JOURNAL OF VIROLOGY, Apr. 2010, p. 3464–3475
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 7
Glycosylation of PrPCDetermines Timing of Neuroinvasion and
Targeting in the Brain following Transmissible Spongiform
Encephalopathy Infection by a Peripheral Route?
Enrico Cancellotti,1* Barry M. Bradford,1Nadia L. Tuzi,1† Raymond D. Hickey,1‡ Debbie Brown,1
Karen L. Brown,1Rona M. Barron,1Dorothy Kisielewski,1Pedro Piccardo,2and Jean C. Manson1
Neuropathogenesis Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh,
United Kingdom,1and Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland2
Received 11 November 2009/Accepted 12 January 2010
Transmissible spongiform encephalopathy (TSE) infectivity naturally spreads from site of entry in the
periphery to the central nervous system where pathological lesions are formed. Several routes and cells within
the host have been identified as important for facilitating the infectious process. Expression of the glycoprotein
cellular PrP (PrPC) is considered a key factor for replication of infectivity in the central nervous system (CNS)
and its transport to the brain, and it has been suggested that the infectious agent propagates from cell to cell
via a domino-like effect. However, precisely how this is achieved and what involvement the different glycoforms
of PrP have in these processes remain to be determined. To address this issue, we have used our unique models
of gene-targeted transgenic mice expressing different glycosylated forms of PrP. Two TSE strains were inoc-
ulated intraperitoneally into these mice to assess the contribution of diglycosylated, monoglycosylated, and
unglycosylated PrP in spreading of infectivity to the brain. This study demonstrates that glycosylation of host
PrP has a profound effect in determining the outcome of disease. Lack of diglycosylated PrP slowed or
prevented disease onset after peripheral challenge, suggesting an important role for fully glycosylated PrP in
either the replication of the infectious agent in the periphery or its transport to the CNS. Moreover, mice
expressing unglycosylated PrP did not develop clinical disease, and mice expressing monoglycosylated PrP
showed strikingly different neuropathologic features compared to those expressing diglycosylated PrP. This
demonstrates that targeting in the brain following peripheral inoculation is profoundly influenced by the
glycosylation status of host PrP.
Transmissible spongiform encephalopathies (TSE) or prion
diseases are a group of fatal neurodegenerative diseases which
include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in
sheep and goats, bovine spongiform encephalopathies (BSE)
in cattle, and chronic wasting disease (CWD) in deer and elk
(30). These diseases can be sporadic, familial, or acquired by
infection, and the common hallmark is a distinct pathology in
the central nervous system (CNS) characterized by neuronal
loss, spongiform degeneration, and gliosis (38, 46).
Expression of the host-encoded cellular PrP (PrPC) is fun-
damental for the onset of disease since PrP-deficient mice are
refractory to TSE infection (11, 31). PrPCis a glycoprotein with
two consensus sites for attachment of N-linked glycans (at
codons 180 and 196 in the mouse) which are variably occupied,
producing di-, mono-, and unglycosylated PrP (43). The diver-
sity in glycosylation, combined with the complexity of added
sugars, results in a large number of glycosylated forms of PrP
(41). A central event associated with TSE infection is the
conformational conversion of PrPCinto an abnormal protease-
resistant form, PrPSc(39). PrPScis deposited in brain and, in
some but not all cases, in peripheral organs of individuals
affected by TSE (21).
Although the pathology associated with TSE is found in the
brain, the periphery is the most natural route of acquiring
infection. Evidence suggests that oral transmission via contam-
inated food is linked with transmission of BSE to humans,
resulting in variant CJD (vCJD) (10, 47), and blood transfusion
has been identified as a probable route of human-to-human
transmission of vCJD (23, 27, 36). Moreover, parenteral ad-
ministration of contaminated human tissue-derived therapeu-
tics has been shown to facilitate iatrogenic spread of these
diseases (8, 46). It is therefore important to understand the
mechanisms that allow the infectious agent to propagate in the
periphery and be transported to the CNS prior to the onset of
neurodegeneration in the brain.
Many studies have been conducted to understand routes of
transmission (for a review see references 1 and 29). Lymphoid
tissues such as the spleen have been shown to play a funda-
mental role in agent replication and propagation in the very
early stages of disease. Indeed, studies of splenectomized and
asplenic mice have shown the lymphoreticular system (LRS) to
be an important site for TSE agent replication (14, 26). The
periphery also appears to have a role in processing the infec-
tious agent following intracerebral (i.c.) inoculation as PrPSc
accumulates in the spleen shortly after inoculation and before
accumulation of the abnormal protein in the brain (15, 17).
Within the LRS, follicular dendritic cells (FDC) have been
* Corresponding author. Mailing address: The Roslin Institute and
R(D)SVS University of Edinburgh, Roslin, Midlothian EH25 9PS,
Scotland, United Kingdom. Phone: 44 131 527 4200. Fax: 44 131 440
0434. E-mail: email@example.com.
† Present address: Biology Teaching Organisation, University of Ed-
inburgh, United Kingdom.
‡ Present address: Department of Molecular and Medical Genetics,
Oregon Health and Science University, Portland, OR.
?Published ahead of print on 27 January 2010.
shown to be important for the uptake of infectivity and subse-
quent spreading toward the CNS (7, 28, 33, 35). Several studies
have also suggested the peripheral nervous systems (PNS) as a
potential route of infectivity to the brain, implicating the vagus
and sciatic nerves in this process (5, 20, 25, 34).
Expression of PrPCin the peripheral tissues appears to be an
important prerequisite for the transport of infectivity to the
CNS following peripheral routes of inoculation. Indeed, it has
been proposed that a continuous chain of cells expressing PrPC
is fundamental for TSE neuroinvasion (6, 40), with overexpres-
sion of endogenous PrP in the PNS greatly facilitating the
spread of infectivity (19). Thus, host PrP appears to have a
fundamental role in the uptake, transport, and replication of
the infectious agent (6). Moreover, it has been suggested that
the different PrPCglycoforms may influence the timing of
neuroinvasion by directly influencing the interaction with the
infectious agent (19). However, the mechanism by which the
different glycoforms are involved in these processes remains to
In order to investigate the role of PrPCglycosylation in TSE
disease after peripheral infection with different TSE strains, we
have used our inbred gene-targeted transgenic mice expressing
different glycosylated forms of PrP. These mice expressed PrP
with no sugars at the first (designated G1/G1 in homozygous
mice) or the second glycosylation site (G2/G2) or both (G3/
G3) under the control of the endogenous PrP promoter (13).
