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Protein Misfolding Cyclic Amplification (PMCA) has proved to be an efficient method mimicking in vitro some of the fundamental steps involved in prion replication in vivo. Thus, it can be used to efficiently replicate a variety of prion strains/species. The in vitro generated prions possess key prion features, i.e., they are infectious in vivo and maintain their strain specificity. One of the big challenges is its use for studying prion transmission barriers. PMCA has been efficiently used for adapting different prion species through a range of species barriers; however its capacity for overcoming purportedly unbreakable species barriers compels us to adapt it in order to use it as a reliable technique. In addition, this in vitro method might be a crucial tool in evaluating the potential risks of different prion strains (natural or experimentally generated in vitro) to humans and animals.
In 1994, Byron Caughey's group published for the first time the cell-free formation of protease-resistant prion protein.1 Seven years later, a similar in vitro phenomenon was carried out in a more efficient manner by Protein Misfolding Cyclic Amplification (PMCA).2 And just a few years later, two crucial studies trying to prove the reliability of the in vitro prion replication positioned the PMCA as one of the most useful techniques in the prion field. Thus (1) the infectivity was amplified in vitro. These studies were done in hamsters,3 mice,4 voles and other wild rodents (Castilla J, personal communication in Symposium on The New Prion Biology Basic Science, Diagnosis and Therapy, Venice 2008), deer,5 sheep and humans.4 In all cases, infectivity was replicated and it correlated with the level of PMCA-amplified PrPSc, (2) PMCA mimicked the prion strain phenomenon. Prions occur in the form of different strains that show distinct biological and physicochemical properties, even though they are encoded by PrP with the same amino acid sequence, most likely with different conformations. In vitro replication presumable mimics the same conformations or strains as in animals in vivo. These studies were done in several mouse strains (301C, 79A, 139A, Me7, RML), human strains4 and deer strains.5 PMCA showed to be a very useful technique for studying two of the most important phenomena in the prion field.
The performance of PMCA related to the transmission barriers was studied for the first time when mouse prions were subjected to serial heterologous PMCA rounds in order to be converted in hamster prions.6 The new in vitro generated hamster prion showed similar but not identical characteristics to other hamster prions (263K, HY and DY). A new hamster strain was created in the same way that new prion strains are generated when a heterologous infection occurs in vivo.6 Again, PMCA showed a promising performance when compared to in vivo studies. Another study related to the transmissibility of prions through different species was accomplished satisfactorily when the prion adaptation phenomenon was mimicked by serial rounds of PMCA. In 1998, mouse and hamster prions where used by Chesebro's group to evaluate how the prion phenomenon of adaptation took place. While these studies took several years for its complete accomplishment,7 just a few weeks through several rounds of PMCA were necessary to show in vitro the same adaptation phenomenon.6
After these results, can we use the in vitro prion replication as a faithful technique for evaluating the transmission barrier? Or, in other words, can we extrapolate the in vitro results studying species and polymorphism barriers to venture risk assessments of certain prion species/strains in humans or animals? In order to answer this question, many other in vitro experiments are being carried out. Although most of them are ongoing, partial results might provide us with an idea about the value of PMCA for evaluating the transmission barriers. Thus, a recently published result shows how RML, a mouse prion strain, generates in vitro and in vivo the same new deer prion strain that when mule deer substrate or deer PrP transgenic mice were used, respectively.5 This data confirmed that the in vitro replication may be useful to predict in a much shorter period of time the features of new prion strains that could be generated in nature. The advantage of this method is the possibility of using a high number of different prion strains and overall different polymorphic versions of PrPs, which is difficult to be done in vivo. In the same way, ongoing experiments trying to find an alternative to the standard strain typing studies for suspect BSE infected samples have shown undistinguishable in vitro and in vivo data when BSE in sheep and classical BSE in vivo infected or in vitro replicated mouse samples were used (J. Castilla, personal communication in Symposium on The New Prion Biology Basic Science, Diagnosis and Therapy, Venice 2008). This result predicts that PMCA will be a good alternative to determine in a more efficient manner and more quickly if the infectious samples have a BSE origin.
