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Keywords:

  • prion;
  • protein misfolding cyclic amplification;
  • species barrier;
  • strain diversity

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Prions are the infectious agents responsible for transmissible spongiform encephalopathy, and are primarily composed of the pathogenic form (PrPSc) of the host-encoded prion protein (PrPC). Recent studies have revealed that protein misfolding cyclic amplification (PMCA), a highly sensitive method for PrPSc detection, can overcome the species barrier in several xenogeneic combinations of PrPSc seed and PrPC substrate. Although these findings provide valuable insight into the origin and diversity of prions, the differences between PrPSc generated by interspecies PMCA and by in vivo cross-species transmission have not been described. This study investigated the histopathological and biochemical properties of PrPSc in the brains of tga20 transgenic mice inoculated with Sc237 hamster scrapie prion and PrPSc from mice inoculated with Sc237-derived mouse PrPSc, which had been generated by interspecies PMCA using Sc237 as seed and normal mouse brain homogenate as substrate. Tga20 mice overexpressing mouse PrPC were susceptible to Sc237 after primary transmission. PrPSc in the brains of mice inoculated with Sc237-derived mouse PrPSc and in the brains of mice inoculated with Sc237 differed in their lesion profiles and accumulation patterns, Western blot profiles, and denaturant resistance. In addition, these PrPSc exhibited distinctive virulence profiles upon secondary passage. These results suggest that different in vivo and in vitro environments result in propagation of PrPSc with different biological properties.

List of Abbreviations: 
AP

alkaline phosphatase

BSE

bovine spongiform encephalopathy

CJD

Creutzfeldt-Jakob disease

CWD

chronic wasting disease

GdnHCl

guanidine hydrochloride

HE

hematoxylin and eosin

HRP

horseradish peroxidase

PK

proteinase K

PMCA

protein misfolding cyclic amplification

PNGase F

peptide: N-glycosidase F

PrP

prion protein

PrPC

host-encoded cellular prion protein

PrPC-res

proteinase K-resistant aggregated PrPC

PrPSc

scrapie form of PrPC

SEM

standard error of the mean

TSE

transmissible spongiform encephalopathy

vCJD

variant Creutzfeldt-Jakob disease

WB

Western blot

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders and include BSE, scrapie in sheep and goats, CWD in deer and elk, and CJD in humans (1). Prions are the infectious agents responsible for TSEs, which are characterized by PrPSc accumulation; PrPSc has a substantially different conformation than that of PrPC (2). Different prion strains have been identified in most species affected by TSE. This strain diversity can be explained by the inherent conformational flexibility of each type of PrPSc, which confers a specific disease phenotype in regard to characteristics such as incubation period, clinical symptoms, and neuropathological characteristics (3, 4). Prion strains are also characterized by the biochemical properties of PrPSc, including their glycosylation profiles, electrophoretic mobility, and resistance to proteases and denaturants (5, 6). However, the mechanism by which a variety of PrPSc are generated from the same primary structure has not yet been elucidated.

Recent studies have reported that PMCA, a highly sensitive method for PrPSc detection (7–9), can overcome the species barrier in several xenogeneic combinations of PrPSc seed and PrPC substrate, such as deer–ferret (10), deer–mouse (11), hamster–mouse, and mouse–hamster (12). Since BSE and vCJD are suspected to be attributable to cross-species transmission of prions (13), interspecies PMCA offers valuable insight into the origin and diversity of prions. However, a detailed comparison of PrPSc generated by interspecies PMCA and PrPSc accumulated in brains following cross-species transmission has not been undertaken because prion diseases are generally less transmissible to heterogeneous species. For example, a hamster scrapie prion strain, Sc237, has been described as non-infectious in wild-type mice because no clinical sign of disease was observed more than 735 days after intracerebral inoculation (14). This phenomenon is known as the “species barrier” (15), and is probably due to the species-specific physicochemical properties of prion proteins (16).

