Advertisement

Central residues in prion protein PrPC are crucial for its conversion into the pathogenic isoform

Open AccessPublished:August 13, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102381
      Conformational conversion of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, is a key pathogenic event in prion diseases. However, the conversion mechanism remains to be elucidated. Here, we generated Tg(PrPΔ91-106)-8545/Prnp0/0 mice, which overexpress mouse PrP lacking residues 91-106. We showed that none of the mice became sick after intracerebral inoculation with RML, 22L, and FK-1 prion strains nor accumulated PrPScΔ91-106 in their brains except for a small amount of PrPScΔ91-106 detected in one 22L-inoculated mouse. However, they developed disease around 85 days after inoculation with bovine spongiform encephalopathy (BSE) prions with PrPScΔ91-106 in their brains. These results suggest that residues 91-106 are important for PrPC conversion into PrPSc in infection with RML, 22L, and FK-1 prions but not BSE prions. We then narrowed down the residues 91-106 by transducing various PrP deletional mutants into RML- and 22L-infected cells and identified that PrP mutants lacking residues 97-99 failed to convert into PrPSc in these cells. Our in vitro conversion assay also showed that RML, 22L, and FK-1 prions did not convert PrPΔ97-99 into PrPScΔ97-99, but BSE prions did. We further found that PrP mutants with proline residues at positions 97 to 99 or charged residues at positions 97 and 99 completely or almost completely lost their converting activity into PrPSc in RML- and 22L-infected cells. These results suggest that the structurally flexible and noncharged residues 97-99 could be important for PrPC conversion into PrPSc following infection with RML, 22L, and FK-1 prions but not BSE prions.

      Keywords

      Abbreviations:

      BSE (bovine spongiform encephalopathy), CJD (Creutzfeldt-Jakob disease), dpi (days postinoculation), IMAC (immobilized metal affinity chromatography), PMCA (protein misfolding cyclic amplification)
      Prions are proteinaceous infectious particles causing prion diseases, a group of devastating neurodegenerative disorders, including Creutzfeldt-Jakob disease (CJD) in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals (
      • Aguzzi A.
      • Baumann F.
      • Bremer J.
      The prion's elusive reason for being.
      ,
      • Prusiner S.B.
      The prion diseases.
      ). They are widely believed to consist, if not entirely, of the abnormally folded, amyloidogenic isoform of prion protein, designated PrPSc, which is produced from the cellular isoform of prion protein, PrPC, through conformational conversion (
      • Aguzzi A.
      • Baumann F.
      • Bremer J.
      The prion's elusive reason for being.
      ,
      • Prusiner S.B.
      The prion diseases.
      ). PrPC is a glycoprotein tethered to the outer cell membrane via a glycosylphosphatidylinositol anchor moiety and expressed most abundantly in the brain, particularly by neurons (
      • Aguzzi A.
      • Baumann F.
      • Bremer J.
      The prion's elusive reason for being.
      ,
      • Prusiner S.B.
      The prion diseases.
      ). It has been assumed that, upon prion infection, PrPSc interacts with PrPC, forcing it to undergo conformational conversion into PrPSc via a seeded protein polymerization mechanism, resulting in propagation of PrPSc or prions. Indeed, mice devoid of PrPC (Prnp0/0) have been shown to be resistant to prion infections, neither propagating PrPSc or prions in their brains nor developing disease even after intracerebral inoculation with the prions (
      • Bueler H.
      • Aguzzi A.
      • Sailer A.
      • Greiner R.A.
      • Autenried P.
      • Aguet M.
      • et al.
      Mice devoid of PrP are resistant to scrapie.
      ,
      • Prusiner S.B.
      • Groth D.
      • Serban A.
      • Koehler R.
      • Foster D.
      • Torchia M.
      • et al.
      Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies.
      ,
      • Manson J.C.
      • Clarke A.R.
      • McBride P.A.
      • McConnell I.
      • Hope J.
      PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology.
      ,
      • Sakaguchi S.
      • Katamine S.
      • Shigematsu K.
      • Nakatani A.
      • Moriuchi R.
      • Nishida N.
      • et al.
      Accumulation of proteinase K-resistant prion protein (PrP) is restricted by the expression level of normal PrP in mice inoculated with a mouse-adapted strain of the Creutzfeldt-Jakob disease agent.
      ), strongly arguing that the conformational conversion of PrPC into PrPSc is a key pathogenic event in prion diseases.
      Many reverse genetic studies using reconstituted Prnp0/0 mice with transgenes encoding various mutant PrPs have been reported to elucidate the conversion mechanism of PrPC into PrPSc and have identified specific sites or amino acid residues in PrPC that are involved in the conversion of PrPC into PrPSc (
      • Fischer M.
      • Rulicke T.
      • Raeber A.
      • Sailer A.
      • Moser M.
      • Oesch B.
      • et al.
      Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.
      ,
      • Uchiyama K.
      • Miyata H.
      • Yano M.
      • Yamaguchi Y.
      • Imamura M.
      • Muramatsu N.
      • et al.
      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.
      ,
      • Hara H.
      • Miyata H.
      • Das N.R.
      • Chida J.
      • Yoshimochi T.
      • Uchiyama K.
      • et al.
      Prion protein devoid of the octapeptide repeat region delays bovine spongiform encephalopathy pathogenesis in mice.
      ,
      • Striebel J.F.
      • Race B.
      • Meade-White K.D.
      • LaCasse R.
      • Chesebro B.
      Strain specific resistance to murine scrapie associated with a naturally occurring human prion protein polymorphism at residue 171.
      ,
      • Saijo E.
      • Kang H.E.
      • Bian J.
      • Bowling K.G.
      • Browning S.
      • Kim S.
      • et al.
      Epigenetic dominance of prion conformers.
      ). We previously generated Tg(PrPΔ91-106)/Prnp0/0 mice, which express mouse PrP lacking residues 91-106, or PrPΔ91-106, on the Prnp0/0 background as low as only 0.4-fold levels of PrPC in wildtype (WT) mice (
      • Uchiyama K.
      • Miyata H.
      • Yamaguchi Y.
      • Imamura M.
      • Okazaki M.
      • Pasiana A.D.
      • et al.
      Strain-dependent prion infection in mice expressing prion protein with deletion of central residues 91-106.
      ). PrPΔ91-106 was predominantly localized at the lipid raft domains of the plasma membrane, similarly to WT PrPC, in mouse neuroblastoma N2a cells and in the brains of Tg(PrPΔ91-106)/Prnp0/0 mice (
      • Uchiyama K.
      • Miyata H.
      • Yamaguchi Y.
      • Imamura M.
      • Okazaki M.
      • Pasiana A.D.
      • et al.
      Strain-dependent prion infection in mice expressing prion protein with deletion of central residues 91-106.
      ). Tg(PrPΔ91-106)/Prnp0/0 mice were markedly resistant to mouse-adapted RML, 22L, FK-1, and BSE prions. They developed disease with a very long incubation time of 538 ± 24 days postinoculation (dpi) with BSE prions with accumulation of PrPScΔ91-106 in their brains, while remained healthy without accumulation of PrPScΔ91-106 in their brains up to at least 604 dpi with FK-1 prions, 765 dpi with RML scrapie prions, and 623 dpi with 22L scrapie prions (
      • Uchiyama K.
      • Miyata H.
      • Yamaguchi Y.
      • Imamura M.
      • Okazaki M.
      • Pasiana A.D.
      • et al.
      Strain-dependent prion infection in mice expressing prion protein with deletion of central residues 91-106.
      ). These results suggested that residues 91-106 could be involved in prion pathogenesis in a strain-dependent manner. However, it remains unclear whether the reduced susceptibility of Tg(PrPΔ91-106)/Prnp0/0 mice to these prions is due to the low expression of PrPΔ91-106 or the lack of residues 91-106 or both. It also remains unknown how residues 91-106 are involved in the conversion of PrPC into PrPSc in prion infection.
      In this study, we generated a new line of Tg(PrPΔ91-106)/Prnp0/0 mice, designated Tg(PrPΔ91-106)-8545/Prnp0/0 mice, which overexpress PrPΔ91-106 in their brains 6-times as high as PrPC in WT mice and intracerebrally inoculated RML, 22L, FK-1, and BSE prions into them. None of the RML, 22L, or FK-1 prion-inoculated Tg(PrPΔ91-106)-8545/Prnp0/0 mice became sick up to at least 337 dpi, without PrPScΔ91-106 accumulation in their brains except for very small amounts of PrPScΔ91-106 detected in one 22L prion-inoculated brain specimen. However, they developed disease at 85 ± 4 dpi with BSE prions with PrPScΔ91-106 accumulation in their brains. These results clearly indicate that residues 91-106 are crucial for PrPC to convert into PrPSc in infection with RML, 22L, and FK-1 prions but not BSE prions. We then narrowed down residues 91-106 by transducing various PrP mutants into RML- and 22L-infected cells, identifying that residues 97-99 are important for PrPC to convert into PrPSc in infection with RML and 22L prions. In vitro protein misfolding cyclic amplification (PMCA) assay also showed that residues 97-99 are important for RML, 22L, and FK-1 prions, but not for BSE prions, to induce the conversion of recombinant PrP into PrPSc. We also found that PrP mutants with proline residues at positions 97 to 99 or charged residues at positions 97 and 99 completely or almost completely lost their converting activity into PrPSc in RML- and 22L-infected cells, suggesting that the structural flexibility and noncharged properties might be important for residues 97-99 to mediate the conversion of PrPC into PrPSc in infection with RML and 22L prions.

