Generation of recombinant human prions

Using our 96-well plate-formatted quaking-induced conversion (QuIC) and the recombinant human full-length prion protein [huPrP(23–231,129 M)] substrate16,28,29, we first systematically screened a broad range of experimental conditions (temperature, time, recHuPrP concentration, different cofactors) for the seeded and unseeded conformational conversion reactions (Supplementary Tables 1, 2, 3, and 4). The use of conformation-dependent immunoassay (CDI) and conformational stability assay (CSA) gave us an opportunity to monitor not only the amount of the conversion product, but also to screen the conformational fidelity of the conversion reactions by comparing the conformational stability of each reaction product with that of the seed. The selection criteria for the conversion products to be used in the subsequent bioassay experiments were as follows: minimized spontaneous conversion in the absence of the seed, high yield of the conversion reaction, and similarity of conformational stability of the product and the seed. As shown in Supplementary Table 3 and Supplementary Fig. 1, in the presence of sCJD MM1 seed the reaction performed at 37 °C in a PBS (pH 6.9) containing 0.1% (w/w) Triton X100, 0.7 mM monosialoganglioside (GM1), and 0.02 mM polyadenylic acid (PolyA) was characterized both by high amplification rate as well as the final product showing conformational stability essentially identical to that of the original sCJD MM1 prion seed. Interestingly, the most efficient cofactor for amplification of sCJD MM2 prions to conformationally similar replicas was not GM1 but another lipid, 1-palmitoyl-2-oleolyl-sn-glycero-3-phospho(1’-rac-glycerol) (Supplementary Fig. 1a and Supplementary Fig. 1b, Supplementary Tables 1, 2, 3, and 4).

Based on these data, and given that sCJD MM1 prions are known to replicate in transgenic mice much more efficiently than the MM2 conterparts30,31,32, we selected for further studies recombinant PrP replicas generated in the reaction seeded with sCJD MM1 prions in the presence of GM1. Importantly, no spontaneous (i.e., non-seeded) zconversion of recHuPrP was observed under the same experimental conditions (Supplementary Fig. 1a). First, we analyzed these replicas by far-UV circular dichroism spectroscopy (Fig. 1a), finding that they are characterized by high content of β-sheet structure (~41% vs. 3% for PrP monomer in the native conformation). Since the starting brain concentrations and purification yelds of sCJD PrPSc are very low (~200-fold lower compared to rodent PrPSc), no high quality CD spectrum could be obtained for the seed. Neverthelless, the content of β-structure in the recombinant PrP sCJD replicas is similar to that previously reported for rodent prions5.

Fig. 1 Structural comparison of rhuPrion and brain-derived sCJD MM1 PrPSc used as an initial seed. a Far UV circular dichroism (CD) spectrum of recombinant human PrP monomer used as a QuIC substrate, and rhuPrion. b Histidine H/D exchange (His-HXMS) for rhuPrion (red) and MM1 rPrPSc (blue). The parameter t 1/2 represents the half-time of exchange reaction for individual His residues. Error bars indicate standard deviation (3 independent experiments). **p < 0.01; ***p < 0.001. c, d Backbone amide H/D exchange (HXMS) data for peptic fragments derived from rhuPrion (red) after 5 min (c) and 24 h (d) incubation in D 2 O. For comparison, previously published data are included for sCJD MM1 rPrPSc (blue) data after 5 min (c) and 10 days (d) incubation in D 2 O16. Error bars indicate standard deviation (3 independent experiments). *p < 0.05; **p < 0.02 Full size image

To further examine the degree of structural fidelity of the replication of sCJD MM1 prions using the recombinant PrP substrate and our present experimental conditions, we used two mass spectrometry-based methods: histidine hydrogen/deuterium exchange mass spectrometry (His-HXMS) and backbone amide hydrogen/deuterium exchange coupled with mass spectrometry (HXMS). These methods have been shown to be uniquely well suited for structural analysis of not only recombinant PrP aggregates but also brain-derived PrPSc 16,33-36.

