Transmission of Murine Scrapie to P101L Transgenic Mice
Rona M. Barron1, Val Thomson1, Declan King1, Jane Shaw1†, David W. Melton2
and Jean C. Manson1
1
Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road,
Edinburgh, UK.
2
Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine
Centre, Western General Hospital, Edinburgh, UK
Corresponding Author
Dr Rona Barron
Neuropathogenesis Unit,
Institute for Animal Health
Ogston Building,
West Mains Road
Edinburgh, EH9 3JF
Tel +44 131 319 8201
Fax +44 131 668 3872
E-mail: rona.barron@bbsrc.ac.uk
Running title: Transmission of mouse scrapie to 101L mice
Summary:
231 words
Text:
4,383 words
† Current Address: Scottish Crop Research Institute, Invergowrie, Dundee.
1
Summary
The PrP protein is central to the Transmissible Spongiform Encephalopathies (TSEs), and the
amino acid sequence of this protein in the host can influence both incubation time of disease
and targeting of disease pathology. The N-terminus of murine PrP has been proposed to be
important in the replication of TSE agents, as mutations or deletions in that region can alter
the efficiency of agent replication.
To address this hypothesis and to investigate the
mechanisms by which host PrP sequence controls the outcome of disease, we have assessed
the influence of a single amino acid alteration in the N-terminal region of murine PrP (P101L)
on the transmission of TSE agents between mice. Mice homozygous for the mutation (101LL)
were inoculated with TSE strains 139A and 79A derived from mice carrying a Prnpa allele,
and 79V and 301V derived from mice carrying a Prnpb allele. Incubation times in 101LL
mice were extended with all four strains of agent when compared to those in the
corresponding mouse genotype from which the infectivity was derived. However the degree
to which the incubation period was increased showed considerable variation between each
strain of agent. Moreover the presence of this single amino acid alteration resulted in a 70
day reduction in incubation time of the 301V strain in Prnpa mice. The effect of the 101L
mutation on murine scrapie incubation time appears therefore to be strain specific.
2
Introduction
The transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative
diseases which include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle
and Creutzfeldt-Jacob disease (CJD) in humans. These diseases are thought to be caused by
the conversion of a normal cellular glycoprotein (PrPC) into an abnormal disease associated
form, PrPSc (Prusiner, 1996). As their name suggests, TSE diseases are transmissible, and
many different TSE agents have been transmitted to and maintained in laboratory mice
(Bruce, 1993, Bruce et al., 2002, Bruce et al., 1991). Mouse passaged TSE strains can be
readily studied in the laboratory, and the identical genetic background of inbred strains of
laboratory mice allows the direct comparison of different TSE agents in each individual strain
of mouse (Bruce et al., 1991). The murine Prnp gene which encodes the PrP protein appears
to be the major but not the only factor controlling the incubation time of TSE disease in mice
(Carlson et al., 1994, Carlson et al., 1988, Lloyd et al., 2001, Manolakou et al., 2001,
Stephenson et al., 2000). Mice that lack PrP protein (PrP null mice) do not develop disease
when inoculated with TSE infectivity (Bueler et al., 1993, Manson et al., 1994a). Transgenic
mice containing multiple copies of the Prnp gene which express elevated levels of PrP
protein, show reduced incubation times with TSE infectivity when compared with wild type
mice (Scott et al., 1989, Westaway et al., 1994, Westaway et al., 1991). This gene dosage
effect is also evident in transgenic mice containing only one copy of the PrP gene, which
express reduced levels of PrP protein and show prolonged TSE incubation times (Manson et
al., 1994b). Different lines of inbred mice can show altered incubation times with the same
TSE agent, and this has been linked to two naturally occurring polymorphisms found in the
mouse PrP protein at amino acids 108 and 189 (Westaway et al., 1987). Mice encoding
108L/189T in PrP (such as C57BL and 129/Ola) contain the murine Prnpa gene, while mice
encoding 108F/189V (such as VM) contain the murine Prnpb gene.
