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Le rôle des ions métalliques dans l'agregation, la production de radicaux et la toxicité du peptide amyloïde-? – Neurometals

Insight into the role of copper and zinc in Alzheimer disease

It was shown that copper and zinc ions play a key role in the aggregation of A? peptides and neuronal toxicity. The purpose of this project was to understand the role of copper and zinc ions in the two key stages of AD development as well as the molecular mechanisms they are associated

New insights into the interaction of copper and zinc with the peptide amyloid-ß and their relation to Alzheimer disease.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly over 65 years old and now affects over 25 million people worldwide. Amyloid plaques are one of the main lesions in AD victims’ brain. A peptide called amyloid ? (A?) is their main component. The process of A? aggregation leading to the formation of amyloid plaques is a central aspect of the development of AD. It was also shown that copper and zinc ions play a key role in the aggregation of A? peptides and neuronal toxicity. <br />The purpose of this project was to understand the role of copper and zinc ions in the two key stages of AD development as well as the molecular mechanisms they are associated with: (i) the coordination of copper and zinc to the Aß peptide and their relationship with aggregation (ii) the toxicity towards neurons of the A? peptide bound to copper via the production of reactive oxygen species (ROS), which are toxic to neurons. For this we have characterized the structure of the complexes between copper and/or zinc ions and A? and we have studied the mechanism of ROS production. <br />This information is important for understanding the role of copper and zinc in conjunction with A? in AD, which helps to address therapeutic strategies against this disease. <br />

Structural information about the complexes between A? and the metal ions (copper (II), copper (I) and zinc (II ), which are the forms of copper and zinc biologically relevant), were obtained by the use of a wide range of different types of spectroscopy including nuclear magnetic resonance, electron paramagnetic resonance, X-ray absorption (at the synchrotrons ESRF in Grenoble and SOLEIL in Saint-Aubin), fluorescence, circular dichroism, etc… The use of various techniques is an important asset to have a coherent overview of the structure of the complexes. This was further coupled to isotope labeling techniques to properly identify the entities without ambiguity.
A multidisciplinary approach using in parallel chemistry techniques and biological effects has been applied to better understand the role of metals in toxicity towards the neurons. Thus, we were able to correlate the mechanism of reactive oxygen species production with toxicity against neurons.

- A detailed description of the structure of complexes between the metal ions copper (I), copper (II) and, to a lesser extent, zinc (II) with the non-aggregated A? peptide.
- A mechanistic understanding of the role of copper (II) and zinc (II) in a model of aggregate
- A better understanding of the so-called copper-A? redox reaction, which seems to be the basis for the production of reactive oxygen species (toxic agent for the cells)

The obtained results of the project will have the following perspectives
- a better understanding of the roles of the metal ions and their interaction with the peptide amyloid-beta in Alzheimer’s disease
- the obtained in vitro result are a good benchmark for studies in a more biological environment, like for instance in Alzheimer’s disease mice
- to conceive more powerful molecules (more fine tuned and more specific) for a therapeutical approach for Alzheimer’s disease based on the role of metal ions.

We have published more than 20 articles in international, peer review journals including several reviews and several in prestigious journals (like Angewandte Chemie Int. Ed., Proc. Nat. Acad. Sci. USA, Chemistry Eur. J.).The majority of the publications concern the interaction in terms of structure and chemical reactivity of copper(I) and copper(II) with the peptide Aß.

Role of Metal Ions in Aggregation, Radical Generation and Toxicity of the Peptide Amyloid-' INTRODUCTION: Amyloid plaques are a hallmark in the brain of Alzheimer's disease (AD) victims. These plaques are mainly constituted by an aggregated peptide called amyloid-' (A'). Since the amyloid plaques occur only in AD patients, the aggregation process from A' to the plaques is considered to be a key event. According to the amyloid cascade hypothesis an increased A' accumulation and aggregation lead first to the formation of A' oligomers and then to amyloid plaques. These oligomers are supposed to provoke neuronal dysfunction and later on dementia. A large body of evidences points to an important role of metal ions (Zn, Cu) in the aggregation of A' and the toxicity towards neurons. Amyloid plaques contain high concentrations (~mM) of these metal ions. Moreover, the redox active Cu+/2+ ion is known for its involvement in the production of reactive oxygen species (ROS, such as HO', H2O2 etc.) that are supposed to be involved in AD, whereas Zn2+ is thought to inhibit ROS production and hence to have a protecting effect. The role of Cu+/2+ ion and ROS can be schematically summarized as follow : Cu + A' -> Cu-A' -> oligomers of Cu-A' -> ROS production -> neuronal death GOAL: The global aim of the project is to better understand the role of the Cu+/2+ and Zn2+ ions in the two key events of AD and their molecular mechanisms: (i) metal-induced A' aggregation, (ii) toxicity of metallated A' aggregates for cells due to ROS production. PROJECT: In the past, we have investigated the coordination of Cu+/2+ and Zn2+ to non-aggregating truncated peptides, models of the native A' [Mekmouche et al. ChemBioChem 2005; Guilloreau et al. JBIC 2006;Talmard et al. ChemBioChem 2007]. In the present project, we plan to investigate the A' aggregation induced by metal ions (Cu+/2+, Zn2+) with the native A'40 and A'42. Native and modified recombinant A' can be produced by overexpression in E. coli (as a fusion protein). They can also be purchased, their price having significantly decreased in the last two years. The coordination chemistry of the involved metal ions and their influence on the A' structure will be studied by physico-chemical techniques (EPR, NMR, Circular dichroism, microcalorimetry, etc.) To get a more detailed knowledge on the Cu2+ binding site in A' at different aggregation states, ENDOR and ESEEM (i.e. advanced electron paramagnetic resonance methods) will be applied. We also plan to focus on the binding of the reduced Cu+ to A', for which very little is known although it likely plays an important role in vivo. That is why we have recently set up a collaboration at the SOLEIL synchrotron (X-Ray Absorption measurements). Subsequently, the metal induced aggregation processes will be studied by methods applicable to aggregates (Transmission Electron Microscopy, chromatography, spectroscopy etc). Particular attention will be paid on the identification of intermediates of the aggregation, which are suggested to be the most toxic species. The ability of the Cu2+-A' complexes to produce ROS (in particular HO') will be investigated in the presence of physiological concentrations of ascorbate, and correlate to their aggregation state. Electrochemistry will be used to study the mechanism of ROS production in connection with the aggregation state. In order to relate this to biology, we plan to measure ROS production in cello and to do toxicity studies on cell cultures (SH-SY5Y) by measuring cell viability. PARTNERS: The project combines three partners from different disciplines and institutes (Partner 1: (bio)inorganic chemistry, Partner 2: molecular and cell biology, Partner 3: advanced EPR spectroscopy). In the project, the native and modified A' peptides will be obtained using recombinant production and site-directed mutagenesis by partner 2 (or will be purchased). Coordination of metal ions to the A' peptide and its impact on the structure of A' will be studied by Partner 1. More specifically, the Cu2+ coordination to the A' peptide and its involvement in the aggregation process will be investigated using advanced EPR techniques by partner 3. Aggregation process in presence of metal ions will be compared to that of peptides only and will require techniques from all partners, in particular PELDOR measurements performed by partner 3. Production of ROS like HO' by the metal ions-A' complexes will be studied by Partner 1. Partner 2 will measure the toxicity towards cell in culture of the different metal ions-A' species. The complementary of these studies should allow getting significant insights into the mechanisms of metal ions induced aggregation of A', ROS production and their biological relevance.

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The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

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