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Published in Volume
125, Issue 1 (January 2, 2015) J Clin Invest. 2015;125(1):350–364.
doi:10.1172/JCI77487.
Copyright © 2015, American Society for Clinical Investigation
Research Article
Prostaglandin signaling suppresses beneficial microglial function in Alzheimer’s disease models
Jenny U. Johansson1, Nathaniel S. Woodling1,2, Qian Wang1, Maharshi Panchal1, Xibin Liang1, Angel Trueba-Saiz3, Holden D. Brown1, Siddhita D. Mhatre1, Taylor Loui1 and Katrin I. Andreasson1
1Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California, USA.
2Neurosciences Graduate Program, Stanford University, Stanford, California, USA.
3Functional and Systems Neurobiology Department, Cajal Institute, CSIC, Madrid, Spain.
Address correspondence to: Katrin I.
Andreasson, Stanford University School of Medicine, 1201 Welch Road,
MSLS P210, Stanford, California 94305, USA. Phone: 650.498.5855; E-mail:
kandreas@stanford.edu.
First published December 8, 2014 Submitted: June 10,
2014; Accepted: October 30,
2014.
Microglia, the innate immune cells of
the CNS, perform critical inflammatory and noninflammatory functions
that maintain normal neural function. For example, microglia clear
misfolded proteins, elaborate trophic factors, and regulate and
terminate toxic inflammation. In Alzheimer’s disease (AD), however,
beneficial microglial functions become impaired, accelerating synaptic
and neuronal loss. Better understanding of the molecular mechanisms that
contribute to microglial dysfunction is an important objective for
identifying potential strategies to delay progression to AD. The
inflammatory cyclooxygenase/prostaglandin E2 (COX/PGE2)
pathway has been implicated in preclinical AD development, both in human
epidemiology studies and in transgenic rodent models of AD. Here, we
evaluated murine models that recapitulate microglial responses to Aβ
peptides and determined that microglia-specific deletion of the gene
encoding the PGE2 receptor EP2 restores microglial chemotaxis
and Aβ clearance, suppresses toxic inflammation, increases
cytoprotective insulin-like growth factor 1 (IGF1) signaling, and
prevents synaptic injury and memory deficits. Our findings indicate that
EP2 signaling suppresses beneficial microglia functions that falter
during AD development and suggest that inhibition of the COX/PGE2/EP2 immune pathway has potential as a strategy to restore healthy microglial function and prevent progression to AD.
Introduction
Alzheimer’s disease (AD), a neurodegenerative disorder associated
with protein misfolding and aggregation in the brain, is the most common
memory disorder, and its prevalence is expected to triple by the year
2050 (1). The widely considered “amyloid hypothesis” of AD causation posits that accumulation of amyloid β42 (Aβ42) triggers inflammation, tau hyperphosphorylation, and synaptic and neuronal loss, leading to cognitive decline (2, 3). Recent studies, however, indicate that brain Aβ42 accumulates in subjects that do not exhibit dementia, which suggests that Aβ42 accumulation may be necessary but not sufficient for development of cognitive impairment (4) and that additional factors are required to tip the balance toward progression to AD dementia.
Recent genetic studies of late-onset AD have identified AD-associated
genes that are involved in the innate immune response and are expressed
in microglia, the resident myeloid cells of the CNS. Microglial genes
associated with AD include CD33 (5–7), TREM2 (8, 9), and CR1 (10, 11); together with additional studies (12), these findings are indicative of an important role of microglia in maintaining local brain homeostasis and preventing Aβ42-mediated synaptic and inflammatory injury. Notably, clearance of accumulating Aβ42 is dependent on effective sensing by microglia (mediated by chemokines), followed by Aβ42 degradation. Moreover, prolonged exposure to proinflammatory cytokines or accumulating Aβ42 peptides cause microglia to lose their normal abilities to clear toxic proteins and control inflammation (13, 14), a detrimental phenotype in the context of age-associated Aβ42
accumulation. Thus, microglia are emerging as critical regulators of
innate immune responses in AD and, more broadly, in other
neurodegenerative disorders, and understanding the molecular and
cellular mechanisms that cause microglial dysfunction may help identify
strategies to restore healthy microglial function and prevent
development of AD.
A longstanding observation in epidemiological studies of normal aging
populations has been that NSAIDs, which inhibit cyclooxygenase-1
(COX-1) and COX-2 and prostaglandin (PG) production, prevent development
of AD (15–18). In addition, early-stage AD is characterized by increased cerebrospinal fluid levels of PGE2 (19, 20), supporting the hypothesis that inflammatory actions of brain COX/PGE2
may underlie preclinical development of AD. Consistently, studies in AD
model mice demonstrate reduced amyloid pathology with global deletion
of individual PGE2 G protein–coupled receptors (21–23), and additional studies have shown a suppressive signaling effect of the PGE2 receptor EP2 on Aβ42 phagocytosis (24, 25).
These studies, along with the recent demonstration of a broad
regulatory function of EP2 signaling on cell cycle, cytoskeletal, and
immune genes in quiescent microglia (26),
suggest that microglial EP2 signaling may be a general suppressor of
immune and nonimmune processes that protect against onset and
progression of AD pathology. To investigate this hypothesis, we used in
vitro and in vivo mouse models that recapitulate acute and chronic
aspects of microglial responses to Aβ peptides. Our findings demonstrate
that microglial EP2 signaling suppresses multiple processes critical to
microglial maintenance of homeostasis in vivo, notably microglial
chemokine generation and chemotaxis, clearance of Aβ peptides,
resolution of innate inflammatory responses to Aβ42, and
trophic factor generation and signaling. We further demonstrate that
ablation of microglial EP2 signaling prevents cognitive impairment and
loss of synaptic proteins in AD model mice.
Results
EP2 signaling exerts age-associated and opposing effects on proinflammatory and chemokine gene expression in response to Aβ42 oligomers. Aβ42 oligomers are early inducers of synaptic and neuronal injury in AD model mice (27). In addition to their direct disruption of synaptic function, Aβ42 oligomers generate a robust NF-κB– and IFN-regulatory factor 1–dependent (IRF1-dependent) inflammatory response (28)
that can secondarily injure synapses and neurons. To determine the
function of EP2 signaling in young and aged immune responses to
oligomeric Aβ42 peptides, we assayed the effects of the selective EP2 agonist butaprost in macrophages stimulated with Aβ42 oligomers; because yields of viable microglia suitable for culture experiments are very low when harvested from adult brain (29),
we examined peritoneal macrophages (which share many properties with
microglia) harvested from both young (4 months) and aged (21 months)
C57B6/J mice. We found that Ep2 mRNA was significantly induced in aged but not young macrophages in response to Aβ42 oligomers (5 μM; Figure 1A). Consistently, Aβ42
oligomers induced a robust inflammatory transcriptional response in
aged but not young macrophages that was further increased by
costimulation with 1 μM butaprost (Figure 1B). Aβ42-induced increases in IL-1β generation and secretion were further amplified with butaprost (Figure 1C and Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI77487DS1),
which suggests that myeloid EP2 signaling increases inflammasome
generation of IL-1β. Conversely, expression of the chemokines MCP-1 and
MIP-1α, which are involved in myeloid cell recruitment to sites of
injury, was suppressed with butaprost both basally and with Aβ42 stimulation (Figure 1D and Supplemental Figure 1, B and C). Finally, expression of Aβ peptide clearance enzymes, notably Neprilysin, Insulysin, and Mmp9, was also suppressed with EP2 activation in Aβ42-stimulated macrophages (Figure 1E). Taken together, these findings demonstrate an age-dependent pattern of gene regulation by EP2 signaling in the context of Aβ42-induced
innate immune responses, with induction of proinflammatory factors
(including IL-1β, COX-2, iNOS, and NADPH oxidase subunits) and
suppression of chemokines and proteases important in microglial
migration and Aβ42 oligomer clearance.
