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Mouse Prion Protein Polymorphism Phe-108/Val-189 Affects the Kinetics of Fibril Formation and the Response to Seeding

EVIDENCE FOR A TWO-STEP NUCLEATION POLYMERIZATION MECHANISM*
  • Leonardo M. Cortez
    Affiliations
    From the Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta T6G 2M8
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  • Jitendra Kumar
    Affiliations
    From the Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta T6G 2M8
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  • Ludovic Renault
    Affiliations
    the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7
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  • Howard S. Young
    Affiliations
    the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7

    the National Institute for Nanotechnology, University of Alberta, Edmonton, Alberta T6G 2M9
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  • Valerie L. Sim
    Correspondence
    To whom correspondence should be addressed: Centre for Prions and Protein Folding Diseases, 204 BARB, University of Alberta, Edmonton, Alberta T6G 2M8, Canada. Tel.: 780-248-1873; Fax: 780-492-1335;
    Affiliations
    From the Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta T6G 2M8

    the Department of Medicine (Neurology), University of Alberta, Edmonton, Alberta T6G 2G3

    the Centre for Neuroscience, University of Alberta, Edmonton, Alberta T6G 2E1, Canada
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Open AccessPublished:January 02, 2013DOI:https://doi.org/10.1074/jbc.M112.414581
      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.

