Prion Hypothesis: The end of the Controversy?

Abstract

Forty-three years have passed since it was first proposed that a protein could be the sole component of the infectious agent responsible for the enigmatic prion diseases. Many discoveries have strongly supported the prion hypothesis, but only recently has this once heretical hypothesis been widely accepted by the scientific community. In the past 3 years, researchers have achieved the holy grail demonstration that infectious material can be generated in vitro using completely defined components. These breakthroughs have proven that a misfolded protein is the active component of the infectious agent and that the propagation of the disease and its unique features depends on the self-replication of the infectious folding of the prion protein. In spite of these important discoveries, it remains unclear whether another molecule besides the misfolded prion protein might be an essential element of the infectious agent. Future research promises to reveal many more intriguing features about the rogue prions.

The mysterious prion diseases

Prion diseases are a group of fatal and infectious neurodegenerative diseases affecting humans and diverse animal species [1]. The infectious nature of prion diseases was first evident more than 70 years ago when accidental transmission of scrapie occurred in sheep. Inoculation against a common virus with a formalin extract of tissue unknowingly derived from an animal with scrapie infected nearly 10% of the flock. Scrapie was subsequently transmitted experimentally to sheep [2] and later to mice [3]. In humans an infectious origin was suspected for kuru, a prion disease identified in some cannibalistic tribes of New Guinea; a formal demonstration came in 1966 with the transmission of kuru to monkeys [4]. These studies were followed by transmission of Creutzfeldt-Jakob disease (CJD) [5] a familial form of prion disease, Gerstmann-Straussler-scheinker syndrome, to primates [6]. These discoveries added to the initial findings indicating that the infectious agent responsible for these diseases was different from any known form of microorganism and led to a plethora of research aimed at understanding the nature of the agent. A second boost in the prion field came when an outbreak of a prion disease in cattle called BSE (Bovine Spongiform Encephalopathy) in Great Britain was mediated by prion infection [7]. Between the discovery of this disease as a prion disorder and the implementation of measures to prevent possible transmission to humans, it is likely that the entire population of UK and a large part of Europe might have consumed infected meat. These initial concerns were confirmed when a new form of human disease, termed variant Creutzfeldt-Jakob disease, appeared and was convincingly linked to BSE [8,9]. This article focuses on discussing recent advances regarding the nature of the infectious agent responsible for prion diseases and the mechanisms of prion propagation.

A Historical Perspective of the Prion Hypothesis

Starting from the initial discoveries in the prion field, it was clear that the infectious agent responsible for prion diseases was different from conventional micro-organisms. Over decades of research, many milestone discoveries have provided crucial evidence in favor of the prion hypothesis, i.e. that a misfolded protein is the main (and perhaps the sole) component of this unorthodox infectious agent called a prion (Fig. 1). In a series of key experiments, Alper and coworkers demonstrated that procedures that obliterate nucleic acids, such as very high doses of ionizing radiation and UV, did not destroy the infectious material [10,11]. The same group also reported that the minimum molecular weight to maintain infectivity (around 2 × 10⁵ Da) was too small to possibly be a virus or other type of micro-organism [12]. Interestingly, this estimate is in the range of the size of the most infectious prion particles obtained with modern sophisticated techniques [13]. Based on these observations, speculation arose that the infectious agent might be protein-based [14]. In 1967, Griffith proposed three scenarios by which a protein could act as the infectious agent causing scrapie [15]. However, little research was done to test this hypothesis until Prusiner and coworkers pioneered an impressive set of discoveries that gave fuel to the prion hypothesis and coined the term prion to refer to this heretical proteinaceous infectious agent [16].

Figure 1. A timeline representation of the major milestones in the prion hypothesis.

Starting with the initial indication that prion diseases can be transmissible, owing to the accidental transmission of scrapie in sheep and ending with the demonstration that infectious material can be generated in vitro using pure recombinant prion protein, the prion field has always been full of unorthodox discoveries.