EP2 ablation increases microglial chemotaxis to nascent amyloid plaques in the APP-PS1 mouse model. Given that EP2 signaling strongly suppressed generation of chemokines in response to oligomeric Aβ42,
we investigated whether EP2 signaling negatively affects microglial
chemotaxis in vivo, using the APP-PS1 (APPSwe-PS1ΔE9) mouse model of
familial AD. This transgenic line coexpresses the human APPSwe and PS1ΔE9
mutant transgenes and exhibits Aβ peptide plaque deposition beginning
at 5 months, with later onset of synaptic loss and spatial memory
deficits after 8–9 months of age (30).
We found that before significant Aβ plaque accumulation had begun at 3
months, a robust microglial response was already underway, characterized
by increased expression of the cytoskeletal protein Iba1 and the lysosomal glycoprotein Cd68 (Figure 2A); the chemokine Mip1a began increasing at 3 months, with a significant rise by 6 months (Figure 2B), presumably in response to accumulating Aβ42 peptide oligomers and fibrils.
Global deletion of Ep2 in APP-PS1 mice (referred to herein as APP-PS1 Ep2–/–) reduced cerebral cortical IL-1β protein at 5 months of age and increased hippocampal Mip1a mRNA, cortical MIP-1α protein, and Insulysin mRNA (Figure 2, C–E), consistent with the in vitro findings shown in Figure 1. Interestingly, APP-PS1 Ep2–/– hippocampus exhibited significantly increased levels of Iba1 and Cd68 compared with control APP-PS1 hippocampus (Figure 2F), suggestive of an altered activation state in microglia lacking Ep2.
We then tested whether EP2-mediated regulation of MIP-1α was
associated with altered chemotaxis of microglia to sites of accumulating
Aβ peptides in APP-PS1 hippocampus (Figure 2G). At 5 months, a time point at which Aβ peptides begin to accumulate in this model, deletion of Ep2 in the APP-PS1 Ep2–/– mice increased microglial recruitment to nascent Aβ plaques, as assayed by quantification of IBA1+ microglia surrounding newly formed Aβ plaques (Figure 2H).
Additional quantification of microglia around small, medium, and large
Aβ plaques demonstrated a significant effect of genotype and plaque size
(Figure 2I). Levels of CD68 were significantly increased in APP-PS1 Ep2–/– mice (Figure 2J). We did not find differences in levels of Aβ42
between genotypes at this age, presumably because Aβ peptide
accumulation is very low at 5 months, in contrast to later ages of 8–9
months, at which APP-PS1 Ep2–/– mice exhibited a
reduction in cumulative Aβ peptide load (Supplemental Figure 2). Taken
together, these findings suggest that at the earliest stages of
pathology in APP-PS1 mice, inhibition of EP2 signaling resulted in
beneficial microglial responses to accumulating Aβ peptides, with
suppressed proinflammatory IL-1β generation and increased MIP-1α
expression and microglial chemotaxis to sites of Aβ peptide
accumulation.
Conditional deletion of Ep2 in microglia increases microglial activation and clearance of Aβ peptides.
We next used a microglial conditional knockout strategy to directly
examine microglial EP2 function in chemotaxis and clearance of Aβ
peptide in vivo. The Cd11b-Cre recombinase line leads to excision
of floxed sequences in cells of the myeloid lineage, including
monocytes, macrophages, and microglia, and has been successfully used to
examine neuroinflammatory responses in brain (26, 29, 31). We injected 17-month-old Cd11b-Cre Ep2fl/fl and control littermate Cd11b-Cre mice intracortically with FITC-conjugated fibrillar Aβ42 peptides and examined them 48 hours later to quantify microglial activation and remaining fluorescent Aβ42 peptide (Figure 3A). The remaining FITC+ staining area and intensity, quantified in a blinded fashion (see Methods), indicated that clearance of Aβ42 peptides was significantly higher in Cd11b-Cre Ep2fl/fl mice than in Cd11b-Cre controls (Figure 3B). Although absolute numbers of microglia were not counted, intensities of IBA1 and CD68 were increased in Cd11b-Cre Ep2fl/fl cortex for any given level of remaining FITC+ Aβ42 area (P < 0.0001 between slopes; Figure 3C). These findings support a primary function of microglial Ep2 in suppressing recruitment and activation of microglia that clear Aβ42 peptides chronically in the APP-PS1 model and acutely after intracortical Aβ42 peptide injection.
Microglial EP2 signaling regulates inflammatory and noninflammatory pathways in vivo in response to Aβ42 peptides.
Although microglia function in innate immune brain responses, they are
also intimately associated with neurons and synapses and perform
essential nonimmune functions important to normal neural function. These
include maintaining structural plasticity by pruning and elimination of
synapses, clearing apoptotic cells, and generating trophic and
neurogenic factors (32). Our findings thus far suggested a harmful function of microglial EP2 signaling both in vitro and in vivo in models of Aβ42
inflammation, with potentiation of proinflammatory responses,
suppression of immune cell trafficking to sites of Aβ peptide
accumulation, and suppression of Aβ peptide clearance. To identify
additional functions of microglial EP2 signaling, we turned to an
unbiased approach and examined microglial-specific gene expression in
response to i.c.v. injection of Aβ42 peptides. Aβ42 peptide injection i.c.v. not only generates a robust, long-lasting innate immune response to Aβ42 (21), but also disrupts memory consolidation (33),
and thus represents a model in which to test effects of microglial EP2
on transcriptional responses and functional outcomes that are altered in
response to Aβ42 peptides. We performed microarray analysis on RNA isolated from adult microglia from 8-month-old Cd11b-Cre Ep2fl/fl and Cd11b-Cre mice 48 hours after injection of i.c.v. Aβ42 peptides.