It is well known that the PrP polymorphisms determine the transmission barrier. There are many examples in ovine where a few amino acids substitutions are implicated in the in vivo susceptibility to different prion strains.8–11 Some of these studies have been confirmed by using transgenic mice which express different polymorphic variants of sheep PrP.12 Again, PMCA is demonstrating its value corroborating these in vivo results and expanding them using a much larger number of polymorphisms and inocula. The most typical case is the ARR/ARR PrP polymorphism in sheep that in vivo shows a high or complete resistance to classical scrapie. These results have been suitably confirmed in vitro proving the reliability of the method (Castilla J, personal communication in Symposium on The New Prion Biology Basic Science, Diagnosis and Therapy, Venice 2008). However, while these studies “black or white” can be easily evaluated in vitro, the ease by which PMCA crosses some transmission barriers might turn a disadvantage for studying other weaker PrP amino acid substitutions. We know that some resistant species barriers can be overcome by serial passages in vivo and this is probably because, although inefficiently, a heterologous conversion process is taking place. This process could become more efficient facilitating its progression by increasing the time through serial passages. In other words, resistant species barriers can also be broken in vivo although harder and much longer than in vitro. In addition, also in vivo, resistant barriers can be crossed by using overexpressing PrP transgenic mice. In this case, the excess of PrP facilitates the replication and the overcoming of the barrier. Thus, transmissibility studies of CWD in mouse succeeded when only Tga20, a mouse PrP overexpressing transgenic mice versus wild type mice were used.13 Another example is the assessment of the species barrier between BSE and humans which, depending on the biological tools that are being used, have dissimilar results and so are their interpretations. Thus, BSE would be infectious in human if overexpressing human PrP transgenic mice are used14 while BSE would be poorly infectious or not infectious at all in humans if knock in transgenic mice expressing a natural amount of human PrP are used.15 How PMCA can help in the inconsistency of these results? PMCA can offer both divergent results: (1) by forcing the in vitro process using serial rounds of PMCA, we have generated human prions starting with BSE inocula and (2) a comparative study using BSE, different human prion strains and classical scrapie in just one round of PMCA has shown that BSE replicates poorly in human substrates (data not shown).
One of the best values of PMCA is the possibility to compare and finally to semi-quantify the strength of the species/polymorphism barriers. However, the in vitro process should be done in a controlled manner since ultimately PMCA might convert any mammalian PrP through the seeding with any kind of prion inocula if enough rounds during the in vitro process are performed. There are two basic ways to control the in vitro prion replication: (1) the number of cycles in a round and (2) the number of rounds. Our results show that no more than one round should be applied to obtain an accurate comparison of transmission barrier strength. The number of cycles is an empirical data that should be obtained by comparison with our own standard. In order to establish the reliability of the method, we wanted to evaluate two different species barriers. The first species barrier corresponds to BSE in mouse versus BSE in bank vole. In both cases, just one round of standard PMCA16,17 was applied. As it is shown in Figure 1A, BSE was able to produce a little amount of new mouse prion material at certain dilution while no amplification at all was obtained when bank vole was used as substrate. The second species barrier studied corresponds to CWD through the same species that have been described above. In this case, while mouse substrate was not able to replicate CWD at all, bank vole substrate showed an efficient replication at different dilutions (Fig. 1B). All these results correlate perfectly with the in vivo results.14 However, in spite of these data, the question about BSE being infectious in bank vole or CWD being infectious in mouse does not have a simple answer. Thus, using the same substrates, bank voles or mice, and using the same prion inocula, BSE and CWD, the answer from in vitro data could be yes if we force the in vitro system by applying a series of rounds of PMCA. Likewise, if we force the in vivo system by doing serial passages of BSE in vole or by using overexpressing mouse PrP transgenic mice (Tga20),14 in both cases a new vole prion can be generated starting with BSE inoculum or a new mouse prion can be generated starting with CWD inoculum. Which method is more reliable to make a risk assessment is still unclear. Fortunately, new tools are emerging to evaluate more precisely the strength of the transmission barrier in prions. These more accurate and sensitive methods will be based on cell-PMCA with specific labels. The testing of these new methods with the most common prions will be enormously useful to make risk assessment particularly for humans. We will be able to quickly answer questions like if atypical BSE or atypical scrapies constitute a risk in humans when these prions enter the food chain directly or through previous in vivo passages in certain sheep or goat PrP polymorphisms.