      Results

      Generation of Tg(PrPΔ91-106)-8545/Prnp0/0 mice

      To clarify the role of residues 91-106 of PrPC in prion infection, we newly generated a PrPΔ91-106–overexpressing line of Tg(PrPΔ91-106)/Prnp0/0 mice, termed Tg(PrPΔ91-106)-8545/Prnp0/0 mice. We noticed that Tg(PrPΔ91-106)-8545/Prnp0/0 mice expressed PrPΔ91-106 at very high levels in their brains. Therefore, to estimate the exact expression levels of PrPΔ91-106 in their brains, we diluted Tg(PrPΔ91-106)-8545/Prnp0/0 brain homogenates with Prnp0/0 brain homogenates 5 and 10 times and compared the signal densities of PrPΔ91-106 in these homogenates to those of PrPC in undiluted C57BL/6 WT brain homogenates on Western blotting with IBL-N anti-PrP antibodies, which were raised against the N-terminal peptide of PrP. PrPΔ91-106 was clearly detected in the brains of Tg(PrPΔ91-106)-8545/Prnp0/0 mice (Fig. 1A). It migrated faster than WT PrPC due to the deletion of residues 91-106, with three major bands similar to WT PrPC, each corresponding to diglycosylated, monoglycosylated, and nonglycosylated forms (Fig. 1A). By densitometric analysis after taking into account the dilution factors of Tg(PrPΔ91-106)-8545/Prnp0/0 brain homogenates, PrPΔ91-106 was estimated to be expressed in the brains of Tg(PrPΔ91-106)-8545/Prnp0/0 mice at levels about 6-times higher than PrPC in WT mice (Fig. 1B). On inspection, no obvious developmental and growth abnormalities were observed in Tg(PrPΔ91-106)-8545/Prnp0/0 mice up to about 2 years after birth, suggesting that PrPΔ91-106 might not be toxic in vivo.
      Figure thumbnail gr1
      Figure 1PrPΔ91-106 expression in the brains of Tg(PrPΔ91-106)-8545/Prnp0/0 mice. A, Western blotting with IBL-N anti-PrP antibodies of brain homogenates from 8- to 10-week-old C57BL/6 WT (n = 3) and Tg(PrPΔ91-106)-8545/Prnp0/0 mice (n = 3). The Tg(PrPΔ91-106)-8545/Prnp0/0 brain homogenates were diluted 5- (5×) and 10-times (10×) with brain homogenates from Prnp0/0 mice. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an internal control. B, signal densities for WT PrPC and PrPΔ91-106 in (A) were measured. The ratio of the PrPΔ91-106 densities against the WT PrPC densities were then calculated after normalization against the GAPDH densities.

      High resistance of Tg(PrPΔ91-106)-8545/Prnp0/0 mice to RML, 22L, and FK-1 prions but not to BSE prions

      We then intracerebrally inoculated RML, 22L, FK-1, and BSE prions into Tg(PrPΔ91-106)-8545/Prnp0/0 mice as well as C57BL/6 WT mice as controls. WT mice developed disease at 167 ± 4, 149 ± 2, 152 ± 7, and 180 ± 5 dpi with RML, 22L, FK-1, and BSE prions, respectively (Table 1). Tg(PrPΔ91-106)-8545/Prnp0/0 mice were highly resistant to RML, 22L, and FK-1 prions, displaying no abnormal clinical signs at least up to 358 dpi with RML prions and 337 dpi with 22L and FK-1 prions (Table 1). In contrast, they developed disease at 85 ± 4 dpi with BSE prions (Table 1). We also investigated PrPScΔ91-106 in the brains of Tg(PrPΔ91-106)-8545/Prnp0/0 mice inoculated with RML, 22L, FK-1, and BSE prions by Western blotting with SAF61 anti-PrP antibody, which recognizes residues 141-152 of mouse PrP (
      • Feraudet C.
      • Morel N.
      • Simon S.
      • Volland H.
      • Frobert Y.
      • Creminon C.
      • et al.
      Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells.
      ). PrPSc was detected in the brains of ill WT mice inoculated with RML, 22L, FK-1, and BSE prions (Fig. 2, AD). However, no PrPScΔ91-106 was detectable in the brains of asymptomatic Tg(PrPΔ91-106)-8545/Prnp0/0 mice, which were sacrificed at 358 dpi with RML prions and 337 dpi with 22L and FK-1 prions, except for very faint signals for PrPScΔ91-106 detected in one 22L-inoculated Tg(PrPΔ91-106)-8545/Prnp0/0 mouse (Fig. 2, AC). In contrast, PrPScΔ91-106 was observed in the brains of BSE-inoculated, ill Tg(PrPΔ91-106)-8545/Prnp0/0 mice (Fig. 2D). Compared to WT PrPSc, PrPScΔ91-106 had lower levels (Fig. 2D). This is consistent with the fact that overexpression of PrP in the brain results in low production of PK-resistant PrP after prion infection (
      • Fischer M.
      • Rulicke T.
      • Raeber A.
      • Sailer A.
      • Moser M.
      • Oesch B.
      • et al.
      Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.
      ). Taken together, these results indicate that residues 91-106 could be crucial for PrPC to convert into PrPSc following infection with RML, 22L, and FK-1 prions but not BSE prions, therefore Tg(PrPΔ91-106)-8545/Prnp0/0 mice being highly resistant to RML, 22L, and FK-1 prions but not to BSE prions.
      Table 1Incubation times in WT and Tg(PrPΔ91-106)-8545/Prnp0/0 mice inoculated with RML, 22L, FK-1, and BSE prions
      PrionsRecipient mouseExpression level of PrP (fold)
      Expression levels of mutant PrP were compared to those of PrPC in WT mice using Western blotting.
      Diseased mice

      /Total mice
      Times to the onset of disease (Days ± SD)p value (log-rank test)
      RMLWT111/11167 ± 4<0.0001
      Tg(PrPΔ91-106)-8545/Prnp0/06.00/7>358
      22LWT110/10149 ± 2<0.0001
      Tg(PrPΔ91-106)-8545/Prnp0/06.00/7>337
      FK-1WT110/10152 ± 7<0.0001
      Tg(PrPΔ91-106)-8545/Prnp0/06.00/7>337
      BSEWT110/10180 ± 5<0.0001
      Tg(PrPΔ91-106)-8545/Prnp0/06.08/885 ± 4
      a Expression levels of mutant PrP were compared to those of PrPC in WT mice using Western blotting.
      Figure thumbnail gr2
      Figure 2PrPScΔ91-106 in the brains of prion-inoculated Tg(PrPΔ91-106)-8545/Prnp0/0 mice. Western blotting with SAF61 anti-PrP antibody of brain homogenates from C57BL/6 WT and Tg(PrPΔ91-106)-8545/Prnp0/0 mice intracerebrally inoculated with or without RML (A), 22L (B), FK-1 (C), and BSE prions (D). Brain homogenates from uninfected mice were used as controls. Date of the sacrifice of BSE-, RML-, 22L-, and FK-1-inoculated WT and Tg(PrPΔ91-106)-8545/Prnp0/0 mice were shown in parenthesis. BSE, bovine spongiform encephalopathy.

      Identification of residues 97-99 as being crucial for PrPC to convert into PrPSc after infection with RML and 22L prions