We examined 3 genetic comparisons (absolute fold change ≥1.5, P < 0.05; Figure 4A). Gene Ontology (GO) expression analysis for the comparison between Aβ- versus vehicle-injected Cd11b-Cre mice showed the Immune System Process as the most highly enriched (enrichment score, 94.42). Expression levels of microglial Ep2 were increased 1.30-fold (P = 0.013) at 48 hours after i.c.v. Aβ in the Cd11b-Cre
control genotype. Unsupervised hierarchical clustering of
differentially expressed genes revealed a striking distinction between
the i.c.v. Aβ and i.c.v. vehicle treatment groups (Supplemental Figure
3A). Ingenuity Pathway Analysis (IPA) of upstream regulatory
transcription factors demonstrated 2 major nodes of inflammatory gene
regulation, Nfkb and Irf7 (Supplemental Figure 3B). In this comparison, Cox2, which is upstream of EP2, was highly induced in vivo in microglia from i.c.v. Aβ42–treated
mice (Supplemental Figure 3C). Application of IPA revealed that many
Aβ-regulated genes were also regulated by COX-2 and PGE2 (Supplemental Figure 3B).
Functional annotation of the 416 transcripts differentially expressed in Aβ- versus vehicle-treated Cd11b-Cre
mice was carried out using the Database for Annotation, Visualization
and Integrated Discovery (DAVID; see Methods). This analysis revealed 20
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were
significantly enriched, almost all of which corresponded to inflammatory
signaling networks (Figure 4B). Comparison of Aβ-treated Cd11b-Cre Ep2fl/fl versus Cd11b-Cre mice revealed 55 regulated genes (Figure 4A), and hierarchical clustering of these genes across conditions demonstrated a clear segregation of Aβ-regulated genes in Cd11b-Cre Ep2fl/fl mice (Figure 4C). Comparison of KEGG pathways revealed shared pathways between the Aβ-treated Cd11b-Cre and vehicle-treated Cd11b-Cre groups and between the vehicle-treated Cd11b-Cre Ep2fl/fl and vehicle-treated Cd11b-Cre groups (Figure 4D). The complete set of enriched KEGG pathways in the Cd11b-Cre Ep2fl/fl versus Cd11b-Cre comparison included cell cycle, proteolysis, and immune pathways (Supplemental Figure 3D).
Interestingly, the majority of differentially regulated genes in the Aβ-treated Cd11b-Cre Ep2fl/fl versus Aβ-treated Cd11b-Cre comparison were not regulated by Aβ, but were specifically changed with microglial Ep2 deletion (38 genes; Figure 4A). This suggested that rather than simply reversing Aβ42-induced inflammatory changes, Cd11b-Cre Ep2fl/fl
microglia engaged alternative response pathways. Functional annotation
of these 38 genes using DAVID revealed an enrichment of PPAR signaling
pathway genes, including retinol dehydrogenase-13 (Rdh13), retinoid X receptor γ (Rxrg), and lipoprotein lipase (Lpl) (Figure 4, D and E). Rdh13
(1.78-fold increase) participates in the endogenous synthesis of
retinoic acid (RA) that binds and activates RXR subunits. PPARγ/RXR
heterodimers inhibit proinflammatory gene expression (reviewed in ref. 34) and increase phagocytosis of Aβ peptides (35). Rxrg, along with RXR and LXR heterodimers, increases expression of the cholesterol transporter ABCA1 (36) and apolipoprotein E (ApoE) (37), proteins that enhance proteolytic degradation of soluble Aβ peptides (38, 39);
recent studies indicate that administration of the FDA-approved RXR
agonist bexarotene (Targretin) reduces interstitial levels of soluble Aβ
peptides and rescues behavioral deficits in AD model mice (40, 41). Lpl,
which functions in lipoprotein remodeling and cholesterol transport,
and whose expression is driven by RA and RXR/LXR transcriptional
activity, was increased 1.52-fold with deletion of microglial Ep2.
The upregulation of these genes is suggestive of induction of
antiinflammatory and Aβ-clearing nuclear hormone receptor signaling
genes in the response of EP2-deficient microglia to Aβ42 peptides in vivo. Added to this was the induction of H+ transporting ATPase (Atp6v0d2; 1.92-fold induction; Figure 4E), a proton pump expressed in lysosomes of myeloid cells. Atp6v0d2 participates in degradation of proteins targeted to the lysosome (42), suggestive of a potential role in Aβ42 degradation.
Insulin-like growth factor 1 (IGF1) is upregulated in vivo in microglia derived from Cd11b-Cre Ep2fl/fl brains. In addition, we found an unexpected increase in Igf1 mRNA levels in microglia derived from i.c.v. Aβ–treated Cd11b-Cre Ep2fl/fl mice (Figure 4E). Whereas at the organismal level, reduced IGF1 signaling increases longevity (43),
at the cellular level, IGF1 promotes cell survival through the PI3K/AKT
pathway and RasGTPase/RAF-1/MEK pathways, and in brain, IGF1 signaling
promotes synaptogenesis, neurogenesis, angiogenesis, and neuroprotection
(44).
Although IGF1 receptors are expressed on all cell types in the CNS, in
general, IGF1 is synthesized in the liver and is transported to the
brain bound to IGF1 binding proteins. Exceptions include postnatal brain
development, where microglia transiently express IGF1 that supports
developing layer V neurons (45), and following brain injury, where microglia express IGF1 and astrocytes and neurons increase IGF receptor expression (44). Validation of the EP2-dependent regulation of IGF1 was carried out in aged primary macrophages, where Igf1 mRNA expression was found to be suppressed by the EP2 agonist butaprost (Figure 4F). Taken together, our unbiased analyses indicated the activation of multiple beneficial pathways in Ep2-deficient
microglia in vivo, including antiinflammatory nuclear hormone, Aβ
clearing, and trophic pathways. Moreover, these pathways were activated
in parallel with suppression of the proinflammatory response (see
below).
Cd11b-Cre Ep2fl/fl mice stimulated i.c.v. with Aβ42 show increased IGF1 receptor signaling and reduced inflammation. We next tested whether microglial Ep2 deletion increased IGF1 signaling following stimulation with i.c.v. Aβ42.
Binding of IGF1 to its tyrosine kinase receptor (IGF1R) leads to
phosphorylation of IGF1R and recruitment of multiple scaffold proteins,
including insulin receptor substrates 1–4 (IRS-1–IRS-4) and Src homology
2 domain–containing transforming protein 1 (44), which bind with different time courses to phosphorylated IGF1R to transduce IGF1 signaling. Here, Cd11b-Cre Ep2fl/fl and Cd11b-Cre mice were treated i.c.v. with vehicle or Aβ42 as above, and hippocampi were analyzed 48 hours later for phosphorylation of IGF1R (Figure 5,
A and B, and Supplemental Figure 4, A and B). Quantification of
immunoprecipitated total and phosphorylated IGF1R (p-IGF1R) demonstrated
a significant increase in the p-IGF1R/total IGF1R ratio in Cd11b-Cre Ep2fl/fl versus Cd11b-Cre mice treated with i.c.v. Aβ42 (Figure 5B).
We also measured levels of IRS-1, one of several scaffolding proteins
that bind to phosphorylated IGF1R, and found a trend toward increased
binding in the Cd11b-Cre Ep2fl/fl genotype (Supplemental Figure 4C). No changes in levels of total IGF1R were noted between genotypes (Supplemental Figure 4D).
