Photodegradation illuminates the role of polyanions in prion infectivity

Understanding the mechanism by which prion infectivity is encoded by the misfolded protein PrP^Sc remains a high priority within the prion field. Work from several groups has indicated cellular cofactors may be necessary to form infectious prions in vitro. The identity of endogenous prion conversion cofactors is currently unknown, but may include polyanions and/or lipid molecules. In a recent study, we manufactured infectious hamster prions containing purified PrP^Sc, co-purified lipid, and a synthetic photocleavable polyanion. The polyanion was incorporated into infectious PrP^Sc complexes, and then specifically degraded by exposure to ultraviolet light. Light-induced in situ degradation of the incorporated polyanion had no effect on the specific infectivity of the samples as determined by end-point dilution sPMCA and scrapie incubation time assays. Furthermore, prion strain properties were not changed by polyanion degradation, suggesting that intact polyanions are not required to maintain the infectious properties of hamster prions. Here, we review these results and discuss the potential roles cofactors might play in encoding prion infectivity and/or strain properties.     

Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle

A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24). To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP). The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 199.7 ± 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 ± 13.5 days). Further, C-BSE was transmitted to wild-type mice with the incubation period of about 409 days, whereas BSE/JP24 prion inoculated mice showed no clinical signs up to 649 days. Severe vacuolation and a widespread and uniform distribution of PrP^Sc were pathologically observed in the brain of BSE/JP24 prion affected TgBoPrP mice. The molecular weight and glycoform ratio of PrP^Sc in BSE/JP24 were different from those in C-BSE, and PrP^Sc in BSE/JP24 exhibited weaker proteinase K resistance than that in C-BSE. These findings revealed that the BSE/JP24 prion has distinct biological and biochemical properties reported for that of C-BSE. Interestingly, a shorter incubation period was observed at the subsequent passage of the BSE/JP24 prion to TgBoPrP mice (152.2 ± 3.1 days). This result implies that BSE/JP24 prion has newly emerged and showed the possibility that L-type BSE prion might be classified into multiple strains.Key words: prion, atypical BSE, PrP^Sc     

The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

Prions are unconventional infectious agents thought to be primarily composed of PrP^Sc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP^Sc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP^Sc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrP^Sc aggregates from PrP^C. The distribution of PrP^Sc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP^Sc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrP^Sc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.     

Evaluation of the Possible Transmission of BSE and Scrapie to Gilthead Sea Bream (Sparus aurata)

In transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders affecting many species, the key event in disease pathogenesis is the accumulation of an abnormal conformational isoform (PrP^Sc) of the host-encoded cellular prion protein (PrP^C). While the precise mechanism of the PrP^C to PrP^Sc conversion is not understood, it is clear that host PrP^C expression is a prerequisite for effective infectious prion propagation. Although there have been many studies on TSEs in mammalian species, little is known about TSE pathogenesis in fish. Here we show that while gilthead sea bream (Sparus aurata) orally challenged with brain homogenates prepared either from a BSE infected cow or from scrapie infected sheep developed no clinical prion disease, the brains of TSE-fed fish sampled two years after challenge did show signs of neurodegeneration and accumulation of deposits that reacted positively with antibodies raised against sea bream PrP. The control groups, fed with brains from uninfected animals, showed no such signs. Remarkably, the deposits developed much more rapidly and extensively in fish inoculated with BSE-infected material than in the ones challenged with the scrapie-infected brain homogenate, with numerous deposits being proteinase K-resistant. These plaque-like aggregates exhibited congophilia and birefringence in polarized light, consistent with an amyloid-like component. The neurodegeneration and abnormal deposition in the brains of fish challenged with prion, especially BSE, raises concerns about the potential risk to public health. As fish aquaculture is an economically important industry providing high protein nutrition for humans and other mammalian species, the prospect of farmed fish being contaminated with infectious mammalian PrP^Sc, or of a prion disease developing in farmed fish is alarming and requires further evaluation.     

Infectivity-associated PrPSc and disease duration-associated PrPSc of mouse BSE prions

ABSTRACT

Disease-related prion protein (PrP^Sc), which is a structural isoform of the host-encoded cellular prion protein, is thought to be a causative agent of transmissible spongiform encephalopathies. However, the specific role of PrP^Sc in prion pathogenesis and its relationship to infectivity remain controversial. A time-course study of prion-affected mice was conducted, which showed that the prion infectivity was not simply proportional to the amount of PrP^Sc in the brain. Centrifugation (20,000 ×g) of the brain homogenate showed that most of the PrP^Sc was precipitated into the pellet, and the supernatant contained only a slight amount of PrP^Sc. Interestingly, mice inoculated with the obtained supernatant showed incubation periods that were approximately 15 d longer than those of mice inoculated with the crude homogenate even though both inocula contained almost the same infectivity. Our results suggest that a small population of fine PrP^Sc may be responsible for prion infectivity and that large, aggregated PrP^Sc may contribute to determining prion disease duration.     

