Keywords: Amino-Terminal APP Intracellular Amyloid Precursor Protein Ascorbate Asn; Aspartate Aβ, Amyloid-β peptide AβDPs, Aβ-degrading proteases Aβox, Oxidized Amyloid-β Peptide C CCA, Coumarin-3-Carboxylic Acid CID, Collision Induced Dissociation CNS, Central Nervous System CTF, Carboxyterminal Fragments Cu

La thèse est une grande aventure faite de rencontres, d'opportunités et de nouveautés. C'est un tout nouveau monde qui s'ouvre à soi, inconnu, inconfortable mais exaltant. Au cours de mon parcours de thèse parfois tumultueux, j'ai eu la chance de faire de belles rencontres scientifiques et humaines.

Je souhaiterais remercier toutes ces personnes et espère n'oublier personne.

m'ont appris tellement… Je n'aurais pas pu être mieux encadrée qu'avec vous ! Merci aussi de m'avoir donné l'opportunité de faire des activités « extra-labos » comme les enseignements, la vulgarisation scientifique, le club jeunes SCF, toutes ces conférences auxquelles j'ai pu assister et présenter, et les sessions à l'ESRF. Ce n'est pas donné à tous les doctorants d'avoir autant d'opportunités et de liberté, et je vous remercie de m'avoir donné cette chance.

Fabrice, tout d'abord merci de m'avoir donné ma chance en me choisissant pour ce projet de thèse.

Merci pour ta disponibilité, tes précieux conseils et pour ton optimisme contagieux. Tu as toujours su partager ta passion pour la chimie mais aussi ta bonne humeur, même quand j'étais au fond du trou, notamment quand le peptide oxydé n'en faisait qu'à sa tête. Je te remercie également pour ta patience et ta pédagogie. Corriger mon premier article en « franglais » tout en faisant attention de ne pas me vexer n'a pas dû être une mince affaire !! Merci pour les longues discussions scientifiques, pour le temps que tu as passé avec moi quand les manips ne fonctionnaient pas (masse ou autre). J'ai passé des supers moments avec toi en conf' et à l'ESRF en mode 007 ! Christelle, je souhaiterais te remercier pour (i) la générosité et la passion avec laquelle tu partages ton savoir scientifique, (ii) ta disponibilité pour discuter de chimie ou de choses plus personnelles et ce, même dans les moments où tu as déjà accordé 110% de ton temps à l'ERC et 90% à l'équipe, (iii) avoir été une super coloc de bureau prête à dégainer du chocolat à tout moment et enfin (iv) ne pas m'avoir viré quand j'ai cassé ta voiture ! Je te remercie de m'avoir fait me sentir comme ton égale lors de nos longues conversations scientifiques pendant lesquelles j'ai pu développer mon sens critique et proposer toutes les idées (quelquefois farfelues) qui me venaient à l'esprit, sans peur du ridicule. Merci également de montrer à toutes les femmes de l'équipe qu'on peut être une super chercheuse et une super maman de 4 bambins en même temps ! Peter, tout d'abord je te remercie de m'avoir accueillie dans ton équipe. Merci pour tes précieux conseils, pour les discussions scientifiques qui m'ont beaucoup apporté mais aussi pour ton enthousiasme permanent. Un grand merci aussi pour la confiance que tu m'as accordée et pour ta disponibilité, même après ton départ pour Strasbourg. Ta passion pour les sciences est vraiment contagieuse, alors propage la autant que tu peux ! Cette thèse n'aurait pu se faire sans l'aide précieuse de :

-Emilien Jamin pour la HRMS, je te remercie pour tes précieux conseils et pour le temps que tu m'as accordé. Et désolée pour la poisse que j'emmenais (souvent) avec moi à l'orbitrap ! -Christian Bijani pour les spectres RMN, merci de ne pas t'être lassé de voir des RMN avec des pics super larges tout le temps !! Et merci d'avoir pris le temps de regarder la partie RMN du manuscrit.

-Lionel Rechignat pour la RPE, merci pour les conseils et pour la conversation sur la théorie de la RPE (toujours aussi obscure pour moi !!).

