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. Author manuscript; available in PMC: 2006 Oct 23.
Published in final edited form as: J Neurochem. 2006 Jan 12;96(4):917–923. doi: 10.1111/j.1471-4159.2005.03586.x

PGH2-derived levuglandin adducts increase the neurotoxicity of amyloid β1–42

Olivier Boutaud *, Thomas J Montine †, Lei Chang ‡, William L Klein ‡, John A Oates *,§
PMCID: PMC1621054  NIHMSID: NIHMS11479  PMID: 16412101

Abstract

The body of evidence indicating that oligomers of amyloid β1–42 (Aβ1–42) produce toxicity to neurons, together with our demonstration that prostaglandin H2 (PGH2) oligomerizes amyloid β1–42, led to the examination of the neurotoxicity of amyloid β1–42 treated with PGH2. The neurotoxic effects of Aβ1–42 incubated with PGH2 was examined in primary cultures of cerebral neurons of mice, monitoring the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an indicator of cell toxicity. Whereas Aβ1–42 itself, incubated for 24 h, has little or no effect on MTT reduction, Aβ1–42 24 h after exposure to PGH2 produced a marked inhibition of MTT reduction, comparable with the inhibition resulting from Aβ1–42 that has been oligomerized by incubation for 6 days. Similar results were obtained when Aβ1–42 was incubated with levuglandin E2 (LGE2), a reactive alde-hyde formed by spontaneous rearrangement of PGH2. The oligomers formed from reaction of Aβ1–42 with LGE2 exhibit immunochemical similarity with amyloid-derived diffusible ligands (ADDLs), as determined by analysis of the products of reaction of Aβ1–42 with LGE2 using western blotting with an antibody that is selective for ADDLs.

Keywords: amyloid, amyloid-derived diffusible ligands, cyclooxygenase, levuglandin, neurotoxicity, prostaglandin H

A contribution of cyclooxygenase (COX) activity to the pathogenesis of Alzheimer's disease (AD) may be inferred from two large prospective studies on individuals with no baseline dementia that found a 60–80% reduction in risk of developing AD associated with the use of non-steroidal anti-inflammatory drugs for at least 2 years (Stewart et al. 1997; in ′t Veld et al. 2001). An alternative explanation of these results is derived from the finding that some non-steroidal anti-inflammatory drugs (NSAIDs) used at high concentrations have been shown to exert COX-independent effects, including alteration of γ-secretase cleavage of the amyloid-precursor protein (Weggen et al. 2001, 2003; Eriksen et al. 2003). The finding of a fivefold elevation of prostaglandin E2 (PGE2) in cerebrospinal fluid of patients with AD reflects increased COX activity in AD (Montine et al. 1999). A link between COX activity and loss of cognitive function is also consistent with data from animal models of dementia. Transgenic mice overexpressing human COX-2 (hCOX-2) in brain develop an age-related loss of cognitive function that is reversed by celecoxib (Andreasson et al. 2001). In a transgenic mouse model of AD with mutations of the amyloid precursor protein (APPswe) and presenilin-1 (A246E), amyloid plaque formation is increased by overexpression of hCOX-2 (Xiang et al. 2002). Moreover, treatment with the non-specific COX inhibitor, ibuprofen, suppresses amyloid plaque formation and ameliorates behavioral changes in an APPswe (Tg2576) transgenic mouse (Lim et al. 2000, 2001).

The product of both COX isoforms is the endoperoxide, prostaglandin H2 (PGH2), which may then serve as a substrate for enzymes that yield prostaglandins and thromboxane A2 as products. PGH2 also rapidly undergoes rearrangement to PGE2 and PGD2, and via ring cleavage to secoprostaglandins that are γ-keto-aldehydes (Salomon et al. 1984). These γ-keto-aldehydes, designated levuglandin (LG) E2 and LGD2, constitute about 20% of the rearrangement products of PGH2 (Salomon et al. 1984; Boutaud et al. 1999). The γ-keto-aldehydes are also formed by free radical oxidation of arachidonic acid in vitro (Brame et al. 1999) and in cell membranes following oxidant injury (Brame et al. 2004). The γ-keto-aldehyde structure is highly reactive and readily forms adducts with amine groups. By adducting the ε-amine of lysine or the N-terminal α-amine, LGs form covalent adducts on proteins (Iyer et al. 1994). Importantly, the initial lysyl-levuglandin adducts themselves are also highly reactive; the Schiff base adduct with lysine can undergo nucleophilic attack (Harada 1970) and the oxidation of the pyrrole adducts generates sites of electrophilic reactivity (Amarnath et al. 1994, 1998). Thus, lysyl-LG adducts can form covalent bonds with other nucleophiles, leading to intermolecular crosslinking. Such crosslinking has been demonstrated between proteins (Iyer et al. 1989; Boutaud et al. 2001).

