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J. Biol. Chem., Vol. 279, Issue 15, 14673-14678, April 9, 2004

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Peroxidase Activity of Cyclooxygenase-2 (COX-2) Cross-links -Amyloid (A) and Generates A-COX-2 Hetero-oligomers That Are Increased in Alzheimer's Disease^*

Seiichi Nagano, Xudong Huang, Robert D. Moir¶, Sandra M. Payton, Rudolph E. Tanzi¶, and Ashley I. Bush||**

From the Laboratory for Oxidation Biology, Genetics and Aging Research Unit, and the Department of Psychiatry, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts 02129, the ^¶Genetics and Aging Research Unit and the Department of Neurology, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts 02129, and the ^||Department of Pathology, University of Melbourne, and Oxidation Disorders Laboratory, Mental Health Research Institute of Victoria, Parkville, Victoria 3052, Australia

Received for publication, December 1, 2003 , and in revised form, December 22, 2003.

	ABSTRACT

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Oxidative stress is associated with the neuropathology of Alzheimer's disease. We have previously shown that human A has the ability to reduce Fe(III) and Cu(II) and produce hydrogen peroxide coupled with these metals, which is correlated with toxicity against primary neuronal cells. Cyclooxygenase (COX)-2 expression is linked to the progression and severity of pathology in AD. COX is a heme-containing enzyme that produces prostaglandins, and the enzyme also possesses peroxidase activity. Here we investigated the possibility of direct interaction between human A and COX-2 being mediated by the peroxidase activity. Human A formed dimers when it was reacted with COX-2 and hydrogen peroxide. Moreover, the peptide formed a cross-linked complex directly with COX-2. Such cross-linking was not observed with rat A, and the sole tyrosine residue specific for human A might therefore be the site of cross-linking. Similar complexes of A and COX-2 were detected in post-mortem brain samples in greater amounts in AD tissue than in age-matched controls. COX-2-mediated cross-linking may inhibit A catabolism and possibly generate toxic intracellular forms of oligomeric A.

	INTRODUCTION

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The neocortical accumulation of -amyloid (A)¹ may play a central role in Alzheimer's disease (AD) (1, 2). Although the mechanism of neuronal damage by A is uncertain, the accumulated A correlates with oxidative damage to lipids, proteins, and nucleic acids in the brains of patients (3–6) and in amyloid precursor protein transgenic mice (7). The toxicity of synthetic human A is exerted by the catalytic generation of H₂O₂ (8, 9), which may be important in designing treatment strategies for AD.

Human A is a metalloprotein that binds Zn(II) and Cu(II) in amyloid plaque (9–11). The ability of A to produce H₂O₂ is mediated by the reduction of Cu(II) and Fe(III) to Cu(I) and Fe(II), respectively (8, 12). Rat A, substituted at three residues compared with human A (Arg⁵ Gly, Tyr¹⁰ Phe, and His¹³ Arg), is less redox active, producing less H₂O₂, and is correspondingly less toxic (8, 9, 12).

Human A is also vulnerable to oxidative damage and cross-linking. Peroxidative activity such as horseradish peroxidase (HRP) or Cu(II)/H₂O₂ generates SDS-resistant oxidized A oligomers linked by dityrosine (DT) bridges (13, 14).

Cyclooxygenases (COX) are members of a heme enzyme family that catalyze the rate-limiting reaction to produce prostaglandins (15). COX-1 is the widely expressed constitutive form, and COX-2 is the inducible form that is up-regulated by cytokines and mitogens. COX-2 may play an important role in AD. Epidemiological studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs), inhibitors of COX, delay the onset of AD (16–18). Ibuprofen, a nonselective COX inhibitor, attenuates plaque pathology in Tg2576 mice (19). Neuronal COX-2 expression is increased in the affected regions of AD brain (20–23), correlating to the severity of AD pathology (24). COX-2 overexpression in primary neurons potentiates A neurotoxicity in vitro (25). Therefore, COX-2 may interact with the metabolism of A in AD.

