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• W2013118051 abstract "Evidence of oxidative stress and the accumulation of fibrillar amyloid β proteins (Aβ) in senile plaques throughout the cerebral cortex are consistent features in the pathology of Alzheimer disease. To define a mechanistic link between these two processes, various aspects of the relationship between oxidative lipid membrane damage and amyloidogenesis were characterized by chemical and physical techniques. Earlier studies of this relationship demonstrated that oxidatively damaged synthetic lipid membranes promoted amyloidogenesis. The studies reported herein specify that 4-hydroxy-2-nonenal (HNE) is produced in both synthetic lipids and human brain lipid extracts by oxidative lipid damage and that it can account for accelerated amyloidogenesis. Aβ promotes the copper-mediated generation of HNE from polyunsaturated lipids, and in turn, HNE covalently modifies the histidine side chains of Aβ. HNE-modified Aβ have an increased affinity for lipid membranes and an increased tendency to aggregate into amyloid fibrils. Thus, the prooxidant activity of Aβ leads to its own covalent modification and to accelerated amyloidogenesis. These results illustrate how lipid membranes may be involved in templating the pathological misfolding of Aβ, and they suggest a possible chemical mechanism linking oxidative stress with amyloid formation. Evidence of oxidative stress and the accumulation of fibrillar amyloid β proteins (Aβ) in senile plaques throughout the cerebral cortex are consistent features in the pathology of Alzheimer disease. To define a mechanistic link between these two processes, various aspects of the relationship between oxidative lipid membrane damage and amyloidogenesis were characterized by chemical and physical techniques. Earlier studies of this relationship demonstrated that oxidatively damaged synthetic lipid membranes promoted amyloidogenesis. The studies reported herein specify that 4-hydroxy-2-nonenal (HNE) is produced in both synthetic lipids and human brain lipid extracts by oxidative lipid damage and that it can account for accelerated amyloidogenesis. Aβ promotes the copper-mediated generation of HNE from polyunsaturated lipids, and in turn, HNE covalently modifies the histidine side chains of Aβ. HNE-modified Aβ have an increased affinity for lipid membranes and an increased tendency to aggregate into amyloid fibrils. Thus, the prooxidant activity of Aβ leads to its own covalent modification and to accelerated amyloidogenesis. These results illustrate how lipid membranes may be involved in templating the pathological misfolding of Aβ, and they suggest a possible chemical mechanism linking oxidative stress with amyloid formation. Alzheimer disease (AD) 3The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β proteins; BHT, 3,5-di-tert-butylhydroxytoluene; CHCA, α-cyano-4-hydroxycinnamic acid; DMPC, 1,2-dimyristoyl-sn-glycerophosphocholine; DTPA, diethylenetriaminepentaacetic acid; HNE, 4-hydroxy-2-nonenal; SAPC, 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine; sinapinic acid, 2,5-dimethoxy-4-hydroxycinnamic acid; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MRM, multiple reaction monitoring; LC/MS/MS, liquid chromatography/tandem mass spectrometry; PATIR-FTIR, polarized attenuated total internal reflection-Fourier transform infrared; GC/MS, gas chromatography mass/spectrometry; CR, Congo Red; PFBHA, O-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine hydrochloride. 