Christelle Hureau

Aβ-mediated ROS production by Cu ions: Structural insights, mechanisms and relevance to Alzheimer's disease

Metal ions are involved in Alzheimers disease (AD) via their ability to induce aggregation of amyloidogenic peptide and production of Reactive Oxygen Species (ROS), two key events in the development of the pathology. Here, we review very recent results concerning the coordination of Cu(I) and Cu(II) ion to the amyloid-beta peptide, the one encountered in AD. Implications of these structural data for the redox chemistry of the Cu(I/II)-Abeta couple are discussed. The different pathways for the ROS generation by the Cu(I/II)-Abeta species are described. In the more relevant one, reduction of dioxygen is realized by a two-electron process involving two Cu(I) in close vicinity, while the production of the hydroxyl radical from hydrogen peroxide is less constrained. A brief summary of how the Abeta peptide is oxidised during the ROS production is also given. Lastly, the pro- vs. anti-oxidant properties of Abeta are commented on.

Role of Metal Ions in the Self-assembly of the Alzheimer’s Amyloid-β Peptide

Aggregation of amyloid-β (Aβ) by self-assembly into oligomers or amyloids is a central event in Alzheimers disease. Coordination of transition-metal ions, mainly copper and zinc, to Aβ occurs in vivo and modulates the aggregation process. A survey of the impact of Cu(II) and Zn(II) on the aggregation of Aβ reveals some general trends: (i) Zn(II) and Cu(II) at high micromolar concentrations and/or in a large superstoichiometric ratio compared to Aβ have a tendency to promote amorphous aggregations (precipitation) over the ordered formation of fibrillar amyloids by self-assembly; (ii) metal ions affect the kinetics of Aβ aggregations, with the most significant impact on the nucleation phase; (iii) the impact is metal-specific; (iv) Cu(II) and Zn(II) affect the concentrations and/or the types of aggregation intermediates formed; (v) the binding of metal ions changes both the structure and the charge of Aβ. The decrease in the overall charge at physiological pH increases the overall driving force for aggregation but may favor more precipitation over fibrillation, whereas the induced structural changes seem more relevant for the amyloid formation.

Deprotonation of the Asp1Ala2 Peptide Bond Induces Modification of the Dynamic Copper(II) Environment in the Amyloid‐β Peptide near Physiological pH

Aggregation of the amyloid-b (Ab) peptide and the production of reactive oxygen species by aggregates are two key features in Alzheimer’s disease. Copper ions have been linked to both of these events, 3] and hence determination of the basic interaction of Cu and Ab is essential for understanding its roles in the development of the pathology. The native Ab peptides consist of 39 to 43 amino acid residues and have been shown to be strongly prone to aggregation (from a few mm concentration). However, the Cu binding site has been localized in the N-terminal part of the peptide encompassing the first 16 amino acid residues (see Scheme S1 in the Supporting Information for the peptide sequence), 5] a truncated peptide that is highly soluble. Hence, this shortened peptide is accepted as a valuable model of Cu coordination to full-length Ab and its high solubility allows classical spectroscopic methods, such as those of the present study, to be used. While most techniques aim at identifying the Cu ligands (for a review, see reference [6] and for very recent reports, see references [7, 8]), NMR spectroscopy is among the few methods also able to reveal dynamical processes in the coordination of Cu to Ab. Indeed, the paramagnetism of the Cu ion induces an enhancement of the relaxation rate of the peptide nuclei, this effect diminishing according to the inverse sixth power of the interatomic distance (for reviews, see references [9, 10]). Consequently, selective broadening of the NMR signals of nuclei spatially close to the metal-ion binding site(s) is observed. In the case of Cu, the line broadening is severe and the effect of the largely substoichiometric ratio of the paramagnetic ion is detectable in the case of fast exchange of the paramagnet between sites. This is also true for C NMR signals despite the lower sensitivity to broadening effects for this nucleus as a result of its lower gyromagnetic ratio compared to that of the proton. As concerns Cu coordination to Ab, only a few NMR studies have been reported and they are limited to H NMR or H–N heteronuclear single quantum correlation (HSQC) experiments. 14] Fast amide proton exchanges are responsible for the loss of the signals of several amino acids (including Asp1 and the three His residues) in apo–Ab peptide in the latter cases, an effect that precludes the analysis of Cu-induced signal broadening. For those reasons, herein we focus on C{H} NMR spectroscopy, which is a straightforward way to inspect the effect of Cu on Ab peptide signals. Furthermore, it is known that near physiological pH, two Cu complexes of Ab coexist, which differ in the protonation state of the peptide and their spectroscopic signatures. 15] They are referred to below as “low-pH” and “high-pH” species. We identify the amino acid residues involved in Cu binding, and give clear-cut evidence for the presence of equilibria between different ligands in both forms. We also give new insights into the dramatic change undergone by the Cu binding sites in Ab between pH values of about 6.6 and 8.7, which arises from the deprotonation and binding of the Asp1 Ala2 peptide bond amide. Figure 1 shows the evolution of the C{H} NMR spectra of the Ab peptide (sequence DAEFRHDSGYEVHHQK) upon addition of 0.1 equivalents of Cu at pH 6.6 and 8.7 (see also Figure S4 in the Supporting Information for spectral domains that concern His residues). 17] Addition of Cu leads to broadening of several signals that is more selective at high pH (right-hand spectra in Figure 1) with only Asp1, Ala2, and the side chain of His mainly affected. More precisely, at pH 6.6 peaks of the carboxylate groups from Asp1, Asp7, Glu3, Glu11, and the unprotected C terminus are significantly broadened with a slight preference for that of Asp7. Only that of Asp1 is broadened at pH 8.7. Peaks of the [*] Dr. C. Hureau, Dr. Y. Coppel, Dr. L. Sabater, Prof. Dr. P. Faller CNRS; LCC (Laboratoire de Chimie de Coordination) 205 route de Narbonne, 31077 Toulouse (France) and Universit de Toulouse; UPS, INPT; LCC 31077 Toulouse (France) Fax: (+ 33)5-6155-3003 E-mail: christelle.hureau@lcc-toulouse.fr peter.faller@lcc-toulouse.fr