COX harnesses two enzymatic activities to produce prostaglandin H₂ (PGH₂). Authentic "cyclooxygenase" activity first converts arachidonic acid to prostaglandin G₂ (PGG₂), and subsequent peroxidase activity reduces PGG₂ to PGH₂. The peroxidase reaction of COX is analogous to that of HRP and can utilize a wide range of hydroperoxides including H₂O₂, rather than PGG₂, as substrates (15). We suspected that the peroxidase activity of COX-2 might induce the formation of A oligomers. To explore this possibility, we examined the effect of COX-2 on A oligomerization and report the formation of A-COX-2 complexes in vitro and in vivo.

	MATERIALS AND METHODS

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Reagents—A peptides were synthesized, purified, and characterized by high pressure liquid chromatography (HPLC), amino acid analysis, and mass spectroscopy by the W. Keck Laboratory of Yale University (New Haven, CT). Ovine COX-2 purified from placenta and polyclonal anti-COX-2 antibody were purchased from Cayman Chemical (Ann Arbor, MI). N-Acetylimidazole and phenylglyoxal were obtained from Acros Organics (Geel, Belgium). Monoclonal anti-A antibodies 4G8 (which detects A residues 18–22) and W0–2 (which detects A residues 5–8) (51) were obtained from Signet Laboratories (Dedham, MA). Horseradish peroxidase-conjugated anti-mouse and rabbit IgG antibodies were from Amersham Biosciences. MagnaBind goat anti-mouse and rabbit IgG beads were from Pierce. The other reagents were obtained from Sigma unless otherwise noted.

Brain Samples—Frozen human post-mortem brain samples (superior temporal cortex, Brodmann area 22, 41/42) from moderately affected AD (Braak stage 3–4 (52), n = 5), severely affected AD (Braak stage 6, n = 4), and control cases (n = 4) were obtained from the Harvard Brain Tissue Resource Center (Belmont, MA). The profile of each case is summarized in Table I. There were no significant differences in the average age or post-mortem interval between the groups (p > 0.5, analysis of variance).

Immunoprecipitation—Solutions reacted in vitro were incubated with 2 µl of anti-COX-2 antibody overnight at 4 °C and with 30 µl of MagnaBind anti-rabbit IgG beads for 1 h at 4 °C subsequently. For co-immunoprecipitation of A or COX-2 from tissue, control and AD brain samples were homogenized by a glass homogenizer in 10 ml/g of ice-cold phosphate-buffered saline containing protease inhibitor mixture (Roche Applied Science). The supernatant was separated by centrifugation at 20,000 x g for 10 min and adjusted to a final protein concentration of 1.75 mg/ml with homogenization buffer. After pretreatment with 10 µl of MagnaBind beads to decrease the nonspecific binding of proteins, the supernatant (0.8 ml) was incubated with 3 µl of anti-A (W0–2) or COX-2 antibody overnight at 4 °C and subsequently with 30 µl of MagnaBind beads for 3 h at 4 °C. For Western blot, the precipitates were washed three times with phosphate-buffered saline and boiled for 5 min at 95 °C with sample buffer containing 5% 2-mercaptoethanol.

Immunoblotting—The samples in sample buffer were loaded onto a NuPAGE 4–12% Bis-Tris gel and separated by electrophoresis at 200 V for 45 min. The gel was then transferred to polyvinylidene difluoride membrane (Bio-Rad) at 75 mA for 90 min. The membrane was blocked with 10% dry milk, 5% bovine serum albumin for anti-A antibodies or 5% dry milk for anti-COX-2 antibody for 1 h at 25 °C. The membrane was then incubated with a primary antibody overnight at 4 °C followed by a horseradish peroxidase-conjugated secondary antibody for 1 h at 25 °C. Antibody labeling was detected by LumiGLO chemiluminescent kit (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The density of each band from the immunoblotting was analyzed by National Institutes of Health Image 1.62 software (National Institutes of Health, Bethesda, MD).