Although generation of a novel PrPSc with high infectivity can be reproduced in just a few days by interspecies PMCA (12), studies on the differences in PrPSc generated in vivo and in vitro are needed to examine whether PMCA is comparable to cross-species transmission of prion diseases in vivo. In this study, we demonstrated that tga20 transgenic mice overexpressing mouse PrPC are susceptible to Sc237 following primary transmission. Therefore, we could compare PrPSc accumulated in the brains of mice inoculated with Sc237 and PrPSc from mice inoculated with Sc237-derived mouse PrPSc generated by interspecies PMCA using Sc237 as PrPSc seed and normal mouse brain homogenate as PrPC substrate. The PrPSc that accumulated in the brains of these mice differed in their histopathological and biochemical properties, and exhibited different virulence patterns upon secondary transmission, suggesting that PrPSc with different biological properties propagate in a heterogeneous environment depending on whether they are produced in vivo or in vitro.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

All animal experiments were performed according to the guidelines of the National Institute of Animal Health.

PMCA

To avoid contamination, normal brain homogenates were prepared in a laboratory that had never contained infected materials. To compare the amplification efficiencies of PrPSc in wild-type mouse- and transgenic mouse-based amplifications, the brains of ICR mice, PrPC-overexpressing tga20 transgenic mice (17), and PrP knockout (PrP0/0) mice were separately homogenized in 20% (w/v) PBS containing complete protease inhibitors (Roche Diagnostics, Mannheim, Germany). The homogenates were stored in small aliquots at −80°C. The homogenates were mixed with an equal volume of elution buffer (PBS containing 2% Triton X-100 and 8 mM EDTA) and incubated at 4°C for 1 hr with continuous agitation. After centrifugation at 4500 g for 5 min, the supernatant of ICR brain homogenate was ready for use as a PrPC substrate. The brain homogenates (10%) of PrP0/0 and tga20 mice were prepared as described above, and mixed in a 5:1 proportion of PrP0/0:tga20. This mixture was also used as a PrPC substrate.

Mouse-adapted scrapie strain Chandler was used as the PrPSc seed. The prion strain was propagated in ICR mice. The brains of mice in the terminal stage of disease were homogenized at a 10% concentration (w/v) in PBS. PMCA was carried out using a fully automatic cross-ultrasonic protein activating apparatus (Elestein 070-GOT, Elekon Science, Chiba, Japan) as reported previously (18). Amplification was performed by 40 cycles of sonication, in which a 3 s pulse oscillation was repeated 5 times at 0.1 s intervals, followed by incubation at 37°C for 1 hr with gentle agitation (19).

Interspecies PMCA

PrPC substrate prepared from ICR mice was used for interspecies PMCA. In addition, PrPC substrate containing digitonin was prepared by adding digitonin (Nacalai Tesque, Kyoto, Japan) to a mixture of mouse brain homogenate and elution buffer at a final concentration of 0.05% prior to incubation and centrifugation. This PrPC substrate was used for the first and second rounds of amplification.

A hamster-adapted scrapie prion strain, Sc237, was propagated in hamsters. When the animals had reached the terminal disease stage, they were killed and their brains pooled and homogenized in 10% (w/v) PBS containing 1% Triton X-100 and 4 mM EDTA. After centrifugation at 4500 g for 5 min, the supernatant was used as the PrPSc seed. The Sc237 seeds were diluted 1:100 in ICR mouse PrPC substrate containing digitonin (total volume, 100 μL) in an electron-beam irradiated polystyrene tube. Amplification was performed by 40 cycles of sonication, in which a 3 s pulse oscillation was repeated five times at 1 s intervals, followed by incubation at 37°C for 1 hr with gentle agitation. After two cycles of 1:10 dilution of the PMCA product and subsequent amplification, the PrPSc signal on WBs was gradually attenuated. The pulse oscillation interval was changed from 1.0 to 0.1 s, and amplification was performed in the absence of digitonin after the second amplification round as reported previously (19). The PMCA products were diluted 1:10 in each of the second through eighth rounds of amplification, and 1:1 000–1:10 000 from the ninth through the 22nd round. The final dilution of the PrPSc seed was 10−39 in the PMCA product of the 22nd amplification round. A negative control reaction (PrPC substrate only) was performed simultaneously in the same manner.

