Hydrogen Peroxide Is Generated during the Very Early Stages of Aggregation of the Amyloid Peptides Implicated in Alzheimer Disease and Familial British Dementia

Abstract

Alzheimer disease and familial British dementia are neurodegenerative diseases that are characterized by the presence of numerous amyloid plaques in the brain. These lesions contain fibrillar deposits of the β-amyloid peptide (Aβ) and the British dementia peptide (ABri), respectively. Both peptides are toxic to cells in culture, and there is increasing evidence that early “soluble oligomers” are the toxic entity rather than mature amyloid fibrils. The molecular mechanisms responsible for this toxicity are not clear, but in the case of Aβ, one prominent hypothesis is that the peptide can induce oxidative damage via the formation of hydrogen peroxide. We have developed a reliable method, employing electron spin resonance spectroscopy in conjunction with the spin-trapping technique, to detect any hydrogen peroxide generated during the incubation of Aβ and other amyloidogenic peptides. Here, we monitored levels of hydrogen peroxide accumulation during different stages of aggregation of Aβ-(1–40) and ABri and found that in both cases it was generated as a short “burst” early on in the aggregation process. Ultrastructural studies with both peptides revealed that structures resembling “soluble oligomers” or “protofibrils” were present during this early phase of hydrogen peroxide formation. Mature amyloid fibrils derived from Aβ-(1–40) did not generate hydrogen peroxide. We conclude that hydrogen peroxide formation during the early stages of protein aggregation may be a common mechanism of cell death in these (and possibly other) neurodegenerative diseases. Alzheimer disease and familial British dementia are neurodegenerative diseases that are characterized by the presence of numerous amyloid plaques in the brain. These lesions contain fibrillar deposits of the β-amyloid peptide (Aβ) and the British dementia peptide (ABri), respectively. Both peptides are toxic to cells in culture, and there is increasing evidence that early “soluble oligomers” are the toxic entity rather than mature amyloid fibrils. The molecular mechanisms responsible for this toxicity are not clear, but in the case of Aβ, one prominent hypothesis is that the peptide can induce oxidative damage via the formation of hydrogen peroxide. We have developed a reliable method, employing electron spin resonance spectroscopy in conjunction with the spin-trapping technique, to detect any hydrogen peroxide generated during the incubation of Aβ and other amyloidogenic peptides. Here, we monitored levels of hydrogen peroxide accumulation during different stages of aggregation of Aβ-(1–40) and ABri and found that in both cases it was generated as a short “burst” early on in the aggregation process. Ultrastructural studies with both peptides revealed that structures resembling “soluble oligomers” or “protofibrils” were present during this early phase of hydrogen peroxide formation. Mature amyloid fibrils derived from Aβ-(1–40) did not generate hydrogen peroxide. We conclude that hydrogen peroxide formation during the early stages of protein aggregation may be a common mechanism of cell death in these (and possibly other) neurodegenerative diseases. There is mounting evidence for the importance of oxidative damage to the brain in a wide range of neurodegenerative diseases based on detection of markers such as elevated levels of redox-active transition metal ions, lipid peroxidation, DNA and protein oxidation, and the introduction of carbonyl groups into proteins (reviewed, for example, in Refs. 1Markesbery W.R. Free Radic. Biol. Med. 1997; 23: 134-147Crossref PubMed Scopus (1985) Google Scholar, 2Miranda S. Opazo C. Larrondo L.F. Munñoz F.J. Ruiz F. Leighton F. Inestrosa N.C. 