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From: TSS (216-119-162-50.ipset44.wt.net)
Subject: BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein (FULL TEXT)
Date: November 28, 2002 at 9:32 am PST
BSE prions propagate as either variant CJD-like or
sporadic CJD-like prion strains in transgenic mice
expressing human prion protein Emmanuel A.Asante, Jacqueline M.Linehan,
Melanie Desbruslais, Susan Joiner,
Ian Gowland, Andrew L.Wood, Julie Welch,
Andrew F.Hill, Sarah E.Lloyd,
Jonathan D.F.Wadsworth and
John Collinge1
MRC Prion Unit and Department of Neurodegenerative Disease,
Institute of Neurology, University College, Queen Square,
London WC1N 3BG, UK
1Corresponding author
e-mail: j.collinge@prion.ucl.ac.uk
Variant Creutzfeldt±Jakob disease (vCJD) has been
recognized to date only in individuals homozygous for
methionine at PRNP codon 129. Here we show that
transgenic mice expressing human PrP methionine
129, inoculated with either bovine spongiform
encephalopathy (BSE) or variant CJD prions, may
develop the neuropathological and molecular phenotype
of vCJD, consistent with these diseases being
caused by the same prion strain. Surprisingly, however,
BSE transmission to these transgenic mice, in
addition to producing a vCJD-like phenotype, can also
result in a distinct molecular phenotype that is indistinguishable
from that of sporadic CJD with PrPSc
type 2. These data suggest that more than one BSEderived
prion strain might infect humans; it is therefore
possible that some patients with a phenotype consistent
with sporadic CJD may have a disease arising
from BSE exposure. Keywords: BSE/Creutzfeldt-Jakob disease/prion/
transgenic Introduction Prion diseases, such as Creutzfeldt-Jakob disease (CJD) in
humans, and scrapie and bovine spongiform encephalopathy
(BSE) in animals, are transmissible neurodegenerative
diseases associated with accumulation of a
disease-associated isoform of host-encoded cellular prion
protein (PrPC), designated PrPSc. PrPSc is thought to
comprise an aggregated form of a conformational isomer
of PrPC. According to the protein-only hypothesis, infectious
prions are composed principally, if not entirely, of an
abnormal isoform of PrP. Distinctive isolates or strains of
prions can be propagated in the same type of host and may
be encoded by differences in PrPSc conformation (Bessen
and Marsh, 1992, 1994; Collinge et al., 1996b; Telling
et al., 1996) and glycosylation (Collinge et al., 1996b).
Variant CJD (vCJD), recognized in 1996, is thought to be
caused by exposure to BSE-like prions (Collinge et al.,
1996b; Lasmezas et al., 1996; Bruce et al., 1997; Hill et al.,
1997). We have previously described four human PrPSc
types in brain tissue from patients with CJD: types 1-3 are
seen in classical (sporadic or iatrogenic) CJD, while type 4
is seen in vCJD (Collinge et al., 1996b). A common polymorphism at codon 129 of the human
PrP gene (PRNP), where either methionine (M) or valine
(V) can be encoded, is a key determinant of susceptibility
to sporadic and acquired prion diseases, and may affect
age at onset in inherited prion disease (Baker et al., 1991;
Collinge et al., 1991; Palmer et al., 1991). To date, all
patients recognized with vCJD have been of the PRNP
129MM genotype (Collinge et al., 1996a; Zeidler et al.,
1997; our unpublished data). PrP polymorphisms are
known to affect prion strain propagation in mice and sheep
(Bruce, 1993). Similarly, codon 129 genotype may play a
role in human prion strain propagation, since certain PrPSc
types are closely associated with codon 129 genotypes. To
date, we have found types 1 and 4 PrPSc only in individuals
of the PRNP 129MM genotype and type 3 PrPSc only in
genotypes MV or VV, while type 2 PrPSc is seen in
association with all three genotypes (Collinge et al.,
1996b; Wadsworth et al., 1999; our unpublished data). We
have previously reported that Tg(HuPrP129V+/+ Prnp0/0)-
152 mice, which express only human PrP V129 (129VV
Tg152 mice), are highly susceptible to infection with
human prions from patients with sporadic and iatrogenic
forms of CJD, regardless of patient genotype at polymorphic
codon 129 (Collinge et al., 1995; Hill et al.,
1997). However, these mice are much less susceptible to
prions from patients with vCJD. Indeed, the transmission
properties of vCJD closely resembled those of BSE, and
these experiments form part of the extensive data arguing
that vCJD is caused by a BSE-like prion strain (Collinge
et al., 1996b; Bruce et al., 1997; Hill et al., 1997). These
mice lacked a species or transmission barrier to classical
CJD prions and were also used to model the transmission
barrier between cattle and humans (Collinge et al., 1995;
Hill et al., 1997). These data were relatively reassuring, in
that transmission of BSE to transgenic mice expressing
only human PrP was inefficient, with <40% of intracerebrally
inoculated mice succumbing to prion disease after
prolonged incubation periods, consistent with the presence
of a substantial transmission barrier. However, an important
caveat with respect to public health considerations was
that vCJD was occurring in humans of the PRNP 129MM
genotype, while these mice expressed human PrP 129V
