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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-009-0633-x) contains supplementary material, which is available to authorized users.

We report the results of solid state nuclear magnetic (NMR) measurements on amyloid fibrils formed by the full-length prion protein PrP (residues 23-231, Syrian hamster sequence). Measurements of intermolecular 13 C-13 C dipole-dipole couplings in selectively carbonyl-labeled samples indicate that β-sheets in these fibrils have an in-register parallel structure, as previously observed in amyloid fibrils associated with Alzheimer's disease and type 2 diabetes and in yeast prion fibrils. Two-dimensional 13 C-13 C and 15 N-13 C solid state NMR spectra of a uniformly 15 N, 13 C-labeled sample indicate that a relatively small fraction of the full sequence, localized to the C-terminal end, forms the structurally ordered, immobilized core. Although unique site-specific assignments of the solid state NMR signals can not be obtained from these spectra, analysis with a Monte Carlo/simulated annealing algorithm suggests that the core is comprised primarily of residues in the 173-224 range. These results are consistent with earlier electron paramagnetic resonance studies of fibrils formed by residues 90-231 of the human PrP sequence, formed under somewhat different conditions, suggesting that an in-register parallel β-sheet structure formed by the C-terminal end may be a general feature of PrP fibrils prepared in vitro.

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.

The transmissible agent of prion disease consists of prion protein in β-sheet rich state (PrPSc), which can replicate its conformation according to a template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide accurately reproduces that of the PrPSc template. Here three conformationally distinct amyloid states were prepared in vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular cofactors. Surprisingly, no signs of prion infection were found in Syrian hamsters inoculated with rPrP fibrils that resembled PrPSc, whereas an alternative amyloid state, with a folding pattern different from that of PrPSc induced a pathogenic process that led to transmissible prion disease. An atypical proteinase K-resistant, transmissible PrP form that resembled the structure of the amyloid seeds was observed during a clinically silent stage before authentic PrPSc emerged. The dynamics between the two forms suggest that atypical PrPres gave rise to PrPSc. While no PrPSc was found in preparations of fibrils using Protein Misfolding Cyclic Amplification with beads (PMCAb), rPrP fibrils gave rise to atypical PrPres in modified PMCAb suggesting that atypical PrPres was the first product of PrPC misfolding triggered by fibrils. The current work demonstrates that a new mechanism responsible for prion diseases different from the PrPSc-templated or spontaneous conversion of PrPC into PrPSc exists. This study provides compelling evidence that non-infectious amyloids with a structure different from that of PrPSc could lead to transmissible prion disease. This work has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.

The question of whether distinct self-propagating structures could be formed within the same amino acid sequence in the absence of external cofactors or templates has important implications for a number of issues, including the origin of prion strains and the engineering of smart, self-assembling peptidebased biomaterials. In the current study, we showed that chemically identical prion protein can give rise to conformationally distinct, self-propagating amyloid structures in the absence of cellular cofactors, post-translational modification, or PrP Scspecified templates. Even more surprising, two self-replicating states were produced under identical solvent conditions, but under different shaking modes. Individual prion conformations were inherited by daughter fibrils in seeding experiments conducted under alternative shaking modes, illustrating the high fidelity of fibrillation reactions. Our study showed that the ability to acquire conformationally different self-propagating structures is an intrinsic ability of protein fibrillation and strongly supports the hypothesis that conformational variation in selfpropagating protein states underlies prion strain diversity.The existence of multiple prion strains is considered to be one of the most puzzling features of prion biology. According to the protein-only hypothesis of prion propagation, the normal cellular isoform of the prion protein, PrP C , can be converted into a range of self-propagating disease-related structures, referred to as strains of PrP Sc (1). Although certain physical properties are common for all PrP Sc strains, each strain also possesses unique strain-specific conformational features. Within the protein-only hypothesis, it is assumed that variation in PrP Sc structure can give rise to a broad range of strain-specific disease phenotypes, including substantial variation in incubation time to disease, in pathology, and in behavioral symptoms.In the past several years considerable evidence has accumulated indicating that prion strains do, indeed, exhibit notable differences in the amount and type of ␤-sheet structure, conformational stability, proteolytic resistance, and surface-exposed epitopes (2-7). Although the results of these aforementioned studies were consistent with the protein-only hypothesis, the primary origin of strain-specific conformational variations remains unclear. What is the possible source of strain-specific structural variation in PrP Sc ? A "unified theory" of prion propagation postulates that the strain-specific features of the prion infectious agent may be encoded by a coprion, or small nucleic acid (8). Alternatively, the ability to adopt multiple self-propagating structures may be an intrinsic property of the fibrillation reaction itself and can be achieved in the absence of a coprion. In the past, two distinct amyloid states were produced from chemically identical A␤ peptides (9); however, this property has never been demonstrated for fibrillation of larger polypeptides or global proteins such as PrP.In the current study...

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The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC) into the disease-associated, transmissible form (PrPSc). PrPC 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 PrPC and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrPC glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrPSc glycoform ratio. The current study demonstrates that undersialylated PrPC is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrPC. As a result, PMCAb-derived PrPSc was less sialylated than brain-derived PrPSc. A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrPC using sialidase was found to increase the rate of PrPSc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrPC 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 PrPC was also found to control PrPSc glycoform ratio. A decrease in PrPC sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrPSc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrPC differed from that of spleen-derived PrPC. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrPC, suggesting that Neu1 is not responsible for desialylation of PrPC. The current work highlights previously unappreciated role of PrPC sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.

Prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded and aggregated form of a sialoglycoprotein called prion protein or PrPC. PrPC has two sialylated N-linked carbohydrates. In PrPSc, the glycans are directed outward, with the terminal sialic acid residues creating a negative charge on the surface of prion particles. The current study proposes a new hypothesis that electrostatic repulsion between sialic residues creates structural constraints that control prion replication and PrPSc glycoform ratio. In support of this hypothesis, here we show that diglycosylated PrPC molecules that have more sialic groups per molecule than monoglycosylated PrPC were preferentially excluded from conversion. However, when partially desialylated PrPC was used as a substrate, recruitment of three glycoforms into PrPSc was found to be proportional to their respective populations in the substrate. In addition, hypersialylated molecules were also excluded from conversion in the strains with the strongest structural constraints, a strategy that helped reduce electrostatic repulsion. Moreover, as predicted by the hypothesis, partial desialylation of PrPC significantly increased the replication rate. This study illustrates that sialylation of N-linked glycans creates a prion replication barrier that controls replication rate and glycoform ratios and has broad implications.