스위스 쮜리히 공대의 아드리아노 아구치(Adriano Aguzzi) 교수와 크리스티나 시구그르손(Christina Sigurdson) 교수가 이끄는 연구팀이 프리온 단백질이 종간장벽을 뛰어넘어 전염되는 원리를 규명하는데 열쇠가 될 수 있는 연구결과를 발표했다는 소식입니다.

프리온 질병을 전염시키는 변형 프리온 단백질이 숙주의 정상 단백질과 만날 때, 정상적인 알파-헬릭스 구조를 베타-시트 구조로 변형시켜 프리온 질병을 전염시키는 열쇠는 아미노산 염기서열의 170번(대부분 S 또는 N) 및 174번(N 또는 T) 위치에 있는 2개의 잔기형태(residue types)에 영향을 받는 것으로 밝혀졌다고 합니다. 연구팀은 특히  아미노산 염기서열 170번의 다형성(170S/N polymorphism)이 종간장벽을 뛰어넘여 이종간 전염에 중요한 역할을 하는 것으로 추정했습니다.

자세한 내용은 아래 기사와 원문을 참고하시기 바랍니다.

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In Prion Disease Transmission Sequence And Structure Is Key

출처 : Medical News Today  Article Date: 15 Jun 2010 – 2:00 PDT
http://www.medicalnewstoday.com/articles/191761.php

Prion diseases are lethal neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD) in humans and bovine spongiform encephalopathy (BSE; commonly known as mad cow disease) in cows. A team of researchers, led by Adriano Aguzzi and Christina Sigurdson, at UniversitätsSpital Zürich, Switzerland, has generated data in mice that provides greater understanding of the factors that determine how easy it is for prion diseases to be transmitted to a new host species. This information provides new insight into a highly important food safety issue; dietary exposure to beef contaminated with the BSE agent is believed to have caused nearly 200 cases of variant CJD in humans.

The key infectious agent in prion diseases is PrPSc, a highly aggregated form of the cellular prion protein (PrPC). The ease with which prions from different species can be transmitted to a new host species varies dramatically. The team found that transmission between species with the same protein building block at position 170 in PrPC was relatively easy while it was relatively difficult between those species with different building blocks at that position. As this protein building block influences the structure of the PrPC protein, the authors suggest that local structure of PrPC affected by the protein building block at position 170 might have a triggering role in prion transmissibility between different species.

Source:
Karen Honey
Journal of Clinical Investigation

TITLE: A molecular switch controls interspecies prion disease transmission in mice

AUTHOR CONTACT:
Adriano Aguzzi
UniversitätsSpital Zürich, Zürich, Switzerland.
Phone: 41.44.255.2107; Fax: 41.44.255.4402;E-mail: adriano.aguzzi@usz.ch.

Christina Sigurdson
University of California at San Diego, La Jolla, California, USA.
Phone: 858.534.0978; Fax: 858.246.0523; E-mail: csigurdson@ucsd.edu.

=========================

Research Article

A molecular switch controls interspecies prion disease transmission in mice

Christina J. Sigurdson^1,2,3, K. Peter R. Nilsson³, Simone Hornemann⁴, Giuseppe Manco³, Natalia Fernández-Borges⁵, Petra Schwarz³, Joaquín Castilla^5,6, Kurt Wüthrich^4,7 and Adriano Aguzzi³

¹Department of Pathology and Department of Medicine, University of California, San Diego, La Jolla, California, USA.
²Department of Pathology, Immunology, and Microbiology, University of California, Davis, Davis, California, USA.
³UniversitätsSpital Zürich, Institute of Neuropathology, Zürich, Switzerland.
⁴Institut für Molekularbiologie und Biophysik, ETH Zürich, Zürich, Switzerland.
⁵CIC BioGUNE, Parque tecnológico de Bizkaia, Bizkaia, Spain.
⁶Ikerbasque, Basque Foundation for Science, Bizkaia, Spain.
⁷Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.

Address correspondence to: Adriano Aguzzi, UniversitätsSpital Zürich, Institute of Neuropathology, Department of Pathology, Schmelzbergstrasse 12, CH–8091 Zürich, Switzerland. Phone: 41.44.255.2107; Fax: 41.44.255.4402; E-mail: adriano.aguzzi@usz.ch. Or to: Christina Sigurdson, Department of Pathology, University of California, San Diego, 9500 Gilman Dr., La Jolla, California 92093, USA. Phone: 858.534.0978; Fax: 858.246.0523; E-mail: csigurdson@ucsd.edu.