The spontaneous and reversible association of amyloid β (Aβ) into dimers led us to consider that this dimerization would energetically favor the crosslinking of Aβ by levuglandins. Accordingly, we examined the effect of PGH2 on Aβ1–42, and demonstrated that PGH2 greatly accelerated the formation of dimers and higher oligomers (Boutaud et al. 2002b). The same marked increase in the rate of formation of oligomers of Aβ was produced by synthetic LGE2. The consequences of accelerated formation of stable Aβ oligomers upon reaction with PGH2 have now been examined by assessing whether the resulting oligomers of Aβ1–42 are neurotoxic. For this purpose, the effect of the oligomers of Aβ produced by PGH2 and by LGE2 on the viability of neurons in primary culture was examined.

Material and methods

Materials

Dulbecco's modified Eagle's medium (DMEM), fetal calf serum and N2 neuron supplement were purchased from Gibco (Rockville, MD, USA). Aβ1–42 was purchased from Sigma (St Louis, MO, USA). PGH2 and LGE2 were prepared as described previously in Boutaud et al. (2002a) and Amarnath et al. (2005), respectively.

Primary cell culture of neurons

Primary dissociated cultures of neurons were prepared according to methods described in Culturing Nerve Cells by Gary Banker and Kimberly Goslin (Banker and Goslim 1998). Wild-type mice on the C57Bl/6J background were used for the neurotoxicity experiments. The pregnant mouse (embryonic day 15, E15) was killed, then the uterus was removed and placed in a sterile Petri dish. The remaining procedures were performed in a laminar flow hood. Brains from E15 mice were dissected under aseptic conditions while immersed in calcium- and magnesium-free Hank's Balanced Salt Solution (HBSS); the cerebral cortex with meninges was removed, transferred to another dish and again immersed in HBSS. All the cerebral cortices from one litter were transferred to a 50 mL conical tube to which 9 mL HBSS and 1 mL 2.5% trypsin were added. After incubation for 15 min at 37 °C, the trypsin-containing solution was removed and replaced by 10 mL HBSS for 5 min. This step was repeated two times. Tissues were then dissociated by triturating first with a Pasteur pipette and then with a Pasteur pipette with the tip fire-polished to about one half the normal diameter. Trituration was continued until no chunks of tissue remained. Cell density was determined with a hemacytometer, and the number of viable cells assessed by Trypan blue exclusion. Cells were then distributed onto poly d-lysine-coated 12-well plates at a density of 3 × 105 cells/cm2 and maintained in DMEM supplemented with 10% fetal calf serum and N2 (Gibco) neuron supplement. Growth medium was changed once weekly and cultures were used between 14 and 16 days in vitro.

Effect of PGH2 on the neurotoxicity of Aβ

Aβ1–42 35 μm, in phosphate-buffered saline (PBS) containing 200 μm EDTA, was incubated for 24 h with PGH2 at a concentration that provides LGE2/D2 in a molar equivalent to each lysine of Aβ1–42, or with the same volume of vehicle (acetone). Aβ1–42 was also incubated for 6 days at 22°C under the same conditions as above to generate the neurotoxic species describes as ‘aged’ amyloid (Simmons et al. 1994; Landolfi et al. 1998). After incubation, 100 μL of the different solutions of Aβ1–42 were pre-incubated at 22°C with 10 μL 10× growth medium for 2 min in order to ‘pre-adduct’ any undegraded LGE2, and added to mouse primary cerebral neurons for determination of the neurotoxicity.

Effect of LGE2 on the neurotoxicity of Aβ

Because of the potential neurotoxicity of PGH2 itself, similar experiments were performed by incubating Aβ1–42 with LGE2. Aβ1–42 35 μm in PBS containing 200 μm EDTA was incubated for 24 h with 0.2 molar equivalents of synthetic LGE2, or with the same volume of vehicle (ethanol). Aβ1–42 in PBS containing 200 μm EDTA was also incubated for 6 days at 22°C to allow formation of toxic oligomers. Because LGE2 might harbor cellular toxicity by itself, we included a control condition in which synthetic LGE2 was incubated in PBS containing 200 μm EDTA for 24 h. After incubation, an aliquot was saved for analysis of oligomers by western blot, and 100 μL of the different solutions of Aβ1–42 were pre-incubated at 22°C with 10 μL 10× growth medium for 2 min in order to ‘pre-adduct’ any undegraded LGE2; these were then added to mouse primary cerebral neurons for determination of the neurotoxicity.