His-HXMS monitors water accessibility of individual His side chains37, providing residue-specific information about the packing and interfaces between β-sheets in ordered protein aggregates16,33,36. As shown in Fig. 1b, four out of five His residues in the PK-resistant region are characterized by distinct environments in brain-derived sCJD MM1 PrPSc and its recombinant PrP replica, as indicated by substantial differences in the rate of H/D exchange for C2 protons in histidine imidazole rings. This clearly indicates distinct packing arrangements within the two types of PrP aggregates examined. Structural differences between sCJD MM1 PrPSc seeds and recombinant PrP products of the conversion reaction are further indicated by HXMS studies that probe the rate of H/D exchange of the backbone amide hydrogens33,34,35,36,38. These structural differences are particularly pronounced in the region between residues ~117 and 144. Peptic fragments from this region in the recombinant PrP product show substantially higher degree of H/D exchange compared to that in the sCJD MM1 seed already after 5 min of incubation in D 2 O (Fig. 1c), and these differences become even more dramatic after longer incubation times. Indeed, in the recombinant PrP product this region shows more than 65% exchange after 24 h, whereas in brain PrPSc it remains highly protected even after 10 days of incubation in D 2 O (Fig. 1d). By contrast, the region between residues ~182 and 205 appears to be less prone to deuterium labeling (and thus more ordered) in the recombinant PrP product than in the brain-derived sCJD MM1 seed. Altogether, these data demonstrate that, in the presence of GM1 as a cofactor, sCJD MM1 prions can efficiently seed the conversion of the recombinant PrP to β-sheet-rich aggregates. However, the structure of this product is not identical to that of the seed. One of the factors contributing to apparently imperfect conformational fidelity of this seeded conversion reaction could be the lack of post-tranlational modifications (GPI anchor, glycosylation of Asn residues) in the recombinant PrP substrate used.

Bioassays of recombinant replicas of human prions

To test the infectivity of the recombinant PrP replicas of sCJD MM1 prions, we performed ten rounds of QuIC (all in the presence of polyA and GM1), initially seeded with 0.1% brain homogenate of sCJD MM1 human brain. The initial reaction concentration of seed PrPSc was 0.126 ng/ml and recombinant PrP 0.1 mg/ml, corresponding to the seed/substrate mass ratio ~1:1,000,000. Each subsequent nine rounds were seeded with 10-fold diluted product from the previous round, resulting in a cumulative 10−13 dilution of the original brain prions and PrPSc seed to 0.1 ag of PrPSc, an amount ~100,000 times below one infectious dose unit of sCJD prions39 (Fig. 2a). Even though the conditions were the same in all cycles, the conformational stability of the reaction products shifted gradualy in each cycle, with Gdn HCl concentration corresponding to the midpoint denaturation moving from 3.06 M for the original sCJD MM1 PrPSc to 2.56 M for the reaction product after 10 rounds of QuIC (Fig. 2a). Furthermore, less cooperative conformational transition was observed with an increase in the number of QuIC rounds. We interpret these observations as indicative of a broadening of the spectrum of conformers with lower average stability than that of the original seed.

Fig. 2 Biochemical properties of rhuPrion. a Conformational stability of serial QuIC products. The reaction in the first round (Rnd1) was seeded with brain-derived sCJD MM1 prions. b Conformational stability profiles of rhuPrion and sCJD prions after the first passage in TgNN6h or Tg40 mice. c Survival curves of rhuPrion, sCJD-S, and sCJD-F prions in the second passage in TgNN6h. d Levels of total PrPSc, protease-resistant PrPSc, and D/N ratio using CDI assay of brain homogenates upon the second passage of different prions in TgNN6h mice. e Western blots of prion isolates probed with antibodies detecting both Type 1 and Type 2 PrPSc (3F4) and antibodies specific for Type 1 (12B2) or Type 2 (1E4) PrPSc: lane 1—MM1 sCJD; lane 2—MM2 sCJD; lane 3—rhuPrion; lane 4—sCJD-F prions; lane 5—sCJD-S prions. f Conformational stability profiles of the original human brain-derived sCJD, rhuPrion, and sCJD prions after the second passage in TgNN6h mice Full size image

