3692?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 119? ? ? Number 12? ? ? December 2009
NSAIDs prevent, but do not reverse,
neuronal cell cycle reentry in a mouse
model of Alzheimer disease
Nicholas H. Varvel,1,2 Kiran Bhaskar,1 Maria Z. Kounnas,3 Steven L. Wagner,3
Yan Yang,2 Bruce T. Lamb,1,2,4 and Karl Herrup5
1Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA. 2Department of Neurosciences,
Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. 3Torrey Pines Therapeutics, Inc., La Jolla, California, USA.
4Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
5Department of Cell Biology and Neuroscience Nelson Biological Laboratories, Rutgers,
The State University of New Jersey, Piscataway, New Jersey, USA.
Alzheimer disease (AD), the most common dementing disorder of
late life, is now the sixth leading cause of death in the United States
(1). The course of the disease is lengthy, and there is currently no
effective treatment. A definitive diagnosis of AD requires postmor-
tem examination of brain tissue for the presence of distinctive AD
histopathology, including neurofibrillary tangles and extracellular
deposits of the β-amyloid peptide (Aβ) in senile plaques. Activation
of microglia and astrocytes, induction of neuronal cell cycle events
(CCEs), and regional cell loss are also observed. Understanding the
relationship between the various neuropathological hallmarks of
AD in the human brain has proven difficult because of a lack of
accurate diagnostic markers; the length of disease progression; and
the considerable variation in the duration, severity, symptoms, age
of onset, and clinical/pathological correlations.
AD is one of many neurodegenerative conditions characterized
by chronic neuroinflammatory processes. Microglia, the resident
immune cells of the brain, are found in a highly activated state
in close anatomical proximity to senile plaques within the AD
brain, where they secrete numerous proinflammatory cytokines
and chemokines (2, 3). Recent studies using in vivo imaging have
demonstrated that microglia rapidly migrate to newly formed Aβ
deposits in mouse models of AD and are capable of removing Aβ
fibrils (4, 5). However, it remains to be determined whether neu-
roinflammatory alterations also contribute to early steps in AD
progression. Retrospective epidemiological studies indicate that
chronic, long-term treatment with nonsteroidal antiinflamma-
tory drugs (NSAIDs) decreases the risk for developing AD, which
suggests that neuroinflammation may play a pivotal role in early
disease processes (6–8). However, thus far, prospective clinical tri-
als with multiple different NSAIDs have failed to demonstrate
significant beneficial effects in individuals with existing cognitive
impairments characteristic of AD (9). At present, the biological
mechanisms underlying the divergent results obtained in the ret-
rospective and prospective NSAID studies remain unclear.
NSAIDs may act via several pathways to influence AD patho-
genesis. First, NSAIDs can reduce neuroinflammation via canoni-
cal antiinflammatory pathways within the brain. Indeed, chronic
administration of NSAIDs reduces neuroinflammation, AD-like
brain pathology, and behavioral impairments in transgenic mouse
models of AD (10–13). Second, NSAIDs can act as γ-secretase
modulators (GSMs). Acute administration of selective NSAIDs
results in production of shorter, less amyloidogenic Aβ peptides
both in vitro and in vivo, likely through interactions with the amy-
loid precursor protein (APP) that influence γ-secretase cleavage
(14–16). Third, NSAIDs may also regulate the levels of β-secretase
through a PPARγ-mediated pathway (17). Finally, NSAIDs may act
to inhibit the formation of Aβ oligomers and deposits through
direct interaction with the Aβ peptide (16).
Increasing evidence suggests that ectopic expression of cell cycle
proteins and DNA synthesis identifies neuronal populations
subject to degeneration in AD (18–21). CCEs are also observed
in mild cognitive impairment, the clinical predecessor to AD
(22). Together, these results suggest the CCEs identify an early
pathogenic process in AD. Highlighting the significance of the
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 119:3692–3702 (2009). doi:10.1172/JCI39716.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
CCEs in the neurobiology of AD, most mouse models that have
been examined exhibit CCEs in a characteristic, age-dependent
pattern (23–26). In the R1.40 AD model (27–29), neuronal CCEs
occur approximately 6 months prior to the first appearance of Aβ
deposits, but lowering or eliminating Aβ production via genetic
means in the R1.40 mouse model causes either a delay or a com-
plete block of neuronal CCEs (30).
The current studies were designed to explore the Aβ-dependent
pathways responsible for induction of neuronal CCEs. We demon-
strated that alterations in brain microglia were coincident with the
first evidence of neuronal CCEs in the R1.40 model and that the
microglial alterations were dependent on Aβ generation. Induc-
tion of systemic inflammation promoted the early appearance of
neuronal CCEs in young R1.40 animals, but not in nontransgenic
controls. In addition, inhibition of neuroinflammation in young
R1.40 animals by chronic administration of 2 commonly used
NSAIDs, ibuprofen and naproxen, blocked alterations in brain
microglia as well as neuronal CCEs in the absence of detectable
alterations in APP processing and Aβ metabolism. Notably, thera-
peutic NSAID treatment of older R1.40 mice did not reverse the
presence of extant CCEs, even after several months of treatment.
These results argue for a role of both Aβ and neuroinflammation
in the induction of neuronal CCEs and provide a potential expla-
nation for the relative successes and failures of the retrospective
and prospective NSAID trials in the treatment of human AD.
Alterations in brain microglia in the R1.40 mouse model of AD. To deter-
mine whether the induction of neuronal CCEs was accompanied
by alterations in brain microglia, we used the genomic-based R1.40
transgenic mouse model of AD, which contains the entire Swedish
mutant human APP (see Methods). Because this model is under
endogenous human regulatory elements, it mimics the correct tem-
poral and spatial expression patterns observed in human brain tis-
sue. The R1.40 mouse, when maintained on the C57BL/6 inbred
genetic background, first begins to exhibit Aβ deposition and neu-
ritic abnormalities at 12–14 months of age (31). In addition, the
R1.40 mouse model exhibits neuronal CCEs in many of the same
brain regions in which they are encountered in human AD (23, 30).
