Acta Neuropathologica

, Volume 135, Issue 2, pp 179–199 | Cite as

Cofactors influence the biological properties of infectious recombinant prions

  • Natalia Fernández-Borges
  • Michele A. Di Bari
  • Hasier Eraña
  • Manuel Sánchez-Martín
  • Laura Pirisinu
  • Beatriz Parra
  • Saioa R. Elezgarai
  • Ilaria Vanni
  • Rafael López-Moreno
  • Gabriele Vaccari
  • Vanessa Venegas
  • Jorge M. Charco
  • David Gil
  • Chafik Harrathi
  • Claudia D’Agostino
  • Umberto Agrimi
  • Tomás Mayoral
  • Jesús R. Requena
  • Romolo Nonno
  • Joaquín CastillaEmail author
Original Paper


Prion diseases are caused by a misfolding of the cellular prion protein (PrP) to a pathogenic isoform named PrPSc. Prions exist as strains, which are characterized by specific pathological and biochemical properties likely encoded in the three-dimensional structure of PrPSc. However, whether cofactors determine these different PrPSc conformations and how this relates to their specific biological properties is largely unknown. To understand how different cofactors modulate prion strain generation and selection, Protein Misfolding Cyclic Amplification was used to create a diversity of infectious recombinant prion strains by propagation in the presence of brain homogenate. Brain homogenate is known to contain these mentioned cofactors, whose identity is only partially known, and which facilitate conversion of PrPC to PrPSc. We thus obtained a mix of distinguishable infectious prion strains. Subsequently, we replaced brain homogenate, by different polyanionic cofactors that were able to drive the evolution of mixed prion populations toward specific strains. Thus, our results show that a variety of infectious recombinant prions can be generated in vitro and that their specific type of conformation, i.e., the strain, is dependent on the cofactors available during the propagation process. These observations have significant implications for understanding the pathogenesis of prion diseases and their ability to replicate in different tissues and hosts. Importantly, these considerations might apply to other neurodegenerative diseases for which different conformations of misfolded proteins have been described.


Cofactors In vitro propagation Infectious recombinant prions Prion strains PMCA TSE 



This work was supported financially by Spanish Government Grants AGL2015-65046-C2-1-R, PCIN‐2013‐065 and BFU2013-48436-C2-1-P, and a Basque Government Grant 2014111157. The authors would like to thank the following for their support: IKERBasque foundation, vivarium and maintenance from CIC bioGUNE and Patricia Piñeiro and Maite Pérez for technical support. Oxford Protein Production Facility UK (OPPF) for the plasmid pOPIN E, Dr. Ester Vázquez Fernández for useful advice in the initial steps. Dr. Mark P. Dagleish (Moredun Research Institute) for useful discussion and advice.

Compliance with ethical standards

Conflicts of interest

The authors declare no conflicts of interest.