The N-Terminal Sequence of Prion Protein Consists an Epitope Specific to the Abnormal Isoform of Prion Protein (PrPSc)

The conformation of abnormal prion protein (PrP^Sc) differs from that of cellular prion protein (PrP^C), but the precise characteristics of PrP^Sc remain to be elucidated. To clarify the properties of native PrP^Sc, we attempted to generate novel PrP^Sc-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP^Sc purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP^Sc from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP^C from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31–39 and 41–47, respectively. This indicates that a PrP^Sc-specific epitope exists in the N-terminal region of PrP^Sc, and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP^Sc. We found that the ratio of proteinase K (PK)-sensitive PrP^Sc to PK-resistant PrP^Sc was constant throughout the disease time course.     

Photodegradation illuminates the role of polyanions in prion infectivity

Understanding the mechanism by which prion infectivity is encoded by the misfolded protein PrP^Sc remains a high priority within the prion field. Work from several groups has indicated cellular cofactors may be necessary to form infectious prions in vitro. The identity of endogenous prion conversion cofactors is currently unknown, but may include polyanions and/or lipid molecules. In a recent study, we manufactured infectious hamster prions containing purified PrP^Sc, co-purified lipid and a synthetic photocleavable polyanion. The polyanion was incorporated into infectious PrP^Sc complexes and then specifically degraded by exposure to ultraviolet light. Light-induced in situ degradation of the incorporated polyanion had no effect on the specific infectivity of the samples as determined by end-point dilution sPMCA and scrapie incubation time assays. Furthermore, prion strain properties were not changed by polyanion degradation, suggesting that intact polyanions are not required to maintain the infectious properties of hamster prions. Here, we review these results and discuss the potential roles cofactors might play in encoding prion infectivity and/or strain properties.Key words: prion, polyanion, photodegradation, incorporation, PrPThe prion diseases are infectious diseases that are believed to be caused by the conformational change of a host-encoded protein, PrP^C, to a pathogenic conformer PrP^Sc. The controversial “protein-only” hypothesis posits that the infectious agent is composed solely of the misfolded conformer PrP^Sc. There have been many attempts to create infectious prions from purified recombinant PrP protein. However, all of the samples generated in these experiments display relatively low levels of specific infectivity when inoculated intracerebrally into wild-type animals.^1–4 Several lines of evidence suggest that cellular cofactors, such as polyanionic molecules, facilitate the formation of the infectious conformation.^5–14The first in vitro PrP conversion assay used radiolabeled PrP^C substrate purified from mammalian cells mixed with a stoichiometric excess of unlabeled PrP^Sc. This cell free assay produced a protease-resistant, radioactive product termed PrP-res.¹⁵ These pioneering studies showed for the first time that PrP could be specifically transformed in vitro, but the yield using purified substrates was low. Using a modification of the cell free assay in which crude brain homogenate replaced purified PrP^C as the substrate, our laboratory was able to amplify PrP^Sc 6-fold over input prion seed, suggesting that non-PrP constituents of crude brain homogenate might be required for efficient PrP^Sc formation in vitro.¹⁶ Using this system, we discovered that nuclease treatment of hamster brain homogenates abolished PrP^Sc amplification in vitro, and that reconstituting the nuclease-treated reactions with purified mammalian RNA rescued the amplification process.⁵ PrP^Sc amplification could also be obtained by adding certain synthetic homopolymeric nucleic acids to immunopurified PrP^C.⁶ Taken together, these surprising results argue that non-proteinaceous, host-encoded cofactors such as RNA molecules might facilitate prion conversion through a structural (as opposed to encoding) mechanism.⁸ The high efficiency of the serial protein misfolding amplification (sPMCA) technique developed by Soto and colleagues has allowed researchers to amplify prion infectivity as well as PrP^Sc molecules.^17,18 Using sPMCA, we showed that infectious PrP^Sc molecules could be formed from immunopurified PrP^C, co-purified lipid and synthetic RNA molecules. Moreover, even unseeded reactions containing these defined components were capable of generating prions with high specific infectivity in a prion-free environment, showing for the first time that wild type infectious prions could be produced de novo.⁷Additional studies in this purified system showed that PrP^C molecules undergo a time-dependent conformational change upon interaction with RNA. When this change occurs, PrP^C adopts an intermediate conformation that mimics some of the characteristics of PrP^Sc, such as detergent insolubility and reactivity to PrP^Sc-specific antibodies, but remains sensitive to proteinase K digestion.⁸ When incubated with a heterogeneous size mixture of homopolymeric [³²P] poly(A) molecules during PMCA, hamster PrP^C molecules incorporated a specific size subset (1–2.5 kb) of the RNA molecules into nuclease-resistant complexes. The physical interaction between RNA and PrP^Sc was confirmed by fluorescence microscopy experiments showing that fluorescein-labeled RNA molecules became integrated into nuclease-resistant complexes with PrP^Sc molecules. Interestingly, neuropathologic analysis of scrapie-infected hamsters revealed that endogenous RNA molecules stained with acridine orange co-localized with large extracellular PrP aggregates.