3The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β proteins; BHT, 3,5-di-tert-butylhydroxytoluene; CHCA, α-cyano-4-hydroxycinnamic acid; DMPC, 1,2-dimyristoyl-sn-glycerophosphocholine; DTPA, diethylenetriaminepentaacetic acid; HNE, 4-hydroxy-2-nonenal; SAPC, 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine; sinapinic acid, 2,5-dimethoxy-4-hydroxycinnamic acid; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MRM, multiple reaction monitoring; LC/MS/MS, liquid chromatography/tandem mass spectrometry; PATIR-FTIR, polarized attenuated total internal reflection-Fourier transform infrared; GC/MS, gas chromatography mass/spectrometry; CR, Congo Red; PFBHA, O-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine hydrochloride. is an age-related neurodegenerative disorder characterized by misfolded and aggregated fibrillar amyloid β proteins (Aβ) in the brain. Among the factors associated with the pathogenesis of AD, oxidative stress is one of the most closely scrutinized (1Markesbery W.R. Free Radic. Biol. Med. 1997; 23: 134-147Crossref PubMed Scopus (1961) Google Scholar, 2Butterfield D.A. Boyd-Kimball D. Brain Pathol. 2004; 14: 426-432Crossref PubMed Scopus (220) Google Scholar). It has been shown, for example, that the brain in AD has increased susceptibility to oxidative stress (3Lovell M.A. Ehmann W.D. Mattson M.P. Markesbery W.R. Neurobiol. Aging. 1997; 18: 457-461Crossref PubMed Scopus (362) Google Scholar, 4Schippling S. Kontush A. Arlt S. Buhmann C. Sturenburg H.J. Mann U. Muller-Thomsen T. Beisiegel U. Free Radic. Biol. Med. 2000; 28: 351-360Crossref PubMed Scopus (167) Google Scholar, 5Montine T.J. Neely M.D. Quinn J.F. Beal M.F. Markesbery W.R. Roberts L.J. Morrow J.D. Free Radic. Biol. 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Aspects Med. 2006; 24: 149-159Crossref Scopus (272) Google Scholar). HNE concentrations in human ventricular fluid are 8–15 μm and elevated in AD (3Lovell M.A. Ehmann W.D. Mattson M.P. Markesbery W.R. Neurobiol. Aging. 1997; 18: 457-461Crossref PubMed Scopus (362) Google Scholar, 18Markesbery W.R. Lovell M.A. Neurobiol. Aging. 1998; 19: 33-36Crossref PubMed Scopus (568) Google Scholar, 19McGrath L.T. McGleenon B.M. Brennan S. McColl D. McIlroy S. Passmore A.P. Q. J. Med. 2001; 94: 485-490Crossref Scopus (193) Google Scholar). HNE has a well known propensity to react with the side chains of various amino acid residues, and HNE-protein adducts have been used as biomarkers of oxidative stress (15Uchida K. Prog. Lipid Res. 2003; 42: 318-343Crossref PubMed Scopus (958) Google Scholar).In light of these observations, it is noteworthy that lipid oxidation products such as HNE modify Aβ and increase Aβ misfolding (20Boutaud O. Ou J.J. Chaurand P. Caprioli R.M. Montine T.J. Oates J.A. J. 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Moreover, the immunoreactivity of antibodies to HNE-modified His residues localizes to amyloid plaques (26Ando Y. Brannstrom T. Uchida K. Nyhlin N. Nasman B. Suhr O. Yamashita T. Olsson T. El Salhy M. Uchino M. Ando M. J. Neurol. Sci. 1998; 156: 172-176Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 27Rofina J.E. Singh K. Skoumalova-Vesela A. van Ederen A.M. van Asten A.J.A.M. Wilhelm J. Gruys E. Amyloid. 2004; 11: 90-100Crossref PubMed Scopus (64) Google Scholar). This suggests that not only does Aβ promote lipid oxidation but that there may also be a mechanistic link between the lipid oxidation products formed during oxidative stress and Aβ misfolding (21Bieschke J. Zhang Q. Powers E.T. Lerner R.A. Kelly J.W. Biochemistry. 2005; 44: 4977-4983Crossref PubMed Scopus (125) Google Scholar). Conversely, several lines of evidence suggest that Aβ contribute to oxidative stress. For example, the overexpression of Aβ in transgenic mice, in Caenorhabditis elegans, and in cell culture results in an increase in biomarkers of oxidative stress and in HNE production (28Wu Y. Luo Y. Curr. Alzheimer Res. 2005; 2: 37-47Crossref PubMed Scopus (70) Google Scholar, 29Pratico D. Uryu K. Leight S. Trojanowswki J.Q. Lee V.M.Y. J. Neurosci. 