We have previously shown that following intracerebral inocu-
lation, all glycotypes are susceptible to infection with at least
one TSE strain and that the type of PrP glycosylation in the
host influenced the incubation period but not the distribution
of pathological lesions in the brain (45). Here, we examine the
influence of host PrP glycosylation on the peripheral acquisi-
tion of infection and demonstrate that, unlike the intracerebral
route, mice without PrP glycosylation were resistant to disease
and that the different glycoforms had a profound influence on
not only the timing of disease but also the type and distribution
of the PrPScdeposits in the brain.
MATERIALS AND METHODS
Transgenic mouse lines. Inbred gene-targeted transgenic mouse lines G1, G2,
and G3, and the corresponding inbred 129/Ola wild-type control line have been
described previously (13). Edinburgh PrP null mice (31) were used as negative
Genotyping of mouse tail DNA. All the transgenic mice used in this study were
genotyped twice: before inoculation and at the end of the experiments. A portion
of tail was removed from each mouse. DNA was prepared from a 1-cm piece of
tail by digestion overnight at 37°C in tail lysis buffer (300 mM sodium acetate, 1%
SDS, 10 mM Tris, pH 8, 1 mM EDTA, 200 ?g/ml proteinase K) and subsequent
extraction with an equal volume of phenol-chloroform. DNA was precipitated
with isopropanol, washed with 70% ethanol, and resuspended in 100 ?l of TE
buffer (10 mM Tris, 1 mM EDTA, pH 7.4). The mismatch PCR method to
identify the different transgenics has been described elsewhere (13).
Preparation of inoculum and injection. Inocula were prepared from the brains
of C57BL mice with terminal ME7 or 79A (mouse-adapted scrapie strains) TSE
disease. A 1% homogenate of each sample was prepared in sterile saline prior to
use as an inoculum. All experimental protocols were submitted to the Local
Ethical Review Committee for approval before mice were inoculated. All exper-
iments were performed under license and in accordance with the United King-
dom Home Office Regulations under the Animals (Scientific Procedures) Act,
Scoring of clinical TSE disease. The presence of clinical TSE disease was
assessed as described previously (16). Animals were scored for clinical disease
without reference to the genotype of the mouse. Genotypes were confirmed for
each animal by PCR analysis of tail DNA at the end of the experiment. Incu-
bation times were calculated as the interval between inoculation and culling due
to terminal TSE disease. Mice were killed by cervical dislocation at the terminal
stage of disease, at termination of the experiment (between 500 and 700 days),
or for welfare reasons due to intercurrent illness.
Lesion profiles. Sections were stained with hematoxylin and eosin and scored
for vacuolar degeneration on a scale of 0 to 5 in nine standard gray matter areas
and on a scale of 0 to 3 in three standard white matter areas as described
Immunohistochemical analysis in brain. Sections were stained for disease
associated PrP using monoclonal antibody 6H4 (1/1,000; Prionics). Antigen re-
trieval by autoclaving at 121°C for 15 min and 10 min in formic acid (98%) was
used to facilitate detection of the antigens. Sections were then blocked with
normal serum prior to incubation with the primary antibody. Antibody binding
was detected with a catalyzed signal amplification system (Dakocytomation) and
visualized with diaminobenzidine (DAB). In all the experiments normal brain
homogenate inoculum and PrP null negative controls were used. Images were
taken using a Nikon Eclipse E800 microscope. Sections were analyzed by an
observer blinded to the mouse genotype and type of inoculum.
PrPScextraction from spleens. Mice were killed by cervical dislocation;
spleens were removed, flash frozen in liquid nitrogen, and stored at ?70°C until
required. Whole spleens were weighed, and PrPScwas extracted using the
method of centrifugal concentration from detergent solution (15). The spleen
was homogenized in 3 ml of 0.2 M potassium chloride and 20 ?l of 100 mM
phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 1,500 rpm for 10 min,
and the subsequent supernatant was centrifuged at 50,000 rpm for 30 min, all at
4°C. This pellet was resuspended in 2 ml of 0.1 M Tris-hydrochloric acid (Tris-
HCl, pH 7.4), and the suspension was divided into two equal parts: one part was
left at 4°C with 40 ?l of 100 mM PMSF for samples that were not digested with
proteinase K (?PK; Sigma), and the other part was incubated with 3 ?l of 18
mg/ml PK at 37°C for the samples digested with PK (?PK), both for 1 h. Twenty
microliters of PMSF, 1 ml of 2% sarcosyl, and 2 ?l of ?-mercaptoethanol were
added, and the samples were incubated at 37°C for an additional 1 h. The
samples were then overlaid on 200 ?l of 20% sucrose and centrifuged at 50,000
rpm for 2 h, and the pellets were stored at ?20°C.