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These are hallmarks of attack by reactive oxygen species (ROS) 3The abbreviations used are:ROSreactive oxygen speciesAβamyloid β-peptideADAlzheimer diseaseFBDfamilial British dementiaABrithe British dementia peptideWTwild-type form of the ABri peptideDMPO5,5-dimethyl-1-pyrroline N-oxideDMPO-OHthe DMPO hydroxyl radical adductAFMatomic force microscopyDETAPACdiethylenetriaminepentaacetic acidPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayEMelectron microscopy , including superoxide, hydrogen peroxide, and the hydroxyl radical. The β-amyloid peptide (Aβ), which is responsible for senile plaque formation in Alzheimer disease (AD), has been reported to generate hydrogen peroxide from molecular oxygen through electron transfer interactions involving bound redox-active metal ions (7Huang X.D. Atwood C.S. Hartshorn M.A. Multhaup G. Goldstein L.E. Scarpa R.C. Cuajungco M.P. Gray D.N. Lim J. Moir R.D. Tanzi R.E. Bush A.I. Biochemistry. 1999; 38: 7609-7616Crossref PubMed Scopus (1030) Google Scholar, 8Huang X.D. Cuajungco M.P. Atwood C.S. Hartshorn M.A. Tyndall J.D.A. Hanson G.R. Stokes K.C. Leopold M. Multhaup G. Goldstein L.E. Scarpa R.C. Saunders A.J. Lim J. Moir R.D. Glabe C. Bowden E.F. Masters C.L. Fairlie D.P. Tanzi R.E. Bush A.I. J. Biol. Chem. 1999; 274: 37111-37116Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar, 9Curtain C.C. Ali F. Volitakis I. Cherny R.A. Norton R.S. Beyreuther K. Barrow C.J. Masters C.L. Bush A.I. Barnham K.J. J. Biol. Chem. 2001; 276: 20466-20473Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 10Opazo C. Barría M.I. Ruiz F.H. Inestrosa N.C. BioMetals. 2003; 16: 91-98Crossref PubMed Scopus (77) Google Scholar). Hydrogen peroxide is readily converted into the aggressive hydroxyl radical by Fenton chemistry and these two ROS could be responsible for some of the oxidative damage seen in the brain in AD. Familial British dementia (FBD) is an inherited neurodegenerative disorder that is strikingly similar in neuropathology to AD, including the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles. FBD is due to a stop codon mutation in the BRI gene, the protein product of which undergoes proteolytic cleavage to release an abnormally long peptide fragment (ABri) that rapidly aggregates in vitro into toxic oligomers (11El-Agnaf O.M.A. Sheridan J.M. Sidera C. Siligardi G. Hussain R. Haris P.I. Austen B.M. Biochemistry. 2001; 40: 3449-3457Crossref PubMed Scopus (43) Google Scholar, 12El-Agnaf O.M.A. Nagala S. Patel B.P. Austen B.M. J. Mol. Biol. 2001; 310: 157-168Crossref PubMed Scopus (71) Google Scholar). Only ABri with an intact intramolecular disulfide bond can do this, whereas the corresponding wild-type peptide (WT) does not aggregate and is non-toxic (11El-Agnaf O.M.A. Sheridan J.M. Sidera C. Siligardi G. Hussain R. Haris P.I. Austen B.M. Biochemistry. 2001; 40: 3449-3457Crossref PubMed Scopus (43) Google Scholar). Because both AD and FBD share similar pathological features, we were interested, as one aspect of our research, in establishing whether or not ABri also has the ability to generate hydrogen peroxide. reactive oxygen species amyloid β-peptide Alzheimer disease familial British dementia the British dementia peptide wild-type form of the ABri peptide 5,5-dimethyl-1-pyrroline N-oxide the DMPO hydroxyl radical adduct atomic force microscopy diethylenetriaminepentaacetic acid phosphate-buffered saline enzyme-linked immunosorbent assay electron microscopy Our method to detect hydrogen peroxide formation employs ESR spectroscopy in conjunction with the spin-trapping technique. The full methodology has been published elsewhere (13Turnbull S. Tabner B.J. El-Agnaf O.M.A. Moore S. Davies Y. Allsop D. Free Radic. Biol. Med. 2001; 30: 1163-1170Crossref PubMed Scopus (178) Google Scholar, 14Turnbull S. Tabner B.J. Brown D.R. Allsop D. Biochemistry. 