      To investigate the mechanism of how residues 91-106 are involved in the conversion of PrPC into PrPSc in prion infection, we determined to identify the residue(s) within residues 91-106 that are crucial for PrPC to convert into PrPSc in RML- and 22L-infected cells. To do this, we first investigated whether residues 91-106 are important for PrPC to convert into PrPSc in RML- and 22L-infected cells, by transducing an expression vector encoding PrPΔ91-106 with the 3F4 epitope or PrPΔ91-106(3F4), into RML- and 22L-infected mouse neuroblastoma N2a cells, termed N2aC24Chm and N2aC24L1-3 cells, respectively. Both infected cells were previously established by incubating N2aC24 cells, which is a clone of N2a cells persistently overexpressing exogenously transduced mouse PrPC, with brain homogenates from RML- and 22L-infected mice and subjecting them to limiting dilution cloning (
      • Fujita K.
      • Yamaguchi Y.
      • Mori T.
      • Muramatsu N.
      • Miyamoto T.
      • Yano M.
      • et al.
      Effects of a brain-engraftable microglial cell line expressing anti-prion scFv antibodies on survival times of mice infected with scrapie prions.
      ). The 3F4 epitope, which is widely used to distinguish transduced PrPs from endogenous PrPC, locates closely to residue 106 (
      • Lund C.
      • Olsen C.M.
      • Tveit H.
      • Tranulis M.A.
      Characterization of the prion protein 3F4 epitope and its use as a molecular tag.
      ), raising the concern that deletion of residue 106 might disrupt the 3F4 epitope, thereby making it impossible to detect PrPΔ91-106 on Western blotting with 3F4 antibody. Therefore, we transduced PrPΔ91-106(3F4) as well as PrPΔ91-105(3F4) and PrPΔ91-104(3F4) into N2aC24Chm and N2aC24L1-3 cells. 3F4 antibody detected PrPΔ91-104(3F4), with slightly lower signal densities than those of WT PrPC(3F4), but not PrPΔ91-106(3F4) and PrPΔ91-105(3F4) (Fig. 3, A and B), indicating that the 3F4 epitope is disrupted by deletion of residues 105 or 106 but only partially by deletion of residue 104. However, no PrPScΔ91-104(3F4) was observed in N2aC24Chm and N2aC24L1-3 cells transduced with PrPΔ91-104(3F4) (Fig. 3, A and B), indicating that residues 91-104 are important for PrPC to convert into PrPSc in RML- and 22L-infected cells.
      Figure thumbnail gr3
      Figure 3Disruption of 3F4 epitope by deletion of residue 106 or 105. Western blotting with 3F4 anti-PrP antibody of the lysates from N2aC24Chm (A) and N2aC24L1-3 cells (B) transduced with expression vectors encoding WT PrPC(3F4), PrPΔ91-106(3F4), PrPΔ91-105(3F4), or PrPΔ91-104(3F4) after treatment with (+) or without (−) PK. Western blotting with SAF61 anti-PrP antibody of the lysates from N2aC24Chm (C) and N2aC24L1-3 cells (D) transduced with control empty expression vector or expression vectors encoding WT PrPC(3F4) or PrPΔ91-104(3F4) after treatment with (+) or without (−) PK. Total PrP levels and PrPSc levels in WT PrPC(3F4)- or PrPΔ91-104(3F4)-transduced N2aC24Chm and N2aC24L1-3 cells were compared to those in control empty vector–transduced N2aC24Chm and N2aC24L1-3 cells. Data were from independent triplicate experiments. NS, not significant; ∗p < 0.05, ∗∗p < 0.01 (Student’s t test).
      We then investigated whether PrPΔ91-104(3F4) could have a transdominant-negative inhibitory activity against the conversion of endogenous PrPC into PrPSc. Western blotting with SAF61 anti-PrP antibody, which detects endogenous PrPC and PrPΔ91-104(3F4), showed that total amounts of PrP were 6.9 and 6.4-times higher in WT PrPC(3F4)- and PrPΔ91-104(3F4)-transduced N2aC24Chm cells and 2.1-times in both WT PrPC(3F4)- and PrPΔ91-104(3F4)-transduced N2aC24L1-3 cells, respectively, than those in empty vector-transduced, control N2aC24Chm and N2aC24L1-3 cells (Fig. 3, C and D). These results indicate that WT PrPC(3F4) and PrPΔ91-104(3F4) are expressed at levels much higher than or comparable to that of endogenous PrPC in N2aC24Chm and N2aC24L1-3 cells, respectively, ruling out the possibility that PrPΔ91-104(3F4) might be insufficiently expressed and therefore fail to convert into PrPScΔ91-104(3F4) in N2aC24Chm and N2aC24L1-3 cells. PrPSc was lower in PrPΔ91-104(3F4)-transduced N2aC24Chm than in empty vector- or WT PrPC(3F4)-transduced N2aC24Chm cells (Fig. 3C). Transfection with lower amounts of the expression vector (2 μg versus 5 μg) for PrPΔ91-104(3F4) downregulated the expression of PrPΔ91-104(3F4) and did not reduce PrPSc in N2aC24Chm cells (Fig. S1). Consistent with this, PrPΔ91-104(3F4)-transduced N2aC24L1-3 cells, which expressed the lower levels of PrPΔ91-104(3F4), exhibited no PrPSc reduction (Fig. 3D). Neither increased expression of PrPΔ91-104(3F4) nor reduced PrPSc were observed in N2aC24L1-3 cells even after transfection with higher amounts of the expression vector (10 μg versus 5 μg) for PrPΔ91-104(3F4) (Fig. S2). Taken together, these results suggest that PrPΔ91-104(3F4) could transdominantly inhibit the conversion of endogenous PrPC into PrPSc in an expression level-dependent manner.
      We then transduced expression vectors encoding a series of PrP mutants with various deletions within residues 91-104, such as PrPΔ91-96(3F4), PrPΔ91-100(3F4), PrPΔ96-100(3F4), PrPΔ96-104(3F4), and PrPΔ100-104(3F4), into N2aC24Chm and N2aC24L1-3 cells. Western blotting with 3F4 antibody showed that only PrPΔ91-96(3F4) was converted into PrPScΔ91-96(3F4) in these cells (Fig. 4, A and B), suggesting that residues other than residues 91-96 are important for the conversion of PrPC into PrPSc. However, PrPΔ100-104(3F4) was hyperglycosylated due to a newly created N-glycosylation site consisting of residues 99(N), 105(K), and 106(T) (Fig. 4, A and B), suggesting the possibility that the unsuccessful conversion of PrPΔ100-104(3F4) into PrPScΔ100-104(3F4) is due to the glycosylation not due to the deletion of residues 100-104. To address this, we transduced an expression vector encoding PrPΔ100-103(3F4), whose deletion does not create a glycosylation site, into N2aC24Chm and N2aC24L1-3 cells. PrPΔ100-103(3F4) was converted into PrPScΔ100-103(3F4) in both cells (Fig. 4, C and D), indicating that the glycosylation in PrPΔ100-104(3F4) is responsible for its unsuccessful conversion into PrPScΔ100-104(3F4) and that residues 100-103 are dispensable for PrPC to convert into PrPSc in infection with RML and 22L prions. Residues 97-99 are missing in the conversion-incompetent PrPΔ91-104(3F4), PrPΔ91-100(3F4), PrPΔ96-100(3F4), and PrPΔ96-104(3F4) but intact in the conversion-competent PrPΔ91-96(3F4) and PrPΔ100-103(3F4). It is thus possible that residues 97-99 could be important for PrPC to convert into PrPSc following infection with RML and 22L prions. To confirm this, we transduced PrP mutants with deletion of each residue at positions 97 to 99, termed PrPΔ97(3F4), PrPΔ98(3F4), and PrPΔ99(3F4), into N2aC24Chm and N2aC24L1-3 cells. PrPΔ97(3F4), PrPΔ98(3F4), and PrPΔ99(3F4) were not converted into PrPSc (Fig. 4, E and F), further supporting that residues 97-99 could play an important role for the conversion of PrPC into PrPSc in infection with RML and 22L prions.
      Figure thumbnail gr4
      Figure 4Crucial role of residues 97-99 in the conversion of PrPC into PrPSc in RML- and 22L-infected cells. Western blotting with 3F4 anti-PrP antibody of cell lysates from N2aC24Chm (A) and N2aC24L1-3 cells (B) transduced with expression vectors encoding WT PrPC(3F4), PrP∆91-104(3F4), PrPΔ91-96(3F4), PrPΔ91-100(3F4), PrPΔ96-100(3F4), PrPΔ96-104(3F4), and PrPΔ100-104(3F4) after treatment with (+) or without (−) PK. Western blotting with 3F4 anti-PrP antibody of cell lysates from N2aC24Chm (C) and N2aC24L1-3 cells (D) transduced with expression vectors encoding WT PrPC(3F4), PrPΔ100-104(3F4), and PrPΔ100-103(3F4) after with (+) or without (−) PK. Western blotting with 3F4 anti-PrP antibody of cell lysates from N2aC24Chm (E) and N2aC24L1-3 cells (F) transduced with expression vectors encoding WT PrPC(3F4), PrPΔ97(3F4), PrPΔ98(3F4), and PrPΔ99(3F4) after treatment with (+) or without (−) PK.

      PrPΔ97-99 is converted into PrPScΔ97-99 in a PMCA assay with BSE prions but not with RML, 22L, and FK-1 prions

      N2a cells or N2a cells overexpressing PrPC are susceptible to RML, 22L, and FK-1 prions (
      • Fujita K.
      • Yamaguchi Y.
      • Mori T.
      • Muramatsu N.
      • Miyamoto T.
      • Yano M.
      • et al.
      Effects of a brain-engraftable microglial cell line expressing anti-prion scFv antibodies on survival times of mice infected with scrapie prions.
      ,
      • Bosque P.J.
      • Prusiner S.B.
      Cultured cell sublines highly susceptible to prion infection.
      ,
      • Enari M.
      • Flechsig E.
      • Weissmann C.
      Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody.
      ,
      • Nishida N.
      • Harris D.A.
      • Vilette D.
      • Laude H.
      • Frobert Y.
      • Grassi J.
      • et al.
      Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein.
      ), but not to BSE prions. Therefore, to investigate the role of residues 97-99 in the strain-dependent conversion of PrPC into PrPSc, we performed an in vitro PMCA assay with baculovirus-derived recombinant mouse WT PrP and PrPΔ97-99 by incubating with brain homogenates from RML-, 22L-, FK-1-, and BSE-infected WT mice. Consistent with our previous results (
      • Uchiyama K.
      • Miyata H.
      • Yamaguchi Y.
      • Imamura M.
      • Okazaki M.
      • Pasiana A.D.
      • et al.
      Strain-dependent prion infection in mice expressing prion protein with deletion of central residues 91-106.
      ), Western blotting of the PK-treated PMCA products showed that WT PrP was converted into PrPSc after incubation with prion-infected brain homogenates to various degrees in a strain-dependent manner (Fig. 5). However, PrPΔ97-99 was converted into PrPScΔ97-99 only after incubation with BSE-infected homogenates, but not with RML-, 22L-, and FK-1-infected homogenates (Fig. 5). No PrPSc and PrPScΔ97-99 was produced from WT PrP and PrPΔ97-99, respectively, when incubated with prion-uninfected control brain homogenates (Fig. 5). These results suggest that residues 97-99 are important for the conversion of PrPC into PrPSc in infection with RML, 22L, and FK-1 prions but not BSE prions.
      Figure thumbnail gr5
      Figure 5PMCA assay of WT PrP and PrPΔ97-99 with RML, 22L, FK-1, and BSE prions. Western blotting of the first round PMCA products with or without PK treatment. PMCA was performed by incubating baculovirus-derived recombinant WT PrP or PrPΔ97-99 with brain homogenates from RML-, 22L-, FK-1-, and BSE-infected WT mice and control prion-uninfected WT mice. BSE, bovine spongiform encephalopathy; PMCA, protein misfolding cyclic amplification.

