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Tyrosine gated electron transfer is key to the toxic mechanism of Alzheimer’s disease ß-amyloid

KEVIN J. BARNHAM^*,1,2, FREDRIK HAEFFNER^,1, GIUSEPPE D. CICCOTOSTO^*, CYRIL C CURTAIN^*,, DEBORAH TEW^*, CHRISTINE MAVROS^*, KONRAD BEYREUTHER, DARRYL CARRINGTON^*, COLIN L. MASTERS^*, ROBERT A. CHERNY^*, ROBERTO CAPPAI^* and ASHLEY I. BUSH^*,||,2

^* Department of Pathology, University of Melbourne, Victoria, and The Mental Health Research Institute of Victoria, Parkville, Australia;
Department of Physics, Stockholm University, AlbaNova University Centre, Stockholm, Sweden;
School of Physics and Materials Engineering, Monash University, Victoria, Australia;
Center for Molecular Biology, The University of Heidelberg, Heidelberg, Germany; and
^|| Laboratory for Oxidation Biology, Genetics and Aging Research Unit and Department of Psychiatry, Harvard Medical School, Massachusetts General Hospital East, Charlestown, Massachusetts, USA

²Correspondence: (KJB) Department of Pathology, University of Melbourne, Victoria 3010, Australia. E-mail: kbarnham{at}unimelb.edu.au; (AIB) Laboratory for Oxidation Biology, Genetics and Aging Research Unit and Department of Psychiatry, Harvard Medical School, Massachusetts General Hospital East, Charlestown, MA 02129, USA. E-mail: bush{at}helix.mgh.harvard.edu

SPECIFIC AIMS

The ß-amyloid peptide (Aß), which plays a central role in the progression of Alzheimer’s disease, is neurotoxic. This toxicity has been linked to generation of H₂O₂. Aß coordinates redox active transition metals, copper, and iron to catalytically generate reactive oxygen species. The chemical mechanism underlying this process is not well defined. We have examined this mechanism to gain a better understanding of factors regulating Aß neurotoxicity.

PRINCIPAL FINDINGS

1. Tyrosine10 (Y10) is a pivotal residue to drive catalytic production of H₂O₂ by Aß/Cu
When Aß binds Cu it activates oxygen and in the presence of a reducing substrate (ascorbic acid is used in this study) catalyzes reduction of oxygen to H₂O₂ by promoting transfer of two electrons and two protons from the substrate to O₂. To identify molecular events underlying Aß/Cu²⁺-catalyzed production of H₂O₂, we performed density functional theory (DFT) calculations.

DFT calculations suggest that Cu²⁺ is reduced to Cu⁺ via proton-coupled electron transfer (PCET) from ascorbate to the side-chain oxygen of the tyrosinate of Y10, which passes (gates) the first electron to Cu²⁺ (Fig. 1 A). Next, dioxygen coordinates to Cu⁺ and is reduced to O₂·^–. The second electron comes when Y10 gives up its side-chain hydroxyl hydrogen atom to O₂·^– via hydrogen atom transfer. Simultaneously, H₃O⁺ donates its proton to O₂·^– via proton transfer, whereupon H₂O₂ and Tyr10 radicals form (Fig. 1B ).

View larger version (21K): [in this window] [in a new window]   Figure 1. Key intermediates involving Y10 in catalytic production of H2O2 by Aß. A) A transition state is formed during the mechanism in which a hydrogen atom is transferred from ascorbate to the side-chain oxygen of Y10. During this process Y10 acts as a gate and passes one electron to Cu2+ that is reduced to Cu+ (Cu, pink; oxygen, red; nitrogen, blue; carbon, green; hydrogen, gray). At the transition state the hydrogen atom (black) is partially transferred from the O2 atom of ascorbate to the side-chain oxygen of Y10, with an O2-H distance of 1.085 Å and an OY10-H distance of 1.351 Å. The spin on copper is 0.33, indicating reduction of Cu2+ is taking place. A total spin of 0.48 has also built up on ascorbate, which is transforming into an ascorbyl radical anion. Tyrosine holds a spin of 0.1. An activation energy computed for this proton-coupled electron transfer (PCET) was only 0.9 kcal/mol. B) An intermediate formed along the reaction path where Y10 has given up its side-chain hydroxyl hydrogen atom to O2·– via hydrogen atom transfer and transformed into a tyrosyl radical. Simultaneously, H3O+ has donated its proton to O2·– via proton transfer and H2O2 has formed. The formed tyrosyl radical and the water molecule are hydrogen bonded to H2O2. The ascorbyl radical anion coordinates via its O1-oxygen anion in an apical position to Cu2+. Cu, pink; oxygen, red; nitrogen, blue; carbon, green; hydrogen, white. Spin on the copper ion is 0.65, indicating Cu2+. The formed tyrosyl radical holds a spin of –1.0.