	RESULTS

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To study the effects of COX-2 on the oligomerization of A, we incubated purified ovine COX-2 (140 nM) with human synthetic A 1–40 ("h40," 2.5 µM) in the presence of H₂O₂ (1 µM) for 2hat37 °C. We observed that the synthetic A alone remained predominantly monomeric upon SDS-PAGE but that the combination of COX-2 and H₂O₂ induced the conspicuous formation of an apparent A dimer, as well as A immunoreactive bands in the range of 50–75 kDa (Fig. 1A). Incubation of A with COX-2 alone induced far lower amounts of the apparent dimer and higher molecular weight A immunoreactive products, but because the incubation was performed under aerobic conditions, small concentrations of H₂O₂ may have been present because of the reduction and subsequent disproportionation of dissolved O₂ (26). This interpretation was indeed supported by the experimental effects of the H₂O₂ scavenger catalase, which completely abolished all but monomeric A immunoreactivity being detected upon co-incubation with A, COX-2, and H₂O₂ (Fig. 1A). A incubated with H₂O₂ alone remained monomeric on SDS-PAGE (Fig. 1B).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Peroxidase activity of COX-2 induces the cross-linking of A. A, human A 1–40 (h40, 2.5 µM) was incubated alone (2 h at 37 °C) or in the presence of COX-2 (140 nM) ± H2O2 (1 µM), as indicated, and Western blotted with 4G8 anti-A antibody. This reaction induced apparent dimerization of A and possible A adduction of COX-2. These apparent cross-links were abolished by co-incubation with catalase (CAT, 10 µg/ml), but not by dimethyl sulfoxide (DMSO, 10 mM) or mannitol (MAN, 10 mM). H2O2 alone incubated with A did not induce apparent cross-linking (see panel B). As a positive control, human A was reacted with HRP (10 µg/ml) to induce dityrosine cross-linking (13). B, human, but not rat, A cross-linking is induced by COX-2/H2O2. A (rat 1–40 (r40) or human 1–40, 2.5 µM), COX-2 (70 nM), and H2O2 (1 µM) were incubated together, or the components were incubated separately as indicated, for 15 h at 37 °C and blotted with 4G8 anti-A antibody. An arrow and an asterisk indicate the apparent dimer of human A and a possible complex of human A with COX-2, respectively. Even after such protracted incubation, the rat homologue of A did not form apparent cross-links.

Alzheimer neuropathology is only seen in animal species that have the human sequence of A (27) and not in rats and mice, which have three amino acid substitutions (Arg⁵ Gly, Tyr¹⁰ Phe, and His¹³ Arg) (28). The tyrosine substitution in the rat/mouse A ("rat A") abolishes the possibility of DT interstrand bridge formation. To explore further whether COX-2 induces DT interstrand bridge formation, we repeated the experiment comparing the products of rat A to human A (Fig. 1B). Although incubation with COX-2/H₂O₂ (but not H₂O₂ alone) induced apparent dimer formation of human A 1–40 as well as high molecular weight immunoreactivity, no such modifications of rat A 1–40 were apparent even after 15 h of incubation (Fig. 1B).

The molecular mass of COX-2 is 70 kDa, and we determined that COX-2 itself did not cross-react with 4G8 antibody (Fig. 1B). Therefore, the A immunoreactive band at 75 kDa when synthetic human A was reacted with COX-2 and hydrogen peroxide (Fig. 1, A and B, asterisk) may indeed be a complex of human A and COX-2. To confirm that this 75-kDa A immunoreactivity reflects A complex formation with COX-2, we immunoprecipitated the incubation products with a polyclonal anti-COX-2 antibody and immunoblotted the immunoprecipitates with anti-A antibody (WO2). We again observed the 75-kDa band from the sample of human A1–40 reacted with COX-2 and hydrogen peroxide (Fig. 2, arrow) but not from the samples of COX-2, human A1–40 alone, or rat A1–40 incubated with COX-2 and hydrogen peroxide. Therefore, human A may have adducted onto the COX-2 in this reaction. In addition, an extra band at 50 kDa was seen only in the precipitate from the sample of human A with COX-2 and hydrogen peroxide (Fig. 2, arrowhead), which may be a complex of human A with a degraded COX-2 fragment.

View larger version (34K): [in this window] [in a new window]   FIG. 2. Confirmation of apparently cross-linked A-COX-2 complexes by immunoprecipitation with COX-2 antibody. A (rat 1–40 or human 1–40, 10 µM), COX-2 (140 nM), and H2O2 (1 µM) were incubated together, or the components were incubated separately as indicated, for 15 h at 37 °C. The samples were immunoprecipitated with anti-COX-2 antibody and then blotted with W0–2 anti-A antibody. An arrow and an arrowhead indicate SDS-resistant complexes of A and COX-2 at approximately 75 and 50 kDa, respectively.

