Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions, but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrPC, into the pathogenic isoform PrPSc, in prion diseases. It has been shown that PrP-knockout (Prnp0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp0/0 mice, neither developed the disease nor accumulated MHM2ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp0/+ mice developed the disease with abundant accumulation of MHM2ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2ScΔ23-88, and that the co-expressing wild-type PrPC can stimulate the conversion of MHM2Δ23-88 to MHM2ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp0/+ mice. However, wild-type PrPSc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp0/+ mice, compared with RML- and 22L-inoculated Prnp0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2ScΔ23-88 without the help of the trans-acting PrPC, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrPC stimulates the conversion of MHM2Δ23-88 into MHM2ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrPC into PrPSc.


Introduction
Prions are causative agents of transmissible spongiform encephalopathies, or prion diseases, a group of fatal neurodegenerative disorders, which include Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals [1,2]. It is believed that prions mainly consist of the abnormally folded, relatively proteinase K (PK)-resistant pathogenic isoform of prion protein, designated PrP Sc [1,2]. PrP Sc is produced by conformational conversion of the normal cellular isoform of PrP, PrP C , via intermolecular interaction of both molecules [1,2]. PrP C is a membrane glycoprotein tethered to the cell surface via a glycosylphosphatidylinositol moiety and expressed most abundantly in the central nervous system, particu-larly by neurons [3,4]. We and others have shown that mice devoid of PrP C (Prnp 0/0 ) are resistant to prions, neither developing the disease nor producing PrP Sc or propagating the prions even after inoculation with the prions [5,6,7,8]. These results indicate that the conversion of PrP C into PrP Sc is an essential event in the pathogenesis of prion disease.
A reverse genetic approach using reconstituted Prnp 0/0 mice with transgenes encoding various deletion mutants of PrP C is useful to investigate the structure-function relationship of PrP C conversion to PrP Sc . Prnp 0/0 mice expressing mouse PrP with Nterminal residues 23-88 deleted, or Tg(PrPD23-88)/Prnp 0/0 mice, developed prion disease after inoculation with RML scrapie prions, with accumulation of PrP Sc D23-88 in their brains [9]. However, onset of the disease was delayed and the conversion of PrPD23-88 into PrP Sc D23-88 was inefficient [9]. These results indicate that the N-terminal residues affect the conversion of PrP C into PrP Sc . Delayed onset of the disease was also observed in Tg(MHM2)/Prnp 0/0 mice inoculated with RML prions [9]. MHM2 is a mouse (M)-hamster (H) chimeric PrP, with hamster PrP-derived methionine residues at 108 and 111 instead of leucine and valine residues of mouse PrP, indicating that the chimeric region also affects the conversion. MHM2D23-88 is chimeric MHM2 with the deletion of N-terminal residues 23-88. Interestingly, Tg(MHM2D23-88)/Prnp 0/0 mice were reported to remain healthy for more than 500 days after inoculation with RML prions [9,10]. No MHM2 Sc D23-88 was accumulated in their brains [9,10]. In contrast, MHM2 Sc D23-88 was easily detectable in the brains of RML-inoculated Tg(MHM2D23-88)/Prnp 0/+ mice [10]. These results indicate that MHM2D23-88 itself might either lose or greatly reduce the converting capacity to MHM2 Sc D23-88, and that the co-expressing wild-type PrP C can stimulate the conversion of MHM2D23-88 to MHM2 Sc D23-88 in trans.
In the present study, we confirmed that Tg(MHM2D23-88)/ Prnp 0/0 mice remained resistant to RML prions for more than 730 days after inoculation. Neither MHM2 Sc D23-88 nor prion infectivity was detected in their brains. However, we found that Tg(MHM2D23-88)/Prnp 0/0 mice were susceptible to 22L scrapie prions, developing prion disease around ,530 days after inoculation. MHM2 Sc D23-88 and prion infectivity were detected in the brains of terminally ill Tg(MHM2D23-88)/Prnp 0/0 mice. These results clearly demonstrate that MHM2D23-88 can convert to MHM2 Sc D23-88 without the help of the co-expressing wildtype PrP C . We also found that the conversion of MHM2D23-88 into MHM2 Sc D23-88 was accelerated and the conversion of wildtype PrP C into PrP Sc was contrarily decelerated in the brains of RML-and 22L-inoculated Tg(MHM2D23-88)/Prnp 0/+ mice. These results indicate that the co-expressing wild-type PrP C stimulates the conversion of MHM2D23-88 into MHM2 Sc D23-88, but to the contrary, the co-expressing MHM2D23-88 disturbs the conversion of wild-type PrP C into PrP Sc , irrespective of prion strains inoculated.