Metal ions and intrinsically disordered proteins and peptides: from Cu/Zn amyloid-β to general principles.

The interaction of d-block metal ions (Cu, Zn, Fe, etc.) with intrinsically disordered proteins (IDPs) has gained interest, partly due to their proposed roles in several diseases, mainly neurodegenerative. A prominent member of IDPs is the peptide amyloid-β (Aβ) that aggregates into metal-enriched amyloid plaques, a hallmark of Alzheimers disease, in which Cu and Zn are bound to Aβ. IDPs are a class of proteins and peptides that lack a unique 3D structure when the protein is isolated. This disordered structure impacts their interaction with metal ions compared with structured metalloproteins. Metalloproteins either have a preorganized metal binding site or fold upon metal binding, resulting in defined 3D structure with a well-defined metal site. In contrast, for Aβ and likely most of the other IDPs, the affinity for Cu(I/II) and Zn(II) is weaker and the interaction is flexible with different coordination sites present. Coordination of Cu(I/II) with Aβ is very dynamic including fast Cu-exchange reactions (milliseconds or less) that are intrapeptidic between different sites as well as interpeptidic. This highly dynamic metal-IDP interaction has a strong impact on reactivity and potential biological role: (i) Due to the low affinity compared with classical metalloproteins, IDPs likely bind metals only at special places or under special conditions. For Aβ, this is likely in the neurons that expel Zn or Cu into the synapse and upon metal dysregulation occurring in Alzheimers disease. (ii) Amino acid substitutions (mutations) on noncoordinating residues can change drastically the coordination sphere. (iii) Considering the Cu/Zn-Aβ aberrant interaction, therapeutic strategies can be based on removal of Cu/Zn or precluding their binding to the peptide. The latter is very difficult due to the multitude of metal-binding sites, but the fast koff facilitates removal. (iv) The high flexibility of the Cu-Aβ complex results in different conformations with different redox activity. Only some conformations are able to produce reactive oxygen species. (v) Other, more specific catalysis (like enzymes) is very unlikely for Cu/Zn-Aβ. (vi) The Cu/Zn exchange reactions with Aβ are faster than the aggregation process and can hence have a strong impact on this process. In conclusion, the coordination chemistry is fundamentally different for most of IDPs compared with the classical, structured metalloproteins or with (bio)-inorganic complexes. The dynamics is a key parameter to understand this interaction and its potential biological impact.

Iron(II) binding to amyloid-β, the Alzheimer's peptide.

Iron has been implicated in Alzheimers disease, but until now no direct proof of Fe(II) binding to the amyloid-β peptide (Aβ) has been reported. We used NMR to evidence Fe(II) coordination to full-length Aβ40 and truncated Aβ16 peptides at physiological pH and to show that the Fe(II) binding site is located in the first 16 amino-acid residues. Fe(II) caused selective broadening of some NMR peaks that was dependent on the Fe:Aβ stoichiometry and temperature. Analysis of Fe(II) broadening effect in the (1)H, (13)C, and 2D NMR data established that Asp1, Glu3, the three His, but not Tyr10 nor Met35 are the residues mainly involved in Fe(II) coordination.