-Vanessa Soldan pour le TEM, je tiens à te remercier pour les précieux conseils et le temps que tu as passé avec moi à sonder l'échantillon à la recherche de fibres (ou d'absence de fibres !).

-Toute l'équipe FAME de l'ESRF. Merci à Isabelle Kieffer pour toutes les explications sur le XAS et pour la visite guidée de la nouvelle ligne. Merci à Denis Testemale pour sa disponibilité et sa patience quand la ligne plantait en notre présence (juré c'est pas de notre faute !) et pour les précieux conseils prodigués en somnambule à toute heure de la nuit !! -Stéphanie Sayen et Emmanuel Guillon lors des sessions à l'ESRF. Merci pour l'aide précieuse apportée, pour toutes les réponses à mes questions floues sur le XANES. Merci aussi d'avoir partagé les sessions blind-test et les bonbons avec moi ! -Petit Poney qui a subi nos expériences plus ou moins douteuses avec l'azote liquide. Merci pour les photos souvenirs ! Coté Pharma-DEV, je remercie Françoise Nepveu, Karine Reybier, Paul-Louis Fabre, Mohamed Haddad, Marieke Vansteelandt, Geneviève Bourdy ainsi que tous les autres membres des équipes Redstress et PEPS, avec qui j'ai eu la chance de travailler, de partager un repas ou bien un café.

Merci à Pierre pour sa gentillesse et sa bonne humeur quotidienne, ainsi que pour l'aide précieuse et les bons conseils qu'il m'a apporté. Merci également à Franck qui a toujours le sourire et qui m'a beaucoup aidé pour les questions administratives. Franck, ton punch est à tomber par terre ! Il me faut la recette ! Merci à tous les stagiaires, doctorants et post-doc de Pharma-DEV qui m'ont permis de travailler dans une super ambiance : Ennaji, Nambinina, Rémi, Thi Thu, Luyen, Solomiia, Filip, Marion, Lucie, Mireia, Laure-Estelle (LEC), Cynthia. Malgré le froid glacial l'hiver, j'ai adoré partager les repas du midi avec vous dans le « couloir-cantine ». Et j'aurais bien besoin de me remettre aux mots croisés du midi !! Un énormissime merci à LEC et Cynthia pour l'accueil très chaleureux que vous m'avez fait à mon arrivée au laboratoire, je me suis sentie bien tout de suite et c'est grâce à vous. Merci pour vos encouragements au quotidien, pour les discussions scientifiques (ou pas !) et pour tous les bons moments passés ensemble. Vous avez été mes modèles de réussite ! Coté LCC, je remercie toute l'équipe F qui m'a chaleureusement accueillie et m'a fait mourir de faim jusqu'à 13h30 pendant mes deux premières années de thèse ! Merci à Manu pour sa bonne humeur et ses super blagues, mais aussi pour les discussions autour des sciences, de l'administration (et des mouches !), de la musique et de l'oenologie ! Merci à Béatrice qui a toujours le sourire, pour sa gentillesse et ses conseils. Laurent, je te remercie de ne pas m'avoir détesté d'office quand j'ai cassé ta voiture ! Je suis très contente d'avoir pu te connaître ces derniers mois et je te remercie pour l'aide et le soutien que tu m'as apporté. Viviane, merci pour ton rire contagieux, ta bonne humeur quotidienne et ta gentillesse ! Je tiens également à remercier tous les étudiants / postdoc actuels ou anciens de l'équipe : Olivia, Hélène, Adam, Olena, Carine, Melisa, Daniel (bon aprèm les garces !), Mireia (mais c'est vrai ça ?!), Megan (so cute !), Gabriel (Mr Marcel), Rufus (c'est qui Clémence ?!), Sara (à ne pas confondre avec Sara !), Marie, Alex (petit puits), Omar (Ahomalll), Elena (a fare l'amore, encore merci pour la chanson !), Valentina (la mammmmmmmmma) et Amandine (ACDC <3). Merci pour la super ambiance que vous apportez dans l'équipe, pour les discussions scientifiques qui permettent de faire avancer nos manips qui rament, mais aussi un grand merci pour les nombreux moments passés ensemble à l'extérieur du labo, les soirées mojitos / danses tahitiennes à La Plage… Le labo a été un super endroit pour se faire des amis ! Mes remerciements s'adressent ensuite à toute l'équipe pédagogique Chimie pharmaceutique aux cotés desquels j'ai eu le plaisir de découvrir l'enseignement pendant mes deux années de DCE : Geneviève Baziard, Salomé El Hage, Barbora Lajoie, Jean-Luc Stigliani, Fatima El Garah, Christelle Recoche-Gueriot et Laurent Amielet. J'ai beaucoup appris auprès de vous et je tiens à vous remercier pour votre accueil et votre bienveillance.

