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The non-COX targets of NSAIDs, lipoxygenases, PPARs, IKK and

NFκκκκB, do not reduce Aββββ42 production

Sarah A. Sagi1, Sascha Weggen1, Jason Eriksen2, Todd E. Golde2 and Edward H. Koo1

From the 1 Department of Neurosciences, University of California San Diego, La Jolla,

CA 921093, USA and the 2Department of Neuroscience and Pharmacology, Mayo Clinic

Jacksonville, Jacksonville, Fl 32224, USA

Address correspondence to: Dr. Edward H. Koo, Department of Neurosciences, University of

California San Diego, La Jolla, CA 92093-0691

Tel: 858-822-1024

FAX: 858-822-1021

Email: edkoo@ucsd.edu

Running Title: Aβ42 reduction is via a novel target of NSAIDs

Abbreviations: AA, arachidonic acid; Aβ, amyloid β; AD, Alzheimer’s disease; APP, amyloid

precursor protein; CHO, Chinese Hamster Ovary; COX, cyclooxygenase; ELISA, enzyme-linked

immunosorbent assay; IKK, Inhibitor of κB kinase; LOX, lipoxygenase; MALDI-TOF, matrix-

assisted laser desorption ionization time of flight mass spectrometry; MEF, mouse embryonic

fibroblast; NFκB, nuclear factor κB; NSAID, non-steroidal anti-inflammatory drug; PLA2,

phospholipase A2; PPAR, peroxisome proliferator-activated receptor

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 12, 2003 as Manuscript M303588200 by guest on June 28, 2020

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Summary

Epidemiological evidence suggests that chronic use of non-steroidal anti-inflammatory drugs

(NSAIDs) reduces the risk of Alzheimer’s disease. Recently, NSAIDs have been shown to

decrease amyloid pathology in a transgenic mouse model of Alzheimer’s disease. This benefit

may be partially attributable to the ability of NSAIDs to selectively reduce production of the

amyloidogenic Aβ42 peptide in both cultured cells and transgenic mice. Although this activity

does not appear require the action of cyclooxygenases in cultured cells, it is not known whether

other NSAID-sensitive targets contribute to this Aβ42 effect. In this study, we have used both

pharmacological and genetic means to determine if other known cellular targets of NSAIDs

could mediate the reduction in Aβ42 secretion from cultured cells. We find that altered

arachidonic acid metabolism via NSAID action on cyclooxygenases and lipoxygenases does not

alter Aβ42 production. Furthermore, we demonstrate that alterations in activity of peroxisome

proliferator-activated receptors, IκB kinase β or nuclear factor κB do not affect Aβ42

production. Thus, NSAIDs do not appear to alter Aβ42 production indirectly through previously

identified cellular targets and may interact directly with the γ-secretase complex itself to affect

amyloid production.

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Introduction

Alzheimer's disease (AD) is the most common form of age-related dementia. Safe

effective treatments are urgently needed. One hallmark of AD is the accumulation of amyloid β-

protein (Aβ), derived from the amyloid precursor protein (APP), in senile plaques in brain.

There are two predominant isoforms of Aβ peptide, Aβ40 and Aβ42, differing in their C-termini:

Aβ42, the longer isoform is more amyloidogenic and toxic to cultured cells. Virtually all

mutations associated with familial AD preferentially increase amount of Aβ42 produced, and

hence the ratio of Aβ42:Aβ40 (1). Thus, decreasing production or increasing the clearance of

Aβ42 may be an effective way to either prevent the development of or treat AD.

Epidemiological studies demonstrate that persons with a history of non-steroidal anti-

inflammatory drugs (NSAIDs) use have a reduced risk of AD (2,3). It is generally assumed that

the chronic inflammatory responses seen in brains of AD individuals play a key role in

neurodegeneration (4,5), but it has not been established that the anti-inflammatory properties of

NSAIDs underlie their apparent neuroprotective effects (6,7). In line with the epidemiological

findings, treatment of APP transgenic mice, which develop Aβ deposits and associated

pathology, with these compounds has proven to be beneficial. Specifically, ibuprofen, curcumin,

and a nitric-oxide derivative of flurbiprofen were recently shown to decrease the formation of

amyloid plaques and reduce inflammatory markers in a transgenic mouse model of AD (8-10).

These and other observations have led to a number of clinical trials to determine if NSAIDs or

immunosuppression may be useful in the treatment of AD.

In our recent studies (11) we found that some NSAIDs, including sulindac, ibuprofen and

indomethacin, lowered the levels of the amyloidogenic Aβ42 isoform, hence reducing the ratio

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of Aβ42:Aβ40, in medium of a variety of cultured cells as well as in brains of APP transgenic

mice. The effective NSAIDs did not grossly affect the production or processing of the amyloid

precursor protein (APP). While these NSAIDs decreased the ratio of Aβ42:Aβ40, they appear to

increase the production of shorter Aβ peptides, such as Aβ38. This suggested that NSAIDs

might subtly alter the production of various Aβ species. To our knowledge, the NSAIDs are the

first class of compounds that specifically reduce Aβ42 production without significant alteration

in Aβ40 levels.