2003; 42: 7675-7681Crossref PubMed Scopus (45) Google Scholar, 15Turnbull S. Tabner B.J. Brown D.R. Allsop D. Neurosci. Lett. 2003; 336: 159-162Crossref PubMed Scopus (63) Google Scholar), but, briefly, any hydrogen peroxide formed during peptide incubation can be converted to hydroxyl radicals via the Fenton reaction upon addition of Fe(II). These radicals are then trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to form the DMPO hydroxyl radical adduct (DMPO-OH), which has a uniquely characteristic 4-line ESR spectrum. Using this technique, we have shown that various amyloidogenic peptides, including Aβ-(1–40), Aβ-(1–42), Aβ-(25–35), as well as α-synuclein (implicated in Parkinson disease and related disorders) and certain toxic fragments of the prion protein (implicated in the transmissible spongiform encephalopathies) can all generate hydrogen peroxide (13Turnbull S. Tabner B.J. El-Agnaf O.M.A. Moore S. Davies Y. Allsop D. Free Radic. Biol. Med. 2001; 30: 1163-1170Crossref PubMed Scopus (178) Google Scholar, 14Turnbull S. Tabner B.J. Brown D.R. Allsop D. Biochemistry. 2003; 42: 7675-7681Crossref PubMed Scopus (45) Google Scholar, 15Turnbull S. Tabner B.J. Brown D.R. Allsop D. Neurosci. Lett. 2003; 336: 159-162Crossref PubMed Scopus (63) Google Scholar, 16Tabner B.J. Turnbull S. El-Agnaf O.M.A. Allsop D. Curr. Top. Med. Chem. 2001; 1: 507-517Crossref PubMed Scopus (151) Google Scholar, 17Tabner B.J. Turnbull S. El-Agnaf O.M.A. Allsop D. Free Radic. Biol. Med. 2002; 32: 1076-1083Crossref PubMed Scopus (227) Google Scholar, 18Tabner B.J. Turnbull S. El-Agnaf O.M.A. Allsop D. Curr. Med. Chem.-Immun. Endoc. & Metab. Agents. 2003; 3: 299-308Crossref Google Scholar). Numerous controls, including reverse and scrambled peptides, nontoxic peptides, and the β- and γ-synucleins were shown to lack this ability when tested under the same experimental conditions, demonstrating the specificity of hydrogen peroxide formation. Furthermore, the ability of these various peptides to generate hydrogen peroxide shows a clear positive correlation with their cytotoxicity. We have noted in all of our experiments in which hydrogen peroxide is formed that the relative intensity of the DMPO-OH ESR spectrum with peptide incubation time always follows a characteristic profile. There is a delay time before the spectrum is first observed after which its intensity rapidly increases to a maximum before slowly decreasing back to zero. The exact time course of the profile varies from peptide to peptide and, to a lesser extent, from batch to batch of the same peptide, but nevertheless each phase of the profile is observed without exception. Here, we have examined how the generation of hydrogen peroxide relates to the extent of peptide aggregation by following the time dependence of hydrogen peroxide accumulation during incubation of a synthetic peptide corresponding to Aβ-(1–40). Peptide aggregation was determined by an immunoassay method for the detection of oligomeric forms of Aβ-(1–40) (19Moore S.A. Huckerby T.N. Gibson G.L. Fullwood N.J. Turnbull S. Tabner B.J. El-Agnaf O.M.A. Allsop D. Biochemistry. 2004; 43: 819-826Crossref PubMed Scopus (49) Google Scholar), the thioflavin T method, which detects β-pleated sheet amyloid fibrils, and by atomic force microscopy (AFM). Similar experiments were also carried out with the ABri and WT peptides. Peptides and Electron Spin Resonance Spectroscopy—The Aβ-(1–40) peptide was purchased from BIOSOURCE International. The ABri and WT peptides were synthesized using a Milligen 9050 peptide synthesizer (PE Applied Biosystems Ltd., Cheshire, UK) (11El-Agnaf O.M.A. Sheridan J.M. Sidera C. Siligardi G. Hussain R. Haris P.I. Austen B.M. Biochemistry. 