In general, strains
3
passaged in Prnpa mice maintain shortened incubation times in Prnpa mice, but show
prolonged incubation times in Prnpb mice, and vice versa. However, some strains passaged in
Prnpb mice (e.g. 79V ) do show shorter incubation times in Prnpa mice (Bruce, 1993). The
different genetic backgrounds of the various inbred lines of mice made studies of the effects
of these polymorphisms difficult. However, introduction of the 108F/189V polymorphisms
into the Prnpa allele of murine PrP by gene targeting (Prnpa(108F/189V) ) has established that it
is indeed the 108/189 polymorphisms which exert the major control over scrapie incubation
time in wild type mice (Moore et al.,1998).
The influence of other amino acid substitutions in the murine Prnp gene on the incubation
time of disease have been previously demonstrated. 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 the wild type (101P) mice. Conversely, a
second human TSE agent, variant CJD (vCJD), displayed prolonged incubation times in
Prnpa101L (101LL) mice when compared to 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 to
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).
4
The mechanism by which these polymorphisms and mutations in murine PrP control
incubation time has not yet been established. The 101 and 108 amino acids are located in the
N-terminal region of PrP (amino acids 21-121) 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.
Materials and Methods
Preparation of Inoculum
Each inoculum was prepared from one mouse brain with terminal TSE disease (139A, 79A,
79V and 301V mouse scrapie). 139A and 79A were prepared from C57BL mice (Prnpa), and
79V and 301V were prepared from VM mice (Prnpb). A 1 % homogenate of each sample
was prepared in normal saline prior to use as an inoculum. Groups of wild type, FV/FV
5
(Moore et al., 1998) and 101LL transgenic mice (Manson et al., 1999) were inoculated
intracerebrally with 20 l of inoculum under fluothane anaesthesia.
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 600-700 days), or due to intercurrent illness. Half
brains were fixed in 10 % formol saline for 48 hours, followed by decontamination in 98 %
formic acid for 1 hour. The remaining half brain was frozen at –70 oC for western analysis.
Fixed brain tissue was dehydrated in alcohol and impregnated in wax during a 7 hour
automated processing cycle.
Sections were cut coronally at 4 levels and mounted on
Superfrost slides.
Lesion Profiles
Sections were H&E 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 6 mice and
plotted with standard error of mean (SEM) against scoring areas to give a lesion profile for
each strain with each agent.
Western Analysis
For the 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
6
10000 g for 15 min at 4 oC to remove cellular debris. Clarified supernates (100 mg/ml wet
weight) were incubated with or without Proteinase K at 20 g/ml for 1 hour at 37 oC, and the
reaction terminated by the addition of PMSF. Samples were mixed with 2x sample buffer
(Novex, Invitrogen) at 10 mg/ml final concentration and incubated at 90 oC 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. 20 l of each
sample (approximately 200 g 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 hours at room temperature with monoclonal antibodies
8H4 or 7A12 (Zanusso et al., 1998) at 50 ng/l concentration. Proteins were visualised with
Horse Radish Peroxidase (HRP) conjugated rabbit anti-mouse secondary antibody (Jackson
Immuno Research Laboratories Ltd), and a chemiluminescence detection kit (Roche
Diagnostics). Membranes were exposed to X-ray film for periods ranging from 10 seconds to
10 minutes.
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 ( 5ATGGCGAACCTTGGCTACTGGCTG-3’; position 107 to 130 Genbank accession number
M18070) and a 3’ primer (5’-TCATCCCACGATCAGGAAGATGAG-3’; position 871 to
848 Genbank accession number M18070). Cycle conditions were: 94 oC for 3 min, followed
by 30 cycles of 30 s at 94 oC, 30 s at 62 oC and 1 min at 72 oC. This was followed by a final
10 min at 72 oC (Biometra Triblock). The presence or absence of a Dde I site within the PCR
product provided a marker for the codon 101P-L alteration.
7
Results and Discussion.
