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From: Terry S. Singeltary Sr. (216-119-143-226.ipset23.wt.net)
Subject: Human Prion Protein with Valine 129 Prevents Expression of Variant CJD Phenotype [FULL TEXT]
Date: January 24, 2005 at 5:18 pm PST

-------- Original Message --------
Subject: Human Prion Protein with Valine 129 Prevents Expression of Variant CJD Phenotype
Date: Mon, 24 Jan 2005 16:56:47 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@LISTSERV.KALIV.UNI-KARLSRUHE.DE


##################### Bovine Spongiform Encephalopathy #####################

REPORTS

Human Prion Protein with
Valine 129 Prevents Expression
of Variant CJD Phenotype

Jonathan D. F. Wadsworth, Emmanuel A. Asante,
Melanie Desbruslais, Jacqueline M. Linehan, Susan Joiner,
Ian Gowland, Julie Welch, Lisa Stone, Sarah E. Lloyd,
Andrew F. Hill,* Sebastian Brandner, John Collinge

Variant Creutzfeldt-Jakob disease (vCJD) is a unique and highly distinctive
clinicopathological and molecular phenotype of human prion disease
associated with infection with bovine spongiform encephalopathy (BSE)-like
prions. Here, we found that generation of this phenotype in transgenic mice
required expression of human prion protein (PrP) with methionine 129.
Expression of human PrP with valine 129 resulted in a distinct phenotype and,
remarkably, persistence of a barrier to transmission of BSE-derived prions on
subpassage. Polymorphic residue 129 of human PrP dictated propagation of
distinct prion strains after BSE prion infection. Thus, primary and secondary
human infection with BSE-derived prions may result in sporadic CJD-like or
novel phenotypes in addition to vCJD, depending on the genotype of the prion
source and the recipient.

Distinct prion strains are associated with
biochemically distinct forms of disease-related
prion protein (PrPSc). Four PrPSc types have
been observed in brain tissue from patients
with distinct Creutzfeldt-Jakob disease (CJD)
phenotypes: types 1 to 3 in classical (sporadic
or iatrogenic) CJD and type 4 in vCJD (1-3).
Polymorphism at residue 129 of human PrP
(where either methionine or valine can be
encoded) powerfully affects genetic suscepti-
bility to human prion diseases (4-7) and ap-
pears to critically influence the propagation of
these human PrPSc types. So far, types 1 and 4
PrPSc have been found only in humans
homozygous for Met129; type 3 PrPSc is seen
almost exclusively in individuals with at least
one valine allele; and type 2 PrPSc has been
commonly observed in all codon 129 geno-
types (1-3). BSE and vCJD prion infection in
transgenic mice expressing human PrP, but not
mouse PrP (1, 8-10), indicates that codon 129
polymorphism determines the ability of human
PrP to form type 4 PrPSc and to generate the
neuropathological phenotype of vCJD.

Challenge of transgenic mice expressing
human PrP Met129 (129MM Tg35 and
129MM Tg45 mice) with BSE and vCJD
prions (11) resulted in faithful propagation of
type 4 PrPSc (10) (Figs. 1 and 2) accompanied
by the key neuropathological hallmark of
vCJD, the presence of abundant florid PrP
plaques (10). However, transgenic mice
expressing human PrP Val129 (129VV Tgl52
mice) responded quite differently. Although
these 129VV Tgl52 mice lack a transmission
barrier to classical forms of CJD, regardless of
the codon 129 genotype of the inoculum (1, 8,
9), the primary challenge with vCJD prions
was characterized by a substantial transmis-
sion barrier to infection (only ~50% of
inoculated mice were infected, compared with
100% of 129MM Tg35 and 129MM Tg45
mice) (Fig. 1; table Sl). In addition, rather
than type 4 PrPSc, vCJD-inoculated 129VV
Tgl52 mice propagated type 5 PrPSc (9),
which shares the same predominance of the
diglycosylated glycoform seen in type 4 PrPSc
but is distinguished by proteinase K digestion
products of greater molecular mass (Fig. 2A),
which closely resemble those seen in human
type 2 PrPSc (9). Type 5 PrPSc is associated
with very weak diffuse PrP deposition in the
brain (9), which contrasts markedly with the
florid PrP plaques associated with the propa-
gation of type 4 PrPSc in humans (12) or
transgenic mice (10). Similar diffuse deposi-
tion of PrP is also observed in clinically
affected BSE-inoculated 129VV Tgl52 mice;
however, type 5 PrPSc is undetectable in brain
homogenate (9).