The focus of these studies was to further investigate the cellular mechanisms responsible

for the Aβ42:Aβ40 reduction by certain NSAIDs. Our initial report established that inhibition of

the cyclooxygenase enzymes COX1 and COX2, the canonical targets of NSAIDs, was not

sufficient to reduce the Aβ42 levels (11). We therefore proposed that NSAIDs affect the

amyloid pathology by lowering the amyloidogenic Aβ42 peptide through a COX-independent

pathway. This hypothesis is not unparalleled, as a dual-action model has been suggested for the

effectiveness of NSAIDs in colorectal cancer studies where both COX-dependent and COX-

independent mechanisms have been described. To further understand the mechanism whereby

NSAIDs lower Aβ42 levels, we examined whether other NSAID-sensitive targets including

arachidonic acid, lipoxygenases, peroxisome-proliferator-activated receptors (PPAR) and nuclear

factor κB influence the generation of Aβ42. In addition, we examined several other compounds

that have been reported to lower risk of AD or reduced amyloid pathology in transgenic mice to

determine whether they also have a previously unrecognized activity against Aβ42.

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Experimental procedures

Cell Lines and Culture Conditions

Chinese hamster ovary (CHO) cells stably expressing APP751 with or without human presenilin 1

M146L (designated APP-WT and PS1ML respectively) were maintained in Dulbecco’s Modified

Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin

(100units/ml) and streptomycin (100µg/ml) in a 370C, humidified incubator with 5% CO2. One

day prior to drug treatment cells were plated at approximately 1x105 cells/ml. Embryonic

fibroblasts from wild-type, IKK2 knockout (kindly provided by I. Verma) or p65/RelA knockout (a

gift from E. Shaulian) mice were maintained as above. Primary cultures of ALOX5 and ALOX15

knockout fibroblasts were generated from neonatal mice resulting from homozygous ALOX5 or

ALOX15 knockout (mice purchased from The Jackson Laboratory) breedings. Fibroblasts were

infected with 60pfu per cell of adenovirus encoding wild-type APP751 for two hours before

treatment with NSAIDs (11).

Drugs and chemicals

Compounds were obtained from Sigma Chemical Co except as noted here. Arachidonic acid,

carbaprostacyclin (cyclic-PgI2) and naproxen - Cayman Chemicals; baicalein, caffeic acid, MK-

886, 8-S-HETE and 15-deoxy-∆-prostaglandin J2 (PgJ2) – Calbiochem; ibuprofen,

indomethacin, sulindac sulfide – Biomol; melittin - ICN Biosciences; S-flurbiprofen – Aldrich;

Aβ1-40, 1-42 standard peptides - American Peptide Inc. Complete protease inhibitor pellet –

Roche; and α-cyano-4-hydroxycinnamic acid solution-Agilent Technologies; GW9662 was

provided by GlaxoSmithKline. LG10305 and BRL49653 were kind gifts from R. Evans. R-

flurbiprofen was supplied by Encore Pharmaceuticals.

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Enzyme-Linked Immunosorbent Assays

For most experiments human Aβ ELISA were performed as described (12,13). For each

experiment duplicate or triplicate samples were analyzed. Each drug was tested at several doses

in a minimum of two independent experiments with sulindac sulfide as a positive control.

Selective reduction in Aβ42 was determined by calculating the Aβ42:Aβ40 ratio for each

sample. These ratios were normalized to vehicle control in each experiment. These normalized

ratios were pooled and ANOVA used to determine if the Aβ42:Aβ40 ratio was significantly

different from vehicle with p<=0.05. Error bars in figures represent standard errors.

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry

MALDI-TOF was performed on Aβ peptides immunoprecipitated from conditioned medium of

CHO cells as described (14) with the following modifications. Complete protease inhibitor,

phosphoramidon and a synthetic Aβ1-22 peptide, that served as an internal control, were added

and all Aβ1-x were immunoprecipitated from conditioned medium by overnight incubation with

anti-mouse IgG agarose beads and 26D6, which recognizes the N-terminus of Aβ. Extraction

from the beads was with formic acid/water/isopropanol 1:4:4 (v/v/v). Eluted material was mixed

1:1 with α-Cyano-4-hydroxycinnamic acid solution prior to spotting for spectrometry. Spectra

shown are representative of at least two experiments performed with duplicate samples.

Treatment-induced changes in Aβ species distribution were determined by normalization of peak

heights to Aβ40.

Bicine/Urea SDS-PAGE and Western Blots

Conditioned medium was immunoprecipitated as for MALDI-TOF analysis, except without the

addition of synthetic Aβ22. Bicine/Urea gels were performed as described in Wiltfang et al (15)

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with the following modifications. The final acrylamide concentration in the separating gel was

10%T,5%C. The comb gel was eliminated from our protocol. In addition, the 30% sucrose and

5% 2-mercaptoethanol were replaced with glycerol and dithiothreitol. Proteins were transferred

to nitrocellulose in 10mM CAPS pH11 with 10% methanol and boiled for 5 minutes.

Immunoblotting was with 26D6 APP antibody and the signal detected by HRP-conjugated goat

anti-mouse IgG followed by enhanced chemiluminescence. Gels shown are representative of a

minimum of three experiments with 2-3 replicates per experiment. Bands intensities were

quantified using CCD camera and software (Syngene).

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Results

Reduction in Aβ42 is not generalized anti-inflammatory actions.