⁸ Taken together, these studies suggest that PrP interacts specifically with polyanionic molecules in vitro and in situ, and raised the possibility that polyanions might be a necessary component of infectious prions.Jeong et al. investigated whether endogenous RNA molecules might be required for prion infectivity by treating scrapie brain homogenates with LiAlH4 (lithium aluminum hydride), a strong reducing agent that can cleave the phosphodiester bond in RNA molecules.¹⁹ Interestingly, treatment of hamster scrapie brain homogenates with LiAlH₄ caused an ∼3-fold increase in scrapie incubation period measured by bioassay, suggesting that RNA may be an important component of infectious prions and therefore may play a role in stabilizing PrP^Sc structure. However, LiAlH₄ is not a specific reagent, and can damage a variety of other macromolecules, including proteins. Therefore, the decrease in infectivity measured in this study cannot be specifically ascribed to degradation of the polyanion.¹⁹We recently reinvestigated the potential role of polyanion in maintaining prion infectivity by using a more targeted approach.²⁰ Specifically, we utilized a synthetic oligonucleotide that could be selectively hydrolyzed by treatment with ultraviolet (UV) light. The photocleavable oligonucleotide was synthesized by inserting a photocleavable linker in between every fives bases of a poly(dT) 100-mer. Exposure to UV light quantitatively converted the oligonucleotide into five base fragments. During incubation with excess recombinant PrP, the photocleavable oligonucleotide became incorporated into a nuclease-resistant nucleoprotein complex, but remained sensitive to photocleavage. This novel system allowed us to study the role of a polyanion molecule incorporated into infectious prions in situ (Fig. 1).Open in a separate windowFigure 1Selective photodegradation of an incorporated polyanion in situ.We used PMCA to create PrP^Sc molecules that contained either the photocleavable oligonucleotide or a non-photocleavable control analog. After treatment with UV light, the infectivity of each sample was measured using a combination of end-point dilution sPMCA and animal bioassays. The end-point dilution PMCA assay showed a ∼1 log decrease in the seeding ability of PrP^Sc samples treated with UV light, but this effect was not specific since a similar decrease was measured in samples containing the control nucleic acid. In the bioassay, there was no change in the incubation periods of animals inoculated with PrP^Sc samples treated either in the presence or absence of UV light. Neuropathological analysis of inoculated animals also showed no differences in neurotropism between the two groups. Degradation of the nucleic acid had no effect on the molecular migration or structural stability of PrP^Sc samples as determined by SDS-PAGE and urea denaturation assays, respectively. There were also no differences in the molecular migration or glycosylation profile of the PrP^Sc molecules produced in the brains of animals inoculated with light- versus mock-treated inocula, and urea denaturation assays showed no differences in PrP^Sc stability. These results collectively demonstrate that the presence of intact polyanion molecules is not required to maintain the infectious, biochemical or strain properties prions generated in vitro.These results are consistent with the stringent “protein-only” hypothesis, but do not yet provide definitive proof. The purified PrP^C molecules used as substrate in these experiments contain a stoichiometric amount of co-purified lipid⁷ that may play a role in the generation of prion infectivity.⁹ Also, although the efficacy of photocleavage conditions was carefully confirmed in control reactions, it is possible that some intact oligonucleotide survived UV treatment at a level below detection. Alternatively, the remnant five base nucleic acid fragments may remain incorporated within the PrP^Sc molecule and play a role in maintaining the infectious conformation. Even in this scenario, our results would place a significant geometric constraint on the role of incorporated polyanion. While polyanions ≥40 bases facilitate the formation infectious prions in vitro,⁸ our results suggest that polyanions >5 bases are not necessary to maintain the infectious properties the prion. The exact role polyanions play in prion formation is still unclear, but it is tempting to speculate that they may serve as scaffolds that facilitate prion conversion by (a) bringing PrP^C and PrP^Sc seed together for templating to occur or (b) acting as a catalyst which is necessary to reduce the activation energy of refolding to the PrP^Sc form. Future studies will need to be performed to differentiate between these two hypotheses. It is also possible that polyanions are completely dispensable for maintaining PrP^Sc structure, and it is the co-purified lipid molecules that serve this role instead. Consistent with this possibility, we recently discovered that mouse PrP^Sc can be serially propagated in vitro in the absence of nucleic acids.²¹ Finally, it is possible that either polyanions or lipids can function equally well as stabilizers of the infectious PrP^Sc conformation. More work is required to distinguish between these possibilities.Generating high levels of specific infectivity solely using purified recombinant PrP remains the ultimate proof of the “protein-only” hypothesis. To date, evidence suggests that cellular cofactors are necessary to create infectious prions but may or may not be required to maintain infectivity once formed. Significantly, Wang et al. showed that bona fide prions could be formed from recombinant PrP, synthetic lipid and RNA molecules.⁹ Although no completely pure preparations of misfolded PrP possessing significant levels of specific infectivity have yet been produced, it should eventually be possible to produce such a preparation if the “protein-only” hypothesis is correct. On the other hand, a rigorous refutation of the hypothesis would require demonstrating that PrP^Sc and infectivity can be dissociated.     

