Novel strain properties distinguishing sporadic prion diseases sharing prion protein genotype and prion type

In most human sporadic prion diseases the phenotype is consistently associated with specific pairings of the genotype at codon 129 of the prion protein gene and conformational properties of the scrapie PrP (PrPSc) grossly identified types 1 and 2. This association suggests that the 129 genotype favours the selection of a distinct strain that in turn determines the phenotype. However, this mechanism cannot play a role in the phenotype determination of sporadic fatal insomnia (sFI) and a subtype of sporadic Creutzfeldt-Jakob disease (sCJD) identified as sCJDMM2, which share 129 MM genotype and PrPSc type 2 but are associated with quite distinct phenotypes. Our detailed comparative study of the PrPSc conformers has revealed major differences between the two diseases, which preferentially involve the PrPSc component that is sensitive to digestion with proteases (senPrPSc) and to a lesser extent the resistant component (resPrPSc). We conclude that these variations are consistent with two distinct strains in sFI and sCJDMM2, and that the rarer sFI is the result of a variant strain selection pathway that might be favoured by a different brain site of initial PrPSc formation in the two diseases.

Nonidet P-40 in 1X PBS pH 7.4. Then, a final wash with 1X PBS pH 7.4 was performed. In cases of PK treatment, the beads were resuspended in 30 µl of LB 100 pH 8.0 and incubated with PK (5 U/ml) for 1 hour at 37˚C constantly mixing; the reaction was blocked with 3 mM PMSF prior to the addition of 2X sample buffer and elution. Two alternative protocols were used for antigen elution of PK-untreated samples: i) incubation of the beads in 1X sample buffer (1.05% SDS, 13.2% glycerol, 0.005% bromophenol blue, 2.5% β -mercaptoethanol, 32.9 mM Tris-HCl, pH 6.8) and heating at 96°C for 6 min (PK-treated samples were directly boiled after addition of 2X sample buffer); ii) incubation of the beads in urea elution buffer (8 M urea, 2% CHAPS, 20 mM Tris pH 8.0) for 1 hour at room temperature with constant mixing. In both cases the beads were then separated from the eluate using a magnet. The eluates in 1X sample buffer were boiled at 100°C for 10 min whereas the samples in urea elution buffer were first reduced with 5 mM TBP, then alkylated with 20 mM iodoacetamide and then subjected to the ReadyPrep 2-D Cleanup Kit which reduces detergents, salts and lipids. The pellets were resuspended in rehydration buffer (7 M urea, 2 M thiourea, 1% DTT, 1% CHAPS, 1% Triton X-100, 0.2 % Bio-Lyte 3/10 ampholyte) for isoelectric focusing (IEF) applications, but where indicated they were resuspended in either 1X sample buffer or 1X Glycoprotein Denaturing Buffer.

Antibody co-elution
To test whether covalent bonding between 8B4 and Dynabeads M-270 Epoxy formed during coupling prevented antibody leakage during the elution, sCJDMM2 and sFI were subjected to IP-8B4; after the last wash with 1X PBS pH 7.4, the beads were separated in two tubes and eluted either in 1X sample buffer or urea elution buffer. For samples eluted in urea elution buffer, ReadyPrep 2-D Cleanup Kit was used and the resulting pellets were resuspended in 1X sample buffer and boiled at 100°C for 10 min. The samples were then subjected to 1-D electrophoresis and immunoblot with i) mAb 3F4; ii) rabbit anti-N-terminal antiserum anti-N; iii) rabbit anti-Cterminal antiserum 2301; iv) goat anti-mouse IgG (Fc) HRP conjugate (that was also used as secondary Ab for 3F4 detection); and v) donkey anti-rabbit IgG HRP-linked whole antibody (also used as secondary Ab for anti-N and 2301 detection).

Immunoprecipitation with OCD4
IP with OCD4 was performed as described 6 , with minor modifications. OCD4-coupled beads were incubated with similar amounts of PrP in each sample. Samples referred to as fractions 4-8 and 17-21 were obtained by combining the contents of the selected fractions obtained with SE experiments from N=2 cases of sCJDMM2 and sFI. S1 and S2 samples (see "Preparation of detergent insoluble fraction") were obtained from a case unaffected by neurological disease.