Neurotoxicity experiments

At day 14 of culture, 100 μL of the different solutions of Aβ1–42 in PBS containing 200 μm EDTA were mixed with 10 μL 10× medium and added to the cells. After 24 h, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 2.5 mg/mL in water was added to the cells for a final concentration of 250 μg/mL and incubated at 37°C for 2 h. At this time, the medium was removed and the purple solid solubilized in 100 μL HCl 0.1 N in isopropanol. The absorbance at 570 nm was monitored in a spectrophotometer.

Analysis of Aβ1–42 oligomers by SDS–PAGE

The presence of oligomers in the solutions of Aβ1–42 incubated with ethanol or 0.1 molar equivalents of synthetic LGE2 for 24 h, or incubated for 6 days, was analyzed by western blot. After incubation, 5 μg peptide were diluted with sodium dodecyl sulfate (SDS) loading buffer containing 15% β-mercaptoethanol and denatured at 70°C for 10 min. After separation by SDS–polyacrylamide gel electrophoresis (PAGE) using 4–12% Tris-Tricine gels, the proteins were transferred onto nitrocellulose membranes 0.2 μm (Bio-Rad Laboratories, Hercules, CA, USA) and immediately boiled for 30 min in Tris-buffered saline. The proteins were then analyzed by western blot with a polyclonal Aβ1–42 antibody (Zymed Laboratories, South San Francisco, CA, USA). Proteins were visualized by horseradish peroxidase-catalyzed chemiluminescence (NEN Life Sciences, PerkinElmer, Boston, MA, USA). In parallel experiments, Aβ1–42 was incubated with concentrations of PGH2 that provide levuglandins in 0.05 or 0.1 molar equivalent to each amyloid. After 24 h at 22°C, 0.8 μg Aβ1–42 was diluted with SDS loading buffer and the proteins were analyzed by western blot as described above.

Analysis of amyloid-derived diffusible ligands (ADDLs) by western blot

Aβ1–42 at 8.8 μm was incubated with ethanol or LGE2 in 1 or 0.1 molar equivalent at 22°C in sodium phosphate buffer, pH 7.4, with 200 μm EDTA for 24 h. After incubation, 0.25 μg peptide was diluted with SDS loading buffer. No heat was applied to the samples prior to loading on the gel. The proteins were separated by SDS–PAGE using 16.5% Tris-tricine gels (Bio-Rad), and were transferred onto nitrocellulose membranes 0.2 μm (Bio-Rad) at 100 V for 60 min at 4°C. The proteins were then analyzed by western blot with a polyclonal antibody specific for ADDLs (M90.2) as described previously (Lambert et al. 2001). Proteins were visualized by horseradish peroxidase-catalyzed chemiluminescence (NEN Life Sciences).

Preparation of ADDLs

Aβ1–42 peptide (California Peptide Research, Napa, CA, USA) was used to prepare synthetic ADDLs according to published protocols (Chang et al. 2003).

Results

The aim of this investigation was to test the hypothesis that Aβ1–42 oligomers formed by reaction with PGH2 were neurotoxic. The neurotoxic effects of PGH2-treated Aβ1–42 on mouse cerebral neurons in primary cultures was determined by monitoring the reduction of MTT (Shearman et al. 1994, 1995; Hughes et al. 2000; Woltjer et al. 2003). Aβ1–42 was incubated for 24 h with or without PGH2 as described in the previous studies in which oligomers were formed within this time period upon exposure to PGH2 (Boutaud et al. 2002b). Whereas Aβ1–42 itself incubated for 24 h had no effect on MTT reduction (Fig. 1), Aβ1–42 24 h after exposure to PGH2 produced a marked inhibition of MTT reduction that was comparable to the neurotoxicity known to result from aggregated amyloid formed by incubation of Aβ1–42 for 6 days (Simmons et al. 1994; Landolfi et al. 1998).

Fig. 1.

Fig. 1

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PGH2 accelerates the neurotoxic properties of Aβ1–42. Aβ1–42 in PBS was incubated for 24 h with PGH2 at a concentration that provides LGE2/D2 in a molar equivalent to each lysine of Aβ1–42, or with the same volume of vehicle (acetone). Aβ1–42 in PBS was also incubated for 6 days at 22°C to allow formation of oligomers. After incubation, 100 μL of the different solutions of Aβ1–42 or of PBS were mixed with 10 μL 10× growth medium and added to mouse primary cerebral neurons at day 14 of culture in 96-well plates. After 24 h, MTT 2.5 mg/mL in water was added to the cells for a final concentration of 250 μg/mL and incubated at 37°C for 2 h. At this time, the medium was removed and the purple crystals were solubilized in 100 μL HCl 0.1 N in isopropanol. The absorbance at 595 nm was monitored in a spectrophotometer. Each data point represents the mean ± SEM of six values; *p < 0.01; **p < 0.005.