(Collinge et al., 1995; Hill et al., 1997). Although classical
CJD from patients with all three PRNP codon 129
genotypes (MM, VV and MV) transmitted efficiently to
these mice, it is possible that part of the transmission
barrier to vCJD infection of these mice resided in the
mismatch at codon 129 between inoculum and host (Hill
et al., 1997). Using the same inocula, we have now
extended these studies to mice expressing human PrP
M129 to further study both the bovine-to-human species
barrier and the propagation of human and BSE prion
strains. Detailed study of the relative transmission barriers
to BSE in transgenic mice expressing human PrP M129
and V129 will be published elsewhere. Here we report the
unexpected finding that BSE prion inoculation can induce
replication of two distinct prion strains in mice expressing
human prion protein. Results Susceptibility of transgenic mice expressing
human PrP M129 to human and bovine prions We produced transgenic mice homozygous for a
human PrP M129 transgene array and murine PrP null
(Bueler et al., 1992) alleles (Prnp0/0), designated
Tg(HuPrP129M+/+ Prnp0/0)-35 (129MM Tg35), with
expression levels of human PrP two times that of pooled
normal human brain (data not shown). These mice were
challenged with prions from cases of sporadic CJD, vCJD
and BSE. 129MM Tg35 mice were highly susceptible to
prions from patients with sporadic CJD of the PRNP
129MM genotype, but were less susceptible to classical
CJD prions from individuals of the PRNP 129VV
genotype (Table I). Transmission of sporadic CJD of the
PRNP 129MV genotype was associated with either
consistent short-duration characteristics as with MM
cases (I024) or long and variable incubation periods
(I020). This may reflect stochastic propagation of either
129M or 129V PrPSc in these patients. This was in contrast
to Tg(HuPrP129V+/+ Prnp0/0)-152 mice, expressing human
PrP V129 (129VV Tg152), which, as we have reported
previously (using the same inocula), are highly susceptible
to classical CJD prions from all three PRNP genotypes
(Collinge et al., 1995; Hill et al., 1997). The presence of a
transmission barrier can be estimated by measuring the fall
in mean incubation period on primary and second passage
in the same host. Second passage of prions from sporadic
CJD (I1202)-inoculated 129MM Tg35 mice resulted in an
incubation period of 249 ± 3 days (4/4 mice), which was
not lower than primary passage [229 ± 5 days (8/8 mice)].
It is possible that the small increase in incubation period
reflects a lower prion titre in mouse than human brain since
affected mice are culled at an early clinical stage.
Consistent short incubation periods on primary passage
with 100% attack rate and no fall in incubation period on
second passage of CJD in these mice, as with our earlier
studies with Tg152 mice (Collinge et al., 1995), are
consistent with lack of a transmission barrier to classical
CJD 129MM prions. However, as with 129VV Tg152
mice (Hill et al., 1997), 129MM Tg35 mice were much
more resistant to vCJD 129MM prions, with only 1/14
mice succumbing to clinical prion disease at a prolonged
incubation period (690 days) (Tables I and II). Indeed, as
judged by development of clinical disease, 129MM Tg35
mice, expressing human PrP 129M, appeared less susceptible
to vCJD than 129VV Tg152 mice, expressing human
PrP 129V (Hill et al., 1997). Similarly, 129MM Tg35 mice
appeared highly resistant to BSE prions, with 6/49
clinically scored transmissions at variable and prolonged
incubation periods (338-492 days) (Tables I and II). Sub-clinical infection in mice expressing human
PrP M129 While by clinical criteria these data might be interpreted as
consistent with the existence of a substantial species
barrier between cattle BSE and transgenic mice expressing
129M human PrP, we investigated all these mice for
evidence of sub-clinical infection. We and others have
previously demonstrated extensive sub-clinical prion
infection in mice inoculated with a strain of hamster
prions (Sc237 or 263K) thought to be non-pathogenic to
wild-type mice (Hill et al., 2000; Race et al., 2001), Table I. vCJD, BSE and sporadic CJD transmissions to transgenic mice
expressing human PrP 129M Inoculum Tg(HuPrP129M+/+ Prnp0/0)-35
Code PRNP129
genotype
Human PrPSc
type
Clinical signs Incubation period
(days ± SEM)
Total affecteda
Sporadic CJD I1199 MM T1 3/3 237 ± 10 3/3
I1202 MM T1 8/8 229 ± 5 8/8
I1196 MM T1 8/8 225 ± 7 8/8
I026 MM T2 7/7 223 ± 1 7/7
I024 MV T2 4/4 241 ± 1 4/4
I022 VV T2 2/6 700, 708 4/6
I020 MV T3 6/7 437 ± 31 7/7
I021 VV T3 2/7 354 3/7
vCJD I336 MM T4 0/2 >600 2/2
I342 MM T4 1/5 690 5/5
I344 MM T4 0/7 >340±720 7/7
BSE I038 MMb 2/20 344, 468 8/20
I060 MMb 0/6 >570 1/6
I062 MMb 2/7 338, 340 3/7
I064 MMb 2/10c 344, 492 2/10
I066 MMb 0/6 >500 0/6 aPositive either by clinical signs, western blotting and/or
immunohistochemistry.
bGenotype at corresponding bovine PrP gene codon.
cOne brain not available for either western blotting or
immunohistochemistry. Transgenic modelling of BSE and vCJD
6359 questioning current definitions of transmission barriers,
which have been conventionally assessed on the basis of
occurrence of clinical disease in inoculated animals.
