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Oxidation of the amyloid beta peptide and implications in the etiology of Alzheimer's Disease. – AlzABox

Oxidation of the amyloid beta peptide and implication in the etiology of Alzheimer’s disease

Oxidative stress involving overproduction of reactive oxygen species (ROS) has been linked to the etiology of Alzheimer’s Disease (AD). This overproduction finally leads to oxidative damage on neuronal lipids, proteins and the amyloid-beta peptide (AB) itself. Understanding how oxidative stress affects AB and what are the consequences appears as promising in deciphering some mechanisms involved in AD etiology.

Elucidate the role of metal-induced reactive oxygen species in AB oxidation, and its consequences in the etiology of Alzheimer’s disease

The global aim of the project AlzABox is to elucidate, at the molecular level, the role of metal-induced reactive oxygen species (ROS) in the oxidation and aggregation of the amyloid-ß peptide (AB), in relation with Alzheimer’s disease (AD). More specifically, we want to understand the molecular mechanisms of the events from metal ions coordination to AB to the neuronal degeneration via ROS production. Main issues are to: i) deeply characterize the metal-induced oxidation of the Aß peptide, in the native Aß40(42)/Cu2+/ascorbate at physiological concentrations (and in truncated in vitro systems, as AB28 or others if necessary), ii) disentangle the consequences of the AB peptide oxidation in the metal ion coordination and in the aggregation process, from both kinetic and thermodynamic views, iii) identify and possibly isolate oxidized oligomeric forms of Aß, iv) finally, better understand the mechanisms and consequences of ROS production and AB oxidation regarding the etiology of AD and the neuronal degeneration.

Based on an innovative and multidisciplinarity approach, the AlzABox project implements a wide range of methodologies and techniques, from chemistry to biology. First, a deep characterization of in vitro model systems of AB submitted to metal-induced oxidative stress conditions will be carried out. It will allows identifying the oxidized amino acid residues and the nature of the oxidation, studying the influence of oxidation time and thus deciphering the early and preferred amino acid residues targeted by the Reactive Oxygen Species (ROS) during oxidation. Advanced mass spectrometric-based coupled techniques will be mainly used, in particular high performance liquid chromatography/tandem mass spectrometry (HPLC/MS-MS), at low and high resolution, and ion mobility coupled to MS. The coordination of metal ions (such as copper or zinc) to oxidized AB will be studied by EPR (electron paramagnetic resonance), NMR (nuclear magnetic resonance) and XAS (X-ray absorption spectroscopy)-based techniques.
In addition, the oxidation of the AB peptide will be characterized in vivo for two transgenic AD mouse models, complemented by post-mortem brain samples from AD patients. The AB peptide species (including truncated and oxidized forms) will be extracted by immunoprecipitation with magnetic beads, by using both N- and C-terminal directed anti-AB antibodies. Particular attention will be given to the analysis of the oxidized species. The results will be compared with those obtained by working with the in vitro model systems.
Finally, the consequences of AB oxidation on aggregation, ROS production and cell toxicity will be studied by various methodologies, including fluorescence-based techniques. The AB oxidized species (in vitro and in vivo samples) will be characterized for their propensity to aggregate, in the presence or absence of metal ion (copper or zinc), their ability to produce metal-induced ROS, and their toxicity towards neuroblastoma SH-SY5Y cells.

The project AlzABox, through its innovative and multidisciplinary approach, can lead to several scientific and technical fallouts, at mid- and long-term. There are three main expected outcomes: i) to develop an in vitro mastered model system suitable for testing compounds for their efficiency in inhibiting ROS production, neurotoxicity and aggregation of AB, ii) to purify the Aßox species from complex samples as a routine work, to possibly identify some of them as early biomarkers of AD development, and potentially to apply the methodology for the development of microfluidics-based diagnosis of AD, and iii) to open ways to novel targeted therapeutic strategies, based on the understanding of AB oxidation as a consequence regarding the mechanisms involved in AD etiology.