Because PGH2 can rearrange to form PGE2 and PGD2 in addition to LGE2, the same experiments were carried out using synthetic LGE2. Also, LGE2 alone was used as a control to exclude direct neurotoxicity, which is considered unlikely after 24 h in the incubation buffer followed by exposure to serum containing amino acids that should ‘pre-adduct’ any undegraded LGE2 before it is added to the neurons. As seen in Fig. 2(a), incubation of Aβ1–42 with LGE2 for 24 h produced the same inhibition of MTT reduction as Aβ1–42 incubated for 6 days. By comparison, Aβ1–42 incubated alone for 24 h had no more effect than PBS or LGE2 added alone to the cells.

Fig. 2.

Fig. 2

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LGE2-derived oligomers of Aβ1–42 are neurotoxic. Aβ1–42 in PBS was incubated for 24 h with 0.2 molar equivalents of synthetic LGE2 or with the same volume of vehicle (ethanol). Aβ1–42 in PBS was also incubated for 6 days at 22°C to allow formation of oligomers, and synthetic LGE2 was incubated in PBS for 24 h. (a) After incubation, 100 μL of the solutions of Aβ1–42, LGE2 or PBS were mixed with 10 μL 10× growth medium and added to mouse primary cerebral neurons at day 14 of culture. After 24 h, MTT was added to the cells for a final concentration of 250 μg/mL and incubated at 37°C for 2 h. At this time, the medium was removed and the purple crystals were solubilized in 100 μL HCl 0.1 N in isopropanol. The absorbance at 595 nm was monitored in a spectrophotometer. Each data point represents the mean ± SEM of six values; **p < 0.005. (b) After incubation, 5 μg Aβ1–42 incubated as indicated in the figure were separated by SDS–PAGE using 4–12% Nupage gel, and the different species of Aβ1–42 were analyzed by western blot.

The species of Aβ present in the conditions associated with neurotoxicity were assessed by western blot analysis after heat denaturation of the peptides in the presence of SDS. In the absence of LGE2, no oligomers were detected by western blot after 24 h, whereas oligomerization was extensive after LGE2 was added for 24 h (Fig. 2b). Similar oligomerization was observed when Aβ1–42 was incubated in PBS for 6 days (Fig. 2b). This electrophoretic mobility of the oligomers is the same as the mobility observed when Aβ1–42 was incubated with PGH2 under the same conditions (Boutaud et al. 2002b). Using western blotting with an antibody that is selective for ADDLs (Chang et al. 2003; Gong et al. 2003), we determined that the products of reaction of LGE2 with Aβ1–42 exhibit immunochemical similarity with ADDLs, and that formation of these oligomers occurred at a molar ratio as little as 0.1 LGE2 per Aβ1–42 (Fig. 3). In these experiments, the samples were not denatured by heat prior to gel migration. It is important to note at this point that when analyzed in mild denaturing conditions (no heat applied to the samples), traces of oligomers were detected in the control conditions by the ADDL-specific antibody (Fig. 3), whereas no oligomers were detected under the same conditions when analyzed by western blot following strong denaturing conditions (Fig. 2b). In contrast, incubation of Aβ1–42 with LGE2 in a molar ratio LGE2/Aβ1–42 as low as 0.05 yielded oligomers that were heat resistant. Similar observations were made when analyzed by electron microscopy, where large fibrils were observed under non-denaturing conditions after 24 h whereas no aggregates were observed by western analysis (Boutaud et al. 2002b). This suggests that oligomers formed spontaneously in 24 h are sensitive to temperature, but that reaction of Aβ1–42 with LGE2 induces a change in the structure of the oligomers formed in 24 h, rendering them resistant to denaturation by heat.

Fig. 3.

Fig. 3

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LGE2 induces formation of oligomers of Aβ1–42 that exhibit immunochemical similarity with ADDLs in a dose-dependant manner. Aβ1–42 in PBS was incubated for 24 h with 1.0 or 0.1 molar equivalents of synthetic LGE2, or with the same volume of vehicle (ethanol). After incubation, 0.25 μg Aβ1–42 incubated as indicated in the figure was separated by SDS–PAGE using 16.5% Tris-tricine gel, and the different species of Aβ1–42 were analyzed by western blot using an antibody specific for ADDLs (M90.2). A control solution of ADDLs was obtained as described previously (Lambert et al. 2001).

Discussion

Soluble oligomers of Aβ are increased in the brain in Alzheimer's disease (Kuo et al. 1996; Hardy and Selkoe 2002; Gong et al. 2003) and these oligomers are neurotoxic (Roher et al. 1996; Lambert et al. 1998; Hartley et al. 1999; Dahlgren et al. 2002; Walsh et al. 2002; Stine et al. 2003; Lacor et al. 2004). We have previously demonstrated that the addition of PGH2 or LGE2 to Aβ markedly accelerates formation of soluble oligomers that have the ultrastructural features of Aβ-derived diffusible ligands (Boutaud et al. 2002b). We now provide evidence that these oligomers are neurotoxic and are recognized by ADDL selective antibodies. These results provide a new molecular link between COX activity and the accumulation of soluble oligomers of Aβ in AD brain.