Je tiens à remercier tout le bureau jeune de la SCF Midi-Py avec qui j'ai eu la chance d'animer des ateliers scientifiques et de monter des projets sympas : Claudia, Cécile, Alix, Morgane, Jérémy, Stéphane, et tous les étudiants de l'équipe F. Les sessions Chimie & Terroir vont me manquer, mais pas les ventes des kits chimistes ! Merci aussi à Lydie Valade et plus largement à Chimie et Société de m'avoir fait partager leur passion pour la vulgarisation scientifique.

Je remercie les organisatrices des fameux et très sélects « Zumbapéro » qui m'ont permis de garder la forme pendant ces années de dur labeur ! Julie, merci d'avoir toujours la pêche et d'avoir été là pour les sessions papotage nécessaires à la survie de la thèse ! Amandine, je ne sais même pas quoi dire… Merci d'avoir été là pour moi pendant les bons mais aussi les mauvais moments, de m'avoir fait rire à en pleurer, d'avoir fait mon copilote pendant les missions… et tant d'autres choses ! Bref, merci d'être toi !! Merci aussi à Laurent de nous avoir supporté toutes les deux, je sais qu'on peut être intenables parfois !! Léa, mon petit, merci d'être là pour moi, avec ta pêche d'enfer et ton hyperactivité !! Merci aussi à ma Flow qui est dans un pays lointain (une fois !). Votre amitié m'a beaucoup aidé à traverser cette thèse.

Merci à mes deux petites angevines Noémie et Marine que j'ai le plaisir de revoir quand je remonte dans le grand nord ! Merci d'être toujours là pour moi après toutes ces années… Pierre, cette thèse est aussi la tienne. Je n'aurais jamais pu aller au bout sans toi. Merci d'avoir été là pendant toutes ces années, de m'avoir soutenu, nourri (j'ai fait des envieux au labo avec mes gamelles du midi !!), remis sur les rails quand je perdais toute motivation. Tu as été d'une patience à toute épreuve et un vrai roc pour moi.

Enfin, un grand merci à toute ma famille qui m'a toujours soutenu pendant mes longues années d'études et m'a encouragé dans les moments de doute. Je sais que je ne suis pas toujours facile à suivre quand je parle de mon travail, c'est comme si je vous parlais chinois, mais vous êtes toujours intéressés et ça me fait très plaisir. Bref, tout ça pour vous dire : 我愛你 (et cette fois-ci c'est vraiment du chinois, pas du langage de chimiste !). Orbitrap [7] .. [10] [19] Hirnrinde" ("On an unusual Illness of the Cerebral Cortex") the uncommon case of a 51-yearold patient who was suffering from memory loss, disorientation, hallucinations and cognitive impairment. After the death of the patient, the post-mortem examination showed an atrophic brain with "striking changes of the neurofibrils" and "minute military foci" caused by the "deposition of a special substance in the cortex". [1] One century later, this "unusual illness" named Alzheimer's Disease (AD) has become the most widespread neurodegenerative disease whose etiology is still unknown. [2] I.A.1. Prevalence

Table of Contents

II.D.1. General principles

II.G.1. X-Ray absorption: general principles

General introduction

General introduction

According to the World Alzheimer Report, [3] 46.8 million people were suffering from dementia worldwide in 2015 and this number is expected to almost double every 20 years.