We inoculated with the amplified material transgenic mice Tg40 that express human PrP(129 M) with native prostranslational modifications (i.e., glycosylation of Asn residues and the GPI anchor)40 as well as transgenic mice TgNN6h that express human PrP(129 M) in which glycosylation was abolished by N181Q and N197Q substitutions12. The latter line of transgenic mice was selected for high levels of cell surface expression of unglycosylated PrPC after monitoring white blood cells by FACS in founder’s mice. Even though it is known that glycosylation per se has no effect on the 3D structure of PrP41, we further verified that the N181Q and N197Q substitutions do not affect the overall folding of the protein. Indeed, recombinant wild-type and mutant proteins show indistinguishable far-UV CD spectra and are characterized by essentially identical thermodynamic stability (Supplementary Fig. 2). It was also verified that the N181Q/N197Q PrPC in TgNN6h mice is cell-surface expressed and contains the GPI anchor (as indicated by immunochemical examination of brain tissue and the release of PrPC into Tx114 aqueous phase upon PIPLC treatment (Supplementary Fig. 3)42. While no disease was observed in Tg40 mice inoculated with the recombinant PrP replica of sCJD MM1 prions, clinical symptoms of prion disease were observed in 60% of TgNN6h mice inoculated with the same preparation, with an average incubation time of 459 days (Table 1). In control experiments it was verified that brain-derived sCJD MM1 prions (used as a seed in our conversion reaction in vitro) triggered disease in both TgNN6h and Tg40 lines, with incubation times of ~417 days and ~169 days, respectively. Neither β-sheet-rich aggregates generated without seeds according to the previously published protocol35, nor native monomers of recHuPrP induced clinical symptoms, and the neuropathology of aged-matched Tg40 and TgNN6h mice showed no abnormalities or deposition of pathogenic prion protein (Table 1). The conformational stability of PrPSc accumulating in Tg40 mice inoculated with sCJD MM1 prions matched the stability of the PrPSc in the inoculum. By contrast, the stability of PrPSc accumulating in the brains of TgNN6h mice inoculated with the same inoculum was significantly reduced, as indicated by lower value of [Gdn HCl] 1/2 (Fig. 2b). TgNN6h mice inoculated with the recombinant replica of MM1 sCJD prions accumulated PrPSc characterized by biphasic denaturation curves, with ~60% of conformers having stability similar to that of PrPSc in sCJD-infected mice and ~40% having higher stability (Fig. 2b). Importantly, all TgNN6h mice inoculated with brain homogenate from the first passage of recombinant PrP prion replicas (rhuPrion) developed clinical symptoms, with the average incubation time of 224 days (Table 1; Fig. 2c). A particularly intriguing phenomenon was observed in the second passage of sCJD MM1 prions in TgNN6h mice, as these mice clearly segregated into two distinct groups: one with the average incubation time of 162 days (denoted sCJD-S, where S stands for slow) and the second one with the average incubation time of 80 days (denoted sCJD-F, where F stands for fast) (Table 1; Fig. 2c). Cumulatively, these experiments indicate that: glycosylation of the host PrPC is important for conformational fidelity of sCJD prions replication in vivo, unglycosylated host PrPC provides an efficient substrate for the replication of synthetic human prions (rhuPrions) generated from an unglycosylated recombinant prion protein, and sCJD prions can propagate in mice expressing unglycosylated PrPC as two distinct strains, one of which is characterized by a remarkably short incubation time. Such subclonining of a single human prion isolate into two or more distinct strains has been described previously in transgenic mice30,32 as well as in cell cultures43 and led to the notion that wild prion isolates actually consist of a mixture (“cloud”) of diverse molecular assemblies10. Whether this strain heterogeneity observed in the present study is at least partly related to the Asn to Gln substitutions positions 181 and 197 necessary to eliminate the two N-linked glycosylation sites on human PrP is not known. However, these amino acid substitutions are generally considered as highly conservative, and circullar dichroism (CD) spectroscopy data indicate no conformational impact on PrPC (Supplementary Fig. 2).

Table 1 Serial transmission experiments in transgenic mice expressing glycosylated and unglycosylated human PrP(129 M) Full size table

In the second passage of rhuPrions in TgNN6h mice, these mice accumulated ~5 and 15-fold more PrPSc than mice infected with sCJD-F and sCJD-S strains, respectively, and the same trends were observed for proteinase-resistant PrPSc (rPrPSc) (Fig. 2d). Different denatured/native (D/N) ratios of PrPSc antibody epitopes (3F4 epitopes 108–112 and 8H4 epitope 175–185) indicate that they are variably exposed in the native state of mice inoculated with different inocula, confirming distinct conformations as detected by CSA (Fig. 2d). Furthermore, Western blot typing with a set of differentiating antibodies revealed that TgNN6h mice inoculated with rhuPrions accumulated Type 1 rPrPSc, whereas those inoculated with sCJD-S and sCJD-F prions accumulate Type 1 and Type 2 rPrPSc, respectively (Fig. 2e). While PrPSc from sCJD-F strain shows CSA profiles superimposable with those for the original human brain-derived sCJD MM1 PrPSc, the nearly superimposable CSA profiles of PrPSc in rhuPrions and sCJD-S prions shifted to the lower stability (Table 1; Fig. 2f). We conclude from these observations that inoculation with rhuPrions leads to the accumulation of very high levels of PrPSc with distinct Type 1 conformational characteristics and stability, whereas the original sCJD MM1 prions cloned in TgNN6h into two isolates, one with short incubation time and Type 2 rPrPSc conformers and the other one with long incubation time and Type 1 rPrPSc.