2001; 21: 4183-4187Crossref PubMed Google Scholar, 30Behl C. Davis J.B. Lesley R. Schubert D. Cell. 1994; 77: 817-827Abstract Full Text PDF PubMed Scopus (2035) Google Scholar). The mechanism underlying this relationship is unknown, but Aβ complexed with Cu(II) promote the oxidation of diverse substances, including cholesterol and phospholipids (31Opazo C. Huang X.D. Cherny R.A. Moir R.D. Roher A.E. White A.R. Cappai R. Masters C.L. Tanzi R.E. Inestrosa N.C. Bush A.I. J. Biol. Chem. 2002; 277: 40302-40308Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 32Murray I.V.J. Sindoni M.E. Axelsen P.H. Biochemistry. 2005; 44: 12606-12613Crossref PubMed Scopus (101) Google Scholar, 33Nelson T.J. Alkon D.L. J. Biol. Chem. 2005; 280: 7377-7387Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 34Puglielli L. Friedlich A.L. Setchell K.D.R. Nagano S. Opazo C. Cherny R.A. Barnham K.J. Wade J.D. Melov S. Kovacs D.M. Bush A.I. J. Clin. Investig. 2005; 115: 2556-2563Crossref PubMed Scopus (123) Google Scholar, 35Yoshimoto N. Tasaki M. Shimanouchi T. Umakoshi H. Kuboi R. J. Biosci. Bioeng. 2005; 100: 455-459Crossref PubMed Scopus (25) Google Scholar). Furthermore, lipid oxidative products and lipid susceptibility to oxidative damage are increased in AD (1Markesbery W.R. Free Radic. Biol. Med. 1997; 23: 134-147Crossref PubMed Scopus (1961) Google Scholar, 3Lovell M.A. Ehmann W.D. Mattson M.P. Markesbery W.R. Neurobiol. Aging. 1997; 18: 457-461Crossref PubMed Scopus (362) Google Scholar, 19McGrath L.T. McGleenon B.M. Brennan S. McColl D. McIlroy S. Passmore A.P. Q. J. Med. 2001; 94: 485-490Crossref Scopus (193) Google Scholar, 36Sayre L.M. Zelasko D.A. Harris P.L.R. Perry G. Salomon R.G. Smith M.A. J. Neurochem. 1997; 68: 2092-2097Crossref PubMed Scopus (917) Google Scholar, 37Bassett C.N. Neely M.D. Sidell K.R. Markesbery W.R. Swift L.L. Montine T.J. Lipids. 1999; 34: 1273-1280Crossref PubMed Scopus (53) Google Scholar, 38Galbusera C. Facheris M. Magni F. Galimberti G. Sala G. Tremolada L. Isella V. Guerini F.R. Appollonio I. Galli-Kienle M. Ferrarese C. Curr. Alzheimer Res. 2004; 1: 103-109Crossref PubMed Scopus (52) Google Scholar, 39Cutler R.G. Kelly J. Storie K. Pedersen W.A. Tammara A. Hatanpaa K. Troncoso J.C. Mattson M.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2070-2075Crossref PubMed Scopus (806) Google Scholar).We have demonstrated previously that oxidatively damaged lipid membranes promote the misfolding and aggregation of amyloid β proteins (Aβ) into fibrils, and that misfolded Aβ promote oxidative damage in synthetic lipid membranes. In further investigations of the phenomena described below, we have verified that Aβ promote oxidative damage in human brain lipids, identified HNE as an oxidation product that by itself mimics the effect of oxidatively damaged membranes on the misfolding and aggregation of Aβ, identified the nature of the chemical reaction between HNE and Aβ, and demonstrated that HNE modification of Aβ promotes misfolding, aggregation, and membrane association. Most significantly, we have demonstrated that Aβ are covalently modified by the HNE that they help produce. This represents a positive feedforward mechanism involving oxidative damage and aggregation, in which Aβ promotes oxidative damage, and the products of oxidative damage promote fibril formation.EXPERIMENTAL PROCEDURESMaterials—Synthetic 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine (SAPC) in chloroform and 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC) powder were obtained from Avanti Polar Lipids (Alabaster, AL). SAPC was packaged as 10-mg aliquots in sealed glass ampoules, under argon, stored at –80 °C, and lyophilized overnight prior to use. Excision grade trypsin and the endoproteinase Asp-N were obtained from Calbiochem. The trypsin stock concentration was 1 μg/μl in 50 mm ammonium bicarbonate at pH 8.5, and the Asp-N stock was 0.04 μg/μl in H2O. 4-Hydroxy-2-nonenal (HNE) and deuterated HNE (d3-HNE) were purchased from Cayman Chemical (Ann Arbor, MI.). 