The searching for a suitable dominant negative PrP which shows a complete blocking effect over the prion replication could be now simpler using PMCA. It is known that certain amino acids are key in the prion replication process but unfortunately we cannot foretell which of them are more relevant simply by comparing PrP amino acid sequences from different species in relation to their relative susceptibility to a diversity of prions. The principal problem to do predictions is that, until now, we had to base our knowledge on in vivo experiments. Thus, we should conclude that rabbit PrP and dog PrP for example had to contain some amino acids in charge of the blockade of prion propagation. Now, according to our preliminary results, we know that this assumption is partially wrong. First of all because the apparent resistance of rabbit might be mediated by other factors since rabbit PrP is a good substrate for being converted by many different prion strains/species (J. Castilla, personal communication in Symposium on The New Prion Biology Basic Science, Diagnosis and Therapy, Venice 2008). In conclusion, the rabbit PrP amino acid sequence and its alleged prion resistance should not be used for designing good dominant negative candidates. In contrast, PMCA is giving valuable information about how efficiently some PrP sequences, natural or experimentally generated, replicate prions. This information is being used by cell-PMCA to generate a large number of substrates containing PrPs which are being strategically designed to carry out critical amino acid substitutions. The inhibitory effects of the new cell-based substrates are being tested in vitro. Our preliminary results show that some cell substrates containing modified PrP with altered amino acids show strong inhibitory effects of the prion replication in vitro. The advantage of these in vitro studies is that these data are based directly on the inhibition of the prion replication more than just infectivity.
Prions have demonstrated to be able to spread naturally or experimentally through a large number of different mammals. Experimental studies have shown that certain prion strains are able to replicate in different species without changing their principal strain features while other may suffer an irreversible change of strain. It is not clear who, strain, host or both should be assigned such a peculiar behavior that in some cases implies a change in the virulence of the newly generated prion strain. Thus, while CWD is not infectious in hamsters18 the same CWD inoculum infects efficiently ferrets and the CWD-ferret is now able to infect hamsters efficiently.19 In a similar way, BSE turns more virulent when is passaged through sheep.20 PMCA is an extraordinary technique to evaluate in a semi- quantitative way the risk of those prions which might change its infectivity characteristics as a consequence of passages in certain hosts.
Before 1996 nobody could predict the spreading of a new prion disease in such a dramatic way. At that time, cattle should be considered a prion free species as many other species nearby to human are currently considered. The absence of natural TSE cases and/or failed experimental transmissions has suggested that certain species could be purportedly resistant to prion diseases. Some of them like rabbits, horses and dogs still have the label of “prion resistant” species. In the case of rabbits, all attempts focused on obtaining rabbit prions through in vivo inoculation of different prion species have been unsuccessful.21,22 Horses, in spite of no reported data against or in favor of their prion susceptibility are still considered a prion free specie and they seem to be in a situation similar to cattle 15 years ago. And dogs, for which few unpublished and incomplete challenges have been reported, are also maintaining the “prion resistant” label because, as in the other two species, there are no natural cases of prion diseases. Is this enough to consider these species prion-free forever? Could some of these species be healthy intermediate host that increase the risk in humans or other animals? Can PMCA answer some of these questions? PCMA is a potent tool that can be forced in order to favor the conversion of any PrPC to PrPSc. Our experiments have shown that all these three species where able to convert their PrP in a protease resistant form. Although the infectivity studies of these rabbits, horses and dogs are ongoing in their respective species (or transgenic variants) we have already obtained positive results when these new prions were inoculated in mice (data not shown). These data indicate that: (1) PMCA is a powerful tool to generate new prion species/strains, (2) The new in vitro generated prions remain infectious (at least in mice), and (3) according to all previous data obtained from in vitro replicated PrPSc, rabbits, horses and dogs should no longer be considered prion resistant species.
Although we were able to obtain new rabbit, horse and dog prions in vitro, the relative efficiency for their generation was clearly different and depended principally on the prion strain used as inoculum. Unfortunately, the use of complete brain as substrate makes difficult the quantification of the strength of the transmission barrier and at this moment we cannot venture to say which of the species is in general more resistant to prion replication. Cell-PMCA, that allows us to use homogeneous substrates, will be the right tool for addressing this question ultimately.
If we take into account that there are not any mammalian substrates that could not be converted in a protease resistant form in our lab, the next and crucial question is about where the limits are. A comparison between mammalian PrP amino acid sequences show that they diverge in not more than 10%. The closest non-mammalian species diverge around 30–35%. Is this too much to overcome those non-mammalian species barriers? In vivo studies have shown that despite several attempts for infecting chickens, they were all negative.23 These negative results in chickens were similar to those also negative experiments focused on infected rabbits. At this moment, just PMCA can answer this open question.
The authors thank the Umberto Agrimi's group, Tomas Mayoral's group, Glenn Telling's group, Francesca Chianini's group, Dr. Suzette Priola's group, Dr. Jean Jewell, Dr. Juan María Torres, Dr. Enric Vidal and Dr. Martí Pumarola for providing samples and ideas for some of the experiments previously described.
Previously published online: www.landesbioscience.com/journals/prion/article/10500