Accelerated Prion Replication in, but
Prolonged Survival Times of, Prion-Infected
CXCR3 −/− Mice
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Constanze Riemer, Julia Schultz, Michael Burwinkel, Anja
Schwarz, Simon W. F. Mok, Sandra Gültner, Theresa
Bamme, Stephen Norley, Frank van Landeghem, Bao Lu,
Craig Gerard and Michael Baier
J. Virol. 2008, 82(24):12464. DOI: 10.1128/JVI.01371-08.
Published Ahead of Print 8 October 2008.
JOURNAL OF VIROLOGY, Dec. 2008, p. 12464–12471
0022-538X/08/$08.00⫹0 doi:10.1128/JVI.01371-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 24
Accelerated Prion Replication in, but Prolonged Survival Times of,
Prion-Infected CXCR3⫺/⫺ Mice䌤
Constanze Riemer,1 Julia Schultz,1 Michael Burwinkel,1 Anja Schwarz,1 Simon W. F. Mok,1
Sandra Gültner,1,2 Theresa Bamme,1 Stephen Norley,3 Frank van Landeghem,4 Bao Lu,5
Craig Gerard,5 and Michael Baier1*
Received 1 July 2008/Accepted 28 September 2008
Prion diseases have a significant inflammatory component. Glia activation, which is associated with increased production of cytokines and chemokines, may play an important role in disease development. Among
the chemokines upregulated highly and early upregulated during scrapie infections are ligands of CXCR3. To
gain more insight into the role of CXCR3 in a prion model, CXCR3-deficient (CXCR3ⴚ/ⴚ) mice were infected
intracerebrally with scrapie strain 139A and characterized in comparison to similarly infected wild-type
controls. CXCR3ⴚ/ⴚ mice showed significantly prolonged survival times of up to 30 days on average. Surprisingly, however, they displayed accelerated accumulation of misfolded proteinase K-resistant prion protein
PrPSc and 20 times higher infectious prion titers than wild-type mice at the asymptomatic stage of the disease,
indicating that these PrP isoforms may not be critical determinants of survival times. As demonstrated by
immunohistochemistry, Western blotting, and gene expression analysis, CXCR3-deficient animals develop an
excessive astrocytosis. However, microglia activation is reduced. Quantitative analysis of gliosis-associated
gene expression alterations demonstrated reduced mRNA levels for a number of proinflammatory factors in
CXCR3ⴚ/ⴚ compared to wild-type mice, indicating a weaker inflammatory response in the knockout mice.
Taken together, this murine prion model identifies CXCR3 as disease-modifying host factor and indicates that
inflammatory glial responses may act in concert with PrPSc in disease development. Moreover, the results indicate
that targeting CXCR3 for treatment of prion infections could prolong survival times, but the results also raise the
concern that impairment of microglial migration by ablation or inhibition of CXCR3 could result in increased
accumulation of misfolded PrPSc.
Transmissible spongiform encephalopathies or prion infections of the central nervous system (CNS) cause a progressive
and ultimately lethal degeneration of neuronal tissue, but the
underlying pathomechanisms are still elusive (13, 51, 57, 67).
Activation of astro- and microglia precedes neuronal death
and is a general hallmark of neurodegenerative protein misfolding diseases (44, 59, 68, 69). Glial activation in prion diseases is most likely a consequence of the accumulation of a
disease-associated isoform(s) of the prion protein (termed
PrPSc or PrPTSE). The reactive gliosis is characterized by increased expression levels of proinflammatory factors, such as
components of the complement system, acute-phase proteins,
cytokines, and chemokines (5, 15, 58, 59, 73). Many of these
factors are thought to contribute to neuronal dysfunction and
degeneration, suggesting that anti-inflammatory therapeutic
approaches may help to fight deleterious effects of the diseaseassociated gliosis. However, the actual role of prion-induced
* Corresponding author. Mailing address: Project Neurodegenerative Diseases, Robert-Koch-Institut, Nordufer 20, 13353 Berlin, Germany. Phone: 49 3045472230. Fax: 49 3045472609. E-mail: adddress:
baierm@rki.de.
䌤
Published ahead of print on 8 October 2008.
glia activation and subsequent chemokine secretion in disease
development is still far from clear.
Elevated chemokine expression levels have been observed in
numerous pathologies of the brain, ranging from viral and
bacterial infections to multiple sclerosis (35, 63, 65), Alzheimer’s disease (18, 71), and stroke (66), indicating important
roles in acute and chronic neurodegeneration (for further reviews see references 54 and 60). During prion infections of the
CNS, chemokines CCL2 (20), CCL3 (41, 58), CCL5 (37, 43),
and CCL6, CCL9, and CCL12 (73) have been found to be
upregulated. Moreover, induction of the chemokines CXCL9
(61) and CXCL10 and CXCL13 (59) is seen at the early,
asymptomatic stages of scrapie infection and is sustained at
high levels until the end, possibly indicating an involvement in
disease progression. In the periphery these chemokines are
potent chemoattractants for T and B cells, respectively (38,
64). However, a significant increase in trafficking of these cell
types into prion-infected brain tissue has never been reported.
Furthermore, upon intracerebral prion inoculation, mice deficient for T and B cells develop disease identical to wild-type
controls, suggesting that these lymphocytes, and adaptive immune responses in general, play no major role in prion pathogenesis in the CNS (36, 39).
The chemokine receptor CXCR3 is widely expressed in
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Project Neurodegenerative Diseases, Robert-Koch-Institut, Nordufer 20, 13353 Berlin, Germany1; Institute of Neuropathology,
Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12203 Berlin, Germany2; Project AIDS—Immunopathogenesis and
Vaccine Development, Robert-Koch-Institut, Nordufer 20, 13353 Berlin, Germany3; Institute of Neuropathology,
Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany4; and
Department of Medicine, Children’s Hospital and Harvard Medical School,
Boston, Massachusetts 021155
PRION INFECTION OF CXCR3⫺/⫺ MICE
VOL. 82, 2008
MATERIALS AND METHODS
Animals and scrapie infections. Generation of CXCR3⫺/⫺ mice has been
described previously (28). CXCR3⫺/⫺ mice and C57BL/6 wild-type controls,
which were outbred from CXCR⫹/⫺ crosses, were kept in the local animal
facilities. Five- to 6-week-old mice were intracerebrally (i.c.) inoculated with 20
␮l of a 10⫺3- or 10⫺4-diluted 10% brain homogenate prepared from a terminally
diseased C57BL/6 wild-type mouse infected with the scrapie strain 139A (courtesy of R. H. Kimberlin, Edinburgh, United Kingdom) as previously described
(61). Mock infections were performed using similarly diluted brain homogenates
obtained from uninfected, healthy wild-type mice. Infected animals were monitored thrice weekly for clinical signs until the symptomatic stage was reached (9)
and daily once disease onset was diagnosed. Mice were sacrificed at 125 days
postinfection (dpi) or at the terminal stage of disease, at which animals would
naturally succumb to the disease within the next 48 h. Analysis and comparisons
at the terminal stage were performed for mice, which were sacrificed on the same
day or less than 5 days apart. The animal experiments and care protocols were
approved by the institutional review committee Landesamt für Gesundheit und
Soziales (Berlin, Germany). Survival times in all groups were statistically analyzed using the unpaired t test and the log rank test.
To determine prion titers an end point titration was carried out in which serial
dilutions of a brain homogenate prepared from a terminally ill wild-type mouse
were inoculated i.c. into tga20 mice (n ⫽ 4 per dilution) (22). The 50% lethal
doses (LD50s) were calculated according to the Spearman-Kaerber method and
plotted against survival times. The resulting curve could be described by the
equation y ⫽ ⫺0.361x3 ⫹ 5.057x2 ⫺ 28.67x ⫹ 123.83, where y is the survival time
(in days postinfection) and x the log LD50 (R2 ⫽ 0.9979). Next, tga20 mice (n ⫽
4 per sample) were i.c. infected with 20 ␮l of 10⫺2-diluted 10% brain homogenates prepared from CXCR3⫺/⫺ or wild-type control mice sacrificed at 125 dpi
or at the terminal stage of the disease. Prion titers (LD50/ml of 10% brain
homogenate) in these samples were then calculated using the survival time/LD50
relationship described above (52).
Histology and immunohistochemistry. Immunohistochemistry was performed
according to previously published procedures (31, 61). Serial sagittal paraffin
sections (6 ␮m) for a minimum of three samples/group and time point were
examined histologically by hematoxylin and eosin staining for spongiform
changes (data not shown). For detection of activated astrocytes, sections were
stained for glial fibrillary acidic protein (GFAP; 1:1,000; Dako, Glostrup, Denmark). Serial sagittal cryo sections (8 ␮m) were examined for microglia activation with an anti-Mac-1 (CD11b) monoclonal antibody (1:200; kindly provided by
B. Engelhardt, Munster, Germany). The total microglia population was stained
with an antibody against ionized calcium binding adapter molecule 1 (Iba-1;
1:100; Dako, Glostrup, Denmark). Stainings were visualized using the ABC
method and diaminobenzidine or 3-amino-9-ethylcarbazol as chromogens. Differences between CXCR3⫺/⫺ and wild-type mice were evaluated by independent
scoring of tissue samples by three investigators without previous knowledge of
the group to which the mice belonged.
Paraffin-embeded tissue (PET) blot analysis. Paraffin sections of formalinfixed brain tissues were transferred onto a nitrocellulose membrane and stained
as previously described (62). The immunodetection of PrPSc was performed with
the anti-PrP antibody 6H4 (1:10,000; Prionics, Zürich, Switzerland) followed by
incubation with an alkaline phosphatase-linked anti-mouse immunoglobulin antiserum (1:2,000; Dako, Glostrup, Denmark). The final staining was performed
with nitro blue tetrazolium–5-bromo-4-chloro-3-indolyl phosphate.
Western blot analysis. For Western blot analysis of proteinase K (PK)-resistant PrPSc, 10% brain homogenates were made in homogenization puffer (phosphate-buffered saline, 0.5% Triton X-100, and 0.05% sodium dodecyl sulfate)
and subjected to PK digestion at a final concentration of 100 ␮g/ml for 1 h at
37°C (1). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
protein transfer, PrPSc bands were detected using the 6H4 antibody (1:5,000;
Prionics, Schlieren-Zurich, Switzerland). For Western blot analysis of differences
in GFAP expression, anti-GFAP antibody (1:5,000; Dako, Glostrup, Denmark)
was used. To control for amounts of loaded protein, membranes were stripped by
incubation in 0.2 M glycine–HCl (pH 2.5), 0.05% Tween 20 for 30 min at room
temperature and reprobed with anti-␤-actin antibody (1:5,000; Sigma, Hamburg,
Germany). Blots were quantified and analyzed using Quantity One software
following the manufacturer’s instructions (Bio-Rad, Munich, Germany). Immunoprecipitations with the PrPSc-specific antibody 15B3 (kindly provided by A.
Raeber, Prionics) prior to Western blot analysis were performed as previously
described (47).
Quantitative real-time PCR. Brains from scrapie-infected CXCR3⫺/⫺ and
wild-type mice as well as from mock-infected and uninfected CXCR3⫺/⫺ and
wild-type mice were removed 125 dpi and at the terminal stage of disease, frozen
in liquid nitrogen, and stored at ⫺80°C. Total RNA from whole brains was
prepared with Trizol reagent (Invitrogen, Karlsruhe, Germany) according to the
manufacturer’s protocol. After digestion with DNase I for 45 min at 37°C, total
RNA was purified with the RNeasy protect mini kit (Qiagen, Hilden, Germany)
and 1.25 ␮g was reverse transcribed using the First-Strand cDNA synthesis kit
(Amersham, Freiburg, Germany). Gene expression levels from each group of
mice were subsequently determined by real-time PCR by employing a GeneAmp
5700 sequence detection system (Perkin-Elmer, Boston, MA) and the Sybr green
PCR kit (Qiagen, Hilden Germany), following data analysis using the ⌬⌬Ct
method. To compensate for variations in amounts of input RNA and efficiencies
of reverse transcription, an endogenous housekeeping gene (glyceraldehyde-3phosphate dehydrogenase) was quantified and results were normalized to these
values. Quantification of signal was achieved by setting thresholds within the
logarithmic phase of PCR and determined by the cycle number at which this
threshold was reached (Ct). The Ct obtained for glyceraldehyde-3-phosphate
dehydrogenase was subtracted from the Ct of the target gene to yield ⌬Ct. The
increase of target gene expression level was calculated according to the formula
2(⌬Ct1 ⫺ ⌬Ct2), where ⌬Ct1 is the normalized test sample value and ⌬Ct2 the
normalized mock-infected control sample value.
Primers. The following primers were used: for GFAP (primer A, GCG GGA
GTC GGC CAG TTA CC; primer B, GAC CTC ACC ATC CC GCA TCT);
2⬘,5⬘-oligoadenylate synthetase (OAS) (primer A, GGT CTC TGA GCT TCA
AGC TGA G; primer B, TAC TGT GGA GGC AAT GGC TTC AA); Spi-2
(primer A, ATC TGC CCT GCT GTC CTC TG; primer B, GCG CTG GCA
TTT CCT GTG TA); CXCL10 (primer A, GCA ACT GCA TCC ATA TCG
ATG AC; primer B, TGT GCG TGG CTT CAC TCC A); CCL2 (primer A,
CCC CAC TCA CCT GCT GCT AC; primer B, ACG GGT CAA CTT CAC
ATT CA); CCL3 (primer A, ACT GCC CTT GCT GTT CTT CT; primer B,
CTG CCG GTT TCT CTT AGT CA); CCL5 (primer A, TGC CCA CGT CAA
GGA GTA TT; primer B, CAG GAC CGG AGT GGG AGT A).
RESULTS
Accelerated accumulation of PrPSc and infectivity but prolonged survival times in the absence of CXCR3. To assess the
role of CXCR3 in a prion disease of the CNS, intracerebral
scrapie infection of mice deficient for CXCR3 was compared
to that in similarly infected wild-type mice. CXCR3⫺/⫺ mice
developed typical scrapie symptoms, including a characteristic
reduction in mobility with progressive ataxia, progressive proprioceptive deficits, poor coat condition, and weight loss (61).
The duration of this clinically overt symptomatic stage was
similar to that in wild-type controls, but the onset of this stage
was delayed (data not shown). CXCR3⫺/⫺ mice survived prion
infections on average 20 to 30 days longer than control mice (P ⬍
0.001) (Fig. 1).
The characteristic accumulation of PK-resistant PrPSc in
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brain tissue and has been found on astrocytes (3, 24), microglia
(3), neurons (72), and oligodendrocytes (49). Established
CXCR3 ligands are CXCL9, CXCL10, and CXCL11 (30). Further chemokines, namely, CCL21 and CXCL13, which are
regular ligands of receptors CCR7 and CXCR5, respectively,
were suggested to recognize CXCR3 as well (33, 56). In vitro
and in vivo disease models have shown that astrocytes, microglia, and neurons may produce the various CXCR3 ligands.
The wide range of cells expressing CXCR3 and/or ligands
thereof points toward a complex function of this system in
glial-glial and glial-neuronal interactions within the CNS. So
far, CXCR3 has been shown in vitro and in vivo to govern
migration but not proliferation of microglia (4, 24, 55, 56).
We characterized the prion infection of CXCR3⫺/⫺ mice in
comparison to wild-type controls to determine the consequences of the impairment of microglial migration on disease
development.
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scrapie infections of infected CXCR3⫺/⫺ and control mice was
analyzed by Western and PET blotting. For both methods,
CXCR3⫺/⫺ mice showed strongly increased PrPSc accumulation at 125 dpi, which appeared, however, to be equivalent at
the terminal stage (Fig. 2 and 3). To assess PrPSc deposition
and distribution, we examined scrapie-infected brains by PET
blot analysis. Deposition started in the medulla oblongata and
thalamus, followed by the hippocampus and cortex. Finally,
typically diffuse PrPSc accumulations appeared in all brain areas, but staining was most intense in the cortex, hippocampus,
and thalamus (Fig. 3). Overall, principal distribution and
spread of PrPSc deposition was similar for both groups. To
address the question of whether the more-rapid accumulation
of PrPSc in receptor-deficient mice correlates with accumulation of prion infectivity, dilutions of 10% brain homogenates
obtained from scrapie-infected CXCR3⫺/⫺ and control mice
were inoculated into tga20 indicator mice. tga20 mice receiving
brain homogenates from control animals obtained at 125 dpi
showed a mean survival time of 117 days. In contrast, tga20
mice exposed to brain homogenates from CXCR3⫺/⫺ mice
from the same stage (125 dpi) survived on average only 89 days
(Table 1). This difference of 28 days corresponds to an at least
20 times higher titer of prion infectivity in the CXCR3⫺/⫺ mice
at 125 dpi and is in agreement with the increased PrPSc accumulation at this time point (Fig. 2 and 3; Table 1). tga20 mice
infected with brain homogenate from terminally ill knockout
and control mice showed no significant difference in survival
times (Table 1). tga20 mice inoculated with brain material
from healthy mock-infected mice never developed disease.
Massive astrocytosis but reduced microglia activation in
CXCR3ⴚ/ⴚ mice. To evaluate differences in the astrocytosis
between CXCR3-deficient and wild-type mice, GFAP expression was determined by immunohistochemistry and Western
blot analysis. CXCR3⫺/⫺ mice showed two- to fourfold-increased GFAP expression in immunohistochemistry at the terminal stage of disease (Fig. 4A to F and I). At the asymptomatic stage (125 dpi), GFAP expression was found to be similar
for CXCR3⫺/⫺ and wild-type animals in the cerebellum, hippocampus, and cortex. However, in close correlation with brain
areas of early PrPSc deposition, activation of astrocytes in
CXCR3⫺/⫺ mice was more pronounced in the medulla oblongata and thalamus (data not shown). Elevated GFAP expression in CXCR3⫺/⫺ mice was confirmed by Western blot analysis,
which showed, in agreement with the immunohistochemistry results, clear differences at the early time point and a sixfold augmentation at the terminal stage of the disease compared to the
wild-type controls (Fig. 4G and H).
At the asymptomatic stage Mac-1-positive activated microglia were found in the midbrain, hippocampus, and striatum of
infected control mice. CXCR3-deficient mice showed comparable Mac-1 staining patterns in the midbrain and striatum,
whereas in the hippocampus numbers of Mac-1-expressing
cells were clearly reduced in comparison to wild-type mice
FIG. 2. Western blot analysis of PrPSc accumulation in control and CXCR3⫺/⫺ mice following i.c. inoculation. (A, upper half) Aliquots of 10%
(wt/vol) brain homogenates obtained at 125 dpi or from terminally ill animals (two mice per time point and group) were treated with PK followed
by Western blotting with the anti-PrP antibody 6H4 and enhanced chemiluminescence. (A, lower half) The same homogenates probed with
antibody 6H4 before PK digestion for detection of total PrP. (B) Densitometric quantitation of PrPSc protein levels shown in panel A.
(C) Detection of total PrPSc using the 15B3 antibody for immunoprecipitation.
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FIG. 1. Significantly prolonged survival times of scrapie-infected
CXCR3⫺/⫺ mice compared to wild-type controls. CXCR3⫺/⫺ and
wild-type-mice survived i.c. prion inoculations of a 10⫺3-diluted brain
homogenate for an average of 215.7 (n ⫽ 19) and 185.3 (n ⫽ 18) days,
respectively (P ⬍ 0.0001) and inoculations of a 10⫺4 dilution for 222.8
(n ⫽ 8) and 203.7 (n ⫽ 15) days (P ⬍ 0.01).
J. VIROL.
PRION INFECTION OF CXCR3⫺/⫺ MICE
VOL. 82, 2008
12467
(data not shown). At the terminal stage controls showed up to
threefold-increased Mac-1-positive cells in the whole brain,
particularly in the hippocampus, striatum, and cortex, with
strongest staining in the cerebellum and thalamus (Fig. 5).
Another characteristic alteration in scrapie infection is the
spongiform vacuolization of brain tissue. To confirm microvacuolization, brain sections of infected animals were subjected to hematoxylin and eosin staining. Interestingly, although there were obvious differences in survival time, PrPSc
accumulation, astrocytosis, and microgliosis, no differences in
the amount or distribution of vacuolization were found between CXCR3⫺/⫺ and wild-type mice at any time point (data
not shown).
For further analysis of disease-associated gliosis and its consequences, quantitative real-time PCR was performed to determine (inflammatory) marker gene mRNA levels in brains
from prion-infected CXCR3⫺/⫺ and wild-type mice as well as
in mock-infected controls (Fig. 6).
As expected, the strongly enhanced astrogliosis in
CXCR3⫺/⫺ mice was also reflected in 30- to 140-fold-elevated
GFAP transcript levels. However, expression levels of all other
markers analyzed were found to be reduced in brain tissue of
CXCR3⫺/⫺ mice at the asymptomatic stage (CXCL10, CCL5,
and OAS) or even at both time points studied (CCL2, CCL3,
and serine protease inhibitor 2 [Spi-2]), indicating a weaker
inflammatory response in these animals.
Expression levels of these marker genes as well as prion
protein and GFAP expression levels were similar for uninTABLE 1. Determination of prion titers in wild-type and CXCR3⫺/⫺
brain tissue in recipient tga20 mice
Donor
genotype
Time after
inoculation of
donor (dpi)
Survival of
recipient tga20
mice (dpi)
Titer
(LD50/ml of 10%
brain homogenate)
Wild type
Wild type
CXCR3⫺/⫺
CXCR3⫺/⫺
125
Terminal
125
Terminal
117
85
89
83
9.0 ⫾ 103
4.2 ⫾ 105
2.1 ⫾ 105
5.8 ⫾ 105
fected wild-type and CXCR3⫺/⫺ mice (data not shown), demonstrating that ablation of CXCR3 per se does not cause a
gliosis or a general imbalance of brain homeostasis, which is in
agreement with a previous study with CXCR3⫺/⫺ mice (55).
DISCUSSION
Overexpression of chemokines is one of the hallmarks of
reactive gliosis in neurodegenerative diseases, including prion
diseases. However, our knowledge concerning the contributions of such inflammatory reactions to prion diseases of the
CNS is still very limited. We therefore characterized the
scrapie infection of mice deficient for CXCR3 to learn more
about a possible role of this chemokine receptor in chronic
neurodegeneration.
Directed migration of microglia along concentration gradients of chemoattractants secreted by astrocytes or neurons is
likely to be a prerequisite for establishing cell-cell interactions
to promote further microglial activation and differentiation in
vivo. Impaired migration toward stressed or injured neurons
reduced the activation of CXCR3⫺/⫺ microglia in an entorhinal cortex lesion model of acute neurodegeneration (55). However, although CXCR3 is required for microglial migration
within brain tissue, it is eventually not critical for recruitment
of peripheral macrophages into the CNS. It has been suggested
that in the terminal stage of prion infections more than 50% of
the Iba-1-positive microglial population is derived from peripheral macrophages which have migrated into the brain (50).
When staining microglia for marker protein Iba-1 expression
we found, however, no differences concerning Iba-1 expression
of microglia between CXCR3⫺/⫺ and control mice (data not
shown). CXCR3 seems therefore not to be involved in directing monocyte migration across the blood-brain barrier, which
is in agreement with a recent study of CXCR3⫺/⫺ mice in an
experimental autoimmune encephalomyelitis model (40).
Microglia activation was clearly reduced in prion-infected
CXCR3⫺/⫺ mice in comparison to wild-type controls (Fig. 5).
The attenuated inflammatory response to the prion infection
in CXCR3⫺/⫺ mice was further reflected by lower mRNA
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FIG. 3. PET blot analysis of PrPSc accumulation. Paraffin sections from wild-type control animals and CXCR3⫺/⫺ mice obtained at 125 dpi, at
the terminal stage of disease, and from a mock-infected control.
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J. VIROL.
expression levels of proinflammatory factors, which have already been shown to play a detrimental role in chronic neurodegeneration (Fig. 6). Ablation of CCL2 delayed disease onset
and prolonged survival times in scrapie-infected mice (20).
Furthermore, CCL3-deficient mice showed a substantial de-
crease of microglia-associated pathology, reduced neuronal
apoptosis, and longer life spans in a murine Sandhoff disease
model (70). Astrocytic overexpression of the human homolog
of Spi-2, ␣1-antichymotrypsin, has been described to enhance
Alzheimer-like pathology in amyloid protein precursor trans-
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FIG. 4. Detection of astrocyte marker GFAP. (A to F) Immunohistochemical staining of GFAP-positive cells. (A, C, and D) Terminal stage
of disease in wild-type mice; (B, E, and F) terminal stage in CXCR3⫺/⫺ mice. Representative sections are shown for the cerebellum (A and B),
cortex (C and E), and hippocampus (D and F). Magnification, ⫻400. (G) Semiquantitative analysis of GFAP staining intensities in various brain
regions. CB, cerebellum; MO, medulla oblongata; MB/T, midbrain/thalamus; HC, hippocampus; Str, striatum; C, cortex. ⫹, low expression level;
⫹⫹, intermediate expression; ⫹⫹⫹, high expression. (H) Western blot analysis of GFAP expression in control and CXCR3⫺/⫺ mice. Aliquots of
10% (wt/vol) brain homogenates obtained at 125 dpi or from terminally ill animals were analyzed using anti-GFAP antibody and enhanced
chemiluminescence (two mice per time point and group). To control for equal amounts of loaded protein, anti-␤-actin antibody was used to analyze
the same homogenates. (I) Densitometric quantitation of the GFAP Western blot assay.
VOL. 82, 2008
PRION INFECTION OF CXCR3⫺/⫺ MICE
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FIG. 6. Gene expression analysis by quantitative real-time PCR.
Results shown are mRNA levels at 125 dpi and the terminal stage of
the disease in C57BL/6 (gray bars) and CXCR3⫺/⫺ (black bars) mice
relative to the expression levels in uninfected control mice. Real-time
PCR results represent the analysis of three mouse brains per group.
genic mice, and ␣1-antichymotrypsin polymorphisms were suggested to modulate age of disease onset and disease duration
for Alzheimer’s patients (26, 46). OAS activates RNase L,
which is involved in apoptotic cell death via its nonspecific
rRNA-degrading activity and may therefore participate in neuronal loss in scrapie (12, 59, 75). Early-stage overexpression of
OAS was previously reported to correlate with shorter survival
times in a murine scrapie model (9).
Impaired microglial migration in CXCR3⫺/⫺ mice may well
contribute to decreased phagocytosis and consequently less
degradation of PrPSc deposits, because only migratory microglia can be expected to efficiently reach deposits of misfolded
protein for subsequent engulfment. A role of phagocytes in
uptake and degradation of PrPSc has been demonstrated in cell
cultures (10, 11, 45) and in prion-infected animals (2, 42).
Moreover, depletion of microglia from prion-infected organotypic brain slices was shown to promote dramatic increases in
PK-resistant PrPSc and prion infectivity (19). We show here
that amounts of PrPSc in the preclinical stage in brains of
CXCR3⫺/⫺ mice were significantly increased compared to the
wild-type controls and infectious prion titers were at least 20fold higher (Fig. 2 and 3 and Table 1). In combination, these
data suggest that microglia negatively affect prion replication
and PrPSc deposition in the CNS and that microglial migration
driven by CXCR3 is critical for this activity.
Because folding intermediates of PrPSc may not necessarily
be PK resistant, we used in addition the PrPSc-specific antibody
15B3 to detect PK-sensitive and -resistant forms of PrPSc si-
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FIG. 5. Immunohistochemical staining for the microglia marker Mac-1. Sections are from animals in the terminal stage of disease in controls
(A, C, and D) and CXCR3⫺/⫺ mice (B, E, and F). Representative sections are shown for the cerebellum (A and B), cortex (C and E), and
hippocampus (D and F). Magnification, ⫻400. (G) Semiquantitative analysis of Mac-1 staining intensities in various brain regions (symbols are as
described for Fig. 4).
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ACKNOWLEDGMENTS
We thank K. Krohn, S. Lichy, and E. Westhäuser for excellent
technical assistance, N. Holtkamp for helpful discussions, and A.
Raeber (Prionics) for his support.
This work was supported in part by grant 01KO0515 from the Federal Ministry for Education and Research, Germany, and by funding
under the Sixth Research Framework Programme of the European
Union, Project AntePrion (LSHB-CT-2006-019090).
REFERENCES
1. Baier, M., S. Norley, J. Schultz, M. Burwinkel, A. Schwarz, and C. Riemer.
2003. Prion diseases: infectious and lethal doses following oral challenge.
J. Gen. Virol. 84:1927–1929.
2. Beringue, V., M. Demoy, C. I. Lasmezas, B. Gouritin, C. Weingarten, J. P.
Deslys, J. P. Andreux, P. Couvreur, and D. Dormont. 2000. Role of spleen
macrophages in the clearance of scrapie agent early in pathogenesis.
J. Pathol. 190:495–502.
3. Biber, K., I. Dijkstra, C. Trebst, C. J. De Groot, R. M. Ransohoff, and H. W.
Boddeke. 2002. Functional expression of CXCR3 in cultured mouse and
human astrocytes and microglia. Neuroscience 112:487–497.
4. Biber, K., A. Sauter, N. Brouwer, S. C. Copray, and H. W. Boddeke. 2001.
Ischemia-induced neuronal expression of the microglia attracting chemokine
secondary lymphoid-tissue chemokine (SLC). Glia 34:121–133.
5. Brown, A. R., S. Rebus, C. S. McKimmie, K. Robertson, A. Williams, and
J. K. Fazakerley. 2005. Gene expression profiling of the preclinical scrapieinfected hippocampus. Biochem. Biophys. Res. Commun. 334:86–95.
6. Brown, D. R. 1999. Prion protein peptide neurotoxicity can be mediated by
astrocytes. J. Neurochem. 73:1105–1113.
7. Brown, D. R., B. Schmidt, and H. A. Kretzschmar. 1996. Role of microglia
and host prion protein in neurotoxicity of a prion protein fragment. Nature
380:345–347.
8. Burwinkel, M., C. Riemer, A. Schwarz, J. Schultz, S. Neidhold, T. Bamme,
and M. Baier. 2004. Role of cytokines and chemokines in prion infections of
the central nervous system. Int. J. Dev. Neurosci. 22:497–505.
9. Burwinkel, M., A. Schwarz, C. Riemer, J. Schultz, F. van Landeghem, and
M. Baier. 2004. Rapid disease development in scrapie-infected mice deficient for CD40 ligand. EMBO Rep. 5:527–531.
10. Carp, R. I., and S. M. Callahan. 1982. Effect of mouse peritoneal macrophages on scrapie infectivity during extended in vitro incubation. Intervirology 17:201–207.
11. Carp, R. I., and S. M. Callahan. 1981. In vitro interaction of scrapie agent
and mouse peritoneal macrophages. Intervirology 16:8–13.
12. Castelli, J. C., B. A. Hassel, K. A. Wood, X. L. Li, K. Amemiya, M. C.
Dalakas, P. F. Torrence, and R. J. Youle. 1997. A study of the interferon
antiviral mechanism: apoptosis activation by the 2-5A system. J. Exp. Med.
186:967–972.
13. Chesebro, B. 1999. Prion protein and the transmissible spongiform encephalopathy diseases. Neuron 24:503–506.
14. Cronier, S., H. Laude, and J. M. Peyrin. 2004. Prions can infect primary
cultured neurons and astrocytes and promote neuronal cell death. Proc.
Natl. Acad. Sci. USA 101:12271–12276.
15. Dandoy-Dron, F., F. Guillo, L. Benboudjema, J. P. Deslys, C. Lasmezas, D.
Dormont, M. G. Tovey, and M. Dron. 1998. Gene expression in scrapie.
Cloning of a new scrapie-responsive gene and the identification of increased
levels of seven other mRNA transcripts. J. Biol. Chem. 273:7691–7697.
16. Diedrich, J. F., P. E. Bendheim, Y. S. Kim, R. I. Carp, and A. T. Haase. 1991.
Scrapie-associated prion protein accumulates in astrocytes during scrapie
infection. Proc. Natl. Acad. Sci. USA 88:375–379.
17. Eikelenboom, P., C. Bate, W. A. Van Gool, J. J. Hoozemans, J. M. Rozemuller, R. Veerhuis, and A. Williams. 2002. Neuroinflammation in Alzheimer’s
disease and prion disease. Glia 40:232–239.
18. Eikelenboom, P., R. Veerhuis, W. Scheper, A. J. Rozemuller, W. A. van Gool,
and J. J. Hoozemans. 2006. The significance of neuroinflammation in understanding Alzheimer’s disease. J. Neural Transm. 113:1685–1695.
19. Falsig, J., C. Julius, I. Margalith, P. Schwarz, F. L. Heppner, and A. Aguzzi.
2008. A versatile prion replication assay in organotypic brain slices. Nat.
Neurosci. 11:109–117.
20. Felton, L. M., C. Cunningham, E. L. Rankine, S. Waters, D. Boche, and V. H.
Perry. 2005. MCP-1 and murine prion disease: separation of early behavioural dysfunction from overt clinical disease. Neurobiol. Dis. 20:283–295.
21. Fioriti, L., N. Angeretti, L. Colombo, A. De Luigi, A. Colombo, C. Manzoni,
M. Morbin, F. Tagliavini, M. Salmona, R. Chiesa, and G. Forloni. 2007.
Neurotoxic and gliotrophic activity of a synthetic peptide homologous to
Gerstmann-Straussler-Scheinker disease amyloid protein. J. Neurosci. 27:
1576–1583.
22. Fischer, M., T. Rulicke, A. Raeber, A. Sailer, M. Moser, B. Oesch, S. Brandner, A. Aguzzi, and C. Weissmann. 1996. Prion protein (PrP) with aminoproximal deletions restoring susceptibility of PrP knockout mice to scrapie.
EMBO J. 15:1255–1264.
23. Florio, T., M. Grimaldi, A. Scorziello, M. Salmona, O. Bugiani, F. Tagliavini,
G. Forloni, and G. Schettini. 1996. Intracellular calcium rise through L-type
calcium channels, as molecular mechanism for prion protein fragment 106126-induced astroglial proliferation. Biochem. Biophys. Res. Commun. 228:
397–405.
24. Flynn, G., S. Maru, J. Loughlin, I. A. Romero, and D. Male. 2003. Regulation
of chemokine receptor expression in human microglia and astrocytes. J. Neuroimmunol. 136:84–93.
25. Forloni, G., R. Del Bo, N. Angeretti, R. Chiesa, S. Smiroldo, R. Doni, E.
Ghibaudi, M. Salmona, M. Porro, L. Verga, et al. 1994. A neurotoxic prion
protein fragment induces rat astroglial proliferation and hypertrophy. Eur.
J. Neurosci. 6:1415–1422.
26. Gopalan, S. M., K. M. Wilczynska, B. S. Konik, L. Bryan, and T. Kordula.
2006. Astrocyte-specific expression of the ␣1-antichymotrypsin and glial
fibrillary acidic protein genes requires activator protein-1. J. Biol. Chem.
281:1956–1963.
Downloaded from http://jvi.asm.org/ on February 28, 2014 by PENN STATE UNIV
multaneously. However, as seen for PK-resistant PrPSc and
infectious prions, this assay demonstrated similarly higher levels of total PrPSc in CXCR3⫺/⫺ mice (Fig. 2). Surprisingly,
neither preclinical PrPSc accumulation nor infectious prion
titers were reflected by the survival times of CXCR3⫺/⫺ mice,
which survived on average up to 30 days longer than the wildtype controls. This observation is not necessarily in conflict
with the described direct neurotoxicity of PK-resistant PrPSc in
vitro (29) but calls into question whether this activity is a key
determinant of survival times in vivo. Similarly, the vastly
higher prion titers in brains of CXCR3⫺/⫺ mice do not argue
for a pronounced direct toxicity of infectious PrPSc conformers. Instead, these results may favor a scenario in which inflammatory glial responses act in concert with PrPSc to cause
neurodegeneration (6–8, 17).
The other major alteration in brain pathology of prioninfected CXCR3⫺/⫺ mice was the excessive increase in GFAPpositive astrocytes (Fig. 4), which may be directly linked to the
more pronounced prion accumulation in these animals. Numerous studies have demonstrated that astrocytes are host
cells for prion replication in vitro and in vivo (14, 16, 32, 34, 48,
53, 74). Moreover, in vitro astrocyte activation and proliferation were previously shown to result directly from exposure to
PrPSc (21, 23, 25). PrPSc additionally renders astrocytes more
responsive to mitogenic factors released by microglia (27, 61).
Such a scenario, in which PrPSc may promote astrocyte proliferation, which in turn supports increased prion replication, was
previously termed a “snowball effect” in a hamster prion model
(72). It is important to note in this context that CXCR3⫺/⫺
mice and wild-type mice showed a similar astrogliosis in a stab
wound model of acute neurodegeneration (55). The overshooting proliferation of astrocytes in CXCR3⫺/⫺ mice seen in our
study is therefore a specific response to the prion infection and
not an intrinsic property of these mice.
Taken together, we show here the development of a qualitatively and quantitatively different gliosis in the absence of
CXCR3, which is characterized by (i) an attenuated microglial
activation, (ii) excessive proliferation of GFAP-positive astrocytes, and (iii) reduced expression of inflammatory and potentially harmful factors. The experimental model identifies
CXCR3 and its ligands as disease-modifying host factors affecting the complex and interlinked process of inflammatory
glia activation and prion replication. Furthermore, our data
suggest that targeting CXCR3 for palliative treatment of prion
infections could prolong survival times but raise the concern
that impairment of microglial migration by ablation or inhibition of CXCR3 may result in increased accumulation of misfolded PrPSc.
J. VIROL.
VOL. 82, 2008
12471
52. Prusiner, S. B., S. P. Cochran, D. F. Groth, D. E. Downey, K. A. Bowman,
and H. M. Martinez. 1982. Measurement of the scrapie agent using an
incubation time interval assay. Ann. Neurol. 11:353–358.
53. Raeber, A. J., R. E. Race, S. Brandner, S. A. Priola, A. Sailer, R. A. Bessen,
L. Mucke, J. Manson, A. Aguzzi, M. B. Oldstone, C. Weissmann, and B.
Chesebro. 1997. Astrocyte-specific expression of hamster prion protein (PrP)
renders PrP knockout mice susceptible to hamster scrapie. EMBO J. 16:
6057–6065.
54. Ransohoff, R. M., L. Liu, and A. E. Cardona. 2007. Chemokines and chemokine receptors: multipurpose players in neuroinflammation. Int. Rev.
Neurobiol. 82:187–204.
55. Rappert, A., I. Bechmann, T. Pivneva, J. Mahlo, K. Biber, C. Nolte, A. D.
Kovac, C. Gerard, H. W. Boddeke, R. Nitsch, and H. Kettenmann. 2004.
CXCR3-dependent microglial recruitment is essential for dendrite loss after
brain lesion. J. Neurosci. 24:8500–8509.
56. Rappert, A., K. Biber, C. Nolte, M. Lipp, A. Schubel, B. Lu, N. P. Gerard, C.
Gerard, H. W. Boddeke, and H. Kettenmann. 2002. Secondary lymphoid
tissue chemokine (CCL21) activates CXCR3 to trigger a Cl⫺ current and
chemotaxis in murine microglia. J. Immunol. 168:3221–3226.
57. Rezaie, P., and P. L. Lantos. 2001. Microglia and the pathogenesis of spongiform encephalopathies. Brain Res. Brain Res. Rev. 35:55–72.
58. Riemer, C., S. Neidhold, M. Burwinkel, A. Schwarz, J. Schultz, J.
Kratzschmar, U. Monning, and M. Baier. 2004. Gene expression profiling of
scrapie-infected brain tissue. Biochem. Biophys. Res. Commun. 323:556–
564.
59. Riemer, C., I. Queck, D. Simon, R. Kurth, and M. Baier. 2000. Identification
of upregulated genes in scrapie-infected brain tissue. J. Virol. 74:10245–
10248.
60. Savarin-Vuaillat, C., and R. M. Ransohoff. 2007. Chemokines and chemokine receptors in neurological disease: raise, retain, or reduce? Neurotherapeutics 4:590–601.
61. Schultz, J., A. Schwarz, S. Neidhold, M. Burwinkel, C. Riemer, D. Simon, M.
Kopf, M. Otto, and M. Baier. 2004. Role of interleukin-1 in prion diseaseassociated astrocyte activation. Am. J. Pathol. 165:671–678.
62. Schulz-Schaeffer, W. J., S. Tschoke, N. Kranefuss, W. Drose, D. HauseReitner, A. Giese, M. H. Groschup, and H. A. Kretzschmar. 2000. The
paraffin-embedded tissue blot detects PrPSc early in the incubation time in
prion diseases. Am. J. Pathol. 156:51–56.
63. Szczucinski, A., and J. Losy. 2007. Chemokines and chemokine receptors in
multiple sclerosis. Potential targets for new therapies. Acta Neurol. Scand.
115:137–146.
64. Taub, D. D., A. R. Lloyd, K. Conlon, J. M. Wang, J. R. Ortaldo, A. Harada,
K. Matsushima, D. J. Kelvin, and J. J. Oppenheim. 1993. Recombinant
human interferon-inducible protein 10 is a chemoattractant for human
monocytes and T lymphocytes and promotes T cell adhesion to endothelial
cells. J. Exp. Med. 177:1809–1814.
65. Tsunoda, I., T. E. Lane, J. Blackett, and R. S. Fujinami. 2004. Distinct roles
for IP-10/CXCL10 in three animal models, Theiler’s virus infection, EAE,
and MHV infection, for multiple sclerosis: implication of differing roles for
IP-10. Mult. Scler. 10:26–34.
66. Wang, Q., X. N. Tang, and M. A. Yenari. 2007. The inflammatory response
in stroke. J. Neuroimmunol. 184:53–68.
67. Weissmann, C. 2004. The state of the prion. Nat. Rev. Microbiol. 2:861–871.
68. Williams, A., A. M. Van Dam, D. Ritchie, P. Eikelenboom, and H. Fraser.
1997. Immunocytochemical appearance of cytokines, prostaglandin E2 and
lipocortin-1 in the CNS during the incubation period of murine scrapie
correlates with progressive PrP accumulations. Brain Res. 754:171–180.
69. Williams, A. E., L. J. Lawson, V. H. Perry, and H. Fraser. 1994. Characterization of the microglial response in murine scrapie. Neuropathol. Appl.
Neurobiol 20:47–55.
70. Wu, Y. P., and R. L. Proia. 2004. Deletion of macrophage-inflammatory
protein 1 alpha retards neurodegeneration in Sandhoff disease mice. Proc.
Natl. Acad. Sci. USA 101:8425–8430.
71. Wyss-Coray, T. 2006. Inflammation in Alzheimer disease: driving force,
bystander or beneficial response? Nat. Med. 12:1005–1015.
72. Xia, M. Q., B. J. Bacskai, R. B. Knowles, S. X. Qin, and B. T. Hyman. 2000.
Expression of the chemokine receptor CXCR3 on neurons and the elevated
expression of its ligand IP-10 in reactive astrocytes: in vitro ERK1/2 activation and role in Alzheimer’s disease. J. Neuroimmunol. 108:227–235.
73. Xiang, W., O. Windl, G. Wunsch, M. Dugas, A. Kohlmann, N. Dierkes, I. M.
Westner, and H. A. Kretzschmar. 2004. Identification of differentially expressed genes in scrapie-infected mouse brains by using global gene expression technology. J. Virol. 78:11051–11060.
74. Ye, X., A. C. Scallet, R. J. Kascsak, and R. I. Carp. 1998. Astrocytosis and
amyloid deposition in scrapie-infected hamsters. Brain Res. 809:277–287.
75. Zhou, A., J. Paranjape, T. L. Brown, H. Nie, S. Naik, B. Dong, A. Chang, B.
Trapp, R. Fairchild, C. Colmenares, and R. H. Silverman. 1997. Interferon
action and apoptosis are defective in mice devoid of 2⬘,5⬘-oligoadenylatedependent RNase L. EMBO J. 16:6355–6363.
Downloaded from http://jvi.asm.org/ on February 28, 2014 by PENN STATE UNIV
27. Hafiz, F. B., and D. R. Brown. 2000. A model for the mechanism of astrogliosis in prion disease. Mol. Cell. Neurosci. 16:221–232.
28. Hancock, W. W., B. Lu, W. Gao, V. Csizmadia, K. Faia, J. A. King, S. T.
Smiley, M. Ling, N. P. Gerard, and C. Gerard. 2000. Requirement of the
chemokine receptor CXCR3 for acute allograft rejection. J. Exp. Med.
192:1515–1520.
29. Hetz, C., M. Russelakis-Carneiro, K. Maundrell, J. Castilla, and C. Soto.
2003. Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of
pathological prion protein. EMBO J. 22:5435–5445.
30. Horuk, R. 2001. Chemokine receptors. Cytokine Growth Factor Rev. 12:
313–335.
31. Jeffrey, M., C. M. Goodsir, A. Holliman, R. J. Higgins, M. E. Bruce, P. A.
McBride, and J. R. Fraser. 1998. Determination of the frequency and distribution of vascular and parenchymal amyloid with polyclonal and N-terminal-specific PrP antibodies in scrapie-affected sheep and mice. Vet. Rec.
142:534–537.
32. Jeffrey, M., C. M. Goodsir, R. E. Race, and B. Chesebro. 2004. Scrapiespecific neuronal lesions are independent of neuronal PrP expression. Ann.
Neurol. 55:781–792.
33. Jenh, C. H., M. A. Cox, W. Hipkin, T. Lu, C. Pugliese-Sivo, W. Gonsiorek,
C. C. Chou, S. K. Narula, and P. J. Zavodny. 2001. Human B cell-attracting
chemokine 1 (BCA-1; CXCL13) is an agonist for the human CXCR3 receptor. Cytokine 15:113–121.
34. Kercher, L., C. Favara, C. C. Chan, R. Race, and B. Chesebro. 2004. Differences in scrapie-induced pathology of the retina and brain in transgenic
mice that express hamster prion protein in neurons, astrocytes, or multiple
cell types. Am. J. Pathol. 165:2055–2067.
35. Kielian, T. 2004. Microglia and chemokines in infectious diseases of the
nervous system: views and reviews. Front. Biosci. 9:732–750.
36. Klein, M. A., R. Frigg, E. Flechsig, A. J. Raeber, U. Kalinke, H. Bluethmann,
F. Bootz, M. Suter, R. M. Zinkernagel, and A. Aguzzi. 1997. A crucial role for
B cells in neuroinvasive scrapie. Nature 390:687–690.
37. Lee, H. P., Y. C. Jun, J. K. Choi, J. I. Kim, R. I. Carp, and Y. S. Kim. 2005.
The expression of RANTES and chemokine receptors in the brains of
scrapie-infected mice. J. Neuroimmunol. 158:26–33.
38. Legler, D. F., M. Loetscher, R. S. Roos, I. Clark-Lewis, M. Baggiolini, and B.
Moser. 1998. B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/
CXCR5. J. Exp. Med. 187:655–660.
39. Lewicki, H., A. Tishon, D. Homann, H. Mazarguil, F. Laval, V. C. Asensio,
I. L. Campbell, S. DeArmond, B. Coon, C. Teng, J. E. Gairin, and M. B.
Oldstone. 2003. T cells infiltrate the brain in murine and human transmissible spongiform encephalopathies. J. Virol. 77:3799–3808.
40. Liu, L., D. Huang, M. Matsui, T. T. He, T. Hu, J. Demartino, B. Lu, C.
Gerard, and R. M. Ransohoff. 2006. Severe disease, unaltered leukocyte
migration, and reduced IFN-gamma production in CXCR3⫺/⫺ mice with
experimental autoimmune encephalomyelitis. J. Immunol. 176:4399–4409.
41. Lu, Z. Y., C. A. Baker, and L. Manuelidis. 2004. New molecular markers of
early and progressive CJD brain infection. J. Cell. Biochem. 93:644–652.
42. Maignien, T., M. Shakweh, P. Calvo, D. Marce, N. Sales, E. Fattal, J. P.
Deslys, P. Couvreur, and C. I. Lasmezas. 2005. Role of gut macrophages in
mice orally contaminated with scrapie or BSE. Int. J. Pharm. 298:293–304.
43. Marella, M., and J. Chabry. 2004. Neurons and astrocytes respond to prion
infection by inducing microglia recruitment. J. Neurosci. 24:620–627.
44. Meda, L., P. Baron, and G. Scarlato. 2001. Glial activation in Alzheimer’s
disease: the role of A␤ and its associated proteins. Neurobiol. Aging 22:885–
893.
45. Mohan, J., J. Hopkins, and N. A. Mabbott. 2005. Skin-derived dendritic cells
acquire and degrade the scrapie agent following in vitro exposure. Immunology 116:122–133.
46. Mucke, L., G. Q. Yu, L. McConlogue, E. M. Rockenstein, C. R. Abraham,
and E. Masliah. 2000. Astroglial expression of human ␣1-antichymotrypsin
enhances Alzheimer-like pathology in amyloid protein precursor transgenic
mice. Am. J. Pathol. 157:2003–2010.
47. Nazor, K. E., F. Kuhn, T. Seward, M. Green, D. Zwald, M. Purro, J. Schmid,
K. Biffiger, A. M. Power, B. Oesch, A. J. Raeber, and G. C. Telling. 2005.
Immunodetection of disease-associated mutant PrP, which accelerates disease in GSS transgenic mice. EMBO J. 24:2472–2480.
48. Ning, Z. Y., D. M. Zhao, H. X. Liu, J. M. Yang, C. X. Han, Y. L. Cui, L. P.
Meng, C. D. Wu, M. L. Liu, and T. X. Zhang. 2005. Altered expression of the
prion gene in rat astrocyte and neuron cultures treated with prion peptide
106–126. Cell. Mol. Neurobiol. 25:1171–1183.
49. Omari, K. M., G. R. John, S. C. Sealfon, and C. S. Raine. 2005. CXC
chemokine receptors on human oligodendrocytes: implications for multiple
sclerosis. Brain 128:1003–1015.
50. Priller, J., M. Prinz, M. Heikenwalder, N. Zeller, P. Schwarz, F. L. Heppner,
and A. Aguzzi. 2006. Early and rapid engraftment of bone marrow-derived
microglia in scrapie. J. Neurosci. 26:11753–11762.
51. Prusiner, S. B. 1998. Prions. Proc. Natl. Acad. Sci. USA 95:13363–13383.
PRION INFECTION OF CXCR3⫺/⫺ MICE