Keywords: polyanion, PrP, purified, spontaneous,de novo

Seeded PrP^Sc Propagation with Purified Substrates.

We previously reported that partially purified hamster PrP^C molecules efficiently convert into a protease-resistant conformation upon incubation with PrP^Sc seed and synthetic polyanionsin vitro (10). To obtain a more homogeneous preparation of hamster PrP^C 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 PrP^C plus equimolar quantities of 20-carbon fatty acids [described insupporting information (SI)Materials and Methods].

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

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, PrP^C alone failed to propagate PrP^Sc, whereas PrP^C plus poly(A) RNA successfully propagated PrP^Sc during all 16 days tested (Fig. 2C). These results show thatin vitro propagation of 139H PrP^Sc 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 PrP^Sc 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 PrP^Sc propagation, we used nuclease-treated, PMCA-generated PrP^Sc molecules to seed subsequent propagation reactions containing either purified PrP^C substrate alone or PrP^C plus poly(A) RNA. These experiments confirmed that polyanions are required to maintain PMCA propagation of PrP^Sc 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 PrP^Sc propagation experiment required to seed PrP^Sc 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 PrP^Sc formation after three successive propagation rounds is ≈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 PrP^Sc molecules present in a sample of undiluted PMCA product to be ≈400 ng/ml as determined by comparison with reference amounts of recombinant PrP. Using this value, we calculate that the minimum number of PrP^Sc molecules required to seed PrP^Sc formation in a three-round PMCA propagation experiment is ≈7 monomers (no. of monomers = reaction volume 100 μl × limiting dilution 10⁻¹¹× estimated PrP^Sc concentration 400 ng/ml × 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 PrP^Sc monomers) (27–29) as well as the minimum number of PrP^Sc monomers (equal to 26) required to initiate serial PMCA propagation reactions in crude homogenates (26). It should be noted that approximately half of the PrP^C 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 PrP^Sc Molecules.

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

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 PrP^Sc molecules described above. Specifically, we purified PrP^C 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 PrP^Sc, indicating that PrP^Sc 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 PrP^Sc formation. The results show that, whereas unseeded PMCA reactions containing both PrP^C and poly(A) RNA could produce PrP^Sc molecules spontaneously, reactions containing PrP^C substrate alone failed to producede novo PrP^Sc molecules. (Fig. 3B). Furthermore, the spontaneous generation of PrP^Sc in experiments containing PrP^C plus poly(A) RNA appeared to be stochastic; PrP^Sc first became detectable during different propagation rounds in different experiments (Fig. 3B andC). The stochastic nature of spontaneous PrP^Sc formation indicates that this process: (i) occurs relatively infrequently (< 1 conversion event per 6 × 10¹¹ input PrP^C molecules per PMCA round) and (ii) is unlikely to be caused by the amplification of preexisting PrP^Sc molecules.

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

In Vitro-Generated, Purified PrP^Sc Molecules Are Infectious.

To test whetherin vitro-generated, purified PrP^Sc 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 thatin vitro-propagated, serially diluted PrP^Sc molecules originally seeded with Sc237 or 139H prions as well asde novo PrP^Sc 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 PrP^Sc (SI Fig. 11) in the brains of hamsters inoculated with either seeded or unseeded PrP^Sc molecules.

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 (≈5 × 10⁴ LD₅₀ per ml) was ≈4-fold lower than the level of infectivity associated with the input Sc237 PrP27-30 seed (≈2 × 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 PrP^Sc seed was diluted >10¹⁵-fold. In contrast, Sc237-seeded samples propagated in either PrP^C 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 PrP^Sc molecules is ≈400 ng/ml in the PMCA product and ≈40 ng/ml in the PrP27-30 seed. Therefore, we calculate that there are ≈1.4 × 10⁷in vitro-propagated PrP^Sc monomers (based on molecular mass of 35,000 Da) and ≈4.3 × 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 “PrP^Sc,” and therefore our method for measuring protease-resistant PrP^Sc molecules should be considered operational.

The infectious titer of the inoculum containingde novo PrP^Sc molecules derived from serial PMCA propagation of PrP^C plus poly(A) RNA substrate mixture for 16 rounds was ≈5 × 10³ LD₅₀ per ml. In contrast, no prion infectivity could be detected in the PrP^C plus poly(A) RNA substrate mixture not subjected to PMCA (Table 1). Taken together, these results confirm that prion infectivity was generatedde novo during the course of the serial PMCA propagation experiment, i.e., in the absence of infectious seed material.

