A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy
1999; Springer Nature; Volume: 18; Issue: 23 Linguagem: Inglês
10.1093/emboj/18.23.6855
ISSN1460-2075
AutoresJean Manson, Elizabeth R. Jamieson, Herbert Baybutt, Nadia L. Tuzi, Rona Barron, I. McConnell, Robert A. Somerville, James W. Ironside, Robert Will, Man Sun Sy, David W. Melton, James Hope, Christopher J. Bostock,
Tópico(s)Trace Elements in Health
ResumoArticle1 December 1999free access A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy Jean C. Manson Corresponding Author Jean C. Manson BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Elizabeth Jamieson Elizabeth Jamieson Institute of Cell and Molecular Biology, Edinburgh University, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author Herbert Baybutt Herbert Baybutt BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Nadia L. Tuzi Nadia L. Tuzi BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Rona Barron Rona Barron BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Irene McConnell Irene McConnell BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Robert Somerville Robert Somerville BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author James Ironside James Ironside CJD Surveillance Unit, Western General Hospital, Crewe Road, Edinburgh, UK Search for more papers by this author Robert Will Robert Will CJD Surveillance Unit, Western General Hospital, Crewe Road, Edinburgh, UK Search for more papers by this author Man-Sun Sy Man-Sun Sy Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author David W. Melton David W. Melton Institute of Cell and Molecular Biology, Edinburgh University, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author James Hope James Hope Institute for Animal Health, Compton Laboratory, Newbury, Berkshire, RG20 7NN UK Search for more papers by this author Christopher Bostock Christopher Bostock Institute for Animal Health, Compton Laboratory, Newbury, Berkshire, RG20 7NN UK Search for more papers by this author Jean C. Manson Corresponding Author Jean C. Manson BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Elizabeth Jamieson Elizabeth Jamieson Institute of Cell and Molecular Biology, Edinburgh University, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author Herbert Baybutt Herbert Baybutt BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Nadia L. Tuzi Nadia L. Tuzi BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Rona Barron Rona Barron BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Irene McConnell Irene McConnell BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author Robert Somerville Robert Somerville BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK Search for more papers by this author James Ironside James Ironside CJD Surveillance Unit, Western General Hospital, Crewe Road, Edinburgh, UK Search for more papers by this author Robert Will Robert Will CJD Surveillance Unit, Western General Hospital, Crewe Road, Edinburgh, UK Search for more papers by this author Man-Sun Sy Man-Sun Sy Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author David W. Melton David W. Melton Institute of Cell and Molecular Biology, Edinburgh University, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK Search for more papers by this author James Hope James Hope Institute for Animal Health, Compton Laboratory, Newbury, Berkshire, RG20 7NN UK Search for more papers by this author Christopher Bostock Christopher Bostock Institute for Animal Health, Compton Laboratory, Newbury, Berkshire, RG20 7NN UK Search for more papers by this author Author Information Jean C. Manson 1, Elizabeth Jamieson2, Herbert Baybutt1, Nadia L. Tuzi1, Rona Barron1, Irene McConnell1, Robert Somerville1, James Ironside3, Robert Will3, Man-Sun Sy4, David W. Melton2, James Hope5 and Christopher Bostock5 1BBSRC Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh, EH9 3JF UK 2Institute of Cell and Molecular Biology, Edinburgh University, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR UK 3CJD Surveillance Unit, Western General Hospital, Crewe Road, Edinburgh, UK 4Case Western Reserve University School of Medicine, Cleveland, OH, USA 5Institute for Animal Health, Compton Laboratory, Newbury, Berkshire, RG20 7NN UK *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:6855-6864https://doi.org/10.1093/emboj/18.23.6855 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A mutation equivalent to P102L in the human PrP gene, associated with Gerstmann–Straussler syndrome (GSS), has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild-type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days. Disease was transmitted from these mice to both wild-type (226 days) and 101LL mice (148 days). In contrast, 101LL mice infected with ME7 had prolonged incubation times (338 days) compared with wild-type mice (161 days). The 101L mutation does not, therefore, produce any spontaneous genetic disease in mice but significantly alters the incubation time of TSE infection. Additionally, a rapid TSE transmission was demonstrated despite extremely low levels of disease-associated PrP. Introduction Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases that are also infectious. It has been hypothesized that these diseases are attributable to a conformational change in the prion protein (PrP), which results in a change from a predominantly α-helical protein to a β-sheet form (Prusiner, 1996; Weissmann, 1996). PrP, which is converted from the normal cellular form of the protein (PrPC) to the protease-resistant, disease-specific form (PrPSc) during the infectious process (Prusiner, 1991), has been proposed to be the infectious agent (Griffith, 1967). The prion hypothesis predicts that PrPSc can propagate its own conversion by acting as a template or a seed (Jarrett and Lansbury, 1993), allowing further conversion of PrPC to PrPSc to occur. Polymorphisms at amino acids 108 and 189 in the murine PrP gene have a major influence on incubation time of scrapie in mice (Moore et al., 1998). Polymorphisms at amino acids 129 and 219 are associated with altered incubation time or susceptibility to human TSEs (Palmer et al, 1991; Goldfarb et al., 1992). Polymorphisms at amino acids 136 and 171 in the PrP gene are associated with susceptibility to TSE disease in sheep (Goldmann et al., 1994). The mechanism by which these mutations lead to altered susceptibility or incubation periods has not been defined, but it has been proposed that the human polymorphisms may be present in a site involved in the conformational transition from PrPC to PrPSc (Glockshuber et al., 1999). In vitro assay systems (Priola et al., 1994) and mouse models (Scott et al., 1992) have suggested that homology between an infectious and endogenous PrP molecule facilitates the efficient conversion of PrPC to PrPSc, allowing TSE disease to develop in the host. By introducing an appropriate PrP gene into transgenic mice, the species barrier can be overcome, as demonstrated with transgenic mice expressing a hamster PrP gene, which were shown to be susceptible to hamster strains of scrapie, in contrast to wild-type mice (Scott et al., 1989). While the above mutations alter the susceptibility of an animal following exposure to an infectious agent, a number of point mutations and insertions in the human PrP gene apparently lead to spontaneous genetic disease (Prusiner, 1997; Parchi et al., 1998), many of which have been transmitted subsequently to rodents and primates (Tateishi and Kitamoto, 1995; Young et al., 1999). It has been suggested that inherited human TSEs result from mutations in the PrP gene leading to amino acid changes that destabilize the three-dimensional structure of the PrP protein (Cohen et al., 1994; Huang et al., 1994; Harrison et al., 1997) and that this inherent instability makes the PrPC protein more likely to convert to and accumulate as PrPSc. A transgenic mouse overexpressing a murine PrP gene with a Leu101 mutation was shown to develop disease spontaneously (Hsiao et al., 1990; Telling et al., 1996), suggesting that the 101L mutation may indeed result in an unstable PrP protein. In addition, the resultant spontaneous disease was transmitted to transgenic mice expressing the same transgene but not to wild-type mice (Hsiao et al., 1994), suggesting a requirement for homology between PrP molecules for transmission of the disease to occur. PrP has also been implicated in the pathological process leading to neurodegeneration. PrPSc accumulates in the brain during disease, but grafting tissue from wild-type mice into the brain of PrP null mice has shown that accumulation of PrPSc is not sufficient to lead to the development of pathology in the brain in the absence of PrPC (Brandner et al., 1996). PrPSc accumulation has not been detected in all TSE disease. Although PrP accumulates in the brains of the 101 transgenic mice described above, it does not have the characteristic protease resistance associated with PrPSc (Hsiao et al., 1990; Telling et al., 1996). Primary transmission of bovine spongiform encephalopathy (BSE) agent to mice has been described in the apparent absence of PrPSc accumulation (Lasmezas et al., 1997). Spontaneous neurodegenerative disease has also been reported in transgenic mice with an AV3 substitution in PrP in which an abnormal transmembrane form of PrP protein accumulates rather than PrPSc (Hedge et al., 1998), although transmissibility of disease from these mice has not been reported. Although certain mutations in PrP have been shown to alter the incubation period of TSE disease (Moore et al., 1998), the mechanism by which this effect is achieved has not been defined. Here we describe the introduction of a 101L mutation into the endogenous murine PrP gene. This mutation does not lead to the development of spontaneous TSE disease in these mice, but dramatically alters the incubation times of disease following exposure of the 101LL mice to different strains of agent. One of the resulting TSE diseases can be transmitted to both wild-type and mutant mice with short incubation periods, despite extremely low amounts of PrPSc in the inoculum. Results Gene expression from the Prnpa101 allele A two-step double replacement gene targeting strategy was used to alter Prnpa exon 3 in HM-1, a 129/Ola murine embryonic stem cell line. Gene targeting was used to alter specifically amino acid 101 (equivalent to amino acid 102 in human PrP) from the wild-type proline (101P) residue to a leucine (101L), to generate in situ a modified Prnpa allele expressing the mutant PrP gene (Moore et al., 1995). The targeted allele has been designated Prnptm1Edin referred to in this text by the more descriptive name Prnpa101L (Figure 1A). HM-1 embryonic stem (ES) clones carrying a Prnpa101L mutant allele were used to produce chimeric mice (Moore et al., 1995). A chimera, which transmitted the mutant allele, was used to generate progeny heterozygous for the Prnpa101L allele (101PL), which were then inter-bred to produce progeny homozygous for the Prnpa101L allele (101LL). Wild-type mice are referred to as 101PP. Figure 1.(A) Gene structure and gene expression from the Prnpa101L allele. Restriction map of the wild-type 129/Ola Prnpa allele and the targeted Prnpa101L alleles. Probes are shown as hatched boxes. The Prnpa allele yields 151 and 613 bp DdeI exon 3 fragments and the Prnpa101L allele yields 464, 151 and 149 bp DdeI fragments from exon 3 due to the additional DdeI site created by the mutation. The following Prnp probes were used: probe a, a 884 bp PCR product of intron 2 from 25 004 to 25 887 bp (DDBJ/EMBL/GenBank accession No. U29186); probe b, a 936 bp KpnI–EcoRI fragment from exon 3; and probe c, a 700 bp EcoRV–BamHI fragment at the 3′ end of the Prnp gene. Abbreviations: BamHI (B), XbaI (X), DdeI (D), EcoRV (RV), E1, exon 1; E2, exon 2; E3, exon 3. (B) Genomic Southern analysis of the Prnpa101L allele. Genomic DNA was isolated from 101LL and 101PP mice and digested with KpnI (K), BamHI (B), EcoRI (E), HindIII (H), XbaI (X) and BstEII (Bs). Following digestion, the fragments were separated on a 1% agarose gel blotted on to Genescreen plus (Dupont) and hybridized with probe c (3′ end of the Prnp gene) and probe a (5′ end of the Prnp gene). These data, together with exon 3 nucleic acid sequence (data not shown), confirm that the targeted Prnpa101L allele is indistinguishable from the parental 129/Ola Prnpa allele, with the exception of the engineered PrP codon 101L alteration. (C) Northern analysis of PrP mRNA in the brain. Total brain RNA (20 μg) probed with a 936 bp KpnI–EcoRI mouse PrP exon 3 DNA probe. Lanes 1 and 2, wild-type 129/Ola (101PP); lane 3, heterozygous PrP null (101P/−) to demonstrate that a 50% reduction in mRNA was detectable; lane 4, PrP−/−; lanes 5–7, mice with two copies of the targeted Prnpa101L allele (101LL); lane 8, mice with one copy of the Prnpa101L allele and a null allele (101L/−). Mouse 18S rRNA reprobe of the membrane. Quantitation of the 18S rRNA signal was determined using PhosphorImager technology (Molecular Dynamics), and these values were used to correct for loading variations. (D) Western blots of crude brain homogenates from mice expressing wild-type Prnpa and gene-targeted Prnpa101L alleles. Samples were resolved on SDS–PAGE and transferred to PVDF membrane. PrP was detected with a rabbit anti-mouse PrP polyclonal serum 1A8 (top) or mouse monoclonal 8H4 (bottom) and visualized with either HRP-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibodies and a chemiluminesence detection kit. Lanes 1–5, mouse with two copies of the Prnpa101L allele (101LL); lanes 7–11, wild-type mouse (101PP). Lanes 1 and 7, 100 μg; lanes 2 and 8, 75 μg; lanes 3 and 9, 50 μg; lanes 4 and 10, 25 μg; and lanes 5 and 11, 1 μg wet weight tissue equivalents. Download figure Download PowerPoint The structure of the targeted Prnpa101L allele in the mice was investigated by Southern blot analysis (Figure 1B) using probes derived from intron 2 and exon 3 (Figure 1A). The 101L mutation changes CCC-Pro to CTC-Leu, creating