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Title: De Novo Generation of Infectious Prions In Vitro Produces a New Disease Phenotype}
Article:
Abstract:
Prions are the proteinaceous infectious agents responsible for Transmissible Spongiform Encephalopathies. Compelling evidence supports the hypothesis that prions are composed exclusively of a misfolded version of the prion protein( PrPSc) that replicates in the body in the absence of nucleic acids by inducing the misfolding of the cellular prion protein( PrPC). The most common form of human prion disease is sporadic, which appears to have its origin in a low frequency event of spontaneous misfolding to generate the first PrPSc particle that then propagates as in the infectious form of the disease. The main goal of this study was to mimic an early event in the etiology of sporadic disease by attempting de novo generation of infectious PrPSc in vitro. For this purpose we analyzed in detail the possibility of spontaneous generation of PrPSc by the protein misfolding cyclic amplification( PMCA) procedure. Under standard PMCA conditions, and taking precautions to avoid cross-contamination, de novo generation of PrPSc was never observed, supporting the use of the technology for diagnostic applications. However, we report that PMCA can be modified to generate PrPSc in the absence of pre-existing PrPSc in different animal species at a low and variable rate. De novo generated PrPSc was infectious when inoculated into wild type hamsters, producing a new disease phenotype with unique clinical, neuropathological and biochemical features. Our results represent additional evidence in support of the prion hypothesis and provide a simple model to study the mechanism of sporadic prion disease. The findings also suggest that prion diversity is not restricted to those currently known, and that likely new forms of infectious protein foldings may be produced, resulting in novel disease phenotypes.
Introduction:
Prions are the proteinaceous infectious agents responsible for Transmissible Spongiform Encephalopathies( TSEs), a group of fatal neurodegenerative disorders, including Creutzfeldt-Jakob disease( CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle and chronic wasting disease in deer[1]. Compelling evidence support the hypothesis that the infectious agent is composed exclusively by a misfolded version of the prion protein( PrPSc) that replicates in the body in the absence of nucleic acids by inducing the misfolding of the cellular prion protein( PrPC)[2]. Recent studies have reported in vitro generation of infectious material by inducing or amplifying PrP misfolding, providing strong support for the prion hypothesis[3]–[5]. In addition of the infectious origin, TSEs can be inherited or appear sporadically. The latter is the most common origin in humans, in which there are no known infectious source and no evidence of the disease in the prior or subsequent generations of the patient's family. Sporadic CJD occurs worldwide with an approximate frequency of 1 new case per million people each year[1]. Molecularly, it is thought that sporadic TSEs arise from the low frequency, spontaneous misfolding of the prion protein which then propagates to other PrPC molecules in a manner similar as in the infectious cases. Based on the knowledge of the molecular mechanism of prion conversion, we have developed the PMCA( Protein Misfolding Cyclic Amplification) technology, designed to mimic PrPSc autocatalytic replication in vitro[6]. In a cyclic manner, minute quantities of PrPSc( as little as one single particle) induce misfolding of large amounts of PrPC in a process catalyzed by ultrasound waves to multiply the number of converting units. We and others demonstrated that PrPSc can be maintained replicating in vitro indefinitively[3],[7]. More importantly, inoculation of in vitro generated PrPSc produced disease in wild type animals[3]. The newly generated protein exhibits the same biochemical, biological, and structural properties as brain-derived PrPSc and maintains the species-barrier and strain-specific characteristics[8]–[10]. These findings represent one of the strongest evidences in favor of the prion hypothesis. However, since infectious material was generated using crude brain homogenates as substrate and starting from small amounts of brain derived infectivity, we cannot absolutely rule out that another agent besides of PrPSc was replicated during the PMCA process. In this sense a great advance was reported by Supattapone and co-workers with the in vitro generation of PrPSc and infectivity by PMCA using purified PrPC and PrPSc with the sole addition of a synthetic polyanion[4]. Moreover, these authors described that misfolded and infectious protein was generated de novo, in hamsters under conditions in which the possibility of cross-contamination was greatly reduced. The result was a hamster strain very similar to the commonly used 263K hamster strain[4]. Spontaneous generation of PrPSc and infectivity was also described in an experiment in which a recombinant mouse PrP fragment( residues 89–230) assembled into amyloid fibrils was found to induce a TSE-like disease with PrPSc formation when injected in transgenic mice overexpressing the same PrP sequence[5]. Although these findings are very interesting, the fact that the disease was transmitted in a first passage only to transgenic animals largely over-expressing the PrP gene and not to wild-type animals is a matter of concern. It is well known that this type of animals develop spontaneously a prion-like disease at advanced age[11]–[13]. In other words, the effect seen might just be an acceleration of the disease process that was set to occur spontaneously at a later time[14]. The main goal of the current study was to identify conditions which permit the de novo generation of PrP and infectivity using PMCA in brain homogenates. Our hypothesis was that if sporadic prion disease originates from a low frequency process of spontaneous PrPC misfolding, we should be able to pick up and propagate this event using PMCA. However, in our experience thousands PMCA experiments have only rarely shown PrPSc formation in control samples without infectious material and in these extremely uncommon cases we could not rule out the possibility of cross-contamination.