3,5-Di-tert-butylhydroxytoluene (BHT), O-2,3,4,5,6-(pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA), diethylenetriaminepentaacetic acid (DTPA), and bis(trimethylsilyl)trifluoroacetamide were obtained from Sigma. α-Cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) (>99% pure) were also obtained from Sigma. Water was purified through an Elix and MilliQ A10 synthesis water purification system (Millipore, Bedford, MA). When needed, Cu(II) was added as CuSO4. Although several experiments were nominally free of Cu(II) ions, no special procedures were employed to remove trace amounts of Cu(II) except where noted.Lyophilized Aβ-(1–40) (Aβ40) and Aβ-(1–42) (Aβ42), both at >95% purity, were obtained from rPeptide (Athens, GA). Aβ-(1–11), Aβ-(10–20), and Aβ-(22–35) were obtained from American Peptide Co. (Sunnyvale, CA). Aβ-(16–20) (KLVFF) (40Tjernberg L.O. Naslund J. Lindqvist F. Johansson J. Karlstrom A.R. Thyberg J. Terenius L. Nordstedt C. J. Biol. Chem. 1996; 271: 8545-8548Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar) was obtained from Bachem Bioscience (King of Prussia, PA). Before use, proteins were stored desiccated at –20 °C. Stock solutions at a concentration of 0.5 mg/ml were prepared by dissolving Aβ40 and Aβ42 in hexafluoroisopropanol, dissolving Aβ-(1–11), Aβ-(10–20), and Aβ-(16–20) in H2O, and dissolving Aβ-(22–35) in 30% acetonitrile with 0.1% trifluoroacetic acid. Hexafluoroisopropanol in the Aβ40 and Aβ42 stocks was evaporated immediately prior to use, and the proteins were redissolved in aqueous buffer at concentrations of 1–5 μm. Aβ-(1–28), Aβ-(1–11), Aβ-(10–20), and Aβ-(22–35) stocks were diluted into aqueous buffer at concentrations of 1–5 μm.Extraction of Brain Lipids—Frozen samples of normal human brain tissue from the temporal cortex were obtained from the Center for Neurodegenerative Disease Research Brain Bank, at the University of Pennsylvania. Lipids were extracted from ∼4-mg specimens using a modified Folch method, described below (41Folch J. Lees M. Stanley G.H.S. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar, 42Christie W.W. Lipid Analysis. 3rd Ed. Oily Press Lipid Library, Bridgewater, UK2003: 96-102Google Scholar). To minimize lipid oxidation during the extraction process, samples and aqueous solvents were purged with argon. The tissue was homogenized with an ultrasonic tip dismembranator in 10 ml/g methanol two times for 10 s on ice. 20 ml/g of chloroform was added, and the tissue was further sonicated two times for 10 s at room temperature. After centrifugation at 5000 × g for 2 min, the supernatant was removed and kept. The pellet was resuspended in 30 ml/g of chloroform:methanol (2:1), sonicated two times for 10 s on ice, incubated at room temperature for 5 min, and centrifuged as before. This supernatant was added to that of the first extract. The combined supernatants were sequentially washed with 0.88% potassium chloride and then methanol:saline (1:1) to remove non-lipid contaminants such as salts, amino acids, sugars, and urea. Following low speed centrifugation after the final wash, the lower organic layer was removed, lyophilized, and extruded into lipid vesicles as described below.Lipid Vesicle Preparation, Oxidation, and Analysis—SAPC, DMPC, and brain lipid extract vesicle suspensions were prepared by extrusion as described previously (32Murray I.V.J. Sindoni M.E. Axelsen P.H. Biochemistry. 2005; 44: 12606-12613Crossref PubMed Scopus (101) Google Scholar). Immediately prior to use, aliquots were mixed with 5 mm HEPES, pH 7.5, and the oxidation was initiated by the addition of ascorbate and Cu(II). Final concentrations in these suspensions were 10 μm SAPC and 25 μm DMPC or 10 μm brain lipid and 1 μm DMPC in a final volume of 400 μl. Where Aβ42 was used, it was premixed with Cu(II) and incubated for 30 min prior to the addition of other reactants as described previously (32Murray I.V.J. Sindoni M.E. Axelsen P.H. Biochemistry. 