Brain sections of adult R1.40 and control mice were examined for
the microglial marker Iba1 via immunohistochemistry at a variety
of ages. R1.40 mice exhibited microglial activation and migration
to sites of neocortical Aβ deposition beginning at about 12–14
months of age. Unexpectedly, however, immunohistochemical
analysis of microglial morphology in 6-month-old R1.40 animals
also demonstrated noticeable alterations (Figure 1, C and D). In
6-month-old nontransgenic animals, microglia displayed a rest-
ing phenotype, with numerous thin branching processes extend-
ing away from a small cell body (Figure 1, A and B). Conversely,
R1.40 microglia exhibited shorter, asymmetrically oriented pro-
cesses as well as swollen cell bodies (Figure 1, C and D). None of
these changes was evident at 4 months of age in R1.40 animals
(data not shown), which suggests that, similar to induction of
neuronal CCEs, alterations in brain microglia occur between 4 and
6 months of age. Notably, the microglia with altered morphologies
in the R1.40 mice were not positive for CD45, a cell surface protein
expressed in highly activated microglia as well as in infiltrating
immune cells (data not shown).
Aβ-dependent alterations in brain microglia in R1.40 transgenic mice.
(A and B) Neocortical microglia in 6-month-old nontransgenic mice
exhibited extensive fine processes with small cell bodies. (C and D)
Age-matched R1.40 animals exhibited reactive neocortical microglia:
thick, asymmetrically oriented processes surrounding a swollen cell
body. (E and F) R1.40;Bace1–/– animals at 6 months of age exhibited
microglia with a resting morphology that was indistinguishable from
that of nontransgenic controls (A and B). (G and H) Cx3cr1+/gfp mice
lacking the R1.40 transgene (G) displayed GFP-expressing microglia
morphologically similar to the Iba1-stained microglia observed in non-
transgenic controls (A and B). Conversely, Cx3cr1+/gfp mice transgenic
mice with the R1.40 transgene (H) exhibited microglia with a reactive
morphology similar to that of 6-month-old R1.40 transgenic mice (C
and D). (I) Morphometric analysis of Iba1-positive microglia revealed
significantly higher FF values in R1.40 animals than in nontransgenic
(nTg) and R1.40;Bace1–/– mice. *P < 0.01; **P < 0.0004. Scale bars:
500 μm (A, C, and E); 100 μm (B, D, F, G, and H).
3694? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
In order to confirm these observations, R1.40 transgenic mice
were crossed to a mouse line in which the Gfp gene is knocked
into the fractalkine receptor locus (Cx3cr1), leading to the spe-
cific labeling of all brain microglia with GFP. We generated mice
heterozygous for the Cx3cr1 knockin (Cx3cr1+/gfp) with and without
the R1.40 transgene and examined their brain sections via fluores-
cent confocal microscopy. Similar to the immunohistochemical
data, GFP in the microglia of 6-month-old Cx3cr1+/gfp mice lacking
the R1.40 transgene exhibited a resting phenotype, with numer-
ous thin, branching processes (Figure 1G). However, microglia in
Cx3cr1+/gfp mice with the R1.40 transgene exhibited a more acti-
vated phenotype, including shortened processes and enlarged cell
bodies (Figure 1H). To obtain a quantitative measure of microg-
lial activation, morphometric analysis of brain microglia was con-
ducted using form factor (FF; see Methods). In general, a higher
FF value is indicative of more bushy, activated microglia, while a
lower FF value is indicative of resting microglia (32–34). Notably,
FF analysis demonstrated that Iba1-positive microglia in 6-month-
old R1.40 animals had a significantly higher mean FF value than
did microglia in age-matched nontransgenic controls (Figure 1I).
To specifically test whether Aβ generation from the R1.40 trans-
gene is required for the alterations in brain microglia observed at
6 months of age, R1.40 transgenic mice were mated with animals
lacking Aβ precursor protein cleavage enzyme 1 (Bace1), the pri-
mary β-secretase required for the first step in the generation of Aβ
from APP. Iba1 immunostaining of cortical microglia in 6-month-
old R1.40 transgenic mice lacking Bace1 (R1.40;Bace1–/– mice)
exhibited a resting phenotype (Figure 1, E and F) resembling that
of age-matched nontransgenic controls
(Figure 1, A and B). These double-mutant
microglia had FF values that were signifi-
cantly lower than those of R1.40 animals,
but indistinguishable from those of age-
matched nontransgenic animals (Figure
1I). Similarly, analysis of fractal dimension,
another morphometric parameter inversely
correlated with microglial activation state
(32–34), was lower in 6-month-old R1.40
mice than in R1.40;Bace1–/– mice or non-
transgenic controls (data not shown).
Together with our previous data demon-
strating that the induction of neuronal
CCEs at 6 months of age is also dependent
on the amyloidogenic processing of APP in
the R1.40 mouse model (30), these results
suggest that alterations in microglial acti-
vation could play a direct role in the Aβ-
dependent induction of neuronal CCEs.
Induction of inflammation promotes microg-
lial activation and neuronal CCEs. Based on
the correlative data linking microglial
alterations and neuronal CCEs, we next
examined whether induction of inflam-
mation is capable of inducing premature
neuronal CCEs in young R1.40 animals.
We intraperitoneally injected 2-month-old
nontransgenic and R1.40 mice with either
LPS (20 μg/animal once daily for 4 days)
or PBS. This LPS injection protocol has
previously been demonstrated to induce
microglia activation within the brain (35). As expected, LPS expo-
sure induced robust microglial activation in both R1.40 mice (Fig-
ure 2E) and nontransgenic controls (Figure 2A) compared with
animals injected with PBS alone (Figure 2I). Additional sections
from these animals were stained for cell cycle proteins and the
neuronal marker NeuN. PBS-injected R1.40 animals did not exhib-
it any evidence of cyclin D–positive neurons in frontal cortical lay-
ers II/III (Figure 2, J–L). Conversely, LPS-injected R1.40 animals
exhibited numerous cyclin D–positive neurons in the same brain
regions (Figure 2, F–H). Staining for cyclin A gave similar results
(data not shown). Notably, LPS-injected nontransgenic controls
exhibited no evidence of neuronal CCEs (Figure 2, B–D). Taken
together, these data suggest that the induction of neuronal CCEs
is dependent on both induction of inflammatory processes and an
R1.40 genotype, and thus, most likely, on Aβ generation.