To examine the effect of increased IGF receptor signaling in Cd11b-Cre Ep2fl/fl
mice, we quantified phosphorylation levels of candidate proteins known
to be phosphorylated downstream of IGF1R/PI3K in cerebral cortex (Figure
5, C
and D, and Supplemental Figure 4, E and F). Levels of AKT phosphorylated
by PDK1 at Thr308 and levels of p-ERK1/2 were significantly increased
in i.c.v. Aβ–stimulated Cd11b-Cre Ep2fl/fl mice compared with Cd11b-Cre
controls. Similarly, phosphorylation of proteins directly targeted by
AKT, including GSK3α and GSK3β, also increased in i.c.v. Aβ–treated Cd11b-Cre Ep2fl/fl mice. Phosphorylation of BAD at Ser112 and ERK1/2 at Thr202/Tyr204 was suppressed in i.c.v. Aβ–treated Cd11b-Cre mice, but preserved in i.c.v. Aβ–treated Cd11b-Cre Ep2fl/fl mice. Taken together, these data indicate that increased IGF1R signaling in Cd11b-Cre Ep2fl/fl mice resulted in enhanced AKT/ERK signaling in the setting of Aβ stimulation in vivo.
As AKT signaling promotes beneficial antiinflammatory effects, we examined the effect of microglial Ep2
deletion on the neuroinflammatory response to i.c.v. Aβ. Levels of
cytokines and chemokines were measured using Luminex multiplex assay in
hippocampi isolated from Cd11b-Cre Ep2fl/fl and Cd11b-Cre mice treated or not with i.c.v. Aβ42 at 48 hours. In Cd11b-Cre controls, i.c.v. Aβ42 broadly significantly upregulated 22 factors, compared with 7 factors differentially regulated in Cd11b-Cre Ep2fl/fl mice (Figure 6A
and Supplemental Figure 5); these included the proinflammatory
cytokines IL-1α, IL-1β, IL-17A, and IL-6. The chemokines MIP-1α, MIP-1β,
and RANTES were also highly regulated by EP2 in this model, but in the
opposite manner to that found in the APP-PS1 model, which suggests that
regulation of chemokines by EP2 in vivo is context specific and may
differ between chronic and acute Aβ42 stimulation. In the i.c.v. Aβ42 model, Aβ42
is administered acutely and initiates a robust inflammatory response,
in contrast to transgenic expression in the APP-PS1 model, which leads
to chronic, low-level production of Aβ peptides. Alternatively, lower
chemokine generation in the i.c.v. Aβ42 model may reflect a more benign inflammatory milieu of Cd11b-Cre Ep2fl/fl
brain at the 48-hour time point. Taken together, the findings of
increased brain IGF1 signaling in combination with reduced production of
inflammatory ILs suggest a beneficial effect of microglial Ep2 deletion in the context of Aβ42-generated immune responses.
Conditional deletion of microglial Ep2 prevents a functional deficit in novel object recognition (NOR).
Neuroinflammatory responses can significantly impair cognitive function
via effects of cytokines, proteases, and oxidative stress on synapses
and neurons. We tested whether microglial Ep2 negatively affects memory function in the setting of Aβ42-mediated
inflammation. Control experiments examining locomotor, anxiety, and
Y-maze performance did not show differences between Cd11b-Cre and Cd11b-Cre Ep2fl/fl
mice (Supplemental Figure 6). Next, we used NOR, a memory task that
relies on the innate preference of mice to spend more time with a novel
rather than a familiar object, which is significantly disrupted in the
i.c.v. Aβ42 model (33).
NOR requires the normal function of the perirhinal and entorhinal
cerebral cortex and the hippocampus. As illustrated in Figure 6B, on day 1 after i.c.v. injection of either vehicle or Aβ42,
mice were habituated to an empty arena and later allowed to briefly
explore 2 identical objects (training session, 0 hours). After 24 hours,
mice were again put in the arena; however, one of the objects used
during training was replaced by a novel object. Recognition memory
(recognition session, 24 hours) of the old versus new object was
assessed as the discrimination index (DI), the ratio of time spent
exploring the old object to time spent exploring both objects. A DI of
~50% is characteristic of the training session, where there is no
preference for either of the 2 objects; with normal memory
consolidation, decreased DI during the recognition session reflects less
time spent with the old object and more time exploring the new object,
as was shown for the i.c.v. vehicle–injected Cd11b-Cre and Cd11b-Cre Ep2fl/fl groups (Figure 6C). These control mice performed normally (P < 0.05, paired t test), in contrast to i.c.v. Aβ42–injected Cd11b-Cre mice, which were not able to recognize the old from the new object at 24 hours. Importantly, this Aβ42-induced NOR deficit was prevented in Cd11b-Cre Ep2fl/fl mice (P < 0.01, paired t test). Taken together, our findings support a highly beneficial effect of microglial Ep2
ablation, resulting in suppression of proinflammatory responses,
increased signaling through the IGF1R pathway, and prevention of NOR
memory deficits.
Conditional deletion of microglial Ep2 reduces spatial memory deficits in APP-PS1 mice. We then tested the effect of deletion of microglial Ep2
in the APP-PS1 model, in which progressive amyloid accumulation and
inflammation lead to synaptic loss and hippocampal-dependent memory
deficits. Male APP-PS1 Cd11b-Cre Ep2fl/fl mice and APP-PS1 Cd11b-Cre
controls were aged to 9 months, the time point at which spatial memory
deficits begin in this line. Hippocampal-dependent spatial memory
performance in the radial arm maze (RAM) was tested (Figure 7A).
Behavior in the RAM was quantified over the last 3 days of a 6-day
period in which mice were evaluated for their ability to locate a new
rewarded choice arm after visiting a previously rewarded sample arm. For
the first 3 days of testing, no significant differences were observed
for any genotype. However, the second 3 days of testing showed a
significant difference between APP-PS1 Cd11b-Cre and APP-PS1 Cd11b-Cre Ep2fl/fl mice for mean number of errors per trial (P < 0.05, APP-PS1 Cd11b-Cre versus APP-PS1 Cd11b-Cre Ep2fl/fl; P = 0.089, APP-PS1 Cd11b-Cre versus Cd11b-Cre; Figure 7A). APP-PS1 Cd11b-Cre mice also showed increased latency to make a correct choice compared with Cd11b-Cre mice (P < 0.05), and this was partially improved with deletion of microglial Ep2 (P = 0.075, APP-PS1 Cd11b-Cre versus APP-PS1 Cd11b-Cre Ep2fl/fl; Figure 7A).
We then assessed effects of microglial Ep2 deletion on synaptic integrity by quantifying levels of candidate synaptic proteins (Figure 7, B and C). The loss of synaptophysin correlates with progression of cognitive decline in AD development (46);
moreover, studies in transgenic mouse AD models have demonstrated that
presynaptic proteins are disrupted early during amyloid accumulation (21, 47), with loss of postsynaptic markers occurring at more advanced stages of pathology (48). At 8–9 months, we found a decrease in levels of the presynaptic proteins synaptophysin and SNAP-25 in APP-PS1 Cd11b-Cre mice compared with Cd11b-Cre controls that was rescued by deletion of microglial Ep2;
no changes were demonstrated in the postsynaptic proteins PSD-95 and
GLUA1 in either group. Thus, the loss of presynaptic markers was
prevented with deletion of microglial Ep2 in the APP-PS1 Cd11b-Cre Ep2fl/fl cerebral cortex.
