PrPSc with Seeding Activity Extensively Overlaps with Proteinase-Resistant PrPSc Rather than Infectious PrPSc

Abstract

The disease-associated prion protein (PrPSc) has the ability to seed the conformational conversion of normal prion proteins into the amyloid fibril form. This prion seeding activity can be measured using an in vitro amplification assay termed real-time quaking-induced conversion (RT-QuIC). There is a strong correlation between RT-QuIC positivity and prion infection; however, the relationship between seeding activity and infectivity remains elusive. In this study, we used endpoint dilution RT-QuIC on the brain homogenates from wild-type mice with mouse-adopted bovine spongiform encephalopathy (mBSE) at defined intervals during the incubation period and evaluated the temporal relationship among prion seeding dose, levels of proteinase-resistant PrPSc (PrPres), and infectious titer. We found that the infectious titer reached a plateau by 100 days postinfection, whereas seeding dose and PrPres levels were continuously elevated. Our calculation showed that the doubling time (dt) for seeding dose from 40 to 100 days postinoculation was closer to the dt for PrPres levels than to the dt for prion titer. Although an uncoupling of seeding doses and PrPres levels was observed at end-stage disease in this model, our findings suggest that there is substantial but not complete overlap between PrPSc with seeding activity and PrPres rather than infectious PrPSc.

Keywords: amyloid; bovine spongiform encephalopathy; hamster; infectivity; kinetics; mouse model; prion; real-time quaking induced conversion (RT-QuIC); scrapie; seeding activity.

Figure 1

Real-time quaking-induced conversion (RT-QuIC) for the detection of prion seeding activity in a brain from a terminally ill CD-1 mouse inoculated with mouse-adapted BSE (mBSE): Syrian hamster recombinant prion protein (amino acids 90–231) was used as a substrate for the RT-QuIC reactions. The seeded brain tissue equivalents (2 µg to 200 fg from an mBSE-inoculated mouse or with 20 ng brain tissue from a normal mouse) are shown at the bottom. (a) Each dot represents the amyloid formation rate (AFR) measured in a 96-well plate rinsed with (pale red) or without (grey) an acetone-ethanol mixture. The 96-well plates from three lot numbers were used for the four analyses. (b) Line plot of Figure 1a. The mean ± SD of the AFR is displayed for each dilution. Significant differences at p < 0.05 (*) between AFR measured in rinsed and non-rinsed 96-well plates are indicated. (c) Each box represents Spearman–Käber estimates of the SD₅₀ per unit per gram of brain tissue measured in a 96-well plate rinsed with (pale red) or without (grey) the acetone-ethanol mixture. Data are derived from quintuplicate wells of four experiments for each brain tissue dilution.

Figure 2

Kinetics of mouse-adopted bovine spongiform encephalopathy (mBSE) prion seeding activity, infectivity, and PrPres (proteinase-resistant disease-associated prion protein (PrPSc)) accumulation in mouse brains during disease progression: (a) Seeding dose (red circle), infectious titer (blue circle), and PrPres accumulation (black circle) in mouse brains are shown. The seeding doses of mBSE prion at each time point were determined using end-point dilution RT-QuIC. Each blue circle represents the mean ± SD of Spearman–Käber estimates of the SD₅₀ per gram of brain and was derived from quadruplicate wells of at least three experiments. The infectious titers of mBSE prion per gram of brain at each time point were estimated using the incubation period bioassay method. The levels of PrPres in 0.1 mg brain equivalents at the same time points were measured using ELISA with the Seprion ligand derived from triplicate wells of two experiments and plotted on a log scale in arbitrary light units. The kinetics of seeding activity, infectivity, and PrPres accumulation are shown as a semilogarithmic plot. A linear relationship was observed from 40 to 100 dpi of seeding activity (red filled circle and dashed line: y = 3,541,353* e^(0.085361x) R² = 0.905892, where y is the seeding dose and x is the survival days), infectivity (blue filled circle and dashed line: y = 296,647* e^(0.056066x) R² = 0.970865, where y is the infectious titer and x is the survival days), and PrPres accumulation (black filled circle and dashed line: y = 19.2724* e^(0.083569x) R² = 0.972250, where y is the relative light units and x is the survival days). (b) Western blot analysis of PrPres in the brains of mice: PrPres was probed with anti-PrP mAb T2. The days postinoculation (dpi) are presented on the top of the blot. NI indicates the uninfected mouse brain. The protein load in each lane is presented on the bottom of the blot. (c) Deposition of PrPSc stained with an SAF84 antibody in coronal sections of the brains of mBSE-inoculated mice in the cerebral cortex upper hippocampal CA1 region. CC, corpus callosum; HP, hippocampus; NC, neocortex.

Figure 3

Comparison of PrPres levels and seeding activity: The 10% (w/v) brain homogenates of mouse-adopted bovine spongiform encephalopathy (mBSE)-affected mice (crude) were centrifuged at 20,000× g for 10 min at 4 °C. After collecting the supernatant (20K-sup), the pellet was reconstituted with the same volume of phosphate buffered saline (PBS) with brief sonication (20K-ppt). (a, panel at left) Western blot analysis of PrPres levels in the samples: PrPres in proteinase K (PK)-treated crude, 20K-ppt, and 20K-sup samples were detected with anti-PrP mAb T2. Brain tissue equivalents (µg) loaded per lane are indicated at the bottom of the blots. Molecular mass standards (kDa) are shown on the right side of the blots. (a, panel at right) Densitometric analysis of the PrPres levels: Box plots depict values from the densitometric analysis of western blots from three mBSE-inoculated mice. (b) RT-QuIC endpoint dilution analysis of the brain samples from three mBSE-inoculated mice. Box plots depict Spearman–Käber estimates of the SD₅₀ per gram brain tissue, which was derived from three different experiments.

Figure 4

Proteinase K (PK) sensitivity of seeding activity in brain homogenates (BHs) from CD-1 mice infected with mouse-adopted bovine spongiform encephalopathy (mBSE): (a) Western blot analysis of PrP from mBSE samples. The mBSE BHs were pretreated with 0.5% triton X-100 (final concentration) and digested with 0, 5, and 40 µg/mL of PK. Bands were probed with anti-PrP mAb T2. (b) End-point dilution RT-QuIC analysis of samples: Each box represents Spearman–Käber estimates of the SD₅₀ per unit per gram of brain tissue from three different experiments.