View larger version (31K): [in this window] [in a new window]   FIG. 3. Characterization of the reaction mechanism for apparent Across-linking induced by COX-2/H2O2. A, effect of peroxidase inhibitors. Human A 1–40 (2.5 µM) and COX-2 (140 nM) ± H2O2 (1 µM) were co-incubated (2 h, 37 °C) as indicated with catalase (CAT, 10 µg/ml), desferroxamine (DFO, 1 mM), azide (2 mM) or cyanide (CYN, 2 mM), and the products were blotted with 4G8 anti-A antibody. B, effect of NSAIDs. Human A 1–40 (2.5 µM) and COX-2 (140 nM) ± H2O2 (1 µM) were co-incubated (2 h, 37 °C) as indicated with indomethacin (IM, 100 µM), ibuprofen (IBU, 500 µM), or aspirin (ASP, 500 µM), and the products were blotted with 4G8 anti-A antibody. As a further negative control, human A was incubated with H2O2 (1 µM) + COX-2 (final concentration, 140 nM) that had been inactivated by boiling (10 min, 95 °C). C, effects of side chain modification. Human A 1–40 (10 µM), COX-2 (140 nM) ± H2O2 (1 µM) were co-incubated (1 h, 37 °C) with diethylpyrocarbonate (DEPC, 3 mM), N-acetylimidazole (NAI, 1 mM), or phenylglyoxal (PG, 1 mM) and blotted with W0–2 anti-A antibody.

Arg⁵, Tyr¹⁰, and His¹³ in human A1–40 are substituted in rat A1–40, which did not polymerize in the presence of COX-2 (Fig. 1B). Therefore, one of these three residues may be the site of dimeric cross-linking induced by COX-2/H₂O₂. As a preliminary analysis to identify the amino acid responsible for the cross-linking, we examined the effect of chemical modifiers for each amino acid (29) on the apparent cross-linking induced by COX-2/H₂O₂ (Fig. 3C). Phenylglyoxal, an arginine modifier, did not have an apparent effect. N-Acetylimidazole, which acetylates tyrosine hydroxyl groups, partially inhibited the apparent cross-linking consistent with inhibiting the formation of dityrosine bridges. Diethylpyrocarbonate, a histidine modifier, did not inhibit the cross-linking but rather promoted the oligomerization of A. This result may indicate that the histidine residues play a role in minimizing radical reactivity or that the diethylpyrocarbonate is acting exceptionally in this system as a chemical cross-linker. Among the caveats with this approach is that the modifications are not completely selective for amino acids. Although future studies of amino acid substituted A will be needed to consolidate these conclusions, these findings do not refute our earlier interpretation (Figs. 1 and 3, A and B) that DT cross-linking could be a consequence of the peroxidative activity of COX-2.

We next explored for evidence that COX-2 might react with A in vivo. There is already abundant evidence for SDS-resistant A dimers and polymers being enriched in AD-affected post-mortem brain tissue (2, 30). Furthermore, there is evidence of elevated H₂O₂ in AD-affected brain tissue (4). Our in vitro data indicated that whenever the combination of COX-2 and H₂O₂ induced A apparent dimeric cross-linking, a proportion of A always adducted to COX-2 itself (Figs. 1, 2, 3). Therefore, if COX-2 indeed contributed to A oligomeric cross-linking in vivo, the presence of such A-COX-2 complexes would be anticipated in post mortem human brain tissue as a biomarker. We investigated this possibility by co-immunoprecipitation of AD and age-matched control post-mortem human brain tissue with anti-A and COX-2 antibodies, as in Fig. 1B. Two major bands were seen at approximately 70 and 50 kDa that were cross-immunoreactive with both antibodies (Fig. 4A). These bands were close to the molecular masses of the two major bands detected in anti-COX-2 immunoprecipitation of human A1–40 incubated with ovine COX-2 and H₂O₂, detected by A immunoblot (Fig. 1B). Again, the 50-kDa band might be a degraded fragment of the complex possibly formed by the attack of free radicals. As a further negative control, we also probed these blots with antibody against human copper/zinc superoxide dismutase, which is widely expressed in the brain, and no immunoreactivity was detected (data not shown).