Western blotting

Samples (10 μL) from each round of amplification were mixed with 10 μL of PK solution (100 μg/mL) and incubated at 37°C for 1 hr. In some experiments, PK-digested samples were treated with PNGase F (New England BioLabs, Ipswich, MA, USA) to remove sugar chains on the PrPSc molecules, according to the manufacturer's instructions. The digested materials were mixed with 20 μL of 2 × SDS sample buffer and incubated at 100°C for 5 min. The samples were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). After blocking, the membrane was incubated for 1 hr with HRP-conjugated T2 monoclonal antibody diluted to 1:10,000. The T2 antibody, which recognizes a discontinuous epitope in amino acid residues 132–156 in the mouse PrP sequence, reacts with both mouse and hamster PrP (20, 21). Anti-PrP monoclonal antibodies 4E10 (HRP conjugated, diluted to 1:10,000) and 3F4 (AP conjugated, diluted to 1:10,000) were also used. The 4E10 antibody recognizes an epitope in amino acid residues 147–158 (RYYRENMYRYPN) of the mouse PrP sequence (22), but does not react with hamster PrP. The 3F4 antibody recognizes an epitope in amino acid residues 110–113 (MKHM) of the hamster PrP sequence (23), but does not react with mouse PrP. After washing, the blotted membrane was developed using Immobilon Western chemiluminescent HRP or AP substrate (Millipore), according to the manufacturer's instructions. Chemiluminescence was analyzed with the Light Capture system (Atto, Tokyo, Japan).

Cross-species transmission of scrapie PrPSc

The 10% homogenate of Sc237-infected hamster brains was injected intracerebrally (20 μL per mouse) into five tga20 transgenic and five Tg52NSE transgenic mice (24), which overexpress mouse PrPC and hamster PrPC, respectively. The PMCA product obtained in the 22nd round (R22) of amplification was diluted 1:10 and injected intracerebrally into five tga20 and five Tg52NSE mice. Densitometric analysis of WBs revealed that the PrPSc signal intensity in the PMCA product was approximately one-eighth of that in a 1% homogenate of Sc237-infected hamster brain. The 10% homogenate of Sc237-infected brains was therefore diluted by 1:800 for injection into the control mice. Animals at the terminal stage of disease were killed and the right hemisphere of the brain stored at −80°C for biochemical analysis. For secondary transmission, brain homogenates (10%) of the Sc237-infected (Sc237/tga #1) and PMCA product-inoculated mice (PMCA/tga #1) were injected intracerebrally (20 μL per mouse) into four and five tga20 transgenic mice, respectively.

Biochemical characterization of scrapie PrPSc that accumulated in the brains of tga20 mice

Resistance to PK digestion and GdnHCl denaturation were examined in the PrPSc that accumulated in the brains of tga20 mice inoculated with Sc237 and PrPSc from the mice inoculated with the R22 PMCA product. To examine PK sensitivity, 10% brain homogenates of Sc237- and PMCA product-inoculated mice were digested with various concentrations of PK (0.05–5 mg/mL) at 37 °C for 1 hr. The digested materials were immediately mixed with an equal volume of 2 × SDS sample buffer and incubated at 100°C for 5 min. The samples were analyzed by WB in three independent experiments. The average intensity of the PrPSc signal in each sample was expressed as the percentage of that in the sample digested with 0.05 mg/mL PK. The results were analyzed by one-way ANOVA and Tukey's multiple comparison test. The PK50 value, which is the concentration of PK needed to reduce the signal intensity by half, was estimated from an approximate curve calculated with the experimental data.