Comparative neuropathology of rhuPrion

Three distinct vacuolization profiles for TgNN6h mice inoculated with rhuPrion and original MM1 sCJD prions were indentified. The rhuPrion pattern exceeded the vauolization profiles of both subclones of MM1 sCJD prions, most prominently in the cortex, hippocampus, subiculum, basal ganglia, and hypothalamus. These distinct lesion profiles found in rhuPrion-inoculated TgNN6h mice were preserved upon second passage (Fig. 3a, b, c), indicating that rhuPrion represents a stable strain that is distinct from two subclones of the original sCJD MM1 prions. Remarkably, both in the first and second passage, CA3 pyramidal layer cells of hippocampus were completely destroyed and replaced by confluent vacuoles. Some of the vacuoles were proximal to large deposits of PrPSc and appeared reminiscent of florid plaques found in vCJD cases. The same hypocampal structures were either intact in sCJD-S subclone or all hypocampal formation structures were affected indiscriminately in sCJD-F subclone (Fig. 3a, b). Moreover, the first and second passage of rhuPrion led to widespread thioflavin S positive plaques of PrPSc, most prominently in the hippocampus and cortex (Fig. 3c, d). In contrast, sCJD-F clone was characterized by fine, evenly distributed deposits of PrPSc with synaptic-like pattern, and the infection with sCJD-S prion clone resulted in small preamyloid deposits of PrPSc in the hippocampus and in the cortex. These data indicate that rhuPrions represent a distinct human prion strain that, apart from cortex and other gray matter, specifically targets cells in the CA3 pyramidal layer of hippocampus and leads to the accumulation of high levels of PrPSc forming amyloid plaques in the hippocampus and cortex.

Fig. 3 Neuropathological profiles of rhuPrion and sCJD prions in TgNN6h mice. a Semi quantitative vacuolization scoring within the indicated brain regions of rhuPrion and sCJD prions in TgNN6h mice: Cx, cortex; Hip, hippocampus; Sub, subiculum; BG, basal ganglia; Th, thalamus; Hy, hypothalamus; Ce, cerebellum; Sep Nlc, septal nuclei; Bs, brain stem. b Neuropathology panels of hippocampus formation (HPF) of age-matched TgNN6h mice inoculated with alpha-helical monomers of human prion protein (A, B), rhuPrion (C, D) in the first and (E, F) second passage; panels G, H, I, J show distinct characteristics of second passage of sCJD-S and sCJD-F prions. c Extensive neuronal loss and vacuolization in the CA3 pyramidal layer of hippocampus (A) and in the cortex (C) in the (first (A, C)) and second (E, G) passage of rhuPrion in contrast to less prominent loss in TgNN6h mice inoculated with sCJD-S (I, K) and sCJD-F (M, O) prions as visualized by H&E staining. Extensive PrPSc depositions in the stratum oriens of CA1 hypocampal formation and cortex following first (B, D) or second (F, H) passage of rhuPrions. Distinctly different patterns of depositions of PrPSc in sCJD-S and sCJD-F prions in both hippocampus (J, N) and cortex (L, P) of TgNN6h mice. d Thioflavin S positive amyloid plaques in the hippocampus and cortex of TgNN6h mice inoculated with rhuPrion. Vacuolization was visualized by H&E staining, and PrPSc deposition was assessed by immunohistochemistry with the antibody 3F4. HPF, Hippocampus formation; DG, granule cell layer of the dentate gyrus; CA3, pyramidal layer of hippocampus Full size image

Second passage rhuPrions from TgNN6h mice brains could be replicated in vitro by protein misfolding cyclic amplification using Tg40 mice brain homogenate as a substrate. The replication rate was paradoxically lower for sCJD-F subclone, and both rhuPrion and sCJD-S replicated with comparable rates (Fig. 4a). These results indicate that unglycosylated prions present in TgNN6h isolates, including rhuPrions, can be replicated using fully posttranslationally processed normal human PrPC substrate. The end-point titration of seeding activity of all three TgNN6h prion isolates in RT QuIC with SHa(PrP90-231) (Fig. 4b) and with Bv(23–231) (Fig. 4c) substrates indicates that rhuPrion has a seeding titer ~100-fold and 1000-fold higher compared to sCJD-S and sCJD-F prions, respectively. Since the concentration of PrPSc in rhuPrions brain homogenates was only ~twofold higher than that of PrPSc in sCJD-S and sCJD-F homogenates (Fig. 4a), this remarkably high seeding potency suggest that rhuPrion is composed of unique conformers with exceptionally high capacity to convert PrP monomers into Thioflavin-positive β-sheet aggregates. The nonequlibrium sedimentation velocity profile of rhuPrion and both sCJD isolates were simillar, with the highest concentration of PrPSc in the botom of the tubes (Fig. 4d, e, f). Based on previous calibration29, we estimate that the prevalevant population of aggregates in rhuPrions has a mass of at least 14 × 106 Da, which corresponds to at least 600 monomers of PrPSc.