Surprisingly, as assessed by histology, immunohistochemistry
and/or the presence of human PrPSc on western
blotting of brain tissue, we found that all (13/13) vCJD-
inoculated 129MM Tg35 mice, which had died apparently
of age-related causes without clinical signs of prion
disease at ages typical for uninoculated or mockinoculated
mice, had pathological (Figure 1A) and/or
biochemical (Figure 2A) evidence of prion infection. Only
a single (1/14) (Table II) 129MM Tg35 mouse challenged
with vCJD developed clinical prion disease. Excluding
those animals that died soon after inoculation, which were
not examined further, all other mice (13/14), which died
of intercurrent or age-related illness between 342 and
726 days post-inoculation without showing clinical signs
of prion disease, were positive for type 4 PrPSc in their
brains. There appeared, therefore, to be a 100% infection
rate of vCJD-inoculated 129MM Tg35 mice (Table I). A
smaller proportion (8/49) of BSE-inoculated 129MM
Tg35 mice also developed sub-clinical disease (Table I;
Figure 2D). Widespread sub-clinical disease was not seen,
however, in vCJD- or BSE-inoculated 129VV Tg152 mice
(Hill et al., 1997). Since the methods used for PrPSc
detection in the current study are more sensitive than
those used in our earlier study (Hill et al., 1997), we reanalysed
the 129VV Tg152 mouse brains (16) for which
frozen tissue remained for study, using the same methods
used here, and found no PrPSc. Sub-passage of infectivity
from both 129V and 129M human PrP-expressing lines of
transgenic mice will be necessary to further characterize
and quantitate these transmission barriers. Fig. 1. Immunohistochemistry of cerebral cortex and hippocampal
regions of transgenic mouse brain showing abnormal PrP
immunoreactivity, including PrP-positive florid plaques
(enlarged in insets). (A) vCJD-inoculated 129MM Tg35 mouse.
(B) BSE-inoculated 129MM Tg35 mouse. (C) vCJDinoculated
129MM Tg45 mouse. (D) BSE-inoculated 129MM Tg45 mouse. (E-G)
Histological analysis showing the thalamus of a BSE-inoculated
129MM Tg35 mouse propagating type 2 human PrPSc with widespread
vacuolation (E; H&E), extensive gliosis (F; GFAP), but no specific
PrP immunoreactive deposits (G; ICSM 35). Scale bar: (A-D) =
100 um; (E), (F) and (G) = 50 um. Table II. Summary of BSE and vCJD transmission to Tg35 and Tg45 Transgenic BSE vCJD
line
Total attack
rate
Clinical
disease
Sub-clinical
infection
Type 2
PrPSc
Type 4
PrPSc
Total attack
rate
Clinical
disease
Sub-clinical
infection
Type 2
PrPSc
Type 4
PrPSc
Tg35 14/49 6/49 8/49 10/11a 1/11a 14/14 1/14 13/14 0/14 14/14
Tg45 9/12 0/12 9/12 0/9 9/9 4/4 1/4 3/4 0/4 4/4
aThree brains not analysed by western blotting (one brain from a
clinically infected animal was unavailable for either western blotting or
immunohistochemistry; single brains from clinically affected and
sub-clinically affected animals were scored positive by
immunohistochemistry). Transgenic mice expressing human PrP M129
develop the neuropathological features and PrPSc
type of vCJD following inoculation with BSE or
vCJD prions Inoculation of vCJD prions into 129MM Tg35 mice
resulted in clinical disease in only a single mouse, but
widespread sub-clinical disease with human PrPSc readily
detectable in brain by western blot analysis. In previous
transmission studies of vCJD prions to 129VV Tg152
mice, a novel type 5 PrPSc pattern was obtained, and
thought to represent a prion strain switch resulting from
mismatch of the codon 129 polymorphism in inoculum and
host human PrP (Hill et al., 1997). A prediction of the
hypothesis that prion strain type is encoded by PrPSc
structural properties, and that the PRNP codon 129
polymorphism plays a key role in human prion strain
propagation, is that transmission of vCJD prions (containing
human PrPSc type 4) to 129MM Tg35 mice would
result in faithful propagation of type 4 PrPSc. This was
indeed what we observed: the PrPSc type seen, as judged by
PrPSc fragment sizes (Figure 2A, compare lanes 1 and 2),
was the type 4 pattern characteristic of vCJD prions in
human brain. The glycoform ratio also closely resembled
that of type 4 PrPSc in human brain (Figure 3). As we have
reported previously, a small difference is seen on glycoform
ratios of the same prion strain propagated in mice and
human brain, presumably reØecting the superimposition of
species-specific glycosylation effects on the prion strainspecific
pattern (Hill et al., 1997). Furthermore, the
neuropathological features in the vCJD-inoculated
129MM Tg35 mice were quite different from those of
129VV Tg152 mice propagating type 5 human PrPSc,
where no PrP immunoreactive plaques were seen (Hill
et al., 1997). Remarkably, the vCJD-inoculated 129MM
Tg35 mice not only developed abundant PrP plaques, an
uncommon feature of prion disease in mice, but many of
these were of the `florid' type (a central plaque core
surrounded by a ring of spongiform vacuoles), which are
characteristic of vCJD in humans (Will et al., 1996)
(Figure 1A) but rarely seen in mice. Florid plaques were
first described in Icelandic scrapie and have also been
described in mice infected with the 111A scrapie strain
(McBride et al., 1988). More recently, florid plaques have
been reported in BSE-inoculated primates (Lasmezas et al.,
1996) and in transgenic mice expressing ovine PrP infected
with sheep-passaged BSE prions (Crozet et al., 2001). Fig. 2. Western blots of proteinase K (PK)-treated brain homogenates