      Characterization of residues 97-99 important for PrPC to convert into PrPSc after infection with RML and 22L prions

      To further investigate the role of residues 97-99 in the conversion of PrPC into PrPSc following infection with RML and 22L prions, we characterized the biochemical properties of residues 97-99 important for the conversion of PrPC into PrPSc in RML- and 22L-infected cells. To do this, we transduced a series of PrP mutants with glutamine (Q) at position 97, tryptophan (W) at 98, and asparagine (N) at 99 mutated to various residues, such as negatively charged glutamate (E) and aspartate (D), positively charged arginine (R), hydrophobic alanine (A), and nonpolar proline (P) into N2aC24Chm and N2aC24L1-3 cells. PrP mutants with charged residues at position 97, termed PrPQ97E(3F4), PrPQ97R(3F4), and PrPQ97D(3F4), completely lost their converting activity into PrPSc. However, a PrP mutant with hydrophobic alanine residue at position 97, PrPQ97A(3F4), was converted into PrPScQ97A(3F4) at very low levels (Fig. 6A). PrP molecules with mutations at position 98, such as PrPW98E(3F4), PrPW98R(3F4), PrPW98D(3F4), and PrPW98A(3F4), were converted into PrPSc at low but easily detectable levels (Fig. 6B). PrPN99A(3F4) was also converted to PrPScN99A(3F4) at low but easily detectable levels (Fig. 6C). However, no or very low levels of PrPSc was produced from PrPN99E(3F4), PrPN99R(3F4), and PrPN99D(3F4), all of which contain negatively or positively charged residues at position 99 (Fig. 6C). Furthermore, PrP mutants with proline residue at each position of residues 97-99, termed PrPQ97P(3F4), PrPW98P(3F4), and PrPN99P(3F4), failed to convert into PrPSc (Fig. 6, AC). These results indicate that mutations with proline residue at positions 97 to 99 and charged residues at positions 97 and 99 completely or almost completely destroyed the converting ability of the mutant PrPs into PrPSc, suggesting that the structural flexibility with noncharged properties of residues 97-99 might be important for the conversion of PrPC into PrPSc after infection with RML and 22L prions.
      Figure thumbnail gr6
      Figure 6Characterization of residues 97-99 important for the conversion of PrPC into PrPSc in RML- and 22L-infected cells. A, Western blotting with 3F4 anti-PrP antibody of cell lysates from N2aC24Chm and N2aC24L1-3 cells transduced with expression vectors encoding WT PrPC(3F4), PrPQ97E(3F4), PrPQ97A(3F4), PrPQ97R(3F4), PrPQ97D(3F4), and PrPQ97P(3F4) after treatment with (+) or without (−) PK. B, Western blotting with 3F4 anti-PrP antibody of cell lysates from N2aC24Chm and N2aC24L1-3 cells transduced with expression vectors encoding WT PrPC(3F4), PrPW98E(3F4), PrPW98A(3F4), PrPW98R(3F4), PrPW98D(3F4), and PrPW98P(3F4) after treatment with (+) or without (−) PK. C, Western blotting with 3F4 anti-PrP antibody of cell lysates from N2aC24Chm and N2aC24L1-3 cells transduced with expression vectors encoding WT PrPC(3F4), PrPN99E(3F4), PrPN99A(3F4), PrPN99R(3F4), PrPN99D(3F4), and PrPN99P(3F4) after treatment with (+) or without (−) PK.