Although Aβ1–42 oligomers have been implicated in memory loss, the understanding of mechanisms that contribute to oligomer formation is still evolving. One proposed mechanism is the covalent modification of the Aβ1–42 peptide following oxidative stress catalyzed by metals such as copper (Atwood et al. 2000; Bush et al. 2003; Sparks and Schreurs 2003). We present evidence for a new mechanism of Aβ1–42 oligomerization which proceeds through formation of covalent adducts of reactive lipids generated by rearrangement of the product of cyclooxygenase.

To ascertain that oligomerization of Aβ by LGE2 occurred by a mechanism that was independent of divalent metal cations, we performed the experiments in a buffer containing 200 μM EDTA; in these conditions we were not able to observe toxicity or SDS-stable oligomers after 24 h of incubation. PGH2 rearranges not only to LG but also to PGE2 and PGD2; PGE2 and PGD2 can be dehydrated to PGA2 and PGJ2, respectively, and the PGE2 and PGA2 series is known to cause neurodegeneration in neuroblastoma cells (Prasad et al. 1998). The potentially confounding effects of these non-LG rearrangement/dehydration products of PGH2 were addressed by demonstrating that Aβ1–42 incubated with LGE2 itself is neurotoxic. Whereas the PGH2 used in these studies has the stereochemical characteristics of that formed enzymatically by the COXs, the synthetic LGE2 comprised several isomers. This indicates that the same neurotoxicity would be expected from iso-levuglandins derived from the series of PGH2 isomers as results from free radical-mediated lipid peroxidation.

Oligomerization of Aβ by LGE2 occurs with ratios of LGE2 : Aβ of only 1 : 10. This suggests that intermolecular crosslinking cannot be the sole mechanism for oligomerization, and raises the possibility that lipid modification of Aβ serves as a seed to accelerate oligomerization, analogous to the lipid modification of Aβ by GM1 ganglioside (Hayashi et al. 2004) or by cholesterol-derived molecules (Zhang et al. 2004).

This demonstration that LGE2 induces formation of neurotoxic oligomers of Aβ1–42 in vitro occurs in the context of evidence that COX activity can engender formation of LG adducts of protein in cells and in vivo. This includes our finding that activation of platelets produces LG adducts of platelet proteins that is COX-1-dependant (Boutaud et al. 2003), and that overexpression of hCOX-2 in mouse neurons increases the formation of LG adducts of protein in brain (Boutaud et al. 2005). More recently, we have demonstrated that levels of LG adducts of protein in the hippocampus of patients with AD are increased 12-fold in comparison with age-matched controls (Zagol-Ikapitte et al. 2005). Moreover, the levels of LG adducts of protein are highly correlated with the pathological evidence of AD severity. These findings in concert provide a rationale for investigations that examine the consequences of COX-dependent oligomerization of Aβ in AD, and in animal models that simulate AD.

Previously, the cellular and biochemical significance of prostanoids has been framed in terms of the stable agents, such as PGE2, PGF2α and PGD2, and their reversible interactions with G-protein receptors. The evidence presented in this report lays the foundation for a new biochemical paradigm for the participation of the cyclooxygenase enzymes in Alzheimer's disease.

Acknowledgements

This work was supported in part by grants from the American Health Assistance Foundation, and from the NIH (GM15431, AG24011, AG11385, AG18877, AG22547 and AG05136). JAO is the Thomas F. Frist, Sr. Professor of Medicine.