Western blotting. Mice were killed by cervical dislocation, and brains and
spleens were removed, flash frozen in liquid nitrogen, and then stored at ?70°C
until required. Spleen preparation has already been described above. Brain
homogenates (10%, wt/vol) were prepared in NP-40 lysis buffer (1% Nonidet
P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and
1 mM PMSF). The homogenate was centrifuged at 16,000 rpm for 10 min at 4°C,
and supernatant was isolated. Total protein was denatured in 1? Novex Tris-
glycine SDS sample buffer (Invitrogen Life Technologies) and 1? NuPage sam-
ple reducing agent (Invitrogen Life Technologies) for 30 min at 95°C. Proteins
were separated by gel electrophoresis at 125 V using 12% Novex Tris-glycine gels
(Invitrogen Life Technologies). Proteins in the acrylamide gel were transferred
to polyvinylidene difluoride (PVDF) membrane at 2 mA/cm2of gel using a
semidry transfer blotter (Bio-Rad) in 1? transfer solution (48 mM Tris, 39 mM
glycine, 0.375% SDS, 20% methanol). Presence of PrP was assessed using the
anti-PrP monoclonal antibodies 8H4 or 7A12 (1/10,000; kind gift of M. S. Sy)
Immunohistochemical analysis of spleens. Tissues were flash frozen in liquid
nitrogen and embedded in optimal cryotomy temperature (OCT) compound
prior to sectioning on a Leica cryostat. Serial sections (10 ?m) were cut and air
dried overnight on SuperFrost Plus slides. Sections were fixed in acetone for 10
min. All labeling was done at room temperature in a humid chamber. For light
microscopy, sections were blocked in normal mouse serum (1/20) for 15 min
prior to incubation for 60 min with the primary antibody: 8H4 for PrP detection
or the rat anti-mouse FDC-M2 monoclonal antibody for FDC (1/800; AMS
Biotechnology). After being washed in Tris (pH 7.6)-bovine serum albumin
(BSA) buffer, sections were further incubated for 60 min in mouse anti-rat
biotinylated serum (1/500; Jacksons). After further washing, sections were
stained with streptavidin-alkaline phosphatase reagent and then Vector red
alkaline phosphatase substrate (Vector) according to the manufacturer’s proto-
cols. Finally, sections were counterstained with hematoxylin and rinsed in Scott’s
tap water prior to dehydration and mounting. Confocal microscopic studies were
performed in sections probed with antibodies CD16/CD32 at a 1/100 dilution
(BD Biosciences). Following washing in Tris (pH 7.6)-BSA buffer, sections were
further incubated for 60 min in mouse anti-rat biotinylated serum (1/500; Jack-
sons). After further washing, sections were incubated with streptavidin-Alexa
Fluor 594 (1/200; Abcam) for 60 min prior to mounting in fluorescent mounting
medium (Dako). For double immunolabeling of FDC and PrPC, sections were
initially blocked in normal goat serum (1/20) for 20 min prior to an overnight
incubation with the anti-PrP antibody 1B3 (1/1,000; kind gift of C. Farquhar),
VOL. 84, 2010 ROLE OF PrP GLYCOSYLATION IN PERIPHERALLY ACQUIRED TSE3465
followed by incubation with the fluorescent secondary antibody goat anti-rabbit
Alexa Fluor 488 (1/200; Abcam) for 60 min. After repeated washings, sections
were subsequently blocked with normal mouse serum (1/20) for 20 min prior to
a 60-min incubation in the second primary antibody, FDC-M2 (1/1,000), followed
by a 60-min incubation in mouse anti-rat biotinylated serum (1/500). Incubation
with the fluorescent antibody streptavidin-Alexa Fluor 594 (1/200; Abcam) for 60
min preceded mounting in fluorescent mounting medium. Control sections, with
normal rabbit or rat serum substituted for the primary antibody, were included
in all immunostaining experiments.
Thioflavin-S treatment. Brain sections were stained with hematoxylin for 30 s
and then washed in water. Sections were treated with a thioflavin-S solution (10
mg/ml) for 5 min and then immersed in 70% ethanol solution for 5 min. After
sections were rinsed in water, they were mounted using fluorescent mounting
medium (Dakocytomation) and analyzed using a Hamamatsu camera and Image
Host PrP glycosylation regulates the timing of TSE neuro-
invasion. To assess the effect of glycosylation in the TSE traf-
ficking following peripheral exposure, wild-type and glycosyla-
tion-deficient mice were inoculated intraperitoneally (i.p.) with
the ME7 or 79A mouse-adapted scrapie strain and monitored
for the appearance of clinical signs of disease (16). G2 homozy-
gous mice (G2/G2) showed a very long incubation period of
disease compared to wild-type mice following i.p. inoculation
with 79A. All G2/G2 animals succumbed to TSE disease, with
an average incubation period of 307 (? 4.5) days, whereas the
average incubation period in wild-type animals was 201 (? 2.2)
days (Table 1). However, inoculation of G2/G2 mice with ME7
produced only a modest increase in the incubation period, with
an average of 280 (?4) days compared to 257 ? 3 days for
wild-type mice inoculated with the same strain (Table 1). Thus,
lack of sugars at the second site delays the onset of disease, but
the extent of this delay is TSE strain specific.
A very long incubation period and low susceptibility were
also observed in G1 homozygous mice (G1/G1) inoculated i.p.
with 79A compared with the wild-type mice. In this case only
11/18 G1 animals succumbed to disease, with an average incu-
bation period of 310 (? 35) days (Table 1), whereas no G1/G1
mice showed any clinical disease signs after more than 700 days
following peripheral administration of ME7. No clinical signs
of disease were observed in G3 homozygous mice (G3/G3)
after i.p. inoculation with either ME7 or 79A (Table 1). There-
fore, host PrP glycosylation clearly influences the incubation
time and susceptibility to disease following an intraperitoneal
Glycosylation state of PrPCin the host determines the tar-
geting of pathological lesions in the brain following peripheral
infection. Western blot analysis using the anti-PrP antibodies
8H4 or 7A12 revealed the presence of PK-resistant unglyco-
FIG. 1. Presence of PrPScin brain of peripherally inoculated mice. In order to detect presence of proteinase K-resistant PrP, Western blotting
using the 7A12 anti-PrP antibody was carried out in brains from clinically positive and clinically negative mice after injections with 79A or ME7.
Brain homogenates from the different genotypes were treated with PK prior to SDS-PAGE analysis and immunoblotting. (A) Brains after
peripheral infection with strain 79A. This analysis revealed presence of PK-resistant PrP in the brain of clinically positive Wt/Wt, G1/G1, G2/G2,
and Wt/G2 mice but not in brains of clinically negative G1/G1 and G3/G3 mice. (B) Mouse brains after peripheral infection with strain ME7. This
analysis revealed the presence of PK-resistant PrP in brains of clinically positive wild-type, G2/G2, and Wt/G2. However, no PK-resistant PrP was
found in the brains of clinically negative G1/G1 and G3/G3 mice. Lane 1, wild type; lane 2, G1/G1; lane 2*, G1/G1 clinically negative; lane 3,
G2/G2; lane 4, Wt/G2; lane 5, G3/G3.