Ethics statements
The Ethics Committee of Animal Care and Experimentation of the University of Occupational and Environmental Health, Kitakyushu, Japan, approved this study (approval number AE08-013). Animals were cared for in accordance with The Guiding Principle for Animal Care and Experimentation of the University of Occupational and Environmental Health and Japanese Law for Animal Welfare and Care.

Prion inoculation
Brains were removed from terminally ill C57BL/6 mice infected with RML or 22L prions. A single brain was homogenized (10%, w/v) in phosphate-buffered saline (PBS) by passing it through 18 to 26 gauge needles and then diluted to 1% with PBS. Four to five week-old mice were intracerebrally inoculated with a 20 ml-aliquot of the homogenates.

Vector constructions
The DNA fragment encoding mouse PrP residues 104-254 with methionine substitutions at codons 108 and 111 was first amplified by polymerase chain reaction (PCR) with a sense primer (59ccaaaaaccaacatgaagcaca-39) and an antisense primer (59-cctctagacctcatcccacgatcaggaaga-39; the underlined sequence is an Xba I site; the bold sequence is a stop codon) using pcDNA3.1-moPrP plasmid [14] as a template. The resulting DNA fragment was then utilized as a 39 primer together with a sense primer (59tcggatccagtcatcatggcgaaccttggc-39; the underlined sequence is a BamH I site; the bold sequence is a start codon) to amplify a DNA fragment encoding full-length mouse PrP with the methionine substitutions using the pcDNA3.1-moPrP template plasmid. After confirmation of the DNA sequences, the DNA fragment was digested by BamH I and Xba I and introduced into a pcDNA3.1 vector (Invitrogen, Carlsbad, CA) to generate pcDNA3.1-MHM2. Finally, to construct E. coli expression vectors for recombinant full-length MHM2 and MHM2D23-88, the corresponding DNA fragments were amplified by Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA) using pcDNA3.1-MHM2 plasmid template with sense primers (59-ctcggatccaaaaagcggccaaagcctgga-39 for MHM2, the underlined sequence is a BamH I site; the italic sequence, residues 23-29 of mouse PrP; 59-ctcggatccggccaaggagggggtacccat-39 for MHM2D23-88, the underlined sequence is a BamH I site; the italic sequence, residues 89-95 of mouse PrP) and an antisense primer (59-ctcaagctttcatcccacgatcaggaagat-39, the underlined sequence is a Hind III site; the bold sequence is a stop codon; the italic sequence, residues 249-254 of mouse PrP). After confirmation of the DNA sequences, the DNA fragments were digested by BamH I and Hind III and introduced into a pQE30 vector (Qiagen, Hilden, Germany) to generate pQE30-MHM2 and pQE30-MHM2D23-88. Pull-down assay Clarified brain homogenate was prepared as follows. 5% (w/v) brain homogenate in PBS was sonicated for 1 min and centrifuged at 5006g for 15 min at 4uC. The supernatant was collected and diluted with PBS to 3 mg-protein/ml. Anti-His4 ab-Protein G beads were prepared as follows. Anti-His4 antibody (Qiagen) (0.3 mg) was incubated with 10 mL of Dynabeads Protein G (Life technologies) in 200 mL of binding buffer (PBS containing 1% Triton X-100 and 1% Tween 20) at RT. After 1 h-incubation, the beads were washed with the binding buffer to remove unbound antibody. The purified His-tagged recombinant PrP was incubated with 75 mg of the clarified brain homogenate in the binding buffer containing 2 M urea at 4uC for 1 h. The anti-His4 ab-Protein G beads were added and the mixture was rotated at 4uC for 4 h. The immune complexes were collected using a magnetic separation rack and washed with wash buffer (PBS containing 0.05% Triton X-100 and 0.05% Tween 20). After washing, the complexes were incubated in 30 mL of lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.5% Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA) containing 50 mg/ml of PK at 37uC for 30 min. The PK-treated samples were subjected to Western blotting.