Consistent with the i.c.v. Aβ42 model, Igf1 mRNA levels were elevated in APP-PS1 Cd11b-Cre Ep2fl/fl versus APP-PS1 Cd11b-Cre hippocampus (Figure 7D). We also assessed levels of chemokine expression and total Aβ42 in APP-PS1 Cd11b-Cre and APP-PS1 Cd11b-Cre Ep2fl/fl mice. For chemokine expression, we found significantly increased Mip1a expression in APP-PS1 Cd11b-Cre Ep2fl/fl versus APP-PS1 Cd11b-Cre 9-month-old male mice (Figure 7E); this was consistent with the increased Mip1a expression observed in APP-PS1 Ep2–/– mice and in vitro data in aged macrophages (Figures 1 and 2). In APP-PS1 Cd11b-Cre Ep2fl/fl cerebral cortex, we found a 23.7% decrease in mean total Aβ42 levels (P = 0.17; Figure 7F), which was not as marked as the effect in APP-PS1 Ep2–/– mice, in which mean cortical levels of Aβ42 were reduced by 36.5% at 8–9 months (P < 0.01; Supplemental Figure 2). The lack of significant decline in Aβ42 levels with microglial Ep2 deletion may be due to incomplete excision of floxed sequences, and we have previously demonstrated that the Cd11b-Cre recombinase line is approximately 50% efficient in excising floxed Ep2 sequences (26). Incomplete excision of floxed sequences is common in many Cre-mediated
systems, in which recombinase activity frequently results in
cell-specific knockdown of gene expression. Alternatively, it is
possible that chronic accumulation of Aβ peptide by 9 months of age in
this transgenic model may overwhelm the ability to clear Aβ, in spite of
the beneficial effects of microglial Ep2 deletion.
To examine the effect of chronic suppression of microglial EP2
signaling in the APP-PS1 model, we again examined phosphorylation of
candidate proteins in the IGF1R/PI3K pathway in cerebral cortex of
9-month-old APP-PS1 Cd11b-Cre Ep2fl/fl, APP-PS1 Cd11b-Cre, and Cd11b-Cre mice (Figure 7G
and Supplemental Figure 7). In this chronic model of Aβ stimulation,
there was a significant induction of AKT signaling, with increased
p-PDK1 (Ser241), p-AKT (Thr308), p-GSK2α (Ser21), p-GSK3β (Ser9), and
p-BAD (Ser112) in APP-PS1 Cd11b-Cre versus Cd11b-Cre mice. Interestingly, APP-PS1 Cd11b-Cre Ep2fl/fl mice exhibited abolished AKT signaling induction, which suggests that in the context of microglial Ep2 deletion, IGF1R/PI3K signaling was more similar to Cd11b-Cre levels. Moreover, as many pathways feed into the AKT pathway, the normalization of AKT signaling to Cd11b-Cre levels in APP-PS1 Cd11b-Cre Ep2fl/fl brain may also reflect chronic effects of multiple beneficial microglial functions activated as a result of microglial Ep2 deletion.
Discussion
Microglial activities represent critical lines of defense against the
development of neurodegenerative disease; microglia clear misfolded
proteins, elaborate trophic and regenerative factors, and regulate and
terminate toxic inflammation. Recent studies point to a steady decline
of these normal microglial functions in aging and in AD. In AD,
microglia not only lose their capacity to clear Aβ peptides but also
develop a persistent proinflammatory phenotype that does not resolve,
accelerating neuronal and synaptic injury (49, 50).
In this study, using in vitro and in vivo genetic strategies, we found
that microglial EP2 activity negatively regulates multiple and distinct
beneficial functions that are critical in opposing the harmful effects
of accumulating Aβ42. Together, our findings in distinct mouse models of Aβ inflammation demonstrated that deletion of microglial Ep2 restores chemotaxis, Aβ clearance, regulation of inflammatory responses, and trophic factor generation and signaling (Figure 8), with behavioral correlates of preventing cognitive deficits and loss of synaptic proteins.
In vitro, aged but not young macrophages increased EP2 expression in response to Aβ42 oligomers. This interesting age-dependent regulation was associated with a robust immune response to Aβ42 that was significantly enhanced with coadministration of EP2 agonist. The observed difference in inflammatory responses to Aβ42
between young and aged macrophages is consistent with observations
demonstrating that innate immune responses increase with age, in part
through increased activation of NF-κB–driven inflammatory pathways (51).
Moreover, the age dependence of EP2 upregulation and EP2-driven
inflammatory gene expression is highly relevant to the pathogenesis of
AD, for which the primary risk factor is age. We also noted a converse
suppressive effect of EP2 signaling on expression of chemokines and
Aβ-degrading enzymes, proteins that are beneficial in controlling
extracellular levels of Aβ peptides in vivo. Thus, in response to Aβ42
oligomers, EP2 signaling upregulated expression of deleterious
oxidative and proinflammatory factors and reduced expression of
beneficial chemotactic and proteolytic genes. This dual regulatory
function suggests that microglial EP2 signaling may be playing a harmful
role in vivo in response to early Aβ42 generation and accumulation. This was confirmed in young 5-month-old APP-PS1 Ep2–/–
mice, which exhibited not only increased chemotaxis of microglia to
sites of nascent amyloid plaques, but increased levels of Aβ proteases
and CD68. Moreover, in Cd11b-Cre Ep2fl/fl mice, microglial deletion of Ep2 significantly accelerated clearance of intracortically injected Aβ peptides.
The opposing regulation of MIP-1α by Aβ peptides and by EP2 signaling
is particularly interesting. Inflammatory chemokines such as MIP-1α,
MIP-1β, and MCP-1 are β chemokines that are important in recruitment of
monocytic cells, including macrophages and microglia. Upon binding to
their G protein–coupled receptors (CCR1 and 5 for MIP-1α), these
chemokines initiate cytoskeletal reorganization and cellular migration.
Of all chemokines, MIP-1α in particular was highly induced by 6 months
in the APP-PS1 model, suggestive of a specific role for microglial
MIP-1α in the inflammatory response to Aβ peptides. Moreover, in
addition to its well-established function of inducing chemotaxis of
immune cells, MIP-1α can also activate immune cells to produce
inflammatory cytokines. Thus, the suppression of MIP-1α expression
coupled to the induction of proinflammatory responses in the context of
Aβ stimulation and EP2 signaling is unexpected, but likely suggests a
complex transcriptional regulation of MIP-1α and cytokine release.
Nonetheless, the functional outcome of EP2 signaling in the context of
Aβ stimulation — worsening of the inflammatory response through
increased proinflammatory gene expression, as well as suppression of
MIP-1α and microglial chemotaxis to sites of Aβ peptide accumulation —
is detrimental.