Abbreviations used

Aβ

amyloid β

AD

Alzheimer's disease

ADDLs

amyloid-derived diffusible ligands

COX

cyclooxygenase

DMEM

Dulbecco's modified Eagle's medium

E

embryonic day

HBSS

Hank's balanced salt solution

LG

levuglandin

MTT

3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide

NSAID

non-steroidal anti-inflammatory drug

PBS

phosphate-buffered saline

PG

prostaglandin

References

  1. Amarnath V, Valentine WM, Montine TJ, Patterson WH, Amarnath K, Bassett CN, Graham DG. Reactions of 4-hydroxy-2 (E)-nonenal and related aldehydes with proteins studied by carbon-13 nuclear magnetic resonance spectroscopy. Chem. Res. Toxicol. 1998;11:317–328. doi: 10.1021/tx970176n. [DOI] [PubMed] [Google Scholar]
  2. Amarnath V, Valentine WM, Amarnath K, Eng MA, Graham DG. The mechanism of nucleophilic substitution of alkylpyrroles in the presence of oxygen. Chem. Res. Toxicol. 1994;7:56–61. doi: 10.1021/tx00037a008. [DOI] [PubMed] [Google Scholar]
  3. Amarnath V, Amarnath K, Masterson TM, Davies SS, Roberts LJI. A simplified synthesis of the diastereomers of levuglandin E2. Synthetic Comm. 2005;35:397–408. [Google Scholar]
  4. Andreasson KI, Savonenko A, Vidensky S, et al. Age-dependent cognitive deficits and neuronal apoptosis in cyclooxygenase-2 transgenic mice. J. Neurosci. 2001;21:8198–8209. doi: 10.1523/JNEUROSCI.21-20-08198.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Atwood CS, Huang X, Khatri A, Scarpa RC, Kim YS, Moir RD, Tanzi RE, Roher AE, Bush AI. Copper catalyzed oxidation of Alzheimer Abeta. Cell. Mol. Biol. 2000;46:777–783. [PubMed] [Google Scholar]
  6. Banker G, Goslim K. Culturing Nerve Cells. 2nd Edn. MIT Press; Cambridge, Massachusetts: 1998. [Google Scholar]
  7. Boutaud O, Brame CJ, Salomon RG, Roberts LJ, 2nd, Oates JA. Characterization of the lysyl adducts formed from prostaglandin H2 via the levuglandin pathway. Biochemistry. 1999;38:9389–9396. doi: 10.1021/bi990470+. [DOI] [PubMed] [Google Scholar]
  8. Boutaud O, Brame CJ, Chaurand P, et al. Characterization of the lysyl adducts of prostaglandin H-synthases that are derived from oxygenation of arachidonic acid. Biochemistry. 2001;40:6948–6955. doi: 10.1021/bi002629k. [DOI] [PubMed] [Google Scholar]
  9. Boutaud O, Aronoff DM, Richardson JH, Marnett LJ, Oates JA. Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H2 synthases. Proc. Natl Acad. Sci. USA. 2002a;99:7130–7135. doi: 10.1073/pnas.102588199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boutaud O, Ou JJ, Chaurand P, Caprioli RM, Montine TJ, Oates JA. Prostaglandin H2 (PGH2) accelerates formation of amyloid β1–42 oligomers. J. Neurochem. 2002b;82:1003–1006. doi: 10.1046/j.1471-4159.2002.01064.x. [DOI] [PubMed] [Google Scholar]
  11. Boutaud O, Li J, Zagol I, Shipp EA, Davies SS, Roberts LJ, 2nd, Oates JA. Levuglandinyl adducts of proteins are formed via a prostaglandin H2 synthase-dependent pathway after platelet activation. J. Biol. Chem. 2003;278:16 926–16 928. doi: 10.1074/jbc.M300940200. [DOI] [PubMed] [Google Scholar]
  12. Boutaud O, Andreasson KI, Zagol-Ikapitte I, Oates JA. Cyclooxygenase-dependent lipid-modification of brain proteins. Brain Pathol. 2005;15:139–142. doi: 10.1111/j.1750-3639.2005.tb00510.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Brame CJ, Salomon RG, Morrow JD, Roberts LJ., 2nd Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts. J. Biol. Chem. 1999;274:13 139–13 146. doi: 10.1074/jbc.274.19.13139. [DOI] [PubMed] [Google Scholar]
  14. Brame CJ, Boutaud O, Davies SS, Yang T, Oates JA, Roden D, Roberts LJ., 2nd Modification of proteins by isoketal-containing oxidized phospholipids. J. Biol. Chem. 2004;279:13 447–13 451. doi: 10.1074/jbc.M313349200. [DOI] [PubMed] [Google Scholar]
  15. Bush AI, Masters CL, Tanzi RE. Copper, beta-amyloid, and Alzheimer's disease: tapping a sensitive connection. Proc. Natl Acad. Sci. USA. 2003;100:11 193–11 194. doi: 10.1073/pnas.2135061100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chang L, Bakhos L, Wang Z, Venton DL, Klein WL. Femtomole immunodetection of synthetic and endogenous amyloid-beta oligomers and its application to Alzheimer's disease drug candidate screening. J. Mol. Neurosci. 2003;20:305–313. doi: 10.1385/JMN:20:3:305. [DOI] [PubMed] [Google Scholar]