2. Tyrosyl radicals are experimentally observed and mutation of Y10 to alanine inhibits reduction of Cu²⁺
We used EPR spectroscopy to ascertain if tyrosine radicals are generated during this reaction with the EPR detectable radical trapping agent 2-methyl-2-nitrosopropane (MNP) that is specific for such radicals. EPR spectra indicated presence of a transiently detectable radical for Aß in the presence of Cu²⁺ and ascorbate. This signal was not observed when an Aß mutant peptide Y10A was used in the same reaction. Reduction of Cu²⁺ to Cu⁺ can be followed by loss of the EPR signal with time. Native Aß42 rapidly reduces Cu²⁺ to Cu⁺ in aqueous solution with near-complete reduction of Cu²⁺ taking 80 min, and the mutation of Y10 to alanine dramatically decreased the ability of Aß to reduce Cu²⁺. After 5 h incubation, Y10A peptide reduced approximately half the amount of Cu²⁺ that was reduced by the native peptide. This confirms the DFT observation that Y10 acts as a gate that facilitates electron transfer to reduce Cu²⁺ to Cu⁺.

3. Mutation of Y10 to alanine inhibits catalytic H₂O₂ production
Catalytic ability of wild-type Aß and mutant Y10A to produce H₂O₂ was determined by DCF assay. V_max for wild-type Aß42 was determined to be 20.3 ± 4 nM H₂O₂/min and was approximately twice that of mutant Y10A peptide (9.6±2 nM H₂O₂/min), confirming a critical role for Y10 in production of H₂O₂. The moderate metal chelator clioquinol (CQ) acted as a noncompetitive inhibitor and Aß/Cu/ascorbate mediated H₂O₂ production, indicating the importance of copper to this process.

4. Y10 is critical to Aß neurotoxicity
The effect of Y10A mutation on neurotoxicity was investigated. Primary cortical neurons were exposed to soluble peptide and cell viability was determined. Soluble Aß42 induced a characteristic neurotoxicity (Fig. 2 A) that was significantly rescued by coincubation with the extracellular H₂O₂ scavenger catalase (2000U/mL, P<0.001) or by coincubation with CQ (2 µM, P<0.001), and also by coincubation with tyrosine radical spin trap MNP (50 µM, P<0.001). CQ is likely to have inhibited toxicity of Aß in the cell culture by interfering with the reaction of the peptide with Cu already present in the culture medium (1.6 µM). Spin trap MNP that inhibited Aß toxicity was the same agent that identified the presence of the Tyr radical in the EPR spectrum, confirming a pivotal role for generation of the Tyr radical in the mechanism of Aß toxicity. Mutant peptide Y10A did not induce neurotoxicity (Fig. 2A ) even when a 2.5-fold higher concentration was used (15 µM, Fig. 2A ). These results confirm a critical role for Y10 radicals in Aß-mediated neurotoxicity.

View larger version (31K): [in this window] [in a new window]   Figure 2. Evidence for Y10 mediating Aß toxicity and cross-linking. A) Survival of primary mouse cortical neuronal culture exposed to Aß (6 µM), Aß plus catalase (2000 U/mL), clioquinol (CQ, 2 µM), or tyrosine-specific spin trap MNP (50 µM). Survival of primary mouse cortical neuronal culture exposed to Y10A mutant (6 µM). Whereas wild-type Aß was found to be toxic, Y10A mutant was not toxic even when tested at concentrations 2.5-fold higher (15 µM). #, P <0.001 vs. vehicle (n=2–4). B) The effect of Cu/H2O2 on oligomerization of Aß monitored by silver staining, shows unreacted Aß is largely monomeric, whereas Y10A exists in a number of discreet oligomeric forms. Addition of copper induces oligomerization of wild-type Aß and Y10A mutant. CQ inhibits the formation of the 55 kDa oligomers and spin trap MNP has no effect on the oligomeric profile. Despite the number of different oligomeric forms, Y10A peptide is not toxic. C) Aß Western blot (WO2) of wild-type Aß compared with Y10A following reaction with Cu/H2O2. In the presence of Cu/H2O2 oligomeric forms of Aß are observed, this oligomerization was inhibited by CQ and MNP, and reduced for Y10A. D) Immunoblotting with dityrosine antibody G6 confirms presence of dityrosine-cross-linked high MW oligomers following coincubation of Aß42 with Cu/H2O2 (left). The metal chelator CQ inhibited dityrosine formation, as did spin trap MNP, whereas Y10A lacking tyrosine at position 10 shows no immunoreactivity (right).