In landmark research, the protease-resistant prion protein (PrP^Sc) was isolated from the infectious material [17], showing that PrP^Sc and infectivity co-purified, and that the protein concentration was proportional to the infectivity titer [18]. These breakthroughs radically changed the prion field, and much effort then turned to study this protein. Infectivity was shown to be retained in highly purified preparations of PrP^Sc, in which no other protein was detectable, although the sample did contain lipids and carbohydrates, and perhaps nucleic acids. Importantly, infectivity was reduced by agents that destroy protein structure and, strikingly by anti-PrP antibodies [18]. The gene encoding PrP (PRNP) was identified in the mammalian genome [19,20], and the corresponding mRNA was shown to be the product of a single host gene, which is expressed mostly in the brain, without significant changes on expression in healthy or infected animals. These findings indicated that the prion protein can exist as both a normal cellular protein (termed PrP^C) and a pathological isoform (termed PrP^Sc) [21]. Surprisingly, no chemical differences can be detected between the two forms of the protein; rather the nature of their distinct properties appears to be enciphered in the conformation adopted by the proteins [22]. Once PRNP was identified, genetic analyses discovered the first PRNP mutation linked to familial prion disease [23]; subsequent studies showed that all of the familial cases of prion disease were linked to PRNP mutations [1]. Moreover, overexpression of mutant Prnp in mice produced a transmissible neurodegenerative disease similar to prion disease [24–28]. Strong evidence in favor of the prion hypothesis came from the demonstration that Prnp^−/− mice were resistant to scrapie infection, developing neither signs of the disease, nor allowing propagation of the infectious agent [29]. The cell-free conversion of PrP^C into PrP^res catalyzed by the pathological protein also provided support for the prion hypothesis. In this system, developed by Caughey and co-workers [30], purified PrP^C mixed with stochiometric amounts of purified PrP^Sc produced a low yield of PrP^res formation under non-physiological conditions that precluded infectivity studies. In 2001 we developed a new system for in vitro replication of prions, termed Protein Misfolding Cyclic Amplification (PMCA) [31]. PMCA reproduces prion conversion in a greatly accelerated and efficient manner [31]. The in vitro-generated material maintains all the biological, biochemical and structural characteristics of in vivo produced prions [32]. These findings confirmed a central tenet of the prion hypothesis, which is that prions can propagate indefinitely and that newly generated PrP^Sc triggers further misfolding to produce an auto-catalytic process of prion replication.

Is the prion hypothesis proven?

Despite the compelling evidence for the prion hypothesis, skeptics have argued that definitive proof cannot be achieved until infectious material is produced in vitro from pure, normal_[SC1] prion protein [33]. This experiment remained elusive for many years. However, in 2004 the production of “synthetic prions” via the in vitro induction of misfolding and aggregation of bacterially expressed recombinant prion protein (rPrP) was reported [34]. Injection of this material into transgenic mice overexpressing PrP induced a transmissible neurodegenerative disease with a very long incubation period. The generated infectious material displays different characteristics compared to natural prions, and the authors proposed that a conformational adaptation must have occurred to stabilize the originally extremely low infectivity of misfolded rPrP, which paradoxically involved a decrease in the thermodynamic stability of the aggregate [35]. These findings and subsequent studies from the same group [35–37], including the formation of many novel synthetic prions and even some composed of protease-sensitive PrP, came tantalizingly close to providing the definitive proof for the prion hypothesis. Unfortunately, the fact that the disease was transmissible only to transgenic animals overexpressing PrP, and not to wild-type animals, is a matter of concern, given the well known propensity for such animals to spontaneously develop a prion-like disease [38–40]. In other words, the disease in these animals might have resulted by acceleration, induced by the injected material, of a pre-existing condition produced by transgenesis, as was the case observed in transgenic mice overexpressing mutant PrP [28]. Recent work, using a different protocol to induce misfolding and aggregation of rPrP has generated infectious prions after various numbers of passages [41]. Importantly, this study demonstrated the induction of disease in wild type animals.