Early NSAID treatment inhibits inflammatory responses and neuronal
CCEs. To determine whether inhibition of the microglial altera-
tions in R1.40 transgenic mice blocks induction of neuronal CCEs,
we placed 3-month-old R1.40 and aged-matched nontransgenic
controls on standard laboratory diets with or without ibuprofen
or naproxen (375 ppm in chow) for 3 months. At 6 months of age,
Iba1 immunohistochemistry of treated R1.40 animals revealed
microglial cells with thin, symmetric processes, indicating that
both ibuprofen and naproxen (Figure 3, C–F) blocked the microg-
lial activation (Figure 3, A and B) to levels resembling the resting
phenotype exhibited by age-matched nontransgenic controls
(Figure 1, A and B). Morphometric analysis of Iba1-positive brain
microglia lent support to these conclusions, as FF was substan-
LPS administration provokes neuroinflammation and neuronal CCEs. (A–D) Nontransgenic
mice at 2 months of age subject to LPS injections exhibited Iba1-immunoreactive neocortical
microglia with an activated morphology (A) with no evidence of expression of cyclin D (B) in
NeuN-positive neurons (C). (E–H) Age-matched R1.40 transgenic animals injected with LPS
exhibited Iba1-positive microglia with an activated morphology (E) as well as expression of
cyclin D (F) in a subset of NeuN-positive cortical layer II/III neurons (G). (I–K) Age-matched
R1.40 animals injected with PBS exhibited Iba1-positive microglia with a resting morphology
(I) and no evidence of expression of cyclin D (J) in NeuN-positive neurons (K). Similar results
were obtained with immunohistochemistry for the cell cycle protein cyclin A (not shown). (D, H,
and L) Merged images. Nuclei were counterstained with DAPI (blue). Arrows indicate cyclin D–
positive neurons. Scale bar: 100 μm.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
tially reduced in the ibuprofen and naproxen treatment groups
compared with untreated R1.40 mice and was indistinguishable
from that of age-matched nontransgenic controls (Figure 3G).
Brain sections from the control and NSAID treatment groups
were also examined for alterations in neuronal CCEs. As previ-
ously reported (30), by 6 months, R1.40 transgenic mice exhib-
ited a substantial number of neurons positive for cyclin A (Figure
4, A–C) and cyclin D (data not shown) in layers II/III of frontal
cortex. These same neuronal populations also exhibited evidence
of DNA replication, as indicated by 3 or 4 spots of hybridization
with a DNA FISH probe for either mouse chromosome 16 (Figure
4C, inset) or the R1.40 human transgene on mouse chromosome
13 (data not shown). Administration of ibuprofen or naproxen
blocked virtually all evidence of CCEs, as measured by cyclin A and
cyclin D expression (Figure 4, D–I) as well as by DNA replication
(Figure 4, F and I, insets). Taken together, these data indicate that
a 3-month treatment of young R1.40 mice with either ibuprofen
or naproxen blocks both microglial activation and development of
neuronal CCEs in the mouse 6 months prior to the initiation of Aβ
deposition in this model.
The effectiveness of the treatments was quantified by determin-
ing the percentage of neurons in frontal cortex layers II/III posi-
tive for cyclin A and cyclin D as well as the percentage exhibiting a
hyperploid phenotype with a DNA probe specific to mouse chro-
mosome 16 or the R1.40 transgene on chromosome 13 (Figure
5, A–D). In 6-month-old R1.40 transgenic mice, 43% of the neu-
rons (NeuN-positive cells) in cortical layers II/III were positive for
cyclin A (Figure 5A). NSAID treatment for 3 months with ibupro-
fen reduced the percentage of cyclin A–positive neurons in corti-
cal layers II/III to approximately 4%, whereas naproxen reduced it
to about 3%. Cyclin D expression was reduced to approximately
5% and 3% by ibuprofen and naproxen, respectively (Figure 5B).
These levels were statistically indistinguishable from those of age-
matched nontransgenic controls (cyclin A, 0.9%; cyclin D, 1.4%).
Quantitative analysis of the percentage of hyperploid neuronal
nuclei in cortical layers II/III was determined after FISH with a
probes specific for R1.40 mouse chromosome 16 or chromosome
13. Untreated R1.40 transgenic mice exhibited hyperploidy for
both probes: 12% for chromosome 16 (Figure 5C) and 9% for chro-
mosome 13 (Figure 5D). After ibuprofen and naproxen treatment,
these percentages were reduced to 0.6% and 0.4%, respectively.
These levels were statistically indistinguishable from those found
in nontransgenic controls.
Lack of effect of chronic NSAID treatment on APP processing and Aβ
metabolism. Considerable evidence suggests that NSAIDs can act
as GSMs in acute treatment paradigms both in vitro and in vivo,
but their potency varies widely. In these short-term assays, ibu-
profen is reported to be most effective, while naproxen lacks any
apparent GSM activity (14, 15). This difference guided our choice
of ibuprofen and naproxen for the current studies as a way of
distinguishing whether any effects we observed were caused by
differences in Aβ generation. Having found no difference in their
ability to inhibit both microglial activation and CCE induction,
we next analyzed our samples to determine whether the impact
of 3 months of chronic NSAID administration differs from the
short-term effects reported previously (14, 15). Brain extracts
from 6-month-old treated and untreated animals were analyzed
via Western blot for levels of holo-APP and APP C-terminal frag-
ments (CTFs). No significant differences in the steady-state levels
of holo-APP were observed in samples from either NSAID treat-
ment group compared with controls (Figure 6, A and B). Western
blot analysis also revealed that the levels of the 2 primary APP
CTFs, CTFα and CTFβ, were unchanged (Figure 6A). Aβ metabo-
lism was similarly unaffected. Finally, the steady-state levels of
both major isoforms of Aβ, Aβ1–40 and Aβ1–42, were determined
by ELISAs from formic acid–treated brain extracts. Neither ibu-
profen nor naproxen treatment led to significant alterations in
Prevention trial of NSAIDs inhibits microglial alterations. (A and B)
R1.40 transgenic mice placed on a control diet for 3 months begin-
ning at 3 months of age exhibited Iba1-immunoreactive neocortical
microglia with an activated phenotype, similar to R1.40 animals at
6 months of age (see Figure 1, C and D). (C–F) R1.40 animals placed
on a 3-month ibuprofen- (C and D) or naproxen-containing (E and F)
diet exhibited Iba1-positive microglia with a resting phenotype resem-
bling that of 6-month-old nontransgenic control and R1.40;Bace1–/–
animals (see Figure 1, A, B, E, and F). Insets show representative
confocal images of Iba1-positive microglia stained with fluorescently
tagged secondary antibodies. (G) Morphometric analysis of Iba1-posi-
tive microglia revealed significantly lower FF values in mice treated
with ibuprofen (IBU) and naproxen (NAP), compared with R1.40 ani-
mals fed a control diet, that were not different from the FF values in
nontransgenic mice. **P < 0.0004; ***P < 0.0002. Scale bars: 500 μm
(A, C, and E); 100 μm (B, D, F, and insets).