from transgenic mice, human cases of variant and sporadic CJD, and lines of wild-type mice. (A) Lane 1, vCJD; lane 2, vCJD-inoculated 129MM Tg35
mouse. (B) Lane 1, vCJD-inoculated 129MM Tg35 mouse; lane 2, BSEinoculated 129MM Tg35 mouse propagating type 2 PrPSc; lane 3, vCJD. (C) Lanes 1 and 2, BSE-inoculated 129MM Tg35 mouse propagating either
type 2 PrPSc (lane 1) or type 4 PrPSc (lane 2). (D) Lane 1,
BSE-inoculated 129MM Tg35 mouse propagating type 2 PrPSc; lane 2, human
sporadic CJD type 2 PrPSc (PRNP genotype 129MM). (E) Lanes 1-3, human sporadic CJDtype 2 PrPSc (PRNP genotype 129MM); lanes 4-6, BSE-inoculated 129MM Tg35 mouse propagating type 2 PrPSc. Samples were PK digested in the absence (lanes 1, 3, 4 and 6) or presence (lanes 2 and 5) of 25 mM EDTA. *Following proteolysis, samples in lanes 3 and 6 were boiled in SDS sample buffer and subsequently adjusted to 25 mM EDTA before electrophoresis. (F) Transmission of vCJD and BSE to 129MM Tg45 mice. Lane 1, vCJD; lane 2, vCJD-inoculated 129MM Tg45 mouse; lane 3, BSEinoculated 129MM Tg45 mouse. (G) Primary transmission of vCJD and BSE to wild-type inbred mice. Lane 1, BSE-inoculated FVB mouse; lane 2,
vCJD-inoculated FVB mouse; lane 3, BSE-inoculated C57BL/6 mouse; lane 4,
BSE-inoculated SJL mouse; lane 5, vCJD-inoculated SJL mouse;
lane 6, BSE-inoculated RIIIS mouse. (H) Secondary transmission of vCJD
and BSE in wild-type inbred mice. Lanes 1-4, BSE was passaged twice in
C57BL/6 mice and then passaged in different wild-type mice: lane 1,
C57BL/6 mouse; lane 2, FVB mouse; lane 3, SJL mouse; lane 4, RIIIS mouse. Lanes 5 and 6, second passage of SJL-passaged BSE in further SJL mice (lane 5) or FVB mice (lane 6). Western blots were analysed by
high-sensitivity ECL using biotinylated anti-PrP monoclonal antibody ICSM 35 (A-D, F-H) or 3F4 (E). BSE prion inoculation of 129MM Tg35 mice also
resulted in both clinical disease and sub-clinical infection
(Tables I and II). In sharp contrast to BSE transmission to
129VV Tg152 mice, where we were unable to detect PrPSc
in brain (Hill et al., 1997), PrPSc was readily detectable in
brains of clinically sick 129MM Tg35 mice and in mice
not showing clinical signs of prion disease when they died
at advanced age (Figures 2C and 3). In one of the eight
sub-clinically affected mice, type 4 human PrPSc was seen
(Figure 2C, lane 2), indistinguishable from that seen in
vCJD-inoculated 129MM Tg35 mice and in human vCJD
itself. In this mouse, neuropathological features were
identical to those of vCJD-inoculated mice, with abundant
Øorid plaques as in human vCJD (Figure 1B). These data
further supported the conclusion that vCJD is caused by a
BSE-like prion strain. However, in all other sub-clinically
affected BSE-inoculated 129MM Tg35 mice (7/8), an
alternate phenotype was observed (Table II). This was also
seen in all clinically affected BSE-inoculated 129MM
Tg35 mice where brain was available for analysis (5/6)
(Table II). Some Tg(HuPrPM129) mice develop a distinct
phenotype following inoculation with BSE prions In 4/6 clinically affected and 6/8 sub-clinically affected
BSE-inoculated 129MM Tg35 mice, a distinctive human
PrPSc type was seen with a quite different fragment size of
unglycosylated PrPSc following proteinase K digestion and
a different ratio of the three glycoforms, monoglycosylated
PrPSc being most abundant (in marked contrast to
type 4 PrPSc, where diglycosylated PrPSc predominates)
(Figure 2B, C and E, compare lanes 1 and 2). Comparison
with known human PrPSc types in CJD indicated that this
type corresponded, both with respect to fragment sizes and
glycoform ratio, to the type 2 PrPSc seen in sporadic and
iatrogenic CJD (Figure 2D, compare lanes 1 and 2, and
Figure 3). Human PrPSc types can also be distinguished by
their metal-binding properties. Both type 1 and type 2
human PrPSc undergo a shift in fragment size following
proteinase K treatment if treated with the metal chelator
EDTA (Wadsworth et al., 1999). Type 3 (also seen in
classical CJD) and type 4 PrPSc do not undergo a metaldependent
shift in proteinase K cleavage site on treatment
with EDTA (Wadsworth et al., 1999). Treatment of BSEinoculated
129MM Tg35 mouse brain homogenates with
EDTA prior to proteinase K cleavage demonstrated that
while that showing a type 4 pattern was unaltered by this
treatment (data not shown), those showing a type 2 pattern
underwent the expected band shift indistinguishable from
type 2 PrPSc from CJD-affected human brain (Figure 2E,
compare lanes 1 and 2 with 4 and 5).
Routinely, in our transmission studies, individual mouse
brains from a group are either frozen for biochemical
studies or fixed for histology; in some mice, one
hemisphere is frozen and the other fixed to allow both
techniques on an individual mouse. Histopathological
analysis on fixed tissue and biochemical analysis on frozen
tissue was only available on a single animal showing type 2
PrPSc. However, neuropathological features of this subclinically
affected, BSE-inoculated 129MM Tg35 mouse,
showing a type 2 PrPSc pattern in the brain, were quite
distinct from those with type 4 PrPSc. There was no
specific PrP immunoreactivity; in particular, there were no
florid or other plaques (Figure 1G). However, widespread
neuronal vacuolation (Figure 1E) and extensive gliosis
(Figure 1F), consistent with spongiform encephalopathy,
clearly confirm sub-clinical disease in this mouse.