Approximately 5% -8% of individuals over age 65, 15% -20% of individuals over age 75, and 25% -50% of individuals over age 85 are affected by dementia. [4] Alzheimer's disease is the most common form of dementia, accounting for 50% -75% of all dementias. [4] Figure I.A-1: Estimated number of people suffering from dementia in each continent in 2015. [3] I.A.2. Clinical signs AD is characterized by a progressive deterioration of cognitive functions and progresses in three stages: mild, moderate and severe. [5] At the early-stage of AD, the patient can have memory lapse, difficulties to complete familiar tasks and be confused with time or place. New problems with words in speaking or writing can arise, and the possible changes in mood can be misinterpreted as depression.

At the middle-stage, the symptoms become stronger, the patient can get frustrated or angry and the personality and behavior are impacted. The memory troubles worsen: the patient can have forgotten events or parts of his personal history.

At the severe stage, the patient requires around-the-clock assistance. He loses the ability to carry on a conversation and to control movement and can be bedbound. Individual is then vulnerable to infections (especially pneumonia) and the cause of death is usually external to the disease.

I.A.3. Histopathological signs a. Brain size [6] In AD, the volume of the brain is significantly reduced compared to normal brain

(Figure I.A-2)
. This atrophy results from the degeneration of synapses and the death of neurons.

Hippocampus, the brain region playing a role in memory and spatial orientation, is particularly affected. The reduction of brain size and the progression of AD are related.

Figure I.A-2: Neuroanatomical comparison of normal brain (A, B) and AD brain (C, D). Prominent atrophy in C compared with A (arrows). B, D: Coronal plane of A and C respectively. Arrow on D shows

an enlargement of the ventricles and a selective hippocampal atrophy. [7] b. Amyloid plaques

The first hallmarks of AD described by Aloïs Alzheimer in 1907 [1] as "minute military foci" caused by the "deposition of a special substance in the cortex" are amyloid plaques (Figure I.A-3a). They are composed of deposits of a peptide named Amyloid-β peptide (Aβ) in aggregated forms [8] mostly fibrils. Those plaques, also named senile plaques, are found in the extracellular media of AD brain and more especially located in the hippocampus region. Aβ is mainly a 40 to 42-amino acid residues peptide originating from the cleavage of Amyloid

Precursor Protein (APP) by two enzymes: β-and γ-secretases (see Section I.B for more details).

c. Neurofibrillary tangles

The other hallmarks of the disease are intracellular neurofibrillary tangles (Figure

I.A-3b
). Those tangles are also observed in Parkinson's disease (PD) [9] and are composed of hyper-phosphorylated Tau proteins. [10] This microtubule-associated protein interacts with tubulin to stabilize microtubules. In AD and PD, the abnormal phosphorylation of Tau induces accumulation as paired helical filaments that aggregate inside neurons in neurofibrillary tangles, making unstable the microtubules. Since microtubules are essential to preserve the structure of the neuron, the neuron loses its functionality. of developing the disease reaches 50% for individuals beyond age 85. [4] As the life expectancy increases with the medicine advances, more and more people are likely to develop AD.

b. Gender

Women are known to have a higher life expectancy than men, thus being more susceptible to suffer from AD. Furthermore, studies suggest that the decrease in estrogen levels due to menopause could increase the risk of having AD. Indeed, clinical trials have shown that women who had been treated with hormone therapy have a lower risk of AD. [12] c. Genetic mutations There are two major forms of AD: the sporadic or late-onset form that is the most common, and the familial or early-onset form, representing less than 5 % of the cases. [13] Individuals living with Down's syndrome (also called trisomy 21) have an increased risk of early-onset AD. Indeed, they carry an extra copy of chromosome 21 in which is located the gene that is responsible for the APP formation. [14] Mutations in genes coding for APP, Presenilin 1 and Presenilin 2 (parts of the γsecretase which is responsible for cleavage of APP in Aβ) and ApoE (involved in Aβ clearance)

genes increase the risk of developing AD as these proteins are involved in Aβ regulation in the brain. [13,15] I.A.