TABLE 1. Incubation periods of Wt and glycosylation mutant homozygous mice infected intraperitoneally with strain 79A or ME7
Result of i.p. inoculationResult of i.c. inoculationa
(avg no. of days ? SEM)
No. of clinically and
total no. of mice
relative to the
(avg no. of days ? SEM)
relative to the
201 ? 2.2
310 ? 35
307 ? 4.5
257 ? 3
280 ? 4
148 ? 2.6
194 ? 21
167 ? 9.3
435 ? 92
163 ? 2
160 ? 2.5
aResults for i.c. inoculation (from reference 45) are shown for comparison.
3466CANCELLOTTI ET AL. J. VIROL.
sylated and monoglycosylated PrPScin brains of all clinically
positive G2/G2 animals inoculated with either ME7 or 79A
(Fig. 1). A PK-resistant PrPSccharacterized by lack of the
diglycosylated band was also detected in 11 out of 18 clinically
positive G1/G1 mice inoculated with 79A but not in the seven
clinically negative G1/G1 animals after inoculation with the
same strain (Fig. 1A). No PrPScwas detected in brain from
G1/G1 mice inoculated with ME7 (Fig. 1B), and this was also
the case for G3/G3 mouse brains after challenge with 79A or
ME7 (Fig. 1).
PrPScdeposition was assessed in the brains of mice inoculated
with 79A and ME7 strains by immunohistochemistry (IHC) using
6H4 antibody. This analysis revealed a remarkable difference in
both the amount and distribution of PrPScdeposition between
wild-type mice and G2/G2 mice injected with 79A. In wild-type
mice a mild to moderate, fine punctate PrPScimmuno-positivity
were also occasionally seen in other brain areas (Fig. 2A to C). In
G2/G2 animals fine punctate PrPScimmuno-positive deposits
were seen in several brain regions: septum, thalamus, hippocam-
pus, hypothalamus, midbrain, and brain stem. In several G2/G2
mice high levels of PrPScaccumulation were also observed in the
had only sparse or no PrPScdeposition (Fig. 2D to F).
FIG. 2. PrPScdeposition in brain of wild-type and transgenic homozygous mice after peripheral administration of 79A. Brains from clinically
positive and clinically negative mice after 79A challenge were immunostained for PrPScwith the monoclonal antibody 6H4 and analyzed by light
microscopy using a Nikon Eclipse E800 microscope. (A to C) Wild-type animal brain showing a characteristic fine punctate PrP deposition. In these
mice little involvement of the hippocampus and cortex areas (A) and of the midbrain (B) was observed, with most of the PrPScdeposition present
in the thalamus (C). (D to F) G2/G2 brains showing a very strong mantle-like PrPScdeposition in several brain regions such as cortex and
hippocampus (D), midbrain (E), and thalamus (F). (G to K) G1/G1 mouse brain showing a very peculiar PrPScdeposition characterized by
aggregates of PrPScin very specific brain regions. In contrast to G2/G2 mice, G1 animals did not show any PrPScdeposition in many brain areas,
including the hippocampus and cortex (G). However, some PrPScdeposition in the form of fine punctuate deposits was observed in the habenula
(H). A closer examination of these brains revealed the presence also of unusual PrPScsmall aggregates in the thalamus (I) that were thioflavin-S
negative (J). These aggregates were PrPScspecific since they were not present in control brain sections treated just with normal mouse serum
without any anti-PrP antibody (K). (L) G3/G3 clinically negative brain showing no PrP deposition in any area of the brain examined. Magnifi-
cations, ?4 (A to G, J, and L), ?10 (H), and ?20 (I).
VOL. 84, 2010 ROLE OF PrP GLYCOSYLATION IN PERIPHERALLY ACQUIRED TSE3467
In ME7-inoculated wild-type animals, fine punctate and
coarse (confluent) PrPScimmuno-positive deposits were seen
in multiple brain areas including the septum, cerebral cortex,
thalamus, hippocampus, hypothalamus, midbrain, brain stem,
and cerebellum (Fig. 3A to C). Similar topographical distribu-
tions of fine punctate and coarse PrPScdeposits were observed
in wild-type and G2/G2 mice. However, PrPScdeposition in the
ME7-infected G2/G2 mice was also characterized by the pres-
ence of large, thioflavin S-positive amyloid plaques in all of the
affected regions. (Fig. 3D to G).
The degree of spongiform degeneration in the CNS as de-
termined by lesion profile analysis is an important parameter
to define TSE disease (9). G2/G2 animals inoculated with 79A
showed a higher degree of spongiform degeneration than wild-
type mice in a number of areas of the gray matter: hypothal-
amus, thalamus, hippocampus, septum, and forebrain cortex.
However, in other regions, such as cerebral cortex or medulla,
no differences were observed. In the white matter areas ana-
lyzed (cerebellar white matter, mesencephalic tegmentum, and
pyramidal tract), G2/G2 mice demonstrated a milder spongi-
osis (Fig. 4A). Differences in spongiform degeneration were
also observed in G2/G2 mice inoculated with ME7, with more
extensive spongiosis in the hypothalamus region in the trans-
genic mice than in wild-type mice and less in other areas such
as the hippocampus, septum, cerebral cortex, and forebrain
cortex in the gray matter and the pyramidal tract of the white
matter (Fig. 4B).
An even more striking difference in pathology was observed
in G1/G1 mice inoculated with 79A compared to wild-type
animals in both the distribution and intensity of the PrPSc
deposition and spongiosis. In the G1/G1 mice sparse, fine
punctate PrPScdeposits were seen in habenula, thalamus, mid-
brain, and brain stem. In addition, coarse punctate PrPScde-
posits were seen in the lateral thalamic region (Fig. 2G to I).
To further investigate if this pattern was composed of amyloid
deposits, infected G1/G1 mouse brain sections were treated
with thioflavin-S (37). This analysis, however, did not reveal
any fluorescent deposits in the parenchyma (Fig. 2J). A further
unusual pathology in brain was also observed in these mice
once a spongiform degeneration profile was performed. In-
deed, in G1/G1 mouse brains spongiosis was restricted to the
hypothalamus and dorsal medulla areas, whereas in wild-type
mice a higher degree of spongiform degeneration was observed
in all the gray and white matter regions analyzed (Fig. 4C). No
spongiform degeneration or PrPScdeposition was found in any
clinically negative G1/G1 and G3/G3 mice after inoculation
with 79A or ME7, supporting the observation that these mice
are resistant to peripheral inoculation with these strains (Fig.
2L and 3H and I).
FIG. 3. PrPScdeposition in brain of wild-type and transgenic homozygous mice after peripheral administration of ME7. Brains from clinically
positive and clinically negative mice after ME7 challenge were immunostained for PrP with the monoclonal antibody 6H4 and analyzed by light
microscopy using a Nikon Eclipse E800 microscope. (A to C) Wild-type animal brains characterized by widespread fine punctate PrPScdeposition
in several brain areas, such as cortex and hippocampus (A), septum (B), and cerebellum (C). (D to F) G2/G2 mice showing a different pattern of
PrPScdeposition characterized by the presence of fine punctuate deposits and amyloid plaques in the cortex (D), septum (E), and cerebellum (F).
Presence of amyloid plaques in these brains was confirmed by thioflavin-S treatment (G). (H and I) Brains of clinically negative G1/G1 (H) and
G3/G3 (I) mice after ME7 challenge showing the absence of PrPScdeposition in every area of the brain examined. In panels H and I, the
hippocampus and cortex areas are represented for both transgenic lines. Magnifications, ?4 (A, B, D, E, and G to I) and ?20 (C and F).
3468 CANCELLOTTI ET AL.J. VIROL.
A single copy of wild-type PrP restores the incubation time
in PrP glycosylation-deficient mice. Heterozygous mice ex-
pressing both wild-type and G2 PrP (Wt/G2) were also inocu-
lated i.p. with 79A or ME7. Wt/G2 mice showed an incubation
time strikingly similar to that of homozygous wild-type mice.
Indeed, Wt/G2 mice inoculated with 79A died in an average
incubation period of 205 (? 3) days, whereas wild-type animals
succumbed with an average incubation period of 201 (? 2.2)
days (Table 2). Similar incubation times were also observed
between heterozygous and wild-type mice inoculated with
ME7 (Wt/G2, 253 ? 5.5 days; Wt/Wt, 257 ? 2.8 days) (Table
2). Thus, a single copy of the wild-type Prnp gene was able to
restore the incubation times of disease observed in the wild-
Western blot analysis performed in brains of the clinically
positive Wt/G2 heterozygous animals, after exposure to 79A or
ME7, revealed the presence of PK-resistant diglycosylated
PrPSc. The glycoprofile of PrPScin these mice was character-
ized by having a monoglycosylated band more intense than the
diglycosylated band (Fig. 1). This differs from the glycoprofile
observed in wild-type homozygous animals characterized by a
predominant diglycosylated band and reflects the glycosylation
status of PrPCin Wt/G2 mice.
Despite identical incubation times, immunohistochemical
analysis of PrPScdeposition in brain sections revealed differ-
ences between wild-type and heterozygous animals. Analysis of
brain sections from Wt/G2 mice inoculated with the 79A strain
showed accumulation of PrPScin several areas of the brain.
However, the severe deposition of PrPScin the cerebral cortex
detected in G2/G2 mice was not apparent in heterozygous
animals (Fig. 5A and B). Analysis of brain sections from
Wt/G2 mice inoculated with the ME7 strain shows fine punc-
tate PrPScdeposits in several areas. Moreover, coarse and
plaque-like PrPScdeposits previously observed in G2/G2 mice
were also present in the heterozygous animals; however, the
size and numbers of these deposits were reduced compared to
those seen in G2 homozygous animals (Fig. 5D and E). The
FIG. 4. Lesion profile analysis of wild-type and PrP glycosylation mutant homozygous mice after peripheral inoculation with 79A or ME7.
Spongiform degeneration in brains of the clinically positive wild-type, G2, and G1 mice was analyzed after peripheral infection with 79A or ME7.
For this purpose nine gray matter areas were scored on a scale of 0 to 5 (y axis). Gray matter areas are represented on the x axis by the following
numbers: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, medial thalamus; 6, hippocampus; 7, septum; 8, cerebral
cortex; and 9, forebrain cortex. Three white matter areas were scored on a scale of 0 to 3 (y axis). White matter areas are represented on the x
axis by the following numbers: 11, cerebellar white matter; 12, mesencephalic tegmentum; and 13, pyramidal tract. The magnitude of spongiform
degeneration in the transgenic mice was then compared with the one in wild-type mice. Graphs show lesion profile analysis of the brains of the
mice indicated on the figure after infection with strains 79A (A and C) and ME7 (B). The mean score for each area is shown (error bars ? standard
error of the mean [SEM]).
TABLE 2. Incubation periods of Wt and heterozygous Wt/G2 mice
infected intraperitoneally with strain 79A or ME7
(avg no. of
days ? SEM)
No. of clinically
no. of mice
to the Wt
201 ? 2.2
205 ? 3
257 ? 3
253 ? 5
VOL. 84, 2010 ROLE OF PrP GLYCOSYLATION IN PERIPHERALLY ACQUIRED TSE3469
immunohistochemical characteristics of PrPScaccumulation in
heterozygous mice can be best described as intermediate be-
tween those seen in G2 and wild-type homozygous animals.
The spongiform degeneration analysis revealed no differences
in the brain areas affected between Wt/G2 and Wt/Wt mice
inoculated with 79A (Fig. 5C). However, Wt/G2 mice inocu-
lated with ME7 have reduced vacuolation in specific brain
areas compared to wild-type mice, e.g., in the hippocampus,
septum, cerebral cortex, forebrain cortex, and the pyramidal
tract (Fig. 5F).
Glycosylation-deficient mice possess mature and functional
FDC that express PrPC. Since FDC have been suggested to be
a pivotal component in the uptake and replication of infectivity
in the periphery, we assessed if the delay or resistance observed
in the glycosylation-deficient mice could be linked to any pos-
sible FDC abnormality.
FDC networks were detected using the FDC-M2 anti-
body. The epitope recognized by this antibody has been
shown to be the activated form of the complement receptor
C4 (44). FDC-M2 labels mature FDC networks within the
germinal center as well as parts of the primary follicle.
Initial light microscopy analysis revealed no overt differ-
ences between the FDC networks of transgenic and wild-
type mice (Fig. 6A). To further explore the functionality of
these networks, immunolabeling was carried out using an
antibody which recognizes an epitope common to both
CD16 and CD32, which are both low-affinity receptors for
the immunoglobulin Fc portion expressed by functional
FDC, as well as other cell types (18). No differences were
evident in the labeling between the different genotypes
It was then determined whether PrPClocalization within
these FDC networks is altered in the transgenic mice com-
pared with wild-type mice. For labeling of PrPC, the rabbit
polyclonal antibody used was 1B3, which targets the amino
acid residues 14 to 36, 83 to 102, 119 to 139, and 188 to 212 on
the PrP molecule (15). Confocal analysis of double immuno-
fluorescent labeling of PrP and FDC networks showed that
PrPCcolocalizes within the FDC networks of each of the trans-
genic genotypes and wild-type samples (Fig. 6C). Thus, the
glycosylation-deficient mice, like wild-type mice, appear to
possess an intact FDC network which expresses PrPC.
Host PrP glycosylation influences the peripheral stages of
TSE infectivity. Following peripheral inoculation of TSE-in-
fected material, accumulation and amplification of infectivity
in the LRS occur rapidly, with infectivity levels in these tissues
reaching a plateau after several weeks (14–15). We therefore
analyzed the presence of PrPScdeposition in spleens of clini-
cally positive or clinically negative glycosylation-deficient
transgenics and wild-type mice after i.p. inoculation with the
79A or ME7 strain by IHC. This analysis showed a correlation
between the amount of PrP deposition in spleen and subse-
quent clinical disease. Clinically positive Wt/Wt and G2/G2
mice after 79A or ME7 infection or G1/G1 mice after 79A
FIG. 5. Brain pathology analysis in G2 heterozygous animals challenged with 79A or ME7. Brains from clinically positive Wt/G2 heterozygous
animals inoculated with 79A or ME7 were immunostained for PrP with monoclonal antibody 6H4 and analyzed by light microscopy using a Nikon
Eclipse E800 microscope. Analysis of the spongiform degeneration in the nine gray matter areas and the three white matter areas described in the
legend of Fig. 4 was also carried out in Wt/G2 brains and compared with spongiform degeneration in Wt/Wt mice. (A and B) Wt/G2 brain after
inoculation with 79A i.p. showed a PrP deposition intermediate between that of wild-type and G2 homozygous animals. PrPScaccumulation was
indeed observed in regions such as thalamus, hippocampus, and cerebral cortex (A) or midbrain (B). However, this deposition was less intense than
the one observed in G2 homozygous animals (compare Fig. 2). (C) Although there were some differences in terms of PrPScdeposition between
Wt/Wt and Wt/G2 mice, no differences were observed between these two lines in terms of spongiosis in all the brain areas analyzed. (D and E)
Wt/G2 mice brains after peripheral infection with ME7 also resembled an intermediate pathology between wild-type and G2 homozygous animals.
PrPScdeposits and small plaques were indeed observed in regions such as the hippocampus (D) and the septum (E) (compare Fig. 3).
(F) Heterozygous animals presented a vacuolation profile that differed from that of wild-type mice infected with ME7, with lower degrees of
vacuolation in several brain areas: 6, hippocampus; 7, septum; 8, cerebral cortex; and 9, forebrain cortex. Magnification, ?4 (A, B, D, and E).
3470 CANCELLOTTI ET AL.J. VIROL.
challenge showed large amounts of PrPScthroughout the
spleen (Fig. 7A to D and F). However, complete absence of
PrP deposition was observed in spleens of clinically negative
G1/G1 mice inoculated with ME7 and of clinically negative
G3/G3 mice challenged with 79A or ME7 (Fig. 7E, G, and H).
Very small PrPScdeposits in the spleen were also observed in
some of the seven G1/G1 mice that were not affected by a
clinical TSE disease after 79A challenge, suggesting that in
these mice PrPScamplification levels were not sufficient to
further elicit TSE clinical disease (Fig. 7I).
Total lack of glycosylation in PrPCprevents trafficking of
PrPScfrom brain to spleen. After intracerebral inoculation,
PrPScis often detected first in the spleen, suggesting that in-
fectivity travels from the site of injection in the brain to the
LRS and replicates in the periphery prior to causing disease in
the CNS (15, 17). To test the influence of host PrP glycosyla-
tion in transport of PrPScfrom brain to spleen, we analyzed by
Western blotting the presence of PrPScin spleens of the three
glycosylation mutant mice infected intracerebrally in previous
experiments (45) with ME7 and 79A. Wild-type and G2 mice
are susceptible to infection with ME7, and PrPScwas detected
in the spleens. However, G1/G1 and G3/G3 mice did not de-
velop any clinical disease after challenge with ME7, and no
PrPScwas detected in spleens (Fig. 8A), suggesting that lack of
replication in the LRS may prevent clinical disease in these
mice, as previously suggested (7). Presence of PrPScwas ob-
served in the spleens of clinically positive G1/G1 and G2/G2
mice infected with 79A, but surprisingly no PrPScwas found in
the spleens of G3/G3 mice that were clinically positive (Fig.
8B). These results suggest that monoglycosylated but not un-
glycosylated PrP is able to sustain transport of infectivity be-
tween the brain and the periphery and that while replication in
the spleen is important for establishing disease with ME7 via
intracerebral or intraperitoneal routes, this step may not be
mandatory for establishing infection with 79A.
The mechanisms regulating peripheral propagation of the
different TSE strains are not yet fully understood. Different
organs, cells, and routes have been shown to be important
according to which strain of agent is infecting a particular host.
Some strains require amplification in the LRS before causing a
productive infection in the CNS, whereas with other strains,
such as naturally occurring BSE in cattle, the involvement of
the LRS is limited (3–4, 12, 22, 42). A number of factors are
likely to contribute to this diversity in neuroinvasion, including
strain, host PrP genotype, and the route of entry. Our results
FIG. 6. Characterization of spleen morphology in glycosylation-deficient mice. In order to assess if a lack of sugars can induce an overt
phenotype in spleen, a number of morphological assays in spleen derived from transgenic, wild-type, and PrP knockout animals (null) were
performed. (A) FDC networks were detected using the FDC-M2 antibody, which labels mature FDC networks within the germinal center. With
light microscopy analysis using a Nikon Eclipse E800 microscope, no differences were highlighted between wild-type and glycosylation-deficient
mice. (B) CD16/CD32 antibody was used to investigate the functionality of FDC by confocal microscopy analysis. Again, there were no differences
evident in the labeling between the different genotypes. Scale bar, 50 ?m. (C) PrP localization within the FDC network was analyzed by confocal
microscopy analysis. Double immunofluorescent labeling of PrP with 1B3 antibody (green) and of FDC networks with FDC-M2 antibody (red)
showed that PrPCcolocalizes within the FDC networks (yellow) of each of the transgenic genotypes and wild-type samples. Scale bar, 50 ?m.
VOL. 84, 2010ROLE OF PrP GLYCOSYLATION IN PERIPHERALLY ACQUIRED TSE3471
now suggest that glycosylation status of the host PrP may be
an important factor in this process and, in particular, in
determining the timing of neuroinvasion and the final tar-
geting in the CNS.
Glycosylated PrPCappears to be required at the early
stages of the infectious process, which may involve uptake
and transport from the site of infection to the spleen or
amplification of infectivity in the spleen. Clinical disease was
observed in each of the PrP glycosylation-deficient mice
following intracerebral inoculation with at least one strain
(45), albeit with very long incubation periods and limited
susceptibility in the G3 mice. In this study this was not the
case as following intraperitoneal inoculation the G3 mice
appeared to show complete resistance to infection with two
different TSE strains.
This may be due to an inability of unglycosylated PrPCto
sustain the transport of infectivity from the periphery to the
brain and/or vice versa. Therefore, PrP glycosylation plays a
central role in the spread of infectivity from the periphery to
the CNS. Moreover, our results suggest that replication or
transport in the LRS is a limiting factor during spreading of
The role played by PrPCglycotypes in the pathogenesis of
disease appears to be strain specific. G2 mice developed dis-
ease after ME7 i.p. inoculation with only a short delay of 21
days compared to wild-type mice. Similarly, when ME7 was
injected directly into the CNS, G2 mice developed TSE disease
with incubation periods similar to those of wild-type mice (45).
Thus, the monoglycosylated G2 PrP protein appears to be able
to replicate and transport the ME7 agent almost as efficiently
as the diglycosylated PrP. However, ME7 fails to establish
disease following peripheral challenge of G1/G1 and G3/G3
mice, and PrPScis absent from spleens and brains of these
mice. This is in accordance with what was observed following
direct inoculation of ME7 into the brain; although these trans-
genic mice did not develop any clinical TSE disease, G3 mice
presented some degree of PrP deposition in the form of amy-
loid plaques (45). Thus, the first glycosylation site appears
critical for the replication of ME7 in both the CNS and pe-
riphery. Failure to establish disease is therefore likely to be due
to a failure of the host to replicate the agent.
G1 and G2 homozygous mice were susceptible to infection
following peripheral challenge with 79A; however, the incuba-
tion time was more than 100 days longer than for wild-type
animals. This delay after peripheral inoculation is much longer
than that observed following inoculation of the same strain
intracerebrally; in G1/G1 and G2/G2 mice the incubation of
clinical prion disease was 40 and 20 days longer, respectively,
than in wild-type mice (45). This suggests either that there is a
difference in efficiency of replication of the agents in the pe-
FIG. 7. PrPScdeposition in spleens of mice after peripheral inoculation with strain 79A or ME7. Spleens from clinically positive and clinically
negative mice were immunostained for PrP with the monoclonal antibody 6H4 and analyzed by light microscopy using a Nikon Eclipse E800
microscope and a 10? objective. PrPScdeposition was observed in spleens of wild-type (A), G1/G1 (B), and G2/G2 (C) clinically positive animals
after inoculation with 79A. PrPScdeposition was also observed in spleens of wild-type (D) and G2/G2 (F) clinically positive animals but not in
G1/G1 clinically negative mice (E) after ME7 infection. PrPScaccumulation was also not observed in G3/G3 clinically negative mice after i.p.
inoculation with either 79A or ME7 (G and H). Slight PrPScdeposition was observed in spleen of some clinically negative G1 mice after challenge
with 79A (I).