To explore the broader regulatory role of microglial EP2 signaling in response to Aβ42, we used an unbiased approach and identified a class of genes upregulated independently of Aβ42 in Cd11b-Cre Ep2fl/fl mice. Among these genes were the nuclear hormone transcriptional pathway components Rxrg, Rdh13, and Lpl,
components of PPAR and RA pathways that promote antiinflammatory,
trophic, and phagocytic microglial activities and that are highly
beneficial in AD models (35).
RXRγ binds the clinical compound bexarotene and increases levels of
ApoE4 and ABCA1 lipidator important for Aβ peptide clearance by ApoE;
activation of RXR/LXRs by bexarotene may lower interstitial soluble Aβ
peptide levels and improve cognitive performance (40, 52).
The regulation of RXRγ in microglia is also interesting in the context
of the circadian clock, which is modulated by retinoid receptors via
regulation of BMAL1 transcription (53).
Thus, it is conceivable that EP2 regulation of RXRγ could modulate the
forward arm of the clock. Recent studies have linked deficient
BMAL:CLOCK/NPAS2 activity to disrupted energy homeostasis, aging, and
neurodegeneration (54–56), which suggests that the prominent increase in EP2 signaling in aging myeloid cells shown in Figure 1 may negatively affect myeloid cellular clock function.
Intriguingly, Ep2-deficient microglia responded to Aβ42
peptides by upregulating IGF1; in brain, this growth factor is not only
potently neuroprotective, neurogenic, and antiinflammatory, but also
enhances synaptic and neuronal plasticity and improves cognitive
function (44).
Although IGF1 can be produced in most tissues, most of it is generated
by the liver and released into the circulation. Circulating IGF1
decreases with age and in AD; moreover, AD is characterized by insulin
resistance and decreased IGF1R signaling (57). In mouse models of AD, circulating serum IGF1 can accelerate clearance of Aβ peptides (58). In our studies of mice acutely administered i.c.v. Aβ42, deletion of microglial Ep2
significantly increased microglial IGF1R signaling in hippocampus.
Examination of protein phosphorylation downstream of IGF1R and PI3K
after Aβ administration revealed marked induction in PI3K/AKT signaling
in Cd11b-Cre Ep2fl/fl mice, which suggests that deletion of Ep2
in microglia preserves and enhances the AKT signaling response to a
noxious stimulus, in this case Aβ peptides. AKT signaling is beneficial
and antiinflammatory; consequently, Cd11b-Cre Ep2fl/fl
mice exhibited a reduced inflammatory response and were also able to
function normally in the NOR memory task. Taken together, these findings
argue for a beneficial protective effect of microglial Ep2 deletion in the context of an acute Aβ stimulus.
Ablation of microglial Ep2 signaling similarly elicited a
beneficial effect in the context of chronic Aβ stimulation, notably in
aging 9-month-old APP-PS1 Cd11b-Cre Ep2fl/fl mice. Spatial memory testing was improved in APP-PS1 Cd11b-Cre Ep2fl/fl mice, as were levels of presynaptic proteins. APP-PS1 Cd11b-Cre Ep2fl/fl
animals also demonstrated increased levels of IGF1 and MIP-1α
expression. Interestingly, in this chronic model, AKT signaling was
broadly induced in APP-PS1 Cd11b-Cre mice compared with Cd11b-Cre controls; however, deletion of microglial Ep2 normalized the APP-PS1 Cd11b-Cre Ep2fl/fl phosphoprotein levels toward Cd11b-Cre levels. This suggests that chronic microglial Ep2
deletion led to a more benign inflammatory and oxidative environment;
this, in addition to the trophic and antiinflammatory effects of chronic
IGF1R/PI3K signaling, might be expected to blunt a reactive increase in
AKT signaling.
Taken together, our findings suggest a broad effect of conditional deletion of Ep2 in microglia in mouse models of AD. This conclusion was based on the selectivity of the Cd11b promoter–driven expression of Cre recombinase in brain to microglia, as demonstrated by Boillee et al.: crosses of Cd11b-Cre mice to Rosa26-lacZ mice showed Cre recombination in the CNS that was restricted to microglia (31). In a previous study using this Cre recombinase line, we observed approximately 50% reduction of floxed Ep2 sequences in microglia and in peritoneal macrophages (26).
Peripherally, CD11b protein is normally expressed in monocytes and
tissue-specific macrophages, and to a lesser extent in subpopulations of
granulocytes, mature B lymphocytes, and CD4+ T lymphocytes (59, 60). Because Cd11b-Cre
recombinase will also reduce levels of peripheral myeloid EP2, we
cannot exclude a potential contribution of peripheral myeloid cell Ep2
deletion to our present findings. There is increasing evidence of
communication between the periphery and the CNS, particularly in
experimental models of aging, where circulating factors can alter
hippocampal neurogenesis, synaptic plasticity, and cognitive function (61, 62). Validation with cell-specific Cre
recombinase lines that are selectively expressed only in brain
microglia or peripherally in macrophages/monocytes will be very helpful
in parsing out the relative contributions of these cell types to models
of AD and aging.
In conclusion, our data demonstrate that microglial EP2 suppresses
multiple beneficial functions that are essential to combat the toxic
effects of Aβ42 peptides on synapses and memory function.
These findings suggest that microglial EP2 activity hastens pathological
progression to AD. By virtue of its broad regulatory effect on
beneficial microglial functions, inhibition of inflammatory EP2
signaling may be a promising strategy to restore healthy microglial
function, arrest the progression of Aβ42-driven pathology, and prevent development of AD.
Methods
Animals. All strains were in the C57BL/6 background. APP-PS1 mice (APPSwe-PS1ΔE9; ref. 63) were crossed to Ep2–/– and Ep2+/+ mice (originally from R. Breyer, Vanderbilt University, Nashville, Tennessee, USA; refs. 64, 65). Cd11b-Cre mice (31),
provided by G. Kollias (Alexander Fleming Biomedical Sciences Research
Center, Vari, Greece) and D. Cleveland (UCSD, La Jolla, California,
USA), efficiently excise floxed sequences in microglia and macrophages (26, 29, 31). C57BL/6 Ep2fl/+ mice have been previously described (26).
All mice were housed in an environment controlled for lighting (12-hour
light/12-hour dark cycle), temperature, and humidity, with food and
water available ad libitum.
Macrophage cell cultures. Peritoneal
macrophages were harvested from young (3–5 months) and aged (19–21
months) female mice (except for MIP-1α ELISA, for which males were
used). Viable and healthy macrophages can be obtained from both young
and aged mice and survive well in culture; this is not the case for
adult-derived microglia, which may not survive consistently in culture
and yield highly variable data. In addition, macrophage yields are
significantly higher per mouse than are microglia yields, thus
minimizing the number of mice required for the study. Mice were injected
with 1.5 ml 3% (w/v) thioglycollate medium (BD Diagnostic Systems) into
the peritoneal cavity, and primary macrophages were isolated 3–4 days
later by flushing with ice-cold HBSS (HyClone). Cells were seeded at a
density of 2 × 106 cells/well onto 12-well plates for RNA and 8 × 104
cells/well in 96-well plates for ELISA measurements in DMEM
supplemented with 10% heat-inactivated FBS (HyClone), 100 U/ml
penicillin and streptomycin, and 1 mM sodium pyruvate and maintained at
5% CO2 at 37°C. After overnight culture, cells were washed twice with medium to remove nonadherent cells before treatment.