  17. Dahlgren KN, Manelli AM, Stine WB, Jr, Baker LK, Krafft GA, LaDu MJ. Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability. J. Biol. Chem. 2002;277:32046–32053. doi: 10.1074/jbc.M201750200. [DOI] [PubMed] [Google Scholar]
  18. Eriksen JL, Sagi SA, Smith TE, et al. NSAIDs and enantiomers of flurbiprofen target gamma-secretase and lower Abeta 42 in vivo. J. Clin. Invest. 2003;112:440–449. doi: 10.1172/JCI18162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gong Y, Chang L, Viola KL, Lacor PN, Lambert MP, Finch CE, Krafft GA, Klein WL. Alzheimer's disease-affected brain: Presence of oligomeric A{beta} ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc. Natl Acad. Sci. USA. 2003;100:10417–10422. doi: 10.1073/pnas.1834302100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harada K. Additions to the azomethine group. In: Patai S, editor. The Chemistry of Carbon-Nitrogen Double Bound. Interscience Publishers; 1970. pp. 255–298. [Google Scholar]
  21. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 2002;297:353–356. doi: 10.1126/science.1072994. [DOI] [PubMed] [Google Scholar]
  22. Hartley DM, Walsh DM, Ye CP, Diehl T, Vasquez S, Vassilev PM, Teplow DB, Selkoe DJ. Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J. Neurosci. 1999;19:8876–8884. doi: 10.1523/JNEUROSCI.19-20-08876.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hayashi H, Kimura N, Yamaguchi H, et al. A seed for Alzheimer amyloid in the brain. J. Neurosci. 2004;24:4894–4902. doi: 10.1523/JNEUROSCI.0861-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hughes E, Burke RM, Doig AJ. J. Biol. Chem. Vol. 275. Inhibition of toxicity in the beta-amyloid peptide fragment beta-(25–35) using N-methylated derivatives: a general strategy to prevent amyloid formation; 2000. pp. 25 109–25 115. [DOI] [PubMed] [Google Scholar]
  25. in ′t Veld BA, Ruitenberg A, Hofman A, Launer LJ, van Duijn CM, Stijnen T, Breteler MM, Stricker BH. Nonsteroidal antiinflammatory drugs and the risk of Alzheimer's disease. N. Engl. J. Med. 2001;345:1515–1521. doi: 10.1056/NEJMoa010178. [DOI] [PubMed] [Google Scholar]
  26. Iyer RS, Ghosh S, Salomon RG. Levuglandin E2 crosslinks proteins. Prostaglandins. 1989;37:471–480. doi: 10.1016/0090-6980(89)90096-8. [DOI] [PubMed] [Google Scholar]
  27. Iyer RS, Kobierski ME, Salomon RG. Generation of pyrroles in the reaction of levuglandin E2 with proteins. J. Org. Chem. 1994;59:6038–6043. [Google Scholar]
  28. Kuo YM, Emmerling MR, Vigo-Pelfrey C, Kasunic TC, Kirkpatrick JB, Murdoch GH, Ball MJ, Roher AE. Water-soluble Abeta (N-40, N-42) oligomers in normal and Alzheimer disease brains. J. Biol. Chem. 1996;271:4077–4081. doi: 10.1074/jbc.271.8.4077. [DOI] [PubMed] [Google Scholar]
  29. Lacor PN, Buniel MC, Chang L, et al. Synaptic targeting by Alzheimer's-related amyloid beta oligomers. J. Neurosci. 2004;24:10191–10200. doi: 10.1523/JNEUROSCI.3432-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lambert MP, Barlow AK, Chromy BA, et al. Diffusible, nonfibrillar ligands derived from Abeta1–42 are potent central nervous system neurotoxins. Proc. Natl Acad. Sci. USA. 1998;95:6448–6453. doi: 10.1073/pnas.95.11.6448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lambert MP, Viola KL, Chromy BA, et al. Vaccination with soluble Abeta oligomers generates toxicity-neutralizing antibodies. J. Neurochem. 2001;79:595–605. doi: 10.1046/j.1471-4159.2001.00592.x. [DOI] [PubMed] [Google Scholar]
  32. Landolfi C, Soldo L, Polenzani L, et al. Inflammatory molecule release by beta-amyloid-treated T98G astrocytoma cells: role of prostaglandins and modulation by paracetamol. Eur. J. Pharmacol. 1998;360:55–64. doi: 10.1016/s0014-2999(98)00663-3. [DOI] [PubMed] [Google Scholar]
  33. Lim GP, Yang F, Chu T, et al. Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer's disease. J. Neurosci. 2000;20:5709–5714. doi: 10.1523/JNEUROSCI.20-15-05709.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lim GP, Yang F, Chu T, et al. Ibuprofen effects on Alzheimer pathology and open field activity in APPsw transgenic mice. Neurobiol. Aging. 2001;22:983–991. doi: 10.1016/s0197-4580(01)00299-8. [DOI] [PubMed] [Google Scholar]