3472 CANCELLOTTI ET AL.J. VIROL.
riphery and the CNS or that lack of diglycosylated protein in
this case is delaying the transport to the CNS. However, the
incubation time in G2 mice was rescued when a wild-type PrP
was expressed alongside the monoglycosylated PrP. In het-
erozygous PrP mice a gene dosage effect on the length of TSE
clinical disease onset was observed due to the contribution of
both alleles. This effect was previously described in wild-type/
knockout PrP heterozygous transgenic mice (PrP?/?) in which
the incubation time was almost doubled compared to incuba-
tion in PrP wild-type homozygous animals (32). In Wt/G2 mice
it was therefore surprising to observe the appearance of clinical
disease at the same time as in Wt/Wt mice when it might have
been expected that incubation periods in these mice would fall
somewhere between those of wild-type and G2 homozygous
mice. This suggests that the mono- and diglycosylated forms of
PrP can act together to facilitate replication and transport of
the agent. This suggestion is also supported by the observation
that heterozygous animals presented a brain pathology that
was an intermediate between that observed in wild-type and
G2 homozygous mice, indicating that both alleles are also
contributing to the final targeting in brain. Thus, the provision
of diglycosylated PrP clearly provides an important function in
the disease process and can overcome the incubation time
delays observed with only monoglycosylated and unglyco-
sylated PrP. The discrepancy between the hybrid pathology
and the wild-type incubation times observed in the heterozy-
gote mice demonstrates that these are an important model for
defining the mechanisms regulating the neuroinvasion of TSE
infectivity, and further studies are under way to address this
Host PrP glycosylation appears to determine the targeting in
the CNS following peripheral challenge. Absence of fully gly-
cosylated PrP had a profound effect in determining the brain
area targeted by PrPScdeposition after infection with 79A. G1
mouse brains were characterized by PrPScaccumulation in very
restricted areas like the habenula and the thalamus. This depo-
sition was remarkably different from the characteristic wide-
spread distribution of PrPScin many brain areas observed in
wild-type mice. G2 homozygous animals also presented
changes in 79A targeting compared to wild-type animals, with
cortex and midbrain heavily affected. This different targeting
may be due to an effect of host PrP glycosylation in determin-
ing routes of propagation of infectivity from the periphery to
the CNS or to the fact that the transgenic mice developed
clinical disease with a much longer incubation time than the
wild types. However, even in cases where there were modest
alterations in the incubation times, in the glycosylation-defi-
cient transgenic mice there were dramatic differences observed
in PrPScdeposition. Absence of a fully glycosylated PrP ap-
pears to facilitate the deposition of amyloid plaque formation
in the brain, compared with fine punctuate staining observed in
wild-type mice after ME7 inoculation. This contrasts with the
targeting in the brain following intracerebral challenge, where
no difference in targeting was observed in the different lines of
mice (45). After inoculation of 79A or ME7 directly into the
brain, clinically positive G1/G1 and G2/G2 mice, indeed, did
not present any differences in terms of spongiform degenera-
tion or PrPScdeposition compared to wild-type animals. Wild-
type and glycosylation-deficient mice had the same vacuolation
profiles and the same widespread, fine punctuate PrPScdepo-
sitions throughout the brain (45). These results may also sug-
gest that the delay observed in the transgenic animals may be
due to an initial targeting of infectivity in different brain areas
that may delay the neurotoxic effect, as previously proposed
(24). Whether differences in routing to the CNS are responsi-
ble for the differences in targeting observed in the glycosyla-
tion-deficient mice remains to be established.
A difference in brain pathology between wild-type and gly-
FIG. 8. Travel of PrPScfrom brain to spleen. In order to assess influence of host PrP glycosylation in trafficking of PrPScfrom the CNS to the
periphery, spleens from glycosylation-deficient mice and wild-type animals inoculated intracranially with ME7 or 79A were tested for presence of
PK-resistant PrP by Western blotting using the anti-PrP antibody 8H4. (A) Western blot analysis of spleen from mice infected i.c. with ME7 after
pretreatment with PK. PK-resistant PrP was observed only in clinically positive wild-type and G2/G2 mice; no PrPScwas found in spleen of clinically
negative G1/G1 and G3/G3 mice. (B) Western blot analysis of spleen from mice infected i.c. with 79A after pretreatment with PK. PrPScwas
recovered in all clinically positive G1/G1, G2/G2, and wild-type mice; however, no PK-resistant PrP was found in clinically positive G3/G3 animals.
VOL. 84, 2010 ROLE OF PrP GLYCOSYLATION IN PERIPHERALLY ACQUIRED TSE3473
cosylation-deficient mice was also confirmed by the analysis of
vacuolation damage in the CNS. Differences were observed in
G1 and G2 mice after infection with both 79A and ME7 in
several brain areas. This analysis also revealed a discrepancy
between PrPScaccumulation and spongiform degeneration.
For example, the strong accumulation of PrPScobserved in the
cortex of G2 but not of wild-type animals was not linked with
a difference in terms of spongiosis in the same areas. This
observation may support previous observations suggesting
that PrPScpresence is not always associated with vacuola-
tion (2, 37).
In summary, we have shown that PrP is a requirement for
the peripheral events in TSE disease. In this process PrP gly-
cosylation is a key component to determine the timing of
neuroinvasion and the targeting in the CNS, and this effect
varies according to TSE strain. Indeed, a fully glycosylated PrP
molecule is required for the most efficient neuroinvasion by
79A. However, ME7, while less dependent on a fully glyco-
sylated PrP, appears to be totally dependent on glycosylation at
the first site. Finally, the results presented here indicating a
role of N-linked glycans in the spread of TSE infectivity may
have some important implications for the development of new
therapeutic approaches. For example, destabilizing sugar-me-
diated interactions between host and the infectious agent
would appear to provide an important approach to block prop-
agation of infectivity.
This work was supported by Biotechnology and Biological Science
Research Council and Medical Research Council. R.D.H. was funded
by Research in Life Sciences Program, University of Edinburgh.
We thank colleagues at The Roslin Institute and in particular all
members of J. C. Manson’s group for the useful discussions about this
work; Robert Somerville and Abigail Diack for critical reading of the
manuscript; Anne Coghill, Sandra Mack, and Gillian McGregor for
assistance with the pathology analysis; Aileen Boyle for lesion profile
analysis; and Irene McConnell, Val Thompson, Simon Cumming,
Leanne Frame, and Kris Hogan for the breeding, care, injections, and
the clinical scoring of the mice. 1B3 antibody was kindly provided by
Christine Farquhar (The Roslin Institute, University of Edinburgh);
8H4 and 7A12 antibodies were kindly provided by Man-Sun Sy (Case
Western Reserve University).
The findings and conclusions in this article have not been formally
disseminated by the Food and Drug Administration and should not be
construed to represent any Administration determination or policy.
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VOL. 84, 2010 ROLE OF PrP GLYCOSYLATION IN PERIPHERALLY ACQUIRED TSE3475