To further evaluate the molecular and
neuropathological phenotype of vCJD- or
BSE-inoculated 129VV Tgl52 mice, we
performed a second passage in the same
breed of mice. Primary transmission of prions
from one species to another is associated
with a species or transmission barrier that is
largely or completely abrogated on second
and subsequent passage in the second species
as the prions adapt to the new host. Second
passage then resembles within-species trans-
mission with a high (typically 100%) attack
rate and much shortened and more consistent
incubation period. It was remarkable, howev-
er, that such adaptation did not occur on
second passage of BSE or vCJD prions in
129VV Tgl52 mice. Brain inocula derived
from four clinically affected BSE-inoculated
129VV Tgl52 mice failed to transmit clinical
disease or asymptomatic prion infection to
additional 129VV Tgl52 mice (Figs. 1 and 3;
table S2). However, two of these inocula
produced clinical prion disease (Fig. 3; table
S2) with abundant PrPSc accumulation (fig.
Sl) on inoculation of wild-type FVB mice
with incubation periods that are not compati-
ble with persistence of the original BSE
inoculum [supporting online material (SOM)
text]. The prion strain generated in BSE-
inoculated 129VV Tgl52 mice was thus
infectious in wild-type FVB mice, but not in
additional 129VV Tgl52 mice.

Valine 129 is unique to human PrP,
and the failure of BSE prions to adapt in
129VV Tgl52 mice on second passage
contrasts sharply with the marked adapta-
tion of BSE prions in FVB mice (Fig. 3;
table S2) or in other murine (13) or primate
(14) hosts that encode methionine at the
corresponding position of PrP. BSE prions
also efficiently adapt on second passage in
129MM Tg35 transgenic mice. Primary
transmission of BSE prions in 129MM
Tg35 mice resulted in bifurcation of prop-
agated strain type and produced either type
2 or 4 PrPSc (Figs. 1 and 2) and neuropa-
thology consistent with human sporadic
CJD or vCJD, respectively (10). These
distinct molecular and neuropathological
phenotypes consistently "breed true" with
very high efficiency on second passage in
additional 129MM Tg35 transgenic mice
(15). These findings contrast sharply with
the complete lack of prion transmission on
second passage of the same BSE inocula in
129VV Tgl52 mice, supporting the inter-
pretation that human PrP Val129 severely
restricts propagation of the BSE prion
strain.

This conclusion was further reinforced
by study of the parameters of second
passage of vCJD prions. As seen with
second passage of BSE prions, clinical
disease was observed only in FVB, and
not in 129VV Tgl52, recipients. Brain
inocula from clinically affected, type 5
PrPSc positive, primary vCJD-inoculated
129VV Tgl52 mice produced clinical prion
disease (Fig. 3; table S2) and PrPSc accu-
mulation (fig. Sl) on subpassage in FVB
mice, but produced only subclinical infec-
tion (with PrPSc accumulation) in 7 out of
11 inoculated 129VV Tgl52 mice (Figs. 1
and 3). Notably, in four of these, high-
sensitivity methods (16) were required to
detect PrPSc in brain homogenate (table
S2). Type 5 PrPSc was faithfully prop-
agated on second passage in 129VV
Tgl52 mice (Fig. 2A). In the three mice
containing the highest levels of type 5
PrPSc, extensive spongiosis was also ob-
served (Fig. 4), and in one of these, in contrast
to the pathology seen on first passage,
numerous PrP plaques were seen (Fig. 4).
Type 4 PrPSc is invariably associated with
prominent florid plaques in the cortex of
human vCJD brain (12) and in vCJD- or
BSE-prion inoculated 129MM Tg35 and Tg45
transgenic mice (10), whereas plaques associ-
ated with type 5 PrPSc were restricted to the
corpus callosum and had a nonflorid morphol-
ogy (Fig. 4). The lack of adaptation of vCJD
prions on second passage in 129VV Tgl52
mice contrasted sharply with the behavior of

Fig. 1. Summary of trans-
missions of vCJD and BSE
prions to transgenic mice.
The total number of prion-
affected mice (both clinical
and subclinical) is reported
for each inoculated group:

129MM Tg45 mice (black),
129MM Tg35 mice (gray),
129W Tgl52 mice (white).
Animals were scored by clin-
ical signs, immunoblotting,
and/or immunohistochemis-
try. Primary transmission
data have been published
previously (9, 10). In trans-
missions that result in bi-
furcation of propagated PrPSc
type, the number of samples positive for type
2 or type 4 PrPSc is reported as a proportion
of the total number of samples examined by
immunoblotting. (*), The occurrence of sub-
clinical prion infection only.

