JOURNAL OF VIROLOGY, Feb. 2011, p. 1484–1494
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 4
Crucial Role for Prion Protein Membrane Anchoring in the
Neuroinvasion and Neural Spread of Prion Infection?
Mikael Klingeborn,1Brent Race,1Kimberly D. Meade-White,1Rebecca Rosenke,2
James F. Striebel,1and Bruce Chesebro1*
Laboratory of Persistent Viral Diseases1and Rocky Mountain Veterinary Branch,2Rocky Mountain Laboratories,
National Institute of Allergy and Infectious Diseases, Hamilton, Montana
Received 14 October 2010/Accepted 19 November 2010
In nature prion diseases are usually transmitted by extracerebral prion infection, but clinical disease results
only after invasion of the central nervous system (CNS). Prion protein (PrP), a host-encoded glycosylphos-
phatidylinositol (GPI)-anchored membrane glycoprotein, is necessary for prion infection and disease. Here, we
investigated the role of the anchoring of PrP on prion neuroinvasion by studying various inoculation routes in
mice expressing either anchored or anchorless PrP. In control mice with anchored PrP, intracerebral or sciatic
nerve inoculation resulted in rapid CNS neuroinvasion and clinical disease (154 to 156 days), and after tongue,
ocular, intravenous, or intraperitoneal inoculation, CNS neuroinvasion was only slightly slower (193 to 231
days). In contrast, in anchorless PrP mice, these routes resulted in slow and infrequent CNS neuroinvasion.
Only intracerebral inoculation caused brain PrPres, a protease-resistant isoform of PrP, and disease in both
types of mice. Thus, anchored PrP was an essential component for the rapid neural spread and CNS
neuroinvasion of prion infection.
Prion diseases, also known as transmissible spongiform en-
cephalopathies (TSE diseases), are fatal neurodegenerative
diseases of humans and animals. TSE diseases include scrapie
in sheep, chronic wasting disease (CWD) in cervids, and bo-
vine spongiform encephalopathy (BSE) in cattle as well as
kuru, Gerstmann-Stra ¨ussler-Scheinker syndrome (GSS), and
familial, sporadic, iatrogenic, and variant forms of Creutzfeldt-
Jakob disease (CJD) in humans. Within a species, TSE dis-
eases are easily transmissible by intracerebral inoculation of
infected tissue homogenates, whereas transmission to a new
species is usually inefficient (16). A hallmark of prion diseases
is the accumulation in infected tissues of a partially pro-
tease-resistant isoform of the prion protein ([PrP] PrPres).
The detection of PrPres by immunoblotting or immunohis-
tochemistry is often used as an important diagnostic feature
of prion disease. PrPres is generated by misfolding and
aggregation of host-encoded protease-sensitive prion pro-
tein, PrPsen, which is attached to the outer leaflet of the
plasma membrane via a glycosylphosphatidylinositol (GPI)
moiety (59). PrPsen is required for susceptibility to prion
infection and for pathogenesis and transmission of prion
diseases (10, 14).
The fastest route of TSE infection is via direct entry to the
central nervous system (CNS) by intracerebral (i.c.) or intraspi-
nal inoculation (39, 42). However, natural and experimental
prion infection models include a number of different routes of
exposure outside the CNS where disease onset is usually
slower. Most notably these include intraperitoneal (i.p.), intra-
venous (i.v), intraneural (i.n.), intratongue (i.t.), subcutaneous,
intranasal, and oral routes of exposure (7, 9, 13, 26, 27, 40, 43,
51). Peripheral infection is often accelerated by local amplifi-
cation of agent in follicular dendritic cells (FDC) of lymphoid
organs, followed by spread via local nerves to the CNS (12, 30,
43, 47, 49, 53). Alternatively, neuroinvasion via peripheral
nerves after i.p., i.v., i.n., i.t., or ocular inoculation may occur
without agent amplification in lymphoid tissues (5, 40, 42,
43, 55). In most cases neurons are the final route of spread
to the CNS, but these neurons must express PrP to be
functional in this respect (8, 55). The mechanism by which
scrapie infectivity is transported along peripheral nerves to
the CNS is not well understood, and some studies suggest
that conventional axonal transport is not the main mecha-
nism (28, 40, 44, 45).
To study if PrPres spread to the CNS in peripheral nerves
was dependent on membrane anchoring of PrP, we compared
wild-type mice expressing anchored PrP with transgenic mice
(tg44?/?) expressing only anchorless PrP, i.e., lacking the usual
GPI membrane anchor (18). Intracerebral scrapie inoculation
of tg44?/?mice leads to high levels of infectivity and amyloid
PrPres in CNS and results in a fatal amyloid brain disease (17).
In these mice the PrP amyloid-associated neuropathology and
cerebral amyloid angiopathy were similar to observations in
several human GSS patients expressing PrP molecules lacking
the GPI anchor (25, 34, 56). Thus, anchorless PrP transgenic
mice appeared to be an excellent model for these unusual
forms of familial prion disease.
In the present experiments we employed five different
routes of inoculation outside the CNS. For all extracerebral
routes of inoculation, GPI anchoring of PrP was required
for efficient spread of infectivity to the brain. Therefore,
anchored PrP appeared to be an essential part of the mech-
anism of rapid neural spread of PrPres and prion infectivity
to the CNS.
* Corresponding author. Mailing address: Laboratory of Persistent
Viral Diseases, Rocky Mountain Laboratories, National Institute of
Allergy and Infectious Diseases, Hamilton, MT 59840. Phone: (406)
363-9354. Fax: (406) 363-9286. E-mail: firstname.lastname@example.org.
?Published ahead of print on 1 December 2010.
MATERIALS AND METHODS
Experimental mice and tissue collection for histochemical and biochemical
analysis. All mice were housed at the Rocky Mountain Laboratories (RML) in
an AAALAC-accredited facility, and research protocols and experimentation
were approved by the NIH RML Animal Care and Use Committee (protocol
number 2007-50). This study was carried out in strict accordance with the rec-
ommendations in the Guide for the Care and Use of Laboratory Animals of the
National Institutes of Health.