      Discussion

      We previously reported that Tg(PrPΔ91-106)/Prnp0/0 mice, which express PrPΔ91-106 in their brains as low as 0.4-fold levels of PrPC in WT mice, were resistant to RML, 22L, and FK-1 prions and only very poorly susceptible to BSE prions. In the present study, we generated a new line of Tg(PrPΔ91-106)/Prnp0/0 mice, termed Tg(PrPΔ91-106)-8545/Prnp0/0 mice, which overexpress PrPΔ91-106 in their brains at levels 6-times higher than PrPC in WT mice and showed that they remained healthy after intracerebral inoculation with RML, 22L, and FK-1 prions, without PrPScΔ91-106 in their brains except for one 22L-inoculated mouse accumulating very small amounts of PrPScΔ91-106 in its brain but developed disease earlier than WT mice after inoculation with BSE prions, with accumulation of PrPScΔ91-106 in their brains. These results clearly indicate that residues 91-106 are crucial for PrPC to convert into PrPSc in infection with RML, 22L, and FK-1 prions but not BSE prions, therefore supporting infection with RML, 22L, and FK-1 prions but not BSE prions.
      Tg(PrPΔ91-106)-8545/Prnp0/0 mice accumulated PK-resistant PrPScΔ91-106 in their brains at levels lower than PK-resistant PrPSc in control WT mice after inoculation with BSE prions. However, BSE-inoculated Tg(PrPΔ91-106)-8545/Prnp0/0 mice developed disease earlier than control WT mice. Similar discrepancy between the levels of PK-resistant PrPSc and the length of incubation times has been reported in Tg mice overexpressing WT PrPC after infection with RML prions (
      • Fischer M.
      • Rulicke T.
      • Raeber A.
      • Sailer A.
      • Moser M.
      • Oesch B.
      • et al.
      Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.
      ). Compared to control WT mice, the Tg mice developed disease with shorter incubation times but accumulated less PK-resistant PrPSc in their brains (
      • Fischer M.
      • Rulicke T.
      • Raeber A.
      • Sailer A.
      • Moser M.
      • Oesch B.
      • et al.
      Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.
      ). It is thus conceivable that the overexpression of PrPΔ91-106, not the lack of residues 91-106, could be responsible for the lower brain accumulation of PK-resistant PrPScΔ91-106 and the shorter incubation times in BSE-inoculated Tg(PrPΔ91-106)-8545/Prnp0/0 mice. It has been suggested that PrPSc molecules are heterogenous in their PK-resistance, including not only PK-resistant but also PK-sensitive PrPSc species (
      • Cronier S.
      • Gros N.
      • Tattum M.H.
      • Jackson G.S.
      • Clarke A.R.
      • Collinge J.
      • et al.
      Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin.
      ,
      • Fang C.
      • Imberdis T.
      • Garza M.C.
      • Wille H.
      • Harris D.A.
      A neuronal culture system to detect prion synaptotoxicity.
      ). It was shown that PK-resistant and PK-sensitive PrPSc molecules were both toxic to primary neurons (
      • Fang C.
      • Imberdis T.
      • Garza M.C.
      • Wille H.
      • Harris D.A.
      A neuronal culture system to detect prion synaptotoxicity.
      ). Thus, the overexpression of PrPΔ91-106 might increase production of PK-sensitive, neurotoxic PrPScΔ91-106 species in BSE-infected Tg(PrPΔ91-106)-8545/Prnp0/0 mice, therefore accelerating disease in these mice.
      We showed that PrPΔ91-104(3F4) failed to convert into PrPScΔ91-104(3F4) in N2aC24Chm and N2aC24L1-3 cells. Western blotting with SAF61 anti-PrP antibody, which recognizes endogenous PrPC and PrPΔ91-104(3F4), showed that compared to PrPSc in empty vector- or WT PrPC(3F4)-transduced N2aC24Chm cells, it was significantly reduced in PrPΔ9-104(3F4)-transduced N2aC24Chm cells in a manner dependent on the expression levels of PrPΔ91-104(3F4). Consistent with this, PrPΔ91-104(3F4)-transduced N2aC24L1-3 cells, which expressed the lower levels of PrPΔ91-104(3F4), did not show PrPSc reduction. These results suggest that PrPΔ91-104(3F4) might have a transdominant-negative inhibitory activity against the conversion of endogenous PrPC into PrPSc in an expression level-dependent manner. It is thus possible that PrPΔ91-106 might also have an inhibitory activity against the conversion of endogenous PrPC into PrPSc in a transdominant-negative way.
      PrPC consists of two structural domains; the flexible, unstructured N-terminal domain, which includes residues 91-106, and the α-helix-rich, globular C-terminal domain (
      • Riek R.
      • Hornemann S.
      • Wider G.
      • Glockshuber R.
      • Wuthrich K.
      NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231).
      ,
      • Donne D.G.
      • Viles J.H.
      • Groth D.
      • Mehlhorn I.
      • James T.L.
      • Cohen F.E.
      • et al.
      Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible.
      ). Upon conversion into PrPSc, PrPC undergoes marked conformational changes in the 2/3 C-terminal part to form a PK-resistant structure (
      • Collinge J.
      • Clarke A.R.
      A general model of prion strains and their pathogenicity.
      ,
      • Wadsworth J.D.
      • Asante E.A.
      • Collinge J.
      Review: contribution of transgenic models to understanding human prion disease.
      ). RML-, 22L-, and FK-1-PrPScs have a PK cleavage site around residues 90 (
      • Mange A.
      • Beranger F.
      • Peoc'h K.
      • Onodera T.
      • Frobert Y.
      • Lehmann S.
      Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein.
      ), thus residues 91-106 are entirely included in their PK-resistant structures. In contrast, BSE-PrPSc has a major PK cleavage site between residues 95 and 96 (
      • Hayashi H.K.
      • Yokoyama T.
      • Takata M.
      • Iwamaru Y.
      • Imamura M.
      • Ushiki Y.K.
      • et al.
      The N-terminal cleavage site of PrPSc from BSE differs from that of PrPSc from scrapie.
      ), indicating that residues 91-106 are only partially included in its PK-resistant structure. It is thus possible that residues 91-106 in PrPC might be involved in the formation of the PK-resistant structure of RML-, 22L-, and FK-1-PrPScs but not BSE-PrPSc, therefore Tg(PrPΔ91-106)/Prnp0/0 mice being highly resistant to RML, 22L, and FK-1 prions but still susceptible to BSE prions.
      PrPSc is abundant in β-sheet contents, in contrast to α-helix-rich PrPC, suggesting that the structural transition of α-helices to β-sheets may be a key mechanism underlying the conversion of PrPC into PrPSc. According to the cryo-electron microscopic analysis of PrPSc fibrils from 263K scrapie prion-infected hamster brains and RML-infected brains of transgenic mice expressing a glycosylphosphatidylinositol-anchorless PrP, PrPSc contained many β-sheets and assembled with parallel in-register intermolecular β-sheet structure to form the fibrils (
      • Kraus A.
      • Hoyt F.
      • Schwartz C.L.
      • Hansen B.
      • Artikis E.
      • Hughson A.G.
      • et al.
      High-resolution structure and strain comparison of infectious mammalian prions.
      ). Other investigators also carried out cryo-electron microscopic analysis for RML-PrPSc purified from the brains of RML-infected WT mice, reporting a similar parallel in-register intermolecular β-sheet structure for RML-PrPSc (
      • Manka S.W.
      • Zhang W.
      • Wenborn A.
      • Betts J.
      • Joiner S.
      • Saibil H.R.
      • et al.
      2.7 Å cryo-EM structure of ex vivo RML prion fibrils.
      ). In RML-PrPSc, residues 95-99 formed the first β-sheet in the PK-resistant structure (
      • Manka S.W.
      • Zhang W.
      • Wenborn A.
      • Betts J.
      • Joiner S.
      • Saibil H.R.
      • et al.
      2.7 Å cryo-EM structure of ex vivo RML prion fibrils.
      ), indicating that residues 91-106 are essential for the formation of the first β-sheet in RML-PrPSc. Given that Tg(PrPΔ91-106)/Prnp0/0 mice were resistant to RML, 22L, and FK-1 prions but not to BSE prions, it is possible that the formation of the first β-sheet might be important for the conversion of PrPC into PrPSc following infection with RML, 22L, and FK-1 prions but not BSE prions. Consistent with this concept, we found that PrP mutants lacking residues 97-99, which comprise a large part of the first β-sheet of residues 95-99, failed to convert into PrPSc in RML-infected N2aC24Chm and 22L-infected N2aC24L1-3 cells. We also found that recombinant PrP lacking residues 97-99 completely or almost completely lost the converting activity into PrPScΔ97-99 after incubation with RML-, 22L-, or FK-1-infected brain homogenates but almost fully converted into PrPScΔ97-99 with BSE-infected brain homogenates in an in vitro PMCA assay.
      We showed that mutant PrPs, which carry proline residue at positions 97 to 99 or charged residues at positions 97 and 99, failed to convert into PrPSc or markedly reduced their converting ability into PrPSc in N2aC24Chm and N2aC24L1-3 cells. Proline is a unique residue with a covalent bond between the carbon in the side chain and the backbone nitrogen, forming a cyclic ring that imposes rigid constraints on a peptide bond and thereby reducing the structural flexibility of the surrounding region. It is thus possible that the structural flexibility of residues 97-99 and noncharged properties of positions 97 and 99 may be important for the formation of the first β-sheet upon the conversion of PrPC into PrPSc after infection with RML and 22L prions. On the other hand, residue 95 is not included in the PK-resistant structure of BSE-PrPSc (
      • Hayashi H.K.
      • Yokoyama T.
      • Takata M.
      • Iwamaru Y.
      • Imamura M.
      • Ushiki Y.K.
      • et al.
      The N-terminal cleavage site of PrPSc from BSE differs from that of PrPSc from scrapie.
      ), suggesting that residues 95-99 might not form a β-sheet in BSE-PrPSc, therefore residues 97-99 being dispensable for the conversion of PrPC into PrPSc following infection with BSE prions. The conformational selection model postulates that inoculated PrPSc could select host PrPC as a substrate for conversion on the basis of its conformational compatibility with the host PrPC (
      • Collinge J.
      • Clarke A.R.
      A general model of prion strains and their pathogenicity.
      ,
      • Wadsworth J.D.
      • Asante E.A.
      • Collinge J.
      Review: contribution of transgenic models to understanding human prion disease.
      ). It is thus alternatively possible that residues 91-106 or residues 97-99 might be important for PrPC to adopt the conformation that is compatible with that of RML-, 22L-, and FK-1-PrPSc. However, BSE-PrPSc could be still structurally compatible even with PrP lacking residues 91-106.
      Many anti-PrP antibodies have been reported to exhibit antiprion activities, clearing both PrPSc and prion infectivity from persistently infected cultured cells and preventing disease progression in animal models of acquired prion diseases (
      • Enari M.
      • Flechsig E.
      • Weissmann C.
      Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody.
      • White A.R.
      • Enever P.
      • Tayebi M.
      • Mushens R.
      • Linehan J.
      • Brandner S.
      • et al.
      Monoclonal antibodies inhibit prion replication and delay the development of prion disease.
      ,
      • Peretz D.
      • Williamson R.A.
      • Kaneko K.
      • Vergara J.
      • Leclerc E.
      • Schmitt-Ulms G.
      • et al.
      Antibodies inhibit prion propagation and clear cell cultures of prion infectivity.
      ), giving rise to the possibility of immunotherapeutics against prion diseases. 6D11 anti-PrP antibody, whose epitope (residues 97–100) includes residues 97-99, was also shown to have an antiprion activity, reducing PrPSc levels in 22L-infected N2a cells and in the spleens of 22L-infected mice (
      • Pankiewicz J.
      • Prelli F.
      • Sy M.S.
      • Kascsak R.J.
      • Kascsak R.B.
      • Spinner D.S.
      • et al.
      Clearance and prevention of prion infection in cell culture by anti-PrP antibodies.
      ,
      • Sadowski M.J.
      • Pankiewicz J.
      • Prelli F.
      • Scholtzova H.
      • Spinner D.S.
      • Kascsak R.B.
      • et al.
      Anti-PrP Mab 6D11 suppresses PrP(Sc) replication in prion infected myeloid precursor line FDC-P1/22L and in the lymphoreticular system in vivo.
      ,
      • Pankiewicz J.E.
      • Sanchez S.
      • Kirshenbaum K.
      • Kascsak R.B.
      • Kascsak R.J.
      • Sadowski M.J.
      Anti-prion protein antibody 6D11 restores cellular proteostasis of prion protein through disrupting recycling propagation of PrP(Sc) and targeting PrP(Sc) for lysosomal degradation.
      ). It has been suggested that 6D11 antibody might prevent the intermolecular interaction between PrPC and PrPSc or the interaction of PrPC with a yet unidentified molecule that is important for its conversion into PrPSc (
      • Pankiewicz J.
      • Prelli F.
      • Sy M.S.
      • Kascsak R.J.
      • Kascsak R.B.
      • Spinner D.S.
      • et al.
      Clearance and prevention of prion infection in cell culture by anti-PrP antibodies.
      ) or might target PrPSc for lysosomal degradation (
      • Pankiewicz J.E.
      • Sanchez S.
      • Kirshenbaum K.
      • Kascsak R.B.
      • Kascsak R.J.
      • Sadowski M.J.
      Anti-prion protein antibody 6D11 restores cellular proteostasis of prion protein through disrupting recycling propagation of PrP(Sc) and targeting PrP(Sc) for lysosomal degradation.
      ), thereby eventually reducing PrPSc levels in prion-infected cells. Our results also suggest another possibility that 6D11 antibody could reduce the structural flexibility of residues 97-99 by binding to its epitope, thereby preventing the conversion of PrPC into PrPSc in prion-infected cells. Thus, agents capable of reducing the structural flexibility of residues 97-99 and consequently disturbing the formation of the first β-sheet upon the conversion of PrPC into PrPSc might be therapeutic against prion diseases, in which residues 97-99 play an important role in the conversion of PrPC into PrPSc. In sporadic CJD of humans, two different types of PrPSc with distinct PK-cleavage site, termed PrPSc types 1 and 2, have been identified (
      • Cali I.
      • Castellani R.
      • Alshekhlee A.
      • Cohen Y.
      • Blevins J.
      • Yuan J.
      • et al.
      Co-Existence of scrapie prion protein types 1 and 2 in sporadic creutzfeldt-jakob disease: its effect on the phenotype and prion-type characteristics.
      ,
      • Notari S.
      • Strammiello R.
      • Capellari S.
      • Giese A.
      • Cescatti M.
      • Grassi J.
      • et al.
      Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease.
      ). PrPSc type 1 is cleaved between residues 81 and 82 and PrPSc type 2 between 96 and 97 (
      • Cali I.
      • Castellani R.
      • Alshekhlee A.
      • Cohen Y.
      • Blevins J.
      • Yuan J.
      • et al.
      Co-Existence of scrapie prion protein types 1 and 2 in sporadic creutzfeldt-jakob disease: its effect on the phenotype and prion-type characteristics.
      ,
      • Notari S.
      • Strammiello R.
      • Capellari S.
      • Giese A.
      • Cescatti M.
      • Grassi J.
      • et al.
      Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease.
      ). It is thus possible that residues 95-99 might form a β-sheet in PrPSc type 1 but not in PrPSc type 2, and also that residues 97-99-targeting agents might be therapeutically effective against PrPSc type 1-associated sporadic CJD.
      In short, we showed here that residues 91-106 are crucial for PrPC to convert into PrPSc after infection with RML, 22L, and FK-1 prions but not BSE prions in mice. We also showed that using RML- and 22L-infected cells, residues 97-99 are particularly important for PrPC to convert into PrPSc in infection with RML and 22L prions probably through their structural flexibility with noncharged properties. An in vitro PMCA assay also supported the important role of residues 97-99 in the conversion of PrPC into PrPSc in infection with RML, 22L, and FK-1 prions but not with BSE prions. Further elucidation of the exact role of residues 97-99 in the conversion of PrPC into PrPSc could be helpful for understanding of the PrP conversion mechanism and the development of therapeutics against prion diseases.