To perform the guanidine denaturation assay, the 10% homogenate of Sc237- and PMCA product-inoculated brains was incubated with agitation at room temperature for 2 hr in various concentrations of GdnHCl ranging from 0 to 4.0 M. The samples were incubated with agitation at 4°C for 30 min in the presence of 10% Sarkosyl, and centrifuged at 100,000 g for 1 hr in a TL-100 ultracentrifuge (Beckman Coulter, Brea, CA, USA). The pellets were dissolved directly in a PK solution (0.05 mg/mL) and incubated at 37°C for 1 hr. The samples were analyzed by WB in three independent experiments. The average intensity of the PrPSc signal in each sample was expressed as a percentage of that in the sample prepared without GdnHCl. The results were statistically analyzed as described above. The GdnHCl50 value, which is the concentration of GdnHCl needed to reduce the signal intensity by half, was estimated from an approximate curve calculated from the experimental data.

Histopathological analysis

The left hemispheres of the brains were fixed in 10% buffered formalin for neuropathological analysis. Coronal slices of the brains were immersed in 98% formic acid to reduce infectivity and embedded in paraffin wax. Sections (4 μm) were cut and stained with HE. The lesion profile was determined by examination of the HE-stained sections and scoring the vacuolar changes in nine standard grey matter areas (25).

For PrPSc immunohistochemistry, sections were pretreated with 3% H2O2 for 10 min at room temperature, and incubated with 10 μg/mL PK in PBS containing 0.1% Triton-X for 10 min, followed by 10 min incubation in 150 mM sodium hydroxide at 60°C. PrPSc was detected in brain sections using anti-PrP monoclonal antibody SAF84 (SPI-Bio, Montigny le Bretonneux, France). The sections were incubated with SAF84 antibody at a concentration of 1 μg/mL for 60 min. Immunoreactions were developed using anti-mouse universal immuno-peroxidase polymer (Nichirei Histofine Simple Stain MAX-PO (M), Nichirei, Tokyo, Japan) as the secondary antibody, and 3–3′diaminobenzidine tetrachloride as the chromogen with a Dako Cytomation Autostainer Universal Staining System (Dako, Carpinteria, CA, USA).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Amplification efficiencies of mouse PrPSc in ICR- and tga20-based amplifications

We compared the amplification efficiencies of Chandler PrPSc using ICR and tga20 brain homogenates as PrPC substrates. For tga20-based amplification, it was necessary to dilute tga20 brain homogenate with PrP0/0 brain homogenate to obtain good amplification. Otherwise, the amplified PrPSc signal was indistinguishable from high background signals from protease-undigested PrP molecules (data not shown). Homogenates (10%) of Chandler-infected brain were diluted 1:100–1: 100,000 with each of the PrPC substrates, and one round of PMCA was performed (Fig. 1). A PrPSc signal was detected in duplicate samples of the 10−5 dilution samples in both ICR- and tga20-based amplifications, and WB profiles of amplified PrPSc from both amplifications were very similar. These observations suggest that wild-type PrPC and diluted transgenic PrPC have similar PrPSc amplification capabilities to Chandler strains in vitro. In the present study, ICR brain homogenate served as the PrPC substrate for convenience and low background signal.

image

Figure 1. Amplification of mouse-adapted Chandler scrapie PrPSc by PMCA. (a) PMCA was performed using ICR brain homogenate as the PrPC substrates. Homogenates (10%) of Chandler-infected brains were diluted 1:100–1:100,000 with the PrPC substrate. Amplification was performed in duplicate except for the negative control. (b) Amplification of Chandler PrPSc was performed using a mixture (1:5) of brain homogenates of tga20 and PrP0/0 mice as the PrPC substrate. After PK digestion, the PMCA products were analyzed by WB using HRP-T2 monoclonal antibody. Arrows indicate the positions of molecular mass markers corresponding to 37, 25, 20, and 15 kDa (Western C standards, Bio-Rad Laboratories, Hercules, CA, USA). N, control in which only PrPC substrate was amplified.