Importance of dynamical processes in the coordination chemistry and redox conversion of copper amyloid-β complexes

Interaction of Cu ions with the amyloid-β (Aβ) peptide is linked to the development of Alzheimer’s disease; hence, determining the coordination of CuI and CuII ions to Aβ and the pathway of the CuI(Aβ)/CuII(Aβ) redox conversion is of great interest. In the present report, we use the room temperature X-ray absorption near edge structure to show that the binding sites of the CuI and CuII complexes are similar to those previously determined from frozen-solution studies. More precisely, the CuI is coordinated by the imidazole groups of two histidine residues in a linear fashion. However, an NMR study unravels the involvement of all three histidine residues in the CuI binding due to dynamical exchange between several set of ligands. The presence of an equilibrium is also responsible for the complex redox process observed by cyclic voltammetry and evidenced by a concentration-dependent electrochemical response.

Cu(II) Affinity for the Alzheimer’s Peptide: Tyrosine Fluorescence Studies Revisited

Copper(II) binding to the amyloid-β peptide has been proposed to be a key event in the cascade leading to Alzheimers disease. As a direct consequence, the strength of the Cu(II) to Aβ interaction, that is, the Cu(II) affinity of Aβ, is a very important parameter to determine. Because Aβ peptide contain one Tyr fluorophore in its sequence and because Cu(II) does quench Tyr fluorescence, fluorescence measurements appear to be a straightforward way to obtain this parameter. However, this proved to be wrong, mainly because of data misinterpretation in some previous studies that leads to a conflicting situation. In the present paper, we have investigated in details a large set of fluorescence data that were analyzed with a new method taking into account the presence of two Cu(II) sites and the inner-filter effect. This leads to reinterpretation of the published data and to the determination of a unified affinity value in the 10(10) M(-1) range.

Electrochemical and homogeneous electron transfers to the Alzheimer amyloid-β copper complex follow a preorganization mechanism

Deciphering the electron transfer reactivity characteristics of amyloid β-peptide copper complexes is an important task in connection with the role they are assumed to play in Alzheimer’s disease. A systematic analysis of this question with the example of the amyloid β-peptide copper complex by means of its electrochemical current–potential responses and of its homogenous reactions with electrogenerated fast electron exchanging osmium complexes revealed a quite peculiar mechanism: The reaction proceeds through a small fraction of the complex molecules in which the peptide complex is “preorganized” so as the distances and angles in the coordination sphere to vary minimally upon electron transfer, thus involving a remarkably small reorganization energy (0.3 eV). This preorganization mechanism and its consequences on the reactivity should be taken into account for reactions involving dioxygen and hydrogen peroxide that are considered to be important in Alzheimer’s disease through the production of harmful reactive oxygen species.

The Catalytically Active Copper‐Amyloid‐Beta State: Coordination Site Responsible for Reactive Oxygen Species Production

Copper-amyloid-β ROS production: Copper ions (red sphere, see picture) have been found to accumulate in amyloid-β plaques and play a role in the generation of reactive oxygen species (ROS) within this context. Mass spectrometry studies were able to detail the sites of oxidation damage and shed new light on the mechanism of ROS production, important for the understanding of the pathogenicity of amyloid-β peptides.

Oxidative stress and the amyloid beta peptide in Alzheimer’s disease

Oxidative stress is known to play an important role in the pathogenesis of a number of diseases. In particular, it is linked to the etiology of Alzheimer’s disease (AD), an age-related neurodegenerative disease and the most common cause of dementia in the elderly. Histopathological hallmarks of AD are intracellular neurofibrillary tangles and extracellular formation of senile plaques composed of the amyloid-beta peptide (Aβ) in aggregated form along with metal-ions such as copper, iron or zinc. Redox active metal ions, as for example copper, can catalyze the production of Reactive Oxygen Species (ROS) when bound to the amyloid-β (Aβ). The ROS thus produced, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative damage on both the Aβ peptide itself and on surrounding molecule (proteins, lipids, …). This review highlights the existing link between oxidative stress and AD, and the consequences towards the Aβ peptide and surrounding molecules in terms of oxidative damage. In addition, the implication of metal ions in AD, their interaction with the Aβ peptide and redox properties leading to ROS production are discussed, along with both in vitro and in vivo oxidation of the Aβ peptide, at the molecular level.