1 Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK
2 Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, Western General Hospital, Edinburgh, UK
Correspondence
Rona Barron
rona.barron{at}bbsrc.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Scottish Crop Research Institute, Invergowrie, Dundee, UK.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The influence of other amino acid substitutions in the murine Prnp gene on the incubation time of disease has been demonstrated previously. The substitution of leucine for proline at amino acid 101 (101L) in the murine Prnpa allele by gene targeting (Prnpa101L) has been shown to shorten TSE incubation times with P102L GSS from humans, 263K from hamsters and SSBP/1 from sheep when compared with wild-type (101P) mice. Conversely, a second human TSE agent, variant CJD (vCJD), displayed prolonged incubation times in Prnpa101L (101LL) mice when compared with wild-type mice (Barron et al., 2001; Manson et al., 1999
). Furthermore, when challenged with the mouse-passaged scrapie strains ME7 (derived from mice carrying the Prnpa gene) and 22A (derived from mice carrying the Prnpb gene), the 101LL mice also displayed increased incubation times when compared with mice homozygous for either Prnpa or Prnpb alleles (Barron et al., 2001
; Manson et al., 1999
). Transgenic mice overexpressing murine PrP containing a methionine substitution at position 108 have also been shown to produce a lengthening in scrapie incubation time compared with mice overexpressing the wild-type gene; however, the effect of mutation and transgene copy number cannot readily be separated in these experiments (Supattapone et al., 2001
).
The mechanism by which these polymorphisms and mutations in murine PrP control incubation time has not yet been established. Amino acids 101 and 108 are located in the N-terminal region of PrP (amino acids 21121), which has been described in NMR structural studies as having no discernible secondary structure (Donne et al., 1997; Hornemann et al., 1997
; Zahn et al., 2000
). However, despite the apparent lack of structure, this region of PrP contains the octapeptide repeat sequences which have been shown to bind copper, and therefore may influence both the structure and the function of the normal protein (Brown et al., 1997
; Brown & Harris, 2002
). Deletion studies have shown also that removing sections of the N-terminal region can cause increased incubation times and reduced efficiency of disease propagation in mice (Flechsig et al., 2000
; Supattapone et al., 2001
), and can reduce efficiency of PrPC conversion in cell-free conversion assays (Lawson et al., 2001
). Due to the close proximity of amino acids 101 and 108, it is possible that similar mechanisms are operating by which changes at these positions in murine PrP control scrapie incubation time in mice. In order to further investigate the role of this region of PrP in influencing TSE disease transmission, we have inoculated 101LL mice with four mouse-passaged TSE strains derived from both Prnpa and Prnpb mice, and compared the disease profile of 101LL mice with that of Prnpa and Prnpb mice of the same genetic background.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Scoring of clinical TSE disease.
The presence of clinical TSE disease was assessed as described (Dickinson et al., 1968). Incubation times were calculated as the interval between inoculation and cull due to terminal TSE disease. Mice were killed by cervical dislocation at the terminal stage of disease, at termination of the experiment (between 600700 days), or due to intercurrent illness. Half brains were fixed in 10 % formol saline for 48 h, followed by decontamination in 98 % formic acid for 1 h. The remaining half brain was frozen at -70 °C for Western blot analysis. Fixed brain tissue was dehydrated in alcohol and impregnated in wax during a 7 h automated processing cycle. Sections were cut coronally at four levels and mounted on Superfrost slides.
Lesion profiles.
Sections were haematoxylin & eosin stained and scored for vacuolar degeneration on a scale of 0 to 5 in nine standard grey matter areas and three standard white matter areas as described previously (Fraser & Dickinson, 1967). Mean scores were produced from a minimum of six mice and plotted with standard error of mean (SEM) against scoring areas to give a lesion profile for each strain with each agent.
Western blot analysis.