an additional DdeI site within exon 3. No other alteration was detected in the targeted allele. The entire Prnp coding region from the 101LL mice was sequenced to confirm that no mutations, other than the desired one, had been introduced during the construction of the mice (data not shown). These studies confirm that the Prnpa101L allele has undergone no detectable deletions, insertions or rearrangements during the gene targeting process or during the production of the mice. The 101PL mice were crossed with CB20 mice to produce an outbred line of mice with the mutant allele as well as being maintained on an inbred 129/Ola background. All TSE inoculation experiments were carried out using inbred mice. The level of expression of the PrP gene from the targeted allele (Prnpa101L) was assessed and compared with the wild-type gene. Northern blot analysis detected similar levels of PrP mRNA in mice with the mutant Prnpa101L allele (101LL) and in wild-type mice (101PP) (Figure 1C). Western blot analysis using both monoclonal and polyclonal PrP antibodies, however, has indicated that the steady-state level of the PrP protein in 101LL mice is apparently lower than that in wild-type mice (Figure 1D). Accurate quantification of the difference in amount of PrP between the two lines of mice has proved difficult by Western blot analysis, but a more quantitative assay system is being developed to address this question. This reduction in PrP in 101LL mice may be a result of altered processing or stability of the mutant protein. Alternatively, altered conformation may lead to differences in the ability of the antibodies to bind to the mutant PrP protein and thus lead to an apparent reduction in the protein levels detected. The different PrP protein levels in the 101LL mice most probably result directly from the 101L mutation, which has been introduced into the murine gene, since sequencing and Southern blot analysis of the PrP gene did not detect any differences between the 101LL and 101PP mice except for the 101L mutation. Additional evidence that suggests that the alteration in PrP protein level may be a specific effect of the 101L mutation is provided by the previous gene targeting experiments in which we introduced alterations into amino acids L108F and T189V. Mice with these alterations were shown to have levels of PrP mRNA and protein identical to the wild-type mice (Moore et al., 1998). Why this mutation should lead to an altered level of PrP is currently under investigation, but for the purposes of assessing TSE incubation times in the 101LL and 101PL mice reported here, it is important to allow for the apparent reduction in level of PrP protein in the 101LL mice. This is because it has been shown that mice with only one copy of the wild-type PrP gene, and thus with reduced levels of PrP protein, have longer incubation times than wild-type mice following inoculation with different strains of TSE (Manson et al., 1994). The Prnpa101L allele is not sufficient for spontaneous neurodegenerative disease Both inbred and outbred mice (>200 animals) carrying one or two copies of the Prnpa101L allele showed no clinical signs of TSE disease up to 899 days of age (63 of which were >700 days old). The brains of all culled mice have been examined for pathological signs of subclinical TSE. Animals over 350 days showed some vacuolation limited to white matter regions of the brain, consistent with ageing vacuolation and typical of control groups. In order to assess whether there had been any accumulation of PrP in the brain, despite the absence of clinical signs of disease, brains from 27 mice both homozygous and heterozygous for the Prnpa101L allele (631–888 days old) were examined by immunocytochemical and Western blot analysis. No PrP deposition was detected in the brains by immunocytochemical analysis (not shown) and no PrPSc was detected by Western blot analysis after long exposure times of immunoblot to X-ray film (Figure 6A). Brains from mice homozygous for the Prnpa101L allele were used as an inoculum to assess whether, despite the absence of clinical or pathological signs of TSE, they carried spontaneously and endogenously generated TSE infectivity. A pool of three brains from mice (101LL) over 700 days old was inoculated intracerebrally into homozygous (101LL), heterozygous (101PL) and wild-type 129Ola (101PP) mice. At the time of writing, these mice have shown no clinical signs of TSE disease (600 days after inoculation). The 101L mutation alters the incubation time of TSE disease Inbred 129/Ola mice carrying no (101PP), one (101PL) or two (101LL) copies of the Prnpa101L allele were inoculated with brain homogenate from a patient who died