1-D electrophoresis and immunoblot
Proteins were separated with 15% Criterion Tris-HCl polyacrylamide precast gels and blotted into Immobilon-FL PVDF membranes for 2 hours at 60V, which were then rinsed with 1X PBS and blocked with Odyssey Blocking Buffer for 1 hour at room temperature before being probed overnight, at 4°C. The primary antibodies were diluted in Odyssey Blocking Buffer with 0.1% Tween 20 as follows: 3F4 (1:40,000 except for SE, SV and CSSA in which it was used at a dilution of 1:4,000); anti-N (1: 2,000); 2301 (1:3,000) and 8B4 (1:10,000, stock 2 mg/ml). After four washes with PBS-T (1X PBS with 0.1% Tween 20), the membranes were incubated for 1 hour with IRDye 800CW goat anti-mouse IgG (1:15,000) or IRDye 680RD goat anti-rabbit IgG PBS, membranes were developed by Odyssey Classic infrared imaging system (LI-COR Biosciences), as described by the manufacturer. Densitometric analysis was performed using the Odyssey application software V3.0 (LI-COR Biosciences). After normalization, the data were plotted and expressed as mean ± standard deviation.
For IP experiments immunoblots were developed by chemiluminescence, requiring the use of a compatible membrane (Immobilon-P PVDF), blocking and antibody incubation buffers (5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T)), HRP-conjugated secondary antibodies and chemiluminescent substrate for developing (further details under 2-D electrophoresis and immunoblot).

2-D electrophoresis and immunoblot
The first dimension (isoelectric focusing) of 2-D PAGE was performed as previously described with minor modifications 11 . Briefly, 11 cm Ready-Strip IPG Strips pH 3-10 were rehydrated for 15 hours with the samples and subjected to isoelectric focusing with PROTEAN IEF Cell (Bio-Rad), for ∼ 40,000 Vh. The IPG Strips were then equilibrated in SDS and reducing/alkylating agents in a two-step process by the addition of 130 mM DTT and 135 mM iodoacetamide, respectively, to the equilibration buffer (6M urea, 2% SDS, 20% glycerol, 0.375 M Tris-HCl pH 8.8), prior to running the second dimension gels. Criterion Tris-HCl polyacrylamide precast gels (8-16%) were used. Proteins were transferred into Immobilon-P PVDF membranes for 2 hours at 60 V; the membranes were then blocked with 5% non-fat dry milk in TBS-T for 1 hour at room temperature before being incubated overnight, at 4°C, with mAb 3F4 (1:40,000). After five washes with TBS-T, the membranes were incubated with goat anti-mouse IgG (Fc)-HRP conjugate (1:5,000) for 1 h at room temperature, washed again and developed by enhanced chemiluminesce reaction using ECL and ECL plus reagents, as recommended by the manufacturer. The signal was captured on Kodak MR and XAR films.

Sodium phosphotungstate precipitation
After addition of a protease inhibitors cocktail (final concentration 1mM PMSF and 5μg/ml aprotinin and leupeptin), the 5% homogenates were incubated with a solution 10% (w/v) NaPTA/85 mM MgCl 2 (final concentration 0.32% NaPTA/2.72 mM MgCl 2 ) for at least 1h at The supernatant obtained, identified as S1, was then ultracentrifuged at 100,000 x g for 1 h at 4°C in a SW55 rotor (Beckman) to generate a supernatant (S2) and pellet (P2) for each sample 12 .
The P2 were resuspended in cold LB 100 pH 8.0; the S2 were stored in -80°C.

Preparation of detergent insoluble fraction under stringent conditions
Stringent conditions were used to maximize PrP C solubility and to guarantee that in the resulting P2 the level of PrP C contamination was less than 1% of the total PrP contained in the BH.
Briefly, BH (20% w/v) in 1X TEND (65 mM NaCl, 0.5 mM EDTA, 10 mM Tris pH 8.0) were mixed with an equal volume of a solution 20% sarkosyl NL in 1X TEND containing a protease inhibitor cocktail, and incubated at 4˚C for 30 min with frequent mixing. S1, obtained by centrifuging the samples at 18,000 rcf for 25 min at 4°C, were subsequently centrifuged at 100,000 x g for 2 h at 4°C in a SW55 rotor to generate S2 and P2 obtained under stringent conditions 13 , with minor modifications. The P2 were either resuspended in LB 100 pH 8.0 or 1% sarkosyl NL TNE pH 7.2 (150 mM NaCl, 5 mM EDTA, 25 mM Tris pH 7.5).

Sedimentation velocity
SV was performed to assess the distribution of PrP Sc aggregates in a gradient according to size 14 .
The protocol used was comparable to that of SE with two major exceptions: i) time of centrifugation (1 hour instead of 19 hours); ii) type of sucrose gradient (5-15% instead of 10-60%). The choice of a different range of sucrose concentrations was based on the results obtained with SE, with the goal of having SV comparisons based primarily on particle size, thus using a gradient less dense than the particles that were attempted to be separated by size.
Incubation of P2 samples in LB containing 1% sarkosyl NL prior to PrP Sc sedimentation did not affect the SV profile.