PMCA has proven to be a very successful technique for generating bona fide infectious prions in vitro. Using PMCA our group was the first to generate in vitro-derived prions in wild type animals which display similar properties to brain-derived infectious material [32]. PMCA has become an invaluable technique to study prions given that it provides the equivalent to a procedure for cultivating prions in vitro. In an extension of this work, Supattapone and colleagues generated infectious material using PMCA and PrP^C and PrP^Sc purified from the brain of healthy and sick animals, respectively [42]. Infectivity was generated in the absence of any other component besides the purified proteins and a synthetic polyanion used to catalyze the reaction. Importantly, this study, as well as a recent report from our group, showed that PMCA can generate, de novo, infectious PrP^Sc in the absence of any infectious brain-derived material to begin the reaction [42,43]. When spontaneously generated prions were injected into wild type animals, the disease exhibited unique clinical, neuropathological and biochemical characteristics which differ from all previous known prions. An important question to be answered by future research is whether the de novo prions were present in the normal brain or were generated during the amplification process. A recent report showing the spontaneous generation of prions amplified by prion replication in cells [44] also highlights this important issue. In this study, infectivity was detectable at low frequency following incubation of uninfected mouse brain homogenate with metal wires that presumably concentrate prions. These remarkable findings lend strong support to the prion hypothesis and provide a mechanistic explanation for the sporadic form of prion diseases. Moreover, they suggest that new forms of prions with different disease characteristics will probably appear in the future. All these PMCA studies were performed using mammalian PrP^C as a substrate. Formation of protease-resistance PrP recombinantly produced in bacteria, seeded by brain PrP^Sc, was first achieved by Atarashi et al., by using an adapted version of PMCA [45]. Although material generated in this way was infectious to wild type animals, it required a long incubation period and resulted in an incomplete attack rate [46], suggesting that purified rPrP, in the absence of other cellular factors, is not capable of producing a highly infectious preparation. However, in a recent and elegant study, Wang et al reported the de novo generation of bona fide infectious prions in vitro by PMCA using exclusively rPrP with the sole addition of RNA and lipids [47]. This study might represent the strongest proof to date for the prion hypothesis.

One argument that has been often used against the prion hypothesis is the existence of prion strains [48]. Nearly all TSEs are known to exhibit various strains characterized by different incubation periods, clinical features and neuropathology [49]. For infectious diseases, different strains generally arise from mutations or polymorphisms in the genetic make-up of the infectious agent. To reconcile the infectious agent composed exclusively of a protein with the strain phenomenon, it has been proposed that PrP^Sc obtained from different prion strains has slightly different conformation or aggregation states that can faithfully replicate at the expense of the host PrP^C [50–52]. This idea is supported by recent studies showing that a group of polythiophene compounds can differentially interact with PrP^Sc associated with distinct strains, thereby enabling them to be structurally distinguished [53]. Additional support for the hypothesis that the strain features are enciphered in the structure of PrP^Sc was provided by our recent report showing that PMCA can faithfully replicate different prion strains in humans and mice, maintaining all their features after multiple passages both in vivo and in vitro [54]. Furthermore, the related phenomena of species barrier, strain adaptation, and molecular memory were also reproduced in vitro by PMCA [55–57], suggesting again that they are dependent purely on PrP^Sc replication. When PrP^C from one species is used to replicate prions from a different species, new strains are generated, pointing to an extremely high flexibility of PrP and raising the possibility of infinite “prionability” of the protein.

Overall, the impressive recent progress in production of infectious material has removed all doubts of the prion hypothesis. In addition, the demonstration that several other proteins in a variety of organisms (such as yeast, fungi and invertebrates) use the prion mechanism to transmit biological information has contributed to and strengthened the prion hypothesis (Box 1). Based on the available data, the idea that prions consist of viruses or any other type of conventional micro-organism is simply untenable. Nevertheless, several key aspects of PrP^Sc infectivity remain unknown, particularly the detailed structure of the infectious protein and whether or not another element besides of the prion protein is required for prion replication.

Box 1. Yeast prions.

The view that the transmission of biological information by propagation of protein misfolding was the exclusive domain of the rogue prion protein changed dramatically in 1994 when a visionary article from Reed Wickner proposed that the yeast proteins Ure2 and Sup35 behave like prions [86]. This article was followed by an impressive collection of studies demonstrating that these proteins indeed acted as prions and the identification of many other prions in yeasts and other fungi [87]. A yeast prion behaves as a non-Mendelian genetic element to transmit biological information in the absence of nucleic acid [88]. In yeast, prions do not kill the cells harboring them, nor do they cause disease; rather, they are inherited by cellular division and usually produce new metabolic phenotypes. However, like their mammalian counterparts, yeast prions are based on the ability of a protein that has acquired an abnormal conformation to influence other molecules of the same protein to adopt the misfolded form. The discovery of the yeast prions was important not only to expand the prion concept towards proteins other than PrP, but also showed that prion transmission can be used for normal biological processes. Currently, it is unknown how many other proteins utilize a prion-based mechanism to perform their biological function, but it seems possible that the efficient conversion of a protein function by transmission of an alternative folding state could provide an excellent way in which to modulate the activity of proteins without the need for genetic changes [87].