To test whether scrapie caused byin vitro-generated PrP^Sc molecules is transmissible, we serially passaged brain homogenates prepared from animals originally inoculated with samples containing Sc237-seeded, 139H-seeded, orde novo PrP^Sc 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 PrP^Sc (SI Fig. 11A) and severe spongiform degeneration (data not shown).

Strain Properties ofin Vitro-Generated Prions.

Originally, we intended to analyze whether inocula derived from Sc237 and 139H could maintain strain differences uponin 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, andSI 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 sPrP^Sc conformers during the PrP27-30 purification procedure (30,31) or the removal of the N-terminal octarepeat region from protease-resistant rPrP^Sc molecules.

We next sought to compare the strain characteristics of animals inoculated with brain-derived PrP27-30 molecules to those of animals inoculated within vitro-generated PrP^Sc molecules. Interestingly, animals inoculated with samples containing PrP^Sc molecules generated byin 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 PrP^Sc molecules had incubation times ≈50 days longer that animals inoculated with brain-derived Sc237 molecules. Animals inoculated with high concentrations of 139H-seededin vitro-generated orde novo PrP^Sc molecules also displayed long scrapie incubation times (Table 1). Allin vitro-generated PrP^Sc inocula displayed shortened incubation times upon second passage (Table 1).

Clinically, hamsters inoculated with Sc237-seeded, 139H-seeded, orde novo PrP^Sc 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 unseededin vitro-generated PrP^Sc inocula, scrapie incubation times were uniformly shortened to ≈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 PrP^Sc 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 otherin vitro-generated PrP^Sc 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 unseededin vitro-generated PrP^Sc molecules (SI Fig. 12B). No neuropathological abnormalities were observed in control animals inoculated with (i) an unseeded PrP^C + poly(A) RNA mixture not subjected to PMCA, (ii) a seeded sample serially propagated by using PrP^C substrate alone, and (iii) a seeded sample serially propagated by using poly(A) RNA alone. (SI Fig. 13 andFig. 4B andC). These negative diagnostic results confirm that the PrP^C 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 PrP^Sc molecules in the brains of hamsters inoculated with seeded and unseededin vitro-generated PrP^Sc molecules as well as brain derived PrP27-30 molecules were nearly indistinguishable (SI Fig. 11B).

Accessory Polyanions Facilitate Prion Formationin Vitro.

Our observation that efficient PMCA propagation of infectious prionsin vitro requires an accessory polyanion raises the possibility that endogenous polyanionic cofactors may participate in prion propagationin vivo, either as catalysts or as scaffolds complexed to PrP^Sc molecules. Further studies are required to distinguish between these possibilities and to identify the specific endogenous polyanions that facilitate prion propagationin 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 PrP^Sc could determine strain properties (45). We observed that two independently generated samples ofde novo PrP^Sc molecules prepared by using purified PrP^C 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 formationin vitro. (i) Recombinant PrP amyloid fibrils may interact with endogenous polyanionsin 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 PrP^C 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 PrP^Sc molecules formed in the presence of polyanions. Thein 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 thein vitro folding experiments reported by Legnameet al. (2).

Spontaneous Generation of Infectious Prions: a Model of Sporadic Prion Disease.

The mechanism by which PrP^Sc 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. PrP^Sc 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 PrP^Sc molecules (48), somatic mutation of PrP sequence in individual brain cells (49), and age-dependent decline in PrP^Sc clearance mechanisms (50). Our results suggest that interactions between PrP^C 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 PrP^Sc molecules were generated in a completely prion-free laboratory, by using only new or prion-free equipment and source materials. (ii) The appearance ofde novo PrP^Sc molecules appears to be stochastic, whereas one might expect that contaminating PrP^Sc molecules would be amplified in a more stereotypic manner during the course serial propagation experiments. (iii)De novo PrP^Sc 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 orin vitro-generated PrP^Sc molecules.

Although less likely, it is also possible that normal hamsters have low levels of endogenous PrP^Sc molecules in their brains. Previous experiments have shown that PrP^Sc molecules can persist chronically in animals without causing disease (51–53). In this scenario, the rate of endogenous PrP^Sc 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 PrP^C preparation plus an accessory polyanion can serve as substrates forin 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 PrP^Sc within infectious prions. We detected approximately equimolar levels of several unsaturated 20-carbon fatty acids, including several isomers of arachidonic acid, in our purified PrP^C 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 PrP^C. 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 PrP^Sc molecules in PMCA reactions.

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