2005; 44: 12606-12613Crossref PubMed Scopus (101) Google Scholar). Quantitative determination of SAPC content relative to a DMPC internal standard was performed by multiple reaction monitoring (MRM) mass spectrometry (LC/MS/MS) as described previously (32Murray I.V.J. Sindoni M.E. Axelsen P.H. Biochemistry. 2005; 44: 12606-12613Crossref PubMed Scopus (101) Google Scholar).HNE Assay—HNE concentrations were determined by following HNE derivatization, organic extraction, and gas chromatography mass/spectrometry (GC/MS) similar to the method described previously (43Meagher E.A. Barry O.P. Lawson J.A. Rokach J. FitzGerald G.A. J. Am. Med. Assoc. 2001; 285: 1178-1182Crossref PubMed Scopus (259) Google Scholar). The samples (400 μl) were analyzed 120 min after initiating oxidation. At this point 10 μm DTPA was added to stop the oxidation, 620 nm d3-HNE was added as an internal standard, and the HNE within the mixture was derivatized with 16 mm PFBHA for 1 h at room temperature. At this point, samples were extracted with 2 volumes of chloroform:methanol (2:1). Following low speed centrifugation, the lower organic layer was removed, passed through a 0.45-μm perfluorocarbon filter, and dried under nitrogen. The dried material was dissolved in 10 μl of pyridine and further derivatized by adding 10 μl of bis(trimethylsilyl)trifluoroacetamide. After 10 min, the samples were dried, dissolved in 100 μl of dodecane, and assayed with the GC/MS in negative ion electron capture ionization mode using ammonia as the collision gas. A temperature program increasing at a rate of 30 °C/min from 190 to 250 °C was used to separate the derivatized d3-HNE and HNE on a DB 35-MS capillary GC column (60 m × 0.25 mm, 0.25 μm coating). The ions monitored were m/z 286.3 for d3-HNE and m/z 283.3 for HNE. Peak areas quantified and expressed as 283.3:286.3 ratios.Mass Spectrometric Analysis of Aβ-HNE Adducts—Aβ40, Aβ42, or a mixture of Aβ-(1–11), Aβ-(10–20), and Aβ-(22–35) (5 μm each) were incubated with 3.8 mm HNE in 10 mm phosphate buffer, pH 7.4, for 1–3 h at 37 °C. Prior to the addition of HNE, 2.5 mm DMPC lipid vesicles and 50 μm of the Aβ aggregation inhibitor Aβ-(16–20) were added to reduce aggregation of HNE-modified Aβ. The reaction of Aβ with HNE was terminated by addition of an equal volume of trifluoroacetic acid, and the samples were lyophilized. For samples subjected to Asp-N digestion, the reaction between HNE and Aβ was terminated by the addition of an equal volume of 100 mm ammonium bicarbonate solution, pH 8.5. The reaction of any excess HNE with ammonium ions allowed for subsequent digestion of the Aβ-HNE adducts with Asp-N. 30 μl of the HNE/Aβ reaction mixture (∼1 μg of Aβ) was quenched with 30 μl of 100 mm ammonium bicarbonate, pH 8.5. 1 μl of Asp-N (0.04 μg) was added to 60 μl of this mixture (∼1 μg of Aβ) and incubated overnight at 37 °C. The enzyme was inactivated by adding 60 μl of trifluoroacetic acid, and the sample was lyophilized. For MALDI-mass spectrometry, the Aβ-HNE adducts or Aβ-HNE adduct digests were dissolved in either 30 μl of a CHCA solution (5 mg of CHCA in 1 ml of 50% ACN with 0.3% trifluoroacetic acid) or sinapinic acid solution (10 mg sinapinic acid in 30% ACN with 0.3% trifluoroacetic acid). MALDI analysis was performed on TOF, Q-TOF, and TOF-Q-TOF instruments (Voyager DE, QStar, and model 4700 mass spectrometers, respectively; Applied Biosystems/MDS Sciex, Foster City, CA).Immunoblot Analysis of Aβ and Aβ-HNE Adducts—Murine anti-His-HNE monoclonal antibody (HNEJ-2) was obtained from Genox Corp. (Baltimore, MD) (44Toyokuni S. Miyake N. Hiai H. Hagiwara M. Kawakishi S. Osawa T. Uchida K. FEBS Lett. 1995; 359: 189-191Crossref PubMed Scopus (180) Google Scholar). 