Transgenic GPI anchorless PrP mice (tg44) expressed the transgene in ho-
mozygous form (?/?) and did not express normal GPI-anchored mouse PrP
(17). Mice were bred and genotyped at RML. Weanling C57BL/10 mice were
obtained from Harlan Laboratories, Indianapolis, IN. Animals were observed
daily for onset and progression of scrapie and euthanized when late-stage scrapie
signs were consistently present. One C57BL/10 mouse from each inoculation
route was euthanized at 100 days postinfection (dpi) for histochemical and
biochemical analyses. Tissues were also collected from C57BL/10 mice when they
were in an advanced stage of scrapie. In a similar fashion, some tg44?/?mice
from each group were euthanized at various days postinfection and at a terminal
stage of disease or no later than 600 dpi for histochemical and biochemical
analyses. A portion of each tissue was flash frozen in liquid nitrogen and kept at
?80°C for future use in biochemical analyses. A second portion was immersed in
10% neutral buffered formalin (3.7% formaldehyde) for histochemistry studies.
At necropsy of mice, brain was collected first to avoid contamination with PrPres
from other tissues.
Scrapie inoculation of mice. Young adult mice were inoculated with an RML/
Chandler scrapie brain homogenate from C57BL/10 mice. For intracerebral,
intraperitoneal, intravenous, and intravenous injection followed with a mock
intracerebral needle stab, 50 ?l of a 1% brain suspension containing 1.0 ? 106
50% infective doses (ID50) was inoculated. One ID50is the dose causing infec-
tion in 50% of C57BL/10 mice. Some routes of inoculation did not allow injection
of large volumes, so we reduced the volume and increased the percentage of
brain suspension to more closely match the ID50inoculated. Mice inoculated by
intrasciatic, perisciatic, intravitreal, and supraciliary routes received 2 ?l of a
10% brain suspension containing 4 ? 105ID50, and mice inoculated in the
tongue received 5 ?l of a 10% suspension containing 1 ? 106ID50.
Route-specific techniques are described below.
For intracerebral inoculation, mice were anesthetized with isoflurane and
injected in the left parietal lobe with a 27-gauge, 0.5-in. needle.
For intravenous injections mice were restrained in a chute and inoculated in
the tail vein. Groups of mice receiving an intracerebral needle stab were anes-
thetized with isoflurane immediately following their i.v. scrapie inoculation, and
a stab wound was made in the left parietal lobe using a sterile 27-gauge needle
with no inoculum (23, 39). The needle was inserted through the skull and
directed into the left parietal lobe; following a slight retraction of the needle, it
was redirected into a second region of the parietal lobe.
For intraneural inoculation mice were anesthetized with an intraperitoneal
injection of a combination of ketamine (80 mg/kg), xylazine (16 mg/kg), and
acepromazine (2 mg/kg). The lateral surface of the left rear leg was clipped and
prepared for surgery. Once mice had reached a medium-to-deep plane of anes-
thesia, a 1-cm incision was made just caudal to the midfemoral region. Blunt
dissection was used to retract muscles and expose the sciatic nerve. After visu-
alization of the nerve, a curved forceps was placed under the nerve and used to
elevate the nerve for easier inoculation. A 30-gauge needle attached to a Ham-
ilton syringe was inserted through the nerve sheath and threaded into the nerve.
The needle was then moved back and forth 1 to 2 mm within the nerve sheath
approximately 10 times (6), and 2 ?l of 10% brain suspension was then injected
within the nerve sheath. The skin incision was closed with 7-mm surgical skin
For perineural inoculation, the same methods described for the intraneural
inoculation were used to expose the nerve, but the nerve sheath was not pene-
trated by the needle, and the inoculum was placed outside the nerve rather than
directly into the nerve sheath.
For tongue inoculation mice were anesthetized as described in the intraneural
section. A 30-gauge needle was directed at an acute angle into the right side of
the tongue to the level of the subepithelium where 5 ?l of inoculum was injected.
For intraocular inoculations mice were anesthetized as described for the in-
traneural nerve inoculations. Inoculum was deposited either in the vitreous
chamber (36) or in the supraciliary space of the eye (1) using a 32-gauge needle
attached to a Hamilton syringe. Results were similar for both ocular routes.
Immunoblotting analysis of PrPres. To detect PrPres by immunoblotting,
tissue homogenates (20%) were prepared in 10 mM Tris-HCl (pH 7.4) using a
Mini-Beadbeater (Biospec products, Bartlesville, OK). All homogenates were
sonicated for 1 min using a Vibracell cup-horn sonicator (Sonics, Newtown, NJ)
and briefly vortexed. PrPres preparation was done as previously described (48).
Briefly, an aliquot of a 20% tissue homogenate was adjusted to 100 mM Tris-HCl
(pH 8.3), 1% Triton X-100, and 1% sodium deoxycholate and treated for 45 min
at 37°C with proteinase K at a final concentration of 50 ?g/ml. The reaction was
stopped by the addition of Pefabloc SC (Roche) to a final concentration of 4 mM,
and the sample was placed on ice for 5 min. An equal volume of 2? Laemmli
sample buffer (Bio-Rad, Hercules, CA) containing 10% ?-mercaptoethanol was
added; samples were boiled for 5 min and then frozen at ?80°C until needed.
Freshly boiled samples were electrophoresed on 16% SDS-PAGE gels (Invitro-
gen, CA). Immunoblots were probed with D13 anti-PrP antibody (InPro Bio-
technology, S. San Francisco, CA) followed by a peroxidase-conjugated anti-
human IgG secondary antibody (Sigma, St. Louis, MO). Bands were detected
using enhanced chemiluminescence substrate (ECL) as directed by the manu-
facturer (GE Healthcare Life Sciences, Piscataway, NJ).