While vCJD prions produce a neuropathological pattern
in 129MM Tg35 mice similar to that seen in human vCJD,
and the characteristic PrPSc type of vCJD is maintained in
all mice, BSE inoculation results in two distinct but highly
consistent phenotypes: one indistinguishable from the
vCJD transmissions, and associated with the characteristic
molecular `signature' of BSE; and a second that resembles
transmission of the commonest molecular sub-type of
classical CJD. vCJD and BSE transmission to a further
HuPrP129M-expressing transgenic line We also inoculated a second transgenic line expressing
HuPrPM129, generated as described for Tg35, with vCJD
and BSE prions. Tg(HuPrP129M+/+ Prnp0/0)-45 (129MM
Tg45) mice were produced similarly to 129MM Tg35
mice, but have a level of expression of human PrP 4-fold
higher than a pooled normal human brain standard
(data not shown). These mice were also highly susceptible
to sporadic CJD, with a 100% attack rate, extremely
short and consistent incubation periods (I024: 7/7 mice
developed disease with an incubation time of 155 ±
5 days), and no fall in incubation period on second
passage, consistent with lack of a transmission barrier to
classical CJD prions. Again, as judged by clinical disease,
we found that these animals were much less susceptible to
vCJD and BSE. However, as seen with BSE- or vCJDinoculated
129MM Tg35 mice, evidence of sub-clinical
prion infection was seen in most clinically unaffected mice
(Table II). While only 1/4 vCJD-inoculated 129MM Tg45 Fig. 3. Scattergraph of proportions of protease-resistant PrP
in higher molecular mass (diglycosylated) and low molecular mass (monoglycosylated) glycoforms seen in brain tissue from sporadic CJD,
vCJD, BSE and in transgenic mice following challenge with CJD,
vCJD and BSE. Data points are plotted as mean ± SEM. Human cases,
indicated as circles: sporadic CJD type 1 PrPSc, light grey (n = 12);
sporadic CJD type 2 PrPSc, mid-grey (n = 49); sporadic CJD type 3
PrPSc, dark grey (n = 22); vCJD type 4 PrPSc, yellow (n = 16). Cattle
BSE, black square (n = 3). Transmissions to 129MM Tg35 mice,
upward triangles: sporadic CJD type 1 PrPSc-inoculated mice, blue
(n = 7); vCJD type 4 PrPSc-inoculated mice, green (n = 10); BSEinoculated mice, red (n = 9; n = 1). Transmissions to 129MM Tg45
mice, inverted triangles: sporadic CJD type 2 PrPSc-inoculated mice,
blue (n = 3); vCJD type 4 PrPSc-inoculated mice, green (n = 4);
BSEinoculated mice, red (n = 4). E.A.Asante et al.
6362 mice developed clinical disease (at 580 days), the
remaining 3/4 mice had neuropathological and biochemical
evidence of prion infection. Again, in close agreement
with the results from 129MM Tg35 mice, analysis of
brains of vCJD-inoculated 129MM Tg45 mice consistently
revealed widespread florid plaque deposition
(Figure 1C) and type 4 PrPSc (Figure 2F, lane 2 and
Figure 3). Similarly, none of the BSE-inoculated 129MM
Tg45 mice developed clinical signs of prion disease for
>700 days, but 9/12 had sub-clinical prion infection
(Table II). Neuropathological examination of BSEinoculated
129MM Tg45 mice revealed closely similar
pathological findings to that of vCJD-inoculated 129MM
Tg45 mice with florid plaques (Figure 1D) and western
blot analysis of brain tissue revealed type 4 PrPSc
(Figure 2F, lane 3 and Figure 3). To date, the alternate
neuropathological pattern associated with type 2 PrPSc has
not been detected in BSE-inoculated 129MM Tg45 mice. vCJD and BSE transmission to various inbred lines
of non-transgenic mice BSE prions transmit readily to wild-type mice but with
prolonged and variable incubation periods. We have
previously reported the PrPSc type of both FVB and
C57BL/6 mice when inoculated with BSE (Collinge et al.,
1996b; Hill et al., 1997). As with other species naturally or
experimentally infected with BSE that we have reported, a
BSE-like pattern is produced with a characteristic PrPSc
fragment size and glycoform ratio. However, these
transmissions involve PrP from another mammalian
species of different molecular mass, such that the proteins
are not directly comparable, as with transmissions of
human prion disease to transgenic mice expressing only
human PrP. This mouse PrPSc pattern is, therefore, referred
to as `diglycosylated dominant'.
In independent experiments to determine the range of
incubation periods of BSE in many inbred mouse lines, as
part of studies to map prion incubation time genes (Lloyd
et al., 2001), we have identified two inbred lines of mice in
which BSE transmission is associated with the production
of a distinctive PrPSc type, with PrPSc glycoform ratios
closely similar to that of human sporadic CJD and referred
to here as a `monoglycosylated dominant' PrPSc pattern.
Interestingly, these lines are also associated with unusually
short incubation periods for BSE (Table III). All four
inbred mouse lines have the same Prnp coding sequence
(Prnp-a; data not shown) and are homozygous for
methionine at codon 128, the corresponding murine
codon to PRNP codon 129. Following inoculation with BSE prions, both FVB and
C57BL/6 mice show the characteristic diglycosylated
dominant PrPSc pattern in the brain (Figures 2G, lanes 1
and 3, and 4A). However, when inoculated with the same
BSE inoculum, SJL and RIIIS mice exhibit a monoglycosylated
dominant PrPSc pattern (Figures 2G, lanes 4
and 6, and 4A). This PrPSc type is stable on further passage
to both SJL and FVB mice (Figures 2H, lanes 5 and 6, and
4C) and is unaffected by EDTA treatment (data not
shown).