      Introduction

      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 (
      • Prusiner S.B.
      Novel proteinaceous infectious particles cause scrapie.
      ). The protein in question is the prion protein (PrP),
      The abbreviations used are: PrP
      prion protein
      PrPC
      cellular form of prion protein
      PrPSc
      disease-associated form of prion protein
      ThT
      thioflavin T
      p-FTAA
      4′,3‴-bis(carboxymethyl)[2,2′;5′,2″,5″,2‴;5‴,2⁗]quinquethiophene-5,5⁗-dicarboxylic acid
      TEM
      transmission electron microscopy
      GdnHCl
      guanidinium hydrochloride
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      a.u.
      arbitrary units
      S
      slow
      F
      fast.
      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 (
      • Bruce M.E.
      • Fraser H.
      Scrapie strain variation and its implications.
      ,
      • Fraser H.
      • Dickinson A.G.
      Scrapie in mice. Agent-strain differences in the distribution and intensity of grey matter vacuolation.
      ,
      • Bruce M.E.
      Scrapie strain variation and mutation.
      ). 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 (
      • Gibbs Jr., C.J.
      • Gajdusek D.C.
      Experimental subacute spongiform virus encephalopathies in primates and other laboratory animals.
      ,
      • Barlow R.M.
      • Rennie J.C.
      The fate of ME7 scrapie infection in rats, guinea-pigs, and rabbits.
      ,
      • Moore R.A.
      • Vorberg I.
      • Priola S.A.
      Species barriers in prion diseases–brief review.
      ). Sometimes this species barrier can be crossed, although generally it is an inefficient process, with newly infected species having prolonged incubation periods (
      • Moore R.A.
      • Vorberg I.
      • Priola S.A.
      Species barriers in prion diseases–brief review.
      ,
      • Chen S.G.
      • Gambetti P.
      A journey through the species barrier.
      ,
      • Hill A.F.
      • Collinge J.
      Prion strains and species barriers.
      ). Interestingly, with sequential passages through the new host species, incubation periods become shorter and stabilize at a new and constant incubation period for that host (
      • Hill A.F.
      • Collinge J.
      Prion strains and species barriers.
      ). During these passages, it is believed that a process of conformational adaptation or a selection of one subtype of PrPSc conformation is taking place (
      • Collinge J.
      • Clarke A.R.
      A general model of prion strains and their pathogenicity.
      ). This adaptation is influenced primarily by the sequence of host PrP (
      • Moore R.A.
      • Vorberg I.
      • Priola S.A.
      Species barriers in prion diseases–brief review.
      ).
      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 (
      • Goldfarb L.G.
      • Petersen R.B.
      • Tabaton M.
      • Brown P.
      • LeBlanc A.C.
      • Montagna P.
      • Cortelli P.
      • Julien J.
      • Vital C.
      • Pendelbury W.W.
      • et al.
      Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism.
      ). The effect of Prnp polymorphism on disease susceptibility can be found in other species too, including codon 132 in cervids (
      • Green K.M.
      • Browning S.R.
      • Seward T.S.
      • Jewell J.E.
      • Ross D.L.
      • Green M.A.
      • Williams E.S.
      • Hoover E.A.
      • Telling G.C.
      The elk PRNP codon 132 polymorphism controls cervid and scrapie prion propagation.
      ) and codons 136/154/171 in sheep (
      • Foster J.D.
      • Parnham D.W.
      • Hunter N.
      • Bruce M.
      Distribution of the prion protein in sheep terminally affected with BSE following experimental oral transmission.
      ,
      • Goldmann W.
      • Hunter N.
      • Smith G.
      • Foster J.
      • Hope J.
      PrP genotype and agent effects in scrapie: change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie.
      ,
      • Houston F.
      • Goldmann W.
      • Chong A.
      • Jeffrey M.
      • González L.
      • Foster J.
      • Parnham D.
      • Hunter N.
      Prion diseases: BSE in sheep bred for resistance to infection.
      ,
      • van Keulen L.J.
      • Vromans M.E.
      • Dolstra C.H.
      • Bossers A.
      • van Zijderveld F.G.
      Pathogenesis of bovine spongiform encephalopathy in sheep.
      ,
      • Westaway D.
      • Zuliani V.
      • Cooper C.M.
      • Da Costa M.
      • Neuman S.
      • Jenny A.L.
      • Detwiler L.
      • Prusiner S.B.
      Homozygosity for prion protein alleles encoding glutamine-171 renders sheep susceptible to natural scrapie.
      ).
      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 (
      • Jarrett J.T.
      • Lansbury Jr., P.T.
      Seeding “one-dimensional crystallization” of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie?.
      ) 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 (
      • Harper J.D.
      • Lansbury Jr., P.T.
      Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins.
      ). PrPC sequence may regulate either or both of these phases.
      In mice, the presence of Prnp allele a (Prnpa, Leu-108/Thr-189) or allele b (Prnpb, Phe-108/Val-189) dramatically influences the incubation period of prion infection (
      • Westaway D.
      • Goodman P.A.
      • Mirenda C.A.
      • McKinley M.P.
      • Carlson G.A.
      • Prusiner S.B.
      Distinct prion proteins in short and long scrapie incubation period mice.
      ,
      • Moore R.C.
      • Hope J.
      • McBride P.A.
      • McConnell I.
      • Selfridge J.
      • Melton D.W.
      • Manson J.C.
      Mice with gene targetted prion protein alterations show that Prnp, Sinc, and Prni are congruent.
      ). Typically, Prnpa mice exhibit shorter incubation times (∼100–200 days) when compared with Prnpb mice (∼255–300 days) (
      • Lloyd S.E.
      • Thompson S.R.
      • Beck J.A.
      • Linehan J.M.
      • Wadsworth J.D.
      • Brandner S.
      • Collinge J.
      • Fisher E.M.
      Identification and characterization of a novel mouse prion gene allele.
      ,
      • Carlson G.A.
      • Goodman P.A.
      • Lovett M.
      • Taylor B.A.
      • Marshall S.T.
      • Peterson-Torchia M.
      • Westaway D.
      • Prusiner S.B.
      Genetics and polymorphism of the mouse prion gene complex: control of scrapie incubation time.
      ,
      • Akhtar S.
      • Wenborn A.
      • Brandner S.
      • Collinge J.
      • Lloyd S.E.
      Sex effects in mouse prion disease incubation time.
      ), although the opposite trend was seen in one study (
      • Moore R.C.
      • Hope J.
      • McBride P.A.
      • McConnell I.
      • Selfridge J.
      • Melton D.W.
      • Manson J.C.
      Mice with gene targetted prion protein alterations show that Prnp, Sinc, and Prni are congruent.
      ). 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 (
      • Lloyd S.E.
      • Onwuazor O.N.
      • Beck J.A.
      • Mallinson G.
      • Farrall M.
      • Targonski P.
      • Collinge J.
      • Fisher E.M.
      Identification of multiple quantitative trait loci linked to prion disease incubation period in mice.
      ,
      • Lloyd S.E.
      • Linehan J.M.
      • Desbruslais M.
      • Joiner S.
      • Buckell J.
      • Brandner S.
      • Wadsworth J.D.
      • Collinge J.
      Characterization of two distinct prion strains derived from bovine spongiform encephalopathy transmissions to inbred mice.
      ).
      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 (
      • Dima R.I.
      • Thirumalai D.
      Probing the instabilities in the dynamics of helical fragments from mouse PrPC.
      ,
      • Tizzano B.
      • Palladino P.
      • De Capua A.
      • Marasco D.
      • Rossi F.
      • Benedetti E.
      • Pedone C.
      • Ragone R.
      • Ruvo M.
      The human prion protein α2 helix: a thermodynamic study of its conformational preferences.
      ,
      • Knaus K.J.
      • Morillas M.
      • Swietnicki W.
      • Malone M.
      • Surewicz W.K.
      • Yee V.C.
      Crystal structure of the human prion protein reveals a mechanism for oligomerization.
      ) or by influencing the susceptibility to prion infection (
      • Foster J.D.
      • Parnham D.W.
      • Hunter N.
      • Bruce M.
      Distribution of the prion protein in sheep terminally affected with BSE following experimental oral transmission.
      ,
      • Goldmann W.
      • Hunter N.
      • Smith G.
      • Foster J.
      • Hope J.
      PrP genotype and agent effects in scrapie: change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie.
      ,
      • Houston F.
      • Goldmann W.
      • Chong A.
      • Jeffrey M.
      • González L.
      • Foster J.
      • Parnham D.
      • Hunter N.
      Prion diseases: BSE in sheep bred for resistance to infection.
      ,
      • van Keulen L.J.
      • Vromans M.E.
      • Dolstra C.H.
      • Bossers A.
      • van Zijderveld F.G.
      Pathogenesis of bovine spongiform encephalopathy in sheep.
      ).
      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.