For detection of PrPSc in transgenic and wild-type mice, 10 % (w/v) homogenates of frozen brain tissue were prepared in NP40 buffer [0·5 % (v/v) NP40, 0·5 % (w/v) sodium deoxycholate, 150 mM NaCl, 50 mM Tris/HCl pH 7·5]. Homogenates were centrifuged at 10 000 g for 15 min at 4 °C to remove cellular debris. Clarified supernates (100 mg wet weight ml-1) were incubated with or without Proteinase K at 20 µg ml-1 for 1 h at 37 °C, and the reaction terminated by addition of PMSF. Samples were mixed with 2x sample buffer (Novex, Invitrogen) at 10 mg ml-1 final concentration and incubated at 90 °C for 20 min. Total protein estimates were not performed, as the effect of the mutation on PrPSc levels was unknown. Samples were therefore loaded as wet weight tissue equivalents. Twenty µl of each sample (approximately 200 µg wet weight tissue) was separated on 12 % Novex Tris/glycine acrylamide gels (Invitrogen). Proteins were transferred onto PVDF membrane by electroblotting, and incubated for 2 h at room temperature with mAbs 8H4 or 7A12 (Zanusso et al., 1998) at 50 ng µl-1 concentration. Proteins were visualized with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse secondary antibody (Jackson ImmunoResearch), and a chemiluminescence detection kit (Roche Diagnostics). Membranes were exposed to X-ray film for periods ranging from 10 s to 10 min.
Genotyping.
All P101L transgenic mice were genotyped after termination of experiments to confirm genotype. A 765 bp fragment containing the Prnp ORF was generated using a 5' primer (5'-ATGGCGAACCTTGGCTACTGGCTG-3'; position 107130, GenBank acc. no. M18070) and a 3' primer (5'-TCATCCCACGATCAGGAAGATGAG-3'; position 871848, GenBank acc. no. M18070). Cycle conditions were: 94 °C for 3 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 62 °C and 1 min at 72 °C. This was followed by a final 10 min at 72 °C (Biometra Triblock). The presence or absence of a DdeI site within the PCR product provided a marker for the codon 101PL alteration.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
For the strains derived from mice homozygous for the Prnpb allele the situation was more complex. Incubation times of 79V are reduced in Prnpa mice (234 days) compared with the expected homologous transmission in VM (Prnpb) mice of 270 days (Bruce, 1993) (data not available from transmission of 79V to Prnpa(108F/189V) mice). However, the incubation time in 101LL mice was found to be extended by over 100 days with respect to both Prnpa and Prnpb genotypes (377 days). With 301V, incubation times in both Prnpa and 101LL mice were extended with respect to Prnpa(108F/189V) mice, but the degree to which the incubation times were increased was greater in Prnpa than in 101LL mice (70 day difference). These results also differed from those obtained previously with 22A (also Prnpb derived) in which incubation times of Prnpa and 101LL mice were increased by approximately the same degree compared with Prnpa(108F/189V) mice (Table 1
, Fig. 1
). However, in transmissions of Prnpb strains of agent to 101LL mice, the observed effects are due to three amino acid changes in host PrP (108, 189 and 101). To truly examine the effect of the 101L mutation on Prnpb-derived strains of agent would require the mutation to be expressed on this allele, as the possibility of combinatory or compensatory mechanisms of the 101L and 108L alterations cannot be ignored during infection with Prnpb strains (Vorberg et al., 2003
). However, the effect of Prnpb strains in Prnpa and 101LL mice (which differ by only one amino acid) was unpredictable. While the 79V strain showed a further lengthening of the Prnpa incubation time in the presence of the 101L mutation, a reduction in the Prnpa incubation time was observed in 101LL mice inoculated with the 301V strain. These results, particularly those produced with the 301V strain, again indicate that the control of incubation time by the 101L mutation is different from that of the 108/189 polymorphisms. While the present results are unable to separate the effect of 108F from that of 189V we are currently addressing this issue in two further lines of gene-targeted mice homozygous for either Prnpa108F/189T or Prnpa108L/189V alleles.
101L mutation can affect PrPSc production
It had been observed previously that brain tissue from 101LL mice infected with ME7 contained lower levels of protease-resistant PrP at the terminal stage of disease than wild-type mice (Manson et al., 1999). To determine whether the 101L mutation in the host PrP gene influenced the level of protease-resistant PrP produced by other strains of mouse scrapie, brain homogenates were prepared from 101LL and wild-type Prnpa mice infected with the Prnpa strains 139A and 79A, and the Prnpb strains 22A, 79V and 301V, and analysed by Western blotting with mAbs 7A12 and 8H4. Preliminary analysis focused on three mice of each genotype with each strain of agent, to assess whether levels of PrPSc in the brain were consistent, or varied between animals. For all transmissions, similar levels of PrPSc were observed between three mice in each group, except for the transmission of 22A to 101LL mice (data not shown). Comparison of 101LL and Prnpa mice inoculated with Prnpa strains showed that heterologous transmissions to 101LL mice produced levels of PrPSc identical to those seen in the homologous transmission to Prnpa mice, despite the difference in incubation times (Table 1
and Fig. 2
). Similarly, transmission of 79V and 301V produced the same PrPSc levels in 101LL and Prnpa mice despite heterologous transmission in the presence of three and two PrP amino acid differences respectively. The difference seen between 101LL and Prnpa mice with 79V on this blot appears, on examination of the lanes without proteinase K treatment, to be due to protein loadings (Fig. 2a
, lanes 58), as protein concentrations were calculated from wet weight of tissue. Previous analysis has shown PrPSc levels in these mice to be similar (data not shown). However, the heterologous challenge of 101LL mice with Prnpb strain 22A did show some degree of variation in the level of PrPSc present. Of three animals examined, one showed similar levels to those found on inoculation of Prnpa mice, but the others contained much lower levels (Fig. 2b
, lanes 510). The presence of the 101L mutation therefore does not affect the level of PrPSc produced by some strains of agent, but can alter PrPSc levels with other strains independent of the Prnp genotype of the source of infectivity, as found here with 22A and previously with ME7 (Manson et al., 1999
).