To confirm that the Aβ42-reducing activity was specific to NSAIDs and not mimicked by

other anti-inflammatory compounds, we investigated the effects of glucocorticoids on Aβ42

levels in CHO cells stably transfected with wild type human APP (APP-WT). In clinical trials,

these compounds did not slow the rate of cognitive decline in AD individuals (16,17). At the

concentrations tested, neither prednisone nor dexamethasone reduced the amount of Aβ42 or the

Aβ42:Aβ40 ratio (Table 1).

Epidemiological studies (18) have also suggested that anti-histamine usage reduces the

incidence of AD. Since histamine responses trigger inflammatory changes, we wanted to

determine if blocking histamine action altered Aβ42 levels. Therefore, we treated Aβ-producing

PS1ML cells with 0.5-20µM of the H-2 histamine receptor antagonist cimetidine. This treatment

did not alter the levels of Aβ detectable by ELISA (Table 1) or bicine/urea gel analysis of

conditioned medium.

Recent studies indicated that the NSAID-like compound curcumin might be beneficial in

a mouse model of AD (9). Curcumin has both anti-inflammatory and anti-oxidant properties.

APP-WT CHO cells were treated with curcumin to determine if it affected the production of Aβ

species. Between 1 and 100µM, there was no reduction in Aβ levels or Aβ42:Aβ40 (Table 1).

Counter to the effect of NSAIDs, there was a slight increase (20%) in at doses between 20µM

and 100µM. This increase in Aβ42:Aβ40 could be attributed to a reduction in Aβ40 levels.

Higher doses were toxic to the cells so Aβ could not be evaluated.

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COX inhibition is not required for NSAID action on Aβ42

We previously reported that the reduction in Aβ42 levels secondary to NSAID treatment

was accompanied by an increase in the amount of Aβ38 species (11). However, several

NSAIDs, such as naproxen and aspirin, did not reduce Aβ42 levels. Whether these NSAIDs

affect shorter Aβ species was not investigated as the inactivity of naproxen in our previous study

had only been determined by ELISA. Here we confirmed the lack of effect on Aβ peptides by

both MALDI-TOF spectrometry and bicine/urea SDS-PAGE methods. In these assays, naproxen

did not reduce Aβ1-42 or increase Aβ1-38, whereas sulindac sulfate, ibuprofen, flurbiprofen and

indomethacin were all effective (Figure 2 and data not shown). In our earlier work, we used

COX-deficient cells to demonstrate that COX enzymes are not required for sulindac sulfide to

reduce Aβ42 levels. However, both of these approaches only concluded that COX inactivation is

insufficient to reduce Aβ42 levels. To address whether COX inhibition is required to alter Aβ

production we used R-flurbiprofen, which does not inhibit COX and is not efficiently isomerized

to S-flurbiprofen. R-flurbiprofen potently reduced Aβ42 levels and increased production of

shorter Aβs (Figure 2C), in agreement with a recent report (19).

Arachidonic acid and lipoxygenases do not affect Aβ42 levels.

NSAIDs inhibit not only COX but can alter the activities of several lipoxygenases, which

could increase arachidonic acid levels and effect the processing of arachidonic acid into HETEs

and leukotrienes (Figure 1). To mimic the effect of NSAIDs on increasing arachidonic acid

levels, we treated the PS1ML cells with exogenous arachidonic acid and assayed the levels of the

various Aβ species by ELISA and bicine/urea gels. We also treated cells with the phospholipase

A2 activator, melittin (20). This treatment mobilizes endogenous arachidonic acid from cellular

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membranes. Arachidonic acid, either added to the extracellular medium or induced by melittin,

increased the total amount of Aβ generated by approximately 20% as compared to control cells.

In the case of arachidonic acid there was a slight reduction in the Aβ42:Aβ40 ratio, due to an

increase in Aβ40 and not a decrease in Aβ42 (Table 2). Melittin treatment had no effect on

Aβ42:Aβ40 ratio.

Several NSAIDs, including ibuprofen and indomethacin, can inhibit the activity of 5-

lipoxygenase (21). 5-lipoxygenase expression may be elevated in aging human brains (22), so

we wanted to determine if the NSAID effect on Aβ42 was mediated by inhibition of this

enzyme. We examined whether other 5-lipoxygenase inhibitors could reduce the Aβ42 levels

detected by our sensitive ELISA assay (Table 2). The doses chosen for these compounds were

above those published to inhibit 5-lipoxygenase, but below toxic concentrations. The inhibitors

nordihydroguiaretic acid, MK-886, and caffeic acid, which preferentially inhibit 5-LOX, were

without effect on total Aβ or Aβ42 production. Since at elevated doses these pharmacological

inhibitors have multiple activities, which could mask the desired effect, we also used a genetic

approach to eliminate 5-lipoxygenase activity. Neonatal fibroblasts from 5-lipoxygenase-

deficient mice were infected with an adenovirus encoding APP695 and then treated with sulindac

sulfide. As shown in Figure 3A, sulindac retained the ability to inhibit Aβ42 production in these

cells, verifying that 5-LOX is not necessary for the NSAID effect on Aβ42.