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Generation and amplification of mouse PrPSc by interspecies PMCA

PrPSc of the hamster prion strain Sc237 was amplified by PMCA with mouse PrPC substrate. Since the 4E10 antibody does not react with hamster PrP because of two amino acid substitutions in the epitope, it is possible to determine the species from which PK-resistant PrP is derived. The generation of mouse PrPSc in the first round of amplification was clearly demonstrated by use of the 4E10 antibody (Fig. 2a). In the presence of digitonin, however, the PrPSc signal gradually decreased from the second to third round of amplification. Although addition of digitonin to the reaction mixture effectively reduces the background signal caused by aggregated PrPC (PrPC-res) in mice (17), digitonin might adversely affect interspecies amplification. The PrPSc molecules can be amplified by omitting digitonin from the amplification buffer (Fig. 2b,c), as described in the Materials and Methods section.

image

Figure 2. Amplification of Sc237 hamster PrPSc by interspecies PMCA. (a) Hamster Sc237 PrPSc was amplified by serial PMCA with mouse PrPC substrate. Amplification was performed in the presence of digitonin, and the R1–R3 samples were analyzed by WB with HRP-4E10 and AP-3F4 antibodies following PK digestion before (–) and after (+) amplification. Lanes labeled “No seed” represent the control reactions, which contained only the PrPC substrate. Numerals in parentheses indicate the Sc237 seed dilution in each sample. Arrows indicate the positions of the 30 and 20 kDa molecular mass markers (MagicMark XP, Invitrogen, Carlsbad, CA, USA). (b) The R2 product in Fig. 1a was amplified by serial PMCA in the absence of digitonin. The R3 and R4 products, labeled “+”, were analyzed by WB with HRP-4E10 and AP-3F4 antibodies following PK digestion. Lanes labeled “N” represent control reactions, these contained only the PrPC substrate. (c) WB profiles of the R3–R22 PMCA products; PrPSc molecules were detected with HRP-T2 antibody. (d) No spontaneous generation of PrPSc was observed; samples 1–10 contained only mouse PrPC substrate and were amplified in the absence of digitonin. The PMCA product was diluted 1:10, amplified, then diluted and amplified again for a total of 10 rounds. After each amplification round, samples (R1–R10) were analyzed by WB with HRP-T2 antibody after PK digestion. Arrows indicate the positions of molecular mass markers corresponding to 37, 25, 20, and 15 kDa.

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In contrast, the presence of the Sc237 seed was confirmed by 3F4 antibody in the first round amplification, and its remnant signal became lower than the minimum limit of detection in WB after the second round of amplification (Fig. 2a,b). PrPSc was detected by T2 antibody, which reacts with both mouse and hamster PrP after the fifth round of amplification (Fig. 2c). From the third to seventh rounds, the amplification efficiency was so low that the PrPSc could barely be maintained during the process of dilution of the PMCA products and its subsequent amplification. After this lag phase in amplification, the PrPSc signal became markedly intense. Although the spontaneous generation of PrPSc molecules has been reported previously (26, 27), no such PrPSc molecules were observed when the mouse PrPC substrate alone was amplified by 10 rounds of serial PMCA (Fig. 2d). Therefore, the signal enhancement was caused by an increase in amplification efficiency after the eighth round of amplification. This high-efficiency amplification continued into the 22nd round of amplification, and a diglycosylated form of PrPSc molecules was predominant over other glycosylated forms throughout the amplification process.

Infectivity of Sc237 and interspecies PMCA-derived mouse PrPSc in tga20 mice

Table 1 shows the results of the bioassay. Contrary to what has previously been believed, the tga20 mice died after an average period of 548 ± 55 days when 10% brain homogenate of Sc237-infected hamsters was administered intracerebrally (Sc237/tga). The latent period in tga20 mice overexpressing mouse PrPC was much longer than that in Tg52NSE mice with hamster PrPC. Infectivity of the PMCA product from the 22nd round of amplification was also examined in these transgenic mice. Although the control mice, which were inoculated with the homogenate diluted 1:800, had not developed the disease after more than 690 days, all tga20 mice inoculated with the R22 product (PMCA/tga) developed the disease after an average period of 345 ± 21 days. A significant difference was observed between the incubation periods of Sc237/tga and PMCA/tga mice. None of the Tg52NSE mice inoculated with the PMCA products developed the disease after more than 720 days.