3696? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
the steady-state levels of Aβ1–40 or Aβ1–42 compared with untreated
transgenic controls (Table 1). These results suggest that chron-
ic administration of NSAIDs blocks microglial activation and
neuronal CCEs by a mechanism independent of their effect on
APP processing and Aβ metabolism.
NSAID treatment cannot reverse previously established neuronal CCEs.
Neuronal CCEs develop in a precise temporal and spatial pattern
in R1.40 mice (30). Neurons in frontal cortical layers II/III first
exhibited CCEs at 6 months of age, whereas neurons in cortical
layers V/VI did not exhibit CCEs until 12 months of age (Figure
7A). Furthermore, after a specific brain region develops CCEs, the
percentage of affected cells remains stable over the lifespan of the
animal (30). This predictable time sequence offered us the oppor-
tunity to determine whether NSAID administration can reverse
the accumulation of neuronal CCEs in cortical layers II/III after
they begin to form. R1.40 transgenic mice were placed on control,
ibuprofen-containing, or naproxen-containing diets (375 ppm in
each) for 6 months beginning at 6 months of age. Brain sections
from the 12-month-old animals were subsequently examined for
the presence of neuronal CCEs via immunohistochemistry and
FISH. Immunohistochemistry for cyclin D and FISH revealed
the presence of numerous immunopositive layer II/III neurons
in control animals as well as in animals treated with ibuprofen
or naproxen (Figure 7, B–G).
Quantification of the data confirmed the visual impressions
from this neuronal population. Similar to the results obtained at 6
months of age (Figure 5), 45%–50% of neurons within frontal corti-
cal layers II/III were positive for cyclin A and cyclin D in 12-month-
old R1.40 transgenic mice (Figure 8, A and B). In addition, the num-
ber of neurons with 3 or 4 spots of hybridization in frontal cortical
layers II/III remained unchanged between 6 and 12 months of age:
10%–15% hyperploidy for both chromosome 16 and chromosome 13
Prevention trial of NSAIDs inhibits neuronal CCEs.
(A–C) R1.40 transgenic mice placed on a control diet
for 3 months beginning at 3 months of age exhibited
expression of cyclin A (A) in numerous NeuN-positive
(B) neurons on the frontal cortex layers II/III, similar
to previously published results (30). Large arrows
indicate cyclin A–positive neurons. (D–I) R1.40 ani-
mals placed on the 3-month ibuprofen- (D–F) or
naproxen-containing (G–I) diet exhibited expression
of cyclin A (D and G) in a subset of NeuN-positive (E
and H) neurons. Similar results were obtained with
immunohistochemistry for the cell cycle protein cyclin D
(not shown). (C, F and I) Merged images. Nuclei were
counterstained with DAPI (blue). FISH with a DNA
probe to mouse chromosome 13 demonstrated a
subset of neurons with 3 or 4 spots of hybridization in
R1.40 animals fed the control diet (C, inset), while the
ibuprofen and naproxen treatment groups exhibited
only 2 spots of hybridization (F and I, insets). Small
arrows indicate FISH signals in the neuronal nucleus.
Scale bars: 100 μm (A–I); 10 μm (insets).
Quantification of inhibition of neuronal CCEs
in prevention trial of NSAIDs. Percentages of
NeuN-positive neurons in cortical layers II/III
exhibiting expression of cyclin A (A) or cyclin D
(B) as well as 3 or 4 spots of hybridization
with FISH for DNA probes from mouse chro-
mosome 16 (C) and 13 (D) was calculated in
R1.40 transgenic mice fed control, ibuprofen-
containing, and naproxen-containing diets as
well as in nontransgenic mice fed the control
diet (n = 5). **P < 0.003, ***P < 0.001 versus
control diet–fed R1.40 transgenic mice.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
(Figure 8, C and D). Although DNA replication is not reversible,
we were surprised to find that after 6 months of treatment with
either ibuprofen or naproxen, there was no significant reduction
in the percentage of cyclin A– or cyclin D–positive neurons (Figure
8, A and B). These data suggest that NSAID administration in a
therapeutic regimen (after the disease has initiated) is incapable of
reversing the previously established neuronal CCEs.
Late NSAID treatment can block the development of additional neuronal
CCEs. Neuronal CCEs first appeared in cortical layers V/VI at
12 months of age in the R1.40 mice (Figure 7H). To determine
whether a 6-month treatment with ibuprofen or naproxen could
block CCEs in this neuronal population, we turned our attention
to the deeper cortical layers of our animals. Unlike the results
obtained for the existing CCEs in frontal cortex layers II/III (Fig-
ure 7, B–G), both ibuprofen and naproxen led to a dramatic reduc-
tion in the number of cyclin A– and cyclin D–positive neurons
in layers V/VI at 12 months of age (Figure 7, J–M). FISH analy-
sis with probes from mouse chromosome 13 (Figure 7, I, K, and
M, insets) and chromosome 16 (data not shown) also revealed a
decrease in the number of hyperploid neuronal nuclei. Quantita-
tive analysis revealed that approximately 50% of the NeuN-positive
cells in cortical layers V/VI were positive for cyclin A and cyclin D
in untreated transgenic mice (Figure 8, A and B). After treatment
with ibuprofen or naproxen, however, there was a significant
reduction in the percentage of cortical layer V/VI neurons express-
ing cell cycle proteins. Quantitative analysis of the percentage of
hyperploid neuronal nuclei by FISH with DNA probes for mouse
chromosome 16 and chromosome 13 revealed a similar picture
(Figure 8, C and D). Thus, long-term administration of NSAIDs
after neuronal CCEs was capable of blocking subsequent events,
but could not reverse the damage in previously affected cells.
