EP2 signaling exerts age-associated and opposing effects on proinflammatory and chemokine gene expression in response to Aβ 42 oligomers. Aβ 42 oligomers are early inducers of synaptic and neuronal injury in AD model mice (27). In addition to their direct disruption of synaptic function, Aβ 42 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 Aβ 42 peptides, we assayed the effects of the selective EP2 agonist butaprost in macrophages stimulated with Aβ 42 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 Aβ 42 oligomers (5 μM; Figure 1A). Consistently, Aβ 42 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). Aβ 42 -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 Aβ 42 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 Aβ 42 -stimulated macrophages (Figure 1E). Taken together, these findings demonstrate an age-dependent pattern of gene regulation by EP2 signaling in the context of Aβ 42 -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 Aβ 42 oligomer clearance.

Figure 1 EP2 signaling modulates inflammatory responses to Aβ 42 oligomers in an age-dependent manner. (A) Peritoneal macrophages of young and aged mice (4 and 21 months, respectively) were stimulated with Aβ 42 oligomers (5 μM). Ep2 mRNA was induced at 4 hours in response to Aβ 42 oligomers in aged macrophages (n = 5–6 per group; effect of Aβ 42 , P = 0.016, ANOVA). (B) Young and aged peritoneal macrophages were stimulated with Aβ 42 oligomers (5 μM) or vehicle, with or without the EP2 agonist butaprost (1 μM), and qPCR was performed at 4 hours (n = 5–6 per group). Aged — but not young — macrophages increased expression of inflammatory genes in response to Aβ 42 oligomers (effect of Aβ 42 in aged macrophages, Cox2, P = 0.013; Inos, P = 0.0056; gp91phox, P = 0.023; p67phox, P = 0.003; p47phox, P = 0.013), and these responses were amplified with butaprost costimulation. (C) Butaprost enhanced Aβ 42 oligomer–mediated IL-1β generation in aged macrophages at 6 hours by ELISA (n = 3 per group, effect of Aβ 42 , P = 0.0006). (D) EP2 agonist suppressed MCP-1 and MIP-1α generation (n = 5 per group; effect of butaprost for MCP-1, P = 0.0062; effect of Aβ 42 and butaprost for MIP-1α, P < 0.0001). (E) Expression of Neprilysin, Mmp9, and Insulysin mRNA was induced in aged macrophages with Aβ 42 oligomers, but suppressed with butaprost (n = 10–11 per group; for Neprilysin, effect of Aβ 42 , P = 0.0005, effect of butaprost, P = 0.0014; for Mmp9, effect of Aβ and butaprost, both P < 0.05; for Insulysin, effect of interaction, P < 0.05). (A–E) *P < 0.05; **P < 0.01, ***P < 0.001 as indicated, Bonferroni post-hoc.

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 Aβ 42 , 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 Aβ 42 peptide oligomers and fibrils.

Figure 2 Microglial chemotaxis to early accumulating Aβ peptides is enhanced with Ep2 deletion in 4- to 5-month-old APP-PS1 mice. (A) qPCR demonstrated preplaque increases in hippocampal Iba1 and Cd68 (effect of genotype, Iba1, P < 0.02; Cd68, P = 0.001; n = 5–9 per group). (B) Mip1a was increased early (effect of genotype, P = 0.01; n = 5–9 per group). (C) Decreased cerebral cortical IL-1β levels in APP-PS1 Ep2–/– versus APP-PS1 mice (n = 14–19 per group). Increased (D) Mip1a hippocampal mRNA (n = 8–13 per group) and MIP-1α cerebral cortical protein (n = 13–21 per group) and (E) Insulysin (n = 8–13 per group) in APP-PS1 Ep2–/– versus APP-PS1 brains. (F) Increased Iba1 and Cd68 mRNA in preplaque 4-month-old APP-PS1 Ep2–/– hippocampus (n = 8–13 per group). (G) Representative Aβ plaque with surrounding IBA1+ and CD68+ microglia from 5-month-old hippocampus. Dashed circle indicates area around plaques used to quantify numbers of microglia and CD68 immunofluorescence. Scale bar: 25 μm. (H) Microglia around hippocampal plaques in 5-month-old mice; average IBA1+ microglia number increased with Ep2 deletion (n = 4–7 mice per group, total 102–122 plaques per group). (I) Ep2 deletion enhanced microglial recruitment to early plaques (effect of genotype, P = 0.0016; effect of plaque size, P < 0.0001; post-hoc P < 0.05 for medium-sized plaque). Small, <250 μm2; medium, 250–575 μm2; large, >575 μm2. (J) Increased CD68 staining in periplaque hippocampal microglia of APP-PS1 Ep2–/– mice (n = 5–6 mice per genotype, total 26–48 plaques per genotype). *P < 0.05, **P < 0.01, ANOVA with Bonferroni post-hoc (A, B, and I) or Student’s t test (C–H and J).