As first outcome, a well-mastered in vitro model would be a powerful tool in developing novel compounds such as platinum or ruthenium complexes that are able to interact with the AB peptide, in competition with the usually associated metallic ions, i.e. copper, iron or zinc. Some complexes are currently studied and reported as promising for their ability to inhibit aggregation of AB in particular. This point could lead to longer-term development of novel drugs. However, such a development should also include drugs targeting oxidation of AB, such as new antioxidant compounds.
Because the development of AD is done on a period of time of several decades and because there is currently no cure for this disease, early diagnosis is currently an important challenge. Another outcome of the project AlzABox is the identification of some AB oxidized species that could be specific enough to be used as biomarkers, at different stages of AD development. At long-term, the magnetic bead immunodepletion strategy proposed for AB purification from complex samples could open ways to the development and optimization of microfluidics-based tools for early and rapid diagnosis.

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Alzheimer’s disease (AD) is the leading cause of dementia in the elderly, by striking 1 over 20 in the World. A defining feature in AD is the post-mortem observation of proteinaceaous plaques, mainly composed of the amyloid-beta peptide (Aß), along with metal ions such as copper, iron or zinc. Aß is found aggregated as amyloid fibrils in the plaques of AD brains, but is present as soluble monomer in healthy subjects. Thus, aggregation of Aß is a critical step and intermediates (often called oligomers) seem to be the most toxic species, reported to mediate oxidative stress by production of reactive oxygen species (ROS). The redox active metal ions (Cu, Fe) are pivotal in the production and defense of ROS (like NADPH oxidase and SOD, respectively), and loosely bound Fe and Cu can catalyze efficiently the generation of ROS. Indeed, in AD more loosely bound Cu and Fe have been detected and there is a large body of evidence that Cu bound to Aß is part if this Cu pool. Cu can also influence the aggregation of Aß. Thus, the complexes formed between metal ions and Aß might be the cross point of ROS production and Aß aggregation linked to Aß-induced toxicity in AD. Their study appears as particularly promising in understanding the mechanisms of AD development in brain.
The project AlzABox aims at studying the relation between metal-induced oxidative stress in AD, the resulting oxidative damages on the Aß peptide and their consequences on the main mechanisms involved in AD etiology, i.e. aggregation, ROS production and cell toxicity. For this purpose, an interdisciplinary approach, at the frontier between chemistry and biology, is proposed to be developed for i) deeply characterizing the metal-induced oxidative modification undergone by the Aß peptide for in vitro and in vivo model systems, ii) understanding the consequences of Aß oxidation regarding the aggregation mechanism, the coordination of metal ions, the metal-induced ROS production and the cell toxicity, iii) establishing biologically relevant mechanisms of oxidation that might be involved in AD development. Mass spectrometric-based analytical methodologies (coupled to chromatography, high resolution), coordination chemistry, cell and living systems biology are among the disciplines that will be implemented to gain knowledge at the molecular level and ensure the biological relevance, for the success of the project.
The novel and original approach developed in the project AlzABox will first lead to improve the knowledge on the oxidative modification undergone by the Aß peptide, submitted to oxidative stress conditions: identification of the oxidized amino acid residues, nature of the chemical modification, preferential amino acid residues targeted by the ROS attack and time-dependency of oxidation, primary events of the oxidation process that impact the Aß sequence, nature of oxidation as the function of the initial aggregation state of the Aß peptide (monomers, oligomers, amorphous aggregates, fibrils)… Then, by putting in perspective the study of the coordination of metal ions (Cu+, Cu2+, Zn2+) to the Aß oxidized species and the propensity of these latter to aggregate, produce ROS or be toxic for cell, a comprehensive mechanism should emerge regarding the deleterious impact of Aß oxidation towards neurons. Comparison with the Aß oxidized species as characterized in the brain of transgenic AD mice, at different levels of AD development, and in the post-mortem brain samples from AD patients will ascertain the biological relevance of the results.
Finally, by gaining significant insights into the mechanisms of Aß oxidation, their consequences on aggregation, ROS production, metal ion binding and cell toxicity, the project AlzABox should contribute significantly to improve the knowledge on the AD pathology and further open ways to the development of of novel therapeutic strategies, targeting the early stages of AD.

Project coordination

fabrice collin (Université)

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.

Partner

LCC LCC, CNRS UPR 8241
CRCA CRCA, UMR 5169 CNRS-UPS, Université Paul Sabatier

Help of the ANR 292,458 euros
Beginning and duration of the scientific project: January 2014 - 36 Months

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