There is a possibility that the different susceptibility of Tg(MHM2D23-88)/Prnp 0/0 mice to RML and 22L prions might not be due to different responsiveness of MHM2D23-88 to RML and 22L prions, but rather due to different infectious doses of RML and 22L prions inoculated. To rule out this possibility, we determined infectious titers in the brain homogenates from RMLor 22L-infected, terminally ill C57BL/6 mice that were similarly prepared as the brain homogenates used as inocula. The serially diluted brain homogenates were intracerebrally inoculated into C57BL/6 mice. No significant difference was detected in the number of diseased mice between the groups inoculated with each dilution of RML-and 22L-infected brain homogenates (Logistic regression analysis, p = 0.083, Table 2), indicating that infectious titers are not different in the RML-and 22L-infected brain homogenates used (Table 2). According to the method of Reed and Muench [13], ID 50 /gram of the brain tissue was calculated as 10 7.7 and 10 8.2 in the RML-and 22L-infected brain homogenates, respectively ( Table 2). These results indicate that the different susceptibility of Tg(MHM2D23-88)/Prnp 0/0 mice to RML and 22L prions is due to different responsiveness of MHM2D23-88 to RML and 22L prions. C57BL/6 mice inoculated with RML and 22L prions developed the disease with similar incubation times (Table 2). However, RML-inoculated Prnp 0/+ mice showed longer incubation times than 22L-inoculated Prnp 0/+ mice (Table 1). These results suggest that RML and 22L prions are affected in their propagation by the expression levels of PrP C , with RML prions being affected more strongly than 22L prions.
We also investigated prion infectivity in the brain of a Tg(MHM2D23-88)/Prnp 0/0 mouse, which was inoculated with Figure 5. Western blotting of the brains from indicator mice inoculated with the brain homogenates from different genotypic mice inoculated with RML and 22L prions. PK-resistant PrP is detectable with M20 anti-PrP antibodies in all inoculated indicator mice, except for the mice sacrificed at 365 days after inoculation with the brain homogenate from the Tg(MHM2D23-88)/Prnp 0/0 mouse, which was sacrificed at 591 days after inoculation with RML prions. doi:10.1371/journal.pone.0109737.g005 RML prions and sacrificed at 591 dpi. None of the indicator mice inoculated with the single brain homogenate from the Tg(MHM2D23-88)/Prnp 0/0 mouse became sick by 365 dpi (Table 3). PrP Sc was also undetectable in the brains of indicator mice sacrificed at 365 dpi (Fig. 5). In contrast, single brain homogenates from RML-inoculated, terminally ill Prnp 0/+ and Tg(MHM2D23-88)/Prnp 0/+ mice caused the disease in indicator mice at 14765 and 14465 dpi, respectively (Table 3), with abundant accumulation of PrP Sc in their brains (Fig. 5). These results are consistent with MHM2 Sc D23-88 being undetectable in the brains of RML-inoculated Tg(MHM2D23-88)/Prnp 0/0 mice.

Discussion
In the present study, we showed that intracerebral inoculation with the brain homogenate from RML-infected, terminally ill wild-type mice never caused the disease in Tg(MHM2D23-88)/ Prnp 0/0 mice for up to 730 days. No MHM2 Sc D23-88 was detectable in their brains. We also failed to detect any prion infectivity in the brain of a Tg(MHM2D23-88)/Prnp 0/0 mouse sacrificed at 591 days after inoculation. These results are consistent with those previously reported by others [9,10]. In contrast, we found that Tg(MHM2D23-88)/Prnp 0/0 mice developed prion disease after intracerebral inoculation with the brain homogenate from 22L-infected, terminally ill wild-type mice, with abundant accumulation of MHM2 Sc D23-88 and high prion infectivity present in their brains. These results show that MHM2D23-88 is converted into MHM2 Sc D23-88 in Tg(MHM2D23-88)/Prnp 0/0 mice after inoculation with 22L prions. We also showed that the similarly prepared brain homogenates from terminally ill, RMLand 22L-inoculated wild-type mice contained similar infectious doses of RML and 22L prions. This excludes the possibility that unsuccessful detection of MHM2 Sc D23-88 in the brains of RMLinoculated Tg(MHM2D23-88)/Prnp 0/0 mice might be due to lower infectious doses of RML prions being inoculated. It rather suggests that MHM2D23-88 is converted into MHM2 Sc D23-88 less efficiently by RML prions than by 22L prions.