Published June 14, 2010
Received for publication December 15, 2009, and accepted in revised form April 28, 2010.

Introduction

Transmissible spongiform encephalopathies (TSEs) are lethal neurodegenerative disorders that include kuru and Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cows, scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids (1). A crucial component of the infectious agent is PrP^Sc, a highly aggregated, β-sheet–rich isoform of the cellular prion protein (PrP^C) (2, 3).

Prion diseases can occur as distinct strains within species expressing identical PrP amino acid sequences (4). Prion strains have originally been defined by transmission to mice, in which they show different incubation periods (ips) and variable brain regions targeted (5–7). Evidence is accumulating that distinct strains represent different PrP^Sc conformations (8–11).

There is evidence that interspecies prion transmission occurs in nature, and it has thus become an important food safety issue. Dietary exposure to beef contaminated with the BSE agent is believed to have caused nearly 200 cases of variant CJD (vCJD) in humans (12–15) as well as spongiform encephalopathies in domestic cats and British zoo animals (16–19). Prion transmission across species barriers has been replicated experimentally and typically leads to incomplete attack rates and to prolonged, highly variable ips (20). Understanding the factors that dictate species barriers is crucial for assessing the risk for human prion disease, particularly as new prion strains may arise in food animals (21–24).

Experimental studies of species and strain barriers in vitro and in vivo indicate that certain amino acid positions have an outstandingly strong influence on prion transmission among species with heterologous prion protein (Prnp) sequences (4, 25–32). For example, hamsters resist infection with mouse prions, although the hamster and mouse Prnp sequences differ at only 9 residues (30–32). Similarly, knock-in mice that only differ from WT mice at codon 101 of the Prnp allele show highly divergent susceptibility to sheep scrapie, BSE, and CJD (33, 34).

Susceptibility to natural prion infections may also be influenced by Prnp polymorphisms. Humans are polymorphic at PRNP codon 129 (methionine/valine). While approximately 38% of Europeans are 129MM homozygous, 100% of the confirmed vCJD cases have occurred in 129MM patients (35), indicating selective susceptibility of this allotype to BSE prions. Since the efficiency of TSE transmission into a new species further appears to vary depending on the prion strain (33, 36, 37), the increased susceptibility of humans carrying the 129MM alleles may not be maintained with future BSE strains.

Although the amino acid sequence of PrP^C is overall highly conserved among mammals, the β2-α2 loop encompassing residues 165–175 is a site of outstandingly high sequence diversity (38). The 3-dimensional structure of PrP^C immediately led to the identification of this loop as a key part of the surface epitope that might influence species barriers (39, 40), and this hypothesis also received support from molecular dynamics simulations (41). Mature PrP^C contains a flexibly unstructured N-terminal 100-residue “tail” and a globular C-terminal domain of similar size, which contains 3 α-helices and a short antiparallel β-sheet (40, 42, 43). The β₂-α₂ loop is structurally polymorphic in that amino acid sequences that contain 170S and 174N form a poorly defined loop in the NMR solution structures at 20°C (44–47), whereas sequences that contain 170N, or 170N and 174T (numbering according to ref. 48) have a well-defined loop (49–51). Here, we used transgenic mice expressing prion proteins that differ in discrete sequence positions of the β₂-α₂ loop to further investigate the effect of the loop structure on interspecies prion transmission. We found that sequence variations affecting only the β₂-α₂ loop conformation can significantly impact cross-species susceptibility to a broad array of prion diseases.

Results

To determine the impact of the loop sequence on interspecies transmission, we used 2 lines of transgenic mice expressing PrP^C variants that differ by 2 amino acid residues. tga20 mice overexpressed WT mouse PrP, whereas tg1020 mice overexpressed a “rigid loop” (RL) variant of PrP with S170N and N174T substitutions (Figure 1A). In both mouse lines, the transgene was expressed in the context of a “half-genomic” prion minigene construct. Expression levels were approximately 6- and 3-fold higher in tga20 and tg1020 mice, respectively, than those for endogenous PrP^C (52, 53).