There is increasing evidence that the neurodegeneration observed
in AD is the result of a pathogenic process initiated well before
the onset of the cognitive symptoms associated with the dis-
ease. Although biochemical, genetic, and pathological studies
clearly implicate alterations in APP processing as being central
to AD pathogenesis, the relationship between Aβ generation and
deposition and the other features of the disease remains unclear.
For example, both cross-sectional pathological studies and PET
studies with the amyloid binding Pittsburgh compound B have
revealed that a relatively large number of aged, cognitively normal
individuals have a substantial amyloid plaque load (36–39). This
has direct implications for both the study of pathogenic mecha-
nisms underlying AD and the various prospective clinical trials
being conducted in human AD.
Our previous studies demonstrated that one of the most reliable
indicators of neuronal populations at risk in AD is ectopic expression
of cell cycle proteins and DNA replication in postmitotic neurons
(18–21). CCEs identify neurons at risk in AD as well as in mild cogni-
tive impairment, which suggests that they represent an early marker
of neuronal vulnerability (21, 40, 41). Their involvement in the core
biology of AD is further documented by their presence in most trans-
genic mouse models of AD that have been examined. In R1.40 ani-
mals, ectopic CCEs first appear 6–8 months prior to Aβ deposition
and subsequently advance in a precise temporal and spatial pattern
in populations of neurons identical to those at risk in the human
disease (23). In addition, our previous studies demonstrated that the
appearance of neuronal CCEs is β-secretase dependent (30).
The current studies expand these insights in several ways.
Although late-stage markers of microglial activation do not
develop until later in disease progression (31), cortical microglia
underwent visible morphological changes between 4 and 6
months of age in R1.40 mice, coincident with the first appear-
ance of neuronal CCEs. This evidence of microglial activation
was dependent on Aβ generation, as it was greatly reduced in
R1.40 animals lacking Bace1. Although alterations in microglia
are typically reported to be elicited in response to the deposition
of Aβ, several recent findings suggest that alterations in microglia
as well as in the production of cytokines and chemokines may
be an early feature that precedes Aβ deposition in other mouse
Chronic dosing with NSAIDs does not alter steady-state brain
n? Aβ1–40?(pmol•g–1)? Aβ1–42?(pmol•g–1)? Aβ1–42/Aβ1–40
10 8.98 (1.86) 3.36 (0.71)
10 9.00 (1.23) 3.09 (0.48)
10 10.57 (2.97) 3.15 (0.71)
B6-R1.40 mice were dosed as described in the text. Values are
expressed as mean (SD).
Lack of effect of chronic NSAID treatments on APP pro-
cessing. (A) Western blots of brain extracts from the
R1.40 animals fed control (C1–C3), ibuprofen-contain-
ing (I1–I3), and naproxen-containing (N1–N3) diets were
probed with antibodies to the C terminus of APP and
subsequently stripped and reprobed with an antibody
against GAPDH as a loading control. Shown on the right
is the approximate size in kDa. (B and C) Relative levels
of holo-APP (B) and CTFβ (C) were quantified (n = 3)
from animals on the control, ibuprofen-containing, and
naproxen-containing diets by normalizing the intensity
values of APP and APP CTFβ to GAPDH. No significant
differences were observed in the relative levels of holo-
APP or CTFβ between treatment groups.
3698? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
models of AD (42, 43). In contrast to recent results suggesting
that peripheral immune cells may play a role in Aβ metabolism
(44–46), we think that this is unlikely to be a major contributor
to our present results, as the Iba1-positive microglia in the R1.40
mice did not stain with CD45, a cell surface protein expressed at
high levels on infiltrating monocytes (data not shown).
This early microglial activation may well play a causative role in
the appearance of neuronal CCEs. We have demonstrated this in
both positive and negative directions. When mice were subjected
to an inflammatory challenge independent of that triggered by Aβ
itself, the appearance of cell cycle changes could be advanced by
months. Significantly, although both nontransgenic and R1.40 mice
exhibited robust microglial activation, only the genetically suscep-
tible R1.40 transgenic mice went on to reveal neuronal CCEs. These
observations imply that both conditions — genetics and inflamma-
tion — must be met to produce neuronal cell cycling.
The causative nature of the inflammatory process was further
suggested by our observation that NSAID treatment could virtually
eliminate the appearance of neuronal
CCEs. Treatment of R1.40 mice with
ibuprofen or naproxen from 3–6
months of age represented the murine
equivalent of a prevention trial. In this
situation, in which therapy was initi-
ated 3 months prior to the first appear-
ance of neuronal CCEs, both ibuprofen
and naproxen were capable of reducing
the microglial morphological altera-
tions and the appearance of CCEs nor-
mally observed at 6 months of age. Our
biochemical analysis suggests that this
effect is independent of any significant
chronic change in APP processing.