Table 1.  Mean incubation time of tga20 and Tg52NSE transgenic mice
Inoculum (dilution of PrPSc seed)tga20Tg52NSE
Primary passageSecondary passagePrimary passage
Transmission rate (total death/total number)Mean incubation time ± SD (days)Transmission rate (total death/total number)Mean incubation time ± SD (days)Transmission rate (total death/total number)Mean incubation time ± SD (days)
  1. , the 10% homogenates of Sc237-infected brains were diluted 1:800 and injected into transgenic mice as controls; ‡,§, the mice indicated by identical superscript symbols had significantly different (P < 0.01) mean incubation times.

Sc237100100% (5/5)548 ± 55‡,§100% (4/4)111 ± 2§100% (5/5)45 ± 2
8 × 10−2†  0% (0/5)>690  100% (5/5)61 ± 1
Interspecies PMCA productR22 (10−40)100% (5/5)345 ± 21100% (5/5)317 ± 6   0% (0/5)>720

Brain homogenates of the primary-passaged mice (Sc237/tga #1 and PMCA/tga #1) were injected intracerebrally into tga20 mice. In the secondary transmission, disease onset in the mice inoculated with brain homogenate of Sc237/tga #1 was significantly accelerated, and these mice (Sc237/tga-2) died after an average of 111 ± 2 days. In contrast, mice inoculated with brain homogenate of PMCA/tga #1 developed the disease after an average of 317 ± 6 days. There was no significant difference between the survival period of primary-passaged mice (PMCA/tga) and that of secondary-passaged mice (PMCA/tga-2).

Histopathological analysis of brains and spleens of tga20 mice inoculated with Sc237 and PMCA-derived mouse PrPSc

To characterize the neuropathological properties of Sc237 and the R22 PMCA-derived mouse PrPSc, we examined the regional profiles of neuronal vacuolation scores in the brains of affected mice (n= 5, Fig. 3). The vacuolation scores of mice inoculated with the PMCA-derived PrPSc were significantly different from those of mice inoculated with Sc237 in six brain regions, namely the midbrain (region 3), hypothalamus (region 4), hippocampus (region 6), paraterminal body (region 7), posterior midline of the cerebral cortex (region 8), and anterior midline of the cerebral cortex(region 9). Furthermore, the vacuolation profiles of PMCA-PrPSc inoculated mice were distinct from those of mice inoculated with mouse scrapie strains such as Chandler, Obihiro, and 22A (unpublished observations). Immunohistochemical results from the brains and spleens of infected mice (n= 5) are shown in Fig. 4. In mice inoculated with Sc237, PrPSc accumulated over the whole region of the interpeduncular nucleus, midbrain, and medulla, diffuse distribution or synaptic-like immunostaining and plaque formation of PrPSc being observed in the corpus callosum. Although spongiform change was less frequent and PrPSc accumulation was not apparent in the brain sections of mice inoculated with the PMCA-derived mouse PrPSc, PrPSc deposition was observed in the spleens of these mice, as it was in the Sc237-inoculated mice.

image

Figure 3. Vacuolation profile in nine different brain areas of tga20 mice inoculated with Sc237 and PMCA. Brain regions are as follows: 1, dorsal medulla; 2, cerebellum; 3, midbrain; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, paraterminal body; 8, posterior midline of the cerebral cortex; 9, anterior midline of the cerebral cortex. The average lesion scores (n= 5 animals/group) and SEM (error bar) are shown in the graph. Asterisks denote significant differences between samples (two-tailed t-test, *, P < 0.05; **, P < 0.001). Closed circles, PMCA product; open circles, Sc237.