The propagation of the monoglycosylated PrPSc glycoform
pattern is established by the host in which primary
passage is carried out, as both SJL and RIIIS mice are
capable of propagating the diglycosylated dominant PrPSc
pattern when challenged with BSE passaged twice in a
C57BL/6 mouse (Figures 2H, lanes 3 and 4, and 4C).
vCJD prions behave in the same way as BSE prions in
FVB mice, producing a prolonged and variable incubation
period and a diglycosylated dominant PrPSc type (Hill
et al., 1997) (Figures 2G, lane 2, and 4A; Table III). vCJD
transmissions to SJL mice also resemble BSE transmissions
to these mice. Inoculation with vCJD gives unusually
short incubation times (Table III) and produces a monoglycosylated
dominant PrPSc pattern which is closely
similar to that produced by BSE transmission (Figures 2G,
lane 5, and 4A) and is unaffected by EDTA treatment (data
not shown). To our knowledge, the PrPSc pattern seen on
BSE or vCJD transmissions to RIIIS and SJL mice used in
our study, or to the RIII strain of mice routinely used for
biological strain typing experiments, has not been reported
previously (Bruce et al., 1997).
The neuropathological features seen in inbred lines of
mice inoculated with the same prion strain vary considerably,
the disease patterns being host, as well as prion
strain, dependent (Bruce, 1993). The neuropathology
observed in SJL and RIIIS mice inoculated with either
BSE or vCJD showed only diffuse staining for PrP without
florid or other PrP immunoreactive plaques (data not
shown). Table III. BSE and vCJD transmissions to inbred lines of mice
Inoculum SJL/OlaHsd RIIIS/J FVB/NHsd C57BL/6/OlaHsd
Incubation Clinical Incubation Clinical Incubation Clinical Incubation
Clinical
time signs time signs time signs time signs
(days ± SEM) (days ± SEM) (days ± SEM) (days ± SEM)
BSE (I783) 196 ± 13 25/40 241 ± 15 20/29 589 ± 21 22/31 710 ± 15 6/25
vCJD (I336) 139 ± 17 6/10 342 ± 31 8/8
vCJD (I344) 256 ± 46 5/7 402 ± 34 7/8
vCJD (I342) 169, 169 2/11 475 ± 68 3/10 Discussion Prion propagation involves recruitment and conversion of
host PrPC into PrPSc, and the degree of primary structural
similarity between inoculated PrPSc and host PrPC is
thought to be a key component of intermammalian
transmission barriers (Prusiner et al., 1990). It is clear,
however, that prion strain type can also be crucial, as
clearly demonstrated by the very distinctive transmission properties of sporadic CJD 129MM and vCJD 129MM
prions (of identical PrP primary structure) in either 129VV
Tg152 (Hill et al., 1997; Collinge, 1999) or 129MM Tg35
mice. Prion strain type may also affect transmission
barriers via an effect on PrPSc tertiary structure and state of
aggregation (Hill et al., 1997; Collinge, 1999).
These 129MM Tg35 mice, in which human PrPSc types
can be propagated, have been used to study the BSE-tohuman
species barrier. The frequent presence of subclinical
prion disease in vCJD- and BSE-inoculated
129MM Tg35 mice further argues for the need to reassess
current definitions of `species' or transmission barriers that
limit prion transmission between different hosts (Hill et al.,
2000). Such barriers have hitherto been quantitated on the
basis of either comparative end-point titrations in the two
respective hosts, or by measuring the fall in incubation
period between primary and subsequent passage as the
prion strain adapts to the new host. Both methods rely on
measurement of time to onset of a clinical syndrome.
Modelling the BSE-to-human barrier in 129MM Tg35
mice would lead to the conclusion, on the basis of induced
clinical disease, that a substantial barrier existed. However,
it is clear that human PrPSc propagation can be
efficiently induced by inoculation with BSE or vCJD
prions, suggesting a smaller barrier to infection (but not to
clinical disease) than hitherto thought (Collinge et al.,
1995) in humans of the PRNP 129MM genotype. Humans
infected with BSE prions, but who became asymptomatic
carriers, may nevertheless pose a threat of iatrogenic
transmission via medical and surgical procedures.
Alternatively, it is possible that the lifespan of the
laboratory mouse is insufÆcient to allow expression of
clinical disease in most inoculated mice, whereas a higher
proportion of infected humans might survive the incubation
period to develop clinical signs of disease. Serial
passage studies and titration of prions in these mice are in
progress to study this further.
These studies further strengthen the evidence that vCJD
is caused by a BSE-like prion strain. Also, remarkably, the
key neuropathological hallmark of vCJD, the presence of
abundant florid PrP plaques, can be recapitulated on BSE
or vCJD transmission to these mice. However, the most
surprising aspect of the studies was the finding that an
alternate pattern of disease can be induced in 129MM
Tg35 mice from primary transmission of BSE, with a
molecular phenotype indistinguishable from that of a subtype
of sporadic CJD. This finding has important potential
implications as it raises the possibility that some humans
infected with BSE prions may develop a clinical disease
indistinguishable from classical CJD associated with type 2
PrPSc. This is, in our experience, the commonest molecular
sub-type of sporadic CJD. In this regard, it is of interest
that the reported incidence of sporadic CJD has risen in the
UK since the 1970s (Cousens et al., 1997). This has been
attributed to improved case ascertainment, particularly as
much of the rise is reported from elderly patients and
similar rises in incidence were noted in other European
countries without reported BSE (Will et al., 1998).
However, it is now clear that BSE is present in many
European countries, albeit at a much lower incidence than
was seen in the UK. While improved ascertainment is
likely to be a major factor in this rise, that some of these
additional cases may be related to BSE exposure cannot be
ruled out. It is of interest in this regard that a 2-fold
increase in the reported incidence of sporadic CJD in 2001
has recently been reported for Switzerland, a country that
had the highest incidence of cattle BSE in continental
Europe between 1990 and 2002 (Glatzel et al., 2002). No
epidemiological case-control studies with stratification of
CJD cases by molecular sub-type have yet been reported.