Tg(MHM2D23-88)/Prnp 0/0 mice inoculated with 22L prions developed the disease with accumulation of MHM2 Sc D23-88 in their brains, indicating that MHM2 Sc D23-88 lacking the Nterminal residues 23-88 is neurotoxic. However, brain spongiosis was milder in terminally ill Tg(MHM2D23-88)/Prnp 0/0 mice than in terminally ill Prnp 0/+ and Tg(MHM2D23-88)/Prnp 0/+ mice, suggesting that MHM2 Sc D23-88 might not be fully toxic. Sonati et al. recently reported that the N-terminal region of PrP C mediates a neurotoxic signal originating from the C-terminal globular domain bound with anti-PrP antibodies [15]. It is thus possible that the Nterminal residues might also play a role in the neurotoxicity of wild-type PrP Sc .
Wild-type indicator mice intracerebrally inoculated with the brain homogenate from a 22L-inoculated, terminally ill Tg(MHM2D23-88)/Prnp 0/0 mouse developed the disease significantly later than those inoculated with the brain homogenates from 22L-inoculated, terminally ill Tg(MHM2D23-88)/Prnp 0/+ and Prnp 0/+ mice. These results indicate that, while MHM2 Sc D23-88 is infectious, it is less infectious than wild-type PrP Sc . Indistinguishable symptoms and PrP Sc with the same PK digestion and glycosylation patterns were observed in all diseased indicator mice. These suggest that biological properties of 22L prions might not be altered even after the passage in Tg(MHM2D23-88)/ Prnp 0/0 mice. Rather, the slightly longer incubation times in indicator mice inoculated with the Tg(MHM2D23-88)/Prnp 0/0 homogenate might be due to the chimeric residues in MHM2 Sc D23-88. Indeed, full-length MHM2 Sc was shown to be slightly less infectious than wild-type PrP Sc in mice overexpressing wild-type mouse PrP C [9]. Alternatively, lack of the N-terminal residues in MHM2 Sc D23-88 might be responsible for the longer incubation times.
Prnp 0/0 mice used in this study ectopically express Dpl in their brains [16,17]. It was reported that transgenic overexpression of Dpl did not modify incubation times and brain pathologies in Prnp +/+ mice infected with RML prions [18]. Prnp 0/+ mice with and without the ectopic expression of Dpl were also reported to display indistinguishable pathologies after infection with 301V BSE prions [19]. It is thus unlikely that Dpl affects the susceptibility of Tg(MHM2D23-88)/Prnp 0/0 mice to RML and 22L prions.
Tg(MHM2D23-88)/Prnp 0/0 mice inoculated with RML and 22L prions showed longer incubation times. Particularly, RMLinoculated Tg(MHM2D23-88)/Prnp 0/0 mice were free of the disease-specific symptoms for up to 730 days. It is known that sequence differences between PrP C in recipient animals and PrP Sc in an inoculum create a prion transmission barrier, causing elongation of incubation times [20]. If a prion transmission barrier is responsible for longer incubation times in primarily inoculated mice, secondarily inoculatied mice with the same genotype may result in shorter incubation times. Tg(PrPD23-31)/Prnp 0/0 , Tg(PrPD32-93)/Prnp 0/0 , and Tg(MHM2)/Prnp 0/0 mice primarily inoculated with RML prions were reported to show prolonged incubation times, but secondarily inoculated mice did not show shorter incubation times [9,21,22]. These results indicate that the N-terminal deletion or the chimeric residues in PrP C does not create a prion transmission barrier for wild-type PrP Sc , suggesting that no prion transmission barrier is present between MHM2D23-88 and wild-type PrP Sc . Rather, we found that, in spite of overexpression of MHM2D23-88, MHM2 Sc D23-88 was detectable in the brains of 22L-inoculated Tg(MHM2D23-88)/Prnp 0/0 mice later than wild-type PrP Sc in the 22L-inoculated Prnp 0/+ mice, indicating that MHM2D23-88 converts into MHM2 Sc D23-88 less efficiently than wild-type PrP C into PrP Sc . It is therefore conceivable that the longer incubation times of Tg(MHM2D23-88)/Prnp 0/0 mice could be due to the decreased conversion of MHM2D23-88 into MHM2 Sc D23-88. We showed that recombinant MHM2D23-88 pulled down PrP Sc (22L) and PrP Sc (RML) less than full-length recombinant MHM2 in a pull-down assay. Thus, the decreased conversion of MHM2D23-88 into MHM2 Sc D23-88 might be attributable to the lower binding of PrP Sc (22L) and PrP Sc (RML) to MHM2D23-88.
