J. Neurochem. (2012) 120, 795–805.
Amyloid-β peptide (Aβ), which is generated by the β- and γ-secretase-mediated proteolysis of β-amyloid precursor protein (APP), plays an important role in the pathogenesis of Alzheimer’s disease (AD). We recently reported that prostaglandin E2 (PGE2) stimulates the production of Aβ through both EP2 and EP4 receptors and that activation of the EP4 receptor stimulates Aβ production through endocytosis and activation of γ-secretase. We here found that transgenic mice expressing mutant APP (APP23) mice showed a greater or lesser apparent cognitive deficit when they were crossed with mice lacking EP2 or EP4 receptors, respectively. Mice lacking the EP4 receptor also displayed lower levels of Aβ plaque deposition and less neuronal and synaptic loss than control mice. Oral administration of a specific EP4 receptor antagonist, AE3-208 to APP23 mice, improved their cognitive performance, as well as decreasing brain levels of Aβ and suppressing endocytosis and activation of γ-secretase. Taken together, these results suggest that inhibition of the EP4 receptor improves the cognitive function of APP23 mice by suppressing Aβ production and reducing neuronal and synaptic loss. We therefore propose that EP4 receptor antagonists, such as AE3-208, could be therapeutically beneficial for the prevention and treatment of AD.
β-amyloid precursor protein
non-steroidal anti-inflammatory drugs
protein kinase A
Alzheimer’s disease (AD) is the most common neurodegenerative disorder of the central nervous system and the leading cause of adult onset dementia, affecting 5% of the population over the age of 65. Pathological characters of AD are accumulation of neurofibrillary tangles and senile plaques and senile plaques are composed of amyloid-β peptides (Aβ), such as Aβ40 and Aβ42 (Mattson 2004). In order to generate Aβ, β-amyloid precursor protein (APP) is first cleaved by β-secretase and then by γ-secretase (Sisodia and St George-Hyslop 2002). Monomeric Aβ easily self-assembles to form oligomers and protofibrils, which play an important role in the induction of the neuronal and synaptic loss that results in cognitive decline (Haass and Selkoe 2007). γ-Secretase is composed of four core components, including presenilin (PS)-1 and PS-2 (Haass 2004). Early onset familial AD is linked to three genes, app, ps1 and ps2 (Haass 2004), strongly suggesting that Aβ is a key factor in the pathogenesis of AD. Consequently, cellular factors that affect the production of Aβ represent good targets for drugs to prevent or treat AD.
It has been suggested that inflammation is important in the pathogenesis of AD; chronic inflammation has been observed in the brains of AD patients, and trauma to the brain and ischemia, both of which can activate inflammation, are major risk factors for the disease (Ikonomovic et al. 2004; Wyss-Coray 2006). Cyclooxygenase (COX), which exists as two subtypes, COX-1 and COX-2, is essential for the synthesis of prostaglandin E2 (PGE2), a potent inducer of inflammation. COX-1 is expressed constitutively, whereas COX-2 expression is induced under inflammatory conditions and is responsible for the progression of inflammation (Srinivasan and Kulkarni 1989; Smith et al. 1998). It has been suggested that the COX-2-mediated production of PGE2 plays an important role in the pathogenesis of AD. For example, elevated levels of PGE2 and over-expression of COX-2 have been observed in AD patient brains (Kitamura et al. 1999; Montine et al. 1999); the extent of COX-2 expression correlates with the degree of progression of AD pathogenesis (Ho et al. 2001); transgenic mice constitutively over-expressing COX-2 show aging-dependent memory dysfunction (Andreasson et al. 2001); PGE2 stimulates the production of reactive oxygen species in microglia and activates β-secretase (Liang et al. 2005); and prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs), inhibitors of COX, delays the onset and reduces the risk of AD (in t’ Veld et al. 2001; Imbimbo et al. 2010). Thus, in order to identify molecular targets for the development of AD drugs, it is important to understand the molecular mechanism involved in the PGE2-mediated progression of the disease.