Supplementary material

401_2017_1782_MOESM1_ESM.pdf (2.3 mb)
Supplementary material 1 (PDF 2304 kb) Fig. S1 Biochemical characterization of two distinct misfolded rec-PrPs. a Western blot representing two duplicates of L-seeded-02 and H-seeded-02, two misfolded rec-PrPs generated after serial PMCA propagation (see Fig. 1). Two distinct migration patterns were observed (Low and High) after digestion with 85 µg/ml of Proteinase-K (PK). b Western blot of amplified L-seeded-02 and H-seeded-02 seeds subjected to a single round of 24-h recPMCA. Each misfolded rec-PrP was subjected to serial dilutions (from 10−1 to 10−8) previous to in vitro propagation. The L-seeded-02 seed was able to amplify until a dilution of 10−5 while the H-seeded-02 seed amplified until dilution of 10−8. All the samples were digested with 85 µg/ml of PK. Despite all the samples were run at the same time, the blot was cropped as indicated by the vertical dotted line to avoid displaying unrelated samples. c PK-resistance assay of the two distinct misfolded rec-PrPs. Representative Western blot of the resistance of L-seeded-02 and H-seeded-02 misfolded rec-PrPs to increasing concentrations of PK. The samples were treated for 1 h at 42 °C with 50, 100, 200, 300, 400, 600, 800, 1600 and 2000 μg/ml of PK. Both misfolded proteins showed a high resistance to PK digestion (up to 400 μg/ml) with L-seeded-02 showing higher resistance (at least 2,000 μg/ml). Membranes were developed with SAF83 monoclonal antibody (1:400). Mw: Molecular weight. Fig. S2 Biochemical analysis of Proteinase K (PK)-resistant PrPSc in brain homogenates from bank vole I109 inoculated with different misfolded rec-PrPs. a The same Western blot with mAb 9A2 (1:400) shown in Fig. 3a is here compared with a replica blot revealed with mAb 12B2 (1:500), as indicated. With both antibodies, the two voles infected with H/L-seeded-03 and having classical PrPSc show clearly different glycosylation patterns. Note that, while the brain-derived classical prion strains, CWD and scrapie, show similar signal intensities with the two antibodies, PrPSc from voles infected with recombinant prions (H/L-seeded-03) seems weaker with 12B2 than with 9A2. As 9A2 and 12B2 maps, two nearby epitopes in the region cleaved by PK (amino acids 99-101 and 89-93, respectively) the relative presence of the 12B2 epitope is a measure of the N-terminal cleavage by PK and thus, indirectly, of conformational differences among PrPSc aggregates. b Measurements of the signal ratio between 9A2 and 12B2 replica blots where a high signal ratio indicates a less N-terminal PK-cleavage site. Note that, the ratio is higher in PrPSc from voles infected with recombinant prions (H/L-seeded-03) than in CWD or scrapie, in keeping with their lower apparent molecular weight, suggesting that N-terminal cleavage by PK is less N-terminal in these samples. PrPSc from the two animals showing a classical PrPres pattern are thus different between them (glycotype) and also from PrPSc in voles infected with CWD or scrapie (differential antibody binding). Fig. S3 Molecular and pathological and phenotypes in vole-adapted prion strains derived from recombinant prions. a Biochemical analysis of PK-resistant PrPSc in brain homogenates from bank vole I109 after second passage of PMCA-derived vole CWD and misfolded rec-PrPs (H-seeded-01, H-seeded-02, L-seeded-Dextran, H-seeded-No cofactor). Representative vole brain homogenates were digested with 200 µg/ml of PK and analyzed by Western blot with monoclonal antibodies 9A2 (1:400) and 12B2 (1:500). Overall, all inocula preserved the original PrPSc type, H-seeded-01 and H-seeded-02 propagating atypical 7 kDa protease-resistant PrPSc, and cofactor-selected recombinant prions propagating classical PrPSc. b As 9A2 and 12B2 maps two nearby epitopes in the region cleaved by PK (amino acids 99-101 and 89-93, respectively), the relative presence of the 12B2 epitope is a measure of the N-terminal cleavage by PK and thus, of conformational differences among PrPSc aggregates. This can be measured by the signal ratio between 9A2 and 12B2 replica blots, where a high signal ratio indicates a less N-terminal PK-cleavage site and correlates with a lower apparent MW. Note that, the ratio is higher in PrPSc from voles infected with recombinant prions than in CWD, suggesting that cleavage by PK is less N-terminal in these samples. Furthermore, a different N-terminal cleavage can be observed even between H-seeded-No cofactor and L-seeded-Dextran. c Patterns of neurodegeneration assessed by lesion profiles (left column) and PET-blot analysis of deposition of PK-resistant PrPSc with monoclonal antibody 6C2 (1:300). H-seeded-01 and H-seeded-02 showed overlapping distribution of spongiform degeneration and PrPSc deposition, characterized by strong PrPSc deposition in white matter tracts, such as the alveus of the hippocampus, corpus callosum and fiber bundles in the striatum and blind involvement of subcortical areas. In contrast, subcortical involvement and grey matter PrPSc deposition were the hallmarks of H-seeded-No cofactor, which closely mirrored the phenotype observed after primary passage of the same inoculum (compare with Fig. 6), despite the dramatic reduction of the incubation times (424 dpi vs. 82 dpi). L-seeded-Dextran showed both, cortical and subcortical involvement. Of note, along with PrPSc deposition in grey matter areas, also white matter deposition was observed in L-seeded-Dextran. Fig. S4 In vitro propagation of unseeded misfolded rec-PrPs (generated in cofactor-containing substrate) using brain-based PMCA. a Rounds (R1-R5) of serial PMCA using bank vole or transgenic mice overexpressing bank vole I109 PrP (TgVole) brain homogenates as substrates. The misfolded rec-PrPs: H-unseeded-No cofactor, L-unseeded-Dextran, H-unseeded-RNA and H-unseeded-Plasmid were used as seeds in replicates of four through five rounds of serial PMCA. Tubes were considered positives if a classical PrPres pattern was observed on Western blot. With the exception of H-unseeded-No cofactor, the rest of the samples were efficiently propagated over a mammalian substrate, with L-unseeded-Dextran and H-unseeded-RNA being particularly efficient seeds. These results correlate strongly with those observed in Fig. 5a. b Four tubes of round 5 of each PMCA-propagated sample were digested with 85 µg/ml of Proteinase-K (PK) and analyzed by Western blot using monoclonal antibody Saf83 (1:400). Both TgVole-based and bank vole-based substrates were similarly efficient. At least two biochemical patterns based on migration properties are shown; a low migration of the L-unseeded-Dextran and a high migration of the H-unseeded-Plasmid. All unseeded samples remained negatives. Control substrates: undigested bank vole or TgVole whole brain homogenates. Mw: Molecular weight. Fig. S5 Electronic microscopy analysis of the cofactor-selected misfolded rec-PrPs. a Cryo-EM images of the cofactor-selected misfolded rec-PrPs. The sample was concentrated 100 times by sedimentation without Proteinase-K (PK)-digestion. Rod-like structures, of 100 to 200 nm in length, are conspicuous. The rods are made up of 10 nm wide fibrils laterally associated or bundled, and are very similar to those seen in preparations of GPI-anchorless PrPSc isolated from brain [64]. Scale bars: 200 nm. b Negative stain TEM images of the dextran-selected misfolded rec-PrP (L-seeded-Dextran) and mouse GPI-anchorless PrPSc. Samples were deposited on freshly glow-discharged carbon-coated gold grids and stained with 5% uranyl acetate. As with cryo-EM images, rods visible in the misfolded rec-PrP samples are very similar to those seen in GPI-anchorless PrPSc samples obtained from brain. Scale bars: 100 and 500 nm. Fig. S6 Modelling the generation and selection of different misfolded rec-PrPs Cartoon showing the putative generation of three different misfolded rec-PrPs (purple, orange and blue figures) after serial rounds of unseeded recPMCA. Different misfolding ratios are obtained as consequence as the presence of brain homogenate (BH) components. A filter representing a specific component (putative cofactor) is drawn as sieve to filter (preferential propagation of) just certain misfolded rec-PrPs. The serial rounds of PMCA select positively the recombinant prion strain that is favoured at expenses of the rest of the strains that could even disappear after a larger number of rounds. Fig. S7 Schematic representation of in vitro propagated recombinant prions and histopathological findings associated to their inoculation in bank vole. An overview of the procedures performed and the results generated along this work. The scheme has been divided in two parts: in vitro and in vivo (bioassay) studies. The in vitro part shows how different types of substrates (Prnp 0/0 brain homogenate and specific cofactors) generated different misfolded rec-PrPs. The bioassay part shows the prion strains resulting in vivo and the major histopathological findings observed after their inoculation in bank vole.


