Schematic diagrams illustrating two alternative hypotheses on origin of prion mutations.

<p>(<b>A</b>) The “cloud” hypothesis proposes that prion isolates are intrinsically heterogeneous and consists of major (red) and minor (various colors) PrP^Sc variants. Changes in the replication environment might provide selective advantages for replication of a minor variant leading to transformation of the PrP^Sc population. (<b>B</b>) The deformed templating model postulates that diverse structural variants are generated as a result of changes in replication environment via numerous PrP^Sc-dependent trial-and-error seeding events. A newly generated variant that fits better than parent PrP^Sc to the altered environment replaces the original PrP^Sc variant.</p

Additional file 1: Figure S1. of Two alternative pathways for generating transmissible prion disease de novo

Establishing a limiting dilution of atypical PrPres in S05 brain material. S05 brain material was serially diluted up to 1013-fold, then each dilution was used to seed serial dgPMCAb; 18 serial dgPMCAb rounds were conducted and analyzed by Western blot. Ten serial PMCAb rounds were sufficient to amplify the highest dilution of brain material that still contains atypical PrPres (109-fold dilution) to the level detectible by Western blot. The reactions seeded with 1010-fold or higher dilutions were all negative regardless of the number of serial dgPMCAb rounds (Fig. 2c). Western blots were stained with SAF-84 antibody. (TIF 1376 kb

Additional file 2: Figure S2. of Two alternative pathways for generating transmissible prion disease de novo

PK-resistance and conformational stability of atypical PrPres and PrPSc. a Analysis of PK-resistance. Brain material from S05-inoculated animals that contained predominantly PrPSc (upper panel) or atypical PrPres (lower panel) were treated with increasing concentration of glycerol-free proteinase and analyzed by Western blot. b Analysis of conformational stability. Brain materials from S05-inoculated animals that contained predominantly PrPSc (upper panel) or atypical PrPres (lower panel) were incubated with increasing concentrations of GdnHCl, digested with PK and analyzed by Western blot. Animals from the second passage of S05 were used. Western blots were stained with 3F4 or SAF-84 antibody as indicated. (TIF 1302 kb

2D analysis of PrP^C and brain- and PMCAb-derived PrP^Sc.

<p><b>A.</b> 2D analysis of Syrian hamster NBH (top panel) and NBH treated with sialidase from <i>A. ureafaciens</i> (bottom gel). <b>B, C.</b> 2D analysis of brain- and PMCAb-derived 263K material (<b>B</b>), and brain- and PMCAb-derived SSLOW material (<b>C</b>). To produce PMCAb-derived material, brain-derived 263K or SSLOW was subjected to 24 serial rounds with 10-fold dilution between rounds. Black and white triangles mark diglycosylated and monoglycosylated glycoforms, respectively, whereas arrows mark the unglycosylated form. D. Migration of PrP^C in 1D SDS-PAGE gel before and after treatment with sialidase. All blots were stained with 3F4 antibody. <b>E.</b> Schematic diagram of PrP^C that illustrates location of sialic acid residues (stars) on N-linked glycans and GPI anchor. Each of the two glycans can carry up to four terminal sialic acid residues.</p

Sialylation of Prion Protein Controls the Rate of Prion Amplification, the Cross-Species Barrier, the Ratio of PrP^Sc Glycoform and Prion Infectivity

<div><p>The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP^C) into the disease-associated, transmissible form (PrP^Sc). PrP^C is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP^C and the strain-specific structure of PrP^Sc. The current work highlights the previously unappreciated role of sialylation of PrP^C glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP^Sc glycoform ratio. The current study demonstrates that undersialylated PrP^C is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrP^C. As a result, PMCAb-derived PrP^Sc was less sialylated than brain-derived PrP^Sc. A decrease in PrP^Sc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP^C using sialidase was found to increase the rate of PrP^Sc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP^C reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP^C was also found to control PrP^Sc glycoform ratio. A decrease in PrP^C sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP^Sc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP^C differed from that of spleen-derived PrP^C. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP^C, suggesting that Neu1 is not responsible for desialylation of PrP^C. The current work highlights previously unappreciated role of PrP^C sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.</p></div