View larger version (36K): [in this window] [in a new window]   FIG. 4. A-COX-2 complexes are elevated in AD-affected brain tissue. A, detection of A-COX-2 complexes by co-immunoprecipitation in human brains. Brain tissues from superior temporal area were homogenized, and the supernatants were immunoprecipitated with anti-COX-2 or A (W0–2) antibody. The precipitates were blotted with anti-A (4G8) or COX-2 antibody, respectively. The background data of the cases are shown in Table I. B, integrated band densities ± S.D. of the data in panel A. IP, immunoprecipitation. *, significantly increased compared with the respective control group (p < 0.05, paired t test).

	DISCUSSION

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Our data indicate that the peroxidative activity of COX-2 induces the dimerization of human A by a H₂O₂-mediated mechanism, and the enzyme itself also cross-links with human A directly. These cross-links were attenuated by a scavenger of H₂O₂ and by peroxidase inhibitors. Our evidence, for the first time, implicates COX-2 activity in the direct oxidation of A, to generate SDS-resistant oligomeric A forms that resemble forms that have previously been reported to be increased in the disease and proposed to mediate pathophysiology.

A is extracted from AD brains as toxic water-soluble, SDS-resistant oligomers (30, 31). Soluble A levels, including the SDS-resistant oligomers, are correlated to the disease severity of AD (32). Diffusible A oligomers are deleterious to hippocampal neurons in vitro (33) and in vivo (34). Recent data have proposed that A oligomers are generated intracellularly and secreted subsequently to form extracellular amyloid fibrils (35). Evidence suggests that the subcellular origin of these oligomers may be microsomes (35), the same compartment that contains COX. To date, none of the reports correlating SDS-resistant apparent oligomers of A with neurotoxicity have established the basis of the SDS resistance. A will form SDS-resistant oligomers to some extent in the absence of any apparent oxidative modification (Fig. 3A). However, our data establish that oxidation will lead to an apparent covalent cross-link that leads to similar SDS resistance. The possibility that oxidized A SDS-resistant oligomers (e.g. produced by COX-2 activity) are more neurotoxic than the nonoxidized oligomers must be considered.

COX possesses both cyclooxygenase and peroxidase activities. The cycle of the peroxidase reaction can occur independently of cyclooxygenase activity, utilizing hydroperoxides such as hydrogen peroxide (15). During the peroxidase reaction, ferric heme of the resting enzyme is oxidized to form ferryl iron and a porphyrin radical (Intermediate I). The porphyrin radical is reduced to form a tyrosyl radical at Tyr³⁸⁵ by an intramolecular electron transfer (Intermediate II). In the presence of reducing co-substrates, Intermediates I and II are returned to resting state (36). Phenolic compounds such as phenol (15), luminol (37), and dopamine (38, 39) are preferred as reducing co-substrates. We hypothesized that the sole tyrosine residue of human A could be a target for the peroxidative activity of COX-2 and that tyrosyl radicals of human A formed by the reduction of oxidized COX-2 will cross-link each other to generate DT (40), emulating the reaction of HRP with A (13). The A-COX-2 complex that we observed may also be formed from the bridging of tyrosyl radicals of oxidized COX-2 and A.

Human A is a strongly redox active peptide that reduces copper and iron ions (8, 12). The same properties of the peptide may lead to redox reactions with COX-2. The redox activity of A also fosters the catalytic generation of hydrogen peroxide (6, 8, 9, 12), which may supply the substrate to COX-2 for peroxidative cross-linking. A 1–40 and A 1–42 have high affinity for Cu²⁺ (41) and are bound to the metal ion in AD-affected brain tissue (9, 11). Therefore, A accumulating in the AD-affected neocortex is very likely to be a source of H₂O₂.H₂O₂ is freely permeable across lipid boundaries, which may contribute to its potency in mediating oxidative attack to neocortical cells (4, 6). Contributions to increased H₂O₂ in AD may also come from failing mitochondrial metabolism and from microglial activation. H₂O₂ generated extracellularly (e.g. from A or microglia) and intracellularly (e.g. from mitochondria) could migrate into microsomal compartments and then act as a cosubstrate for COX-2-mediated cross-linking of A. Pharmacological removal of excess copper and iron or scavenging of hydrogen peroxide may therefore be therapeutic strategies to prevent A oxidation by COX-2. Such a mechanism may contribute to the efficacy of clioquinol, which blocks hydrogen peroxide production by A by binding to copper and iron at its active site (42).