Different strain-specific susceptibility was also reported in Prnp 0/0 mice transgenically expressing mouse PrP with a serine residue at codon 170, PrP-170S [23], or ovine PrP with a valine residue at codon 136, OvPrP-V136 [24]. Tg(PrP-170S)/Prnp 0/0 mice were highly resistant to RML and 79A prions, but susceptible to 22L and ME7 prions [23]. Only a very small number of the mice inoculated with RML and 79A prions showed brain accumulation of PrP Sc -170S [23]. In contrast, all mice inoculated with 22L and ME7 prions accumulated PrP Sc -170S in their brains [23]. Tg(OvPrP-V136)/Prnp 0/0 mice were susceptible to SSBP1 prions, but resistant to CH1641 prions [24]. OvPrP Sc -V136 was accumulated in the brains of the mice inoculated with SSBP1 prions [24]. These results suggest that strain-specific differential susceptibility in these mice is also due to different conversion efficiency of the host PrP C s into their PK-resistant isoforms.
The primary sequence of PrP Sc is the same from different prion strains. Therefore, the different susceptibility of Tg(MHM2D23-88)/Prnp 0/0 , Tg(PrP-170S)/Prnp 0/0 , and Tg(OvPrP-V136)/ Prnp 0/0 mice to different prions cannot be explained by sequence differences between PrP Sc and the host PrP C , or that there is a prion transmission barrier. Several lines of evidence indicate that the conversion of PrP C into PrP Sc involves interaction of PrP C with the inoculated PrP Sc [25,26]. We showed that in a pull-down assay with recombinant MHM2D23-88, only a tiny amount of PrP Sc (RML) was pulled down while a considerably higher amount of PrP Sc (22L) was pulled down, suggesting that different binding of MHM2D23-88 to PrP Sc (RML) and PrP Sc (22L) might be involved in different conversion efficiency of MHM2D23-88 into MHM2 Sc D23-88 in RML-and 22L-inoculated Tg(MHM2D23-88)/Prnp 0/0 mice. It was reported that PrPD23-28 reduced the binding to PrP Sc (RML), and PrPD23-31 was insufficiently converted into PrP Sc D23-31 in mice inoculated with RML prions [21]. Miller et al. also reported that PrPD23-28 bound less PrP Sc (RML) and was converted very inefficiently to PrP Sc D23-28 in an in vitro assay [27]. MHM2D23-88 lacks residues 23-31, suggesting that the reduced binding of MHM2D23-88 to PrP Sc s might be partly due to the lack of residues 23-31. However, Tg(PrPD32-93)/Prnp 0/0 and Tg(MHM2)/Prnp 0/0 mice were also reported to have reduced susceptibility to RML prions [9], suggesting that the other deleted region(s) and the chimeric region also might be relevant to the binding of MHM2D23-88 to PrP Sc . It is thus of interest to compare the binding potential of PrP-170S and OvPrP-V136 to PrP Sc from different prion strains.