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 Aβ 42 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 Aβ 42 peptides and examined them 48 hours later to quantify microglial activation and remaining fluorescent Aβ 42 peptide (Figure 3A). The remaining FITC+ staining area and intensity, quantified in a blinded fashion (see Methods), indicated that clearance of Aβ 42 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+ Aβ 42 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 Aβ 42 peptides chronically in the APP-PS1 model and acutely after intracortical Aβ 42 peptide injection.

Figure 3 Conditional deletion of microglial Ep2 accelerates clearance of intracortically injected Aβ peptide. Cd11b-CreEp2fl/fl and Cd11b-Cre mice (17 months old, n = 6–7 per group) underwent intracortical injection of 185 pmol fibrillar Aβ 42 or vehicle, and cortical tissue was analyzed 48 hours later. (A) Representative images of sections at the injection site containing Aβ 42 deposits visualized by FITC fluorescence and immunostained for microglial markers IBA1 and CD68. Scale bar: 100 μm. (B) Remaining FITC-labeled Aβ peptide at 48 hours after injection was significantly reduced in Cd11b-Cre Ep2fl/fl versus Cd11b-Cre cerebral cortex (*P < 0.05, Student’s t test). (C) Relationship between intensity of microglial markers IBA1 and CD68 and the remaining FITC+ area showed a significant correlation (Cd11b-Cre, IBA1, r2 = 0.88, CD68, r2 = 0.93; Cd11b-Cre Ep2fl/fl, IBA1, r2 = 0.88, CD68, r2 = 0.88). Correlation of microglial IBA1 and CD68 levels with remaining FITC+ area showed significant difference between genotypes (P < 0.0001), with higher IBA1 and CD68 intensity present in Cd11b-Cre Ep2fl/fl sections per given FITC+ area.

Microglial EP2 signaling regulates inflammatory and noninflammatory pathways in vivo in response to Aβ 42 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 Aβ 42 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 Aβ 42 peptides. Aβ 42 peptide injection i.c.v. not only generates a robust, long-lasting innate immune response to Aβ 42 (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 Aβ 42 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. Aβ 42 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. Aβ 42 –treated mice (Supplemental Figure 3C). Application of IPA revealed that many Aβ-regulated genes were also regulated by COX-2 and PGE 2 (Supplemental Figure 3B).

Figure 4 Microglial EP2 signaling regulates distinct immune and non-immune pathways in response to i.c.v. Aβ 42 . Adult microglia were harvested for microarray analysis from brains of 8-month-old Cd11b-Cre and Cd11b-Cre Ep2fl/fl mice 48 hours after i.c.v. administration of either vehicle or Aβ 42 fibrillar peptides. (A) Venn diagram of the 3 comparisons. (B) KEGG pathways significantly enriched in Aβ 42 - versus vehicle-treated Cd11b-Cre mice. (C) Hierarchical clustering of 55 genes differentially regulated in the i.c.v. Aβ 42 –treated Cd11b-Cre Ep2fl/fl group versus the i.c.v. Aβ–treated Cd11b-Cre group. (D) KEGG pathways shared between comparisons, with the PPARγ pathway represented in 38 uniquely regulated genes in the Cd11b-Cre Ep2fl/fl i.c.v. Aβ 42 versus Cd11b-Cre i.c.v. Aβ comparison. Pathways that are not shared are shaded gray. (E) Expression levels for regulated genes compared between Cd11b-Cre Ep2fl/fl and Cd11b-Cre groups treated with i.c.v. Aβ 42 include genes encoding PPARγ signaling (Rxrg and Lpl), microglial lysosomal (Atp6v0d2), and trophic (Igf1) proteins. P values for main effect of Aβ 42 treatment are shown in the figure (Bonferroni post-hoc between genotypes, *P < 0.05, **P < 0.01, ***P < 0.001; n = 3–7 per group). (F) Butaprost suppressed expression of Igf1 in peritoneal macrophages of 15- and 21-month-old mice stimulated with Aβ 42 oligomers at 4 hours (*P < 0.05, **P < 0.01, Student’s t test; n = 5–6 per group).

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 Aβ 42 -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 Aβ 42 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 Aβ 42 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 Aβ 42 show increased IGF1 receptor signaling and reduced inflammation. We next tested whether microglial Ep2 deletion increased IGF1 signaling following stimulation with i.c.v. Aβ 42 . 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 Aβ 42 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. Aβ 42 (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).