We recently reported that PGE2 stimulates the production of Aβ in cells stably expressing a form of APP with two mutations (K651N/M652L; APPsw) that elevates cellular and secreted levels of Aβ (Hoshino et al. 2007). Using agonists and antagonists specific for each of the four PGE2 receptors (EP1, EP2, EP3 and EP4 receptors), we found that both EP2 and EP4 receptors are involved in the PGE2-stimulated production of Aβin vitro (Hoshino et al. 2007). With respect to the mechanism underpinning this stimulation, we also recently demonstrated that activation of the EP2 receptor stimulates the production of Aβ through activation of adenylate cyclase, an increase in the cellular level of cAMP and activation of protein kinase A (PKA) (Hoshino et al. 2009). In contrast, EP4 receptor activation causes its co-internalization with PS-1 (γ-secretase) into endosomes, a process that activates γ-secretase (Hoshino et al. 2009). Furthermore, we showed that deletion of the EP2 or EP4 receptor decreases brain levels of Aβ in transgenic mice expressing APPsw (APP23, a mouse model for AD), suggesting that EP2 or EP4 receptor activation stimulates the production of Aβin vivo (Hoshino et al. 2007). These previous results suggest that EP2 and/or EP4 receptors could represent valuable molecular targets for the treatment of AD. However, the effect of deletion of these receptors on other AD-related phenotypes, such as neuronal and synaptic loss and cognitive deficits, has not been tested. The effect on cognitive performance is particularly important, because functional phenotypes (cognitive dysfunction) and pathological phenotypes (such as an increase in the brain level of Aβ) are not always directly linked (Roberson et al. 2007; Kanninen et al. 2009). In this study, we therefore examined the effect of EP2 and EP4 receptor inhibition on cognitive function in APP23 mice, revealing that genetic inhibition of the EP4 receptor but not the EP2 receptor not only suppresses neuronal and synaptic loss but also improves cognitive performance. Similarly, oral administration of AE3-208, an EP4 receptor-specific antagonist, improved the cognitive function of the APP23 mice. These results suggest that the EP4 receptor is a valuable molecular target for the development of drugs to prevent or treat AD.
Materials and methods
Materials and animals
See Appendix S1.
Morris water maze test
The Morris water maze test was conducted in a circular 90- or 150-cm diameter pool filled with water at a temperature of 22.0 + 1°C, as described previously (Kobayashi et al. 2000; Huang et al. 2006), with some minor modifications. Details are described in Appendix S1.
ELISA for Aβ and β- and γ-secretase-mediated peptide cleavage assay
Aβ40 and Aβ42 levels and β- and γ-secretase activity in the brain were determined as described previously (Hoshino et al. 2007). Details are described in Appendix S1.
Thioflavin-S staining and immunohistochemical and immunofluorescence analyses
Thioflavin-S staining and immunohistochemical and immunofluorescence analyses were performed as detailed in Appendix S1.
All values are expressed as the mean ± standard error of the mean (SEM). One- or two-way anova followed by the Tukey test was used to evaluate differences between more than two groups. The Student’s t-test for unpaired results was used for the evaluation of differences between two groups. Differences were considered to be significant for values of p < 0.05.
Effect of deletion of EP2 or EP4 receptor on cognitive function in APP23 mice
We first used a Morris water maze to compare the spatial learning and memory of 6-month-old APPsw/EP2−/− and APPsw/EP4−/− mice with that of APPsw/EP2+/+ and APPsw/EP4+/+ mice, respectively. Mice were trained for 7 days to learn the location of a hidden platform, and the time required to reach the platform (escape latency) was measured. As shown Fig. 1(a), APPsw/EP2−/− mice required a longer time than APPsw/EP2+/+ animals to reach the platform, suggesting that EP2 receptor deletion exacerbates the cognitive deficit in the APP23 mice. In contrast, the APPsw/EP4−/− mice tended to take less time to reach the platform than the corresponding control animals (APPsw/EP4+/+) (Fig. 1b), suggesting that deletion of the EP4 receptor ameliorates the cognitive deficit. These differences did not reflect differences in swimming ability, because swimming speed and the ability to locate a visible platform were similar between the groups (data not shown).