The conformational selection model of prion strains also has been proposed as a mechanism to explain strain-specific susceptibility [28,29]. This model postulates that the inoculated PrP Sc selects the host PrP C as a substrate for conversion due to its conformational compatibility with the host PrP C [28,29]. Conformational incompatibility between the inoculated PrP Sc and the host PrP C thus leads to unsuccessful or insufficient conversion of the host PrP C into its PK-resistant isoform. Indeed, accumulating lines of evidence suggest that PrP Sc is folded in a strain-specific conformation [30]. Nuclear magnetic resonance studies of recombinant PrPs suggest that the N-terminal domain, including the deleted residues and chimeric residues in MHM2D23-88, confers structural stability within the C-terminal globular domain [31,32]. It is thus possible that MHM2D23-88 adopts a different conformation from that of wild-type PrP C , and that the adopted conformation of MHM2D23-88 still remains compatible with PrP Sc (22L), but not with PrP Sc (RML). The conformational selection model might also explain the different responsiveness of OvPrP-V136 and PrP-170S to different prion strains [23,24].
It was shown that the region corresponding to the chimeric region is exposed in PrP C molecules, but hidden in PrP Sc molecules [33,34,35], and that the octapeptide repeat region of residues 51-90 is trypsin-sensitive in PrP C , but trypsin-resistant in PrP Sc [36]. These results indicate that upon the conversion of PrP C into PrP Sc , a marked conformational change occurs within the N-terminal domain. Interestingly, the N-terminal domain of PrP C is highly flexible and displays a marked conformational heterogeneity [32,33,37]. It is thus also possible that lack of residues 23-88 and insertion of the chimeric residues might reduce the N-terminal conformational heterogeneity of MHM2D23-88, and that the reduced conformational heterogeneity might render MHM2D23-88 resistant to RML but still susceptible to 22L prions.
The N-terminal domain was shown to be important for internalization of PrP C [38]. The conversion of PrP C into PrP Sc has been suggested to occur on the cell surface and/or along the endocytic pathway to lysosomes [39,40]. Thus, defective internalization of MHM2D23-88 might lead to the insufficient conversion of MHM2D23-88 to MHM2 Sc D23-88. However, it remains unknown how internalized PrP C can undergo strain specific conversion.
What is the mechanism for MHM2D23-88 to decelerate the conversion of wild-type PrP C into PrP Sc ? Two possibilities have been proposed for the trans-action of PrP C on the conversion of MHM2D23-88 to MHM2 Sc D23-88 [10]. One is that PrP C might bind to MHM2D23-88 and then promote the conversion of MHM2D23-88 into MHM2 Sc D23-88. In this scenario, MHM2D23-88 having increased conversion competence with help from the co-expressing PrP C might compete with the coexpressing PrP C for an as yet unidentified factor(s) important for the conversion, such as a conjectural molecule protein X, thereby decelerating the conversion of PrP C into PrP Sc . The other is that PrP C is first converted into PrP Sc and the nascent PrP Sc then acts on MHM2D23-88 to convert it into MHM2 Sc D23-88. In this case, since MHM2D23-88 converts into MHM2 Sc D23-88 very slowly, PrP Sc participating in the conversion of MHM2D23-88 into MHM2 Sc D23-88 could not engage in other conversion events until MHM2D23-88 is converted into MHM2 Sc D23-88, ultimately causing the decreased conversion of PrP C into PrP Sc . Elucidation of the trans-action of PrP C on the conversion of MHM2D23-88 into MHM2 Sc D23-88 and the trans-inhibition of MHM2D23-88 on the conversion of wild-type PrP C into PrP Sc would be worthy for understanding the conversion mechanism of PrP C into PrP Sc . Figure S1 Higher magnification images of HE-stained brain sections from different genotypic mice inoculated with prions. (A) Spongiosis is milder in the cerebral cortex, hippocampus, thalamus and cerebellum from 22L-inoculated, terminally ill Tg(MHM2D23-88)/Prnp 0/0 mice than in 22Linoculated, terminally ill Prnp 0/+ and Tg(MHM2D23-88)/Prnp 0/+ mice. (B) Spongiosis is observed in the cerebral cortex, hippocampus, thalamus and cerebellum from RML-inoculated, terminally ill Prnp 0/+ and Tg(MHM2D23-88)/Prnp 0/+ mice, but not from RMLinoculated, symptom-free Tg(MHM2D23-88)/Prnp 0/0 mice. Scale bar, 100 mm. (TIF)