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James A Duce,
Andrew Tsatsanis,
Michael A Cater,
Simon A James,
Elysia Robb,
Krutika Wikhe, Su Ling Leong,
Keyla Perez,
Timothy Johanssen,
Mark A Greenough, [......],
Denise Galatis,
Robert D Moir,
Colin L Masters,
Catriona McLean,
Rudolph E Tanzi,
Roberto Cappai,
Kevin J Barnham,
Giuseppe D Ciccotosto,
Jack T Rogers,
Ashley I Bush
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ABSTRACT: Alzheimer's Disease (AD) is complicated by pro-oxidant intraneuronal Fe(2+) elevation as well as extracellular Zn(2+) accumulation within amyloid plaque. We found that the AD β-amyloid protein precursor (APP) possesses ferroxidase activity mediated by a conserved H-ferritin-like active site, which is inhibited specifically by Zn(2+). Like ceruloplasmin, APP catalytically oxidizes Fe(2+), loads Fe(3+) into transferrin, and has a major interaction with ferroportin in HEK293T cells (that lack ceruloplasmin) and in human cortical tissue. Ablation of APP in HEK293T cells and primary neurons induces marked iron retention, whereas increasing APP695 promotes iron export. Unlike normal mice, APP(-/-) mice are vulnerable to dietary iron exposure, which causes Fe(2+) accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD postmortem neocortex is inhibited by endogenous Zn(2+), which we demonstrate can originate from Zn(2+)-laden amyloid aggregates and correlates with Aβ burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD.
Cell 09/2010; 142(6):857-67. · 32.40 Impact Factor
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ABSTRACT: The deposition of alpha-synuclein (alpha-syn) aggregates in dopaminergic neurons is a key feature of Parkinson's disease. While dopamine (DA) can modulate alpha-syn aggregation, it is unclear which other factors can regulate the actions of DA on alpha-syn. In this study, we investigated the effect of solution conditions (buffer, salt and pH) on the oligomerization of alpha-syn by DA. We show that alpha-syn oligomerization is dependent on the oxidation of DA into reactive intermediates. Under acidic pH conditions, DA is stable, and DA-mediated oligomerization of alpha-syn is inhibited. From pH 7.0 to pH 11.0, DA is unstable and undergoes redox reactions, promoting the formation of SDS-resistant soluble oligomers of alpha-syn. We show that the reactive intermediate 5,6-dihydroxylindole mediates the formation of alpha-syn soluble oligomers under physiological conditions (pH 7.4). In contrast, under acidic conditions (pH 4.0), 5,6-dihydroxylindole promotes the formation of SDS-resistant insoluble oligomers that further associate to form sheet-like fibrils with beta-sheet structure that do not bind the dye thioflavin T. These results suggest that distinct reactive intermediates of DA, and not DA itself, interact with alpha-syn to generate the alpha-syn aggregates implicated in Parkinson's disease.
Journal of Molecular Biology 05/2009; 387(3):771-85. · 4.00 Impact Factor
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ABSTRACT: alpha-Synuclein is the major component of the intracellular Lewy body inclusions present in Parkinson disease (PD) neurons. PD involves the loss of dopaminergic neurons in the substantia nigra and the subsequent depletion of dopamine (DA) in the striatum. DA can inhibit alpha-synuclein fibrillization in vitro and promote alpha-synuclein aggregation into soluble oligomers. We have studied the mechanism by which DA mediates alpha-synuclein aggregation into soluble oligomers. Reacting alpha-synuclein with DA increased the mass of alpha-synuclein by 64 Da. NMR showed that all four methionine residues were oxidized by DA, consistent with the addition of 64 Da. Substituting all four methionines to alanine significantly reduced the formation of DA-mediated soluble oligomers. The (125)YEMPS(129) motif in alpha-synuclein can modulate DA inhibition of alpha-synuclein fibrillization. However, alpha-synuclein ending before the (125)YEMPS(129) motif (residues 1-124) could still form soluble oligomers. The addition of exogenous synthetic YEMPS peptide inhibited the formation of soluble oligomers and resulted in the YEMPS peptide being oxidized. Therefore, the (125)YEMPS(129) acts as an antioxidant rather than interacting directly with DA. Our study defines methionine oxidation as the dominant mechanism by which DA generates soluble alpha-synuclein oligomers and highlights the potential role for oxidative stress in modulating alpha-synuclein aggregation.