Recent reports indicate that some NSAIDs activate the murine leukocyte-type 12-

lipoxygenase and its human homologue 15-lipoxygenase (23,24), so we examined the effect of

modulating 15-lipoxygenase on Aβ42:Aβ40. The 15-lipoxygenase inhibitor baicalein had no

effect on Aβ42 levels (Table 2). As above, we cultured fibroblasts from L-12-lipoxygenase

deficient mice and assayed Aβ levels after adenoviral infection. Once again we saw a marked

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reduction in Aβ42 with sulindac treatment (Figure 3B), indicating that this enzyme is also not

required for the amyloid-altering effects of NSAIDs.

Peroxisome-proliferator-activated receptors do not alter Aβ42 levels

The peroxisome-proliferator-activated receptors (PPARs) are sensitive to NSAIDs and

the eicosanoids produced by arachidonic acid metabolism. Recent studies have also indicated

that PPAR activation inhibits microglial activation and the production of proinflammatory

molecules and may attenuate Aβ toxicity (25,26). NSAIDs activate PPARα and PPARγ (27),

and inhibit PPARδ (28). Each or these PPARs must heterodimerize with the RXR nuclear

transcription factor to bind DNA and alter transcription. If NSAIDS reduce Aβ42 by activating

PPARα then the PPARα agonist 8(S)-HETE should also reduce Aβ42 levels from APP-

expressing cells. However, 8(S)-HETE did not reduce Aβ42 levels secreted from APP-WT cells.

Likewise the PPARγ agonists ciglitazone, 15-deoxy-∆-prostaglandin J2 or BRL49653 (29) failed

to reduce Aβ42 levels (Table 3). In addition, we examined the ability of a PPARγ antagonist,

GW9662 (30), to block the response to sulindac sulfide. This antagonist had no effect on the

ability of sulindac to reduce Aβ42 levels (Figure 4A and B). We also tested the Aβ42-reducing

potential of the RXR agonist LG10305, which was ineffective in reducing Aβ42 levels.

To confirm the pharmacological approach, APP-WT CHO cells were transfected with

either PPARγ or PPARδ cDNAs and then stimulated with 15-deoxy-∆-prostaglandin J2 or cyclic-

prostaglandin I2, respectively. Overexpression of these receptors, with or without activator, did

not alter the ratio of Aβ42:Aβ40 (Figure 4C). Because pharmacologic and genetic

manipulations of the various PPARs had no affect on Aβ42:Aβ40, these receptors are unlikely to

mediate the NSAID Aβ42 response.

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NFκB is not required to mediate the sulindac sulfide-induced reduction in Aβ42

Several NSAIDs can inhibit the activity of the NFκB transcription complex at one or

more levels: the inhibition of IKK and the prevention of NFκB DNA binding (31,32). Inhibition

of IKK would prevent IκB phosphorylation and degradation and prevent translocation of NFκB

to the nucleus. The NFκB complex is a dimer that can be composed of several proteins. The

most common NFκB complex contains p65/RelA and p50. Tomita et al (33) reported that

overexpression of p65/RelA increased production of Aβ42 but not Aβ40 indicating a potential

Aβ42 specific signaling pathway that can be modulated by NSAIDs. Because elimination of

p65/RelA by genetic knockout prevents induction of most NFκB-responsive genes (34), we used

mouse embryonic fibroblasts from IKKβ or p65-knockout mouse embryos to test whether the

NFκB pathway was required for the NSAID-mediated reduction in Aβ42. As before, the MEFs

were infected with APP695 adenovirus and then treated with sulindac sulfide. In both cell lines,

sulindac inhibited the production of Aβ42 similar to control fibroblasts (Figure 3), indicating that

these proteins are not required to mediate the NSAID response.

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Discussion

The unexpected finding that NSAIDs reduce the production of Aβ42 in vitro in cell

culture medium and in vivo in brains of transgenic mice opened two key questions. The first

question was whether the effect was mediated by the anti-inflammatory action of these

compounds, even if not due to COX inhibition. The second is the mechanism by which NSAIDs

lower Aβ42 levels. The goal of this study is to examine a number of candidate cellular pathways

that may be responsible for this NSAID effect on Aβ42 generation.

NSAIDs are potent anti-inflammatories that are primarily known to inhibit COX

enzymes. However, our initial report (11) demonstrated that Aβ42 was reduced by sulindac in

cells lacking COX enzymes. Furthermore, several NSAIDs that inhibited COX could not reduce

Aβ42:Aβ40. These findings indicate that inhibition of COX was not sufficient to reduce Aβ42

levels. Further confirmation of this hypothesis was obtained from a study treating cultured cells

with the R-enantiomers of flurbiprofen and ibuprofen (19) and from our results with R-

flurbiprofen using both MALDI-TOF and western blotting analyses. The fact that R-

enantiomers of these NSAIDs, which are inactive against COX, were able to reduce Aβ42

production argues against COX inhibition as the basis for Aβ42 reduction.