      DISCUSSION

      In mice, the polymorphisms at positions 108 and 189 are major determinants of prion disease incubation time (
      • Westaway D.
      • Goodman P.A.
      • Mirenda C.A.
      • McKinley M.P.
      • Carlson G.A.
      • Prusiner S.B.
      Distinct prion proteins in short and long scrapie incubation period mice.
      ,
      • Moore R.C.
      • Hope J.
      • McBride P.A.
      • McConnell I.
      • Selfridge J.
      • Melton D.W.
      • Manson J.C.
      Mice with gene targetted prion protein alterations show that Prnp, Sinc, and Prni are congruent.
      ), 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 (
      • Padrick S.B.
      • Miranker A.D.
      Islet amyloid: phase partitioning and secondary nucleation are central to the mechanism of fibrillogenesis.
      ,
      • Ferrone F.
      Analysis of protein aggregation kinetics.
      ). Both PrPa and PrPb had prolonged flat lag phases in unseeded conditions. This better fits a double-nucleation mechanism (
      • Padrick S.B.
      • Miranker A.D.
      Islet amyloid: phase partitioning and secondary nucleation are central to the mechanism of fibrillogenesis.
      ). 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 (
      • Baskakov I.V.
      • Bocharova O.V.
      In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features.
      ), 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. (
      • Alvarez-Martinez M.T.
      • Fontes P.
      • Zomosa-Signoret V.
      • Arnaud J.D.
      • Hingant E.
      • Pujo-Menjouet L.
      • Liautard J.P.
      Dynamics of polymerization shed light on the mechanisms that lead to multiple amyloid structures of the prion protein.
      ) 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 (
      • Serio T.R.
      • Cashikar A.G.
      • Kowal A.S.
      • Sawicki G.J.
      • Moslehi J.J.
      • Serpell L.
      • Arnsdorf M.F.
      • Lindquist S.L.
      Nucleated conformational conversion and the replication of conformational information by a prion determinant.
      ).
      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.
      Figure thumbnail gr9
      FIGURE 9Proposed mechanism of PrPb fibril formation in unseeded and delayed seeding conditions. A, the unseeded reaction is shown, where monomers (dark triangles) first form ThT-negative (p-FTAA-positive) oligomers of increasing size and ultimately form conformationally active oligomers (light triangles, ThT-low, p-FTAA-positive) followed many hours later by receptive substrates (white squares, ThT-high), which go on to form fibrils. B, a typical seeded reaction is shown, with seed added at time 0. When monomers bind seed, they are immediately converted into a conformationally active state (ThT-low, p-FTAA-positive). As more monomers bind and are converted, a sufficient number or size is reached such that receptive substrates form. C and D, with delayed seeding, larger ThT-negative (p-FTAA-positive) oligomers are already present when seed is added, so immediate conversion of the larger oligomers occurs (becoming ThT-low, p-FTAA-positive), giving a greater linear increase in ThT fluorescence signal over time and allowing receptive substrates to form in a shorter time period after 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 (
      • Ferrone F.A.
      • Hofrichter J.
      • Eaton W.A.
      Kinetics of sickle hemoglobin polymerization. II. A double nucleation mechanism.
      ,
      • Ruschak A.M.
      • Miranker A.D.
      Fiber-dependent amyloid formation as catalysis of an existing reaction pathway.
      ). Such lateral association is a recognized process in recombinant PrP fibril formation (
      • Anderson M.
      • Bocharova O.V.
      • Makarava N.
      • Breydo L.
      • Salnikov V.V.
      • Baskakov I.V.
      Polymorphism and ultrastructural organization of prion protein amyloid fibrils: an insight from high resolution atomic force microscopy.
      ).
      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 (
      • Moore R.C.
      • Hope J.
      • McBride P.A.
      • McConnell I.
      • Selfridge J.
      • Melton D.W.
      • Manson J.C.
      Mice with gene targetted prion protein alterations show that Prnp, Sinc, and Prni are congruent.
      ,
      • Lloyd S.E.
      • Thompson S.R.
      • Beck J.A.
      • Linehan J.M.
      • Wadsworth J.D.
      • Brandner S.
      • Collinge J.
      • Fisher E.M.
      Identification and characterization of a novel mouse prion gene allele.
      ,
      • Carlson G.A.
      • Goodman P.A.
      • Lovett M.
      • Taylor B.A.
      • Marshall S.T.
      • Peterson-Torchia M.
      • Westaway D.
      • Prusiner S.B.
      Genetics and polymorphism of the mouse prion gene complex: control of scrapie incubation time.
      ,
      • Akhtar S.
      • Wenborn A.
      • Brandner S.
      • Collinge J.
      • Lloyd S.E.
      Sex effects in mouse prion disease incubation time.
      ,
      • Lloyd S.E.
      • Onwuazor O.N.
      • Beck J.A.
      • Mallinson G.
      • Farrall M.
      • Targonski P.
      • Collinge J.
      • Fisher E.M.
      Identification of multiple quantitative trait loci linked to prion disease incubation period in 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.

      Acknowledgments

      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.

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