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From the Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta T6G 2M8the Department of Medicine (Neurology), University of Alberta, Edmonton, Alberta T6G 2G3the Centre for Neuroscience, University of Alberta, Edmonton, Alberta T6G 2E1, Canada
Prion diseases are fatal neurodegenerative disorders associated with the polymerization of the cellular form of prion protein (PrPC) into an amyloidogenic β-sheet infectious form (PrPSc). The sequence of host PrP is the major determinant of host prion disease susceptibility. In mice, the presence of allele a (Prnpa, encoding the polymorphism Leu-108/Thr-189) or b (Prnpb, Phe-108/Val-189) is associated with short or long incubation times, respectively, following infection with PrPSc. The molecular bases linking PrP sequence, infection susceptibility, and convertibility of PrPC into PrPSc remain unclear. Here we show that recombinant PrPa and PrPb aggregate and respond to seeding differently in vitro. Our kinetic studies reveal differences during the nucleation phase of the aggregation process, where PrPb exhibits a longer lag phase that cannot be completely eliminated by seeding the reaction with preformed fibrils. Additionally, PrPb is more prone to propagate features of the seeds, as demonstrated by conformational stability and electron microscopy studies of the formed fibrils. We propose a model of polymerization to explain how the polymorphisms at positions 108 and 189 produce the phenotypes seen in vivo. This model also provides insight into phenomena such as species barrier and prion strain generation, two phenomena also influenced by the primary structure of PrP.
Background: Alleles Prnpa and Prnpb of mouse prion protein (PrP) influence the incubation period of prion disease.
Results: PrPa and PrPb, products of these alleles, aggregate differently in vitro.
Conclusion: The polymorphism at 108/189 influences the oligomeric stages of PrP polymerization.
Significance: Elucidating the mechanism of PrP aggregation is relevant to understanding prion disease susceptibility, prion strains, and species barriers.
Prion diseases are protein folding disorders that include Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids, and scrapie in sheep. These diseases have several similarities to other protein folding neurodegenerative disorders, such as Alzheimer disease, but the hallmark of prion diseases is the infectious nature of the protein responsible for the neurodegeneration (
and its infectious form is generated when the monomeric, predominantly α-helical, and soluble form (PrPC) is structurally converted into an amyloid structure, oligomeric in nature, high in β sheet, and partially protease-resistant (PrPSc). Many different forms of PrPSc can be generated, and this structural promiscuity is thought to be the molecular basis for prion “strains,” defined by distinct incubation time, phenotype, and/or pathology (
). Although certain structures of PrPSc may be better able to convert PrPC from select species, once the process has begun, it is relentless. More PrPC is converted into PrPSc, PrPSc accumulates, and neuronal death follows.
The primary structure of the host PrPC is a major determinant of the host prion disease susceptibility. For example, subtle differences in PrPC sequence are sufficient to render some mammals, such as rabbits and horses, immune to prion disease (
The PrP sequence can also vary within a species; these polymorphisms too can influence disease susceptibility or even phenotype. In humans, methionine homozygosity at codon 129 of the prion protein gene (Prnp) increases susceptibility to prion disease. Codon 129 also determines the phenotype of a genetic form of Creutzfeldt-Jakob disease caused by the D178N mutation (
Given the clear influence of PrPC sequence on prion disease, the next question is by what mechanism is this influence conferred? The PrPC sequence may dictate which PrPSc conformations are permissive or preferred, or it may influence how readily the conversion process can occur under the guidance a given PrPSc structure. Although the mechanism of prion conversion into amyloids remains obscure, the canonical model proposed for amyloid formation is nucleated polymerization (
) consisting of two phases: (i) a nucleation phase where monomers undergo conformational change and self-associate to form oligomeric nuclei; and (ii) an elongation phase, in which nuclei rapidly grow by the further addition of monomers, forming larger fibrils until saturation (
). The reason for this discrepancy is not clear, but different prion strains were used; in the latter study, the strain used for inoculation was first passaged in Prnpb mice, whereas Prnpa mice were used as the source of strains for the other studies (
As the differences in the primary structure of PrPa and PrPb are the main distinguishing features in these studies, it follows that this polymorphism is the major factor determining the incubation period in vivo. Both residues have been implicated in prion disease, either by playing a role in the initial stages of PrPC conversion (
We hypothesized that different polymerization kinetics could explain the different incubation periods of these two alleles and that we would be able to detect these kinetic differences in vitro by putting recombinant mouse PrPa or PrPb into fibril-forming assays. Having found this to be the case, we then proceeded to use this in vitro model of conversion to explore the potential mechanisms of fibril formation process in each allele type.