Discussion:
In this study we report spontaneous generation of PrPSc in vitro using a modified PMCA procedure with brain homogenate substrate. Misfolded protein was produced in two different animal species without the need to add seeds of in vivo generated PrPSc. At present it is unclear whether PMCA replicated a PrPSc intermediate already present in the brain homogenate or rather “spontaneous” misfolding was induced by the amplification cycles. In this context, a recent study reported the presence of small quantities of a PrPSc-like protein in healthy brains[20], but it is yet unknown whether this protein is infectious. Although de novo prion formation was achieved before by PMCA using purified proteins in the presence of synthetic polyanions[4], the purpose of our study was to evaluate whether de novo generation of prions is possible under standard PMCA conditions. This is important to assess the validity of using PMCA for prion diagnosis. Another aim was to study whether different species of animals have a distinct propensity to form spontaneous prions under a given set of conditions. Finally, we aimed to characterize the newly generated infectivity and to show that the diversity of prions is perhaps larger than we currently think. Our results indicate that standard PMCA rounds consisting of 144 cycles( or less) did not show spontaneous formation of PrPSc, but longer rounds of 240 cycles were successful in replicating or generating misfolded protein. These findings suggest that de novo formation of PrPSc can be experimentally distinguished from replication of pre-formed PrPSc, indicating that the biochemical, conformational or stability properties of the PrP structures involved in both processes are probably different. Therefore, PMCA can be adapted to produce de novo generated PrPSc and it is likely that more drastic modifications of the procedure may lead to higher rates of spontaneous prion formation. Indeed, the higher rate of de novo PrPSc formation reported in the study by Deleault and colleagues, who used only 48 PMCA cycles per round[4], indicates that using polyanions and purified PrPC may favor spontaneous prion formation, suggesting that some factors in brain homogenate may prevent PrP misfolding. Our data support the use of PMCA,( under conditions in which PrPSc de novo generation does not occur), for biochemical detection of prions and its potential application for TSE diagnosis. The usefulness of PMCA for highly sensitive and specific detection of prions in biological fluids, has been demonstrated by studies from us and other groups[21]–[25]. Strikingly, the rate and number of PMCA cycles required to produce de novo PrPSc was variable in distinct species. Considering that from the species studied humans are the only one in which sporadic disease is known to occur, we were surprised that de novo PrPSc was never obtained with human brain homogenates under the experimental conditions used. This contrast with the high levels of amplification of human PrPSc observed in samples seeded with vCJD( Fig. S1) or various sCJD brain homogenates( data not shown). The absence of spontaneous formation of PrPres in humans may be due to the different conditions for the preparation of the tissue, including several hours of post-mortem delay or the lack of perfusion before collection of the brain. However, this is unlikely, because similar results were obtained using brains of transgenic mice expressing human PrP subjected to the same conditions to prepare brain than the other rodent species. Our interpretation of these results is that human PrPC has a lower propensity to initiate misfolding than the rodent protein. The alternative explanation that factors present in the human brain may prevent conversion is unlikely considering the experiments with humanized transgenic mice. The appearance of sporadic disease in humans may simply reflect the longer life span that provides greater chances for stochastic processes of spontaneous misfolding. However, it is also possible that sporadic disease is actually more frequent in animals, because the rate of spontaneous illness in animals has not been systematically studied. Several lines of evidence enable us to rule out the possibility that de novo PrPSc was the result of cross-contamination. First of all, conventional PMCA that allows us to amplify as little as a single particle of PrPSc did not show spontaneous formation of misfolded protein, even after extensive PMCA cycling( Fig. 1). Second, the rate of spontaneous generation of prions in different species does not correlate with the availability of infectious material in our lab. Moreover, the variable rate was surprisingly constant among different experiments, arguing that it is dependent on some intrinsic properties of the protein rather than a stochastic event as cross-contamination. Third, an experiment done using strictly prion-free equipment and reagents, performed in a new laboratory that was never exposed to prions and done by a person who has never been in contact with prion material showed very similar results as