Mechanism of prion replication and potential roles of co-factors in prion infectivity

Prion replication begins when PrP^Sc in the infectious material interacts with host PrP^C, thereby catalyzing its conversion to the pathogenic form of the protein. The precise molecular mechanism of PrP^C → PrP^Sc conversion is not completely understood, but available data support a seeding/nucleation model in which infectious PrP^Sc is an oligomer that acts as a seed to bind PrP^C and catalyze its conversion into the misfolded form by incorporation into the growing polymer [58–60]. At some point, the long PrP^Sc polymers break into smaller pieces driven either by a mechanical force or catalyzed by a yet unknown process. This fragmentation increases the number of effective nuclei that can direct further conversion of PrP^C. Because polymer fragmentation is probably a rate-limiting event in prion replication, it is very important to understand how this occurs in vivo and whether another cellular factor participates in this process. The seeding-nucleation model provides a rational and plausible explanation for the infectious nature of prions. Infectivity relies on the capacity of preformed stable misfolded oligomeric PrP^Sc to act as a seed to catalyze the misfolding and aggregation process [60]. Indeed, the PMCA technology is based on the assumption that prion replication depends on the formation and multiplication of oligomeric seeds [31,59].

An open question in the field is whether or not prion replication requires a cellular co-factor. Several pieces of evidence indicate that co-factor(s) might participate in prion replication [61]. The existence of a host-encoded conversion factor was first suspected from experiments with transgenic mice expressing chimeric prion proteins from two different species [62]. Based on these findings, Prusiner coined the term “protein X” to refer to this factor; however, there is no formal proof that the accessory molecule is indeed a protein. Further evidence for the existence of conversion factors came from PMCA studies of PrP^C → PrP^Sc conversion. Purified hamster PrP^C was not converted when mixed with highly purified PrP^Sc; however, conversion was restored when the complete brain homogenate was added to the sample [63–65]. These results suggest that unknown factors present in brain homogenate are essential for prion conversion. Supattapone’s group showed that natural or synthetic RNA can act as conversion factors and catalyze prion replication in hamster [66], but surprisingly not in mouse [67]. It was proposed that RNA might serve both as scaffold for efficient conversion and a necessary molecular complex-pair as the minimal infectious unit [68]. These data suggest that nucleic acids may after all be involved in prion replication. However, an alternative possibility is that RNA only accelerates or stabilizes an otherwise experimentally-inaccessible PrP^Sc-like conformation. The same group has shown that it is the negative charge of RNA that is responsible for the interaction with PrP, given that several other natural or synthetic polyanions can have the same activity [64]. These results, however, do not fit with the initial finding that only eukaryotic RNA, and not prokaryotic RNA, assist prion conversion [66]. In more recent studies, replication of PrP^Sc and infectivity stemming from recombinant PrP required in addition to RNA, synthetic anionic phospholipids [47]. The requirement for lipids is consistent with previous studies reporting higher infectivity with lipid membrane–associated PrP^Sc [69,70]. In spite of these findings, it is possible that RNA and lipids are merely in vitro mimics of the activity of one or more unknown in vivo facilitating factors. Recent attempts to identify the mysterious co-factor, using a complementation PMCA assay and fractionation techniques, have shown that it is present in all major organs of diverse mammalian species, and is predominantly located in the lipid raft fraction of the cytoplasmic membrane [65]. However, conversion factor activity is not present in the lower organisms tested (yeast, bacteria and flies), suggesting that prion replication is a feature only present in mammals. Surprisingly, treatments that eliminate nucleic acids, proteins or lipids do not prevent prion replication in vitro [65]. One possible interpretation of these findings is that various different compounds might act as a conversion factor in vitro, such that elimination of only some does not prevent prion replication. Indeed, the addition of various classes of molecules (synthetic nucleic acids, heparin, albumin or fatty acids) produced a small, but detectable effect on enhancing prion replication in vitro [66].

If co-factors are indeed necessary for prion replication, it is possible to imagine at least five possible roles played by these accessory molecules (Fig. 2):

Figure 2. Possible roles by which co-factors may participate in prion replication.

At least five different scenarios can be proposed for the involvement of cellular co-factors in prion propagation. i, The co-factor might integrate into the infectious agent, alter PrP^Sc folding and provide biological information to the infectivity process, perhaps by determining strain characteristics. ii, The co-factor might act as an essential catalyst for prion replication, perhaps by interacting with PrP^C, altering its folding and permitting its interaction with PrP^Sc. iii, Through binding and integration into the PrP^Sc polymer, the co-factor might help to stabilize the conformation of PrP^Sc. iv, The co-factor might participate in the key process of fragmenting PrP^Sc polymers to produce smaller structures and multiplying the number of seeds to allow the continuation of prion replication. v, The co-factor might bind to PrP^Sc, thus increasing its biological stability, reducing its clearance in vivo and increasing its chances to reach target organs. It is important to highlight that these possibilities are not mutually exclusive and indeed, it is likely that a co-factor could be involved in several of these processes simultaneously.