To examine whether NSAIDs may act through a general anti-inflammatory mechanism

unrelated to COX inhibition, we screened several compounds unrelated to conventional NSAIDs

with known anti-inflammatory activity. One of the proposed bases for the neuroprotective

benefits of NSAIDs is to inhibit the inflammatory responses seen in brains of AD individuals,

thus we thought it is pertinent to analyze this potential mechanism. This notion led to a

treatment trial in AD individuals with the corticosteroid, prednisone, which was found to be

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without benefit (17). In our cultured cell system, we found that glucocorticoids, including

prednisone and dexamethasone, did not reduce Aβ42 levels. On the other hand, our finding that

curcumin does not alter Aβ42 levels is notable in light of the recent report showing a significant

reduction in amyloid pathology in transgenic mice treated with curcumin chronically (9). As

curcumin has multiple properties, these findings taken together suggest that curcumin may

reduce Aβ load in brain by a mechanism distinct from the Aβ42 property seen in NSAIDs.

Although there was initial suggestion that H2 antagonists, such as cimetidine, lowered the risk of

AD, a recent analysis of the Cache county epidemiological data and a placebo-controlled study

concluded that use of these drugs did not affect AD risk or progression (35,36). These more

recent analyses are more aligned with our finding that cimetidine had no effect on Aβ42.

However, as our cell culture experiments are performed under basal conditions, we cannot draw

any conclusion as to the benefit of the aforementioned anti-inflammatory compounds in cells

already challenged by inflammatory stimuli. It is possible that mediation of inflammatory

responses may be beneficial in treating AD, and this benefit could be dependent or independent

of Aβ42 production. These findings therefore highlight the fact that there likely are multiple

activities from these anti-inflammatory compounds and that multiple mechanisms can account

for the reduction in Aβ levels, amyloid pathology, and/or ameliorate AD risk.

A major focus of this study is to test candidate cellular pathways that may be responsible

for the observed reduction in Aβ42 by examining the non-COX pathways known to be affected

by NSAIDs. Although the primary target of NSAIDs is COX inhibition, NSAIDs are pleiotropic

compounds with other known effects. These pathways are summarized in the schematic diagram

in figure 1. For example, one such pathway is through altered AA metabolism. Inhibition of

COX blocks the conversion of AA to prostaglandin H2 and result in a build up of AA that has

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been mobilized from the plasma membrane by the action of phospholipase A2 (37).

Phospholipase A2 has been reported to increase APPs secretion, which presumably would lower

Aβ release (38). In addition, NSAIDs regulate arachidonic acid not only by inhibiting COX

enzymes but also via interaction with lipoxygenases. 5-Lipoxygenase activity increases in the

brain with age, is inhibited by indomethacin, ibuprofen, and sulindac (21,22). On the other hand,

some NSAIDs increase activity and expression of 15-lipoxygenase. Consequently, we

investigated the role of lipoxygenases and AA metabolism in Aβ42 generation via

pharmacologic and genetic means. Mobilization of AA by supplementation of culture medium

with exogenous AA or by activation of PLA2 by melittin did not reduce Aβ42 levels but showed

a slight increase in total Aβ levels. In our studies, neither 5- nor 15-LOX appeared to influence

Aβ42:Aβ40 in conditioned medium. We cannot exclude the possibility that inflammatory

leukotrienes generated by lipoxygenases are an exacerbating factor in Alzheimer’s disease, but

this does not appear to be manifested in Aβ42 levels.

PPARs are another important class of NSAID targets that have been the focus of some

attention, especially in the cancer-related literature. The PPAR family of nuclear receptors

consists of PPARα, PPARγ and PPARδ (also denoted as PPARβ). The NSAIDs indomethacin,

flufenamic acid, fenoprofen and ibuprofen appear to directly activate the transcriptional activity

of PPARα and PPARγ (27). PPARs also have important roles in modulating lipid and glucose

metabolism. In this regard, γ-secretase activity can be strongly influenced by the cholesterol

content of the plasma membrane (39), and insulin affects not only glucose metabolism but also

APP processing and Aβ levels (40). In addition, PPAR-γ agonists have anti-inflammatory

properties, and in particular, these agonists inhibited Aβ stimulated release of proinflammatory

products from microglia (25). Despite interesting potential links to amyloid or NSAID activity,

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our studies described here were not able to observe any selective effects of PPAR

overexpression, activation or inhibition on lowering Aβ42 levels.

Lastly, PPARs also regulate transcriptional activation by STAT, AP-1 and NFκB

transcription factors. In this context, overexpression of NFκB/p65 has been shown to increase

the proportion of Aβ1-42 (33). Moreover, there is evidence that NSAIDs can antagonize NFκB

more directly. NFκB may have profound effects in Alzheimer’s disease progression by elevating

levels of inducible nitric oxide synthase, tumor necrosis factors, interleukins and complement

factors, all of which are known to be elevated in AD brains (3). Our results showed that NSAID-

induced Aβ42 effects were intact in IKKβ or p65-deficient fibroblasts, much as has been seen in

COX-deficient MEFs, thereby arguing against this pathway as a major contributor to the Aβ42

reduction.