Although this prevention paradigm
holds out great hope for clinical appli-
cation, the NSAID therapeutic trial is
a sobering reminder of the complexity
of the disease. NSAID treatment begun
after the first appearance of neuronal
CCEs could prevent the appearance of
neuronal CCEs that normally appear
in the deeper cortical layers V/VI at 12
months of age, but this treatment was
incapable of reversing the neuronal
CCEs already present in cortical layers
II/III. The failure to reduce the levels
of cell cycle proteins in these animals
is especially noteworthy. It would
be expected that the long period of
drug treatment should substantially
reduce the inflammation caused by
the APP transgene and its Aβ byprod-
uct. Despite this, 6 months of therapy
(one-fifth to one-quarter of a mouse
lifetime) was incapable of restoring the
normal cell cycle protein status of the
There are considerable data regard-
ing the efficacy of NSAIDs in the pre-
vention and/or treatment of AD. Ret-
rospective epidemiological studies suggest that a wide variety of
NSAIDs may significantly reduce one’s lifetime risk of developing
AD (6), although the effect may not be felt rapidly (8). Long-term
use of NSAIDs is associated with protection from the development
of AD (47) and reduction in Aβ deposition in mouse models of AD
(10, 11, 48, 49) and is also positively correlated with a reduction
of plaque-associated microglia in both humans (50, 51) and mice
(10, 11, 48, 49). Inflammatory events found in mouse models of
AD — namely, microglial activation and expression of proinflam-
matory molecules — are significantly reduced by NSAID treatment
(11, 48, 49, 52, 53). Yet in opposition to these findings of efficacy
are the disappointing outcomes of prospective trials with NSAIDs
in subjects with mild to moderate AD. No detectable effects on
a variety of clinical outcome measures of AD progression were
found in controlled trials of naproxen, celecoxib, and rofecoxib (9,
54). Indeed, a recent trial of more than 2,500 individuals reported
no significant cognitive improvement after 4 years of treatment
with either naproxen or celecoxib (9). In addition, a large phase III
Therapeutic trial of NSAIDs inhibits subsequent, but not extant, neuronal CCEs. (A) Neuronal
CCEs were first observed in frontal cortical layers II/III at 6 months of age and persisted for 2 or
more years in the R1.40 animals. Neuronal CCEs were not observed in deeper cortical layers
V/VI until 12 months of age. (B–M) R1.40 transgenic mice at 6 months of age were fed control (B,
C, H, and I), ibuprofen-containing (D, E, J, and K) or naproxen-containing (F, G, L, and M) diets
for 6 months. (B and H) Control diet–fed mice exhibited expression of cyclin D (large arrows) in
NeuN-positive neurons in frontal cortex layers II/III and layers V/VI. (D, F, J, and L) Ibuprofen- or
naproxen-containing diet–fed mice exhibited expression of cyclin D in a subset of NeuN-positive
neurons in layers II/III (D and F), with minimal expression of cyclin D in layers V/VI (J and L). (C,
E, G, I, K, and M) Merged images. Sections were stained with NeuN (red), and nuclei were coun-
terstained with DAPI (blue). FISH analysis with a DNA probe specific for mouse chromosome 16
demonstrated the presence of a subset of neuronal nuclei with 3 or 4 spots of hybridization (small
arrows) in all treatment groups in cortical layers II/III (C, E, and G, insets) and only the control diet
group in cortical layers V/VI (I, K, and M, insets). Scale bars: 100 μm (B–M); 10 μm (insets).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
clinical trial of flurbiprofen was recently completed without any
evidence of cognitive improvement in AD subjects (55). The inabil-
ity of NSAID treatment of R1.40 mice to reverse CCEs once they
begin to appear is consistent with the human trial data and sug-
gests that, unlike initiation, the progression of AD may be inde-
pendent of the presence of inflammation in the brain.
It must be considered that the NSAIDs are acting in multi-
ple different ways in these studies. First, the NSAIDs may act
to block microglial activation via inhibition of cyclooxygenase
directly in the brain, a mechanism that is supported by our pres-
ent findings. Second, NSAIDs are well recognized to also inhibit
cyclooxygenase in the systemic immune system, and this could
have secondary consequences for altered infiltration and/or acti-
vation of peripheral immune cells within the brain. Although
we cannot rule this out, Iba1-positive microglia in 6-month-old
R1.40 mice (with or without NSAIDs) did not stain with CD45
(data not shown). Third, NSAIDs may have off-target effects.
Some studies have demonstrated that an acute, relatively short-
term administration of selected NSAIDs to AD mouse models
results in decreased production of longer Aβ peptides, in favor of
shorter, less-amyloidogenic isoforms (56), although not all stud-
ies have observed this effect (57). This would offer a potential
nonimmune mechanism of NSAID action.
Unlike these previous reports, we found that chronic NSAID
treatment of predepositing R1.40 mice resulted in no significant
alterations in the steady-state levels of Aβ1–40, Aβ1–42 or in their
ratio. Many factors might explain these differences; we believe the
main factor is the long-term nature of our trials in animals that are
predepositing. Most of the chronic NSAID studies in mouse mod-
els of AD have focused on Aβ deposition as the primary outcome
measure, which is supported by our recent finding of reduced Aβ
deposition in the R1.40 mouse model of AD when treated with
375 ppm ibuprofen from 15 to 24 months of age (N.H. Varvel, G.E.
Landreth, B.T. Lamb, and K. Herrup, unpublished observations).
Our data are also consistent with recent studies demonstrating
that treatment of the Tg2576 mouse model of AD with identical
doses of ibuprofen and naproxen for 30 days in predepositing ani-
mals does not alter Aβ levels (48).
It has also been suggested that NSAIDs regulate Bace1 expres-
sion levels through a PPARγ-mediated pathway. Certain NSAIDs,
including ibuprofen, are agonists for PPARγ (58). Activation of
PPARγ with the selective agonist pioglitazone reduces Bace1 levels
as well as steady-state levels of CTFβ in a mouse model of AD (17),
but any direct relationship between ibuprofen administration
and suppression of Bace1 levels remains unresolved. In our stud-
ies, we found no differences in CTFβ levels between the NSAID
treatment groups, confirming our conclusion that the primary
effect of the drugs in reducing the cell cycle response of the R1.40
neurons is to inhibit inflammatory processes and not to suppress
or change APP processing.
In summary, neuronal CCEs are among the earliest manifesta-
tions of AD pathogenesis, and we argue that their presence rep-
resents a valuable biomarker for risk of neurodegeneration. The
induction of these events was blocked by the chronic adminis-
tration of 2 commonly used NSAIDs, ibuprofen and naproxen,
when administered to animal models of the disease prior to the
initial induction of neuronal CCEs. Once neuronal CCEs were ini-
tiated, however, NSAIDs were incapable of reducing the levels of
cell cycle proteins within the affected cells. Our present findings
suggest that successful human therapies for AD are best begun at
the earliest possible times.