Diagnosis

The diagnosis of AD is realized at 70% accuracy with clinical examination and neuropsychological assessment of the patient combined with brain imaging techniques. [7] However, the definitive diagnosis of AD requires histopathologic confirmation and is made post-mortem, based on the observation of specific pathological lesions: intracellular neurofibrillary tangles and senile plaques. [11] a proposed diagnostic criteria for dementia and for AD. [16] Medical history, clinical examination, neuropsychological testing and laboratory assessment are the recommended standard methods of examination. Criteria are made for several stages: probable AD dementia, possible AD dementia and probable or possible AD dementia with evidence of the AD pathophysiological process. The diagnostic criteria have been revised in 2011 [17] and are still used as no specific marker for AD is known. However, they are realized in combination with brain imaging techniques.

b. Neuroimaging [18] Structural magnetic resonance imaging (MRI) and positron emission tomography (PET) are two brain imaging techniques clinically used for the detection of abnormalities in the brain. [19] Structural MRI is commonly used to visualize brain atrophy caused by neuronal and dendritic losses. However, the cerebral atrophy is not specific to AD and can be the result of another pathology. Structural MRI has thus limitations in AD recognition as it cannot detect the histopathological hallmarks of AD (amyloid plaques and neurofibrillary tangles).

Fluoro-deoxy-D-glucose (FDG) PET is used as an indicator of brain metabolism by measuring the synaptic activity. Nevertheless, as metabolism can be disrupted for different reasons, FDG is not a specific marker for AD either.

Specific biomarkers of AD hallmarks are now under focus. Recently, the European Medicines Agency granted marketing authorization for the Florbetapir F18 ( 18 F-AV-45) (also called Amyvid), a PET marker with a good affinity for Aβ plaques (Kd = 3.7 nM [20] ). This active substance can be used to detect amyloid plaques on living patient. [20][21] This is a great advance for AD diagnosis.

I.A.6. Current treatments

There is currently no cure for AD, only symptomatic treatments. The U.S Food and Drug Administration (FDA) has approved four medications that are marketed in France and classified in two groups: acetylcholinesterase inhibitors (Donepezil, Rivastigmine and Galantamine) and N-Methyl-D-Aspartate (NMDA) receptor antagonists (Memantine).

Apart from amyloid plaques and neurofibrillary tangles, AD is characterized by a deficit of acetylcholine, a neurotransmitter that diffuses signal across the synapse, between two neurons. Acetylcholinesterase inhibitors avoid acetylcholine degradation by inhibiting acetylcholinesterase, an enzyme that catalyzes the breakdown of acetylcholine. The level of acetylcholine thus remains stable, allowing neurotransmission.

An over-concentration of glutamate is also observed in synaptic clefts of AD patients.

This molecule is an excitatory neurotransmitter that plays a role in neural activation but it can also lead to neuronal death if physiologically over-concentrated. NMDA receptor antagonists block the glutamate receptors to avoid the loss of neurons.

These symptomatic treatments only slow down the cognitive deterioration and have no effect on patients in the severe stage of AD. Researchers particularly focus on amyloid plaques and neurofibrillary tangles to find new therapeutic pathways that could prevent or stop the progression of the disease. [22] I.B. Aβ and the amyloid plaques formation APP is a type-1 trans-membrane protein expressed in various tissues of the organism, especially in the central nervous system (CNS). [23] Its major neuronal isoform encompasses 695 amino acid residues. [24] Although its physiological function is still unclear, APP would play an important role in brain development, memory and synaptic plasticity. [24] Two different pathways of APP metabolism can occur, as shown in Thus, Aβ peptides are the product of a minor pathway of APP metabolism, [26] released in the extracellular space of healthy brain during neuronal activity, without leading necessarily to Alzheimer's pathology. Aβ is subject to a proteolytic degradation by Aβ-degrading proteases (AβDPs), which regulates Aβ levels in the brain. [27] Its functions in the brain are still unknown, although Aβ could play a role in the synaptic plasticity and the memory. [28]