The amount of brain PrPres detected by immunoblotting was defined relative
to the signal detected in i.c. inoculated tg44?/?mice at terminal stage, and
scoring was as follows: 3, 25 to 100% of terminal mouse; 2, 5 to 24% of terminal
mouse; 1, 1 to 4% of terminal mouse; 0, no detectable signal (?1% of signal in
Neuropathology and IHC. Tissues were removed and placed in 10% neutral
buffered formalin (3.7% formaldehyde) for 3 to 5 days before dehydration and
embedding in paraffin. Serial 5-?m sections were cut using a standard Leica
microtome, placed on positively charged glass slides, and dried overnight at 56°C.
Slides were then deparaffinized using standard procedures, rehydrated to aque-
ous conditions, and processed for standard hematoxylin and eosin (H&E) stain-
ing or immunohistochemistry (IHC) analysis. For immunohistochemical detec-
tion of PrPres using Fast Red chromogen (Ventana, Tucson, AZ), tissue sections
were pretreated in citrate buffer, pH 6.0, and heated to 120°C at 20 lb/in2for 20
min in a Decloaking Chamber (Biocare, Walnut Creek, CA) for antigen re-
trieval, followed by staining in the Ventana automated NexES stainer. Staining
for PrP was done using human anti-mouse PrP antibody D13 at a dilution of
1:500 (InPro Biotechnology) at 4°C for 16 h, followed by a biotinylated anti-
human IgG at 1:500 (Jackson ImmunoResearch, West Grove, PA) and strepta-
vidin-alkaline phosphatase with Fast Red chromogen.
Immunohistochemical detection of PrPres using diaminobenzidine (DAB)
chromogen (DAB Map kit; Ventana) was done on selected slides at later times
in the course of the experiments as follows. Antigen retrieval and staining were
performed using a Ventana automated Discovery XT stainer. PrPres antigens
were exposed by incubation in CC1 buffer (Ventana) containing Tris-borate-
EDTA, pH 8.0, for 188 min at 95°C. Staining for PrP was done using human
anti-mouse PrP antibody D13 at a dilution of 1:500 (InPro Biotechnology) at
37°C for 2 h, followed by a biotinylated anti-human IgG at 1:500 (Jackson
ImmunoResearch) and avidin-horseradish peroxidase with DAB as chromogen.
The staining for PrPres was observed using an Olympus BX51 microscope using
Microsuite FIVE software.
Immunofluorescence microscopy. For immunofluorescence staining for PrPres
and astroglia or PrPres and microglia, tissue sections were pretreated in citrate
buffer, pH 6.0, and heated to 120°C at 20 lb/in2for 20 min for PrPres antigen
retrieval. Staining was performed using D13 human anti-mouse PrP (1:500),
rabbit anti-glial fibrillary acidic protein ([GFAP] 1:1,000; Dako, Carpinteria,
CA), and rabbit anti-Iba1 (1:1,000; Wako, Richmond, VA). D13 plus either
anti-GFAP or anti-Iba1 antibodies was coincubated for 2 h at room temperature,
followed by coincubation with Alexa-Fluor 568-labeled anti-human IgG plus
Alexa-Fluor 488-labeled anti-rabbit IgG (Invitrogen, Carlsbad, CA) for 30 min at
room temperature in darkness. Both secondary antibodies were used at a 1:200
dilution. After samples were rinsed with double-distilled H2O (ddH2O), cover-
slips were applied to tissue sections with ProLong Gold antifade reagent with
4?,6?-diamidino-2-phenylindole (DAPI; Invitrogen) to stain nuclei and protect
fluorescence from fading. The staining for PrPres, astroglia, and microglia was
observed using an Olympus BX51 microscope using Microsuite FIVE software.
Slow neural spread after intraneural scrapie inoculation. In
mice and hamsters, i.n. inoculation in the sciatic nerve is
known to be an efficient extracerebral route of scrapie infec-
tion, which results in rapid neuroinvasion and clinical scrapie
(2, 6, 40). In agreement with these results, we found similar
rapid development of clinical brain disease after both i.c. and
i.n. inoculation in C57BL/10 mice expressing anchored PrP;
VOL. 85, 2011PRION NEUROINVASION1485
however, after perineural inoculation clinical disease was sig-
nificantly delayed (Table 1). To test whether PrP anchoring
had a role in neural spread and neuroinvasion, we compared
i.c. and i.n. routes in homozygous anchorless PrP mice, line
tg44?/?(17). In these transgenic mice the two routes were
quite different. After i.c. inoculation PrPres amyloid deposition
was detected in the brain starting at 146 dpi (Fig. 1A), and
clinical neurological disease was seen at 298 to 321 dpi (Fig.
1A). In contrast, after i.n. inoculation, mice had no PrPres
amyloid in brain or clinical brain disease even after 600 dpi
(Fig. 1A). However, i.n. inoculation resulted in some neural
spread and accumulation as PrPres was detected by immuno-
blotting (Fig. 1B) and by IHC (Fig. 1C to F) in spinal cord and
sciatic nerve and nerve roots on the ipsilateral (inoculated)
side at late times postinfection. Furthermore, a higher fre-
quency of PrPres detection was found in nerve and lumbar
spinal cord than in thoracic and cervical spinal cord regions,
which suggested slow progression of PrPres up the nerve and
the spinal cord (Table 2). In addition, between 500 and 600
dpi, clinical signs of rear leg weakness and/or paralysis were
noted on the ipsilateral side, indicating that significant damage
might have been caused by the local PrPres deposition in these
nerves or in lumbar spinal cord (Fig. 1C to F). However, the
lack of PrPres in brain and the small amount in upper spinal
cord following i.n. inoculation, in spite of extensive PrPres
deposition in the sciatic nerve, indicated that neural spread of
PrPres was slow in tg44?/?mice. Therefore, GPI-anchored
PrP was associated with rapid neural spread and brain infec-
tion following scrapie inoculation via sciatic nerve, whereas
anchorless PrP was not.