6E10 antibodies with specificity for Aβ residues 4–9 and 4G8 antibodies with specificity for residues 18–22 were obtained from Senetek (Maryland Heights, MO) by a kind gift from Domenico Pratico. Donkey anti-mouse antibody conjugated to horseradish peroxidase (SC2314) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).Slot blots were used to detect the modification of Aβ by endogenous HNE generated during lipid oxidation. 200 μl of the Aβ oxidation reaction solution containing 4.5 μg of protein was mixed with 200 μl of 100 mm ammonium bicarbonate, pH 7.5. Samples were adsorbed onto 0.2-μm nitrocellulose membranes by slot blot microfiltration (Bio-Dot SF microfiltration apparatus), and the wells were washed twice with 50 mm Tris, 150 mm NaCl, pH 7.4. Membranes were blocked with a 5% solution of powdered fat-free milk (BLOTTO in TTBS; BioRad). The slot blot membranes were incubated overnight with 15 μg/ml of the antibody to HNE-modified histidine (HNE-J) at 4 °C. Following washes in TTBS and incubation of the membranes with secondary antibody (diluted 1:1000) for 1 h, the blot was incubated with Renaissance Luminol reagents (PerkinElmer Life Sciences). X-Omat Blue XB-1 film (Eastman Kodak Co.) was exposed to blots of synthetic lipids for 10 s and to blots of brain-derived lipids for 10 min. Quantification of the slot blot was performed by digitally scanning the X-Omat film and measuring film background and slot blot image densities with ImageQuant TL software (Amersham Biosciences).Western blots were used to detect Aβ and modification of Aβ with excesses of synthetic HNE. Both synthetic Aβ and HNE-modified Aβ were electrophoresed on a Tris-Tricine 10–20% polyacrylamide gel (72 ng of protein per well) and were transferred to 0.2-μm nitrocellulose membranes. The membranes were removed and washed with Tween in Tris-buffered saline (TTBS, 0.1% Tween 20 in 150 mm NaCl, 20 mm Tris, pH 7.6). Aβ epitope retrieval was performed by boiling the unblocked membrane in PBS for 5 min. The membranes were immunoblotted overnight at 4 °C with the HNEJ-2 antibody and Aβ antibodies (4G8, 1:200 and 6E10, 1:500). They were then washed, incubated with Luminol reagents, and exposed in the same manner as the slot blots.Congo Red (CR) Binding Assay—Aβ40 (10 μm) or Aβ42 (2 μm) was incubated at a 1:1 mole ratio with DMPC vesicles containing 3 mol % HNE in PBS (150 mm NaCl, 10 mm sodium phosphate, pH 7.4) at room temperature with continuous gentle agitation. The HNE concentrations were therefore 0.3 and 0.06 μm for Aβ40 and Aβ42, respectively. At intervals, aliquots of Aβ40 (112 μl) and Aβ42 (98 μl) were added to 28 or 42 μl, respectively, of 10 μm CR in PBS and incubated for 30 min at room temperature. Fibril formation was assayed by measuring the ratio of sample absorption at 541 and 403 nm, which are the wavelengths of maximum difference and of an isosbestic point for fibril-bound CR and unbound CR, respectively.Infrared Spectroscopy—Polarized attenuated total internal reflection Fourier transform infrared (PATIR-FTIR) spectra were collected in rapid-scanning mode as 1024 co-added interferograms using a Bio-Rad FTS-60A spectrometer, a liquid nitrogen-cooled MCT detector, an aluminum wire grid polarizer, a resolution of 2 cm–1, scanning speed of 20 kHz, triangular apodization, and one level of zero filling. Supported lipid monolayers, composed of either DMPC or brain lipid extract, were prepared in a Langmuir trough by applying ∼5 nmol of DMPC in a mixture of hexane:ethanol (9:1 by volume) to the surface of a subphase buffer. An enclosure around the Langmuir trough is filled with argon to avoid spontaneous air oxidation of lipids at the air-water interface, and all studies were performed at ∼21 °C. The subphase buffer contained 30 mm HEPES buffer in D2O at pD 7.4. When indicated, an aliquot of HNE in ethanol was lyophilized, resuspended in subphase buffer, and injected into the subphase before forming the monolayer. For DMPC membranes, 5 μl of 28 mm HNE stock was injected into 6 ml of subphase to yield an HNE concentration of 23 μm in the subphase. For brain lipid extracts, 10 μl of a 32 mm HNE stock solution was injected into a 2.5-ml subphase volume to yield an HNE concentration of 128 μm in the subphase.The monolayer was compressed to a surface pressure of 20 dynes/cm and applied onto a silane-treated germanium internal reflection crystal as described previously (45Koppaka V. Axelsen P.H. Biochemistry. 