I.B.2. APP and Aβ mutations

Familial AD are caused by mutations on a gene of APP, Presenilin 1 (PSEN1) or Presenilin 2 (PSEN2). PSEN1 and PSEN2 are two subunits of γ-secretase. The mutations on both PSEN1 and PSEN2 lead to a higher Aβ production, PSEN1 mutations specifically conducting to an increased Aβ1-42 formation. [13] For APP, 65 mutations are indexed in the Alzheimer Disease & Frontotemporal Dementia Mutation Database, with only 15 being non-pathogenic. [29] The mutations are divided in three categories: mutations at the β-secretase cleavage site, at the γ-secretase cleavage site and in the mild-domain amyloid-β region. [30] The mutations at the γ-secretase cleavage site can alter the cleavage position and lead to an increase of the Aβ1-42/Aβ1-40 ratio. The mutations at the β-secretase cleavage site increase the rate of APP proteolysis by the β-secretase. The mutations in the mild-domain of Aβ region in APP alters Aβ assembly by increasing the propensity of Aβ to form oligomers and fibrils. [31] As APP mutations can occur in the Aβ (1-letter code). [13] I.B.3. Amyloid cascade hypothesis AD is a multifactorial disease and the multiple mechanisms related to the disease are unclear. However, since Aβ has been found in healthy brain on soluble form but on aggregated form in AD patient's brain, [8] a hypothesis has been proposed to explain the formation of senile plaques composed of aggregated Aβ. The amyloid cascade hypothesis (Figure I.B-4) formulated in the early 1990s [32][33][34][35] has become the dominant model for AD pathogenesis, [36] although still controversial. [37][38] It is proposed that an abnormal extracellular increase of Aβ levels in brain could lead to its aggregation in β-sheet rich structures. [39] The aggregation starts with the formation of oligomers species that are reorganized into protofibrils and fibrils, which are found in amyloid plaques, a hallmark of AD. In particular, oligomers accumulated in AD patient brains [40] are proposed to be the more toxic species for cells [41][42] as they can permeabilize cellular membranes, thus initiating a series of events leading to cell dysfunction and death. [43] According to this hypothesis, the others events such as the intracellular formation of neurofibrillary tangles and the disruption of synaptic functions would ensue from this early and key event. Metal ions such as zinc, iron and copper ions have been found in amyloid plaques. [44] In addition, Cu and Zn are exchanged within the synaptic cleft of some neurons. They are supposed to play an important role in the aggregation according to the amyloid cascade hypothesis. [45] Actually, metal ions can bind Aβ and thus modulate the aggregation process.

They act either on the kinetics or on the thermodynamics by impacting the morphology of the formed aggregates. [46] Furthermore, amyloid aggregates with entrapped redox-active metal ions such as copper ions are considered as more toxic since they can produce Reactive Oxygen Species (ROS), deleterious for the biomolecules. [47] I.B.4. Aggregation

Aβ is a natively unfolded peptide with no defined 3D structure. As this peptide is highly flexible and unstructured, it can easily undergo aggregation to amorphous or ordered structures (Figure I.B-5, green and blue pathways respectively), the β-sheet rich structures being thermodynamically the most stable. [48][49] The fibrils formed during the aggregation process are organized in stacked parallel or anti-parallel β-sheets structures. In Aβ40 fibrils, the 12-24 and 30-40 residues would be responsible for β-sheet formation. [50] The aggregation process of Aβ in β-sheet structures is dynamic and complex, consisting in multiple self-assembly steps. Two different steps are observed over time: nucleation and elongation. . Picture from reference [46] .

During nucleation, the unstructured monomers in solution cluster in small aggregates called oligomers that further form nuclei (red pathway in Figure I.B-5). Nucleation is the slower and limiting step of aggregation since association of monomers that occurs during nucleation is not thermodynamically favorable. [46] The nucleation phase is then followed by a rapid elongation phase in which protofibrils and finally fibrils are formed from nuclei (orange pathway in Figure

I.B-5).
Several techniques have been developed to monitor Aβ aggregation. [46,51] Among them, fluorescence of Thioflavin T (ThT) dye is widely used. ThT interacts with β-sheet and upon interaction undergoes a strong fluorescence enhancement. [52] This allows to monitor the aggregation kinetics. Atomic force microscopy (AFM) and transmission electron microscopy Metal ions such as zinc, iron and copper are present in the brain. They are necessary and required to regulate the neuronal activity in the synapses and involved in biological functions of metallo-proteins. In several diseases such as AD, the metal ion homeostasis is disrupted and the concentration is very far from the physiological one, with Cu and Zn levels that can reach up to three times the control levels. [53] Moreover, high content of these metal ions is found in amyloid plaques extracted from AD brains. [44] In addition, such ions can bind to Aβ under physiological concentrations. Thus, knowing the coordination mode of these metal ions with