      Experimental procedures

      Ethics statements

      The Ethics Committees of Animal Care and Experimentation of the University of Occupational and Environmental Health (approval number AE08–013, March 18, 2019) approved this study. 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. Every effort was made to reduce distress and the number of animals used.

      Generation of Tg(PrPΔ91-106)-8545/Prnp0/0 mice

      The transgene used for Tg(PrPΔ91-106)-8545/Prnp0/0 mice was constructed in elsewhere (
      • Uchiyama K.
      • Miyata H.
      • Yamaguchi Y.
      • Imamura M.
      • Okazaki M.
      • Pasiana A.D.
      • et al.
      Strain-dependent prion infection in mice expressing prion protein with deletion of central residues 91-106.
      ). In brief, a DNA fragment encoding mouse PrPΔ91-106 was first constructed using polymerase chain reaction (PCR) and then inserted into a unique Sal I site of the Syrian hamster PrP cosmid vector, CosSHa.tet (InPro Biotechnology, Inc) to enable PrPΔ91-106 to be expressed under the control of the promoter/enhancer of the hamster Prnp. The transgene was injected into the zygotes of Prnp0/0 mice, as described elsewhere (
      • Brinster R.L.
      • Chen H.Y.
      • Trumbauer M.E.
      • Yagle M.K.
      • Palmiter R.D.
      Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.
      ,
      • Wilmut I.
      • Hooper M.L.
      • Simons J.P.
      Genetic manipulation of mammals and its application in reproductive biology.
      ), after the removal of the cosmid-derived sequences, resulting in a line of Tg(PrPΔ91-106)-8545/Prnp0/0 mice. Genotyping was performed by PCR as described elsewhere (
      • Nishida N.
      • Tremblay P.
      • Sugimoto T.
      • Shigematsu K.
      • Shirabe S.
      • Petromilli C.
      • et al.
      A mouse prion protein transgene rescues mice deficient for the prion protein gene from purkinje cell degeneration and demyelination.
      ).

      Prion inoculation

      10% brain homogenates (w/v) were first prepared in phosphate-buffered saline (PBS) by passing brains from terminally ill C57BL/6 mice infected with RML, 22L, FK-1, and BSE prions through 18 to 26 gauge needles and then diluted to 1% with PBS. A 20 μl-aliquot of the homogenates was intracerebrally inoculated into 4- to 5-week-old Tg(PrPΔ91-106)-8545/Prnp0/0 and C57BL/6 mice (CLEA Japan). Mice were diagnosed as disease when they developed more than five of the following clinical signs: emaciation, decreased locomotion, ruffled body hair, ataxic gait, kyphosis, priapism, upright tail, and paralysis of the hind legs, as described elsewhere (
      • Sakaguchi S.
      • Katamine S.
      • Yamanouchi K.
      • Kishikawa M.
      • Moriuchi R.
      • Yasukawa N.
      • et al.
      Kinetics of infectivity are dissociated from PrP accumulation in salivary glands of Creutzfeldt-Jakob disease agent-inoculated mice.
      ).

      Construction of expression vectors

      DNA fragments encoding the N-terminal parts of mutant mouse PrPs with the 3F4 epitope were first amplified by PCR with a sense primer BamHI-PrP(ATG)-S (Table 2) and specific antisense primers (Table 2) using 3F4-tagged mouse WT PrPC-encoding pcDNA3.1-moPrP(3F4) (
      • Yamaguchi Y.
      • Miyata H.
      • Uchiyama K.
      • Ootsuyama A.
      • Inubushi S.
      • Mori T.
      • et al.
      Biological and biochemical characterization of mice expressing prion protein devoid of the octapeptide repeat region after infection with prions.
      ) as a template. Together with an antisense primer PrP(stop)-XbaI-AS (Table 2), the resulting DNA fragments were then utilized as a 5′ primer to amplify DNA fragments encoding full-length 3F4-tagged mutant PrPs using pcDNA3.1-moPrP(3F4) as a template. After DNA sequence confirmation, the amplified DNA fragments were inserted into BamH I/Xba I-digested pcDNA3.1(+) (Invitrogen).
      Table 2Primers and their sequences used for construction of the expression vectors in this study
      Sense/antisensePrimer nameSequence (5′→3′)
      SenseBamHI-PrP(ATG)-Stcggatcccgtcatcatggcgaac (Underline, BamH I; Bold, start codon)
      AntisensePrP(stop)-XbaI-AScctctagagctcatcccacgatcag (Underline, Xba I; Bold, stop codon)
      Specific antisensePrPΔ91-106-ASatgcttcatgttttggccccat (Italic, downstream from codon 107; Not italic, upstream from codon 90)
      PrPΔ91-105-AScttcatgttggtttggccccat (Italic, downstream from codon 106; Not italic, upstream from codon 90)
      PrPΔ91-104-AScatgttggttttttggccccat (Italic, downstream from codon 105; Not italic, upstream from codon 90)
      PrPΔ91-96-AScttgttccactgttggcccccatcc (Italic, downstream from codon 97; Not italic, upstream from codon 90)
      PrPΔ91-100-AStggtttgctgggttggcccccatcc (Italic, downstream from codon 101; Not italic, upstream from codon 90)
      PrPΔ96-100-AStggtttgctgggatgggtaccccc (Italic, downstream from codon 101; Not italic, upstream from codon 95)
      PrPΔ96-104-AScatgttggttttatgggtaccccc (Italic, downstream from codon 105; Not italic, upstream from codon 95)
      PrPΔ100-104-AScatgttggttttgttccactgatt (Italic, downstream from codon 105; Not italic, upstream from codon 99)
      PrPΔ100-103-AScatgttggtttttgggttccactgatt (Italic, downstream from codon 104; Not italic, upstream from codon 99)
      PrPΔ97-ASgctgggcttgttccaattatgggtaccccc (Italic, downstream from codon 98; Not italic, upstream from codon 96)
      PrPΔ98-AStttgctgggcttgttctgattatgggtacc (Italic, downstream from codon 99; Not italic, upstream from codon 97)
      PrPΔ99-AStggtttgctgggcttccactgattatgggt (Italic, downstream from codon 100; Not italic, upstream from codon 98)
      PrPΔ97-99-AStggtttgctgggcttattatgggtaccccc (Italic, downstream from codon 100; Not italic, upstream from codon 96)
      PrPQ97E-ASgggcttgttccattcattatgggtacc (Bold, mutated codon)
      PrPQ97A-ASgggcttgttccaagcattatgggtacc (Bold, mutated codon)
      PrPQ97R-ASgggcttgttccaacgattatgggtacc (Bold, mutated codon)
      PrPQ97D-ASgggcttgttccaatcattatgggtacc (Bold, mutated codon)
      PrPQ97P-ASgggcttgttccaaggattatgggtacc (Bold, mutated codon)
      PrPW98E-ASgctgggcttgttttcctgattatgggt (Bold, mutated codon)
      PrPW98A-ASgctgggcttgttagcctgattatgggt (Bold, mutated codon)
      PrPW98R-ASgctgggcttgttacgctgattatgggt (Bold, mutated codon)
      PrPW98D-ASgctgggcttgttatcctgattatgggt (Bold, mutated codon)
      PrPW98P-ASgctgggcttgttaggctgattatgggt (Bold, mutated codon)
      PrPN99E-AStttgctgggcttttcccactgattatg (Bold, mutated codon)
      PrPN99A-AStttgctgggcttagcccactgattatg (Bold, mutated codon)
      PrPN99R-AStttgctgggcttacgccactgattatg (Bold, mutated codon)
      PrPN99D-AStttgctgggcttatcccactgattatg (Bold, mutated codon)
      PrPN99P-AStttgctgggcttaggccactgattatg (Bold, mutated codon)
      PrPK100E-ASgtttgctgggttcgttccactgattat (Bold, mutated codon)
      PrPK100A-ASgtttgctgggagcgttccactgattat (Bold, mutated codon)
      PrPK100R-ASgtttgctgggacggttccactgattat (Bold, mutated codon)
      PrPK100D-ASgtttgctgggatcgttccactgattat (Bold, mutated codon)
      PrPK100P-ASgtttgctgggagggttccactgattat (Bold, mutated codon)
      Primers for baculovirus expression vectorsMet forward sense primercgggatccgccaccatggcgaaccttggctac (underlined sequence, BamH I site; bold sequence, start codon)
      Stop reverse antisense primercccaagctttcatcccacgatcaggaag (underlined sequence, Hind III site; bold sequence, stop codon)
      PrPΔ97-99-AStggtttgctgggcttattatgggtaccccc (Italic, downstream from codon 100; Not italic, upstream from codon 96)
      For the construction of a baculovirus expression vector for PrPΔ97-99, pFastBac-moPrPΔ97-99, a DNA fragment encoding the residues 1 to 96 fused with residues 100 to 104 of mouse PrP was first amplified by PCR with a Met forward sense primer (Table 2) and a PrPΔ97-99-AS primer using pFastBac-moPrP (
      • Uchiyama K.
      • Miyata H.
      • Yamaguchi Y.
      • Imamura M.
      • Okazaki M.
      • Pasiana A.D.
      • et al.
      Strain-dependent prion infection in mice expressing prion protein with deletion of central residues 91-106.
      ) as a template. The resulting DNA fragment was then utilized as a 5′ primer to amplify another DNA fragment encoding PrPΔ97-99 together with a Stop reverse antisense primer (Table 2) using pFastBac-moPrP as a template. After DNA sequence confirmation of the fragment, they were inserted into BamH I/Hind III-digested pFastBac1 (Thermo Scientific Inc), resulting in pFastBac-moPrPΔ97-99.