|
|
The effect of the 101L mutation on murine scrapie incubation times largely parallels the effect of the 108/189 polymorphisms in murine PrP, as incubation times are extended in 101LL mice compared with the homologous transmission in either Prnpa or Prnpb mice. However, the degree to which incubation times are affected by 101L is distinct from 108/189, and is also very unpredictable with Prnpb-derived strains. Specific differences between 101L and 108/189 are also evident with some Prnpa-derived strains. The related strains 139A and 79A produce almost identical incubation times and lesion profiles in Prnpa mice, but can be distinguished by their incubation times in Prnpb and Prnpa(108F/189V) mice, where the 79A incubation time is approximately 140 days longer than 139A (Table 1, Fig. 1
). On inoculation of 101LL mice, the 139A and 79A strains of agent produced almost identical incubation times and alterations in lesion profile (Table 1
; Figs 1 and 3
), proving that the 101L mutation alters the disease profile of these agents in a manner that is indeed distinct from that of 108/189. Future experiments involving mice expressing Prnpa(108F/189T) will allow us to examine the influence of L108F alone on TSE disease progression, and to directly compare the effect of the L108F and P101L alterations. These experiments may reveal how mutations in this unstructured N-terminal region of PrP can have dramatic effects on disease phenotype.
Other mutations in the N-terminal region of murine PrP have also been shown to affect incubation times of murine scrapie strains. The introduction of methionine at positions 108 and 111 in murine PrP (to create the epitope for mAb 3F4) increased the incubation times of four mouse-passaged scrapie strains in transgenic mice overexpressing Prnpa(108M/111M) compared with mice overexpressing Prnpa (Supattapone et al., 2001). The mutations caused prolonged incubation times (ranging from 1·6- to 2·8-fold) for each of the scrapie strains investigated, independent of the Prnp genotype from which the strain was derived. Although the precise effects of the 108M/111M mutations are difficult to quantify in these experiments due to the differences in Prnp gene copy number and genetic background between control and mutant mice, the results suggested that mutations in this region of PrP may extend TSE incubation times with all mouse-passaged TSE isolates. However, in this experiment incubation times with 301V were extended in Prnpa(108M/111M) mice compared with the transgenic mice overexpressing Prnpa. This was not observed in 101LL mice, where a shortening of 301V incubation time occurred with respect to the wild-type Prnpa mice. These observations indicate that the effect on incubation time of introducing the 3F4 epitope is distinct from that caused by the presence of 101L in murine PrP.
The shorter incubation time of 301V (Prnpb mouse-passaged BSE) in 101LL mice, when compared with the wild-type Prnpa mice, is in complete contrast with previous experiments which have shown longer incubation times in 101LL mice inoculated with vCJD than in 101PP mice (Barron et al., 2001). Strain characteristics of this particular TSE agent have been show to remain remarkably consistent after transmission through several different species (Bruce et al., 1997
). However, vCJD (which is thought to be human-passaged BSE) produced increased incubation times and altered lesion profiles in 101LL mice compared with wild-type mice (Barron et al., 2001
), while BSE transmitted through a Prnpb mouse (301V) produced shortened incubation times (Table 1
, Fig. 1
) and unaltered lesion profiles in 101LL mice compared with wild-type mice (Fig. 3
). Adaptation to mouse with this strain of agent therefore increases the efficiency of transmission to 101LL mice, even in the presence of three polymorphisms in murine PrP, and this may in some way be related to why this human TSE agent alone transmits efficiently to wild-type mice (Bruce et al., 1997
; Tateishi, 1996
). These issues are currently being addressed by transmitting and subpassinging human TSE isolates in P101L mice (unpublished data).