the one carried out in our standard facilities. Finally, the biochemical, structural and biological properties of de novo generated PrPSc in hamster were substantially different to those of various conventional hamster prion strains. Although all these evidences strongly indicate that the results are not due to cross-contamination, this possibility cannot entirely be ruled out. Inoculation of wild type hamsters with de novo generated PrPSc produced disease in all animals. Strikingly, many of the disease characteristics were substantially different to those observed with several other hamster prion strains. In this study we directly compared PGP-h1 with 3 well-established strains( 263K, HY and DY) and literature comparisons with other strains such as Sc237, SHa( Me7), MT-C5, SHa( RML), 139H and Me7-H, also show substantial differences[26],[27]. It is important to note that PMCA amplification of PrPSc from various strains( of hamsters, mice, human or deer origin) faithfully propagates the strain characteristics[8]–[10],[28],[29]. Thus, the differences observed in the de novo generated material are not attributable to PMCA amplification, but to intrinsic differences of this new prion strain. In our direct comparisons the incubation time of PGP-h1 was significantly larger than 263K and HY strains, but much shorter than DY. Clinical signs were also clearly distinguishable from those observed in animals affected by other strains( see Video S1 and Video S2 in supporting online material). The animals inoculated with PGP-h1 were not hyperactive or aggressive, but not lethargic either. They exhibited a social withdrawal and lack of interest for the surroundings, which reflected in a much reduced horizontal and vertical activity and increased extent of inactive time in an open field test. The clinical differences on the PGP-h1 induced disease were likely the result of the severe brain damage produced, including extensive spongiform degeneration, PrPSc accumulation and brain inflammation. Remarkably, the brain areas targeted by vacuolation damage were significantly different from all other hamster strains studied and included a substantially greater extent of spongiosis in colliculus and a much reduced level of injury in cerebellum and cortex. The biochemical characteristics of PrPSc were also different in PGP-h1, mostly in terms of the higher quantity of PrPSc accumulated in the brain and the lower relative resistance to proteolytic degradation than the other hamster strains analyzed. Taken together this data demonstrate that de novo generated prions correspond to a novel strain of infectious material, able to generate a new disease in wild type animals. We are currently assessing the infectious properties of some of the other de novo generated PrPSc obtained in this study to examine whether or not new strains and new diseases are produced also in other species. Our findings provide a model to study the possible origins of sporadic TSEs and a new avenue to investigate the mechanism and factors controlling spontaneous formation of infectious material. For example, using the modified PMCA reaction described in this study we could assess the sequence determinants of the variable propensity of PrPC in distinct species to undergo conversion into the pathological form. We could also study the contribution of other factors in brain to alter the rate of de novo generation of PrPSc. Finally, our data provides further support for the prion hypothesis, since misfolded and infectious protein was generated in vitro, without the need for addition of pre-existing PrPSc. The fact that the de novo generated PrPSc corresponds to a new strain of infectious material suggest that the diversity of alternative and transmissible foldings that PrPSc can adopt is much larger than usually thought. This is worrisome, because it raises the possibility that novel and perhaps more aggressive infectious prion foldings may spontaneously originate in diverse species, leading to the emergence of new and unpredictable forms of transmissible diseases.
Prions are the unprecedented infectious agent associated with prion diseases , which are composed exclusively of a misfolded protein . In this study we report the de novo formation of infectious prion protein in vitro , which when inoculated into wild type animals is able to induce a new disease phenotype with unique clinical , neuropathological and biochemical characteristics . The findings described in this article are very important , because they support the prion hypothesis and provide a simple model for studying the mechanism responsible for the appearance of sporadic prion disease . The data also suggest that the universe of possible prions is not restricted to those currently known , but that likely many new forms of infectious protein foldings may be produced . This is worrisome , because it raises the possibility that novel and perhaps more aggressive infectious prion foldings may spontaneously originate in diverse species , leading to the emergence of new and unpredictable forms of transmissible diseases .