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Natalia Fernández-Borges
    • 1
  • Michele A. Di Bari
    • 2
  • Hasier Eraña
    • 1
  • Manuel Sánchez-Martín
    • 3
    • 4
  • Laura Pirisinu
    • 2
  • Beatriz Parra
    • 5
  • Saioa R. Elezgarai
    • 1
  • Ilaria Vanni
    • 2
  • Rafael López-Moreno
    • 1
  • Gabriele Vaccari
    • 2
  • Vanessa Venegas
    • 1
  • Jorge M. Charco
    • 1
  • David Gil
    • 1
  • Chafik Harrathi
    • 1
  • Claudia D’Agostino
    • 2
  • Umberto Agrimi
    • 2
  • Tomás Mayoral
    • 5
  • Jesús R. Requena
    • 6
  • Romolo Nonno
    • 2
  • Joaquín Castilla
    • 1
    • 7
    Email author
  1. 1.CIC bioGUNE, Parque tecnológico de BizkaiaDerioSpain
  2. 2.Department of Veterinary Public Health and Food SafetyIstituto Superiore di SanitàRomeItaly
  3. 3.Servicio de Transgénesis, Nucleus, Universidad de SalamancaSalamancaSpain
  4. 4.IBSAL, Instituto de Investigación Biomédica de SalamancaSalamancaSpain
  5. 5.Laboratorio Central de Veterinaria (LCV)MadridSpain
  6. 6.CIMUS Biomedical Research Institute & Department of Medical SciencesUniversity of Santiago de Compostela-IDISSantiago de CompostelaSpain
  7. 7.IKERBASQUE, Basque Foundation for ScienceBilbaoSpain

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