The interaction between different strains of TSE agent and host PrP is a complex process, and alterations in the host PrP protein can have a dramatic effect on TSE disease progression. If the infectious process is dependent on interaction between PrP molecules, it is unclear why in 101LL mice the Prnpb-derived strains behave in a more unpredictable manner than the Prnpa-derived strains. It may be due to the presence of three amino acid changes between the 101LL host and the Prnpb line in which the agent was propagated. It is also possible that Prnpb strains have more diverse PrPSc conformations than the Prnpa strains we have examined. Alternatively, a direct interaction between PrPC and PrPSc may not define the infectious process. Other molecules may be involved in this process, and their interaction with PrPC may define the importance of PrP sequence in transmission of disease (Kaneko et al., 1997; Telling et al., 1995
). We now aim to further define the nature of the interaction between the N terminus of host PrP and the infectious agent through the study of the other mutations in this region of PrP.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brown, L. R. & Harris, D. A. (2002). The prion protein and copper: what is the connection? pp. 103113. Edited by E. J. Massaro. Totowa, NJ: Humana Press.
Brown, D. R., Qin, K. F., Herms, J. W. & 10 other authors (1997). The cellular prion protein binds copper in vivo. Nature 390, 684687.[CrossRef][Medline]
Bruce, M. E. (1993). Scrapie strain variation and mutation. Br Med Bull 49, 822838.[Abstract]
Bruce, M. E., McConnell, I., Fraser, H. & Dickinson, A. G. (1991). The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. J Gen Virol 72, 595603.[Abstract]
Bruce, M. E., Will, R. G., Ironside, J. W. & 10 other authors (1997). Transmissions to mice indicate that new variant CJD is caused by the BSE agent. Nature 389, 498501.[CrossRef][Medline]
Bruce, M. E., Boyle, A., Cousens, S., McConnell, I., Foster, J., Goldmann, W. & Fraser, H. (2002). Strain characterization of natural sheep scrapie and comparison with BSE. J Gen Virol 83, 695704.
Bueler, H., Aguzzi, A., Sailer, A., Greiner, R. A., Autenried, P., Aguet, M. & Weissmann, C. (1993). Mice devoid of PrP are resistant to scrapie. Cell 73, 13391347.[Medline]
Carlson, G. A., Goodman, P. A., Lovett, M., Taylor, B. A., Marshall, S. T., Petersontorchia, M., Westaway, D. & Prusiner, S. B. (1988). Genetics and polymorphism of the mouse prion gene-complex control of scrapie incubation-time. Mol Cell Biol 8, 55285540.[Medline]
Carlson, G. A., Ebeling, C., Yang, S. L., Telling, G., Torchia, M., Groth, D., Westaway, D., Dearmond, S. J. & Prusiner, S. B. (1994). Prion isolate specified allotypic interactions between the cellular and scrapie prion proteins in congenic and transgenic mice. Proc Natl Acad Sci U S A 91, 56905694.[Abstract]
Dickinson, A. G., Meikle, V. M. & Fraser, H. (1968). Identification of a gene which controls the incubation period of some strains of scrapie agent in mice. J Comp Pathol 78, 293299.[Medline]
Donne, D. G., Viles, J. H., Groth, D., Mehlhorn, I., James, T. L., Cohen, F. E., Prusiner, S. B., Wright, P. E. & Dyson, H. J. (1997). Structure of the recombinant full-length hamster prion protein PrP(29231): the N terminus is highly flexible. Proc Natl Acad Sci U S A 94, 1345213457.
Flechsig, E., Shmerling, D., Hegyi, I., Raeber, A. J., Fischer, M., Cozzio, A., von Mering, C., Aguzzi, A. & Weissmann, C. (2000). Prion protein devoid of the octapeptide repeat region restores susceptibility to scrapie in PrP knockout mice. Neuron 27, 399408.[Medline]
Fraser, H. & Dickinson, A. G. (1967). Distribution of experimentally induced scrapie lesions in the brain. Nature 216, 13101311.[Medline]
Hornemann, S., Korth, C., Oesch, B., Riek, R., Wider, G., Wuthrich, K. & Glockshuber, R. (1997). Recombinant full-length murine prion protein, mPrP(23231): purification and spectroscopic characterization. FEBS Lett 413, 277281.[CrossRef][Medline]
Kaneko, K., Zulianello, L., Scott, M., Cooper, C. M., Wallace, A. C., James, T. L., Cohen, F. E. & Prusiner, S. B. (1997). Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proc Natl Acad Sci U S A 94, 1006910074.