Mice. The R1.40 transgene is a full genomic copy of human APP (a 650-kb
insert from a yeast artificial chromosome clone) carrying the Swedish
(K670M/N671L) mutation associated with early-onset familial AD. Creation
of the R1.40 transgenic mouse strain and subsequent backcrossing to inbred
strains has been described previously (27–29). Age- and gender-matched
nontransgenic C57BL/6J animals served as controls in all analyses. Homo-
zygous R1.40 animals, maintained on the C57BL/6J genetic background,
were crossed to Bace1–/– animals (provided by R. Yan, Cleveland Clinic;
ref. 59), also maintained on the C57BL/6J genetic background, to generate
F1 R1.40;Bace1+/– animals. F1 animals were intercrossed to generate animals
homozygous for the R1.40 transgene and homozygous for the Bace1 knock-
out allele as well as Bace1–/– animals lacking the R1.40 transgene. Homozy-
gous R1.40 animals, maintained on the C57BL/6J genetic background, were
also crossed to Cx3cr1gfp/gfp animals (provided by R.M. Ransohoff, Cleve-
land Clinic; ref. 60), on the C57BL/6J genetic background, to generate F1
R1.40;Cx3cr1+/gfp animals. F1 animals were intercrossed to generate animals
homozygous for the R1.40 transgene and heterozygous for the Cx3cr1 knock-
out allele as well as Cx3cr1+/gfp animals lacking the R1.40 transgene. Animals
were housed at the Cleveland Clinic Biological Resources Unit, a facility fully
Quantification of inhibition of neuronal CCEs in
NSAID therapeutic trial. Percentages of NeuN-pos-
itive neurons in cortical layers II/III and layers V/VI
exhibiting expression of cyclin A (A) or cyclin (B) as
well as 3 or 4 spots of hybridization with FISH for
DNA probes from mouse chromosome 16 (C) and
13 (D) were calculated in R1.40 transgenic mice
fed control, ibuprofen-containing, and naproxen-
containing diets as well as in nontransgenic mice
fed the control diet (n = 5). *P < 0.001 versus con-
trol diet–fed R1.40 transgenic mice.
3700? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
accredited by the Association of Assessment and Accreditation of Laboratory
Animal Care. All procedures were approved by the Institutional Animal Care
and Use Committee of the Cleveland Clinic.
NSAID treatments. Ibuprofen and naproxen were obtained from Sigma-
Aldrich. These compounds were formulated into standard, color-coded ani-
mal chow by Research Diets at a final concentration of 375 ppm ibuprofen
or naproxen. Male and female R1.40 mice at 3 and 6 months of age and age-
matched nontransgenic controls were fed drug-supplemented or control
chow ad libitum for either 3 or 6 months. During each trial, animals were
weighed on a weekly basis. Mice were sacrificed following the experimental
period and processed for either histological or biochemical analyses.
LPS administration. Male and female R1.40 mice and age-matched non-
transgenic controls (2 months old) were injected with LPS (20 μg/animal,
L2880; Sigma-Aldrich) or PBS via intraperitoneal injection. Animals were
subject to LPS or PBS once a day for 4 consecutive days and sacrificed 4
hours after the final injection (35).
Antibodies. Antibodies used for these studies include rabbit polyclonal
cyclin A antibody (ab 7956; Abcam), specific for the C-terminal domain of
cyclin A2; mouse monoclonal cyclin D antibody (ab 31450; Abcam); mouse
monoclonal NeuN antibody (Chemicon), used as a neuronal-specific marker;
and CT-15, specific for the C-terminus of APP (diluted 1:5,000, provided by
E.H. Koo, UCSD, San Diego, California, USA). Secondary antibodies were
conjugated to various fluorescent Alexa Fluor dyes (Invitrogen).
Histology and immunohistochemistry. Animals were prepared for histology
analysis as described previously (23). Animals were deeply anesthetized with
Avertin, a mixture of 2,2,2-tribromoethanol (Sigma-Aldrich) and 2-methyl-
2-butanol (Sigma-Aldrich) dissolved in water at a final ratio of 1:2:80 (0.02
cc/mg body weight); they were perfused transcardially with 0.1 M sodium
phosphate buffer, pH 7.4, followed by 4% paraformaldehyde in 0.1 M
sodium phosphate buffer. The brain was dissected, immediately removed
from the cranium, and transferred to fresh 4% paraformaldehyde at 4°C
overnight. To perform double fluorescent immunohistochemistry, sections
were first rinsed in PBS containing 0.1% Triton X-100 (PBST). Sections were
subsequently incubated for 1 hour at room temperature in 10% goat serum
in PBS to block nonspecific binding. All primary antibodies were diluted
in PBST and applied overnight at 4°C. After rinsing in PBS, the slides were
incubated for 2 hours with a secondary antibody, which was conjugated
with various fluorescent Alexa Fluor dyes (diluted 1:1,000; Invitrogen). The
sections were then rinsed in PBS followed by incubation in the 10% goat
serum blocking solution for 1 hour and incubation with the second primary
antibody (raised in a different species than the first primary antibody) over-
night at 4°C. Sections were subsequently rinsed in PBS, and the second sec-
ondary antibody, conjugated with a difference fluorescent dye, was applied
to sections for 2 hours at room temperature. After rinsing, all sections were
mounted in DAPI Hardest Reagent (Vector Laboratories) under a glass cov-
erslip. Antibody concentrations used for immunohistochemistry were as
follows: rabbit anti-cyclin A2 (diluted 1:200; Abcam), mouse anti-cyclin D
(diluted 1:200; Abcam), mouse anti-NeuN (diluted 1:500; Chemicon), and
fluorescently tagged secondary antibodies (diluted 1:1,000; Invitrogen).
FISH was performed as described previously (61) using a mouse-specific
DNA probe (480C6, from the RPCI-22 BAC library) containing 150 kb of
genomic sequence from the region that encodes the endogenous Sim2 gene
located on mouse chromosome 16. Confocal images were acquired with a
Zeiss LSM 510 META laser scanning confocal microscope (Carl Zeiss Micro-
Imaging) using an argon laser (excitation, 488 nm), a diode laser (excitation,
405 nm), and a 100× Plan Apo, NA 1.4, oil immersion objective. The image
represents a maximum intensity projection of a z stack consisting of 14
images, each with an optical slice of 0.8 μm.