2001; 40: 3449-3457Crossref PubMed Scopus (43) Google Scholar). Oxidized and reduced forms of ABri and WT were prepared (11El-Agnaf O.M.A. Sheridan J.M. Sidera C. Siligardi G. Hussain R. Haris P.I. Austen B.M. Biochemistry. 2001; 40: 3449-3457Crossref PubMed Scopus (43) Google Scholar). Solutions (100 μm) of Aβ-(1–40), ABri, and WT in phosphate-buffered saline (PBS), pH 7.4, were prepared as described O.M.A. Nagala S. Patel B.P. Austen B.M. J. Mol. Biol. 2001; 310: 157-168Crossref PubMed Scopus (71) Google Scholar, S. Tabner B.J. El-Agnaf O.M.A. Moore S. Davies Y. Allsop D. Free Radic. Biol. Med. 2001; 30: 1163-1170Crossref PubMed Scopus (178) Google and in in the incubation a of peptide was and of DMPO diethylenetriaminepentaacetic acid and then were after the was to an ESR for detection of the DMPO-OH spectrum. were as (13Turnbull S. Tabner B.J. El-Agnaf O.M.A. Moore S. Davies Y. Allsop D. Free Radic. Biol. Med. 2001; 30: 1163-1170Crossref PubMed Scopus (178) Google Scholar, 14Turnbull S. Tabner B.J. Brown D.R. Allsop D. Biochemistry. 2003; 42: 7675-7681Crossref PubMed Scopus (45) Google Scholar, 15Turnbull S. Tabner B.J. Brown D.R. Allsop D. Neurosci. Lett. 2003; 336: 159-162Crossref PubMed Scopus (63) Google Scholar). of or metal to ABri was carried out as described for Aβ (13Turnbull S. Tabner B.J. El-Agnaf O.M.A. Moore S. Davies Y. Allsop D. Free Radic. Biol. Med. 2001; 30: 1163-1170Crossref PubMed Scopus (178) Google Scholar). Because and the same for the addition to each of peptide the intensity of the ESR spectrum the of DMPO-OH formed each time that the intensity of the ESR spectrum increases with hydrogen peroxide Mature Aβ-(1–40) fibrils were prepared as described with incubation for the of the incubation the was in a for The was and the was in and was before in the of for Aβ was in pH and of this was to each well of a was then with a and acid of The was then with on a pH 7.4, and was and the was for was then with of the to be tested were to in were to each and the was for with in was and the was for a The was then with with in and for the was with before were after the to for are the of T of Aβ-(1–40) and ABri (100 in were after incubation for various These were into of thioflavin was in using a with and for the intensity of a was from all are the of were for various time as These were in as the of each to The were found with Aβ-(1–40) when the were and with ABri when the were was with the was prepared for by of the Aβ-(1–40) or of the ABri a metal was into a and to in to of the was using a with a in in with was used to the of deposits early were on each and the then were for to the experimental aggregation were from the for a process. of this the and the of peptide and and and their the of The were by these and the for the reaction different a of the for example, Rev. 1997; Scopus Google The same was used to the for the relative hydrogen peroxide Hydrogen peroxide was by redox-active transition metal ions present in the The the “burst” of hydrogen peroxide were by of the relative of and Hydrogen for was μm) in for of to We of our ESR on the first of incubation with our published (13Turnbull S. Tabner B.J. El-Agnaf O.M.A. Moore S. Davies Y. Allsop D. Free Radic. Biol. Med. 2001; 30: 1163-1170Crossref PubMed Scopus (178) Google Scholar, 17Tabner B.J. Turnbull S. El-Agnaf O.M.A. Allsop D. Free Radic. Biol. Med. 2002; 32: 1076-1083Crossref PubMed Scopus (227) Google Scholar, 18Tabner B.J. Turnbull S. El-Agnaf O.M.A. Allsop D. Curr. Med. Chem.-Immun. Endoc. & Metab. Agents. 2003; 3: 299-308Crossref Google Scholar), that the DMPO-OH spectrum was after was monitored by immunoassay and and thioflavin T and the profile observed with Aβ and other Rev. 1997; Scopus Google Scholar, Y. K. M. S. A. J. Biol. 