Given that the above results suggest that the EP4 receptor may represent the better potential molecular target for the development of AD drugs, we next compared AD-related phenotypes, such as the formation of plaques and neuronal and synaptic loss, between four strains of mice (WT/EP4+/+, WT/EP4−/−, APPsw/EP4+/+ and APPsw/EP4−/−). We first repeated the Morris water maze test using 6-month-old mice, under slightly different experimental conditions (such as the size of swimming pool and tracking period). As shown Fig. 2(a), APPsw/EP4+/+ mice required a longer time than WT/EP4+/+ mice to reach the platform and this result is consistent with previous reports (Van Dam et al. 2003). Again, this difference did not reflect reduced swimming ability, as the swimming speed and the ability to locate a visible platform were similar between the four strains (data not shown). APPsw/EP4−/− mice required a shorter time to reach the platform than APPsw/EP4+/+ mice (Fig. 2a). Furthermore, there was no significant difference in the escape latency between APPsw/EP4−/− and WT/EP4+/+ mice (Fig. 2a). These results suggest that the expression of APPsw disturbs spatial learning and memory and this effect can be ameliorated by deletion of the EP4 receptor. WT/EP4−/− mice took a significantly shorter time to reach the platform than WT/EP4+/+ mice; however, the difference was observed only at day 0 (Fig. 2a).
We then did a transfer test to examine the spatial memory of platform location. After a 7-day training period as described above, each mouse was subjected to a Morris water maze test where the platform was removed and we measured the per cent search time for each quadrant. As shown in Fig. 2(b), the percentage of time spent in the trained quadrant was lower for the APPsw/EP4+/+ group than for either the WT/EP4+/+ or the APPsw/EP4−/− mice. The crossing time of the area where the platform had been located was lower in the APPsw/EP4+/+ group than in the WT/EP4+/+ and APPsw/EP4−/− cohorts (Fig. 2c). There was no significant difference between APPsw/EP4−/− and WT/EP4+/+ mice or between WT/EP4−/− and WT/ EP4+/+ mice for these indices (Fig. 2b and c). These results showed that deletion of the EP4 receptor ameliorates the spatial memory deficits of APP23 mice.
We then examined EP4 receptor expression in the brain of 18-month-old mice (hippocampal CA3 region) by immunofluorescence analysis. As shown in Fig. 3, expression of the receptor was clearly observed in the brains of WT/EP4+/+ and APPsw/EP4+/+ mice. Staining with antibody against neuronal nuclei (NeuN) confirmed expression of the EP4 receptor in neurons (Fig. 3), consistent with previous results (Choi et al. 2006). However, co-staining with antibody against NeuN and that against glial fibrillary acidic protein (a maker for astrocytes) or F4/80 (a maker for microglia) was not so clear (Fig. S1).
Effect of deletion of EP4 receptor on Aβ plaque deposition and neuronal and synaptic loss in APP23 mice
We have previously reported that the levels of Aβ40 and Aβ42 in soluble and insoluble brain fractions prepared from 6-month-old APPsw/EP4−/− mice are lower than those from APPsw/EP4+/+ mice (Hoshino et al. 2007), a finding that we confirmed here (Fig. 4a). We also examined Aβ plaque deposition by thioflavin-S staining using 18-month-old mice. As shown in Fig. 4(b) and (c), in both the hippocampus and the cerebral cortex, the level of Aβ plaque deposition was much lower in APPsw/EP4−/− mice than in APPsw/EP4+/+ animals.