      Protein misfolding cyclic amplification

      Recombinant baculoviruses-derived WT PrP and PrPΔ97-99 (Bac-WT PrP and Bac-PrPΔ97-99) were purified by immobilized metal affinity chromatography (IMAC), as reported previously (
      • Imamura M.
      • Kato N.
      • Okada H.
      • Yoshioka M.
      • Iwamaru Y.
      • Shimizu Y.
      • et al.
      Insect cell-derived cofactors become fully functional after proteinase K and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and BSE prion proteins.
      ). In brief, pFastBac-moPrP and pFastBac-moPrPΔ97-99 were first introduced into DH10 Bac E. coli and then transfected into Spodoptera frugiperda 21 insect cells with a bacmid DNA-Cellfectin mixture (Thermo Scientific Inc) to generate recombinant baculovirus encoding WT PrP or PrPΔ97-99. The recombinant baculoviruses were then infected into High Five cells (Thermo Scientific Inc) for 72 h at 27 °C to produce Bac-WT PrP and Bac-PrPΔ97-99, and the cell lysate was subjected to IMAC purification of Bac-WT PrP and Bac-PrPΔ97-99. PMCA was then performed as described elsewhere (
      • Imamura M.
      • Kato N.
      • Okada H.
      • Yoshioka M.
      • Iwamaru Y.
      • Shimizu Y.
      • et al.
      Insect cell-derived cofactors become fully functional after proteinase K and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and BSE prion proteins.
      ). The PMCA reaction mixture was prepared by mixing 4 μl of IMAC-purified Bac-WT PrP or PrPΔ97-99 (approximately 100 ng/μl), 10 μl of the PK- and heat-treated High Five cell lysate as a conversion inducer, 85 μl of 1× PBS containing 4 mM EDTA and 0.25% Triton X-100, and 1 μl of the 1% brain homogenates infected with RML, 22L, FK-1, and BSE prions. PMCA was performed using the automatic cross-ultrasonic protein activating apparatus (Elestein 070-GOT; Elekon Science Corp) as previously reported (
      • Imamura M.
      • Kato N.
      • Okada H.
      • Yoshioka M.
      • Iwamaru Y.
      • Shimizu Y.
      • et al.
      Insect cell-derived cofactors become fully functional after proteinase K and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and BSE prion proteins.
      ). Thirty-two cycles of sonication (pulse oscillation for 3 s was repeated five times at 0.1-s intervals) were followed by incubation at 37 °C for 30 min with gentle agitation. The resulting PMCA products were treated with 40 μg/ml of PK for 1 h at 37 °C and subjected to Western blotting with horseradish peroxidase–conjugated T2 anti-PrP antibody, which recognizes residues 132-156 and 212-217 of mouse PrP (
      • Hayashi H.
      • Takata M.
      • Iwamaru Y.
      • Ushiki Y.
      • Kimura K.M.
      • Tagawa Y.
      • et al.
      Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein.
      ). Signals were visualized using chemiluminescent detection reagent, EZWestLumi plus (ATTO) with a chemiluminescence imaging system (LuminoGraph I, ATTO).

      Cell cultures

      N2aC24L1-3 and NaC24Chm cells (
      • Uchiyama K.
      • Muramatsu N.
      • Yano M.
      • Usui T.
      • Miyata H.
      • Sakaguchi S.
      Prions disturb post-Golgi trafficking of membrane proteins.
      ) were cultured in Dulbecco’s Modified Eagle Medium High Glucose (Wako Pure Chemical Industries) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific) and 1× penicillin–streptomycin mixed solution (×100) (Wako Pure Chemical Industries). Cells were maintained in a 5% CO2 in air humidified incubator at 37 °C and passaged at a 1:10 ratio every 3 to 4 days.

      Transfection

      N2aC24L1-3 and NaC24Chm cells were cultured in a 6-well plate at a density of 5 × 105 cells/well. Cells were transfected with 5 μg plasmid DNA using Lipofectamine 2000 (Invitrogen) as recommended in the instruction manual. Two days later, the cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 2 mM EDTA) and microcentrifuged at 15,000 rpm for 5 min at 4 °C. The supernatants were subjected to protein concentration measurement using the bicinchoninic acid protein assay kit (Nacalai Tesque) and then to Western blotting.

      Brain homogenization

      Brain homogenates were prepared using a Multi-beads shocker (Yasui Kikai Co) in the lysis buffer and microcentrifuged at 3000 rpm for 5 min at 4 °C. The supernatants were subjected to protein concentration measurement using the bicinchoninic acid protein assay kit (Nacalai Tesque) and then to Western blotting.

      Western blotting

      For detection of PrPSc, brain homogenates and cell lysates treated with PK (20 μg PK/mg proteins; Wako Pure Chemical Industries) at 37 °C for 30 min. Total proteins were separated by 15% SDS-polyacrylamide gels and then electrically transferred onto an Immobilon-P PVDF membrane (Millipore Corp). The membrane was blocked with 5% nonfat dry milk in TBST (0.5% Tween-20, 150 mM NaCl, 10 mM Tris-HCl, pH7.4) for 1 h at room temperature and then incubated with SAF61 mouse monoclonal antibody (Bertin Pharma), 3F4 mouse monoclonal anti-PrP antibody (BioLegend), IBL-N rabbit polyclonal anti-PrP antibodies (Immuno-Biological Laboratories), and anti-GAPDH antibody (Santa Cruz Biotechnology) overnight at 4 °C. After washed three times in TBST, the membrane was then incubated with HRP-conjugated anti-mouse IgG secondary antibody (GE Healthcare), anti-rabbit IgG antibody (GE Healthcare) in 1% nonfat dry milk-containing TBST for 1 h at room temperature. The immunoreactive signals were detected using Immobilon Western Chemiluminescent HRP substrate (Millipore) and a chemiluminescence image analyzer (LAS-4000 mini; Fujifilm Co). Densitometric analysis was performed using Image Gauge software (Fuji Film).

      Data availability

      All data are available in the main article or the supporting information.

      Supporting information

      This article contains supporting information.

      Conflict of interests

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Stanley B. Prusiner for providing Prnp0/0 mice.

      Author contributions

      S. S. methodology; A. D. P., H. M., J. C., H. H., M. I., R. A., and S. S. investigation; A. D. P., H. M., J. C., H. H., M. I., R. A., and S. S formal analysis; S. S. and A. D. P. writing-original draft.

      Funding and additional information

      This research was supported in part by JSPS KAKENHI (grand number 19H03548) to S. S. A. D. P. was supported by the OTSUKA Toshimi Scholarship Foundation (No. 20–66, No. 21–8).