The 101L mutation alters murine scrapie incubation times
Transgenic mice homozygous for the 101 leucine mutation (101LL) and wild type mice
(101PP) were inoculated with infectivity from three sources of murine scrapie (139A, 79A,
79V) and one source of mouse-passaged BSE (301V). TSE strains 139A and 79A were both
derived from mice homozygous for Prnpa, whereas 79V and 301V were derived from mice
homozygous for Prnpb. The incubation times obtained for each transmission are shown in
Table 1 and Figure 1. The analysis of these results was complex due to transmission of TSE
strains derived from both Prnpa and Prnpb mice. Incubation times produced by Prnpa derived
strains in 101LL mice could be directly compared with the homologous transmission in Prnpa
mice, as the 101L mutation was introduced into the Prnpa gene by gene targeting. This
comparison would reveal the specific effect of the 101L mutation on TSE disease progression.
In addition, comparison of 101LL mice with Prnpa(108F/189V) mice (which also contain the
mutations in the Prnpa gene) would demonstrate whether the 101L and the 108F/189V
mutations influenced incubation time in a similar manner. However with Prnpb derived
strains, the effects seen in 101LL mice were most likely due to the presence of 108L/189T
and 101L. The interpretation of the Prnpb strain data therefore depends on whether the 101LL
incubation times are compared with the homologous strain transmission to Prnpb mice, or the
heterologous strain transmission to Prnpa mice. Comparison of 101LL mice with Prnpb mice
shows the effect of three amino acid changes in host PrP (108, 189 and 101) on transmission
of homologous strains of agent, whereas comparison with Prnpa mice examines the precise
effect of the 101L mutation on the transmission of non-homologous strains of agent. The
following interpretation of the results aims to address both of these points.
8
When inoculated with 139A and 79A, 101LL mice produced extended incubation times
which were approximately double those obtained in Prnpa mice. This observation was similar
to that obtained previously with ME7 (Manson et al., 1999). In order to assess whether this
mechanism of control on incubation time could be similar to that of the 108F/189V
polymorphisms, we compared the incubation times of these agents in Prnpa(108F/189V) mice,
which are the same genetic background as 101LL mice. The results showed that 79A and
139A incubation times were increased in both 101LL and Prnpa(108F/189V) mice when compared
to Prnpa mice (Table 1, Figure 1). However, the degree to which incubation times were
extended varied with the strain of agent. With the 79A strain of agent the 108F/189V
alterations lead to a more prolonged incubation time than the 101L mutation, whereas the
opposite was true for the 139A strain. This suggested that the interaction with each strain of
agent and control of incubation time differs with 108F/189V and 101L.
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 to 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 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 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 to
Prnpa(108F/189V) mice (Table 1, Figure 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
9
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 the 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
10
showed that heterologous transmissions to 101LL mice produced identical levels of PrPSc to
those seen in the homologous transmission to Prnpa mice, despite the difference in incubation
times (Table 1 and Figure 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 (Figure 2, lanes 5 to 8), as protein concentrations
were calculated from wet weight of tissue. Previous analysis has shown PrP Sc 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 (Figure 2(b), lanes 5 to 10). 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).
Targeting of Vacuolation
The effect of the 101L mutation in host PrP on targeting of pathology was assessed by
comparing the degree of vacuolation in mice carrying the 101L mutation to those carrying
wild type 101P PrP (Prnpa). The degree of vacuolation in specific brain areas is characteristic
of individual mouse passaged scrapie strains. Vacuolation is scored on a scale of 0 to 5 in
nine grey matter and three white matter areas, and average scores (± SEM) plotted to give a
lesion profile, which is essentially a fingerprint for each strain (Fraser & Dickinson, 1967).
Lesion profiles were produced for each strain of mouse scrapie, comparing mean vacuolation
scores from 101LL and Prnpa mice displaying both clinical and pathological signs of TSE
11
disease (Figure 3). The heterologous transmission to 101LL mice of Prnpa derived strains
139A and 79A produced significant variation in the targeting of vacuolation when compared
with the corresponding homologous transmission in Prnpa mice. The 139A and 79A Prnpa
derived mouse scrapie strains share a common lineage, and produce identical lesion profiles
in wild type mice of both Prnp genotypes. These strains can however be distinguished by
their incubation times in Prnpb (or Prnpa(108F/189V)) mice (Table 1, Figure 1). Both of these
strains displayed increased levels of vacuolation in areas 1 (dorsal medulla), 3 (superior
colliculus), 4 (hypothalamus) and 7 (septum), but decreased levels of vacuolation in the white
matter regions (1* cerebellar white matter, 2* midbrain white matter, 3* cerebral peduncle) of
101LL mice. The change in vacuolar profile and incubation time of these two related strains
in 101LL mice appears identical (Figure 1, 3 & Table 1) indicating a similar interaction
between each strain and the 101L mutation in PrP.