Primary microglia culture. Primary
microglia were isolated from the brains of postnatal day 7 C57BL/6J
mouse pups obtained from Charles River Laboratories. Primary microglia
were isolated using the Neural Tissue Dissociation Kit (P), MACS
Separation Columns (LS), and magnetic CD11b Microbeads from Miltenyi
Biotec. Microglia were grown in culture for 3–5 days before being
treated in each experiment as previously described (26).
Preparation of oligomeric and fibrillar Aβ42. Oligomeric and fibrillar Aβ peptide species were generated as previously described (21, 66).
Quantitative real-time PCR (qPCR). RNA
isolation, cDNA production, and SYBR-Green-based qPCR (QuantiTect SYBR
Green Kit; Qiagen) were performed as previously described (29) using the standard curve method and normalizing to 18S and Gapdh.
Primers were designed by PrimerQuest (Integrated DNA Technologies) and
synthesized by Integrated DNA Technologies. Primer sequences are listed
in Supplemental Table 1.
Intracortical and i.c.v. injection of Aβ42 peptides. FITC-conjugated fibrillar Aβ42 peptides (185 μM; rPeptide) or vehicle were administered intracortically to 17-month-old female and male Cd11b-Cre and Cd11b-Cre Ep2fl/fl littermates (67, 68). Mice were under isoflurane anesthesia during surgery. 1 μl fibrillar Aβ42
(185 pmol) or vehicle was delivered to opposite hemispheres of the
cerebral cortex over 10 minutes using a 32-gauge Hamilton syringe; after
injection, the needle was left in place for 5 minutes and then
withdrawn over 4 minutes to prevent backflow. The stereotaxic injections
were placed at the following coordinates from the bregma: mediolateral,
1.2 mm; anteroposterior, 1.2 mm; dorsoventral, 1.5 mm. After injection,
each mouse recovered spontaneously on a heated pad. At 48 hours after
injection, mice were euthanized, and brains were rapidly harvested.
Intracortical Aβ42–injected brains were sectioned coronally
on a freezing microtome at 40-μm intervals. From each mouse, 30 sections
were chosen for immunostaining with IBA1 and CD68 antibodies, with
sections covering the span of the needle injection track and at least 5
sections anterior and posterior to the injection track. FITC+
sections (average, 15 per mouse) were analyzed in a blinded manner by
FITC intensity or area covered above a threshold level, and mean IBA1
and CD68 intensities were determined within the FITC+ area. For i.c.v. Aβ peptide injections, fibrillar Aβ peptide species (40 pmol) or vehicle (PBS) were administered to Cd11b-Cre and Cd11b-Cre Ep2fl/fl mice as previously described (69).
Stereotaxic injections were placed at the following coordinates from
the bregma: mediolateral, –1.0 mm; anteroposterior, –0.3 mm;
dorsoventral, –2.5 mm. After injection, each mouse recovered
spontaneously on a heated pad.
Microglia quantification. Images were
acquired in the hippocampal area by a blinded experimenter at ×400
magnification of all identifiable hippocampal plaques with surrounding
microglia, and with 5 z planes spaced 5 μm apart covering the
plaque and microglia. A circle centered on the plaque with diameter of
75 μm was stamped, and the cropped image was analyzed. IBA1+ microglia were manually counted going through the z
planes and included if they were touching the plaque with either the
cell body or processes. Plaques were grouped into small (<250 μm2), medium (250–575 μm2) and large (>575 μm2)
sizes. Percent CD68 coverage area above threshold level was determined
in the cropped image for the extended focus, including all z planes. 5 sections (average, 10.6 ± 1.1 plaques) were analyzed per mouse.
Immunostaining. Immunostaining of paraformaldehyde-fixed mouse sections was carried out as previously described (21).
Antibodies used include anti-rat CD68 (1:1,000 dilution; AbD Serotec),
anti-rabbit IBA1 (1:2,000 dilution; Wako), and anti-mouse 6E10 for human
Aβ (1:2,000 dilution; Covance). Secondary antibodies and detection
reagents included donkey FITC-conjugated anti-mouse, Cy3-conjugated
donkey anti-rabbit, and DyLight 649–conjugated anti-rat antibody
(Jackson ImmunoResearch Laboratories). Biotinylated secondary antibodies
(Vector Labs) were used at a dilution of 1:250. Rabbit and mouse sera
(Jackson ImmunoLabs) were used as negative controls in place of primary
antibodies on adjacent sections.
Image acquisition for immunostaining.
Imaging of immunostained sections was done using a Nikon Eclipse E600
microscope (Nikon Instruments) and a Hamamatsu Orca-ER digital camera
(Hamamatsu Photonics). Images were analyzed using the measurements
module of Volocity 4.3.2 image analysis software (Improvision).
Western analysis and ELISA and multi-antibody array measurement of phosphoproteins.
For quantification of phosphorylated IGF1R, hippocampi were homogenized
in 10 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,
1% Nonidet P-40, 1 mM sodium orthovanadate, and protease inhibitors.
After centrifugation to remove insoluble material, supernatants were
incubated overnight with bead-conjugated antibody against IGF1R (Santa
Cruz), and immunocomplexes were collected with centrifugation; beads
were washed in homogenization buffer before separation by SDS-PAGE.
Blots were incubated with primary antibodies against phosphotyrosine
(4G10, Platinum, Millipore) to detect p-IGF1R; membranes were stripped
and reprobed with anti–IGF-I Receptor β (Cell Signalling). Levels of
MIP-1α and MCP-1 (R&D Systems) and IL-1β (BD Biosciences) were
measured by ELISA. For quantification of phosphoproteins, 36 μg cortical
lysates were applied in 60 μl to the PathScan AKT Signaling Antibody
Array kit (Chemiluminescent Readout, Cell Signaling Technology); the
assay was carried out according to the manufacturer’s instructions. This
assay simultaneously detects 16 phosphoproteins, many belonging to the
AKT signaling cascade. Capture antibodies specific for the
phosphorylated proteins were spotted on nitrocellulose-coated glass
slides, and lysates were incubated overnight at 4°C. After washing,
arrays were incubated with a biotinylated antibody, followed by
chemiluminescent film detection. Spot intensities were quantified using
ImageJ.
Aβ peptide quantification. 5M guanidine-extracted Aβ42 peptides were precipitated with ethanol to remove the guanidine, and Aβ42 peptides were measured by ELISA as previously described (22, 70).