of Gerstmann–Straussler syndrome (GSS) (Table I). The entire coding region of the PrP gene of this patient was sequenced and was shown to be heterozygous for the 102L mutation, homozygous for 129M and carried no other mutation in the PrP gene. Only one of eight wild-type mice showed clinical signs of disease 456 days after inoculation. Vacuolation was apparent in the cerebellar and midbrain white matter regions in the brain of this mouse, but no significant grey matter vacuolation was detected. The remaining seven wild-type mice showed no signs of clinical disease or significant vacuolar pathology in either white or grey matter regions of the brain up to 701 days post-infection. The brains of these mice were also devoid of PrP by immunocytochemical analysis and no PrPSc was detected by Western blot analysis (Figure 6B). Table 1. Incubation time of disease following inoculation with GSS, GSSLL and ME7 Inoculum Mouse strain No. with clinical TSE No. without clinical TSE Incubation time ± SEM GSS (10−1) 101PP 0 1 101PL 4 0 479 ± 29 101LL 2 0 254 ± 0 GSS (10−2) 101PP 1 6 456 101PL 10 8 450 ± 12 101LL 15 0 288 ± 4 GSSLL (10−2) 101PP 16 0 226 ± 3 101PL 20 0 201 ± 3 101LL 18 0 148 ± 2 ME7 (10−2) 101PP 10 0 161 ± 2 101PL 29 0 353 ± 4 101LL 18 0 338 ± 8 Mice with no (101PP), one (101PL) and two (101LL) copies of the Prnpa101L allele were inoculated with primary GSS (GSS), GSS passed through a 101LL mouse (GSSLL) and ME7. The inoculum was injected intracerebrally at 10−1 or 10−2 dilution. Mice carrying two copies of the Prnpa101L allele (101LL) all developed clinical signs of disease and were culled between 254 and 317 days after infection. The clinical phase extended over 4–6 weeks and included ataxia, hind limb paralysis and marked kyphosis. Vacuolar pathology of both grey and white matter was evident in the brains of these mice (Figure 2A). After extensive immunocytochemical analysis of sections at different levels throughout the brain, using two different polyclonal antibodies (1B3 and 1A8), abnormal accumulation of PrP could not be detected in five of the brains (Figure 3B, Table II). This was despite the fact that all cases had marked vacuolation in the thalamus, septum and hypothalmus and severe white matter vacuolation of the midbrain and cerebellar regions. Minor diffuse deposits of PrP were detected in five other cases, restricted to the thalamus and inner cortical layer. Two of these cases also showed aggregates of PrP in the corpus callosum near the site of injection, perhaps due to retention of inoculum in this region. Figure 2.Mice with one and two copies of the Prnpa101L allele and wild-type mice were inoculated with brain homogenate from: (A) a GSS patient with the 102L mutation; (B) a 101LL mouse terminally infected with GSS; (C) ME7. The extent of vacuolar change in the brain was assessed semi-quantitatively in nine areas of grey matter and three of white matter by lesion profiling as described (Fraser and Dickinson, 1967, 1968). Circles (green) are lesion profiles of mice at the terminal stages of disease with one copy of the gene-targeted allele Prnpa101L (101PL), triangles (red) are mice with two copies of the Prnpa101L allele (101LL) and diamonds (blue) are wild-type (101PP) mice. Lesion profiles were constructed (using a minimum of 10 animals in each group) on a scale of 0–5, and mean scores for each area are shown graphically (error bars ± SEM). Note: lesion scores without error bars represent scoring areas where there was no variation in lesion scores. Lesion profile scoring areas: grey matter areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, medial thalamus; 6, hippocampus; 7, septum; 8, cerebral cortex; 9, forebrain cerebral cortex. White matter areas: 1*, cerebellar white matter, 2*, midbrain white matter 3*, cerebral peduncle. Download figure Download PowerPoint Figure 3.Mice with one and two copies of the Prnpa101L allele and wild-type mice were inoculated with brain homogenate from a GSS patient with the 102L mutation. The brains of the animals were examined either at the terminal stage of the disease for animals which developed TSE disease or at the end of their lifespan for animals with no clinical signs of disease. PrP deposition in the hippocampus and thalamus detected by immunocytochemical analysis using two polyclonal antibodies 1A8 (not shown) and 1B3 (Farquhar et al., 1989). (A) PrP was detected in the thalamus of 101PL mice inoculated with GSS in both clinically positive and negative (as shown here at 639 days) animals, but (B) was barely detectable at the terminal stage of disease (254 days) in 101LL mice inoculated with GSS. Bar, 100 μm. Download figure Download PowerPoint Table 