The tyrosine of human A is thought to coordinate the metals that interact with the peptide (43, 44), and we hypothesize that it may inappropriately coordinate with the metal active site of COX-2 (Fig. 5). The DT bond that may be formed in this reaction is chemically stable and resistant to proteolytic cleavage, and so the catabolism of DT cross-linked proteins or peptides is inhibited (45). The total DT content is elevated in the regions affected by A pathology in AD brains, in accordance with the regions where COX-2 expression is up-regulated (46). A polyclonal antibody raised against what may have been a DT-A antigen detected highly abundant immunoreactivity in neuritic plaques (14).

View larger version (15K): [in this window] [in a new window]   FIG. 5. Model for A oligomerization and A-COX-2 cross-linking.

Me₂SO and mannitol, scavengers of hydroxyl radicals, did not inhibit the cross-linking of A (Fig. 1A). This is consistent with the oxidative reaction of the peroxidase, which is not mediated by the production of hydroxyl radicals. Both sodium azide and sodium cyanide are general inhibitors of peroxidase. However, cyanide is known to have much stronger affinity to the COX peroxidase site than azide (15). Consistent with that, cyanide a showed stronger inhibitory effect on the cross-linking of A (Fig. 3A).

Interestingly, nonselective NSAIDs attenuated the A dimerization that was induced by COX-2 and COX-2 complex formation with human A (Fig. 3B). NSAIDs inhibit cyclooxygenase activity more than the peroxidase activity of COX. However, NSAIDs such as indomethacin and flurbiprofen specifically suppress the radical formation at Tyr³⁸⁵ of COX (36), suggesting that NSAIDs inhibited COX-2-A cross-linking (Fig. 3B) by decreasing tyrosyl radicals in COX-2. Alternatively, this inhibitory effect of NSAIDs may be a consequence of the direct interactions of NSAIDs with A peptide that have been described (49, 50). The concentrations of indomethacin and ibuprofen used in this study are similar to those in the report by Weggen et al. (50), who demonstrated that some NSAIDs decrease the production of human A1–42. The prevention of A cross-linking would facilitate A clearance and may be a novel mechanism for the proposed protective effect of NSAIDs on AD.

We detected the possible complexes of A and COX-2 from human brains by co-immunoprecipitation with antibodies against each component (Fig. 4). These complexes are increased in AD brains, especially in severely affected cases (Fig. 4). This may be due to the increased activity of COX-2 promoting more cross-linking of COX-2 and A or due to diminished clearance of the complexes in AD. The 50-kDa band of fragmented COX-2 that we observed (Fig. 4A) has also previously been detected in AD brains (21). This is consistent with our current data (Fig. 2) indicating that reaction with A may lead to the fragmentation of COX-2 into such a 50-kDa species that is adducted to A, whose concentrations appear to be increased in AD brain (Fig. 4).

The significance of the COX-2-A complex is not clear, although we hypothesize that abnormal A reaction with COX-2 may interfere with COX-2 integrity, function, or metabolism. More importantly, we noted that COX-2 mediates the production of A dimers, accompanied by the invariable generation of such COX-2-A complexes (Figs. 1, 2, 3, 4). Therefore, the detection of COX-2-A complexes in human tissue is evidence that these two proteins are in biochemical proximity in vivo and that therefore the peroxidation of A by COX-2 in AD may indeed contribute to the A oligomeric forms that are increasingly considered to mediate the disease process. If the intracellular oligomerization of A by COX-2 is an early event in the pathology of AD, its biochemical intervention may be a treatment strategy to halt or delay the disease progression at early stages.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant R01-1912686, the Alzheimer's Association, and the National Health and Medical Research Council (to A. I. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of Uehara Memorial Foundation Postdoctoral Fellowship.

^** To whom correspondence should be addressed: Laboratory for Oxidation Biology, Genetics and Aging Research Unit, Massachusetts General Hospital, Bldg. 114, 16th St., Charlestown, MA 02129. Tel.: 617-726-8244; Fax: 617-724-1823; E-mail: bush{at}helix.mgh.harvard.edu.

¹ The abbreviations used are: A, -amyloid; AD, Alzheimer's disease; COX, cyclooxygenase; HRP, horseradish peroxidase; DT, dityrosine; NSAID, nonsteroidal anti-inflammatory drug; PG, prostaglandin; HPLC, high pressure liquid chromatography.

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