  35. Montine TJ, Sidell KR, Crews BC, Markesbery WR, Marnett LJ, Roberts LJ, 2nd, Morrow JD. Elevated CSF prostaglandin E2 levels in patients with probable AD. Neurology. 1999;53:1495–1498. doi: 10.1212/wnl.53.7.1495. [DOI] [PubMed] [Google Scholar]
  36. Prasad KN, Hovland AR, La Rosa FG, Hovland PG. Prostaglandins as putative neurotoxins in Alzheimer's disease. Proc. Soc. Exp. Biol. Med. 1998;219:120–125. doi: 10.3181/00379727-219-44323. [DOI] [PubMed] [Google Scholar]
  37. Roher AE, Chaney MO, Kuo YM, et al. Morphology and toxicity of Abeta-(1–42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease. J. Biol. Chem. 1996;271:20 631–20 635. doi: 10.1074/jbc.271.34.20631. [DOI] [PubMed] [Google Scholar]
  38. Salomon RG, Miller DB, Zagorski MG, Coughlin DJ. Solvent-induced fragmentation of prostaglandin endoperoxides. New aldehyde products from PGH2 and a novel intramolecular 1,2-hydride shift during endoperoxide fragmentation in aqueous solution. J. Am. Chem. Soc. 1984;106:6049–6060. [Google Scholar]
  39. Shearman MS, Ragan CI, Iversen LL. Inhibition of PC12 cell redox activity is a specific, early indicator of the mechanism of beta-amyloid-mediated cell death. Proc. Natl Acad. Sci. USA. 1994;91:1470–1474. doi: 10.1073/pnas.91.4.1470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shearman MS, Hawtin SR, Tailor VJ. The intracellular component of cellular 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction is specifically inhibited by beta-amyloid peptides. J. Neurochem. 1995;65:218–227. doi: 10.1046/j.1471-4159.1995.65010218.x. [DOI] [PubMed] [Google Scholar]
  41. Simmons LK, May PC, Tomaselli KJ, et al. Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro. Mol. Pharmacol. 1994;45:373–379. [PubMed] [Google Scholar]
  42. Sparks DL, Schreurs BG. Trace amounts of copper in water induce beta-amyloid plaques and learning deficits in a rabbit model of Alzheimer's disease. Proc. Natl Acad. Sci. USA. 2003;100:11 065–11 069. doi: 10.1073/pnas.1832769100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stewart WF, Kawas C, Corrada M, Metter EJ. Risk of Alzheimer's disease and duration of NSAID use. Neurology. 1997;48:626–632. doi: 10.1212/wnl.48.3.626. [DOI] [PubMed] [Google Scholar]
  44. Stine WB, Jr, Dahlgren KN, Krafft GA, LaDu MJ. In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J. Biol. Chem. 2003;278:11 612–11 622. doi: 10.1074/jbc.M210207200. [DOI] [PubMed] [Google Scholar]
  45. Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 2002;416:535–539. doi: 10.1038/416535a. [DOI] [PubMed] [Google Scholar]
  46. Weggen S, Eriksen JL, Das P, et al. A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature. 2001;414:212–216. doi: 10.1038/35102591. [DOI] [PubMed] [Google Scholar]
  47. Weggen S, Eriksen JL, Sagi SA, Pietrzik CU, Ozols V, Fauq A, Golde TE, Koo EH. Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid {beta}42 production by direct modulation of {gamma}-secretase activity. J. Biol. Chem. 2003;278:31831–31837. doi: 10.1074/jbc.M303592200. [DOI] [PubMed] [Google Scholar]
  48. Woltjer RL, Maezawa I, Ou JJ, Montine KS, Montine TJ. Advanced glycation endproduct precursor alters intracellular amyloid-beta/A beta PP carboxy-terminal fragment aggregation and cytotoxicity. J. Alzheimers Dis. 2003;5:467–476. doi: 10.3233/jad-2003-5607. [DOI] [PubMed] [Google Scholar]
  49. Xiang Z, Ho L, Yemul S, et al. Cyclooxygenase-2 promotes amyloid plaque deposition in a mouse model of Alzheimer's disease neuropathology. Gene Expr. 2002;10:271–278. doi: 10.3727/000000002783992352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zagol-Ikapitte I, Masterson TS, Amarnath V, Montine TJ, Andreasson KI, Boutaud O, Oates JA. Prostaglandin HH-derived adducts of proteins correlate with Alzheimer's disease severity. J. Neurochem. 2005;94:1140–1145. doi: 10.1111/j.1471-4159.2005.03264.x. [DOI] [PubMed] [Google Scholar]
  51. Zhang Q, Powers ET, Nieva J, et al. Metabolite-initiated protein misfolding may trigger Alzheimer's disease. Proc. Natl Acad. Sci. USA. 2004;101:4752–4757. doi: 10.1073/pnas.0400924101. [DOI] [PMC free article] [PubMed] [Google Scholar]

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