Protease-Sensitive Synthetic Prions

Prions arise when the cellular prion protein (PrP^C) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP^Sc. Frequently, PrP^Sc is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164), denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174) did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP^Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600–750 days in Tg4053 mice, which exhibited sPrP^Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP^Sc.     

Lack of Prion Infectivity in Fixed Heart Tissue from Patients with Creutzfeldt-Jakob Disease or Amyloid Heart Disease

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP^Sc) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP^Sc in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP^Sc. Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.     

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions,but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (Prnp^0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp^0/0 mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp^0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp^0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice, compared with RML- and 22L-inoculated Prnp^0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the trans-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.     

Prion Seeding Activities of Mouse Scrapie Strains with Divergent PrPSc Protease Sensitivities and Amyloid Plaque Content Using RT-QuIC and eQuIC

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrP^Sc) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrP^Sc propagation involves conversion from its normal isoform, PrP^C, by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrP^Sen) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrP^Sc (PrP^Res). Scrapie brain dilutions up to 10⁻⁸ and 10⁻¹³ were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrP^Res levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrP^C. Although the brains of 263K-affected mice had little immunoblot-detectable PrP^Res, RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrP^Res strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrP^Res levels. We also found that eQuIC, which incorporates a PrP^Sc immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrP^Sc.     

Evidence That Bank Vole PrP Is a Universal Acceptor for Prions

Bank voles are uniquely susceptible to a wide range of prion strains isolated from many different species. To determine if this enhanced susceptibility to interspecies prion transmission is encoded within the sequence of the bank vole prion protein (BVPrP), we inoculated Tg(M109) and Tg(I109) mice, which express BVPrP containing either methionine or isoleucine at polymorphic codon 109, with 16 prion isolates from 8 different species: humans, cattle, elk, sheep, guinea pigs, hamsters, mice, and meadow voles. Efficient disease transmission was observed in both Tg(M109) and Tg(I109) mice. For instance, inoculation of the most common human prion strain, sporadic Creutzfeldt-Jakob disease (sCJD) subtype MM1, into Tg(M109) mice gave incubation periods of ∼200 days that were shortened slightly on second passage. Chronic wasting disease prions exhibited an incubation time of ∼250 days, which shortened to ∼150 days upon second passage in Tg(M109) mice. Unexpectedly, bovine spongiform encephalopathy and variant CJD prions caused rapid neurological dysfunction in Tg(M109) mice upon second passage, with incubation periods of 64 and 40 days, respectively. Despite the rapid incubation periods, other strain-specified properties of many prion isolates—including the size of proteinase K–resistant PrP^Sc, the pattern of cerebral PrP^Sc deposition, and the conformational stability—were remarkably conserved upon serial passage in Tg(M109) mice. Our results demonstrate that expression of BVPrP is sufficient to engender enhanced susceptibility to a diverse range of prion isolates, suggesting that BVPrP may be a universal acceptor for prions.     