Free radical biology & medicine 03/2009; 46(10):1328-37. · 5.42 Impact Factor
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ABSTRACT: The interaction of the small (140 amino acid) protein, alpha-synuclein (alphaS), with Cu(2+) has been proposed to play a role in Parkinson's disease (PD). While some insight from truncated model complexes has been gained, the nature of the corresponding Cu(2+) binding modes in the full length protein remains comparatively less well characterized. This work examined the Cu(2+) binding of recombinant human alphaS using Electron Paramagnetic Resonance (EPR) spectroscopy. Wild type (wt) alphaS was shown to bind stoichiometric Cu(2+) via two N-terminal binding modes at physiological pH. An H50N mutation isolated one binding mode, whose g parallel, A parallel, and metal-ligand hyperfine parameters correlated well with a {NH2, N(-), beta-COO(-), H2O} mode previously identified in truncated model fragments. Electron spin-echo envelope modulation (ESEEM) studies of wt alphaS confirmed the second binding mode at pH 7.4 involved coordination of His50 and its g parallel and A parallel parameters correlated with either {NH2, N(-), beta-COO(-), N(Im)} or {N(Im), 2 N(-)} coordination observed in alphaS fragments. At pH 5.0, His50-anchored Cu(2+) binding was greatly diminished, while {NH2, N(-), beta-COO(-), H2O} binding persisted in conjunction with another two binding modes. Metal-ligand hyperfine interactions from one of these indicated a 1N3O coordination sphere, which was ascribed to a {NH2, CO} binding mode. The other was characterized by a spectrum similar to that previously observed for diethylpyrocarbonate-treated alphaS and was attributed to C-terminal binding centered on Asp121. In total, four Cu(2+) binding modes were identified within pH 5.0-7.4, providing a more comprehensive picture of the Cu(2+) binding properties of recombinant alphaS.
Journal of the American Chemical Society 07/2008; 130(24):7766-73. · 9.91 Impact Factor
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09/2007; , ISBN: 9780470028636
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ABSTRACT: The deposition of α-synuclein (α-syn) aggregates in dopaminergic neurons is a key feature of Parkinson's disease. While dopamine (DA) can modulate α-syn aggregation, it is unclear which other factors can regulate the actions of DA on α-syn. In this study, we investigated the effect of solution conditions (buffer, salt and pH) on the oligomerization of α-syn by DA. We show that α-syn oligomerization is dependent on the oxidation of DA into reactive intermediates. Under acidic pH conditions, DA is stable, and DA-mediated oligomerization of α-syn is inhibited. From pH 7.0 to pH 11.0, DA is unstable and undergoes redox reactions, promoting the formation of SDS-resistant soluble oligomers of α-syn. We show that the reactive intermediate 5,6-dihydroxylindole mediates the formation of α-syn soluble oligomers under physiological conditions (pH 7.4). In contrast, under acidic conditions (pH 4.0), 5,6-dihydroxylindole promotes the formation of SDS-resistant insoluble oligomers that further associate to form sheet-like fibrils with β-sheet structure that do not bind the dye thioflavin T. These results suggest that distinct reactive intermediates of DA, and not DA itself, interact with α-syn to generate the α-syn aggregates implicated in Parkinson's disease.
Journal of Molecular Biology.
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[show abstract]
[hide abstract]
ABSTRACT: α-Synuclein is the major component of the intracellular Lewy body inclusions present in Parkinson disease (PD) neurons. PD involves the loss of dopaminergic neurons in the substantia nigra and the subsequent depletion of dopamine (DA) in the striatum. DA can inhibit α-synuclein fibrillization in vitro and promote α-synuclein aggregation into soluble oligomers. We have studied the mechanism by which DA mediates α-synuclein aggregation into soluble oligomers. Reacting α-synuclein with DA increased the mass of α-synuclein by 64 Da. NMR showed that all four methionine residues were oxidized by DA, consistent with the addition of 64 Da. Substituting all four methionines to alanine significantly reduced the formation of DA-mediated soluble oligomers. The 125YEMPS129 motif in α-synuclein can modulate DA inhibition of α-synuclein fibrillization. However, α-synuclein ending before the 125YEMPS129 motif (residues 1–124) could still form soluble oligomers. The addition of exogenous synthetic YEMPS peptide inhibited the formation of soluble oligomers and resulted in the YEMPS peptide being oxidized. Therefore, the 125YEMPS129 acts as an antioxidant rather than interacting directly with DA. Our study defines methionine oxidation as the dominant mechanism by which DA generates soluble α-synuclein oligomers and highlights the potential role for oxidative stress in modulating α-synuclein aggregation.
Free Radical Biology and Medicine.