In summary, our studies failed to identify a potential candidate cellular pathway, known

to be affected by NSAIDs, that mediates the Aβ42 effect. Our studies therefore reinforce the

concept that the ability to reduce Aβ42 production is not shared by all NSAIDs, but rather it is

due to a novel secondary activity. Our studies did not address whether NSAIDs can directly

target either APP or the γ-secretase complex itself. In view of our results, it is tempting to

suggest that NSAIDs may conformationally alter γ-secretase activity, much as presenilin

mutations preferentially increase Aβ42 levels. This notion would be consistent with the subtle

switch in Aβ peptides following treatment of cultured cells with certain NSAIDs to favor the

shorter species. In related studies we have observed that the Aβ42 lowering activity can be

modified by presenilins and that Aβ42 generation from an in vitro γ-secretase assay can be

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modified by NSAIDs 1. A recent report by Takahashi et al. using in vitro assay also suggested

that NSAIDs alter the activity of γ-secretase (41). Taken together, these observations lead us to

favor the notion that NSAIDs alter Aβ42 production by subtly modifying APP cleavage either at

the level of the substrate or a component of the γ-secretase complex, rather than indirectly, such

as by modulating cellular signaling pathways. Identification of the molecular target, however,

will be difficult for two reasons. First, the precise nature of γ-secretase remains to be elucidated

and second, NSAIDs reduce Aβ42 production with low affinity. Nevertheless, the findings

presented here are an important first step in the long term goal of identifying compounds that

selectively lower Aβ42 in vivo but have negligible COX inhibition to limit the well known

gastrointestinal side effects as potential candidates for AD treatment.

1 Weggen, S. et al. submitted to JBC

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Acknowledgements

We thank E. Shaulian for p65 knockout MEFs, Dr. I. Verma for IKKβ knockout MEFs, Drs. C.

Glass and M. Ricote kindly provided PPAR plasmids, Dr. Numa Gottardi-Littell for APP695

adenovirus, W. Wechter of Encore Pharmaceuticals for R-flurbiprofen. Compounds LG10305

and BRL49653 were kindly provided by Dr. R. Evans. Compound GW9662 was kindly

provided by Glaxo Smith Kline Pharmaceuticals. We are grateful to R. Wang and E. Komives

for providing training and advice for MALDI-TOF. We also wish to thank T. Souder and T.

Monnier for their efforts in performing Aβ ELISA assays. This work is supported by NIH

Grants AG 20206 (TEG and EHK) and 2T32 AG00216 (SAS).

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References

1. Selkoe, D. J. (2001) Physiol Rev. 81, 741-766

2. Zandi, P. P. and Breitner, J. C. (2001) Neurobiol.Aging 22, 811-817

3. Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G. M., Cooper, N. R.,

Eikelenboom, P., Emmerling, M., Fiebich, B. L., Finch, C. E., Frautschy, S., Griffin, W. S.,

Hampel, H., Hull, M., Landreth, G., Lue, L., Mrak, R., Mackenzie, I. R., McGeer, P. L.,

O'Banion, M. K., Pachter, J., Pasinetti, G., Plata-Salaman, C., Rogers, J., Rydel, R., Shen, Y.,

Streit, W., Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F. L., Veerhuis, R., Walker, D.,

Webster, S., Wegrzyniak, B., Wenk, G., and Wyss-Coray, T. (2000) Neurobiol.Aging 21,

383-421

4. Aisen, P. S. and Davis, K. L. (1994) Am.J.Psychiatry 151, 1105-1113

5. Aisen, P. S. (2000) Neurobiol.Aging 21, 447-448

6. Wyss-Coray, T. and Mucke, L. (2000) Nat.Med. 6, 973-974

7. Golde, T. E. (2002) Nat.Med. 8, 936-938

8. Lim, G. P., Yang, F., Chu, T., Chen, P., Beech, W., Teter, B., Tran, T., Ubeda, O., Ashe, K.

H., Frautschy, S. A., and Cole, G. M. (2000) J.Neurosci. 20, 5709-5714

9. Lim, G. P., Yang, F., Chu, T., Gahtan, E., Ubeda, O., Beech, W., Overmier, J. B., Hsiao-

Ashec, K., Frautschy, S. A., and Cole, G. M. (2001) Neurobiol.Aging 22, 983-991

10. Jantzen, P. T., Connor, K. E., DiCarlo, G., Wenk, G. L., Wallace, J. L., Rojiani, A. M.,

Coppola, D., Morgan, D., and Gordon, M. N. (2002) J.Neurosci. 22, 2246-2254

11. Weggen, S., Eriksen, J. L., Das, P., Sagi, S. A., Wang, R., Pietrzik, C. U., Findlay, K. A.,

Smith, T. E., Murphy, M. P., Bulter, T., Kang, D. E., Marquez-Sterling, N., Golde, T. E., and

Koo, E. H. (2001) Nature 414, 212-216

by guest on June 28, 2020http://w

ww

.jbc.org/D

ownloaded from

20

12. Suzuki, N., Cheung, T. T., Cai, X. D., Odaka, A., Otvos, L., Jr., Eckman, C., Golde, T. E.,

and Younkin, S. G. (1994) Science 264, 1336-1340

13. Murphy, M. P., Uljon, S. N., Fraser, P. E., Fauq, A., Lookingbill, H. A., Findlay, K. A.,

Smith, T. E., Lewis, P. A., McLendon, D. C., Wang, R., and Golde, T. E. (2000)

J.Biol.Chem. 275, 26277-26284

14. Uljon, S. N., Mazzarelli, L., Chait, B. T., and Wang, R. (2000) Methods Mol.Biol. 146, 439-

452

15. Wiltfang, J., Smirnov, A., Schnierstein, B., Kelemen, G., Matthies, U., Klafki, H. W.,

Staufenbiel, M., Huther, G., Ruther, E., and Kornhuber, J. (1997) Electrophoresis 18, 527-