In this work, we compare for the first time the kinetics of amyloid fibril formation of recombinant mouse PrPa and PrPb. From these results, together with the conformational and structural analysis of the formed fibrils, we propose different mechanisms of polymerization for these two isoforms of mouse PrP.
In mice, the polymorphisms at positions 108 and 189 are major determinants of prion disease incubation time (
), but the molecular basis for this phenomenon is unclear. We hypothesized that the effect of host PrP sequence on incubation time was related to the mechanism of prion conversion, involving the oligomerization of PrPC into PrPSc. In this study, we provide evidence that PrP aggregation does not follow the canonical nucleation polymerization and that in vivo findings can be explained by different polymerization kinetics and seeding responses of the two mouse PrP isoforms.
PrPa and PrPb differ only at residues 108 and 189, and we did not detect differences in α-helical content or thermal/chemical stability between the two monomers. Instead, the primary differences were found between their kinetic and seeding behaviors.
Based on our kinetic data, neither PrP isoform follows true nucleation polymerization. This model predicts that fibril mass is proportional to t2 during nucleation, meaning that there is never a flat lag phase (
). Also, PrPa concentration had a linear effect on elongation rate and lag phase, as opposed to the exponential effect predicted by nucleation polymerization. PrPb concentration did not correlate at all with lag phase, rate of elongation, or maximum ThT fluorescence. In fact, at highest PrPb concentration, a paradoxical increase in lag phase was observed. This is possibly explained by an accumulation of off-pathway oligomers that interfered with the aggregation process (
), although we were not able to detect these by TEM or in supernatants of centrifuged samples after aggregation.
Other aspects of PrPb kinetics also support an alternate model. Our data indicate that the PrPb starting monomers, in 2 m GdnHCl, cannot be directly incorporated into fibrils. This is why the lag phase persists despite the addition of seed. Therefore, it is a different component, which we call a receptive substrate, that is incorporated into the fibrils during exponential growth. How then do the starting monomers become receptive substrates? When unseeded, the starting monomers do eventually form receptive substrates and become fibrils, but the process is extremely long with greater variation in kinetic profile and fibril structure. This variability suggests that a number of pathways to receptive substrate formation are possible and that several different receptive substrates can be produced, each of which then produces a different kinetic profile and fibril product. Alvarez-Martinez et al. (
) have explained variation in aggregation kinetics by proposing the existence of “conformationally active monomers,” which randomly form different nuclei. Whichever nucleus forms first is the one that dictates the dynamics of the aggregation process and the resulting fibril structure. This model could explain the variability seen in our data, but not why we cannot eliminate the lag phase with seeding nor why we do not see a correlation between PrPb kinetics and monomer concentration. Rather than monomers, our data suggest that conformationally active oligomers may be the necessary precursors to the formation of receptive substrates.