2000; 39: 10011-10016Crossref PubMed Scopus (116) Google Scholar). At this point, 5 μl of 28 mm HNE stock in ethanol was lyophilized, resuspended in the subphase buffer, and injected into the subphase. After collecting a base-line spectrum, 500 ng of samples of Aβ40 or Aβ42 in 30 mm NaDPO4 buffer, pD 11.9, were injected into the continuously stirred subphase to yield a protein concentration of 300 nm in experiments with DMPC and 230 nm in experiments with brain lipid extracts. Sample spectra were collected over 90 and 125 min, as indicated. A flat base-line correction was performed on sample spectra assuming zero absorbance at 1800 cm–1, but no water vapor subtraction or smoothing manipulations were performed. The spectra were fitted using IRfit, a procedure that fits a limited set of component bands simultaneously to several spectra with a minimum number of adjustable parameters (46Silvestro L. Axelsen P.H. Biochemistry. 1999; 38: 113-121Crossref PubMed Scopus (41) Google Scholar). In this study, one simultaneous fit was performed on 12 spectra from three independent experiments. Only the spectra region between 1700 and 1600 cm–1 was fitted.RESULTSAβ42 and Brain Lipid Oxidation—The ability of Aβ42 to promote the oxidation of SAPC in human brain lipid extracts was measured using an MRM-LC/MS/MS assay as described previously (32Murray I.V.J. Sindoni M.E. Axelsen P.H. Biochemistry. 2005; 44: 12606-12613Crossref PubMed Scopus (101) Google Scholar). This assay is superior to many other assays of oxidative damage in that it yields precise and unambiguous quantitative information about oxidative loss of a specific substrate. Two adaptations of the conditions described previously for synthetic lipids were necessary for the application of this assay to human brain lipid extracts. First, brain vesicle suspensions with phosphate concentrations of 10 μm contained virtually no DMPC by MRM-LC/MS/MS. Therefore, 1 μm DMPC was added to all brain lipid vesicles as an internal standard. The SAPC concentration in these extracts was 0.34 ± 0.1 μm. Second, 1.5 mm MgSO4 was added to minimize sequestration of Cu(II) by anionic lipids.Ascorbate (50 μm) and Cu(II) (0.5 μm) reduced SAPC to 79 ± 19% of original levels after 120 min (Fig. 1). The addition of 5 μm Aβ42 decreased it to 38 ± 16% of its original level over the same time interval. These results are consistent with previously reported results from synthetic SAPC and DMPC vesicles, although the variability of the data was considerably larger in these human lipid extracts (32Murray I.V.J. Sindoni M.E. Axelsen P.H. Biochemistry. 2005; 44: 12606-12613Crossref PubMed Scopus (101) Google Scholar). The data in Fig. 1 only represent four of the five extracts that were examined. Lipids from brain specimen 3 (Table 1) had only about half of the SAPC content as the other samples, and it was completely resistant to oxidation with or without Aβ42 present. Therefore, this sample was excluded from our analysis.TABLE 1Characteristics of frozen human brain tissues from which brain lipid vesicles were preparedCase no.DatePMIAge at death1200387321996992319936744199047251989232 Op" @default.
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• W2013118051 title "Membrane-mediated Amyloidogenesis and the Promotion of Oxidative Lipid Damage by Amyloid β Proteins" @default.
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• W2013118051 doi "https://doi.org/10.1074/jbc.m608589200" @default.
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