Heterologous challenges with Prnpb
derived strains (79V, 22A and 301V) produced less variability between Prnpa and 101LL
mice, despite differing from the source of infectivity by two and three PrP amino acids
respectively. Identical lesion profiles were obtained in 101LL and Prnpa mice challenged
with 301V, and although 79V showed reduced levels of vacuolation in areas 6 to 9
(hippocampus, septum, cerebral cortex and forebrain cerebral cortex) of the 101LL brain, the
overall pattern was the same as in Prnpa mice.
The 22A strain showed slightly more
variability due to altered levels of vacuolation in areas 4 (hypothalamus), 7 (septum) and 8
(cerebral cortex), but all other areas of grey and white matter showed identical targeting of
vacuolation in both lines of mice. The 101L mutation therefore exerts a strain specific effect
on vacuolar targeting ranging from little or no alteration with heterologous challenges
differing at three amino acids (108L, 189T and 101L), to major alterations with heterologous
challenges differing at only one amino acid (101L), which was also observed previously with
ME7 (Manson et al., 1999).
12
Conclusions
Transmission of TSE disease is thought to be most efficient when identical PrP sequences are
present in both the donor and recipient of infectivity. The differences in PrP sequence
between species are thought to reduce the efficiency of conversion of PrPC to PrPSc, and result
in low transmission frequencies and long incubation periods. However, TSE infectivity from
human, hamster and sheep brain tissue has previously been transmitted to 101LL mice with
reduced incubation times compared to wild type mice, despite the lack of PrP sequence
identity between species (Barron et al., 2001, Manson et al., 1999). The PrP sequence of
C57BL mice (Prnpa) in which ME7, 139A and 79A are propagated differs from that of 101LL
mice by only one amino acid (101P), yet incubation times in 101LL mice are two fold greater
than in wild type mice. The PrP sequence of VM mice (Prnpb) in which 79V, 301V and 22A
are propagated differs from 101LL mice at three residues (101P, 108F and 189V), however
the effect in this case is far less predictable. With Prnpb derived strains, incubation times
observed in 101LL mice were also extended when compared with Prnpb mice, but were found
to be both shorter (301V) and longer (79V and 22A) when compared with the Prnpa mice.
Thus within the mouse species the presence of the 101L alteration prolongs the scrapie
incubation time normally observed in the donor genotype. However for agents from other
species, the effect of the 101L mutation appears in most cases to be to facilitate the
transmission of disease, perhaps indicating that the 101L mutation allows more efficient
adaptation of an agent in mice.
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 to the homologous transmission in either Prnpa or Prnpb mice. However the
13
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, Figure 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, Figures 1 and 2), proving that the
heterologous challenge caused by the 101L mutation alters the disease profile of these agents
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
such 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 monoclonal antibody 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 - 2.8 fold) for each of the
scrapie strains investigated, independent of the Prnp genotype from which the strain was
derived. Although the precise effect 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
14
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 to
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. This
observation indicates 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 to wild type
mice (Barron et al., 2001), while BSE transmitted through a Prnpb mouse (301V) produced
shortened incubation times (Table 1, Figure 1) and unaltered lesion profiles in 101LL mice
compared to wild type mice (Figure 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
15
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
The authors would like to thank Man-Sun Sy for the 7A12 and 8H4 antibodies, A. Boyle and
W-G Lui for lesion profile analysis, A. Suttie and S. Mack for tissue processing and
sectioning, S. Dunlop and E. Murdoch for care and scoring of the animals, and W. Goldmann
for review and comment on the manuscript. This work was supported by a grant from
BBSRC.
16
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19
Table 1.
Transmission of murine scrapie strains to Prnpa101L and Prnpa(108F/189V) mice.
