely hyperactivity and the propensity to climb and hang vertically on cage cover bars. In contrast, these clinical signs were seldom observed in animals inoculated with brain-derived PrP27-30. Upon serial passage of Sc237-seeded, 139H-seeded, and unseeded in vitro-generated PrPSc inocula, scrapie incubation times were uniformly shortened to {approx}90 days (Table 1), and inoculated animals did not exhibit vertical climbing activity.

We also examined the neuropathology of hamsters inoculated with Sc237-seeded, 139H-seeded, and two independently generated samples of unseeded PrPSc molecules (SI Fig. 12A). The severity of vacuolation produced by one of the unseeded inocula (prepared completely in a prion-free environment and indicated by #) was significantly lower (P < 0.05) than the 139H-seeded inoculum in five of five brain regions, the Sc237-seeded inoculum in four of five brain regions, and the other unseeded inoculum (indicated by filled triangles) in one of five brain regions (cerebellum). Furthermore, the regional pattern of vacuolation produced by this inoculum differed from the patterns produced by other in vitro-generated PrPSc inocula in that relatively little vacuolation was observed in the frontal cortex and hippocampus, compared with other brain regions (SI Fig. 12A). The severity of vacuolation produced by the other unseeded inoculum (indicated by filled triangles) more closely resembled those caused by the seeded inocula (SI Fig. 12A). There were no statistically significant differences in the PrP deposition scores measured by immunohistochemistry between any of the groups inoculated with seeded or unseeded in vitro-generated PrPSc molecules (SI Fig. 12B). No neuropathological abnormalities were observed in control animals inoculated with (i) an unseeded PrPC + poly(A) RNA mixture not subjected to PMCA, (ii) a seeded sample serially propagated by using PrPC substrate alone, and (iii) a seeded sample serially propagated by using poly(A) RNA alone. (SI Fig. 13 and Fig. 4 B and C). These negative diagnostic results confirm that the PrPC and poly(A) RNA substrates are not themselves contaminated with prions or any other neurotropic infectious agents.

Biochemically, the guanidine denaturation profiles of protease-resistant PrPSc molecules in the brains of hamsters inoculated with seeded and unseeded in vitro-generated PrPSc molecules as well as brain derived PrP27-30 molecules were nearly indistinguishable (SI Fig. 11B).


   Discussion
Top
 Abstract
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Accessory Polyanions Facilitate Prion Formation in Vitro. Our observation that efficient PMCA propagation of infectious prions in vitro requires an accessory polyanion raises the possibility that endogenous polyanionic cofactors may participate in prion propagation in vivo, either as catalysts or as scaffolds complexed to PrPSc molecules. Further studies are required to distinguish between these possibilities and to identify the specific endogenous polyanions that facilitate prion propagation in vivo. Several classes of negatively charged macromolecules could potentially serve as cofactors for prion propagation, including: nucleic acids (17, 32–38), glycosaminoglycans (14–16), phospholipid-rich membranes (39–42), and chaperone proteins (43). Interestingly, it has been proposed that polyanions could play a broad role in protein folding and misfolding, and the ability of polyanions to facilitate prion conversion may represent a specific example of that general concept (44). It has also been proposed that binding of specific RNA molecules to PrPSc could determine strain properties (45). We observed that two independently generated samples of de novo PrPSc molecules prepared by using purified PrPC plus synthetic poly(A) RNA substrates produced moderately different regional vacuolation profiles in hamsters. This result indicates that, although polyanions might constrain the folding potential of PrP molecules, purified prions formed spontaneously in the presence of poly(A) RNA can display at least some degree of conformational heterogeneity. More work is required to determine whether polyanions can influence prion strain properties.

It has been previously reported that refolding pure, recombinant PrP into amyloid fibers in the absence of polyanions could produce synthetic mammalian prions (2). Several possible explanations could account for the discrepancy between the results of that study and our demonstration that polyanions are necessary for prion formation in vitro. (i) Recombinant PrP amyloid fibrils may interact with endogenous polyanions in situ after inoculation. An observation consistent with this possible explanation is that synthetic prions formed from recombinant PrP molecules display unique biochemical and neuropathological strain properties upon initial inoculation into transgenic mice expressing truncated PrPC molecules but subsequently cause typical scrapie (resembling infection with the murine RML prion strain) upon serial passage into wild-type mice (2, 46). (ii) Polyanions may not be absolutely required to form an infectious prion, but may increase the efficiency of prion conversion. High concentrations of recombinant PrP were used to produce disease with very long incubation times in Tg mice overexpressing PrP, and therefore it is possible that the specific infectivity of PrP amyloid formed without polyanions may be substantially lower than the specific infectivity of PrPSc molecules formed in the presence of polyanions. The in vitro-prions generated in our experiments also produced longer initial incubation times than brain-derived prions; one possible explanation for this effect is that poly(A) RNA may be an imperfect cofactor for forming hamster prions. (iii) Differences in specific experimental conditions (e.g., PrP preparation method, buffer composition, reaction pH, or the presence of denaturants) could account for the apparent difference in polyanion requirement between the two systems. More work is required to determine the reason that polyanions are required to produce infections prions in PMCA experiments but not in the in vitro folding experiments reported by Legname et al. (2).