2. PrP deposition detected by immunocytochemistry in GSS primary transmission Mouse genotype No. Clinical TSE Vacuolar pathology PrP deposition 101PP 7 − − − 101PP 1 + − − 101PL 1 − − − 101PL 1 − − + 101PL 3 − + + 101PL 6 + + + 101LL 5 + + − 101LL 5 + + + Mice with no (101PP), one (101PL) and two (101LL) copies of the Prnpa101L allele inoculated with infectious material from a patient with GSS were examined for PrP accumulation by immunocytochemistry using two polyclonal antibodies (1B3 and 1A8). The amount of PrP deposited in the positive 101PL cases was variable. Only very low levels of PrP were detected in the positive 101LL cases. Although no PrP deposition was detected immunohistochemically in five of the 101LL mice, the analysis may not have been sensitive enough to detect very low levels of PrP in the brain. The histochemical processes required here for the treatment of the tissues (e.g. formic acid treatment) have been reported to lead to a loss of PrPC from some tissues (Kitamoto et al., 1991) and may also lead to some loss of PrPSc. Western blot analysis of the brains of the 101LL mice infected with GSS readily detected PrPC. Polyclonal antisera did not detect any protease-resistant PrP in these brains on Western blots (data not shown). However, use of a monoclonal antibody (8H4) and prolonged exposure times of the immunoblot to X-ray film did detect very low levels of protease-resistant PrP in the 101LL GSS-infected mice (Figure 6B). This protease-resistant PrP was not present in uninfected or aged 101LL mice after similarly long exposure times and was also not detected in PrP null mice (Figure 6B). It is therefore likely to represent extremely low levels of PrPSc in the infected brains. Accumulation of disease-specific PrP does not appear to be a significant feature of the TSE pathology in this experimental model. Mice carrying one copy of the wild-type and one copy of the mutant allele (101PL) varied considerably in terms of clinical and pathological TSE (Tables I and II). One group showed no clinical signs of TSE disease (death between 537 and 701 days from other causes) and had no significant vacuolar pathology; a second group showed no clinical signs of disease (death between 537 and 646 days from other causes) but had significant grey and white matter vacuolation. A third group developed TSE disease between 411 and 540 days and had severe vacuolation in grey and white matter regions (Figure 2A). Immunocytochemical analysis of clinically negative 101PL mice revealed one animal with no detectable PrP and one with PrP deposition. All clinically positive mice showed PrP immunostaining. The amount of PrP deposited in the 101PL mice varied from minor cortical PrP to severe deposition throughout the thalamus and cortex and did not appear to relate to the clinical status of the animals (Figure 3A, Table II). PrPSc was detected by Western blot analysis in all clinically positive 101PL mice inoculated with GSS but not in the two clinically negative animals examined (Figure 6B). In mice with only one copy of the mutant gene (101PL), the incubation period of disease is less constant than in the homozygous mice (101LL). Prolonged and variable incubation periods in the heterozygous mice may reflect a copy number effect, with a single copy of the mutant gene leading to longer incubation times. The presence of the wild-type gene in these mice may be interfering with the disease process associated with the mutant allele. The mutant allele (Prnpa101L) has now been crossed on to a PrP null background so that the effect of a single copy of the mutant gene can be assessed in the absence of the wild-type gene. Disease can be transmitted from a GSS-infected 101LL mouse to mice with or without the Prnpa101L allele A brain homogenate was prepared from a 101LL mouse infected with GSS that had been culled at the terminal stage of disease (254 days) with no apparent PrP deposition. The brain used to prepare the inoculum is that shown in Figure 3B. The inoculum (GSSLL) was injected intracerebrally into mice of the three genotypes (101PP, 101PL and 101LL). Disease was transmitted to all three groups of animals, with homozygous 101LL mice having the shortest incubation period. Significantly, even the wild-type (101PP) mice developed TSE disease within 226 days (Table I). Thus despite the failure to detect any PrP accumulation by immunohistochemical analysis and the extremely low levels of protease-resistant PrP detected by Western blot analysis in the brains of 101LL mice infected with GSS, the GSSLL inoculum prepared from it was able to transmit TSE disease rapidly to mice both with
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