We next determined the number of neurons in the hippocampal CA3 region by NeuN staining using 18-month-old mice. As shown in Fig. 4(d) and (e), the number of NeuN-positive cells (neurons) was significantly higher in the WT/EP4+/+ and APPsw/EP4−/− brain sections than in the APPsw/EP4+/+ tissue, suggesting that the neuronal loss induced by Aβ was ameliorated by deletion of the EP4 receptor. Similar results were observed for the hippocampal CA1 region (Fig. S2). We also estimated the number of synapses by synaptophysin staining using 18-month-old mice. The level of synaptophysin was higher in sections from both WT/EP4+/+ and APPsw/EP4−/− mice than in those from APPsw/EP4+/+ mice (Fig. 4f and g), indicating that Aβ-induced synaptic loss was suppressed by deletion of the EP4 receptor. Taken together, these results suggest that deletion of the EP4 receptor decreases the level of Aβ and Aβ plaque deposition in the brain and protects against Aβ-induced neurodegeneration. To confirm this further, stereological quantification of cell number that is more reliable should be performed in future studies.
Effect of oral administration of AE3-208 on AD-related phenotypes in APP23 mice
The results described above suggest that pharmacological inhibition of the EP4 receptor ameliorates AD-related phenotypes in APP23 mice. In order to test this, we used an EP4 receptor-specific antagonist, AE3-208. The Ki values of AE3-208 obtained by competition binding assay are 1.3, 30, 790 and 2400 nM for EP4, EP3, FP and TP, respectively, and more than 10 μM for the other prostanoid receptors (Kabashima et al. 2002). We have previously reported that AE3-208 suppresses the PGE2-stimulated production of Aβin vitro (Hoshino et al. 2007). APP23 and wild-type mice were fed either AE3-208-supplemented chow or a control diet between the ages of 3 and 6 months (the average dose of AE3-208 was calculated to be 17.8 mg/kg body weight/day). No significant differences were observed in the amount of chow consumed by the four groups of mice (APP23 or wild-type mice fed AE3-208-supplemented or control chow) during the experimental period. We then examined the spatial learning and memory of the 6-month-old animals in a Morris water maze test. Swimming speed and ability to locate a visible platform were indistinguishable between the four groups (data not shown). However, APP23 mice fed AE3-208-supplemented chow took significantly less time to find the hidden platform than the mice fed control chow (Fig. 5a). No significant difference in the escape latency was recorded between wild-type mice fed AE3-208-supplemented chow and wild-type mice fed control chow (Fig. 5a). These results suggest that the deficit in spatial learning and memory in the APP23 mice can be ameliorated by oral administration of AE3-208.
As shown in Fig. 5(b), the amount of time spent in the trained quadrant showed a tendency to be greater for the APP23 mice fed AE3-208-supplemented chow than for the mice fed control chow. Furthermore, the crossing time of the area where the platform had been located was significantly greater in the former case (Fig. 5c). However, the difference in Fig. 5(b) was not statistically significant and we have no clear explanation for the discrepancy between the ‘time in quadrant’ and ‘platform crossings’ outcomes.
In order to test whether pharmacological inhibition of the EP4 receptor ameliorates AD-related pathological phenotypes in APP23 mice, we compared the amount of Aβ40 and Aβ42 in soluble and insoluble fractions prepared from the brains of APP23 mice fed either AE3-208-supplemented or control chow using 6-month-old mice. As shown in Fig. 6(a), the levels of Aβ40 and Aβ42 in the insoluble brain fractions from the former group were significantly lower. However, no significant difference was observed in the case of the soluble fractions (Fig. 6a).
We have previously reported that EP4 receptor activation increases Aβ levels through its co-internalization into endosomes with PS-1 (γ-secretase), with resulting activation of γ-secretase in vitro (Hoshino et al. 2009). This finding was supported by our previous in vivo demonstration that brain γ-secretase activity is lower in APPsw/EP4−/− mice than in APPsw/EP4+/+ animals, and that the co-localization of PS-1 with Rab7 (a marker of late endosomes and lysosomes) is not as apparent in the former group (Hoshino et al. 2009). In the present study, we examined the effect of oral administration of AE3-208 on the activity and localization of γ-secretase using 6-month-old mice. As shown in Fig. 6(b), the activity of γ-secretase, but not that of β-secretase, was lower in the brains of APP23 mice fed AE3-208-supplemented chow than in those of mice fed control chow. Furthermore, we found that the co-localization of PS-1 with Rab7 was not as apparent in the former group (Fig. 6c and d). We quantitatively examined the effect of AE3-208 on the expression of PS-1 staining and found that the effect was not statistically significant (data not shown).
