Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of ...PrP(C) into PrP(Sc) in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP(C) may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrP(C) into PrP(Sc) from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrP(Sc) by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 10¹²-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrP(C) susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.
Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrP
Sc
). 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 PrP
Sc
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 PrP
Sc
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.
The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrP(Sc)), 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 PrP(Sc) template. Here we report that authentic PrP(Sc) and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrP(Sc) and lack any detectable PrP(Sc) 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 PrP(Sc) evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrP(Sc) 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 PrP(Sc) 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.
Previous studies established that transmissible prion diseases could be induced by in vitro -produced recombinant prion protein (PrP) fibrils with structures that are fundamentally different from ...that of authentic PrP scrapie isoform (PrPSc ). To explain evolution of synthetic prions, a new mechanism referred to as deformed templating was introduced. Here, we asked whether an increase in expression level of the cellular form of PrP (PrPC ) speeds up the evolution of synthetic strains in vivo . We found that in transgenic mice that overexpress hamster PrPC , PrPC overexpression accelerated recombinant PrP fibril-induced conversion of PrPC to the abnormal proteinase K-resistant state, referred to as atypical PrPres, which was the first product of PrPC misfolding in vivo . However, overexpression of PrPC did not facilitate the second step of synthetic strain evolution-transition from atypical PrPres to PrPSc , which is attributed to the stochastic nature of rare deformed templating events. In addition, the potential of atypical PrPres to interfere with replication of a short-incubation time prion strain was investigated. Atypical PrPres was found to interfere strongly with replication of 263K in vitro ; however, it did not delay prion disease in animals. The rate of deformed templating does not depend on the concentration of substrate and is hence more likely to be controlled by the intrinsic rate of conformational errors in templating alternative self-propagating states.
Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform ...of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.
A new mechanism for transmissible prion diseases Makarava, Natallia; Kovacs, Gabor G; Savtchenko, Regina ...
The Journal of neuroscience,
2012-May-23, 2012-05-23, 20120523, Letnik:
32, Številka:
21
Journal Article
Recenzirano
Odprti dostop
The transmissible agent of prion disease consists of prion protein (PrP) in β-sheet-rich state (PrP(Sc)) that 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 PrP(Sc) 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 PrP(Sc), whereas an alternative amyloid state, with a folding pattern different from that of PrP(Sc), 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 PrP(Sc) emerged. The dynamics between the two forms suggest that atypical proteinase K-resistant PrP (PrPres) gave rise to PrP(Sc). While no PrP(Sc) 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 PrP(C) misfolding triggered by fibrils. The current work demonstrates that a new mechanism responsible for prion diseases different from the PrP(Sc)-templated or spontaneous conversion of PrP(C) into PrP(Sc) exists. This study provides compelling evidence that noninfectious amyloids with a structure different from that of PrP(Sc) could lead to transmissible prion disease. This work has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.
Transmission of prions to a new host is frequently accompanied by strain adaptation, a phenomenon that involves reduction of the incubation period, a change in neuropathological features and, ...sometimes, tissue tropism. Here we show that a strain of synthetic origin (SSLOW), although serially transmitted within the same species, displayed the key attributes of the strain adaptation process. At least four serial passages were required to stabilize the strain-specific SSLOW phenotype. The biological titration of SSLOW revealed a correlation between clinical signs and accumulation of PrPSc in brains of animals inoculated with high doses (10−1-10−5 diluted brain material), but dissociation between the two processes at low dose inocula (10−6-10−8 diluted brain material). At low doses, several asymptomatic animals harbored large amounts of PrPSc comparable with those seen in the brains of terminally ill animals, whereas one clinically ill animal had very little, if any, PrPSc. In summary, the current study illustrates that the phenomenon of prion strain adaptation is more common than generally thought and could be observed upon serial transmission without changing the host species. When PrPSc is seeded by recombinant PrP structures different from that of authentic PrPSc, PrPSc properties continued to evolve for as long as four serial passages.
Background: Strain adaptation accompanies cross-species transmission of prions.
Results: Adaptation of a strain of synthetic origin was observed within the same host species.
Conclusion: When induced by recombinant PrP fibrils, PrPSc properties evolve over multiple serial passages within the same host.
Significance: The phenomenon of prion strain adaptation is more common than generally thought.
Bioassay by end-point dilution has been used for decades for routine determination of prion infectivity titre. Here we show that the new protein misfolding cyclic amplification with beads (PMCAb) ...technique can be used to estimate titres of the infection-specific forms of the prion protein with a higher level of precision and in 3-6 days as opposed to 2 years, when compared with the bioassay. For two hamster strains, 263 K and SSLOW, the median reactive doses determined by PCMAb (PMCAb(50)) were found to be 10(12.8) and 10(12.2) per gram of brain tissue, which are 160- and 4,000-fold higher than the corresponding median infectious dose (ID(50)) values measured by bioassay. The 10(2)- to 10(3)-fold differences between ID(50) and PMCAb(50) values could be due to a large excess of PMCAb-reactive prion protein seeds with little or no infectivity. Alternatively, the differences between ID(50) and PMCAb(50) could be due to higher rate of clearance of infection-specific prion protein seeds in animals versus PMCAb reactions. A well-calibrated PMCAb reaction can be an efficient and cost-effective method for the estimation of infection-specific prion protein titre.
In 1999, the UK implemented universal leucoreduction as a precaution against transmission of variant Creutzfeldt-Jakob disease by transfusion of domestic blood or red blood cells. We aimed to assess ...how effectively leucoreduction reduced infectivity of transmissible spongiform encephalopathies (TSEs) in blood. 450 mL of whole blood collected and pooled from scrapie-infected hamsters was leucoreduced with a commercial filter. Blood cell concentrations were quantified, and infectivity titres measured. Blood cell recovery and white blood cell removal complied with American Association of Blood Banks standards. Leucofiltration removed 42% (SD 12) of the total TSE infectivity in endogenously infected blood. Leucoreduction is necessary for the removal of white-cell-associated TSE infectivity from blood; however, it is not, by itself, sufficient to remove all blood-borne TSE infectivity.
The route of transmission of most naturally acquired transmissible spongiform encephalopathy (TSE) infections remains speculative. To investigate urine as a potential source of TSE exposure, we used ...a sensitive method for detection and quantitation of TSE infectivity. Pooled urine collected from 22 hamsters showing clinical signs of 263K scrapie contained 3.8 +/- 0.9 infectious doses/mL of infectivity. Titration of homogenates of kidneys and urinary bladders from the same animals gave concentrations 20,000-fold greater. Histologic and immunohistochemical examination of these same tissues showed no indications of inflammatory or other pathologic changes except for occasional deposits of disease-associated prion protein in kidneys. Although the source of TSE infectivity in urine remains unresolved, these results establish that TSE infectivity is excreted in urine and may thereby play a role in the horizontal transmission of natural TSEs. The results also indicate potential risk for TSE transmission from human urine-derived hormones and other medicines.
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