5. Oxidative modifications to Y10 lead to formation of soluble aggregated forms of Aß
A possible indirect mechanism for H₂O₂-mediated toxicity is via covalent cross-linking of Aß as a result of oxidative modifications. The presence of tyrosine radicals and hydroxyl radicals could induce oxidative modification of Aß peptide, including formation of dityrosine and hydroxytyrosine (Dopa) adducts of Aß.

Silver staining indicated that while Aß was primarily present as a monomer, Y10A exhibited SDS-resistant oligomers (Fig. 2B ). Addition of Cu²⁺ induced further oligomerization in both peptides with a prominent band at 55 kDa observed for both. Coincubation with CQ reduced the intensity of these bands indicating that their formation was metal dependent. Spin trap MNP appeared to have little effect on these aggregation profiles. These data also show that despite the prevalence of oligomeric forms (Fig. 2B ), Y10A is not toxic (Fig. 2A ).

Probing the gels with antibody WO2 (epitope residues 5–8 of Aß) (Fig. 2C ) showed that Aß underwent SDS-resistant oligomerization and that this oligomerization was inhibited by CQ and, to a lesser extent, by spin trap MNP. Significantly less oligomerization was observed for Y10A. The blot was probed for formation of dityrosine immunoreactive oligomers (Fig. 2D ), which were observed only for wild-type Aß in the presence of Cu²⁺/H₂O₂ but were completely inhibited by addition of CQ and partially inhibited by spin trap MNP. As expected, Y10A showed no dityrosine immunoreactivity upon incubation with Cu²⁺/H₂O₂ (Fig. 2D ). Dityrosine formation is dependent on a metal-induced tyrosine radical and was inhibited by CQ and MNP (Fig. 2D ), as was toxicity of Aß (Fig. 2A ). These data support tyrosine covalent bridging as a possible event that might contribute to the aspect of Aß toxicity that is abolished by Y10A mutation.

CONCLUSIONS AND SIGNIFICANCE

Aß neurotoxicity is inhibited by catalase, indicating that H₂O₂ plays a critical role in the toxicity. Aß coordinates Cu²⁺ or Fe³⁺ and generates H₂O₂ catalytically. Catalytic reduction of O₂ to H₂O₂ requires the Aß/Cu catalytic unit to act as a two-electron redox unit during the catalytic cycle. This is achieved by having two distinct one-electron acceptors, Cu(II) and the peptide free radical localized on Y10. Catalytic activity of Aß/Cu most closely resembles that of galactose oxidase.

Soluble oligomers of Aß have been reported to be more toxic than monomeric Aß. These oligomers are SDS stable and oligomerization is resistant to the harsh conditions of 50% formic acid and 8M urea, suggesting that the oligomers are covalently cross-linked. Our data identify a likely mechanism for formation of such linkages via oxidative modification, including tyrosine cross-links. Our data showing the prevalence of Y10A oligomers that are nontoxic, identifies dityrosine cross-linked oligomers as the toxic species rather than oligomers per se. A likely mechanism for formation of such tyrosine cross-linkages is via oxidative modification. Stable dimers and trimers of oligomeric Aß have been isolated from AD brain tissue and there is a general increase of dityrosine adducts in AD brains.

Our findings define a chemical mechanism at a quantum theory level that clarifies how Aß/metal interactions catalytically generate H₂O₂. This model is consistent with the current understanding of metal-mediated generation of H₂O₂ as an essential component of Aß neurotoxicity. Generation of H₂O₂ causes oxidative modification to Aß-generating soluble aggregated forms of Aß, a process rescued by CQ (Fig. 3 ). In a recent phase II proof of concept clinical trial, CQ inhibited cognitive decline in moderate to severe AD patients and concomitant reduction of plasma Aß42 levels.

View larger version (18K): [in this window] [in a new window]   Figure 3. Aß neurotoxicity. Scheme depicting how Aß coordinates Cu, generates H2O2, and induces toxicity. In generating H2O2, a radical is generated on Y10 that facilitates further oxidative modifications resulting in soluble aggregated forms of Aß that subsequently induce toxicity. Metal chelator CQ, catalase, and MNP, which traps the radical preventing further reaction, inhibit generation of these modified forms of Aß. Replacement of Y10 by an alanine inhibited the formation of these oxidatively modified forms of Aß and rendered it not toxic.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1890fje;

¹ These authors contributed equally to the work.

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