If the production of conformationally active oligomers is the rate-limiting step, dependent on structural change from the monomer state, it follows that changes in monomer concentration will not facilitate the process of fibril growth, as found in our study. Also, the addition of seed to starting monomers does not immediately lead to exponential growth in PrPb because starting monomers must first form conformationally active oligomers and then receptive substrates; only the receptive substrates are incorporated into fibril growth. The presence of these early oligomers (ThT-negative and ThT-low oligomers) was confirmed by p-FTAA fluorescence. A similar mechanism, “nucleated conformational conversion,” has been described for the yeast prion element [PSI+] where nuclei are formed by conformational rearrangement of less structured oligomeric intermediates that are in equilibrium with monomers (
Of note, although seeding does not eliminate PrPb lag phase, seed addition does greatly accelerate the time to exponential growth phase, meaning that the seed must be affecting the process in some manner. Seed addition produces an immediate and rapid increase in p-FTAA fluorescence and a slower linear increase in ThT fluorescence (prior to the exponential growth phase), with later addition yielding both a higher rate of ThT increase and a shorter time to onset of exponential growth phase. Given this, we propose the following mechanism (Fig. 9). Starting monomers gradually aggregate into larger and larger oligomers (which are ThT-negative but p-FTAA-positive). When these ThT-negative oligomers bind seeds, they become conformationally active and acquire some low ThT fluorescence (ThT-low, p-FTAA-positive), but do not yet become fully receptive substrates (and therefore do not undergo exponential growth). As larger ThT-negative oligomers are expected to accumulate over time, seeds that are added to the process later will bind larger ThT-negative oligomers that in turn become conformationally active, thus producing higher rates of ThT increase. The larger oligomers may also be closer to adopting a receptive substrate conformation, which would explain why there is an apparent acceleration to exponential phase after delayed seed addition.
It should be noted that PrPa could fundamentally share the mechanism that we propose for PrPb and still display the kinetic and seeding tendencies we observed. If PrPa converts to conformationally active oligomers and then to receptive substrates more efficiently, and is not rate-limiting, the conformationally active oligomers would not be detectable in our seeded kinetic studies. The efficient formation of receptive substrates might correlate with less off-pathway aggregation, explaining the shorter lag phases and reduced variability seen in unseeded PrPa reactions. It would also explain why PrPa is not overly influenced by seed type; there is less time for seed and oligomer to interact.
We are proposing that the seed induces structural change without immediately triggering polymerization. Such a process has been described in “surface-catalyzed nucleation,” where monomers nonspecifically bind the lateral aspects of fibrils with subsequent conformational rearrangement to form on pathway oligomers, and ultimately nuclei, which can bind the ends of fibrils and proceed with exponential growth (
Somewhat unexpectedly, our conformational stability assay and TEM experiments revealed that PrPb may be structurally influenced by seeds to a greater extent than PrPa; the morphology and stability of PrPa fibrils did not vary as much as PrPb fibrils. Also, PrPb more closely mimicked the kinetics of the seeds used.
The purpose of this study was to gain insight into how polymorphisms of PrP translate into phenotypic differences in prion disease by studying kinetic profiles of fibril formation in vitro. The long lag phases for PrPb correlate with long incubation times in Prnpb mice (
). In addition, if conformationally active oligomers are part of the PrP aggregation mechanisms, they may present a new therapeutic target, one that lies structurally in between monomeric and fibrillar states. Finally, our data may also inform the study where a shorter incubation time was seen in Prnpb mice. Here, strain properties may be key, and we have found that PrPb appears to be more strongly influenced by the type of seed used. Importantly, this seed influence could have risk implications as hosts presumed to be less susceptible based on PrP sequence may simply need to be exposed to the appropriate strain. A real life example of this can be found in the rise of chronic wasting disease. At present, chronic wasting disease does not appear to cross the species barriers into humans, but there is growing evidence for distinct strains of chronic wasting disease, and the influence of human PrP polymorphism on their transmission characteristics is unknown.
We thank Byron Caughey, David Westaway, and Holger Wille for the critical reading of this manuscript, David Wishart for providing the DNA constructs for PrP expression, and Peter Nilsson for the provision of p-FTAA dye.