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

Abstract

Prion diseases are fatal neurodegenerative disorders associated with the polymerization of the cellular form of prion protein (PrP(C)) into an amyloidogenic β-sheet infectious form (PrP(Sc)). The sequence of host PrP is the major determinant of host prion disease susceptibility. In mice, the presence of allele a (Prnp(a), encoding the polymorphism Leu-108/Thr-189) or b (Prnp(b), Phe-108/Val-189) is associated with short or long incubation times, respectively, following infection with PrP(Sc). The molecular bases linking PrP sequence, infection susceptibility, and convertibility of PrP(C) into PrP(Sc) remain unclear. Here we show that recombinant PrP(a) and PrP(b) aggregate and respond to seeding differently in vitro. Our kinetic studies reveal differences during the nucleation phase of the aggregation process, where PrP(b) exhibits a longer lag phase that cannot be completely eliminated by seeding the reaction with preformed fibrils. Additionally, PrP(b) 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.

FIGURE 1.

Time courses of PrP^a and PrP^b fibril formation monitored by ThT fluorescence. PrP (22 μm) was incubated in 50 mm phosphate buffer, pH 7.0, 2 m GdnHCl at 37 °C and 500 rpm in the absence or in the presence of seeds. A and B, kinetics of unseeded (A) and seeded (B) polymerization reactions of PrP^a (empty circles) and PrP^b (filled circles). C, lag phases for the kinetics of PrP^a (empty bars) and PrP^b (filled bars) plotted in A and B. D, lag phase of PrP^a (left) and PrP^b (right) aggregation process in the presence of 0.01, 0.1, 0.5, or 2% preformed homologous fibrils. The fibril formation process was followed by ThT fluorescence, and the lag phase was estimated as the intersection of quadratic and exponential fitting curves as described under ”Experimental Procedures.“ Three replicates were plotted for each reaction. Error bars indicate ± S.E.

FIGURE 2.

Time courses of PrP^a and PrP^b fibril formation in the presence of homologous and heterologous seeds. Monomeric PrP (22 μm) was seeded with 0.5% (v/v) of fibrils, and the kinetics were followed by ThT fluorescence. A, monomeric PrP^a (empty circles) and PrP^b (filled circles) were aggregated in the presence of preformed fibrils of homologous PrP (PrP^a with PrP^a fibrils; PrP^b with PrP^b fibrils). B, monomeric PrP^a (empty circles) and PrP^b (filled circles) were aggregated in the presence of preformed fibrils of heterologous PrP (PrP^a with PrP^b fibrils; PrP^b with PrP^a fibrils). C, the lag phases for PrP^a and PrP^b in the presence of homologous (Homo.) and heterologous (Hete.) seeds were calculated as described under ”Experimental Procedures.“ The average of three replicates was plotted for each reaction. Error bars indicate ± S.E.

FIGURE 3.

Kinetics of PrP^b aggregation seeded at different times. A, monomeric PrP^b (22 μm) was aggregated in the absence (filled circles) or in the presence (empty squares, triangles, and circles) of 0.5% (v/v) homologous seeds. The seeds were added at the beginning of the reaction (squares), after 5 h (triangles), or after 10 h (empty circles). The kinetics were normalized, and the percentage of ThT fluorescence was graphed. B, the same data were plotted as absolute fluorescence using a different y scale to focus on the processes happening before the exponential phase. The slopes were calculated by fitting the data with linear regression. The arrows in A and the dotted lines in B indicate the times at which the seeds were added. Three replicates were averaged for each reaction. Error bars indicate ± S.E.

FIGURE 4.

PrP^b prefibrillar (ThT-negative) oligomer formation monitored by p-FTAA. Monomeric PrP^b (20 μm) was put into aggregation assays in 1.5-ml test tubes. 50 μl was withdrawn at different times and incubated with p-FTAA (squares) or ThT (circles) to monitor the formation of prefibrillar oligomers and fibril formation, respectively. The assay was carried out in the absence (filled symbols) or in the presence (empty symbols) of 1% (v/v) of seeds.

FIGURE 5.

PrP aggregation at different monomeric concentrations. A and B, PrP^a (A) and PrP^b (B) were put into aggregation assays at three different concentrations: 27 μm (squares), 16 μm (circles), and 11 μm (triangles). Three replicates were averaged for PrP^a (A), but individual replicates were plotted for PrP^b (B) to better display the high heterogeneity observed for these reactions. C, the lag phases for PrP^a (empty bars) and PrP^b (filled bars) under the different PrP monomer concentrations were calculated from the intersection of quadratic and exponential fitting curves, as described under ”Experimental Procedures.“ Error bars indicate ± S.E.

FIGURE 6.

Conformational stability assays of PrP fibrils formed under seeded and unseeded conditions. A–F, Western blot of PrP^a (A–C) and PrP^b (D–F) fibrils formed in the absence (A and D) or in the presence of homologous (B and E) or heterologous (C and F) seeds and subjected to GdnHCl-induced denaturation and proteinase K digestion. 12 μl of proteinase K-treated samples was analyzed by Western blotting using SAF83 antibody, as described under “Experimental Procedures.” MW, molecular weight markers.

FIGURE 7.

Influence of slow and fast seeds on the kinetics of PrP fibril formation. A and B, 22 μm PrP^a (A) or PrP^b (B) was incubated in the absence (A, open triangles) or the presence of 0.5% (v/v) slow (filled circles) or fast (filled squares) seeds. The original kinetics of the reactions that produced the S and F seeds are shown for comparison, as a dotted line (F) and dashed line (S). n = 3 for unseeded reactions; n = 2 for seeded reactions. Error bars indicate ± S.E.

FIGURE 8.

Influence of different seeds on the structure of PrP fibrils. a–f, images of PrP^a (a–c) and PrP^b (d–f) fibrils generated in the absence of seeds (a and d) or in the presence of fast (b and e) or slow (c and f) seeds were obtained by TEM. Scale bars, 100 nm.

FIGURE 9.

Proposed mechanism of PrP^b 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.