1. The co-factor might contribute biological information to the infectious material. If the accessory molecule gets incorporated into the PrP^Sc particles it is possible that it could help to determine the folding characteristics of prions. In addition, it might facilitate the interaction with cellular components and modify the tropism of the infectious agent. In this case, the co-factor could have an important role in modulating the properties of prion strains. A recent report provides indirect evidence for this possibility [71]. Experiments using a panel of cells to replicate mouse prions showed that infection of different cell types leads to phenotypic “mutation” and selection of prion strains. One possible explanation for these results is that cellular diversity in co-factors could direct prion strain changes, given that no differences in PrP were detected between the different cells [72].

2. The co-factor might act as an essential catalyst for prion replication. The conversion factor could facilitate the PrP^C to PrP^Sc conversion, perhaps by directing the interaction of the two proteins or through binding to PrP^C and inducing a partial unfolding of the protein necessary to adopt the misfolded conformation. The idea that co-factors facilitate prion replication is supported by in vitro conversion experiments, showing that the PrP^C to PrP^Sc conversion is more efficient in the presence of accessory molecules [47,65,67].

3. The co-factor might help to stabilize the conformation of PrP^Sc. In this model, although the co-factor does not directly participate in the conformational conversion, it might contribute to stabilization of the newly formed misfolded structure. This view is supported by neuropathological data, especially from other neurodegenerative diseases, which shows that attachment of a variety of molecules (e.g. proteins, nucleic acids, metal ions, proteoglycans, etc.) to cerebral protein aggregates makes them much more compact and stable structures [73–75].

4. The co-factor might participate in the fragmentation of PrP^Sc polymers. As described above, prion replication follows a seeding-nucleation process of polymerization. For this process of seeding-nucleation to efficiently propagate prions, it is necessary not only for PrP^Sc to be able to seed the misfolding and aggregation of PrP^C, but also for long polymers to be fragmented into smaller pieces as a way to increase the effective number of seeds catalyzing prion replication. Indeed, in yeast prions (Box 1), the chaperone Heat shock protein 104 (Hsp104) probably fulfills this role [76,77] given that the replication of most yeast prions depends on this factor and that inhibition of Hsp104 activity precludes prion propagation [78].

5. The co-factor might increase the biological stability of prions, thereby reducing their in vivo clearance. Another possibility is that the co-factor increases the pharmacokinetic and bioavailability properties of PrP^Sc. In this sense, it is important to emphasize that the ability of an infectious agent to propagate disease depends not only on its successful replication, but also on its ability to remain intact in the body and reach the target location. This is a challenging task for an infectious agent composed solely of a protein. Recent studies have identified a possible pathway for prion clearance involving Mfge8-mediated phagocytosis of prions by microglia [79]. In this context a cofactor could reduce the affinity of PrP^Sc for Mfge8, thereby decreasing the ability of microglia to engulf and destroy prions. The possibility that the co-factor contributes to the biological stability of PrP^Sc is in agreement with results showing that attachment of prions to nitrocellulose paper, soil particles or steel wires increases prion infectivity [80–82].

One of the most important issues related to the existence of possible component(s) other than PrP involved in prion transmission is to distinguish between factors contained in the infectious particle and host factors that are involved in the conformational change. This distinction is very relevant; if additional factors must be part of the infectious particle, then the infectious units would not be composed solely of PrP^Sc. Alternatively, additional factors might need to be present in the host cells to sustain efficient prion replication. These factors could be normal cellular components, presumably engaged in other functions in the infected cells, that accidentally participate in prion conversion. In the latter case, these additional factors should not be considered part of the infectious particle, but rather host-encoded molecules that facilitate prion replication.

Concluding remarks

The hypothesis that a single protein is the sole component of the infectious agent responsible for prion diseases has been highly controversial for the past 30 years. This is not surprising considering that the prion hypothesis claims that a protein behaves like a living micro-organism to infect an individual by various routes (even oral administration), survive metabolic clearance, self-replicate in the body, reach the target organ and induce a cascade of neurodegenerative damage, resulting in disease and ultimately death of the infected individual. Moreover, the prion agent exists as multiple strains leading to diseases with sometimes subtle and othertimes overtly different clinical and neuropathological characteristics. Prion propagation also respects the still incompletely understood rules of species barriers and exhibits characteristics of strain adaptation and memory. All of these features can be easily explained by an agent containing genetic material, but a protein-based infectious agent makes an explanation more challenging. For this reason, although compelling evidence accumulated steadily over the years to support the prion hypothesis, the majority of scientists remained skeptical. However, exciting research in the past 3 years has demonstrated that infectious material can be generated with defined components in vitro and that the information for strains and the species barrier appears to be exclusively contained in the folding of PrP^Sc. These findings have proven beyond any doubt that the prion hypothesis was indeed correct. This does not mean that everything is known regarding prions. On the contrary, there are many outstanding questions still unanswered (Box 3). Among them, it is particularly important to understand whether or not other cellular factors play a role in prion replication. This is not simply a matter of curiosity; it is crucial to understand the nature of the infectious agent. In this context we are still uncertain whether the prion hypothesis is equivalent to a PrP^Sc-only hypothesis. Although it is clear that PrP^Sc is the main informational molecule in the infectious agent, it remains possible that a co-factor is required, perhaps to achieve efficient propagation in the in vivo setting. In the latter scenario a molecule other than PrP^Sc would be essential for infectivity. Nevertheless, the putative existence of a co-factor, and its potential role in the process, does not negate the protein-based mechanism of transmission by which prions produce disease. Another important area of research is to elucidate the 3-dimensional structure of PrP^Sc and determine the structural features of prion strains. The remarkable progress in the generation of infectious prions from purified components, owing to the successful application of the PMCA technique, opens the possibility to produce large amounts of chemically homogeneous and metabolically labeled infectious PrP^Sc, paving the way to fine structural studies.

Box 3. Outstanding questions.

• Is PrP^Sc the only component of the infectious prion agent?

• What is the nature and role of cellular co-factors in prion replication?

• What is the 3-dimensional structure of PrP^Sc?

• What is the normal function of PrP^C? Does it have anything to do with the conformational flexibility of the protein?

• How is the prion strain phenomenon enciphered in the structure of PrP^Sc?

• How does PrP^Sc induce brain degeneration?

• To what extent does the prion principle operate in other diseases of protein misfolding?

• How common is the prion phenomenon in nature?

• Do prion-like proteins exist in mammals which confer advantageous properties to their infected hosts?”

Interestingly, the advances in the prion hypothesis have paralleled the expansion of the prion concept beyond prion diseases. Indeed, recent years have ushered in the end of a period of skepticism which surrounded the possibility that a single protein could act as an infectious agent and the beginning of an era in which the prion phenomenon is being explosively expanded to some of the most prevalent human diseases (Box 2) [60,83–85]. If further research proves that several other diseases can be transmitted by the propagation of misfolded proteins, prions could turn out to be more than a rare caprice of nature.

Box 2. Transmission of other diseases by a prion-based mechanism.

Misfolded protein aggregates are associated not only with prion diseases, but also with a large group of diseases known as protein misfolding disorders (PMDs), which include some highly prevalent and insidious illnesses such as Alzheimer’s disease, Parkinson’s disease, type 2 diabetes and over 20 additional human maladies [89,90]. Compelling evidence suggests that the accumulation of misfolded protein aggregates triggers tissue damage, organ dysfunction and disease in all of these PMDs. To date, only prion disorders are considered to be transmissible. However, the molecular mechanisms responsible for protein misfolding and aggregation are remarkably similar in all PMDs [60]. Indeed, the formation of misfolded aggregates in all cases follows a seeding-nucleation model, which can be accelerated by the addition of pre-formed seeds. The ability of seeds to template misfolding and aggregation is the key element of prion transmissibility, and this similarity suggests that all PMDs have the intrinsic potential to be transmissible [60]. Strikingly, reports from the past three years, using cellular and/or animal models, have provided evidence suggesting that the transmission of protein misfolding by a prion-like mechanism might be at the heart of the most common PMDs, including Alzheimer’s, Parkinson’s and Huntington’s diseases [84,91–94]. The exciting goal of future research is to determine if misfolded proteins implicated in PMDs are infectious and if they transmit disease under natural conditions. In other words, we need to carefully assess whether misfolded proteins are transmitted between individuals and propagate within communities as conventional infectious agents.