Conformational stability and solubility assay
Five aliquots of P2 in LB 100 pH 8 (30µl), from the same brain equivalents (w/v), were diluted 1:1 with GdnHCl solutions to obtain final GdnHCl concentrations ranging between 0 M and 4 M.
The samples were incubated at 37˚C for 1 h while mixing and then centrifuged at 22˚C for 20 min at 16,000 x g. Since insoluble PrP Sc was the focus of the investigation, pellets were resuspended in 1X sample buffer and boiled for 10 min at 100˚C. In addition, and different from the original protocol, the assay was also applied to PK-treated P2 samples, to compare the solubility curves of resPrP Sc with those of totPrP Sc . PK digestion (PK 5 U/ml) was performed before incubation with GdnHCl solutions 15 . Finally, all samples were subjected to immunoblotting with 3F4. After densitometry analysis using Odyssey application software V3.0 and normalization, individual dose-response best-fitted denaturation curves and [GdnHCl] 1/2 values were determined using GraphPad Prism 6 (GraphPad Software). The mean [GdnHCl] 1/2 values for each group ± standard error were determined and plotted.

SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure S1. Western blot profiles and glycoform ratios of PK-treated and untreated PrP Sc as well as presumed PrP C from thalamus of sCJDMM2 and sFI. BH from thalamus of sCJDMM2 (N=3) and sFI (N=3) were processed as described in Figure 1. Analyses of totPrP Sc (A), resPrP Sc (B) and "PrP C " (C) reveal that the differences in glycoform ratios between sCJDMM2 and sFI (D-F) are comparable to those observed in the frontal cortex ( TotPrP Sc from sCJDMM2 and sFI cases were centrifuged at high speed for 19 hours in a 10-60% sucrose gradient. Identical aliquots were collected from each fraction and processed for WB (A).
B: Quantitative analyses of the totPrP Sc glycoforms was carried out in the low-density (fractions 1-7 for sCJDMM2 and 3-9 for sFI) and high-density peaks (fractions 16-20). In sFI, the totPrP Sc glycoform ratio in the low-density fractions differs from that in high-density fractions (P < 0.05 for di-and un-glycosylated conformers) while in sCJDMM2 the glycoform ratio does not significantly change along the gradient. As a result of this divergence, glycoform ratios differ between sFI and sCJDMM2 in the low-density fractions but not in the high-density fractions. In sCJDMM2, glycoform ratios are 20:48:32 in peak 1-7 and 13:46:41 in peak 16-20; in sFI they are 41:50:9 in peak 3-9 and 24:45:31 in peak 16-20.
Supplementary Figure S4. Immunoprecipitation of sedimentation equilibrium fractions with the conformational antibody OCD4 that selectively immunoreacts with misfolded PrP.
A: Relatively large quantities of PrP, likely totPrP Sc , are recovered from sCJDMM2 and sFI fractions 4-8 (low-density) and 17-21 (high-density) but not from S1 and S2 fractions of a negative case, harboring mostly PrP C . Note the weak signal in the S1 sample, likely representing insoluble PrP 11 . B: Shorter exposure demonstrates the presence of the h.m.w components (arrows) in both sets of sedimentation fractions from sCJDMM2, but only in the 17-21 fractions from sFI, as well as the prominence of the di-glycosylated form in fractions 4-8 from sFI (arrowhead).
Supplementary Figure S5. Glycoform ratios of resPrP Sc following sedimentation equilibrium centrifugation. Individual fractions of totPrP Sc obtained from sCJDMM2 and sFI were digested with PK (10 U/ml) and processed for WB (A). A non-specific band of ~32 kDa is also observed (double arrow). B: The resPrP Sc glycoform ratios in fractions 16-20, although apparently similar in profile, differ in their relative amounts of mono-and un-glycosylated forms (28:58:14 for sCJDMM2 and 28:43:29 for sFI).
Supplementary Figure S6. Sedimentation equilibrium of resPrP Sc generated by PK treatment of totPrP Sc from sCJDMM2 and sFI before fractionation. A: Sedimentation profile of resPrP Sc demonstrating two partially overlapping but distinct peaks that populate fractions of lower density in sFI than in sCJDMM2 (8-18 in sFI vs. 14-21 in sCJDMM2). B: