The Journal of Neuroscience, August 20, 2003, 23(20):7504-7509
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Anti-Inflammatory Drug Therapy Alters 
-Amyloid Processing and Deposition in an Animal Model of Alzheimer's Disease
Qiao Yan,1
Jianhua Zhang,1
Hantao Liu,1
Safura Babu-Khan,1
Robert Vassar,1
Anja Leona Biere,1
Martin Citron,1 and
Gary Landreth2
1Department of Neuroscience, Amgen Inc., Thousand
Oaks, California 91320, and 2Alzheimer Research
Laboratory, Department of Neurosciences, Case Western Reserve University,
Cleveland, Ohio 44106
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Abstract
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Alzheimer's disease (AD) is characterized by a microglial-mediated
inflammatory response elicited by extensive amyloid deposition in the brain.
Nonsteroidal anti-inflammatory drug (NSAID) treatment reduces AD risk, slows
disease progression, and reduces microglial activation; however, the basis of
these effects is unknown. We report that treatment of 11-month-old Tg2576 mice
overexpressing human amyloid precursor protein (APP) with the NSAID ibuprofen
for 16 weeks resulted in the dramatic and selective reduction of SDS-soluble
-amyloid (A)42, whereas it had smaller effects on
SDS-soluble A40 levels. Ibuprofen treatment resulted in 60%
reduction of amyloid plaque load in the cortex of these animals. In
vitro studies using APP-expressing 293 cells showed that ibuprofen
directly affected APP processing, specifically reducing the production of
A42. Ibuprofen treatment resulted in a significant reduction
in microglial activation in the Tg2576 mice, as measured by CD45 and CD11b
expression. NSAIDs activate the nuclear hormone receptor peroxisome
proliferator-activated receptor 
(PPAR); however, a potent
agonist of this receptor, pioglitazone, only modestly reduced SDS-soluble
A levels and did not affect amyloid plaque burden or microglia
activation, indicating that PPAR activation is not involved in the
A lowering effect of NSAIDs. These data show that chronic NSAID
treatment can reduce brain A levels, amyloid plaque burden, and
microglial activation in an animal model of Alzheimer's disease.
Key words: Alzheimer's disease; NSAIDs; -amyloid; PPAR; inflammation; microglia
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Introduction
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Alzheimer's disease (AD) is characterized by the deposition of fibrillar
forms of -amyloid (A) in the brain and the compaction of the
A fibrils into senile plaques
(Akiyama et al., 2000
). Several
isoforms of A are proteolytically generated when -secretases and
-secretases cleave the large amyloid precursor protein (APP). Although
A40 is the predominant cleavage product in vitro and
in vivo, the less abundant and less soluble A42,
which contains two additional C-terminal hydrophobic amino acids, is generally
considered the key pathogenic A species, and modulation of its
production may have therapeutic value (for review, see
Citron, 2002). The focal
deposits of A elicit a significant microglial-mediated inflammatory
response. Microglia associate with the fibrillar A, invest the plaque
with their processes (Stalder et al.,
2001), and undergo a phenotypic activation. Microglial activation
is accompanied by the elaboration of a wide range of proinflammatory molecules
that mediate the fulminating autoactivation of these cells and a concomitant
astrocytosis. The inflammatory response is associated with the demise of
neurons adjacent to the plaques (Kalaria,
1999). Murine models of AD that overexpress APP and develop
A plaques also exhibit proinflammatory activation of microglial cells
(Frautschy et al., 1998;
Benzing et al., 1999;
Stalder et al., 1999;
Mehlhorn et al., 2000;
Bornemann et al., 2001).
There is compelling epidemiological evidence that long-term nonsteroidal
anti-inflammatory drug (NSAID) therapy has a dramatic effect on the incidence
of AD (McGeer et al., 1996),
resulting in a reduction of risk by as much as 60-80%
(Stewart et al., 1997;
in t' Veld et al., 2001).
Moreover, NSAID treatment delays disease onset, acts to ameliorate symptomatic
severity, and slows disease progression
(Rich et al., 1995;
Stewart et al., 1997). In
humans, NSAID therapy results in a substantial reduction in the number of
microglia associated with senile plaques, supporting the view that these cells
are the targets of drug action (Mackenzie
and Munoz, 1998). These findings have stimulated substantial
interest in the biological basis of the effect of this class of drugs,
especially in view of evidence that the accepted targets of the action of
NSAIDs, the cyclooxygenases (COXs), are unlikely to mediate these effects
(Sainati et al., 2000;
Landreth and Heneka, 2001;
Tegeder et al., 2001;
Weggen et al., 2001).
Lim and colleagues (2000)
reported the provocative finding that 6 months of treatment of a transgenic
animal model of AD with the NSAID ibuprofen resulted in a significant
reduction of amyloid plaque burden and in total A peptide levels. These
studies also demonstrated that ibuprofen treatment led to a reduction of
plaque-associated microglia and a corresponding reduction in proinflammatory
cytokine levels in the brain. Other NSAIDs have recently been reported to
exhibit similar effects on amyloid pathology
(Jantzen et al., 2002).
The mechanisms through which NSAIDs act to achieve these effects are
presently unclear. In a compelling recent study, Weggen et al.
(2001) reported that a subset
of NSAIDs, including ibuprofen, acted to selectively suppress the production
of the highly amyloidogenic A42 species both in
vitro and in an acute treatment paradigm in vivo. NSAIDs also
have poorly defined effects on intracellular signaling pathways
(Tegeder et al., 2001),
including those used by cytokines (Baek et
al., 2002). A newly recognized target of NSAID action is the
nuclear receptor and transcription factor peroxisome proliferator-activated
receptor (PPAR) (Lehmann et
al., 1997; Berger and Moller,
2002). The binding of NSAIDs to PPAR results in the
inhibition of proinflammatory gene expression
(Delerive et al., 2001). We
suggested previously that NSAIDs may act to elicit the anti-inflammatory
effects in the AD brain through an ability to bind to and activate
PPAR. PPAR activation leads to inhibition of microglial-mediated
neurotoxicity and cytokine expression elicited by A fibrils both in
vitro (Combs et al., 2000)
and in vivo (Heneka et al.,
2000).
The aim of this study was to test whether the NSAID ibuprofen and the
PPAR agonist pioglitazone acted to suppress the development of amyloid
pathology and inflammatory responses in the brains of APP-expressing Tg2576
transgenic mice. We report that 4 months of ibuprofen treatment resulted in a
reduction in the plaque burden in these mice and a reduction in microglial
activation. Significantly, ibuprofen treatment selectively reduced the levels
of SDS-soluble A42 in the brains of the APP-overexpressing
mice. Pioglitazone had only modest effects on total A levels, and it did
not alter microglial activation, nor did it significantly affect amyloid
plaque burden.
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Materials and Methods
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Materials. Ibuprofen was purchased from Sigma (St. Louis, MO).
Pioglitazone HCl (ACTOS; Takeda Pharmaceuticals, Osaka, Japan) was obtained
from a local pharmacy. These compounds were formulated into standard,
color-coded, AIN-76A rodent diet by Research Diets (New Brunswick, NJ) at a
final drug concentration of 120 ppm pioglitazone and 375 ppm ibuprofen. The
dose of pioglitazone was selected on the basis of its ability to stimulate the
transcriptional activation of PPAR-responsive genes in rodents. The
dose of ibuprofen was the same as that used by Lim et al.
(2000).
Drug treatment of Tg2576 mice. APP transgenic Tg2576 mice at 11
months old were fed the drug-supplemented chow ad libitum for 16
weeks. There were five animals in each treatment group. Animals were housed
singly in individual cages, and their body weight and food consumption were
monitored weekly. There were no significant differences in the amount of chow
consumed or in weight of the mice during the experimental period, either
within or between treatment groups. The food consumption of animals in this
experiment was 
5 gm of rodent chow per day per animal, resulting in a
final dose of pioglitazone of 20 mg · kg-1 ·
d-1 and ibuprofen of 62.5 mg · kg-1 ·
d-1. At the end of the experimental period, animals were killed by
inhalation of CO2. The brain was dissected and the hemispheres
separated along the midline. Half of the brain was frozen on dry ice for
A ELISA analysis. The other hemisphere was frozen in OCT medium for
histological study.
Immunohistochemistry. Coronal sections (14 µM) were
cut on a cryostat microtome. Sections were thaw mounted onto Fisher
"plus" microscope slides, air-dried, and then stored at -20°C
until use. Sections were warmed to room temperature and fixed in 4%
paraformaldehyde/0.1 M phosphate buffer, pH 7.2, for 1 hr. The
endogenous tissue peroxidase activity was quenched by incubation with 3%
H2O2 in PBS for 20 min. For A
immunohistochemistry, sections were subsequently incubated with 88% formic
acid for 20 min to expose the A epitope. Sections were then incubated
with blocking solution (3% normal goat serum, 5% normal horse serum, 0.25%
carrageenan lambda, 0.1% Triton X-100 in PBS) for 1 hr. The primary antibodies
used were biotinylated mouse anti-human A monoclonal antibody 4G8
(Signet Pathology System, Dedham, MA) at 0.5 µg/ml, rat anti-CD45
monoclonal antibody (MCA 1388; Serotec, Raleigh, NC) at 0.2 µg/ml, rat
anti-CD11b monoclonal antibody (MAB 1387Z; Chemicon, Temecula, CA) at 5
µg/ml, biotinylated mouse anti-phosphotyrosine monoclonal antibody (B-1531;
Sigma) at 1 µg/ml, a control biotinylated mouse myeloma IgG at 1 µg/ml
(Sigma), or biotinylated lectin BS1 (L-5391; Sigma) at 1 µg/ml in the
blocking solution overnight at 4°C. The antigen was detected by secondary
antibody where needed and ABC-DAB method, as reported previously
(Yan et al., 1997). Sections
were dehydrated and coverslipped with mounting medium.
Morphological data analysis. Stained sections were examined under
a light microscope. Digital images were obtained and analyzed with Meta-Morph
software (Universal Imaging, West Chester, PA). Sections from each animal
containing cingulate cortex (between bregma 1.1 mm and -0.1 mm)
(Franklin and Paxinos, 1996)
were used for the analysis. The area of interest was manually outlined under
4x magnification. The software was programmed to measure the numbers of
plaques, the average size of plaques, and the integrated plaque-staining gray
scale. The percentage of area covered by plaques (plaque burden) was
calculated by multiplying the number of plaques with the average size of
plaques divided by the area of interest. For the analysis of microglia
activation, the digital images of CD45 stained rostral hippocampus and CD11b
stained frontal cortex were obtained under 10x magnification and
analyzed with MetaMorph software. Two sections per animal were used for this
analysis. Because the individual microglial cells were difficult to identify
with the current immunostaining protocol, the CD45 or CD11b positive stained
profiles registered by MetaMorph software were likely containing a cluster of
intermingled microglial cells. All of the quantitative morphological data were
analyzed by one-way ANOVA followed by Dunnett's t test.
Brain preparation for A ELISA. Half of the brain
was homogenized in 1 ml of PBS with 3x protease inhibitor mixture
(Boehringer Mannheim, Mannheim, Germany). For detection of total A
(soluble and insoluble), 200 µl of the above homogenates was mixed with 800
µl of 88% formic acid. Homogenates were stirred constantly overnight at
room temperature to solubilize plaques. The homogenates were centrifuged at
100,000 x g at room temperature in an ultracentrifuge. The
supernatants were collected and neutralized with 20 vol of 1 M
Tris, pH 11, and 10 vol of SuperBlock-Tris-buffered saline (SB/TBS) containing
3x protease inhibitor mixture (Boehringer Mannheim) for ELISA. For the
detection of SDS-extractable A (nonformic acid extraction) in the brain,
200 µlof the initial brain homogenate in PBS was mixed with 200 µl of 50
mM Tris, 150 mM NaCl, 5 mM EDTA, 2% SDS,
0.05% sodium azide, pH 7.5, and 1x protease inhibitor mixture
(Boehringer Mannheim). Homogenates were centrifuged at 16,000 x
g for 10 min at room temperature, and the supernatants were diluted
1:10 with SB-TBS. The assay was standardized by addition of Ab42
and A40 peptides to a nontransgenic mouse brain homogenate
processed in the same manner as the experimental samples. Sandwich ELISA
assays for A42 and A40 were performed as
described previously (Vassar et al.,
1999). The data were analyzed by one-way ANOVA and then Dunnett's
t test.
In vitro APP processing assay. Human embryonic kidney (HEK) 293
cells stably expressing full-length human APP695 were maintained in DMEM and
10% FBS. The cells were plated 24 hr before treatment at 60,000 cells per well
in 96-well plates. Ibuprofen and pioglitazone stock solutions were prepared in
DMSO and diluted in DMEM just before treatment. Cells were treated for 18-24
hr and the medium was collected. A42, A40,
and 
APPs levels were quantitated by standard sandwich ELISA as
described previously (Vassar et al.,
1999). All measurements were done in duplicate.
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Results
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The observation that chronic NSAID treatment could diminish AD risk
(Stewart et al., 1997;
in t' Veld et al., 2001) and
alter A plaque pathology in transgenic mice
(Lim et al., 2000) led us to
examine in greater detail the regulation of A metabolism and deposition
by this class of drugs. Moreover, we also wished to test whether drugs that
target the nuclear receptor PPAR could elicit similar effects.
Examination of amyloid plaque deposition in the cingulate cortex of Tg2576
mice revealed that treatment with ibuprofen reduced A deposition and
plaque burden (Fig. 1).
Quantitation of amyloid deposition demonstrated that ibuprofen treatment
resulted in a 60% (p < 0.05) decrease in the area occupied by
plaques compared with control animals (Fig.
1D). This effect was primarily a consequence of a
reduction in the number of plaques, which was diminished by 50%
(Fig. 1F). We observed
an overall reduction in the size of the individual plaques by 24% (p
< 0.01) (Fig. 1E)
in the ibuprofen-treated animals. Pioglitazone-treated animals exhibited no
significant changes in any of these parameters.
We investigated whether the drug-mediated amelioration of plaque pathology
was linked to changes in APP processing and A levels in the brain.
Analysis of Tg2576 mice that were treated for 4 months with either ibuprofen
or the PPAR agonist pioglitazone revealed that the total amount of
formic acid-solubilized A40 or A42 was
modestly reduced. The ibuprofen-treated animals exhibited a 26% reduction in
A40, whereas pioglitazone treatment resulted in a 21%
reduction in A40; however, neither reached statistical
significance (Fig.
2A). However, quantitation of SDS-soluble A levels
in the brains of these mice demonstrated that both ibuprofen and pioglitazone
reduced A40 levels by 19%, a difference that was
statistically significant compared with control animals (p < 0.05)
(Fig. 2B).
Importantly, there was a striking reduction in the levels of SDS-soluble
A42 after drug treatment. Ibuprofen treatment reduced
SDS-soluble A42 levels by 63% (p < 0.05).
Pioglitazone-treated animals exhibited an overall reduction of SDS-soluble
A42 levels of 50%, but this effect did not reach statistical
significance. These data demonstrate that anti-inflammatory drug therapy
alters the levels of amyloidogenic A peptides in the brain.
It has been argued that the principal cellular target of NSAIDs are
microglia that are phenotypically activated as a consequence of amyloid
deposition. We evaluated microglia activation and abundance by staining for
cell surface markers, which are elevated after activation of these cells.
Ibuprofen treatment significantly reduced the number of CD45-positive
microglia in hippocampus (39% reduction; p < 0.05)
(Fig. 3A,B). CD45 is a
tyrosine phosphatase that plays critical roles in immune receptor signaling.
CD45 expression levels are elevated in reactive microglia in the AD brain
(Masliah et al., 1991) and in
murine models of the disease (Wilcock et
al., 2001). We also found that there was an ibuprofen-mediated 49%
reduction (p < 0.05) in the expression of the complement receptor
3 subunit CD11b (Fig. 4). CD11b
(also known as the m2 integrin and Mac1) is
a sensitive marker of microglial activation in the AD brain
(Mackenzie and Munoz, 1998)
and in murine models of the disease
(Sturchler-Pierrat et al.,
1997). Pioglitazone treatment did not result in a significant
change in the microglial expression of either CD45 or CD11b. We were unable to
observe a significant change in microglial phosphotyrosine levels in response
to ibuprofen, as reported previously by Lim and colleagues
(2001) (data not shown).
Activated microglia are found associated with amyloid plaques, thus the
decrease in the number of reactive cells is correlated with reduced plaque
burden.
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Figure 3. Ibuprofen reduces the number of CD45-positive microglia. The brains of
control Tg2576 mice (A) and those treated for 4 months with ibuprofen
(B) or pioglitazone (C) were stained for CD45. There was a
39% reduction in the number of CD45-positive microglial cells in the
hippocampus of ibuprofen-treated mice (*p < 0.05). The
number of animals used is indicated in each column (D). Ibu,
Ibuprofen; Piog, pioglitazone.
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Figure 4. Ibuprofen reduces the number of CD11b-positive microglia. The brains of
control Tg2576 mice (A) and those treated for 4 months with ibuprofen
(B) or pioglitazone (C) were stained for CD11b. There was a
49% reduction (*p < 0.05) in the number of
CD11b-positive microglial cells in the frontal cortex of ibuprofen-treated
mice. The number of animals used is indicated in each column (D).
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A number of NSAIDs, including ibuprofen, have recently been reported to
selectively regulate the processing of APP, and it has been argued that this
effect may underlie their beneficial effects in AD
(Weggen et al., 2001). NSAID
treatment of APP-overexpressing cells was reported to result in a preferential
reduction in the production of A42 and a parallel increase in
A38, although it had no effect on A40
(Weggen et al., 2001). We
tested whether ibuprofen and pioglitazone altered A production in HEK293
cells overexpressing human APP. Ibuprofen treatment resulted in a dramatic
reduction in the levels of A42 secreted into the medium,
although it had no effect on A40 levels
(Fig. 5A). Ibuprofen
had no effect on the -secretase cleavage of APP, which was used as an
indirect measure of drug effects on cellular viability and metabolism.
Pioglitazone had no significant effect on A production
(Fig. 5B).
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Figure 5. Effect of anti-inflammatory drugs on A40 and
A42 generation in APP-overexpressing cells. Human embryonic
kidney 293 cells stably expressing full-length human APP695 were treated for
18-24 hr with the indicated concentration of ibuprofen or pioglitazone, and
the medium was collected. A42, A40, and
APPs levels were quantitated by standard sandwich ELISA. A,
Ibuprofen, A40 ( ), A42 ( ), and
APPs ( ), levels are shown ± SD. B, Pioglitazone,
A40 (), A42 (), and APPs
() levels are shown ± SD.
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Discussion
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The mechanisms by which anti-inflammatory agents affect AD risk in humans
and amyloid pathology in animal models of the disease are likely to be complex
and diverse. We report that treatment of Tg2576 mice with ibuprofen during the
period in which A is deposited in the brain resulted in significant
alterations in amyloid plaque pathology, A production, and microglial
activation. These data provide strong evidence that ibuprofen, and likely
other NSAIDs, have multiple cellular and molecular targets.
The widely accepted target of the action of NSAIDs are the cyclooxygenases,
and much of the discussion on both of the mechanisms underlying the effects of
these drugs and the design of clinical trials has focused on these enzymes and
their products (Yermakova and O'Banion,
2000; Pasinetti,
2001; Aisen, 2002).
Despite considerable investigative activity, there are few compelling data to
indicate that these enzymes play critical roles in eliciting neuronal death or
otherwise exacerbate the pathophysiology of AD. The recent report of the
failure of clinical trials using COX-2-specific inhibitors in AD patients has
served to further diminish confidence that cyclooxygenases are the
biologically relevant target of NSAID action in AD
(Sainati et al., 2000). It has
been demonstrated conclusively that these enzymes are not involved with the
selective reduction in A42 levels
(Weggen et al., 2001).
Moreover, it is unlikely that the broad range of anti-inflammatory actions of
NSAIDs could arise simply from COX inhibition. Alternate mechanisms of NSAID
actions have been reported. Ibuprofen and other NSAIDs have been found to
inhibit several distinct signal transduction cascades
(Tegeder et al., 2001;
Baek et al., 2002). These
effects have not been examined extensively, and no consistent conclusions can
be drawn from the existing data. A more detailed examination of these
mechanisms is clearly required.
The principal effect of ibuprofen treatment in Tg2576 mice was a very
significant reduction in plaque burden after 4 months of treatment. The
magnitude of the decrease in the number of plaques that we observed was
similar to those reported by Lim et al.
(2000) (52.6 vs 51%), as was
the reduction in area occupied by amyloid plaques (55.9 vs 60%). The reduction
in amyloid burden observed here is also consistent with that reported recently
by Jantzen et al. (2002) in
mice treated with ibuprofen or a flurbiprofen derivative.
We found that ibuprofen treatment resulted in only a modest and
statistically insignificant decrease in total insoluble A in the brains
of Tg2576 mice. This finding is quantitatively different from that reported by
Lim et al. (2000). These
authors reported a 40% decrease in total 2% SDS-insoluble A in
"residual" regions of the cortex with smaller reductions ranging
from 20 to 35% in hippocampus, entorhinal cortex, and piriform cortex.
Notably, they reported a 34% decrease in soluble A in these animals;
however, these changes did not reach statistical significance. These authors
have subsequently reported that a 3 month treatment of Tg2576 mice with
ibuprofen resulted in an 50% reduction in total soluble A, total
insoluble A, and A42; however, this change was
restricted to the entorhinal cortex. A levels in other brain regions
were not significantly different (Lim et
al., 2001). Thus, whereas our data on plaque pathology are similar
to those reported previously, we obtained a quite different outcome with
respect to alterations in the levels of the individual A species that
contribute to the plaque. Although our measurements were performed using the
entire cerebral hemisphere rather than subregions of the brain, it seems
unlikely that this can account for the different outcomes of the experiments.
We found that although we observed a 60% reduction in plaque burden by
ibuprofen, there was only a 26% decrease in total A levels. This finding
suggests that not all A is plaque-associated in these animals. A similar
disparity between amyloid burden and total A peptide levels has also
been observed by Janus and colleagues
(2000) after A
immunization. The basis of this effect is not clear but may represent the
redistribution of A from plaques into a soluble pool.
The primary source of A in the brain are neurons, and the present
data demonstrate that ibuprofen treatment dramatically affects the buildup of
the highly amyloidogenic A42 species produced by these cells.
Importantly, there was no significant change in SDS-soluble
A40 levels. Our observation of a selective decrease in
A42 levels after ibuprofen treatment of transgenic mice is
consistent with data recently reported by Weggen et al.
(2001). These authors found
that a 3 d treatment of Tg2576 mice with ibuprofen resulted in an 40%
decrease in soluble A42 levels in the brains of these mice
without a significant change in A40 levels. We also
demonstrate, using in vitro assays in cells overexpressing APP, that
ibuprofen affects APP processing, resulting in a dramatic and selective
reduction in the levels of A42 released by the cells but
having no effect on A40 production. These latter observations
serve to verify the findings reported by Weggen et al.
(2001) using a similar assay
system. This selective reduction in A42 levels and a
concomitant increase in A38 has been postulated to arise from
a drug-mediated alteration in -secretase specificity, although the
precise mechanisms subserving this effect are presently unclear.
Microglial activation has been argued to contribute to the progressive
course of AD because of elaboration of a large array of proinflammatory
molecules that mediate, in part, the neuronal loss observed in the disease
(Kalaria, 1999;
Akiyama et al., 2000). We found
that ibuprofen treatment of Tg2576 mice resulted in a 40% reduction in
CD45-positive microglia and a 49% decrease in CD11b expression. The induction
of CD45 and CD11b expression accompanies the phenotypic activation of these
cells in the AD brain (Masliah et al.,
1991, Mackenzie et al., 1998). These data are consistent with the
findings of Lim et al. (2000),
who reported an ibuprofen-mediated decrease in microglial activation, as
reflected by phosphotyrosine staining, another marker of microglial
activation. We were unable to observe analogous effects with this marker. The
ibuprofen-mediated suppression of microglial activation observed here and by
Lim et al. (2000) is in
contrast to data reported by Jantzen et al.
(2002), who reported that mice
doubly transgenic for APP and PS1 exhibited a small but not significant
increase in major histocompatibility complex II-positive profiles when treated
with ibuprofen for 5 months. These data were interpreted as reflective of
microglia activation by the ibuprofen treatment.
It is not obvious how ibuprofen elicits effects on the microglia. We argued
that ibuprofen may suppress microglial activation through its ability to
activate the nuclear receptor PPAR
(Landreth and Heneka, 2001).
This study evaluated the effects of the highly specific and potent PPAR
agonist pioglitazone on the basis of our previous observations that
PPAR activation robustly antagonized the A-mediated activation of
microglia and the production of a number of proinflammatory and neurotoxic
molecules (Combs et al., 2000).
Pioglitazone treatment effected a change in A peptide levels; however,
the magnitude of this change was too small to translate to a significant
effect on plaque pathology. Specifically, we observed that pioglitazone had
only a modest effect on total A levels and had no detectable effect on
microglial activation in the Tg2576 mice. Pioglitazone has been reported to
pass the blood-brain barrier (Kiyota et
al., 1997); however, it seems likely that the dose used in this
study was insufficient to result in levels of the drug in the brain required
to elicit the robust activation of this nuclear receptor (G. Landreth,
unpublished observations). In the cell-based assay, which circumvents problems
of in vivo pharmacokinetics and blood-brain barrier penetration,
pioglitazone did not show the specific A42-lowering effect
observed by Weggen et al.
(2001) for NSAIDs. At high
pioglitazone concentrations, a reduction in all secreted APP metabolites,
which we interpret as nonspecific toxicity was observed (data not shown).
Therefore, if the amyloid burden reduction in ibuprofen-treated mice is indeed
the result of a reduction in A42 production, this is unlikely mediated
through the PPAR pathway. In summary, these data support the use of
anti-inflammatory drug treatment in AD, at least for those agents that effect
a reduction in the levels of amyloidogenic peptides and the proinflammatory
response of microglial cells. The additional mechanisms of the action of
ibuprofen remain unclear, and it remains a formal possibility that ibuprofen
affects the clearance of A from the brain or intervenes in processes
mediating the fibrilization and deposition of it. However, the selective
ibuprofen effect on the A42 isoform, both in tissue culture
and in our chronic in vivo treatment, is explained most
parsimoniously by a modulation of -secretase activity, which leads to
reduction in A42 production.
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Footnotes
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Received Jan. 21, 2003;
revised May. 29, 2003;
accepted Jun. 3, 2003.
We thank the members of the Amgen animal facility for invaluable assistance
with this study.
Correspondence should be addressed to Dr. Martin Citron, Amgen, M/S 29-2-B,
One Amgen Center Drive, Thousand Oaks, CA 91320-1799. E-mail:
mcitron{at}amgen.com.
R. Vassar's present address: Feinberg School of Medicine, Northwestern
University, Chicago, IL 60611.
Copyright © 2003 Society for Neuroscience
0270-6474/03/237504-06$15.00/0
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Top
Abstract
Introduction