Mice were inoculated with prions derived from 5 species (Figure 1A), for which PrP^C 3-dimensional structures have been fully characterized by solution NMR spectroscopy: sheep, cattle, cervid, hamster, and mouse (Figure 1B). The overall structure of the C-terminal globular domain is nearly identical among these 5 species (Figure 1B), yet they differ in the local conformations of the β₂-α₂ loop (Figure 1C) encompassing residues 165–175 (40, 44–47, 50, 51).

Inoculation with mouse scrapie strains. All prion strains used for the inoculations were proteinase K (PK) resistant. Therefore, the criterion used for determining transmission to tg1020 and tga20 mice was the presence of PK-resistant PrP^Sc in the brain of the recipients as well as development of a rapidly progressive neurologic disease with widespread spongiosis. To assess the susceptibility of WT-PrP and RL-PrP mice to mouse-adapted scrapie, we inoculated tga20 and tg1020 mice intracerebrally with 10⁴ LD₅₀ of Rocky Mountain Laboratory (scrapie strain passage 5, referred to herein as RML5) prions (Figure 2A). tga20 mice developed terminal prion disease in 74 ± 6 days, whereas tg1020 mice developed disease after a much longer and more variable ip (323 ± 92 days; unpaired, 2-tailed Student’s t test, P = 0.002; Figure 2B and Table 1). The prion-containing brain homogenate recovered from the latter mice was termed RL-RML¹ (RL-passaged RML, first passage). WT mice developed scrapie in 170 ± 12 days after inoculation with 10³ log LD₅₀ of the same inoculum (Figure 2B), indicating that RL-PrP mice developed disease with a 47% increase in the ip compared with the WT mice.

RML-PrP^Sc aggregates in brain displayed similar morphologies in tga20 and tg1020 mice, consisting of widespread deposits visible by immunohistochemistry (Figure 2C). Elderly uninoculated tg1020 mice developed a spontaneous transmissible prion disease, with focal cerebral PrP plaques (53), yet the morphology of these spontaneous deposits was profoundly different from the widespread diffuse deposits developing in inoculated mice. Also, N-terminally cleaved PrP never acquired PK resistance of all 3 glycoforms in spontaneously sick tg1020 mice. Therefore, the PK-resistant PrP^Sc detected in tg1020 and tga20 mice indicates that transmission of RML prions had occurred in both strains of host mice.

Inoculation of RL-RML¹ homogenate into a further generation of tg1020 mice resulted in a shortening of the ip and a reduction of variability by nearly 50%, to 171 ± 11 days (Figure 2, A and B, and Table 1). Hence, the substitutions of residues 170 and 174 had created a species barrier for RML prions, which was overcome by serial passaging. Conversely, back passage of the RL-RML¹ inoculum into tga20 mice led to a more than 50% increase in the ip and to a 5-fold increase in variability (186 ± 33 days; Figure 2B and Table 1). The latter results point to a species barrier erected by the WT-Prnp sequence against RL-PrP prions. In conclusion, mouse prions could be adapted to hosts expressing either WT or RL Prnp; both resulting inocula were readily propagated in hosts expressing the same Prnp variant but were much less amenable to propagation in hosts expressing the other variant. This fulfills the definition of a species barrier.

RL-RML¹ PrP^Sc aggregates were sparsely distributed in tga20 mice but were widespread in tg1020 mice, as shown by immunohistochemistry and histoblotting (Figure 2D). Neither RML nor RL-RML¹ tissue deposits were stained by Congo red (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI42051DS1). We stained brain sections with the fluorescent amyloidotropic dye, polythiophene acetic acid (PTAA) (54). PTAA stains PrP^Sc deposits and emits distinct fluorescence spectra that report on the supramolecular arrangement of protein aggregates.

All RML-infected tg1020 brains, but none of the tga20 brains, contained PTAA⁺ plaques (Figure 2C). We compared PTAA emissions by plotting the fluorescence intensity ratio at 531 and 642 nm (R_531/642) against the ratio between the intensity at 531 nm and at the emission maximum (R_531/Emax). The resulting values are independent of absolute fluorescence intensity and can discriminate among multiple prion strains (55, 56). PTAA emission characteristics were highly variable among animals and sometimes even within individual animals, suggesting highly polymorphic PrP^Sc conformations in the first passage of RML (Figure 2E). However, none of the tga20 and tg1020 mice inoculated with RL-RML¹ prions had PTAA⁺ plaques. This finding suggests that the supramolecular arrangements of PrP^Sc had changed drastically upon passage.

We then compared brain homogenates of tga20 and tg1020 mice inoculated with RL-RML¹ by PK digestion followed by Western blotting. The concentration of PrP^Sc was relatively homogenous in tg1020 mice but highly variable in tga20 mice (Figure 2F). We concluded that the S170N and N174T substitutions created a species barrier to RML prions, which was eventually overcome in the RL mice through multiple passages.

Inoculation with CWD infectious brain homogenate. We then inoculated tg1020 and tga20 mice with CWD prions from a terminal, naturally infected mule deer. WT mice are resistant to infection with CWD prions (57, 58), whereas overexpression of WT Prnp renders tga20 mice susceptible to CWD — albeit after long ips (59). The mature PrP^C sequences of mice and mule deer differ at 23 residues, but the 165–175 loop sequence in tg1020 mice is identical to that of deer and elk (Figure 1A).

CWD infection led to accelerated disease in tg1020 mice compared with tga20 mice (279 ± 48 days and 543 ± 72 days, respectively; Figure 3A and Table 1) (unpaired, 2-tailed Student’s t test, P = 0.001). We detected abundant fine and clumped aggregates in hippocampus and cerebral cortex by using immunohistochemistry for PrP in tg1020 mice. The PTAA emission spectra were similar for all histologically visible aggregates in the first CWD passage, with little variability within and between tg1020 animals.

Surprisingly, the second passage of RL-CWD in tg1020 mice resulted in a slight lengthening of the ip (Figure 3A and Table 1). Using immunohistochemistry for PrP and PTAA emission spectra, 2 conspicuously differing PrP^Sc aggregate morphologies emerged: (a) a noncongophilic type, with a low R_531/642 similar to the first passage, and (b) a rare, 50- to 100-μm dense congophilic type, with a high R_531/642 (Figure 3B). The R_531/642, which depends on fluorescence resonance transfer between PTAA molecules, indicated that the LCP chains were tightly packed in the noncongophilic aggregates and loosely packed in the congophilic aggregates (Figure 3C). Maybe the conformational variants were interfering with the conversion of each other, thereby resulting in an unexpectedly long ip.

We then investigated the biochemical profile of CWD-induced PrP^Sc in tg1020 brain homogenates using PK digestion and PrP immunoblotting. The first passage of CWD in tg1020 mice yielded barely detectable PK-resistant PrP^Sc. By the second passage, PK-resistant PrP showed the typical shift in electrophoretic mobility, suggesting that a PK-resistant form had become more abundant (Figure 3D).

We then assessed the effect of the PrP^Sc primary amino acid sequence on the strain properties by comparing a mouse-adapted CWD (M-CWD) strain with mule deer CWD in tg1020 mice. There was no significant difference in the ips: M-CWD–infected tg1020 mice (Figure 3E) and mule deer CWD–infected tg1020 mice (Figure 3A) had ips of 252 ± 51 days and 279 ± 48 days, respectively (Student’s t test, P = 0.45).

PrP aggregates in the M-CWD–inoculated tg1020 brains histologically appeared large and congophilic (Figure 3F), similar to the rare congophilic plaques seen in the RL-CWD–infected tg1020 mice and the M-CWD–infected tga20 mice (Figure 3, B and F). The LCP emission spectra R_531/642 and R_531/Emax were also similar (Figure 3G), with values that are indicative of loosely packed PTAA chains.

The above data indicate that after primary deer CWD inoculation, RL-PrP may give rise to morphologically small, noncongophilic aggregates, leading to tight packing of PTAA, or large, congophilic plaques, leading to looser packing of PTAA. In contrast, the M-CWD strain gave rise to a single morphology in both tga20 and tg1020 mice, which appeared similar to the large congophilic plaques described above.

Inoculation of hamster scrapie into RL-PrP and WT-PrP mice. Mice were initially believed to be completely resistant to infection with particular strains of hamster prions (60), but an extensive series of experiments revealed that mice can replicate hamster prions subclinically (32). The hamster PrP^C β₂-α₂ loop sequence is homologous with the RL-PrP sequence at position 170N and with the WT mouse sequence at 174N. To determine whether either WT-PrP or RL-PrP, or both, could support hamster prion replication, we inoculated tga20 and tg1020 mice with the Sc237 strain of hamster prions. The ips were consistently more than 400 days for all mice, but the attack rate varied markedly. Whereas 6 out of 6 tg1020 mice showed N-terminally cleaved, PK-resistant PrP^Sc accumulation in histoblot and Western blot assays (Figure 4, A and B), versus a background of 0 out of 13 uninoculated tg1020 mice (Supplemental Figure 2), only 1 out of 4 tga20 mice had any detectable PrP^Sc, even following concentration of PrP^Sc by sodium phosphotungstic acid precipitation (Figure 4B). tg1020 mice, but not tga20 mice, showed conspicuous spongiform encephalopathy, which was particularly evident in the cerebral cortex (Figure 4C). PrP^Sc aggregates were finely granular and diffusely distributed (Figure 4A). The Congo red, PTAA, and polythiophene methyl imidazole (PTMI) stains were negative (Supplemental Figures 1 and 3), precluding emission spectra comparisons with RL-RML or RL-CWD. We concluded that hamster scrapie could be consistently transmitted to tg1020 but not tga20 mice. The long ip suggests either inefficient transmission or generation of PrP^Sc aggregates with low toxicity.

Inoculation with BSE and sheep scrapie. We inoculated tg1020 and tga20 mice with BSE and sheep scrapie isolates (Swiss BSE and Colorado sheep scrapie from single animals). Cattle, sheep, and mouse PrP protein sequences are identical at the β₂-α₂ loop, and both BSE and sheep scrapie isolates can infect tga20 mice, as previously reported (56). After inoculation with BSE or sheep scrapie, aged tg1020 mice (>300 days old) developed weight loss and mild paraparesis as well as rare RL-PrP plaques in the hippocampus. However, in the tg1020 mice there was no evidence for BSE or sheep scrapie transmission by any method. PrP histoblots and Western blots were consistently negative for PK-resistant PrP^Sc (Supplemental Figures 2 and 4).

In vitro cross-species PrP^Sc amplification using protein misfolding cyclic amplification. To assess whether the transmission barrier induced by the S170N and N174T substitutions could be reproduced in vitro, we performed protein misfolding cyclic amplification (PMCA) experiments, using the tg1020 and tga20 brain as a substrate. We seeded the PMCA reaction with RML mouse prions, 263K hamster scrapie, deer CWD, and Colorado sheep scrapie. Six separate samples of tg1020 and tga20 brain were seeded with each prion strain and amplified for 5 rounds (Table 2 and Supplemental Figure 5). We found that by the fifth round, tg1020 brain homogenates yielded PK-resistant PrP in 100% of samples seeded with hamster scrapie and 83% of samples seeded with CWD, compared with only 17% and 50% of tga20 brain homogenates seeded with hamster scrapie and CWD, respectively. tg1020 and tga20 brain homogenate led to PK-resistant PrP in all samples seeded with RML but in no samples seeded with sheep scrapie. Therefore, the PMCA experiments largely reproduced our in vivo findings; the main difference occurred with sheep scrapie, in which PK-resistant PrP^Sc was not detected in any of the tga20 and tg1020 samples. Although we have shown that the tga20 mice and tg1020 mice were both susceptible to RML infection, the difference in the ip was not revealed by differences in seeding ability using PMCA.

Discussion

170 and 174 are positions that can modulate prion transmission. The S170N and N174T substitutions in the β₂-α₂ loop of PrP^C erected a complete barrier to TSEs derived from 2 different host species, cattle and sheep (Table 3). Conversely, the very strong barrier of mice against hamster prions was completely ablated, as hamster prions infected all mice expressing RL-PrP^C (Table 3). The potent impact on species barriers caused by 2 amino acid substitutions indicates that prion species barriers may be profoundly altered in humans or animals expressing polymorphisms or mutant PrP molecules. Thus, human or animal TSEs may be replicated by mutant molecules, even when otherwise strong species barriers are known to exist.

TSE infection in the tg1020 mice. We have previously reported that tg1020 mice spontaneously develop a new strain of TSE (53). The resulting disease may confound the assessment of TSE transmission to prion-inoculated tg1020 mice. On the other hand, several traits allowed for unequivocal differentiation between transmitted and spontaneous prion disease. First, immunoblots showed the canonical “shift” in electrophoretic mobility in PrP^Sc recovered from TSE-infected tg1020 mice after PK digestion. This shift has never been detected in spontaneously sick tg1020 mice. Second, inoculated mice showed widespread deposition of PrP^Sc in the brain, which extended to regions, such as the thalamus, in which aggregates have never been observed in spontaneous disease. Third, the staining profiles of specific LCP dyes and their fluorescence emission spectra differed vastly between inoculated and spontaneously sick tg1020 mice. For example, PTAA failed to stain any brain sections of RL-RML² and RL hamster strains in tg1020 mice, whereas it consistently labeled spontaneously appearing prion aggregates. Taken together, these features support that the instances of disease reported in this study represent bona fide transmission of TSE rather than spontaneous disease typical of noninoculated tg1020 mice.

tga20 mice express 2-fold more PrP^C than the tg1020 mice and would therefore have been expected to develop prion disease after a shorter ip if there was no transmission barrier induced by the S170N and N174T substitutions. However, the tga20 mice only developed prion disease with a shorter ip after inoculation with RML prions and actually had a longer ip after inoculation with CWD prions. With RML infection, the tg1020 mice showed clear evidence of a transmission barrier, in that the ip exceeded that of lower expressing WT mice and shortened by 50% upon second passage. Together, the data clearly support that the differences in prion susceptibility are due to the S170N and N174T substitutions and not due to the differences in PrP^C expression.

Consistency of in vivo and in vitro studies of species barriers. Two decades of published studies indicate that the penetrance of cross-species transmission is affected by the sequence similarity between the host PrP^C and the incoming PrP^Sc (61). Even single–amino acid polymorphisms can radically alter the transmissibility profiles of individual prion strains (62–69). Scrapie infection of cells expressing variant sequences of PrP^C has also revealed the striking effects of mutations on the efficiency of conversion of PrP^C to PrP^Sc (70).

Hamster prions were successfully transmitted only to tg1020 mice and not to tga20 mice. The hamster PrP sequence is homologous to RL-PrP at position 170 and to WT-PrP at position 174, suggesting that homology at 170 was critical for transmission. Studies of prion transmission in bank voles and PMCA experiments with CWD in a panel of mammalian species also implicate residue 170 as a key position for influencing species barriers (37, 71). These results can be rationalized by classifying TSE hosts into 2 groups: 170S animals that include cattle, sheep, and mice and 170N animals that include elk, deer, hamsters, and RL mice.

Here, we show that susceptibility to a number of prion strains seemed to be driven by homology at position 170, although further experiments with single mutants would be necessary to fully clarify the relative contributions of residues at positions 170 and 174. tga20 mice could readily convert other 170S prions, such as cattle BSE and classical sheep scrapie, but not the 170N prions, such as hamster scrapie. While the overexpressing tga20 mice could convert 170N mule deer CWD prions after a long ip (59), WT mice are resistant to CWD (57, 58). Conversely, tg1020 mice could convert 170N hamster scrapie and mule deer CWD but completely resisted infection with 170S sheep scrapie and BSE (Table 3). While tg1020 mice may have developed spontaneous infectivity, which would have been detected on further passages, this possibility would not have interfered with the above interpretation.

The above results prompted us to reinterpret published prion transmission experiments and epidemiologic studies in the context of the 170S/170N transmission barrier. In accordance with our predictions, prions are readily transmitted within 170S mammals: sheep scrapie prions infect cattle (72–74) and mice (75, 76), and cattle BSE prions readily infect sheep (77–79) and mice (14, 80). BSE (170S) has also been transmitted to other 170S species, including greater kudu, nyala, eland, scimitar-horned oryx, bison, rhesus macaque, domestic cat, and mink (13, 16, 81, 82). Conversely, prions generated in 170S animals are poorly transmissible to 170N recipients and vice versa. Transgenic mice overexpressing bovine or ovine PrP completely resisted infection to 9 different CWD isolates (58), and WT mice do not develop clinical prion disease after infection with elk CWD or hamster 263K scrapie (57, 58, 60). Furthermore, cattle and sheep are poorly infectible with mule deer CWD, since few animals develop disease after prolonged ips of 6 years, and only after intracerebral inoculation (83, 84). Among 170N animals, prions are also transmissible, as elk CWD is infectious to hamsters (85), albeit not very efficiently. Collectively, most historical data from studies on species barriers support the model that similarity at the loop region, and particularly the 170S/N switch, impacts transmission barriers in a broad variety of species. As a possible exception to these observations, cattle may be susceptible to CWD from white-tailed deer (86). The latter finding suggests that specific prion strains can overrule the codon 170 homology requirement.

Does the β₂-α₂ loop direct prion strain conformation? What are the structural consequences of the substitutions at residues 170 and 174, and how might they explain the important role of these substitutions in interspecies prion transmission? Crystallographic investigations of PrP peptides revealed that adjacent β₂-α₂ loops can engage in a “dry steric zipper” interface, which was proposed to represent the elemental backbone of many amyloids. Peptide crystal structures encompassing the β₂-α₂ loops bearing the 170S/174N and 170N/174T substitutions were arranged in a P1 or a P2₁ crystal space group, respectively (87). These observations suggest striking differences in the β-sheet alignment of PrP^Sc aggregates between prion-infected 170S and 170N animals and may provide a plausible starting point for clarifying the structural basis of prion species barriers that are highly relevant to public health, including the potential transmissibility of bovine and cervid prions to humans.

Methods

Prion inoculations. tga20 or tg1020 (RL) transgenic mice, which overexpressed murine PrP (52, 53), were intracerebrally inoculated in the left parietal cortex with 30 μl of brain homogenate. This was either a 5%–10% brain homogenate from a terminally sick, naturally CWD-infected mule deer (provided by M. Miller, Colorado Division of Wildlife, Fort Collins, Colorado, USA), an uninfected control deer (reported in ref. 59), a 5% brain homogenate from a terminal, naturally scrapie-infected Suffolk sheep (n = 4 mice) (provided by T. Spraker, Colorado State University, Fort Collins, Colorado, USA), or a 5% brain homogenate from a naturally BSE-infected cow (n = 4 mice) (provided by A. Zurbriggen, University of Bern, Bern, Switzerland). Mice were monitored every second day, and TSE was diagnosed according to clinical criteria, including ataxia, kyphosis, and hind leg paresis. Mice were sacrificed at the onset of terminal disease. Mice were maintained under specific pathogen–free conditions. All of the present studies were reviewed and approved by the animal care and use committee (Kantonales Veterinäramt Zürich) in the Zurich Cantonal Veterinary Office, Switzerland.

Western blots. The 10% brain or spleen homogenates were prepared in PBS, using a Precellys 24 Tissue Homogenizer. Extracts of 50–90 μg protein were diluted with a Tris-based buffer (10 mM Tris, 10 mM EDTA, 100 mM NaCl, 0.5% NP40, and 0.5% DOC) and digested with 100 μg/ml PK for 30 minutes at 37°C. A SDS-based buffer was then added, and the samples were heated to 95°C for 5 minutes, prior to electrophoresis through a 12% Bis-Tris precast gel (Invitrogen), followed by transfer to a nitrocellulose membrane by wet blotting. Proteins were detected with anti-PrP POM1 antibody (epitope in the globular domain, amino acids 121–231) (88). For secondary detection, we used an HRP-conjugated anti-mouse IgG antibody (Zymed, Invitrogen). Signals were visualized with the ECL detection kit (Pierce). Sodium phosphotungstic acid (NaPTA) precipitation was used when noted to enrich for PrP^Sc, prior to Western blotting, using published methods (89).

Histoblots. Histoblots were performed as reported in Taraboulos et al. (90), using up to 100 μg/ml of PK for the digestion of PrP^C. Histoblots were developed using the anti-PrP POM1 antibody (88).

PTAA staining of frozen tissue sections. Frozen sections from mouse brain were dried for 1 hour and fixed in 100% and 70% ethanol for 10 minutes each. After washing with deionized water, sections were equilibrated in 100 mM sodium carbonate at pH 10.2 for 30 minutes. The PTAA was diluted in the sodium carbonate buffer (1 μg per 100 μl buffer) and added to the sections. The sections were incubated with PTAA for 30 minutes at room temperature and washed with sodium carbonate buffer. The spectra in the tissue were recorded with a Zeiss Axioplan 2 microscope, fitted with a Spectraview 4.0 (Applied Spectral Imaging) and a Spectra-Cube (interferometrical optical head SD 300) module with cooled CCD camera, through a 470 nm/40 nm (LP 515) bandpass filter (Zeiss) in steps of 10 nm. The data were processed with SpectraView 3.0 EXPO. Spectra were collected from 10 individual spots within 3–4 plaques, from a minimum of 3 different mice of each prion strain.

PTAA staining of formalin-fixed tissue sections. Paraffin-embedded, formalin-fixed, and formic-acid treated brain sections were deparaffinized and treated with PK (10 μg/ml in PBS) for 10 minutes prior to staining. PTAA staining was performed as described above.

Histopathology and immunohistochemical stains. Two-μm-thick sections were cut onto positively charged silanized glass slides and stained with H&E or immunostained using antibodies for PrP (SAF84), for astrocytes (GFAP), or microglia (Iba1). For PrP staining, sections were deparaffinized, incubated for 6 minutes in 98% formic acid, and then washed in distilled water for 5 minutes. Sections were heated to 100°C in a pressure cooker in citrate buffer (pH 6.0), cooled for 3 minutes, and washed in distilled water for 5 minutes. Immunohistochemical stains were performed on an automated Nexus staining apparatus (Ventana Medical Systems), using an iVIEW DAB Detection Kit (Ventana Medical Systems). After incubation with protease 1 (Ventana Medical Systems) for 16 minutes, sections were incubated with anti-PrP SAF-84 (SPI-bio; 1:200) for 32 minutes. Sections were counterstained with hematoxylin. GFAP immunohistochemistry (1:1,000 for 24 minutes; DAKO) for astrocytes and Iba1 immunohistochemistry (1:2,500 for 32 minutes; Wako Chemicals) for microglia were similarly performed, but with antigen retrieval, by heating to 100°C in EDTA buffer (pH 8.0).

Serial automated PMCA procedure. The whole process of in vitro prion replication, including the PrP^Sc detection of amplified samples, was performed following the basic conditions described previously (91–93). The optimization of the technique was performed empirically, taking into account all modifiable parameters. Our optimal results were obtained using the following settings: 60 μl of sample, 70% of power of sonication, 30 minutes of incubation time, 20 seconds of constant sonication, 180 ml of water over the horn plate sonicator, and 37°C–38°C in the water of sonication.

Statistics. Continuous data are presented as mean ± SD. The ips, from inoculation to terminal prion disease, in the tga20 and tg1020 mice were compared using Student’s unpaired t test. Two-tailed P values of less than 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism.

Supplemental data

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Acknowledgments

We thank our histopathology and animal care staff for technical support. This study was supported by the European Union (TSEUR to A. Aguzzi and UPMAN to K. Wüthrich), the Swiss National Science Foundation, the National Competence Centers for Research on Neural Plasticity and Repair (to A. Aguzzi) and on Structural Biology (to K. Wüthrich), NIH grants K08-AI01802 and 5R21NS055116 (to C.J. Sigurdson), the Foundation for Research at the University of Zürich (to C.J. Sigurdson), the US National Prion Research Program (to C.J. Sigurdson and A. Aguzzi), the Knut and Alice Wallenberg Foundation (to K.P.R. Nilsson), and the ETH Zürich (to K. Wüthrich). K. Wüthrich is the Cecil H. and Ida M. Green Professor of Structural Biology at The Scripps Research Institute.

Footnotes

Conflict of interest: Adriano Aguzzi receives funding from the Novartis Foundation.

Citation for this article: J Clin Invest doi:10.1172/JCI42051.

K. Peter R. Nilsson’s present address is: Department of Chemistry, IFM, Linköping University, Linköping, Sweden.

Simone Hornemann’s present address is: UniversitätsSpital Zürich, Institute of Neuropathology, Zürich, Switzerland.

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