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Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). examined after incubation for after a in with mature amyloid fibrils of the ability of formed amyloid fibrils to generate hydrogen peroxide, the Aβ-(1–40) peptide was under the same as before for a of The characteristic DMPO-OH spectrum was in after and and mature amyloid fibrils were observed by electron microscopy and the of this incubation not The fibrils were by by of the and a of fibrillar peptide was for to in DMPO-OH spectrum was from during this incubation and tested using our ESR of and Hydrogen for the and reduced forms of ABri and of the WT peptide were μm) in as described ESR were from the form of ABri with an intact intramolecular disulfide bond were observed with the other related peptide after incubation a for Aβ (13Turnbull S. Tabner B.J. El-Agnaf O.M.A. Moore S. Davies Y. Allsop D. Free Radic. Biol. Med. 2001; 30: 1163-1170Crossref PubMed Scopus (178) Google Scholar), the formation of the DMPO-OH spectrum from the form of ABri was by or that hydrogen peroxide is formed by a this peptide, ESR were a experiments employing this peptide that this was the during which the DMPO-OH ESR spectrum was The time course for hydrogen peroxide formation and the characteristic noted this the and of the DMPO-OH spectrum was than that observed during the aggregation of Aβ-(1–40) with the ESR spectrum after of were time to on the of the aggregates present after various incubation corresponding to hydrogen peroxide forms of which were into short in were present incubation when hydrogen peroxide levels of aggregates were seen with evidence of protein fibrils. The thioflavin T assay was used to these aggregates contain the characteristic β-pleated sheet of shown in the aggregates formed by ABri in did not to thioflavin was found to be the case after a incubation of to The of these was to on how the of hydrogen peroxide during incubation of both Aβ-(1–40) and ABri relates to the extent of peptide and shows that for Aβ-(1–40) the time which the generation of hydrogen peroxide to a when the the presence of structures resembling described Y. Y. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The to detect a DMPO-OH spectrum during the incubation of mature Aβ-(1–40) amyloid fibrils and the reduced levels of hydrogen peroxide observed during the stages of amyloid both the that hydrogen peroxide generation is an early Our that hydrogen peroxide is not generated the aggregation of Aβ-(1–40) but as a short “burst” early on during the peptide incubation when mature amyloid fibrils are not Numerous studies have shown that Aβ that has been to aggregate similar to by addition to cells is The molecular mechanisms responsible for this are but the addition of Aβ to cells oxidative radical and and it has been reported that toxicity can be by radical or C.S. Perry G. Smith M.A. Rev. 2003; 43: PubMed Scopus Google for is with of cells to hydrogen peroxide or other There is clear on the of the toxic form of Aβ, but has on early protein or as the Y. Y. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, R. Glabe 2003; PubMed Scopus Google Scholar, B.M. J. M. 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Our the that this could be of the generation of hydrogen peroxide during the early stages of Aβ before the formation of of senile the presence of redox-active metal ions, hydrogen peroxide is readily converted via the Fenton reaction into the reactive hydroxyl which could be responsible for of this early oxidative Because oxidative can the of Aβ C.S. Perry G. Smith M.A. Rev. 2003; 43: PubMed Scopus Google Scholar), the presence of hydrogen peroxide (and the hydroxyl could a positive mechanism Aβ oligomers their The of hydrogen peroxide generated by Aβ can be by with a C. Cherny R.A. Moir R.D. R. Masters C.L. Tanzi R.E. Inestrosa N.C. Bush A.I. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M.P. Free Radic. Biol. Med. 2004; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). this is the product is which is and could also to oxidative brain damage J. Biol. Chem. 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Neurosci. 8: PubMed Scopus Google Scholar). these have not been shown to to cell and the of our to these is not we that hydrogen peroxide can by as a A. M. Mol. Neurobiol. 2004; PubMed Scopus Google Scholar). We have also that hydrogen peroxide is generated by the form of ABri but not by the reduced form of this peptide or by the corresponding WT well with the toxicity of these peptides to studies with the same of ABri and WT peptides as used have shown that the form of ABri is toxic to cells with the WT peptide and the reduced form of ABri non-toxic (11El-Agnaf O.M.A. Sheridan J.M. Sidera C. Siligardi G. Hussain R. Haris P.I. Austen B.M. Biochemistry. 2001; 40: 3449-3457Crossref PubMed Scopus (43) Google Scholar, 12El-Agnaf O.M.A. Nagala S. Patel B.P. Austen B.M. J. Mol. Biol. 2001; 310: 157-168Crossref PubMed Scopus (71) Google Scholar). Our experiments with other amyloidogenic proteins and peptides, including Aβ, and various forms of the prion protein have also a correlation the generation of hydrogen peroxide and (13Turnbull S. Tabner B.J. El-Agnaf O.M.A. Moore S. Davies Y. Allsop D. Free Radic. Biol. Med. 2001; 30: 1163-1170Crossref PubMed Scopus (178) Google Scholar, 14Turnbull S. Tabner B.J. Brown D.R. Allsop D. Biochemistry. 2003; 42: 7675-7681Crossref PubMed Scopus (45) Google Scholar, 15Turnbull S. Tabner B.J. Brown D.R. Allsop D. Neurosci. Lett. 2003; 336: 159-162Crossref PubMed Scopus (63) Google Scholar, 16Tabner B.J. Turnbull S. El-Agnaf O.M.A. Allsop D. Curr. Top. Med. Chem. 2001; 1: 507-517Crossref PubMed Scopus (151) Google Scholar, 17Tabner B.J. Turnbull S. El-Agnaf O.M.A. Allsop D. Free Radic. Biol. Med. 2002; 32: 1076-1083Crossref PubMed Scopus (227) Google Scholar, 18Tabner B.J. Turnbull S. El-Agnaf O.M.A. Allsop D. Curr. Med. Chem.-Immun. Endoc. & Metab. Agents. 2003; 3: 299-308Crossref Google Scholar). studies on the form of ABri have that it is toxic during the early stages of aggregation when oligomers are present than after incubation (11El-Agnaf O.M.A. Sheridan J.M. Sidera C. Siligardi G. Hussain R. Haris P.I. Austen B.M. Biochemistry. 2001; 40: 3449-3457Crossref PubMed Scopus (43) Google Scholar, 12El-Agnaf O.M.A. Nagala S. Patel B.P. Austen B.M. J. Mol. Biol. 2001; 310: 157-168Crossref PubMed Scopus (71) Google Scholar). Our that oligomers of ABri are present during the phase of hydrogen peroxide formation are in with The of the ABri oligomers by is similar to that observed by O.M.A. Nagala S. Patel B.P. Austen B.M. J. Mol. Biol. 2001; 310: 157-168Crossref PubMed Scopus (71) Google Scholar). incubation in we found that ABri formed that did not to thioflavin a lack of β-pleated sheet fibrillar with ABri does not aggregate into amyloid fibrils but forms aggregates with reduced thioflavin T R. K. J. R.E. J. Mol. Biol. 2003; PubMed Scopus Google and a lack of O.M.A. Nagala S. Patel B.P. Austen B.M. J. Mol. Biol. 2001; 310: 157-168Crossref PubMed Scopus (71) Google Scholar). the of the aggregates seen is similar to that reported using with in the by El-Agnaf O.M.A. Nagala S. Patel B.P. Austen B.M. J. Mol. Biol. 2001; 310: 157-168Crossref PubMed Scopus (71) Google The that hydrogen peroxide was formed from ABri oligomers under these the that hydrogen peroxide formation does not to be with the presence of mature amyloid fibrils. we that of the may not be to AD and FBD but could well have to other neurodegenerative diseases with protein We for to the of the aggregation process.