Genesis of Mammalian Prions: From Non-infectious Amyloid Fibrils to a Transmissible Prion Disease

The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrPSc), which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrPSc template. Here we report that authentic PrPSc and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrPSc and lack any detectable PrPSc particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres) before authentic PrPSc evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrPSc and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as “deformed templating” postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrPSc can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases

Recombinant prion protein induces a new transmissible prion disease in wild-type animals

Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrPSc). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP that had been converted into the cross-β-sheet amyloid form and subjected to annealing. Serial transmission gave rise to a disease phenotype with highly unique clinical and neuropathological features. Among them were the deposition of large PrPSc plaques in subpial and subependymal areas in brain and spinal cord, very minor lesioning of the hippocampus and cerebellum, and a very slow progression of disease after onset of clinical signs despite the accumulation of large amounts of PrPSc in the brain. The length of the clinical duration is more typical of human and large animal prion diseases, than those of rodents. Our studies establish that transmissible prion disease can be induced in wild-type animals by inoculation of rPrP and introduce a valuable new model of prion diseases

Molecular Structure of Amyloid Fibrils Controls the Relationship between Fibrillar Size and Toxicity

According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells.For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein (PrP(C)) at high levels confirming that cytotoxicity was in part PrP(C)-dependent. Silencing of PrP(C) expression by small hairpin RNAs designed to silence expression of human PrP(C) (shRNA-PrP(C)) diminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrP(C)-mediated and PrP(C)-independent mechanisms depends on the structure of the aggregates.This work provides a direct illustration that the relationship between an amyloid's physical dimension and its toxic potential is not unidirectional but is controlled by the molecular structure of prion protein (PrP) molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrP(C) expression can be exploited to reduce their deleterious effects

Preserving prion strain identity upon replication of prions in vitro using recombinant prion protein

Last decade witnessed an enormous progress in generating authentic infectious prions or PrPSc in vitro using recombinant prion protein (rPrP). Previous work established that rPrP that lacks posttranslational modification is able to support replication of highly infectious PrPSc with assistance of cofactors of polyanionic nature and/or lipids. Unexpectedly, previous studies also revealed that seeding of rPrP by brain-derived PrPSc gave rise to new prion strains with new disease phenotypes documenting loss of a strain identity upon replication in rPrP substrate. Up to now, it remains unclear whether prion strain identity can be preserved upon replication in rPrP. The current study reports that faithful replication of hamster strain SSLOW could be achieved in vitro using rPrP as a substrate. We found that a mixture of phosphatidylethanolamine (PE) and synthetic nucleic acid polyA was sufficient for stable replication of hamster brain-derived SSLOW PrPSc in serial Protein Misfolding Cyclic Amplification (sPMCA) that uses hamster rPrP as a substrate. The disease phenotype generated in hamsters upon transmission of recombinant PrPSc produced in vitro was strikingly similar to the original SSLOW diseases phenotype with respect to the incubation time to disease, as well as clinical, neuropathological and biochemical features. Infrared microspectroscopy (IR-MSP) indicated that PrPSc produced in animals upon transmission of recombinant PrPSc is structurally similar if not identical to the original SSLOW PrPSc. The current study is the first to demonstrate that rPrP can support replication of brain-derived PrPSc while preserving its strain identity. In addition, the current work is the first to document that successful propagation of a hamster strain could be achieved in vitro using hamster rPrP.Peer Reviewe

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

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

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