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image

Figure 4. Vacuolation (upper panels) and PrPSc accumulation (lower panels) in four different brain areas and spleens of tga20 mice inoculated with Sc237 (Sc237/tga) and PMCA-derived PrPSc (PMCA/tga). The control for histological analysis was a normal tga20 mouse.

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Biochemical properties of PrPSc that accumulated in the brains of tga20 mice inoculated with Sc237 and the PMCA-derived mouse PrPSc

The WB profile of the PrPSc molecules that accumulated in the brains of tga20 mice inoculated with Sc237 (Sc237/tga) and the R22 PMCA products (PMCA/tga) was compared to the profiles of the Sc237 mouse scrapie prion strains (Obihiro, ME7 and Chandler), and the PrPSc generated by interspecies PMCA (R22 PMCA). The R22 PMCA PrPSc resembled the Sc237 rather than the mouse scrapie strains in the ratio of the three glycosylated forms of PrPSc, but the diglycosylated form of PrPSc was 0.7 kDa smaller than that of the Sc237 PrPSc (Fig. 5a). The size of the unglycosylated PrPSc molecules of PMCA/tga #1 was 0.9 kDa smaller than that of the Sc237/tga #1 mouse (Fig. 5a,b). This difference in molecular weight was generally observed between the PMCA/tga and Sc237/tga mice (Fig. 5c). In the secondary passaged mice (PMCA/tga-2 and Sc237/tga-2), this difference was preserved in the WB profiles (Fig. 5b,d).

image

Figure 5. Comparison of WB profiles of PrPSc molecules. (a) WB profiles of PrPSc from mouse scrapie strains (Obihiro, ME7 and Chandler), hamster scrapie strain (Sc237), the brains of tga20 mice inoculated with Sc237 (Sc237/tga #1), the PMCA product obtained in the 22nd round of interspecies amplification (R22 PMCA), and the brains of tga20 mice inoculated with the R22 PMCA products (PMCA/tga #1). After PK digestion, PrPSc was detected with HRP-T2 antibody. (b) Unglycosylated PrPSc accumulated in the brains of mice inoculated with Sc237/tga #1, Sc237/tga-2 #1, PMCA/tga #1, and PMCA/tga-2 #1. The PK-digested samples were analyzed after treatment with PNGase F. (c) WB profiles of PrPSc accumulated in the brains of all affected mice in the primary transmission (n= 5 in each group). (d) WB profiles of PrPSc accumulated in the brains of all affected mice in the secondary transmission (n= 5 in PMCA/tga-2 and n= 4 in Sc237/tga-2). Arrows indicate the positions of the 30 and 20 kDa molecular mass markers.

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PrPSc accumulated in the Sc237/tga and PMCA/tga mice showed similar PK resistance and PK50 values (Fig. 6, left panel). In contrast, a distinct difference was observed in the GdnHCl denaturation assay (Fig. 6, right panel). PrPSc in Sc237/tga and PMCA/tga mice exhibited increased signal intensities after treatment with GdnHCl at 0.5–1.5 M. However, PrPSc signals in PMCA/tga mice were significantly higher than in Sc237/tga mice (2.0–3.5 M). These results suggest that the PrPSc that accumulated in the mice inoculated with the PMCA-derived mouse PrPSc was more resistant to GdnHCl denaturation than the PrPSc from the mice inoculated with Sc237.