532

16. Koch, H. J. and Szecsey, A. (2000) Neurology 55, 1067

17. Aisen, P. S., Davis, K. L., Berg, J. D., Schafer, K., Campbell, K., Thomas, R. G., Weiner, M.

F., Farlow, M. R., Sano, M., Grundman, M., and Thal, L. J. (2000) Neurology 54, 588-593

18. Anthony, J. C., Breitner, J. C., Zandi, P. P., Meyer, M. R., Jurasova, I., Norton, M. C., and

Stone, S. V. (2000) Neurology 54, 2066-2071

19. Morihara, T., Chu, T., Ubeda, O., Beech, W., and Cole, G. M. (2002) J.Neurochem. 83,

1009-1012

20. Hassid, A. and Levine, L. (1977) Res.Commun.Chem.Pathol.Pharmacol. 18, 507-517

21. Kolasa, T., Brooks, C. D., Rodriques, K. E., Summers, J. B., Dellaria, J. F., Hulkower, K. I.,

Bouska, J., Bell, R. L., and Carter, G. W. (1997) J.Med.Chem. 40, 819-824

22. Manev, H., Uz, T., Sugaya, K., and Qu, T. (2000) FASEB J. 14, 1464-1469

23. Vanderhoek, J. Y., Ekborg, S. L., and Bailey, J. M. (1984) J.Allergy Clin.Immunol. 74, 412-

417

by guest on June 28, 2020http://w

ww

.jbc.org/D

ownloaded from

21

24. Shureiqi, I., Chen, D., Lee, J. J., Yang, P., Newman, R. A., Brenner, D. E., Lotan, R.,

Fischer, S. M., and Lippman, S. M. (2000) J.Natl.Cancer Inst. 92, 1136-1142

25. Combs, C. K., Johnson, D. E., Karlo, J. C., Cannady, S. B., and Landreth, G. E. (2000)

J.Neurosci. 20, 558-567

26. Combs, C. K., Bates, P., Karlo, J. C., and Landreth, G. E. (2001) Neurochem.Int. 39, 449-457

27. Lehmann, J. M., Lenhard, J. M., Oliver, B. B., Ringold, G. M., and Kliewer, S. A. (1997)

J.Biol.Chem. 272, 3406-3410

28. He, T. C., Chan, T. A., Vogelstein, B., and Kinzler, K. W. (1999) Cell 99, 335-345

29. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., and

Kliewer, S. A. (1995) J.Biol.Chem. 270, 12953-12956

30. Leesnitzer, L. M., Parks, D. J., Bledsoe, R. K., Cobb, J. E., Collins, J. L., Consler, T. G.,

Davis, R. G., Hull-Ryde, E. A., Lenhard, J. M., Patel, L., Plunket, K. D., Shenk, J. L.,

Stimmel, J. B., Therapontos, C., Willson, T. M., and Blanchard, S. G. (2002) Biochemistry

41, 6640-6650

31. Yamamoto, Y., Yin, M. J., Lin, K. M., and Gaynor, R. B. (1999) J.Biol.Chem. 274, 27307-

27314

32. Straus, D. S., Pascual, G., Li, M., Welch, J. S., Ricote, M., Hsiang, C. H.,

Sengchanthalangsy, L. L., Ghosh, G., and Glass, C. K. (2000) Proc.Natl.Acad.Sci.U.S.A 97,

4844-4849

33. Tomita, S., Fujita, T., Kirino, Y., and Suzuki, T. (2000) J.Biol.Chem. 275, 13056-13060

34. Beg, A. A., Sha, W. C., Bronson, R. T., Ghosh, S., and Baltimore, D. (1995) Nature 376,

167-170

by guest on June 28, 2020http://w

ww

.jbc.org/D

ownloaded from

22

35. Zandi, P. P., Anthony, J. C., Hayden, K. M., Mehta, K., Mayer, L., and Breitner, J. C. (2002)

Neurology 59, 880-886

36. Carlson, M. C., Tschanz, J. T., Norton, M. C., Welsh-Bohmer, K., Martin, B. K., and

Breitner, J. C. (2002) Alzheimer Dis.Assoc.Disord. 16, 24-30

37. Chan, T. A., Morin, P. J., Vogelstein, B., and Kinzler, K. W. (1998)

Proc.Natl.Acad.Sci.U.S.A 95, 681-686

38. Emmerling, M. R., Moore, C. J., Doyle, P. D., Carroll, R. T., and Davis, R. E. (1993)

Biochem.Biophys.Res.Commun. 197, 292-297

39. Wahrle, S., Das, P., Nyborg, A. C., McLendon, C., Shoji, M., Kawarabayashi, T., Younkin,

L. H., Younkin, S. G., and Golde, T. E. (2002) Neurobiol.Dis. 9, 11-23

40. Watson, G. S. and Craft, S. (2003) CNS.Drugs 17, 27-45

41. Takahashi, Y., Hayashi, I., Tominari, Y., Rikimaru, K., Morohashi, Y., Kan, T., Natsugari,

H., Fukuyama, T., Tomita, T., and Iwatsubo, T. (2003) J.Biol.Chem. 278, 18664-18670 by guest on June 28, 2020http://w

ww

.jbc.org/D

ownloaded from

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Figure legends

Figure 1: Schematic diagram highlighting the multiple pathways downstream of

arachidonic acid known to be influenced by NSAIDs. The cellular components indicated in

bold type are activated by NSAIDs while those in italics are inhibited by NSAIDs.