Agent
Mouse line
Challenge
Amino acid
differences*
Incubation Time
(days ±SEM)
ME7
Prnpa
Homologous
None
161 ± 2†
Prnpa101L
Heterologous
101
338 ± 8†
Prnpa(108F/189V)
Heterologous
108/189
275 ± 3
Prnpa
Homologous
None
147 ± 2
Prnpa101L
Heterologous
101
306 ± 7
Prnpa(108F/189V)
Heterologous
108/189
240 ± 2
Prnpa
Homologous
None
139 ± 2
Prnpa101L
Heterologous
101
298 ± 3
Prnpa(108F/189V)
Heterologous
108/189
382 ± 12
Prnpa
Heterologous
108/189
493 ± 6‡
Prnpa101L
Heterologous
101/108/189
527 ± 28‡
Prnpa(108F/189V)
Homologous
None
227 ± 3
Prnpa
Heterologous
108/189
234 ± 6
Prnpa101L
Heterologous
101/108/189
377 ± 10
Prnpa(108F/189V)
Homologous
None
ND
Prnpa
Heterologous
108/189
253 ± 2
Prnpa101L
Heterologous
101/108/189
181 ± 1
Prnpa(108F/189V)
Homologous
None
134 ± 1
139A
79A
22A
79V
301V
* PrP amino acid positions which differ in host and source of infectivity
†Data shown from Manson et al, 1999
‡Data shown from Barron et al, 2001
ND no data available.
20
Incubation Time (days)
600
Prnpb strains
Prnpa strains
500
400
300
200
100
0
ME7
139A
79A
22A
79V
301V
Strain of Agent
Figure 1.
Transmission of murine scrapie strains to Prnpa101L and Prnpa(108F/189V) mice.
Incubation times (±SEM) of six mouse passaged TSE agents in Prnpa mice (dotted bars),
Prnpa101L mice (hatched bars) and Prnpa(108F/189V) mice (solid bars). No incubation time data
was available for Prnpa(108F/189V) mice with 79V. All incidence of disease was 100%, except
101LL/79V (14/15) and 101LL/22A (9/16).
ME7 and 22A 101LL incubation times taken from Manson et al 1999, and Barron et al 2001.
21
Figure 2.
(a)
1 2 3 4 5 6 7 8 9 10 11 12
30kDa
P
P
L L P P
79A
(b)
L L P
79V
P
L L
139A
1 2 3 4 5 6 7 8 9 10
30kDa
P P
L L P
301V
P L L L L
22A
Western analysis of brain homogenates for the presence of PrPSc, using monoclonal antibody
7A12. (a) lanes 1-4, 79A; lanes 5-8, 79V; lanes 9-12, 139A. (b) lanes 1-4, 301V; lanes 5-10,
22A. P represents 101PP mice, L represents 101LL mice. Samples in lanes 7-10 show two
101LL mice with differing levels of PrPSc. All samples were incubated either with (even
lanes) or without (odd lanes) proteinase K at 20g/ml prior to electrophoresis.
22
Figure
Figure2 3.
(a)
(b)
79A
5
4
4
3
3
Score
Score
5
2
2
1
1
0
0
1 2 3 4 5 6 7 8 9
139A
1 2 3 4 5 6 7 8 9
1* 2* 3*
Area
Area
5
(d)
79V
5
4
4
3
3
Score
Score
(c)
2
1
301V
2
1
0
0
1 2 3 4 5 6 7 8 9
1* 2* 3*
Area
5
(e)
1* 2* 3*
1
2
3
4
5
6
7
8
9
1* 2* 3*
Area
22A
Score
4
3
2
1
0
1
2 3
4 5
6
7 8
9
1* 2* 3*
Area
The extent of the vacuolar change in the brain was scored on a scale of 0-5 in nine areas of
grey matter and 3 areas of white matter, and mean scores (calculated from a minimum of 6
animals) are shown graphically (error bars +/- SEM). Grey matter scoring 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
scoring areas: 1*, cerebellar white matter: 2*, midbrain white matter: 3*, cerebral peduncle.
23
Separate profiles are shown for each mouse TSE strain; (a) 79A, (b) 139A, (c) 79V, (d) 301V,
(e) 22A. Blue diamonds represent 101PP mice, pink squares represent 101LL mice.
24