Spontaneous Generation of Infectious Prions: a Model of Sporadic Prion Disease. The mechanism by which PrPSc molecules and infectious prions originate in sporadic Creutzfeldt–Jakob disease (sCJD) is not known, and no experimental model of this disease has previously been described. PrPSc molecules invariably accumulate in the brains of patients with sCJD, and the disease can be experimentally transmitted to normal primates (47). Several hypotheses have been proposed to explain the etiology of sCJD, including: stochastic formation of PrPSc molecules (48), somatic mutation of PrP sequence in individual brain cells (49), and age-dependent decline in PrPSc clearance mechanisms (50). Our results suggest that interactions between PrPC molecules and endogenous polyanions may contribute to the relatively infrequent process of prion formation in patients with sCJD.

Several lines of evidence indicate that prions form spontaneously at low frequency in unseeded experiments, rather than by amplification of preexisting prions. (i) Most importantly, de novo PrPSc molecules were generated in a completely prion-free laboratory, by using only new or prion-free equipment and source materials. (ii) The appearance of de novo PrPSc molecules appears to be stochastic, whereas one might expect that contaminating PrPSc molecules would be amplified in a more stereotypic manner during the course serial propagation experiments. (iii) De novo PrPSc molecules generated in a prion-free environment were infectious, whereas various negative control samples were not. Interestingly, this sample produced a unique regional profile characterized by relatively mild vacuolation in the frontal cortex and hippocampus, compared with other samples containing either PrP27-30 or in vitro-generated PrPSc molecules.

Although less likely, it is also possible that normal hamsters have low levels of endogenous PrPSc molecules in their brains. Previous experiments have shown that PrPSc molecules can persist chronically in animals without causing disease (51–53). In this scenario, the rate of endogenous PrPSc production in normal animals might be balanced by a putative clearance mechanism, preventing accumulation.

Limiting the Possible Composition of Infectious Prions. The results presented in this article indicate that a purified PrPC preparation plus an accessory polyanion can serve as substrates for in vitro prion propagation. Therefore, we can logically limit the possible composition of the scrapie agent to, at most, these defined components. More work is required to determine whether the accessory polyanion component functions as an unbound catalyst or is physically complexed to PrPSc within infectious prions. We detected approximately equimolar levels of several unsaturated 20-carbon fatty acids, including several isomers of arachidonic acid, in our purified PrPC preparations. The only fatty acid previously identified as a component of the PrP GPI anchor is stearic acid, a saturated 18-carbon compound (54), and therefore it is likely that the copurified fatty acids are not covalently bound to PrPC. Phospholipids have been shown to influence the folding of recombinant PrP molecules (40–42), and it will be interesting in future studies to determine whether lipids might also regulate the formation of PrPSc molecules in PMCA reactions.


   Materials and Methods
Top
 Abstract
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
PrPC and PrP27-30 molecules were purified by using modified versions of previously published protocols (55). PMCA and serial dilution/propagation experiments were performed as described (22) by using purified substrates instead of brain homogenates. Standard bioassay and neuropathology techniques were used to measure prion infectivity and strain properties (56). PrPSc stability was assayed as described (46). Detailed descriptions for each of these techniques, statistical methods, and chemical analyses are provided in SI Materials and Methods.


   Acknowledgements
Top
 Abstract
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Eve Kemble, Karlina Merkens, Ryan van Hoff, Maudine Waterman, and Wendy Wells (Pathology Department, Dartmouth Medical School) for expert assistance with preparation of histological sections; Todd Bissonnette, Jeremy Brown, Eric DuFour, and David McGuire (Dartmouth Animal Resource Center) for expert veterinary care and assistance with tissue collection; Ta Yuan Chang and Cathy Chang for making their laboratory available for experiments, assuring lack of contamination; James Geoghegan for performing the radioactive T4 kinase reactions; Susan Kennedy and Steve Bobin (Dartmouth Molecular Biology and Proteomics Core Facility) for assistance with peptide mass spectroscopic analysis; Angela LaCroix-Fralish and Brian Jackson (Dartmouth Trace Element Analysis Core Facility) for assistance with ICPMS analysis; James and Barbara Evans for assistance with GCMS lipid analysis; and Jiang Gui for expert advice on statistical methods. This work was supported by The Burroughs Wellcome Fund and National Institutes of Health Grant NS046478.


   Footnotes
 

Abbreviations: PMCA, protein misfolding cyclic amplification; sCJD, sporadic Creutzfeldt–Jakob disease.

To whom correspondence should be addressed at: Department of Biochemistry, 7200 Vail Building, Dartmouth Medical School, Hanover, NH 03755. E-mail: supattapone{at}dartmouth.edu

Author contributions: N.R.D. and S.S. designed research; N.R.D., B.T.H., and S.S. performed research; N.R.D., B.T.H., J.R.R., and S.S. analyzed data; and N.R.D., B.T.H., J.R.R., and S.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

See Commentary on page 9551.

This article contains supporting information online at www.pnas.org/cgi/content/full/0702662104/DC1.

© 2007 by The National Academy of Sciences of the USA


   References
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 Abstract
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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