Fig. 2. Molecular strain typing of
vCJD and BSE prion transmissions
in transgenic mice. (A to D)
Immunoblots of proteinase K-
treated brain homogenates from
variant and sporadic CJD (PRNP
129 MM genotype) and transgenic
mice were analyzed by enhanced
chemiluminescence with either
monoclonal antibody 3F4 against
PrP (A) or biotinylated monoclo-
nal antibody ICSM 35 against PrP
(B to D). The identity of the brain
sample is designated above each
lane with the type of PrPSc present
in the sample designated below.
Transmissions that result in the propagation of either type 2 or type 4 PrPSc.

vCJD prions in wild-type FVB mice, where
typical adaptation was observed on second
passage with 100% clinical prion disease
with abundant PrPSc accumulation (fig. Sl) at
markedly reduced incubation periods (Fig. 3;
table S2).

Fig. 3. Summary of transmissions
of vCJD and BSE prions to trans-
genic and wild-type FVB mice. The
total number of prion-affected
mice (both clinical and subclinical)
is reported for each inoculated
group: 129VV Tg152 mice (white),
wild-type FVB mice (gray). Ani-
mals were scored by clinical signs,
immunoblotting, and/or immuno-
histochemistry. Data are derived
from tables S1 and S2. (*), The
occurrence of subclinical prion
infection only.


Fig. 4. Neuropathological
analysis of transgenic
mouse brain. Primary
transmission of vCJD
prions in 129VV Tgl52
mice produces type 5
PrPSc that is maintained
after secondary passage
in 129VV Tg152 mice but
induces propagation of
either type 2 or type 4
PrPSc after passage in
129MM Tg35 mice. Im-
munohistochemistry
(PrP) shows abnormal
PrP immunoreactivity,
including PrP-positive
plaques, stained with
monoclonal antibody 3F4
against PrP. Sections
stained with hematoxylin-
and-eosin (H&E) show
spongiform neurodegen-
eration (left, corpus cal-
losum; middle and right,
parietal cortex). Scale
bar, 100 um. Lower pan-
els show the regional
distribution of abnormal
PrP deposition. Green
boxes in the sketches
denote the area from
which PrP-stained sec-
tions are derived.


Both BSE and vCJD prions failed to
propagate efficiently on either primary or,
remarkably, second passage, in 129VV
Tgl52 mice in sharp contrast to 129MM
Tg mice or wild-type animals, and where
detectable, infection was associated with a
distinct PrPSc type and pathological pheno-
type. Thus, human PrP Val129 appears not
to be a compatible substrate for propagation
of the prion strain seen in vCJD. This inter-
pretation was supported by the transmission
properties of 129VV Tgl52-passaged vCJD
prions in 129MM Tg35 mice. Here, 14 out
of 15 129MM Tg35 mice inoculated with
isolates containing type 5 PrPSc showed
PrPSc accumulation (Fig. 1; table S3), typi-
cally to much higher levels than seen in
129VV Tgl52 mice receiving the same
inocula. However, the PrPSc seen was not
of the type 5 pattern but instead these trans-
missions mirrored the behavior of BSE
prions in 129MM Tg35 mice (10), where,
instead, either type 4 or type 2 PrPSc were
seen (Figs. 1 and 2, B to D). Thus human
PrPSc types 4 and 5 are restricted to propa-
gating in mice expressing human PrP
Met129 or Val129, respectively.

Neuropathologically, type 4 PrPSc was
associated with relatively little spongiosis
and abundant florid plaques (Fig. 4) typical
of vCJD in humans (12), whereas type 2
PrPSc was associated with much higher
levels of vacuolation in many areas of the
brain, accompanied by generally diffuse
PrP deposition and occasional small, non-
florid plaques (Fig. 4) that closely resem-
bled human sporadic CJD with type 2
PrPSc PRNP (human prion protein gene)
129MM (3). Clinical prion disease was
observed in all 129MM Tg35 mice propa-
gating type 2 PrPSc, whereas mice propa-
gating type 4 PrPSc were subclinically
infected (table S3).

In conclusion, we have demonstrated
that BSE and vCJD prion infection in
transgenic mice can result in the propaga-
tion of distinct molecular and neuropatho-
logical phenotypes dependent on host PrP
residue 129 and possibly other, as yet
unidentified, disease modifying loci (10).
These data predict a critical role for PRNP
codon 129 in governing the thermodynamic
permissibility of human PrPSc conformation
that can be interpreted within a conforma-
tional selection model of prion transmission
barriers (17-19) (SOM text) and suggest
that there is no overlapping preferred
conformation for Val129 and Met129 human
PrP that can be generated as a result of
exposure to the vCJD/BSE prion strain.
Biophysical measurements suggest that this
powerful effect of residue 129 on prion
strain selection is likely to be mediated by
means of its effect on the conformation of
PrPSc or its precursors or on the kinetics of
their formation, as it has no measurable
effect on the folding, dynamics, or stability
of the normal cellular prion protein PrPC
(20).