Image analysis. Morphometric analysis of the activation state of microglia
was performed on Iba1-stained images acquired at a ×10 objective using
Image-Pro Plus software (Media Cybernetics). Images were captured with
a pixel depth of 8 bits, RGB, and TIFF format. The sections from differ-
ent animals corresponding to the same level were imaged with identical
exposure settings. At least 3 images taken at random planes covering the
frontal cortex from each animal and from 3 animals per group were used
for the analysis. Several earlier reports have used FF as a means to classify
microglia or macrophages based on their appearance and morphological
characteristics (32–34). FF measures contour irregularity of a cell by the for-
mula (4π × Ac)/Pc2, where Ac and Pc represent the cell’s area and perimeter,
respectively. We selected 1–100 as a filter range. FF value is higher in bushy
cells, characterized by large bodies and numerous rather sparsely ramified
processes, whereas cells with small, round bodies and abundant systems of
regularly branched processes classified as ramified return lower FF values
(32–34). In total, 583 (nontransgenic), 352 (R1.40), 188 (R1.40;Bace1–/–),
555 (ibuprofen), and 686 (naproxen) Iba1-immunoreactive microglial cells
per experimental group were analyzed.
As another morphometric criteria for microglial activation, the mass-radi-
us fractal dimension was also calculated (32–34). In contrast to FF analysis,
in fractal dimension analysis, cells with more ramified processes exhibit
higher fractal values than bushy cells with fewer ramified processes.
Neuronal cell counts. For each treatment group, we examined 5 animals at
each age. A total of 5 evenly spaced sections containing the frontal cortex
were double stained for the neuronal marker NeuN and for cyclin A or
cyclin D. The area located between 2.5 mm and 3.4 mm anterior to the
bregma was identified in each section analyzed. We scored NeuN-positive
cells within cortical layers II/III or V/VI for the presence or absence of the
cell cycle marker. Only cells with a discernible portion of their nucleus
in the section were scored. For each of the 5 sections, the percentage of
NeuN-positive cells exhibiting immunoreactivity for the cell cycle marker
was tabulated, and the percentages for the 5 sections analyzed in each ani-
mal were averaged. For each age and treatment group, the percentages were
then averaged over all 5 animals. Adjacent sections that had undergone
processing for FISH were tabulated in a similar fashion, in which neurons
were scored for the presence or absence of 3 or 4 spots of hybridization. All
counts were performed in a blinded fashion, and data were analyzed with
the Student’s t test, 2-tailed (GraphPad Prism, GraphPad Software).
Western blot of tissue homogenates. Mice were killed by cervical disloca-
tion, and their brains were removed, divided sagittally (after removing
cerebellum), and snap frozen. Tissues were subsequently homogenized in
10 volumes of Tris-buffered saline (50 mM Tris, pH 7.4; 150 mM NaCl;
1 mM EDTA; and 0.1% Triton-X) with protease inhibitor cocktail (Sigma-
Aldrich). Total brain homogenates were subsequently sonicated to shear
DNA and centrifuged at 14,000 g for 30 minutes at 4°C to remove nuclei
and cell debris. Total protein concentrations were determined using the
Pierce bicinchoninic acid (BCA) Protein Assay Kit (Pierce). Brain protein (30
μg) was run on a Novex NuPage, 4%–12% Bis-Tris gel (Invitrogen), and then
transferred to a PVDF membrane. The Western blots were subsequently
incubated with antibody CT-15 against the C terminus of APP (62).
Aβ ELISA. Mouse brain hemispheres were extracted with 70% formic
acid, homogenized at 100,000 g, and neutralized (to pH 7.5) with 10× 2 M
Tris (pH. 11.0). Brain extracts were frozen once and then tested for Aβ1–40
and Aβ1–42 levels. Aβ1–40 and Aβ1–42 were measured in brain extracts using
specific sandwich ELISAs developed for mouse brain tissue containing the
human APP transcript. Aβ peptides were captured using either the Aβ1–40-
selective monoclonal antibody B113 or the Aβ1–42-selective monoclonal
antibody A387. Bound peptides were detected using alkaline phosphatase
mAb B436 (raised against Aβ1–12) combined with a CSPD-Sapphire II
Luminescence substrate (Applied Biosystems) in order to quantify alka-
line phosphatase activity. Relative luminescent units were measured using
a standard 96-well luminometer. Each sample was assayed in triplicate
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 12 December 2009
at appropriate dilutions so that relative luminescent units were in the
standard curve range included on each plate. Standard curves were con-
structed with synthetic Aβ1–40 or Aβ1–42 peptides (Bachem) spiked into
C57BL/6J brain extract and diluted in a manner similar to that for the
samples using medium.
Statistics. Data are presented as mean ± SEM unless otherwise specified.
Differences between groups were determined by unpaired Student’s t test,
2-tailed. A P value less than 0.05 was considered significant.
This work was supported by NIH grants AG023012 (to B.T. Lamb)
and AGO24494 (to K. Herrup and B.T. Lamb), the Alzheimer’s
Association (to B.T. Lamb), and an anonymous foundation. We
thank R.M. Ransohoff for Cx3cr1gfp/gfp mice and R. Yan for Bace1–/–
mice. We thank Maryanne Pendergast and the Case Western
Reserve University Neurosciences Imaging Center for assistance
with the confocal microscopy.
Received for publication May 1, 2009, and accepted in revised form
September 16, 2009.
Address correspondence to: Karl Herrup, Rutgers, The State Uni-
versity of New Jersey, Department of Cell Biology and Neurosci-
ence, Nelson Biological Laboratories, Busch Campus, 604 Allison
Road, Piscataway, New Jersey 08854-8082, USA. Phone: (732) 445-
1794; Fax: (732) 445-3306; E-mail: email@example.com.
Or to: Bruce T. Lamb, Lerner Research Institute, Cleveland Clinic,
Department of Neurosciences NC3-164, 9500 Euclid Avenue,
NC30, Cleveland, Ohio 44195-0001, USA. Phone: (216) 444-3592;
Fax: (216) 444-7927; E-mail: firstname.lastname@example.org.
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