image

Figure 6. Biochemical characteristics of PrPSc accumulated in the brains of Sc237/tga and PMCA/tga mice. Resistance of PrPSc to treatment with various concentrations of PK (left panels) or GdnHCl (right panels) was compared. The samples were analyzed by WB with HRP-T2 antibody in three independent experiments; typical results are shown in the upper panels. Arrows indicate the positions of the 30 and 20 kDa molecular mass markers. The average relative intensity of the PrPSc signal and SEM of each reagent concentration are represented graphically in the lower panels. The numerals in parentheses in the upper panels indicate the PK50 and GdnHCl50 values estimated from approximate curves generated from the experimental data. The asterisks at the top of the graph indicate significant differences (P < 0.05) between these mice in the average relative intensities of PrPSc signals at each concentration of GdnHCl. Closed circles, Sc237/tga; closed triangles, PMCA/tga; error bars, SEM.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Contrary to what has previously been believed, we have demonstrated the development of prion disease in transgenic mice overexpressing mouse PrP in the primary passage of Sc237 transmission after a latent period of over 500 days. Although no clinical sign of disease has previously been observed in wild-type mice after intracerebral inoculation with Sc237 (28), the disease has been found to progress to the subclinical stage of infection in the primary passage in the form of occasional PrPSc accumulation has in the brain, however pathogenicity has not previously been detected until the secondary passage (29, 30). Because tga20 mice express approximately 10-fold more PrPC in their brains than wild-type mice, the course of disease development might be accelerated by a quantitative effect of PrPSc molecules accumulated in the brains.

In the homogeneous combination of mouse PrPSc and PrPC substrate, PMCA products preserve the histopathological and biochemical characteristics of the original PrPSc in mice (31), suggesting that PMCA is comparable to in vivo systems in generating PrPSc. However, the properties of PrPSc accumulated in the brains of Sc237/tga and PMCA/tga mice are considerably different. That is, the lesion profiles and PrPSc accumulation patterns in the brains of PMCA/tga mice are different from those found in Sc237/tga mice, and PrPSc accumulated in the brains of these mice differs in WB profiles and resistance to GdnHCl denaturation. These results suggest that alteration of mouse PrPC induced by interspecies PMCA is different from that in cross-species transmission in vivo. In addition, the results of secondary transmission indicate that Sc237/tga PrPSc undergoes a significant change in pathogenicity in the mouse brain but PMCA/tga PrPSc retains its pathogenicity at the level of primary transmission. These observations strongly support the presumption that a different kind of PrPSc is propagated in the brains of PMCA/tga and Sc237/tga mice. More detailed information on PrPSc in these mice will be obtained from histopathological and biochemical analysis of brain samples of mice after secondary passage.

Sheep scrapie can be experimentally transmitted to goats, rats, hamsters, and mice and several scrapie prion strains have been established by serial transmission in these animals (32, 33). In these instances, the scrapie prion protein is thought to contain a molecular ensemble of heterogeneous PrPSc which are maintained in infected sheep (34), and PrPSc that fit the given environment will be established as a prion strain by selection or mutation in the process of overcoming the “species barrier” (35). According to this idea, a possible explanation for our results is that characteristic selection or mutation of PrPSc might have occurred during interspecies PMCA with detergents and ultrasonic treatment, and the mouse PrPSc that was suitable for in vitro amplification was able to propagate in the brains of PMCA/tga mice. The observation that unglycosylated PrPSc of a similar size to that of the PMCA product can be reproduced in PMCA/tga and PMCA/tga-2 mice (Fig. 5a,b) may support the idea that molecular diversity of PrPSc in a prion strain is changeable by interspecies PMCA.

In conclusion, we have revealed that the biological properties of PrPSc in mice inoculated with Sc237 are different from those in mice inoculated with its interspecies PMCA product. These observations suggest that in vitro amplification of PrPSc by PMCA is not necessarily equivalent to in vivo propagation of PrPSc in the case of interspecies amplification, and PrPSc with different biological properties propagate in a heterogeneous environment depending whether that environment is in vivo or in vitro. Identification of the factors affecting PrPSc characteristics under artificial conditions may enable the interspecies PMCA technique to contribute to our understanding of the mechanism by which PrPSc diversity is generated.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

We wish to thank Ms. Noriko Mishima-Yoshida for her assistance. We also thank Dr. Kentaro Masujin and Dr. Yoshifumi Iwamaru for their helpful suggestions. This study was supported by a Grant-in-Aid from the Bovine Spongiform Encephalopathy Control Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES