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1 Centro de Investigación en Sanidad Animal, INIA, Valdeolmos, 28130 Madrid, Spain
2 Departamento de Reproducción Animal y Conservación de Recursos Zoogenéticos, 28049 Madrid, Spain
Correspondence
Juan M. Torres
jmtorres{at}inia.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Infectology, The Scripps Research Institute, 5353 Parkside Drive, Jupiter, FL 33458, USA. 
Present address: Plum Island Animal Disease Centre, ARS, USDA, PO Box 848, Greenport, NY 11944, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Bovine PrP is the product of a single Prnp gene, which encodes a glycosylphosphatidylinositol-attached lipid raft-associated membrane glycoprotein of 250 aa (Oesch et al., 1985). A characteristic stretch of repeats of an octapeptide motif is present close to the N terminus of the protein. In cattle, some degree of polymorphism in the number of recurrent octapeptides has been described (Goldmann et al., 1991; Yoshimoto et al., 1992; Hunter et al., 1994; Neibergs et al., 1994; Schlapfer et al., 1999; Walawski & Czarnik, 2003). Previously, we and others have shown that the number of octapeptide repeats (ORs) can be inversely associated with incubation times after experimental intracerebral inoculation (Chiesa et al., 1998; Castilla et al., 2004, 2005). Thus, transgenic mice bearing a bovine 7OR- or 10OR-PrP showed reduced survival times compared with mice bearing the wild-type bovine 6OR protein. As the 5OR polymorphism is also present in cattle populations, we decided to assess the effect, if any, of this allelic variant on susceptibility to BSE infection. For this purpose, we generated transgenic mice lines expressing a bovine 5OR-PrPC, characterized the expression of 5OR-PrPC in these mice and performed infectivity studies to evaluate their susceptibility to disease. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), previously digested with XhoI. This vector contains the murine PrP promoter, exon 1, intron 1, exon 2 and 3' untranslated sequences. Transgenic mice were generated by microinjection of mouse oocytes with linearized plasmid MoPrP.Xho as reported previously (Castilla et al., 2003, 2004, 2005). The presence of MoPrP.Xho bovine transgenes were identified by a PCR assay using specific primers for the mouse PrP exon 2 and bovine PrP ORF. The primers used were 5'-CCAGCCTCCACCACCATGTGGC-3' and 5'-CATTCTGCCTTCCTAGTGGTACC-3'. The presence of a 291 bp PCR product was indicative of a MoPrP.Xho 5OR transgene. The absence of the endogenous murine PrP ORF was confirmed by PCR using primers 5'-ATGGCGAACCTTGGCTACTGGC-3' and 5'-GATTATGGGTACCCCCTCCTTGG-3'.
Source of inoculum: preparation of brain homogenates.
BSE-infectious material for inoculation purposes was supplied by the Veterinary Laboratory Agency (VLA, Surrey, UK). A single BSE-infected brainstem (VLA reference RQ 225 : PG1199/00), designated BSE2, was used for comparative studies of infection in transgenic mice expressing either 5OR- or 6OR-PrPC. A pool of BSE material obtained from the brainstem of 49 BSE-infected cattle (reference TSE/08/59), designated BSE1, was also used. The infectious titre of BSE1 was 
108 ID50 (g bovine brainstem)–1 (Castilla et al., 2003). No estimation of infectious titre was available from BSE2, but it contained 8- to 16-fold more PrPres than the BSE1 inoculum as judged by immunoblot analysis (Castilla et al., 2004). A brain homogenate from a healthy cow diagnosed as negative for PrPres was used as a negative control. Brain homogenates (10 %, w/v) were prepared in sterile PBS without Ca2+ or Mg2+ using mechanical homogenization (OMNI International). To minimize the risk of bacterial infection, all inocula were pre-heated for 10 min at 70 °C before inoculation into mice.
Deglycosylation assay.
Brain homogenates (10 % in PBS without Mg+2 and Ca+2) were centrifuged for 5 min at 20 000 g in a benchtop refrigerated microfuge. Pellets were then resuspended in 20 mM potassium phosphate buffer (pH 7.8) to reconstitute the initial 10 % homogenate. After the addition of SDS to a final concentration of 1 %, samples were incubated for 15 min in a boiling water bath. Samples were then diluted with 4 vols deglycosylation buffer (20 mM potassium phosphate, 30 mM EDTA, 1.2 % Triton X-100, 1 % SDS, 0.8 % NP-40, 1.2 % 2-mercaptoethanol) and incubated at 37 °C for 20 h in the presence or absence of 50 U N-glycosidase F (PNGase F; Roche) ml–1. Proteins were precipitated with cold methanol, solubilized in Laemmli sample buffer and subjected to SDS-PAGE, Western blotting and immunoblotting as described below.
Detection of PrPC and PrPSc by Western blotting and ELISA.
Analysis of PrPC expression in transgenic mice was performed as follows. Brain homogenates from transgenic (Tg) mice were extracted with an equal volume of 10 % Sarkosyl in PBS. Samples were pre-cleared by centrifugation at 2000 g for 5 min and either treated with 20 µg proteinase K (PK; Roche) ml–1 at 37 °C for 60 min or left untreated. Brains from infected mice were homogenized in 5 % Sarkosyl in PBS. Samples (100 µl) were pre-cleared by centrifugation at 2000 g. Soluble and insoluble fractions were obtained by centrifugation at 25 000 g for 30 min. Samples were treated with 20 µg PK ml–1 at 50 °C for 60 min or left untreated. An equal volume of 2x Laemmli sample buffer was added to all samples and each one was boiled for 5 min before loading on an SDS/12 % polyacrylamide gel. For immunoblotting, mAb 2A11 (Brun et al., 2004) was used at a 1 : 1000 dilution and mAbs Sha31, 12B2 and 9A2 (Féraudet et al., 2004; Yull et al., 2006) were used at a concentration of 1 µg ml–1. Immunocomplexes were detected using a horseradish peroxidase-conjugated anti-mouse IgG. Development of immunoblots was carried out using enhanced chemiluminescence. ELISA was used for the detection of PrPSc in brains using the commercial TeSeE assay (Bio-Rad). A modification of this assay in which the PK treatment and protein precipitation steps were omitted was used for measuring PrPC content in transgenic brain samples.
Infection experiments.
Groups of 8–12 mice (6–7 weeks old, weighing approximately 20 g) were housed following the guidelines of the Code for Methods and Welfare Considerations in Behavioural Research with Animals (Directive 86/609EC). Inoculations were performed into the right parietal lobe using a 25-gauge disposable hypodermic needle. Twenty microlitres of 10 % brain homogenate was delivered to each animal. When progression of the disease was evident, animals were sacrificed and their brains were removed for analysis. These samples were used to determine spongiform degeneration by histopathology and PrPres accumulation in brain preparations by immunohistochemistry and Western blot.
Histopathology and immunohistochemistry.
Formalin-fixed brains were cut into four pieces and immersed in 98 % formic acid for 1 h before routine processing and embedding in paraffin wax. Serial sections of 4 µm nominal thickness were cut and stained with haematoxylin and eosin (H&E) for routine histopathological examination and for immunohistochemical techniques. Sections were taken from (a) the medulla oblongata at the level of the obex (myelencephalon); (b) the cerebellum; (c) the thalamus/hypothalamus (diencephalon); (d) the hippocampus; (e) the cerebral cortex (frontal and occipital areas); (f) the corpus striatum; and (g) the midbrain (mesencephalon). Sections were scored blindly for spongiform changes, gliosis (astrocytosis and microgliosis), eventual neuronal changes and PrP deposits at any level. The avidin–biotin–peroxidase complex technique was used for the immunohistochemical study of PrPres and glial fibrillary acid protein (GFAP). After dewaxing and dehydration, endogenous peroxidase was quenched by incubation with 3 % hydrogen peroxide in methanol for 30 min at room temperature. Samples for PrPres labelling were rehydrated, pre-treated with 98 % formic acid for 15 min at room temperature and 4 M guanidine isothiocianate for 2 h at 4 °C, and treated with PK (4 µg ml–1 in Tris/HCl, pH 7.8) for 15 min at 37 °C. Tissue sections were rinsed in PBS (pH 7.4) and blocked with 10 % normal goat serum (Sigma) for 30 min at room temperature. Samples were incubated overnight at 4 °C with mAb 2A11 (tissue culture supernatant diluted 1 : 400 in PBS). A secondary biotinylated goat anti-mouse IgG (Dako) diluted 1 : 200 in PBS and an avidin–peroxidase complex (Vector) were used. The reaction was visualized by the application of 3,3'-diaminobenzidine (1 mg ml–1; Sigma) and H2O2, before counterstaining with Harris’s haematoxylin, dehydration and routine assembly. Samples for GFAP labelling were rehydrated without pre-treatment and blocked as described above. Sections were incubated overnight at 4 °C with primary anti-bovine GFAP polyclonal antibody (Dako) diluted 1 : 500 in PBS. A secondary goat anti-rabbit IgG (Vector) diluted 1 : 20 in PBS was used, and the rest of the technique was carried out as described above. Tomato (Lycopersicum esculentum) lectin was used for histochemical detection of glial cells. After dehydration and quenching of endogenous peroxidase, sections were incubated overnight with biotin-conjugated L. esculentum lectin (6 µg ml–1 in 10 % normal goat serum; Sigma) at 4 °C. Sections were then incubated with avidin–peroxidase complex (Sigma) for 1 h at room temperature, washed three times in PBS and the rest of the technique carried out as described above. Specific primary antibodies were replaced by PBS, and non-immune mouse serum or non-immune rabbit serum in tissue sections was used as the negative control. All histological changes were graded semi-quantitatively on a scale of 0–3.
Statistical analysis.
Data handling, analysis and graphical representation were performed using PRISM 2.01 (GraphPad Software). Statistical differences were determined using Student’s t-test for non-paired variants. Differences were considered significant at a value of P<0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Transgenic PrPC expression in homozygous mice was then checked by Western blotting using a specific anti-PrP monoclonal antibody (2A11). Equivalent total brain protein samples, confirmed by checking the actin content, were separated by SDS-PAGE (Fig. 1a). In all mouse lines tested, the SDS-PAGE banding pattern of PrP proteins was similar, although PrP expression levels varied among the different lines obtained. PrP levels from lines BoPrP5OR-Tg008 and BoPrP5OR-Tg004 were compared with those of mouse lines BoPrP6OR-Tg022 and BoPrP6OR-Tg078 expressing a bovine 6OR-PrP (Castilla et al., 2003). Whereas BoPrP6OR-Tg078 expressed higher levels of PrP, both BoPrP6OR-Tg022 and BoPrP5OR-Tg004 showed similar levels, although lower that those of BoPrP5OR-Tg008. The relative levels of BoPrP6OR-Tg022 were established previously to be half that of cattle brain PrP (Castilla et al., 2003). Therefore, for comparison purposes, we concluded that BoPrP5OR-Tg004 PrP expression levels were 0.5-fold, whilst those of BoPrP5OR-Tg008 were 1-fold. This conclusion was further supported using a brain PrPC capture ELISA using serial dilutions of equivalent total protein concentrations (data not shown).
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). As expected, following SDS-PAGE, brain PrP expressed in BoPrP5OR-Tg mice displayed a lower apparent relative molecular mass than BoPrP6OR-Tg mice. Moreover, the reduction of one OR did not alter the biochemical properties of PrP; it showed no increased solubility and showed a similar sensitivity to protease. These results indicated that PrP from BoPrP5OR-Tg mice is likely to be processed in a similar way to PrP from BoPrP6OR-Tg mice.
Susceptibility of BoPrP5OR-Tg mice to BSE infection
To asses the effect of OR reduction on susceptibility to BSE prions, mice were inoculated with a 10 % brain homogenate of 6OR-PrPSc-containing BSE2 inoculum into the right parietal lobe. As a control, mice were also inoculated with a homogenate prepared from a healthy cow brain that showed no detectable PrPSc. Whereas BoPrP5OR-Tg004 mice survived for 635±31 days after inoculation (mean±SEM), BoPrP6OR-Tg022 mice expressing similar levels of a bovine 6OR-PrP transgene survived for only 475±24 days (Table 1). Thus, 6OR-PrPSc propagation in 5OR-PrPC-expressing mice was not as efficient as it was in 6OR-PrPC mice. Kaplan–Meier representation of survival times reflected significant differences between both mouse groups (Fig. 2a). In contrast, no significant differences in survival times were found when comparing BoPrP5OR-Tg004 mice inoculated with BSE2 (635±31 days) with those inoculated with the healthy brain control (622±34 days) (Table 1 and Fig. 2b). These results were supported by the fact that only 50 % of 5OR mice showed detectable levels of PrPres in their brains (Table 1). In agreement with these results, BoPrP5OR-Tg004 mice inoculated with the inoculum containing less PrPSc (BSE1) survived for 691±24 days with a similar attack rate (60 %) (Fig. 2c and Table 1).
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and Table 1). Moreover, and supporting our data, independent of the inoculum used, the attack rate in BoPrP5OR-Tg008 mice was lower (75 %) than in BoPrP6OR-Tg022 mice (100 %) (Table 1). Thus, we suggest that propagation of 6OR BSE prions in 5OR-PrP mice is not as efficient as in 6OR-PrP mice.
Immunohistochemical analysis
Western blotting and immunohistology showed similar numbers of BoPrP5OR-Tg animals with PrPSc. BSE-infected BoPrP5OR-Tg mice showed bilateral and symmetrical vacuolization in all of the analysed areas. Lesion profiles were similar in both BoPrP5OR-Tg004 and BoPrP5OR-Tg008 mouse lines. The distribution of the vacuolation pattern in these mice mainly targeted the midbrain, thalamus and medulla oblongata at the level of the obex and showed no differences when compared with BoPrP6OR-Tg022 mice. Spongiosis was always found associated with gliosis, either astrocytosis or microgliosis; immunolabelling with anti-GFAP antibody [Fig. 3a(ii, vi and x)] and with L. esculentum [Fig. 3a(iii, vii and xi)] revealed that these cells were associated with PrPSc deposits [Fig. 3a(iv, viii and xii)]. Fine to coarse granular deposits of PrPSc were scattered in the perikarya of neurons along with larger ovoid deposits in the cytoplasm of glial cells, mainly in the brain stem (data not shown). Punctate neuropil labelling was observed in several brain sites [Fig. 3a(xii)]. However, the most prominent labelling was in the form of plaque-like deposits [Fig. 3a(iv and xii), arrowheads], which appeared scattered in all brain areas except the cerebral cortex.
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). We observed that PrPres deposition patterns for BSE-infected BoPrP5OR-Tg mice were strikingly similar to those of infected BoPrP6OR-Tg mice. No mice showed either ependymal or vascular patterns, and glial-associated PrPres staining was very light. In contrast, intracellular PrPres and neuropil deposits were abundant. Both 5OR-PrPC mouse Tg lines showed accumulation of large amounts of amyloid plaque-like deposits. Overall, no differences in neuropathological findings could be found in the infected BoPrP5OR-Tg mice compared with infected BoPrP6OR-Tg mice.
Characterization of new Bo5OR-PrPSc
The newly formed Bo5OR-PrPSc was analysed biochemically for insolubility and PK sensitivity. No differences in insolubility or PK sensitivity were found (data not shown). To analyse in more detail the length of the protease-resistant fragment after PK treatment, different antibodies mapping near to the N-terminal site of the PK-resistant PrP core were used (Yull et al., 2006). As shown in Fig. 4(a), PrPres from both BoPrP5OR-Tg and BoPrP6OR-Tg mice was readily detected by mAb Sha31, which recognizes an epitope within the resistant core of PrPres, and mAb 9A2, which recognizes an epitope at the N terminus of the PrPres fragment (Fig. 4b). However, little or no signal in either BoPrP5OR-Tg or BoPrP6OR-Tg mice was found when mAb 12B2 was used, indicating that PK cleavage resulted in loss of the 12B2 epitope seen in both sets of animals (mAb 12B2 recognizes the epitope in bovine PrPC) (data not shown). These results indicated that both 5OR-PrPSc and 6OR-PrPSc are similarly processed after PK treatment and, therefore, a similar structure is likely to be shared by PrPSc aggregates from both 5OR and 6OR mice. Moreover, analysis of the PrPres glycoform ratios did not reveal any significant differences (Fig. 4c), indicating that both proteins retained similar post-translational modifications.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Such polymorphic point mutations have not been associated with bovine breeds, but the presence of polymorphic variations in the number of ORs within the Prnp gene has been reported. Both in observed clinical cases and by the use of transgenic mice, it has been shown that the number of ORs affects either spontaneous onset of the disease or strongly influences incubation times (Goldfarb et al., 1991; Poulter et al., 1992; Chiesa et al., 1998; Vital et al., 1998; Castilla et al., 2004, 2005). Thus, an inverse relation might exist in which the higher the number of ORs, the shorter the incubation/survival time. Mouse models have proved very useful in understanding inter- and/or intraspecific transmission barriers to prions. It is known that overexpression of PrP transgenes in mouse models can shorten the length of incubation times considerably and, consequently, shorten survival times. Our BoPrP5OR-Tg mouse lines expressed low levels of 5OR-PrPC contributing to lengthy survival times. However, when compared with other mouse Tg lines expressing similar, near to physiological, low levels of 6OR-PrPC, the survival times were significantly increased, in agreement with reported incubation times for gene-targeted bovine Tg mice (Bishop et al., 2006). Thus, considering expression levels as a major factor affecting the length of incubation/survival times, it was possible to associate the extended survival rate with the presence of 5OR-PrPC. One explanation for this could be that the transformation rate of 5OR-PrPC to 5OR-PrPSc by means of an incoming 6OR-PrPSc is less efficient than that for 6OR-PrPC; this is also supported by the limited number of inoculated BoPrP5OR-Tg mice that scored positive for PrPres. The biochemical properties of 5OR-PrPSc were the same as 6OR-PrPSc in terms of insolubility, PK sensitivity, PrPres glycoform ratio and PrPres epitope mapping. Moreover, on the basis of immunohistological examination, it was not possible to determine differences in the pattern of brain lesions between BoPrP5OR-Tg and BoPrP6OR-Tg mice. In spite of the lack of biochemical and pathological differences between 5OR-PrPSc and 6OR-PrPSc, it remains to be determined whether the infectivity of both types of prion in homologous PrPC-expressing mice is similar. In other words, do the survival and attack rates for 5OR-PrPSc in BoPrP5OR-Tg mice differ from those for 6OR-PrPSc in BoPrP6OR-Tg mice? Experiments are in progress to assess the infectivity of both 5OR and 6OR bovine prions in homozygous 5OR/5OR and 6OR/6OR mice, as well as in heterozygous 5OR/6OR Tg mice.
In cattle, the presence of five, six and seven ORs in the Prnp gene has been reported (Goldmann et al., 1991; Brown et al., 1993; Neibergs et al., 1994; Ferguson et al., 1997; Schlapfer et al., 1999). Due to scarce epidemiological data, it remains to be established whether or not there is an association between these polymorphisms and the prevalence of BSE in cattle. However, some epidemiological considerations might support the evidence for a genetic basis in the reported BSE incidence. The dispersal of BSE cases has been shown to be different depending on the country affected (Hagenaars et al., 2000). In contrast, homozygous animals bearing 5OR Prnp alleles have never been found in BSE-affected cows (Goldmann et al., 1991; Yoshimoto et al., 1992; Neibergs et al., 1994), although the frequency of this genotype is usually very low, particularly in the Frisona-Holstein breeds (Brown et al., 1993). The allele encoding 6OR-PrP seems to be the most frequent in cattle. Based on these data, it is reasonable to assume that the presence in nature of 5OR prions may be less frequent than 6OR prions.
The significant delay in survival times and the reduced attack ratio observed in BoPrP5OR-Tg mice after inoculation of a BSE inoculum reflects a lower efficiency of prion replication, which is probably due to a reduced ability for transformation of 5OR-PrPC into 5OR-PrPSc. This fact might substantiate the genetic basis for the low incidence of BSE in 5OR-PrPC-expressing cattle.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 September 2006;
accepted 12 February 2007.
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-helix structure and filled boxes represent predicted 
-sheet structures. (c) Representation of Western blot profiles of the relative ratios of PrPres detected with mAb Sha31 in brain extracts from the indicated BSE-infected transgenic lines. Data shown are the means of six or more measurements obtained from density scans in at least two different Western blots. To interpret the plot, read the values for the diglycosyl, monoglycosyl and aglycosyl fractions along the bottom, right and left axes of the triangle respectively. For each point, the sum of the three values is 100.