We have previously reported that deletion of the EP4 receptor in APP23 mice does not affect the modification of APP or α- and β-secretase activity (Hoshino et al. 2009), both of which are important for Aβ production. Here, we examined the effect of oral administration of AE3-208 on these processes using 6-month-old mice. We could separate by sodium dodecyl sulfate–polyacrylamide gel electrophoresis between the mature (N- and O-glycosylated) and immature (N-glycosylated alone) forms of APP (mAPP and imAPP, respectively) (Tomita et al. 1998). As shown in Fig. 6(e), the total amount of APP and the ratio of mAPP and imAPP were similar between APP23 mice fed AE3-208-supplemented chow and those fed control chow, suggesting that the administration of AE3-208 does not affect APP modulation. We also found that the administration of AE3-208 did not affect the level of PS-1 (Fig. 6e). We then examined α- and β-secretase activity by comparing the level of secreted C-terminal fragment (CTF), representing an indirect index of secretase activity. We could not detect a CTFγ band under our experimental conditions. However, as shown in Fig. 6(f), CTFα and CTFβ were detected in the APP23 mice and the amounts of CTFα and CTFβ were indistinguishable between APP23 mice fed AE3-208-supplemented chow and those fed control chow, thereby suggesting that the administration of AE3-208 does not affect α- or β-secretase activity.
Taken together, these results suggest that the improvement in the cognitive function of the APP23 mice orally administered AE3-208 is mediated by a decrease in the brain levels of Aβ through suppression of co-internalization of the EP4 receptor with γ-secretase into endosomes, thereby inhibiting the activation of γ-secretase.
We have previously suggested that EP2 and EP4 receptors represent valuable molecular targets for the development of drugs to prevent or treat AD by showing that the amount of Aβ in the brains of APPsw/EP2−/− and APPsw/EP4−/− mice is lower than that in the respective control mice (Hoshino et al. 2007). However, among the antagonists specific for either the EP2 or EP4 receptor, or both, which type offers the most therapeutic potential? In order to address this issue, we herein compared the cognitive performance of APPsw/EP2−/− or APPsw/EP4−/− mice with that of their respective wild-type counterparts. This approach was adopted because, although AD is characterized by cognitive impairment, the functional (cognitive) phenotypes and pathological phenotypes (such as an increase in Aβ levels and Aβ plaque deposition) of the disease are not always directly linked. For example, some conditions ameliorate cognitive dysfunction in AD model mice without affecting the pathological phenotypes (Roberson et al. 2007; Kanninen et al. 2009). Our results suggested that APPsw/EP4−/− mice but not APPsw/EP2−/− mice display a higher level of cognitive function (spatial learning and memory) than their respective wild-type controls, suggesting that inhibition of the EP4 receptor might prove the better therapeutic option.
We have previously reported that PGE2-stimulated production of Aβin vitro is partially mediated by EP2 receptor-dependent activation of the cAMP–PKA pathway (Hoshino et al. 2009), and that the amount of Aβ in the brains of APPsw/EP2−/− mice is lower than that in control mice (Hoshino et al. 2007). Another group has also shown that deletion of the EP2 receptor in AD model mice reduces Aβ plaque deposition (Liang et al. 2005). Thus, it is surprising that deletion of this receptor exacerbates cognitive dysfunction in APP23 mice, suggesting that deletion of the EP2 receptor impaired cognitive performance through an Aβ-independent mechanism. It has previously been reported that Aβ inhibits long-term potentiation (LTP) through inhibition of the cAMP–PKA pathway (Vitolo et al. 2002), and that inhibition of the EP2 receptor also suppresses LTP via a similar mechanism (Akaneya and Tsumoto 2006). Thus, deletion of the EP2 receptor may exacerbate cognitive dysfunction in APP23 mice through inhibition of LTP, a process known to be important for memory formation. It was recently reported that deletion of the gene encoding EP2 receptor in mice without the expression of APPsw have behavioral deficits (Savonenko et al. 2009), thus it is unclear whether the observed effects of EP2 receptor deletion in this study are specific to the AD model. However, it was previously reported that siRNA for EP4 did not affect LTP (Akaneya and Tsumoto 2006).