Figure 2: Aββββ42 lowering potential of NSAIDs does not correlate with the ability to inhibit

COX. A: MALDI-TOF analysis of Aβ reveals that not all NSAIDs alter Aβ42 and Aβ38 levels.

PS1ML cells were treated with 300µM naproxen, 350µM ibuprofen, 250 µM flurbiprofen or

DMSO control. Ibuprofen and flurbiprofen reduced Aβ42 and increased Aβ38 species while

naproxen had no effect on any of the Aβ species. B: Bicine-urea SDS gel analysis of Aβ from

NSAID-treated cells. Results from naproxen, ibuprofen, and flurbiprofen treatment were similar

to that obtained MALDI-TOF. Migration of Aβ standard peptides is indicated on the right. C:

Bicine-urea SDS gel analysis of Aβ with flurbiprofen enantiomers. Cells were treated with

250µM racemic, purified S- or R-flurbiprofen. Both enantiomers reduced Aβ42 as effectively as

racemic compound.

Figure 3: The Aββββ42 lowering potential of NSAIDs does require lipoxygenase 5,

lipoxygenase 15, IKK2 or p65RelA. ELISA analysis of Aβ in the media of sulindac-treated

fibroblasts genetically deficient in various components. A Fibroblasts from neonatal LOX5-/-

mice. B Fibroblasts from LOX15-/- neonatal mice. C IKK2-/- MEFs, D p65/RelA-/- MEFs.

Shown are averages ± S.E.

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Figure 4: PPARs do not alter Aββββ42 production. A and B: Inhibition of PPARγ with GW9662

does not affect the NSAID response. PS1ML cells were treated with the indicated concentrations

of the PPARγ inhibitor GW9662 with or without 75µM sulindac. Reduction in Aβ42 was

unaffected by PPARγ inhibition as shown in a representative experiment. Migration of Aβ38,

Aβ40 and Aβ42 standard peptides is indicated on the right. Quantitation of the GW9662

treatment studies is shown in panel B. C: Overexpression of PPARγ or PPARδ does not alter

Aβ40 or Aβ42 levels. APP-WT CHO cells were transiently transfected with PPARγ or PPARδ,

treated with the indicated agonists and Aβ levels determined by ELISA. Shown are averages ±

S.D.

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Table 1: Anti-inflammatory treatment does not reduce Aββββ42:Aββββ40. Effects of indicated

compounds on Aβ42:Aβ40 as determined by ELISA.

Compound Concentrations tested

Selective reduction in Aβ42

Prednisone 0.1-10µM None Dexamethasone 5nM-5µM None Cimetidine 1-20µM None Curcumin 1-50µM None* *Aβ42:Aβ40 was slightly increased at curcumin doses 20µM and above.

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Table 2: Altered arachidonic acid metabolism does not explain the NSAID effect on Aββββ42.

Aβ42 and Aβ40 levels were determined by ELISA.

Compound Primary Action Concentrations tested

Selective reduction in Aβ42

Arachidonic acid ↑PLA2 25-250µM None** Melittin ↑PLA2 0.1-5.0µg/ml None MK886 ↓5-LOX 0.1-1µM None Caffeic acid ↓5-LOX 5-50µM None Baicalein ↓15-LOX 5-50µM None ** Arachidonic acid caused a slight decrease in Aβ42:Aβ40, due to an increase in Aβ40 but did

not decrease Aβ42.

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Table 3: Peroxisome proliferator-activated receptors do not alter Aββββ42:Aββββ40. Aβ42 and

Aβ40 levels were determined by ELISA.

Compound Receptor target Concentrations tested

Selective reduction in Aβ42

8-(S)-HETE ↑PPARα 0.1−1µΜ None Ciglitazone ↑PPARγ 5-50µM None Prostaglandin J2 ↑PPARγ 0.1−10µM None BRL ↑PPARγ 0.1−1µM None LG 10305 ↑RXR 0.1−10µM None

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Figure 1

Phospholipase A2

Arachidonic Acid

5-LipoxygenasesCyclooxygenases 1122//1155--LLiippooxxyyggeennaasseess

PPPPAARRγγγγγγγγ

Prostanoids 5-HETEs Leukotrienes 12/15-HPETE

IKK/NFκB PPPPAARRαααααααα

Insulin resistanceCholesterol by guest on June 28, 2020

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control

ibuprofen

naproxen

flurbiprofen

Figure 2

Aβ40

Aβ42

Aβ40

Aβ42

Aβ40Aβ42

A

B C

Aβ40Aβ42

Aβ38

Aβ38

Aβ38

Aβ38

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Sarah A. Sagi, Sascha Weggen, Jason Eriksen, Tood E. Golde and Edward H. Koo42 productionβreduce A

B, do notκThe non-COX targets of NSAIDs, lipoxygenases, PPARs, IKK and NF

published online June 12, 2003J. Biol. Chem. 

  10.1074/jbc.M303588200Access the most updated version of this article at doi:

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