Although caution must be exercised in
extrapolating from animal models, even
where, as here, faithful recapitulation of
molecular and pathological phenotypes is
possible, our findings argue that primary
human BSE prion infection, as well as sec-
ondary infection with vCJD prions by iatro-
genic routes, may not be restricted to a single
disease phenotype. These data, together with
the recent recognition of probable iatrogenic
transmission of vCJD prions to recipients of
blood {21, 22), including a PRNP codon 129
Met/Val heterozygous individual (22), re-
iterate the need to stratify all human prion
disease patients by PrPSc type. This surveil-
lance will facilitate rapid recognition of novel
PrPSc types and of any change in relative
frequencies of particular PrPSc subtypes in
relation to either BSE exposure patterns or
iatrogenic sources of vCJD prions.

References and Notes

1. J. Collinge, K. C. L. Sidle, J. Meads, J. Ironside, A. F.
Hill, Nature 383, 685 (1996).

2. J. D. F. Wadsworth et al., Nature Cell Biol;. 1, 55 (1999).

3. A. F. Hill et al., Brain 126, 1333 (2003).

4. J. Collinge, M. S. Palmer, A. J. Dryden, Lancet 337,
1441 (1991).

5. M. S. Palmer, A. J. Dryden, J. T. Hughes, J. Collinge,
Nature 352, 340 (1991).

6. H. S. Lee et al., J. Infect. Dis. 183, 192 (2001).

7. S. Mead et al., Science 300, 640 (2003).

8. J. Collinge et al., Nature 378, 779 (1995).

9. A. F. Hill et al., Nature 389, 448 (1997).

10. E. A. Asante et al., EMBO J. 21, 6358 (2002).

11. Materials and methods are available as supporting
material on Science Online.

12. R. G. Will et al., Lancet 347, 921 (1996).

13. M. Bruce et al., Philos. Trans. R. Soc. London Ser. B.
343, 405 (1994).

14. C. I. Lasmezas et al., Proc. Natl. Acad. Sci. U.S.A. 98,
4142 (2001).

15. E. A. Asante, J, D. F. Wadsworth, J. Collinge,
unpublished observations. These data will be
reported in full elsewhere.

16. J. D. F. Wadsworth et al, Lancet 358, 171 (2001).

17. J. Collinge, Lancet 354, 317 (1999).

18. J. Collinge, Annu. Rev. Neurosci. 24, 519 (2001).

19. A. F. Hill, J. Collinge, Trends Microbiol. 11, 578 (2003).

20. L. L Hosszu et al.,J. Biol Chem. 279, 28515 (2004).

21. C. A. Llewelyn et al., Lancet 363, 417 (2004).

22. A. H. Peden, M. W. Head, D. L Ritchie, J. E. Bell, J. W.
Ironside, Lancet 364, 527 (2004).

23. We thank C. Brown and his team for animal care,
R. Young for preparation of figures, and K. Fox and
S. Cooper for technical assistance. We especially
thank all patients and their families for generously
consenting to use of human tissues in this re-
search, 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 Veterinary Laboratories
Agency for providing BSE tissues. This work was
funded by the UK Medical Research Council and
European Commission. One of the routine antibodies
used in this work (ICSM 35) is marketed by D-Gen
Ltd., an academic spin-off company. J.C. is a director
of D-Gen and J.C., J.D.F.W., and A.F.H. are shareholders
and consultants of D-Gen.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1103932/DC1

Materials and Methods
SOM Text
Fig. S1
Tables S1 to S3
References and Notes

11 August 2004; accepted 21 October 2004
Published online 11 November 2004;


Medical Research Council (MRC) Prion Unit and
Department of Neurodegenerative Disease, Institute
of Neurology, University College London, Queen
Square, London WC1N 3BG, UK.

*Present address: Department of Biochemistry and
Molecular Biology and Department of Pathology,
University of Melbourne, Parkville, Victoria 3010,
Australia.

To whom correspondence should be addressed.
E-mail: j.collinge@prion.ucl.ac.uk

www.sciencemag.org SCIENCE VOL 306 3 DECEMBER 2004


TSS

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