We have previously reported that EP4 receptor activation stimulates the production of Aβ through its co-internalization with γ-secretase into endosomes, leading to the activation of γ-secretase (Hoshino et al. 2009). We also showed that there are lower levels of Aβ and less endosomal localization of γ-secretase in the brains of APPsw/EP4−/− mice than in those of APPsw/EP4+/+ animals (Hoshino et al. 2007, 2009). Furthermore, in the present study, we have demonstrated that APPsw/EP4−/− mice display lower levels of Aβ plaque formation and neuronal and synaptic loss than APPsw/EP4+/+ mice. These results suggest that deletion of the EP4 receptor ameliorates cognitive dysfunction in APP23 mice by decreasing brain levels of Aβ and suppressing neurodegeneration.
The findings of the present study also demonstrate that oral administration of the EP4 receptor-specific antagonist, AE3-208, ameliorates the spatial learning and memory deficits of APP23 mice. AE3-208 has been shown to have some therapeutically beneficial effects, including suppression of tumor growth (Terada et al. 2010) and suppression of autoimmune encephalomyelitis (Yao et al. 2009). However, it has been reported that AE3-208 exacerbates dextran sodium sulfate-induced colitis, an animal model for ulcerative colitis (Kabashima et al. 2002), and that a specific agonist for the EP4 receptor stimulates bone formation and prevents bone loss (Yoshida et al. 2002), suggesting that EP4 receptor antagonists, including AE3-208, have adverse effects on colitis and osteoporosis, possibilities that must be considered if these agents are to be developed for the clinical treatment of AD. Although the transitional character of orally administered AE3-208 to the brain has not yet been examined, the results of the present study suggest that it can pass the blood–brain barrier. AD is a chronic disease that requires long-term drug treatment in order to produce therapeutic effects. Thus, this property of AE3-208 would be of great advantage for its clinical use. As for the mechanism underpinning the amelioration of cognitive dysfunction in the APP23 mice following the administration of AE3-208, we believe that this is mediated by a similar mechanism to EP4 receptor deletion, given that oral administration of AE3-208 decreases levels of Aβ and γ-secretase activity and inhibits the localization of γ-secretase in endosomes. The soluble Aβ level was reduced in APPsw/EP4−/− mice but not in mice administered with AE3-208. This difference would be because of the difference in extent of the inhibition; deletion of the gene encoding EP4 receptor completely inhibits the function of this protein, whereas administration of the drug may cause partial inhibition. As for the difference between soluble and insoluble Aβ for the modulation by administration of AE3-208, we have no clear explanation at present. One possible explanation is that the temporal alteration in synthesis of Aβ may affect more drastically soluble Aβ level than insoluble one.
Although this study focused on how inflammation affects the pathogenesis of AD through PGE2 but not on how inflammation is induced in association with AD progression, we examined the effect of inhibition of EP4 receptor on the activation of astrocytes. As shown in Fig. S3, the expression of glial fibrillary acidic protein (a maker for the activity of astrocytes) was higher in 18-month-old APPsw/EP4+/+ mice than in WT/EP4+/+ and APPsw/EP4−/− mice. As for AE3-208, because we used 6-month-old mice, the activation of astrocytes by the expression of APPsw was not so clear; however, the activity was a little lower in drug-treated mice than in control mice (Fig. S3). These results suggest that the inhibition of EP4 receptor suppresses APPsw-mediated activation of astrocytes (inflammation). Based on previously reported results, the activation of EP4 receptor seems to affect immune systems both positively and negatively. For example, EP4 receptor-stimulated differentiation of TH1 cells and production of IL-23 in dendritic cells and resulting inflammation in experimental autoimmune encephalomyelitis were reported (Yao et al. 2009). However, in microglia, the activation of EP4 receptor was reported to suppress the LPS-stimulated production of pro-inflammatory cytokines (Shi et al. 2010).