Prion replication environment defines the fate of prion strain adaptation

<div><p>The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Successful cross-species transmission is often accompanied by strain adaptation and result in stable changes of strain-specific disease phenotype. Amino acid sequences of host PrP^C and donor PrP^Sc as well as strain-specific structure of PrP^Sc are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult if possible at all. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster strain 263K was propagated under normal or RNA-depleted conditions using serial Protein Misfolding Cyclic Amplification (PMCA) conducted first in mouse and then hamster substrates. We found that 263K propagated under normal conditions in mouse and then hamster substrates induced the disease phenotype similar to the original 263K. Surprisingly, 263K that propagated first in RNA-depleted mouse substrate and then normal hamster substrate produced a new disease phenotype upon serial transmission. Moreover, 263K that propagated in RNA-depleted mouse and then RNA-depleted hamster substrates failed to induce clinical diseases for three serial passages despite a gradual increase of PrP^Sc in animals. To summarize, depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging of PMCA-derived materials in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation.</p></div

De-sialylation of PrP^C increases the rate of PrP^Sc amplification in PMCAb.

<p><b>A.</b> 263K, HY, Drowsy or SSLOW scrapie brain materials were diluted 10³–10¹⁰-fold into 10% NBH, dsNBH or mock treated NBH (NBH incubated with a buffer for enzymatic de-sialylation in the absence of sialidase) as indicated and subjected to a single PMCAb round. Undigested 10% NBH was used as reference. <b>B.</b> Analysis of PrP^Sc amplification fold. Scrapie brain materials were diluted 10⁴-fold for 263K or 10³-fold for SSLOW into 10% NBH or dsNBH as indicated and subjected to four serial PMCAb rounds. The material amplified in each round was diluted to a specified dilution fold into 10% NBH or dsNBH for the next PMCAb round as indicated. Unamplified seeds are shown as round 0. Prior to electrophoresis, samples were treated with PK. All blots were stained with 3F4 antibody.</p

De-sialylation of PrP^C reduces species barrier in PMCAb.

<p><b>A.</b> 263K or HY brain materials were diluted 10³-fold into 10% mouse dsNBH or NBH and subjected to ten serial PMCAb rounds with 5-fold dilution between rounds. Prior to electrophoresis, samples were treated with PK. Blots were stained with Ab3531 antibody. <b>B.</b> 22L or ME7 brain material was diluted 10³-fold into 10% hamster dsNBH or NBH and subjected to ten serial PMCAb rounds with 5-fold dilution between rounds. Prior to electrophoresis samples were treated with PK. Blots were stained with 3F4 antibody. Black and white triangles mark diglycosylated and monoglycosylated forms, respectively. <b>C.</b> PK-resistance profile illustrating relative representation of di-, mono-, and unglycosylated glycoforms in 263K-seeded PMCAb products produced in three reactions with mouse NBH (bold solid lines) or dsNBH (dashed lines).</p

2D analysis of brain- and spleen-derived PrP^C.

<p>2D analysis of Syrian hamster spleen (<b>A</b>) and brain homogenates (<b>B</b>). Diglycosylated and monoglycosylated full-length PrP^C (FL) are marked by black and white triangles, respectively. Di-, mono- and unglycosylated C2 fragments (C2) in spleen or C1 fragments (C1) in brain are marked by bold, medium and thin arrows, respectively. PrP^C, C1 or C2 dimers (D) are encircled. M stands for a marker lane: brain or spleen samples were diluted 10-fold and used as references for 2D gels. Blots were stained with SAF-84 antibody.</p

Atomic Force Microscopy imaging of R- and S-fibrils.

<p>Phase AFM images of intact R- and S-fibrils (A and B, respectively), or R- and S-fibrils after fragmentation by ultrasound treatment (C and D respectively). Scale bars = 0.5 µm.</p