It will be important to review the incidence of sporadic
CJD associated with PrPSc type 2 and other molecular subtypes
in both BSE-affected and unaffected countries in the Fig. 4. Scattergraph of proportions of protease-resistant PrP in higher
molecular mass (diglycosylated) and low molecular mass (monoglycosylated) glycoforms seen in sporadic CJD, vCJD, BSE and in
wild-type mice following challenge with vCJD and BSE. Data points
are plotted as mean ± SEM. (A-C) Human cases indicated as circles:
sporadic CJD type 1 PrPSc, light grey (n = 12); sporadic CJD type 2
PrPSc, mid-grey (n = 49); sporadic CJD type 3 PrPSc, dark grey
(n = 22); vCJD type-4 PrPSc, yellow (n = 16). Cattle BSE, black square
(n = 3). (A) Primary transmission of vCJD and BSE to wild-type mice:
vCJD-inoculated FVB mice, green diamond (n = 19); vCJD-inoculated
SJL mice, green triangle (n = 4); BSE-inoculated FVB mice, red
diamond (n = 12); BSE-inoculated SJL mice, red triangle (n = 7);
BSE-inoculated RIIIS mice, red star (n = 4); BSE-inoculated C57BL/6
mice, inverted red triangle (n = 3). (B) Transmission of SJL-passaged
BSE to further wild-type mice: SJL-passaged-BSE-inoculated FVB
mice, blue diamond (n = 4); BSE passaged twice in SJL mice, blue
triangle (n = 3); BSE passaged three times in SJL mice, open triangle
(n = 3). (C) Transmission of BSE passaged twice in C57BL/6 mice to
further wild-type mice: C57BL/6-passaged BSE to FVB mice, orange
diamond (n = 3); C57BL/6-passaged BSE to SJL mice, orange triangle
(n = 4); C57BL/6-passaged BSE to RIIIS mice, orange star (n = 3);
C57BL/6-passaged BSE to C57BL/6 mice, inverted orange triangle
(n = 3). E.A.Asante et al. light of these findings. If human BSE prion infection can
result in propagation of type 2 PrPSc, it would be expected
that such cases would be indistinguishable on clinical,
pathological and molecular criteria from classical CJD. It
may also be expected that such prions would behave
biologically like those isolated from humans with sporadic
CJD with type 2 PrPSc. The transmission properties of
prions associated with type 2 PrPSc from BSE-inoculated
129MM Tg35 mice are being investigated by serial
passage.
We consider these data inconsistent with contamination
of some of the 129MM Tg35 mice with sporadic CJD
prions. These transmission studies were performed according
to rigorous biosafety protocols for preparation of
inocula and both the inoculation and care of mice, which
are all uniquely identified by sub-cutaneous transponders.
However, crucially, the same BSE inocula have been used
on 129VV Tg152 and 129MM Tg45 mice, which are
highly sensitive to sporadic CJD but in which such
transmissions producing type 2 PrPSc were not observed.
Furthermore, in an independent experiment, separate
inbred lines of wild-type mice, which are highly resistant
to sporadic CJD prions, also propagated two distinctive
PrPSc types on challenge with either BSE or vCJD. No
evidence of spontaneous prion disease or PrPSc has been
seen in groups of uninoculated or mock-inoculated aged
129MM Tg35 mice.
While distinctive prion isolates have been derived from
BSE passage in mice previously (designated 301C and
301V), these, in contrast to the data presented here, are
propagated in mice expressing different prion proteins
(Bruce et al., 1994). It is unclear whether our Ændings
indicate the existence of more than one prion strain in
individual cattle with BSE, with selection and preferential
replication of distinct strains by different hosts, or that
`mutation' of a unitary BSE strain occurs in some types of
host. Western blot analysis of single BSE isolates has not
shown evidence of the presence of a proportion of
monoglycosylated dominant PrPSc type in addition to the
diglycosylated dominant pattern (data not shown).
Extensive strain typing of large numbers of individual
BSE-infected cattle either by biological or molecular
methods has not been reported.
Presumably, the different genetic background of the
different inbred mouse lines is crucial in determining
which prion strain propagates on BSE inoculation. The
transgenic mice described here have a mixed genetic
background with contributions from FVB/N, C57BL/6 and
129Sv inbred lines; each mouse will therefore have a
different genetic background. This may explain the
differing response of individual 129MM Tg35 mice, and
the difference between 129MM Tg35 and 129MM Tg45
mice, which are, like all transgenic lines, populations
derived from single founders. Indeed, the consistent
distinctive strain propagation in FVB and C57BL/6 versus
SJL and RIIIS lines may allow mapping of genes relevant
to strain selection and propagation, and these studies are in
progress.
That different prion strains can be consistently isolated
in different inbred mouse lines challenged with BSE
prions argues that other species exposed to BSE may
develop prion diseases that are not recognizable as being
caused by the BSE strain by either biological or molecular
strain typing methods. As with 129MM Tg35 mice, the
prions replicating in such transmissions may be indistinguishable
from naturally occurring prion strains. It
remains of considerable concern whether BSE has transmitted
to, and is being maintained in, European sheep
flocks. Given the diversity of sheep breeds affected by
scrapie, it has to be considered that some sheep might have
become infected with BSE, but propagated a distinctive
strain type indistinguishable from those of natural sheep
scrapie. Materials and methods Generation of transgenic mice The 759 bp human PrP ORF was amplified by PCR with pfu polymerase
from genomic DNA encoding methionine at codon 129, using forward
primer 5'-GTCGACCAGTCATTATGGCGAACCTT-3' and reverse
primer 5'-CTCGAGAAGACCTTCCTCATCCCACT-3'. Restriction
sites SalI and XhoI (underlined) were introduced in the forward and
reverse primers, respectively, for cloning. The sequence was confirmed
and ligated into the cosmid vector CosSHaTet (Scott et al., 1989).