PrPres deposition in nerves and muscles of the tongue. We
next tested tongue inoculation as this extraneural route is known
to result in CNS neuroinvasion via the hypoglossal nerve in ani-
mals expressing anchored PrP (4, 5, 7, 50). In C57BL/10 mice we
also found 100% incidence of clinical scrapie after inoculation
via the i.t. route (Table 1). In contrast, in tg44?/?mice, i.t.
inoculation resulted in deposition of PrPres in tongue muscle
and nerves (Fig. 2A and B). However, no PrPres was found in
brain (Fig. 2C), and no clinical signs were observed in any of
the six mice analyzed from 354 to 600 dpi. The absence of CNS
neuroinvasion, together with the presence of PrPres in nerve
branches in the tongue, suggested that spread of infectivity by
cranial nerves was very inefficient in tg44?/?mice compared to
spread in C57BL/10 mice. This conclusion was in agreement
with results from sciatic nerve inoculation.
Infection of retina and brain after ocular scrapie inocula-
tion. Ocular (i.o.) scrapie inoculation was also tested as this
route is known to produce significant local infection as well as
CNS neuroinvasion via the optic nerve and optic tract in mice
expressing anchored PrP (22, 24, 42, 57). In C57BL/10 mice we
found 100% incidence of clinical scrapie and abundant PrPres
in brain at 231 ? 32 days after inoculation via the i.o. route
(Table 1). In contrast, in tg44?/?mice, after i.o. inoculation
PrPres was not detected in brain until 357 dpi. PrPres amyloid
plaques were found in the brains of 8 of 11 tg44?/?mice
between 357 and 604 dpi (Fig. 3A and B). Two of the eight
positive mice also had clinical signs of CNS disease (Fig. 3A).
Interestingly, the majority of PrPres plaques in brain were not
located in visual areas but were in cerebellum and cerebral
cortex, often associated with the pia mater of the meninges and
adjacent leptomeningeal blood vessels entering the brain (Fig.
3C and D).
After i.o. inoculation of tg44?/?mice, large perivascular
PrPres amyloid deposits were found in the inoculated eye by
immunohistochemistry at 232 dpi (Fig. 4A and C). The most
extensive PrPres accumulation was found surrounding blood
vessels in the retinal ganglion layer (Fig. 4A, B, F, and G). At
the site where the optic nerve enters the retina, PrPres depo-
sition was readily detected in optic nerve at 232 dpi (Fig. 4C to
E) and was extracellular but closely associated with GFAP-
expressing astrocytes (Fig. 4E and F) and Iba1-positive acti-
vated microglia and/or macrophages with large plump cell bod-
ies and thickened processes (Fig. 4G). PrPres was also found in
close association with the pia mater in optic nerve (Fig. 4D and
E). From this location PrPres might spread from the pia to the
cerebrospinal fluid (CSF) in the adjacent subarachnoid space
and subsequently via CSF to cortical and cerebellar meninges
(Fig. 3C and D). The relatively high incidence of neuroinvasion
in i.o. inoculated tg44?/?mice indicated that spread of infec-
tivity from retina to brain was more efficient than spread from
sciatic nerve or tongue inoculation sites, but the progression
was still very slow compared to that in mice with anchored PrP
Limited brain infection after intravenous or intraperitoneal
scrapie inoculation. In a previous study, i.c. and i.p. inoculated
heterozygous tg44?/?mice had readily detectable levels of
infectivity in blood plasma (54). This finding together with the
prominent perivascular distribution of PrPres in brain after i.c.
inoculation (17, 18) prompted us to investigate the possibility
of hematogenous neuroinvasion in tg44?/?mice. In C57BL/10
mice, i.v. and i.p. scrapie inoculation caused 100% incidence of
brain disease at 203 ? 6 and 193 ? 5 dpi, respectively (Table
1), and accumulation of high levels of PrPres was detected
in brain at similar times (Fig. 5B, lanes 2 and 3). However,
perivascular PrPres accumulation was not a prominent feature
in C57BL/10 mice. Therefore, there was no obvious morpho-
logical evidence for hematogenous neuroinvasion in C57BL/10
mice. Furthermore, previous experiments by several laborato-
ries using mice expressing anchored PrP suggested that neu-
roinvasion after either i.v. or i.p. inoculation proceeds by am-
plification in lymphoid tissues, followed by spread to the CNS
TABLE 1. Influence of inoculation route on incidence and
incubation period of scrapie disease in C57BL/10 mice
i.v. ? i.c. stab
154 ? 6
156 ? 2
233 ? 7
198 ? 12
231 ? 32
193 ? 5
203 ? 6
190 ? 6
aThe ID50dose of RML scrapie inoculated via the different routes is de-
scribed in Materials and Methods. i.c., intracerebral; i.n., intraneural; p.n., per-
ineural; i.t., intratongue; i.o., intraocular; i.p., intraperitoneal; i.v., intravenous.
bNumber of days postinoculation when mice were euthanized due to clinical
scrapie. Values are means ? 1 standard deviation.
cNumber of mice positive for clinical scrapie/total number of mice in group.
1486 KLINGEBORN ET AL.J. VIROL.
by peripheral nerves rather than by hematogenous neuroinva-
sion (8, 12, 29, 30, 43, 47, 49, 53).
In contrast to mice with anchored PrP, tg44?/?mice inoc-
ulated by i.v. or i.p. routes had no brain PrPres until after 384
dpi (Fig. 5A). However, by 600 dpi, 3 of 12 i.p. inoculated mice
and 3 of 11 i.v. inoculated mice had PrPres detectable by
immunoblotting in brain (Fig. 5A). Two of these i.v. inoculated
mice had clinical signs at late times (Fig. 5A). Representative
immunoblots are shown in Fig. 5B. By immunohistochemistry,
PrPres was found primarily adjacent to meninges of cerebel-
FIG. 1. PrPres deposition in sciatic nerve and CNS following intracerebral and sciatic nerve scrapie inoculation of tg44?/?mice. (A) Relative
levels of PrPres deposition detected by immunoblotting in brain of tg44?/?mice after scrapie inoculation via intracerebral (i.c.), intraneural (i.n.),
and perineural (p.n.) routes. Solid symbols represent mice with clinical neurological signs as previously described (17). Relative amount of brain
PrPres detected by immunoblotting is defined in Materials and Methods. (B) Immunoblot of PrPres in sciatic nerve and CNS following i.c. (lanes
1 to 4) and i.n. (lanes 5 to 10) scrapie inoculation. Brain samples from clinical C57BL/10 mice (WT) inoculated via indicated routes are in lanes
1 and 5. Tissues from an i.c. inoculated tg44?/?mouse are in lanes 2 to 4 (308 dpi). Brain and/or lumbar spinal cord samples from two i.n.
inoculated tg44?/?mice are in lanes 6 and 7 (600 dpi) and lane 8 (600 dpi). Ipsilateral sciatic nerves are shown for two i.n. inoculated tg44?/?mice
in lanes 9 and 10 (both 600 dpi). Lane 8 was overexposed to show PrPres in lumbar spinal cord. Lanes 1, 2, and 5 were loaded with 0.25 mg, and
all other lanes were loaded with a 1-mg tissue equivalent. Approximate molecular sizes of 18 and 22 kDa are indicated. (C to F) Immunohisto-
chemical detection of PrPres deposits in sciatic nerve and lumbar spinal cord following sciatic nerve scrapie inoculation in tg44?/?mice.
(C) Extensive PrPres deposition was found in the ipsilateral sciatic nerve at 454 dpi. (D) Numerous amyloid plaques (arrows) seen by hematoxylin
and eosin staining of the sciatic nerve seen in panel C. (E) PrPres deposition was found in nerve roots (N) and white matter tracts (WM) but only
rarely in gray matter tracts (GM) on the ventral aspect of the lumbar spinal cord at 600 dpi. (F) The boxed area in panel E magnified. Arrows show
blood vessels surrounded by PrPres. Bars, 100 ?m (C, D, and E) and 50 ?m (F).
VOL. 85, 2011 PRION NEUROINVASION1487
lum and cerebrum (Fig. 5C and D) and in rare cases was also
seen in the choroid plexus (Fig. 5C). This was similar to the
meningeal PrPres seen after i.o. inoculation (Fig. 3C and D).
The low incidence and slow tempo of CNS infection after i.v.
and i.p. scrapie inoculation in mice expressing anchorless PrP
compared to mice expressing anchored PrP supported our
previous conclusions that anchored PrP had an important in-
fluence on neuroinvasion after peripheral prion inoculation.
To investigate the role of the blood-brain barrier (BBB) in
hematogenous spread of scrapie to the brain, we mechanically
disturbed the BBB at the time of i.v. inoculation. Mice were
inoculated by the i.v. route with scrapie, and within 2 min
thereafter a needle stab to the brain was made with a clean
needle (23, 39). In C57BL/10 mice the mean incubation period
was 190 dpi after i.v. inoculation with the i.c. stab compared to
203 dpi without the i.c. stab, which was a small but statistically
significant difference (Mann-Whitney; 95% confidence interval
[CI], P ? 0.0024). In tg44?/?mice, the combined i.v. and stab
(i.v.?stab) method of infection compared to i.v. inoculation
alone increased the incidence of neuroinvasion (Fig. 6A). Fur-
thermore, PrPres deposition was clearly evident in the brain
along the i.c. stab needle track at 225 dpi (Fig. 6B and C),
suggesting that scrapie invasion of the brain occurred via leak-
age from blood into the stab wound. Thus, these experiments
supported the conclusion that the BBB was an obstacle to early
neuroinvasion by blood infectivity in both C57BL/10 mice and
Spread of PrPres to extraneural tissues after peripheral
infection routes in tg44?/?mice. In several previous studies
after either i.c. or i.p. scrapie infection of heterozygous tg44?/?
mice, extensive prion infectivity and/or PrPres deposition was
detectable by immunoblotting or IHC at extraneural sites such
as lymphoid tissues, brown and white fat, heart, tongue, skel-
etal muscle, and plasma (18, 54, 60). In the present studies
after inoculation of tg44?/?mice by five peripheral routes
described above, brown fat had prominent PrPres detectable
starting at 146 to 230 dpi (Table 3). PrPres was also found in
heart, spleen, white fat, and colon (lamina propria) at times
prior to detection in brain. Thus, in the absence of PrP an-
choring, spread of PrPres to extraneural sites was rapid, in
marked contrast to the slow and inefficient spread to the CNS
found in these same mice. Therefore, spread to these different
locations appeared to be mediated by distinct mechanisms.
Most cases of natural transmission of prion infection are
believed to occur at peripheral sites, followed by spread to the
CNS. In the present study we investigated the role of PrP
membrane anchoring in scrapie neuroinvasion using periph-
eral sites of infection which mimic various types of natural or
iatrogenic infection. Five different routes of scrapie inoculation
outside the brain, i.n., i.t., i.o., i.p., and i.v., were used in
C57BL/10 mice expressing anchored PrP and in tg44?/?trans-
genic mice that express only PrP lacking the GPI anchor. In all
FIG. 2. (A) Immunohistochemical detection of PrPres deposits in
tongue nerves at 600 days following scrapie inoculation in the tongue
of a tg44?/?mouse. Arrows indicate PrPres deposits in close associa-
tion with neurolemma. (B) Widespread PrPres accumulation mostly
along capillaries between muscle cells in tongue of mouse from panel
A. (C) Sagittal section of medullar area of brain stem of the same
mouse showing no detectable PrPres. Cerebellum and fourth ventricle
are visible in the upper left corner. Inset shows higher magnification of
boxed area. Light brown diffuse staining is PrPsen, which is also seen
in uninoculated animals (data not shown), and is easily distinguished
from PrPres (panels A and B) in tg44?/?mice. Bars, 50 ?m (A, B, and
inset in C) and 500 ?m (C).
TABLE 2. PrPres deposition in brain, three levels of spinal cord,
and ipsilateral sciatic nerve following sciatic nerve scrapie
inoculation of tg44?/?micea
Frequency of PrPres deposition (no. of positive
samples/total no. of samples tested) at:
155–230 dpi349–454 dpi 544–600 dpi
aMice were inoculated in sciatic nerve with RML scrapie at 4 ? 105ID50.
bAt 15 dpi, sciatic nerve near the inoculation site was negative for PrPres by
immunoblotting and IHC (data not shown), suggesting that PrPres detected at
later times was newly generated.
1488 KLINGEBORN ET AL.J. VIROL.
of these routes, neuroinvasion was severely affected by the lack
of anchored PrP. Strikingly, in tg44?/?mice, three routes (i.n.,
i.t., and i.o.) resulted in no or slow neuroinvasion of the brain
(Fig. 1A, 2C, and 3A), whereas these same routes gave efficient
neuroinvasion via neural spread in mice with anchored PrP
(Table 1) (5, 22, 40). However, the poor neuroinvasion of brain
after i.n., i.t., and i.o. inoculation in tg44?/?mice was not due
to a lack of PrPres generation in nerves as PrPres could be
detected in sciatic nerve, branches of the hypoglossal nerve in
tongue, and optic nerve at later times after inoculation. Thus,
a lack of anchored PrP appeared to affect the speed and effi-
ciency of neuroinvasion via nerves in tg44?/?mice but did not
block accumulation of PrPres in peripheral nerves.
Another possible interpretation of these findings is that
presence of anchorless PrP, rather than absence of anchored
PrP, might alter neuroinvasion. However, in our previous ex-
periments using mice coexpressing anchored and anchorless
PrP, i.p. scrapie inoculation resulted in CNS neuroinvasion in
100% of mice between 275 and 357 dpi (18; also and B. Race
and B. Chesebro, unpublished data). Therefore, lack of neu-
roinvasion after peripheral scrapie inoculation in tg44?/?mice
appears to depend more on the absence of anchored PrP
rather than on the presence of anchorless PrP.
Two other caveats might also influence the interpretation
of our results. First, peripheral nerves in mice expressing no
PrPsen (Prnp?/?mice) or mice expressing only anchorless
PrPsen (heterozygous tg44?/?mice) were recently found to
develop abnormal axonal morphology at around 60 weeks of
age (11), which might influence spread of PrPres. However,
this axonal problem was not detected in younger mice such as
those used for our infections and thus would not be expected
to influence the tempo of neuroinvasion in our experiments.
Second, PrPsen expression levels in tg44?/?mice are 8-fold
lower than in C57BL/10 mice (17), and this lower expression
level might also influence the rate of neuroinvasion by neural
and other peripheral routes tested here. However, in other
transgenic mice expressing anchored PrP at 10-fold lower lev-
els than C57BL/10, we previously found that i.p. scrapie infec-
tion gave 90 to 100% incidence of neuroinvasion and clinical
scrapie with incubation periods somewhat longer than the i.c.
FIG. 3. PrPres deposition in brain and eyes following ocular scrapie inoculation in tg44?/?mice. (A) Relative amount of PrPres deposition
detected in brain by immunoblotting at various times after inoculation. Solid symbols represent mice with clinical neurological signs. (B) Immu-
noblot of PrPres in brain and eyes following ocular scrapie inoculation. High levels of PrPres can be seen at terminal stage of disease in brains (Br)
of an i.o. inoculated C57BL/10 (WT) mouse (lane 1), an i.c. inoculated tg44?/?mouse at 308 dpi (lane 2), and an i.o. inoculated tg44?/?mouse
at 469 dpi (lane 3), whereas no PrPres was detected in a 520-day-old uninoculated (un) tg44?/?mouse (lane 4). In lanes 5 to 10, brains and eyes
from two other i.o. inoculated tg44?/?mice are shown (animal 286, 441 dpi; animal 291, 435 dpi). Ipsilateral (I) eyes (lanes 7 and 10) have high
levels of PrPres, while the contralateral (C) eyes (lanes 6 and 9) have undetectable levels of PrPres. Brains had either moderate (lane 8) or low
(lane 5) levels of PrPres. Lanes 1 to 3 were loaded with 0.25 mg, and all other lanes were loaded with a 1-mg tissue equivalent. Approximate
molecular sizes of 18 and 22 kDa are indicated by the two bars on the right hand side. (C) Immunohistochemical staining using monoclonal
antibody D13 shows PrPres amyloid plaque deposition in a sagittal section of the cerebellum of a tg44?/?mouse at 357 days after ocular scrapie
inoculation. (D) Higher magnification of panel C shows perivascular PrPres plaques in the molecular layers (M), as well as in meninges between
two lobules, around blood vessels (arrows) and in the granular layer (G) of the third cerebellar lobule. Bars, 500 ?m (C) and 100 ?m (D).
VOL. 85, 2011 PRION NEUROINVASION1489
FIG. 4. Immunohistochemical and immunofluorescence detection of PrPres deposition in eye and optic nerve at 232 dpi after ocular scrapie
inoculation in several tg44?/?mice. (A) Extensive PrPres deposition (red, Fast Red staining) in retinal ganglion layer at 232 dpi. The eye was cut
at an angle tangential to the ganglion layer, resulting in the appearance of a flap of ganglion layer extending into the vitreal space in the center
of the section. Fewer PrPres deposits are present in the inner nuclear layer (INL) and only rarely were deposits detected in the outer nuclear layer
(ONL) or other retinal cell layers outside INL. (B) At higher magnification of the boxed area in panel A, PrPres deposits are evident in the ganglion
layer, and many PrPres plaques have a distinct perivascular appearance (arrows). (C) In a different mouse at 232 dpi the section shows widespread
PrPres accumulation (brown, DAB staining) in the optic nerve entering the retina as well as in the ganglion layer of the retina and in the vitreous
fluid. (D) At higher magnification of the boxed area in panel C, PrPres can be seen in optic nerve (ON), vitreous fluid (VF), ganglion layer (G),
and inner nuclear layer (INL) and around two retinal blood vessels (arrows). PrPres plaques adjacent to the pia mater of optic nerve are indicated
1490 KLINGEBORN ET AL.J. VIROL.
route (median of 360 days versus 260 days) (35). Therefore,
lack of PrP anchoring, rather than low PrPsen levels, appears
to account for the low incidence of neuroinvasion seen after
i.p. infection in tg44?/?mice.
Slow neural spread of PrPres in tg44?/?mice was particu-
larly apparent after i.n. inoculation, where the earliest detec-
tion of PrPres in lumbar spinal cord was at 349 dpi (Table 2).
With an average distance of 20 mm from the inoculation site in
the sciatic nerve to lumbar spinal cord, the rate of neural
PrPres spread was calculated to be roughly 0.06 mm per day.
Previous studies have reported rates of spread of scrapie in-
fectivity and/or PrPres ranging from 0.5 to 3.3 mm per day in
mice and hamsters with anchored PrP (6, 28, 40, 41, 46). Thus,
the rate of neural PrPres spread in tg44?/?mice was approx-
imately 10 to 50 times lower than in mice and hamsters with
anchored PrP, demonstrating that neural PrPres spread was
severely affected in the absence of the PrP GPI anchor. In both
tg44?/?mice and mice with anchored PrP, neural spread of
by arrowheads. (E) Double immunofluorescence staining of the optic nerve of mouse shown in panels C and D at the level of entry into the retina.
PrPres deposits (red) and an extensive network of GFAP-expressing astrocytes (green) can be seen in the optic nerve. This astrocyte network was
also seen in optic nerve from uninfected tg44?/?mice (data not shown). Arrows indicate PrPres plaques in close proximity to the pia mater on
the edge of the nerve. (F) Double immunofluorescence staining of retina at high magnification shows amyloid PrPres deposits (red), mostly
perivascular and in close proximity to perivascular GFAP-expressing astrocytes (green). Close to the lower edge of the picture, the outer nuclear
layer of the retina stains strongly with the nuclear stain DAPI (blue). (G) Iba1-positive microglia or macrophages (green) were detected in close
proximity to PrPres deposits (red) in the retinal ganglion layer. Most of the Iba1-positive cells have large plump cell bodies (arrows) and thickened
processes, suggesting an activated phenotype. Bars, 500 ?m (A and C), 100 ?m (D and E), and 50 ?m (B, F, and G).
FIG. 5. (A) Relative amount of PrPres deposition detected by immunoblotting in brain of tg44?/?mice after intravenous (i.v.) and intraper-
itoneal (i.p.) scrapie inoculation. Solid symbols represent mice with clinical neurological signs. (B) Immunoblot of PrPres in brain and after i.v.
or i.p. scrapie inoculation. Brains from three C57BL/10 (WT) mice (lane 1, i.c. at 144 dpi; lane 2, i.v. at 197 dpi; lane 3, i.p. at 188 dpi) and a tg44?/?
mouse (lane 4, i.c. at 308 dpi) at terminal stage of disease are shown. Brain from i.v. inoculated tg44?/?mice at 603 dpi (lane 5), 560 dpi (lane
6), and 483 dpi (lane 7) showed various levels of PrPres. Brains of i.p. inoculated tg44?/?mice showed undetectable PrPres (lane 8, 601 dpi) or
very low PrPres (lanes 9 and 10, 601 and 580 dpi, respectively). Lanes 5 and 6 and 8 to 10 were loaded with 1.0 mg, and all other lanes were loaded
with 0.25-mg tissue equivalents. Approximate molecular masses of 18 and 22 kDa are indicated on the right-hand side. (C and D) Immunohis-
tochemical staining using monoclonal antibody D13 shows PrPres amyloid plaque deposition in brain after intraperitoneal scrapie inoculation of
tg44?/?mice. (C) PrPres plaques (red, Fast Red) were detected in the choroid plexus (arrow) and radiating out from blood vessels (arrowheads)
in the meninges of the 10th cerebellar lobule and the fissure between the 9th and 10th cerebellar lobules at 740 days after i.p. inoculation. (D) In
another mouse at 601 dpi after i.p. inoculation, PrPres deposits (brown, DAB stain) were found only along the meninges in the posterior portion
of the cerebral cortex. Bar, 200 ?m.
VOL. 85, 2011 PRION NEUROINVASION 1491
PrPres was much slower than conventional retrograde axonal
transport (i.e., 85 mm to 430 mm per day ). The very slow
neural spread in tg44?/?mice might be due to limited and slow
diffusion of both inoculated and newly formed PrPres in inter-
stitial spaces of the nerve. In contrast, in the presence of
anchored PrP, neural spread is likely to be a faster cell-asso-
ciated process. Our data show that anchored PrP is a vital part
of this process of neural transmission after infection at periph-
eral sites. The exact details of this process remain unclear (33),
but they appear to differ from the mechanisms involved in
neuroinvasion by A?-amyloid as this material showed no evi-
dence for neuroinvasion after inoculation by i.v., i.o., oral, and
intranasal routes (20). However, in a more recent report, neu-
roinvasion after i.p. inoculation of A?-amyloid was observed
Our results show that both i.v. and i.p. routes of scrapie
inoculation in tg44?/?mice resulted in slow and inefficient
neuroinvasion compared to mice expressing anchored PrP
(Fig. 5A). Previous studies have shown that PrP expression in
nerves is necessary for neuroinvasion of the CNS after both of
these routes of scrapie inoculation in mice (8, 28, 42, 55). After
both i.v. and i.p. scrapie inoculation in mice with anchored PrP,
CNS neuroinvasion is thought to occur via splanchnic nerves
innervating lymphoid tissues, where scrapie infectivity is am-
plified (3, 19, 29). Thus, the poor neuroinvasion of the CNS in
tg44?/?mice after i.v. and i.p. inoculation might be due to
poor spread of PrPres in splanchnic nerves, similar to sciatic
and cranial nerves innervating the tongue. Similar results were
seen in recent studies of G3 transgenic mice which produce
only unglycosylated PrP (15). In G3 mice PrP is GPI anchored
to membranes in the Golgi apparatus inside the cell rather
than on the plasma membrane. G3 mice are susceptible to i.c.
infection by 79A scrapie, but after i.p. inoculation they do not
develop clinical disease or PrPres in brain. These results sug-
gest that PrP must be anchored to the cell plasma membrane
for effective CNS neuroinvasion.
As has been discussed in a preceding paper, lack of PrP
anchoring in tg44?/?mice leads to extensive formation of
PrPres in an amyloid form (17). Possibly, large aggregates of
amyloid PrPres might spread from peripheral inoculation sites
to CNS less efficiently than the usual PrPres aggregates found
in typical scrapie disease in mice, sheep, and other models.
However, this conclusion seems unlikely for several reasons.
First, tg44?/?mice were inoculated with brain homogenates
from C57BL/10 mice expressing anchored PrP, and the inoc-
ulum contained little, if any, amyloid PrPres. Second, in
tg44?/?mice only CNS neuroinvasion was slow, whereas
PrPres and infectivity appeared to spread efficiently in extran-
eural tissues such as brown fat, heart, colon, and spleen (Table
3). Since plasma of infected tg44?/?mice also contained sig-
nificant amounts of infectivity (54), it is possible that extran-
eural spread in tg44?/?mice is hematogenous or lymphatic.
Interestingly, in mice with anchored PrP, there is also evi-
dence against the hematogenous route of neuroinvasion. After
i.p. and i.v scrapie inoculation, CNS neuroinvasion was not
observed in mice lacking PrP expression in nerves (8) or after
i.p. inoculation of sympathectomized mice (29). In contrast,
studies in sheep and goats (32, 58) found that initial sites of
PrPres deposition in CNS were near the circumventricular
organs (CVO) of the brain, which might indicate their role as
a possible site of entry of prions from blood. However, the
fenestrated capillaries of the CVO, which allow passage of
small peptides (up to 50 amino acids), would not be likely to
allow penetration by full-length PrPsen or larger multimers of
TABLE 3. Spread of PrPres to brain and brown fat after
inoculation of tg44?/?mice with scrapie brain
homogenate by various routes
Earliest PrPres detection (dpi) by inoculation routea
i.c.i.n. i.t.i.o.i.p. i.v. i.v. ? stab
aAs detected by immunoblotting or IHC. Inoculation details are presented in
the Materials and Methods section. See Table 1 for abbreviations of the inocu-
bPrPres was usually seen first in brown fat and subsequently in white fat, heart,
spleen, and colon.
cAfter i.t. inoculation, earliest PrPres was found in white fat.
FIG. 6. The effect of blood-brain barrier manipulation on PrPres
deposition in brain of tg44?/?mice after intravenous scrapie inocula-
tion. (A) Relative amount of PrPres detected by immunoblotting in
brains of tg44?/?mice after intravenous scrapie inoculation followed
by a mock intracerebral needle stab (i.v. ? stab) was higher and
detected earlier than after intravenous inoculation alone. Solid sym-
bols represent mice with clinical neurological signs, and open symbols
represent mice without clinical signs. (B) Immunohistochemical stain-
ing with monoclonal antibody D13 at the site of the mock needle stab
in the cerebral cortex at 225 dpi after i.v.?stab inoculation in a tg44?/?
mouse. A large PrPres plaque is evident at the dorsal surface adjacent
to the pia mater. (C) H&E staining shows disruption of several neu-
ronal cell layers along the needle track corresponding to locations of
PrPres amyloid plaques seen in panel B. Bar, 200 ?m.
1492KLINGEBORN ET AL.J. VIROL.
PrPres (52). After i.v. and i.p. inoculation of tg44?/?mice, no
PrPres deposition was found in CVO regions (data not shown).
The PrPres found in brain of the few positive i.v. or i.p. inoc-
ulated mice was mostly in close proximity to the meninges of
the cerebral cortex (Fig. 5D) and occasionally also in the cho-
roid plexus (Fig. 5C), suggesting that spread of infectivity from
blood to CSF was a possible route of neuroinvasion at very late
times after i.p. or i.v. inoculation.
Ocular inoculation of tg44?/?mice resulted in more fre-
quent CNS invasion than with the other peripheral routes
tested. Extensive PrPres was found in the retina and optic
nerve of i.o. inoculated tg44?/?mice (Fig. 4). In mice and
hamsters with anchored PrP, the optic tract is thought to be the
main route for cerebral neuroinvasion of scrapie after i.o.
inoculation (22, 24, 42, 57). However, in tg44?/?mice inocu-
lated by the i.o. route, the majority of PrPres plaques detected
at early stages of neuroinvasion were associated with meninges
and submeningeal brain parenchyma and were not associated
with the optic tract in the brain (Fig. 3C and D). This suggested
that neural spread via the optic tract was not the main route of
spread to the CNS in these mice. Alternatively, spread from
retina to brain might be via CSF, which is in the subarachnoid
space of the optic nerve and can flow between the optic nerve
and the brain (37, 38). If PrPres could cross the pial membrane
to the subarachnoid space (Fig. 4D and E, arrows), it could
enter the CSF and spread to other pial surfaces in brain.
This appeared to occur after i.o. inoculation of tg44?/?mice
as most PrPres plaques in the brain were found in close
proximity to the meninges in the cerebellum and cerebral
cortex (Fig. 3D).
In summary, the present data appeared to support the con-
clusion that the presence of GPI-anchored PrP is an important
factor in facilitating efficient CNS scrapie infection after inoc-
ulation by several different peripheral routes. Anchored PrP
might be part of the transport process of PrPres by peripheral
nerves to the CNS. However, the precise mechanism of this
process remains to be elucidated. The current experiments also
suggested that CNS neuroinvasion by the hematogenous route
is unlikely to occur at high incidence in tg44?/?mice because
after i.p. inoculation these mice have significant blood infec-
tivity (54) but only rare late neuroinvasion. Thus, the BBB
seems to be a barrier to prion infectivity in tg44?/?mice, and
this is similar to previous conclusions from studies in nontrans-
genic mice with anchored PrP (8, 29).
We thank Dan Long and Lori Lubke for histological technical sup-
port and Lisa Kercher for technical assistance with intraocular scrapie
inoculations. We also thank Byron Caughey, Karin Peterson, Andrew
Timmes, and John Portis for critical evaluation of the manuscript,
Lynne Raymond for assistance in breeding the transgenic mice, and Ed
Schreckendgust for animal husbandry.
This research was supported by the intramural research program of
the National Institute of Allergy and Infectious Diseases, National
Institutes of Health.
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