Lawson, V. A., Priola, S. A., Wehrly, K. & Chesebro, B. (2001). N-terminal truncation of prion protein affects both formation and conformation of abnormal protease-resistant prion protein generated in vitro. J Biol Chem 276, 3526535271.
Lloyd, S. E., Onwuazor, O. N., Beck, J. A., Mallinson, G., Farrall, M., Targonski, P., Collinge, J. & Fisher, E. M. C. (2001). Identification of multiple quantitative trait loci linked to prion disease incubation period in mice. Proc Natl Acad Sci U S A 98, 62796283.
Manolakou, K., Beaton, J., McConnell, I., Farquar, C., Manson, J., Hastie, N. D., Bruce, M. & Jackson, I. J. (2001). Genetic and environmental factors modify bovine spongiform encephalopathy incubation period in mice. Proc Natl Acad Sci U S A 98, 74027407.
Manson, J. C., Clarke, A. R., Hooper, M. L., Aitchison, L., McConnell, I. & Hope, J. (1994a). 129/Ola mice carrying a null mutation in PrP that abolishes messenger-RNA production are developmentally normal. Mol Neurobiol 8, 121127.[Medline]
Manson, J. C., Clarke, A. R., McBride, P. A., McConnell, I. & Hope, J. (1994b). PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology. Neurodegeneration 3, 331340.[Medline]
Manson, J. C., Jamieson, E., Baybutt, H. & 10 other authors (1999). A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy. EMBO J 18, 68556864.
Moore, R. C., Hope, J., McBride, P. A., McConnell, I., Selfridge, J., Melton, D. W. & Manson, J. C. (1998). Mice with gene targetted prion protein alterations show that Prnp, Sinc and Prni are congruent. Nat Genet 18, 118125.[Medline]
Prusiner, S. B. (1996). Molecular biology and pathogenesis of prion diseases. Trends Biochem Sci 21, 482487.[CrossRef][Medline]
Scott, M., Foster, D., Mirenda, C. & 9 other authors (1989). Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell 59, 847857.[Medline]
Stephenson, D. A., Chiotti, K., Ebeling, C., Groth, D., DeArmond, S. J., Prusiner, S. B. & Carlson, G. A. (2000). Quantitative trait loci affecting prion incubation time in mice. Genomics 69, 4753.[CrossRef][Medline]
Supattapone, S., Muramoto, T., Legname, G., Mehlhorn, I., Cohen, F. E., DeArmond, S. J., Prusiner, S. B. & Scott, M. R. (2001). Identification of two prion protein regions that modify scrapie incubation time. J Virol 75, 14081413.
Tateishi, J. (1996). Transmission of human prion diseases to rodents. Semin Virol 7, 175180.[CrossRef]
Telling, G. C., Scott, M., Mastrianni, J., Gabizon, R., Torchia, M., Cohen, F. E., Dearmond, S. J. & Prusiner, S. B. (1995). Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 83, 7990.[Medline]
Vorberg, I., Groschup, M. H., Pfaff, E. & Priola, S. A. (2003). Multiple amino acid residues within the rabbit prion protein inhibit formation of its abnormal isoform. J Virol 77, 20032009.
Westaway, D., Goodman, P. A., Mirenda, C. A., McKinley, M. P., Carlson, G. A. & Prusiner, S. B. (1987). Distinct prion proteins in short and long scrapie incubation period mice. Cell 51, 651662.[Medline]
Westaway, D., Mirenda, C. A., Foster, D. & other authors (1991). Paradoxical shortening of scrapie incubation times by expression of prion protein transgenes derived from long incubation period mice. Neuron 7, 5968.[Medline]
Westaway, D., Dearmond, S. J., Cayetanocanlas, J., Groth, D., Foster, D., Yang, S. L., Torchia, M., Carlson, G. A. & Prusiner, S. B. (1994). Degeneration of skeletal muscle, peripheral nerves, and the central nervous system in transgenic mice overexpressing wild-type prion proteins. Cell 76, 117129.[Medline]
Zahn, R., Liu, A., Luhrs, T. & 7 other authors (2000). NMR solution structure of the human prion protein. Proc Natl Acad Sci U S A 97, 145150.
Zanusso, G., Liu, D. C., Ferrari, S. & 11 other authors (1998). Prion protein expression in different species: analysis with a panel of new mAbs. Proc Natl Acad Sci U S A 95, 88128816.
Received 6 February 2003;
accepted 16 July 2003.