Figure 5 Inhibition of microglial EP2 signaling increases brain IGF1/PI3K signaling after i.c.v. Aβ 42 administration. (A) Cd11b-Cre and Cd11b-Cre Ep2fl/fl mice were administered vehicle or Aβ 42 peptides i.c.v., and hippocampi were examined for IGF1R activation at 48 hours. Hippocampal lysates were immunoprecipitated with anti-IGF1R antibody and blotted against p-Tyr to detect p-IGF1R, IRS-1, and total IGF1R. A representative blot is shown. (B) Increased p-IGF1R/total IGF1R ratio in Cd11b-Cre Ep2fl/fl hippocampus after i.c.v. Aβ 42 administration (n = 6–9 per group; *P < 0.05, Student’s t test). (C) Diagram of the IGF1R/AKT pathway. (D) Quantification of phosphoproteins downstream of PI3K/AKT in cerebral cortex showed increased AKT and ERK1/2 signaling in i.c.v. Aβ–treated Cd11b-Cre Ep2fl/fl mice (n = 6–9 per group; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test).

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. Aβ 42 at 48 hours. In Cd11b-Cre controls, i.c.v. Aβ 42 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 Aβ 42 stimulation. In the i.c.v. Aβ 42 model, Aβ 42 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. Aβ 42 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 Aβ 42 -generated immune responses.

Figure 6 Inhibition of microglial EP2 signaling decreases inflammation and rescues memory following i.c.v. Aβ 42 administration. (A) Cluster analysis producing separation for genotype and treatment is shown for 22 immune factors in cerebral cortex that were significantly upregulated in hippocampus 48 hours after i.c.v. Aβ 42 . Asterisks denote factors that were differentially regulated by microglial EP2 (see Supplemental Figure 5). (B) Overview of the NOR memory test, with the novel object shaded green. (C) DI (comparing 0-hour training session with 24-hour recognition session) demonstrated normal memory consolidation in control i.c.v. vehicle–treated Cd11b-Cre and Cd11b-Cre Ep2fl/fl mice, and absence of memory consolidation in the i.c.v. Aβ 42 –treated Cd11b-Cre cohort, which was significantly rescued by microglial Ep2 deletion in the i.c.v. Aβ 42 –treated Cd11b-Cre Ep2fl/fl cohort (n = 8–15 mice per group; **P < 0.01, *P < 0.05, paired 1-tailed Student’s t test).

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 Aβ 42 -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. Aβ 42 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 Aβ 42 , 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. Aβ 42 –injected Cd11b-Cre mice, which were not able to recognize the old from the new object at 24 hours. Importantly, this Aβ 42 -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).

Figure 7 Effects of microglial Ep2 deletion in 9-month-old male APP-PS1 mice on spatial memory performance, presynaptic protein levels, and PI3K/AKT signaling. (A) Cd11b-Cre, APP-PS1 Cd11b-Cre, and APP-PS1 Cd11b-Cre Ep2fl/fl cohorts were assessed for spatial memory performance in the RAM, using mean errors per trial and latency to make a correct choice as outcome measures (n = 7–11 per group). Whereas APP-PS1 Cd11b-Cre Ep2fl/fl and Cd11b-Cre animals made similar numbers of errors per trial during the course of testing, APP-PS1 Cd11b-Cre mice made significantly more (*P < 0.05, Mann-Whitney U test). Cd11b-Cre and APP-PS1 Cd11b-Cre Ep2fl/fl mice also made the correct choice more quickly than did APP-PS1 Cd11b-Cre mice (P < 0.05). (B and C) The loss of synaptophysin and SNAP-25 observed in APP-PS1 Cd11b-Cre mice was reversed in APP-PS1 Cd11b-Cre Ep2fl/fl mice (*P < 0.05, **P < 0.01, Student’s t test; n = 5–6 per group). The postsynaptic proteins PSD-95 and GLUA1 were not changed. (D) Increased Igf1 mRNA in APP-PS1 Cd11b-Cre Ep2fl/fl versus APP-PS1 Cd11b-Cre mice and in APP-PS1 Cd11b-Cre versus Cd11b-Cre mice (n = 6–10 per group; *P ≤ 0.05, Student’s t test). (E) Increased Mip1a mRNA expression in APP-PS1 Cd11b-Cre Ep2fl/fl versus APP-PS1 Cd11b-Cre hippocampus (n = 8–13 per group; ***P < 0.0001; Student’s t test). (F) Cerebral cortex from APP-PS1 Cd11b-Cre and APP-PS1 Cd11b-Cre Ep2fl/fl mice was assayed for Aβ 42 levels by ELISA (P = 0.17, Mann-Whitney 2-tailed t test; n = 5–6 per group). (G) Quantification of PI3K/AKT phosphoproteins in cerebral cortex of Cd11b-Cre, APP-PS1 Cd11b-Cre, and APP-PS1 Cd11b-Cre Ep2fl/fl mice showed significant induction of the AKT signaling pathway in APP-PS1 Cd11b-Cre mice that was absent in APP-PS1 Cd11b-Cre Ep2fl/fl mice (*P < 0.05, **P < 0.01, Student’s t test; n = 4–5 per group).

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. Aβ 42 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 Aβ 42 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 Aβ 42 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 Aβ 42 were reduced by 36.5% at 8–9 months (P < 0.01; Supplemental Figure 2). The lack of significant decline in Aβ 42 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.