Microinjection of the purified DNA was carried out according to the
standard protocol into single cell eggs of a strain of mice (FVB/
N 3 Sv129 3 C57BL/6) in which the murine PrP gene has been ablated
(Bueler et al., 1992). Genotyping was performed by PCR, and PrP
expression levels estimated by western blot analysis. Transmission studies Strict biosafety protocols were followed. Inocula were prepared, using
disposable equipment for each inoculum, in a microbiological containment
level 3 laboratory and inoculations performed within a class 1
microbiological safety cabinet. Five separate BSE inocula, each derived
from single natural BSE-affected cow brainstems (I060, I062, I064, I066,
I783), and a separate inoculum prepared from a pool of five natural BSE
brainstems (I038) were studied. Aliquots of these (except I783) have been used in previously published studies (Collinge et al., 1995; Hill et al., 1997). BSE tissues were collected under strict aseptic conditions using sterile instrumentation, specifically for transmission studies, by the UK Central Veterinary Laboratory [now the Veterinary Laboratories Agency (VLA)]. The BSE pool homogenate was titrated into RIII wild-type mice at VLA with a resultant titre of 103.3 mouse intracerebral LD50 units/g of tissue. Sporadic and vCJD inocula were prepared from brain tissue from neuropathologically confirmed cases. Consent for use of tissues for research was obtained. The genotype of each transgenic mouse was confirmed by PCR of tail DNA prior to inclusion and all mice were uniquely identiÆed by sub-cutaneous transponders. RIIIS/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and SJL/ OlaHsd mice were obtained from Harlan UK Ltd (Bicester, UK). Disposable cages were used, and all cage lids and water bottles were also uniquely identified by transponder and remained with each cage of mice throughout the incubation period. Care of the mice was according to institutional guidelines. Both transgenic and wild-type mice were anaesthetized with a mixture of halothane and O2, and intracerebrally inoculated into the right parietal lobe with 30 ul of a 1% brain homogenate prepared in PBS. Thereafter, all mice were examined daily for clinical signs of prion disease. Mice were killed if they were
exhibiting any signs of distress or once a diagnosis of prion disease was established. Criteria for clinical diagnosis of scrapie in mice were as described previously (Carlson et al., 1986). Neuropathology and immunohistochemistry Mice were killed using CO2 asphyxiation, brains fixed in 10% buffered
formol±saline and then immersed in 98% formic acid for 1 h and paraffin
wax embedded. Serial sections of 4 um were pre-treated with autoclaving,
formic acid and 4 M guanidine thiocyanate. Abnormal PrP accumulation
was examined using an anti-PrP monoclonal IgG antibody raised against
recombinant human PrP (ICSM 35; A.Khalili-Shirazi, unpublished data),
followed by a biotinylated anti-mouse IgG secondary antibody and an
avidin-biotin-horseradish peroxidase conjugate before development with
3',3-diaminobenzedine tetrachloride as the chromogen. The extent of
gliosis was determined by GFAP (Dako) staining. Slides were pre-treated
by heating in the microwave (900 W) in citrate buffer pH 6.0 for 25 min,
followed by overnight incubation (1:1000). Biotinylated swine anti-rabbit Transgenic modelling of BSE and vCJD
6365 immunoglobulins and avidin-biotin complex were applied as described
above. Harris haematoxylin was used as the counterstain. Appropriate
controls were used throughout. Western blotting Preparation of brain homogenates (10% w/v in PBS), proteinase K
digestion (50 or 100 mg of proteinase K for 1 h at 37°C) and subsequent
western blotting were performed as described previously (Wadsworth
et al., 2001). For primary screening of both transgenic and wild-type
mouse brain homogenates, blots were probed with a biotinylated anti-PrP
monoclonal antibody which recognizes both human and mouse PrP
(biotinylated-ICSM 35) in conjunction with an avidin-biotin-alkaline
phosphatase conjugate (Dako) and development in chemiluminescent
substrate (CDP-Star; Tropix Inc.). Primary screening of brain
homogenates was performed blind to sample identity. Quantitation and analysis of PrP glycoforms Western blotting was performed as above but using different primary and
secondary detection reagents. For transgenic mice expressing human PrP,
blots were incubated with anti-PrP monoclonal antibody 3F4 (Kascsak
et al., 1987), whereas for wild-type mice expressing mouse PrP, blots
were incubated with anti-PrP monoclonal antibody 6H4 (Prionics,
Switzerland), followed by incubation with goat anti-mouse IgG-
alkaline phosphatase conjugate (Sigma) and development in chemifluorescent substrate (AttoPhos; Promega) and visualization on a Storm 840 PhosphorImager (Molecular Dynamics). Quantitation of PrPSc
glycoforms was performed using ImageQuaNT software (Molecular
Dynamics). Acknowledgements We thank G.Mallinson for biotinylation of antibodies, S.Brandner for
neuropathological opinion, R.Bond and team for animal care, and
R.Young for preparation of figures. We specially thank all patients and
their families for generously consenting to use of human tissues in
this research, and the UK neuropathologists who have kindly helped
in providing these tissues. We thank R.Bradley, D.Matthews,
S.A.C.Hawkins and colleagues at the UK VLA for providing BSE
tissues. We thank C.Weissmann for critical review of the manuscript.
This work was supported by the Wellcome Trust, Medical Research
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Received August 1, 2002; revised September 24, 2002;
accepted October 17, 2002
E.A.Asante et al.
6366TSS Diagnosis and Reporting of Creutzfeldt-Jakob Disease T. S. Singeltary,
Sr; D. E. Kraemer; R. V. Gibbons, R. C. Holman, E. D. Belay, L. B.
Schonberger http://jama.ama-assn.org/issues/v285n6/ffull/jlt0214-2.html CJD WATCH http://www.fortunecity.com/healthclub/cpr/349/part1cjd.htm Terry S. Singeltary Sr., Bacliff, Texas USA
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