Isolation of primary microglia from adult mouse brain. For isolation of microglia, 8- to 9-month-old Cd11b-Cre and Cd11b-Cre Ep2fl/fl mice were administered i.c.v. injection of Aβ42
fibrils or saline. At 48 hours after surgery, mice were sacrificed by
transcardiac perfusion with cold heparinized 0.9% NaCl. Brains were then
removed from the mice and pooled, 2 brains of the same genotype per
sample, to ensure adequate cell and RNA yield. The brains were then
enzymatically dissociated and isolated using magnetic CD11b microbeads
(Miltenyi Biotec) according to the manufacturer’s protocol.
RNA isolation and microarray. RNA purification from primary microglia of adult Cd11b-Cre and Cd11b-Cre Ep2fl/fl mice was performed using TRIzol
(Life Technologies) followed by the RNeasy Mini Kit (Qiagen). RNA
quality was assessed using a BioAnalyzer (Agilent) and determined to be
sufficient for microarray analysis (RNA Integrity Number >7.0 for all
samples). cDNA synthesis, labeling, hybridization, and scanning were
performed by the Stanford Protein and Nucleic Acid (PAN) Facility using
GeneChip Mouse Gene 2.0 ST arrays (Affymetrix). Microarray data were
statistically analyzed using Partek software (Partek) to identify
differentially expressed genes between groups by ANOVA using an
unadjusted P value of <0.05. Genes that had a fold change of
>1.5 between genotypes were used for unsupervised hierarchical
clustering analysis. Database for Annotation, Visualization and
Integrated Discovery (DAVID) functional annotation software (version
6.7; NIAID, NIH) was used to identify KEGG molecular pathways
significantly overrepresented among lists of differentially expressed
genes. Data were deposited in GEO (accession no. GSE57181).
NOR. The NOR task, based on the ability
of mice to show preference for novel versus familiar objects when
allowed to explore freely (71), is disrupted after i.c.v. administration of Aβ peptides and fibrils (33).
NOR was performed during the light cycle. Mice were individually
habituated to an open arena (50 cm × 50 cm, dim light, 24°C) on day 1.
During the subsequent training session, 2 identical objects were placed
into the arena, and exploratory behavior was monitored for 5 minutes. On
day 2, mice were placed back into the same arena, in which one of the
objects used during training was replaced by a novel object of similar
dimensions but a different shape/color, and exploratory behavior was
monitored for 5 minutes. Digital video tracking (using an infrared
camera and vplsi Viewpoint software) of body movements and head position
was used to quantify locomotor and exploratory activities around the
objects (2-cm zone around the objects). Exploration behavior was
assessed by calculating DI, the ratio of time spent exploring the old
object to time spent exploring both objects (expressed as a percentage).
A DI of ~50% is associated with correct training and no object
preference; a significant decrease in DI is characteristic of
recognition of the novel object. To evaluate memory, comparisons were
made for each group between the recognition (24 hours) and training (0
hours) sessions. Paired t tests were performed between time points. Behavioral testing was performed by experimenters blinded to genotype.
RAM. The RAM is a spatial memory test
that assesses working and reference memory over several consecutive
days; the protocol was adopted from that of Clelland et al. (72).
Previous experiments demonstrated that no deficits exist in nonmnemonic
behaviors (visual and sensory-motor abilities) in APP-PS1 mice (30).
Mice were food restricted for 1 week prior to testing and maintained at
85%–90% body weight for the duration of testing, and water was provided
ad libitum. All testing occurred at the beginning of the dark phase of
the diurnal light/dark cycle or in dim light during the end of the light
cycle. The testing apparatus consisted of a wooden 8-arm RAM with
Plexiglas walls, elevated 50 cm from the floor and surrounded by visual
cues. 3 days before testing, mice were habituated to the maze. Day 1 of
the habituation phase consisted of a 10-minute group exposure (by cage)
to the RAM, in which all arms were unblocked but no food was provided.
The second day, mice were individually exposed twice to the maze for 5
minutes, with each arm baited with food rewards. This was repeated on
the third day, but with only 3 doors open. Following habituation, mice
were tested for their ability to separate sample (familiar) from choice
(new) arms in the RAM. Cage order was randomized throughout testing.
Mice received 2 trials per day (consisting of 1 sample phase and 1
choice phase) for 6 consecutive days. During the sample phase, all arms
except the start arm and the sample arm were blocked off. The mouse was
permitted to visit the sample arm and retrieve the food reward. Mice
were retrieved from the maze after either (a) spending 10 seconds in the
sample arm after retrieving the pellet or (b) exiting the sample arm.
During the choice phase, arms in the start and sample (unrewarded)
locations and an additional correct (rewarded) location were open. Mice
that entered the correct arm were considered to have made correct
choices. Mice that made incorrect choices (i.e., entered the sample arm
or reentered the start arm) were allowed to self-correct, enter the
correct arm, and retrieve the pellet before being removed from the maze.
Sample and correct arms were randomized for each combination, such that
sample arm was either to the left or right of the start arm, and for
each combination, the start arm was located in 1 of 2 locations
perpendicular to either the correct or the sample arm.
Statistics. Data are expressed as mean ± SEM. Statistical comparisons were made with GraphPad Prism software using Student’s t
test (2-tailed unless otherwise indicated; for 2 groups meeting normal
distribution criteria by Shapiro-Wilk normality test), Mann-Whitney U
test (for 2 groups not meeting normal distribution criteria), or 2-way
ANOVA with Bonferroni multiple-comparison tests (for groups across 2
variables, with multiple comparisons between groups). Linear regression
was used to analyze microglial markers in the FITC-Aβ intracortical
experiment. The linear model was chosen for goodness-of-fit of the
dataset (r2 = 0.88 and 0.93). Slopes were compared
using F test. For Luminex multiplex analysis, fluorescence intensities
that reached statistical significance with i.c.v. Aβ42 were transformed to relative concentrations (median z
score). Cluster analysis (Gene Cluster 3.0 and Java TreeView 1.0.13)
produced a separation of samples according to treatment and genotype.
For NOR analyses, paired t test was used to compare performance
between the 0- and 24-hour time points. Data were subjected to Grubbs’
test to identify the presence or absence of outlier data points for
exclusion from analysis. For all tests, a P value of 0.05 or less was considered significant.
Study approval. This study was conducted in accordance with NIH guidelines, and protocols were approved by the IACUC at Stanford University.
Supplemental data
Acknowledgments
This work was supported by NIH grant
RO1AG030209 (to K.I. Andreasson), NIH grant R21AG033914 (to K.I.
Andreasson), Alzheimer’s Association (to K.I. Andreasson), Swedish
Research Council (to J.U. Johansson), National Science Foundation (to
N.S. Woodling), and NRSA grant F31AG039195 (to N.S. Woodling). The
authors thank Damien Colas, Bayara Chuluun, Grace Hagiwara, and Craig
Heller for assistance in behavioral experiments; Frank Longo, Theo
Palmer, and Tony Wyss-Coray for use of equipment; and Suraj Pradhan and
the Stanford Human Immune Monitoring Center for Luminex measurements.
Footnotes
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