As described in the introduction, NSAIDs have attracted considerable attention as a new class of drugs for the treatment and prevention of AD, although it should also be noted that some clinical studies have recorded negative results (Imbimbo et al. 2010). NSAIDs can be classified into two groups: newly developed COX-2-specific NSAIDs (such as celecoxib) and classical NSAIDs without COX-2 specificity (such as indomethacin). The clinical use of classical NSAIDs is associated with gastrointestinal side effects (Hawkey 2000), as a result of the strong protective effect of prostaglandins on the gastrointestinal mucosa (Vane and Botting 1996). Given that it is mainly COX-1, which is expressed in this mucosa, COX-2-specific NSAIDs cause less of an effect on prostaglandin levels in this region, and therefore produce fewer gastrointestinal side effects than classical NSAIDs. However, it has recently been shown that clinical use of COX-2-specific NSAIDs is associated with cardiovascular thrombotic side effects (Ray et al. 2004; Singh 2004). These side effects of NSAIDs are likely to prove problematic if the drugs are used long term for the prevention or treatment of AD.
Compared with NSAIDs, we consider that EP4 receptor-specific antagonists have advantages in relation to both safety and efficacy, based on the following lines of evidence. EP1 and EP3 receptors have been reported to be involved in PGE2-mediated protection of the gastrointestinal mucosa by stimulating the production of bicarbonate and gastric mucosal blood flow, respectively (Takeuchi et al. 1997; Araki et al. 2000). Therefore, antagonists specific for the EP4 receptor would be gastrointestinally safer than NSAIDs. However, it is now believed that inflammation has both positive and negative effects in relation to the progression of AD; for example, inflammation activates the phagocytosis of Aβ by microglia (Shaftel et al. 2007; Chakrabarty et al. 2010). However, NSAIDs that inhibit overall inflammation inactivate microglial phagocytosis (Yan et al. 2003). Therefore, compared with general anti-inflammatory agents, inhibitors that specifically act on the inflammation-mediated progression of AD may be more effective. NSAIDs suppress inflammation through both COX-dependent and COX-independent mechanisms, such as activation of the peroxisome proliferators activated receptor-γ and inhibition of nuclear factor-kB (Tegeder et al. 2001), with COX-mediated inhibition and the resulting decrease in PGE2 levels seen to play a major role in the anti-AD activity of NSAIDs (Qin et al. 2003; Heneka et al. 2005). Furthermore, it was recently reported that the ability of NSAIDs to decrease PGE2 levels is important in NSAID-dependent protection of hippocampal LTP against Aβ toxicity and restoration of Aβ-mediated suppression of synaptic plasticity and memory function (Kotilinek et al. 2008). Based on the findings of the present study, we consider that PGE2 impairs cognitive performance at least partly through activation of the EP4 receptor. Thus, we propose that EP4 receptor-specific antagonists, such as AE3-208, will prove therapeutically more effective than NSAIDs as a result of their greater safety and efficacy. However, although we previously suggested that EP1 and EP3 receptors are not involved in PGE2-stimulated production of Aβin vitro, it is not clear whether activation of EP1 and EP3 receptors affect cognitive performance. Furthermore, modulation of COX-2 expression by activation of EP4 receptor was also suggested (Shi et al. 2010). Therefore, the mechanism by which PGE2 modulates cognitive performance is unclear at present and understanding of such mechanism is important for the identification of other targets of AD drugs.
We thank Dr M. Staufenbiel (Novartis Institutes for BioMedical Research) for providing transgenic mice. We also thank Ono Pharmaceutical Co. (Osaka, Japan) for providing AE3-208. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Health, Labour, and Welfare of Japan, as well as the Japan Science and Technology Agency, Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Authors have no conflicts of interest.