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

During prion infection, the normal, protease-sensitive conformation of prion protein (PrP^C) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP^Sc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP^Sc in prion-infected brain homogenates as an initiating seed to convert PrP^C and trigger the self-propagation of PrP^Sc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP^Sc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP^Sc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP^Sc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.     

Transmissibility of H-Type Bovine Spongiform Encephalopathy to Hamster PrP Transgenic Mice

Two distinct forms of atypical bovine spongiform encephalopathies (H-BSE and L-BSE) can be distinguished from classical (C-) BSE found in cattle based on biochemical signatures of disease-associated prion protein (PrP^Sc). H-BSE is transmissible to wild-type mice—with infected mice showing a long survival period that is close to their normal lifespan—but not to hamsters. Therefore, rodent-adapted H-BSE with a short survival period would be useful for analyzing H-BSE characteristics. In this study, we investigated the transmissibility of H-BSE to hamster prion protein transgenic (TgHaNSE) mice with long survival periods. Although none of the TgHaNSE mice manifested the disease during their lifespan, PrP^Sc accumulation was observed in some areas of the brain after the first passage. With subsequent passages, TgHaNSE mice developed the disease with a mean survival period of 220 days. The molecular characteristics of proteinase K-resistant PrP^Sc (PrP^res) in the brain were identical to those observed in first-passage mice. The distribution of immunolabeled PrP^Sc in the brains of TgHaNSE mice differed between those infected with H-BSE as compared to C-BSE or L-BSE, and the molecular properties of PrP^res in TgHaNSE mice infected with H-BSE differed from those of the original isolate. The strain-specific electromobility, glycoform profiles, and proteolytic cleavage sites of H-BSE in TgHaNSE mice were indistinguishable from those of C-BSE, in which the diglycosylated form was predominant. These findings indicate that strain-specific pathogenic characteristics and molecular features of PrP^res in the brain are altered during cross-species transmission. Typical H-BSE features were restored after back passage from TgHaNSE to bovinized transgenic mice, indicating that the H-BSE strain was propagated in TgHaNSE mice. This could result from the overexpression of the hamster prion protein.     

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrP^Sc. A critical prediction of the protein-only hypothesis is that autocatalytic PrP^Sc molecules should be infectious. However, some autocatalytic recombinant PrP^Sc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >10⁵-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrP^C substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrP^C. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrP^C molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrP^Sc structure.     

Characterization of Syrian hamster adapted prions derived from L-type and C-type bovine spongiform encephalopathies

Atypical forms of bovine spongiform encephalopathy (BSE) may be caused by different prions from classical BSE (C-BSE). In this study, we examined the susceptibility of mice overexpressing mouse and hamster chimeric prion protein (PrP) to L-type atypical BSE (L-BSE). None of the transgenic mice showed susceptibility to L-BSE, except mice overexpressing hamster PrP. We also examined the transmission properties of L-BSE in hamsters. The incubation period of hamsters intracerebrally inoculated with L-BSE was 576.8 days, and that of the subsequent passage was decreased to 208 days. Although the lesion and glycoform profiles and relative proteinase K resistant core fragment of the abnormal isoform of PrP (PrPcore) of L-BSE were similar to that of C-BSE, the deposition of the abnormal isoform of PrP (PrP^Sc) and the molecular weight of PrPcore of L-BSE was different from than that of C-BSE. In hamster models, some prion strain characteristics of L-BSE were indistinguishable from those of C-BSE.Key words: prion, atypical, L-BSE, PrPcore, hamster, transmission     

Generation of antisera to purified prions in lipid rafts

Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrP^C into alternately folded PrP^Sc. Immunoassays toward pre-clinical detection of infectious PrP^Sc have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrP^Sc selective antibodies. We report a method to purify infectious PrP^Sc from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrP^Sc was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM >20-fold and purified DRM immunogen >40-fold) relative to 1% crude brain homogenate. Purification of PrP^Sc from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrP^Sc antigen was performed using wild-type (wt) and Prnp^0/0 mice, both on Balb/cJ background. A robust immune response against PrP^Sc was observed in all inoculated Prnp^0/0 mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrP^C and PrP^Sc, while binding to other brain-derived protein was not observed. In contrast, the PrP^Sc inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.Key words: prion, scrapie, Prnp0/0 mice, purification methodology, antibody, antisera, lipid-rafts, detergent resistant membranes, neuroscience, immunization, diagnostic     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP^Sc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP^res isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP^Sc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.