      Supporting information

      References

        • Aguzzi A.
        • Baumann F.
        • Bremer J.
        The prion's elusive reason for being.
        Annu. Rev. Neurosc. 2008; 31: 439-477
        • Prusiner S.B.
        The prion diseases.
        Brain Pathol. 1998; 8: 499-513
        • Bueler H.
        • Aguzzi A.
        • Sailer A.
        • Greiner R.A.
        • Autenried P.
        • Aguet M.
        • et al.
        Mice devoid of PrP are resistant to scrapie.
        Cell. 1993; 73: 1339-1347
        • Prusiner S.B.
        • Groth D.
        • Serban A.
        • Koehler R.
        • Foster D.
        • Torchia M.
        • et al.
        Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10608-10612
        • Manson J.C.
        • Clarke A.R.
        • McBride P.A.
        • McConnell I.
        • Hope J.
        PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology.
        Neurodegeneration. 1994; 3: 331-340
        • Sakaguchi S.
        • Katamine S.
        • Shigematsu K.
        • Nakatani A.
        • Moriuchi R.
        • Nishida N.
        • et al.
        Accumulation of proteinase K-resistant prion protein (PrP) is restricted by the expression level of normal PrP in mice inoculated with a mouse-adapted strain of the Creutzfeldt-Jakob disease agent.
        J. Virol. 1995; 69: 7586-7592
        • Fischer M.
        • Rulicke T.
        • Raeber A.
        • Sailer A.
        • Moser M.
        • Oesch B.
        • et al.
        Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.
        EMBO J. 1996; 15: 1255-1264
        • Uchiyama K.
        • Miyata H.
        • Yano M.
        • Yamaguchi Y.
        • Imamura M.
        • Muramatsu N.
        • et al.
        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.
        PLoS One. 2014; 9e109737
        • Hara H.
        • Miyata H.
        • Das N.R.
        • Chida J.
        • Yoshimochi T.
        • Uchiyama K.
        • et al.
        Prion protein devoid of the octapeptide repeat region delays bovine spongiform encephalopathy pathogenesis in mice.
        J. Virol. 2018; 92e01368-17
        • Striebel J.F.
        • Race B.
        • Meade-White K.D.
        • LaCasse R.
        • Chesebro B.
        Strain specific resistance to murine scrapie associated with a naturally occurring human prion protein polymorphism at residue 171.
        PLoS Pathog. 2011; 7e1002275
        • Saijo E.
        • Kang H.E.
        • Bian J.
        • Bowling K.G.
        • Browning S.
        • Kim S.
        • et al.
        Epigenetic dominance of prion conformers.
        PLoS Pathog. 2013; 9e1003692
        • Uchiyama K.
        • Miyata H.
        • Yamaguchi Y.
        • Imamura M.
        • Okazaki M.
        • Pasiana A.D.
        • et al.
        Strain-dependent prion infection in mice expressing prion protein with deletion of central residues 91-106.
        Int. J. Mol. Sci. 2020; 21: 7260
        • Feraudet C.
        • Morel N.
        • Simon S.
        • Volland H.
        • Frobert Y.
        • Creminon C.
        • et al.
        Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells.
        J. Biol. Chem. 2005; 280: 11247-11258
        • Fujita K.
        • Yamaguchi Y.
        • Mori T.
        • Muramatsu N.
        • Miyamoto T.
        • Yano M.
        • et al.
        Effects of a brain-engraftable microglial cell line expressing anti-prion scFv antibodies on survival times of mice infected with scrapie prions.
        Cell Mol. Neurobiol. 2011; 31: 999-1008
        • Lund C.
        • Olsen C.M.
        • Tveit H.
        • Tranulis M.A.
        Characterization of the prion protein 3F4 epitope and its use as a molecular tag.
        J. Neurosci. Met. 2007; 165: 183-190
        • Bosque P.J.
        • Prusiner S.B.
        Cultured cell sublines highly susceptible to prion infection.
        J. Virol. 2000; 74: 4377-4386
        • Enari M.
        • Flechsig E.
        • Weissmann C.
        Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9295-9299
        • Nishida N.
        • Harris D.A.
        • Vilette D.
        • Laude H.
        • Frobert Y.
        • Grassi J.
        • et al.
        Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein.
        J. Virol. 2000; 74: 320-325
        • Cronier S.
        • Gros N.
        • Tattum M.H.
        • Jackson G.S.
        • Clarke A.R.
        • Collinge J.
        • et al.
        Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin.
        Biochem. J. 2008; 416: 297-305
        • Fang C.
        • Imberdis T.
        • Garza M.C.
        • Wille H.
        • Harris D.A.
        A neuronal culture system to detect prion synaptotoxicity.
        PLoS Pathog. 2016; 12e1005623
        • Riek R.
        • Hornemann S.
        • Wider G.
        • Glockshuber R.
        • Wuthrich K.
        NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231).
        FEBS Lett. 1997; 413: 282-288
        • Donne D.G.
        • Viles J.H.
        • Groth D.
        • Mehlhorn I.
        • James T.L.
        • Cohen F.E.
        • et al.
        Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457
        • Collinge J.
        • Clarke A.R.
        A general model of prion strains and their pathogenicity.
        Science. 2007; 318: 930-936
        • Wadsworth J.D.
        • Asante E.A.
        • Collinge J.
        Review: contribution of transgenic models to understanding human prion disease.
        Neuropathol. Appl. Neurobiol. 2010; 36: 576-597
        • Mange A.
        • Beranger F.
        • Peoc'h K.
        • Onodera T.
        • Frobert Y.
        • Lehmann S.
        Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein.
        Biol. Cell. 2004; 96: 125-132
        • Hayashi H.K.
        • Yokoyama T.
        • Takata M.
        • Iwamaru Y.
        • Imamura M.
        • Ushiki Y.K.
        • et al.
        The N-terminal cleavage site of PrPSc from BSE differs from that of PrPSc from scrapie.
        Biochem. Biophys. Res. Commu. 2005; 328: 1024-1027
        • Kraus A.
        • Hoyt F.
        • Schwartz C.L.
        • Hansen B.
        • Artikis E.
        • Hughson A.G.
        • et al.
        High-resolution structure and strain comparison of infectious mammalian prions.
        Mol. Cell. 2021; 81: 4540-4551
        • Manka S.W.
        • Zhang W.
        • Wenborn A.
        • Betts J.
        • Joiner S.
        • Saibil H.R.
        • et al.
        2.7 Å cryo-EM structure of ex vivo RML prion fibrils.
        bioRxiv. 2021; ([prerint])https://doi.org/10.1101/2021.12.13.472424
        • White A.R.
        • Enever P.
        • Tayebi M.
        • Mushens R.
        • Linehan J.
        • Brandner S.
        • et al.
        Monoclonal antibodies inhibit prion replication and delay the development of prion disease.
        Nature. 2003; 422: 80-83
        • Peretz D.
        • Williamson R.A.
        • Kaneko K.
        • Vergara J.
        • Leclerc E.
        • Schmitt-Ulms G.
        • et al.
        Antibodies inhibit prion propagation and clear cell cultures of prion infectivity.
        Nature. 2001; 412: 739-743
        • Pankiewicz J.
        • Prelli F.
        • Sy M.S.
        • Kascsak R.J.
        • Kascsak R.B.
        • Spinner D.S.
        • et al.
        Clearance and prevention of prion infection in cell culture by anti-PrP antibodies.
        Eur. J. Neurosci. 2006; 23: 2635-2647
        • Sadowski M.J.
        • Pankiewicz J.
        • Prelli F.
        • Scholtzova H.
        • Spinner D.S.
        • Kascsak R.B.
        • et al.
        Anti-PrP Mab 6D11 suppresses PrP(Sc) replication in prion infected myeloid precursor line FDC-P1/22L and in the lymphoreticular system in vivo.
        Neurobiol. Dis. 2009; 34: 267-278
        • Pankiewicz J.E.
        • Sanchez S.
        • Kirshenbaum K.
        • Kascsak R.B.
        • Kascsak R.J.
        • Sadowski M.J.
        Anti-prion protein antibody 6D11 restores cellular proteostasis of prion protein through disrupting recycling propagation of PrP(Sc) and targeting PrP(Sc) for lysosomal degradation.
        Mol. Neurobiol. 2019; 56: 2073-2091
        • Cali I.
        • Castellani R.
        • Alshekhlee A.
        • Cohen Y.
        • Blevins J.
        • Yuan J.
        • et al.
        Co-Existence of scrapie prion protein types 1 and 2 in sporadic creutzfeldt-jakob disease: its effect on the phenotype and prion-type characteristics.
        Brain. 2009; 132: 2643-2658
        • Notari S.
        • Strammiello R.
        • Capellari S.
        • Giese A.
        • Cescatti M.
        • Grassi J.
        • et al.
        Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease.
        J. Biol. Chem. 2008; 283: 30557-30565
        • Brinster R.L.
        • Chen H.Y.
        • Trumbauer M.E.
        • Yagle M.K.
        • Palmiter R.D.
        Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.
        Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4438-4442
        • Wilmut I.
        • Hooper M.L.
        • Simons J.P.
        Genetic manipulation of mammals and its application in reproductive biology.
        J. Reprod. Fertil. 1991; 92: 245-279
        • Nishida N.
        • Tremblay P.
        • Sugimoto T.
        • Shigematsu K.
        • Shirabe S.
        • Petromilli C.
        • et al.
        A mouse prion protein transgene rescues mice deficient for the prion protein gene from purkinje cell degeneration and demyelination.
        Lab. Invest. 1999; 79: 689-697
        • Sakaguchi S.
        • Katamine S.
        • Yamanouchi K.
        • Kishikawa M.
        • Moriuchi R.
        • Yasukawa N.
        • et al.
        Kinetics of infectivity are dissociated from PrP accumulation in salivary glands of Creutzfeldt-Jakob disease agent-inoculated mice.
        J. Gen. Virol. 1993; 74: 2117-2123
        • Yamaguchi Y.
        • Miyata H.
        • Uchiyama K.
        • Ootsuyama A.
        • Inubushi S.
        • Mori T.
        • et al.
        Biological and biochemical characterization of mice expressing prion protein devoid of the octapeptide repeat region after infection with prions.
        PLoS One. 2012; 7e43540
        • Imamura M.
        • Kato N.
        • Okada H.
        • Yoshioka M.
        • Iwamaru Y.
        • Shimizu Y.
        • et al.
        Insect cell-derived cofactors become fully functional after proteinase K and heat treatment for high-fidelity amplification of glycosylphosphatidylinositol-anchored recombinant scrapie and BSE prion proteins.
        PLoS One. 2013; 8e82538
        • Hayashi H.
        • Takata M.
        • Iwamaru Y.
        • Ushiki Y.
        • Kimura K.M.
        • Tagawa Y.
        • et al.
        Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein.
        J. Vet. Med. Sci. 2004; 66: 515-520
        • Uchiyama K.
        • Muramatsu N.
        • Yano M.
        • Usui T.
        • Miyata H.
        • Sakaguchi S.
        Prions disturb post-Golgi trafficking of membrane proteins.
        Nat. Commun. 2013; 4: 1846