Aβ is a pre-requisite to understand their role in AD.

a. Zn(II) coordination to the Aβ peptide Zn ion exists only as Zn(II) and its coordination to Aβ is still not well-established. [54][55] Although it is consensual that a complex 1:1 is formed, [55] the nature of the amino acid residues involved in the coordination sphere is still under debate. A novel binding model has been recently proposed, based on Nuclear Magnetic Resonance (NMR) and X-ray Absorption Spectroscopy (XAS) studies of Zn coordination with mutated and N-terminal acetylated peptides (Figure I.C-1). [56] In this model, Zn(II) would be bound by imidazole rings of His6 and either His13 or His14 residues, the carboxylate group of Glu11 and the carboxylate group of Asp1, Glu3 or Asp7. b. Cu(II) coordination to the Aβ peptide

The Cu(II) coordination to Aβ has been widely studied for years and is challenging as several species are formed depending on the pH. Numerous studies have been realized in the past decade and the results have been recently reviewed, [54,[57][58][59] leading to a consensual model with different Cu(II) binding modes depending on the pH. The four binding modes observed for pH values higher than 6. His14. [60][61][62][63][64] For component II, two distinct models have been proposed. In the first one, Cu(II) is bound via the carbonyl function from Ala2-Glu3 and the imidazole rings of the three His. [62,64] In the second one, Cu(II) is bound to the N-terminal amine of Asp1, the amidyl function of Asp1-Ala2, the carboxylate group of Ala2 and the imidazole ring of one His. [60][61]65] The first model does not explain the effect of pH on the coordination as all the residues involved in Cu(II) coordination that can undergo deprotonation are already deprotonated. The second model explains the change of Cu(II) binding mode that occurs around pH 7.8 with the deprotonation of the Asp1-Ala2 amide function, leading to its coordination. Furthermore, Electron Nuclear Double Resonance (ENDOR), Hyperfine Sublevel Correlation (HYSCORE) and NMR studies highlight the involvement of both the NH2 terminus of Asp1 and the deprotonated Asp1-Ala2 amide bond, favoring the second model. [57] Thus, the second proposed model (illustrated in The other two components (called III and IV) are formed at higher pH with the deprotonation of the Ala2-Glu3 and Glu3-Phe4 amide functions, respectively. [65] Cu(II) is bound via the -NH2 terminus, the two amidyl functions between Asp1 and Glu3 and one His residue in component III and via the NH2 terminus and the three amidyl functions between Asp1 and Phe4 in component IV.

A carboxylate group has also been proposed to be involved in apical position for several components, coming from Asp1 [60][61][62] or from Glu3, Asp7 and Glu11 carboxylates in equilibrium with Asp1 for component I. [61] c. Adapted from reference [66] .

Model A proposes a linear binding of histidines with a dynamic exchange between His6, His13 and His14. Model B involves an equilibrium between the His dyad and the His triad.

NMR studies have shown the implication of the three histidines in the Cu(I) coordination with a dynamic exchange, in line with the two proposed models. [66] However, XAS studies [66][67] and a comparison of synthetized Cu(I) complexes HisHis dipeptides and Cu(I) complexes with truncated Aβ6-14 and Aβ10-14 peptides [68][69] highlight a linear binding mode with 2 histidines, corroborating the model A.

In addition, according to a tandem mass spectrometry (MS/MS) study on Cu(I)-Aβ structure, the two histidines mostly involved in Cu(I) coordination would be His13 and His14 [70] . Thus, evidences suggest that Aβ is bound to Cu(I) by histidine residues in a linear fashion with a dynamic exchange between His6, His13 and His14, the major form being His13 and His14 dyad. This is in line with affinity studies realized on three Cu(I) complexes with one His-Ala mutation on Aβ peptide (named H6A, H13A and H14A) [71][72][73] that point out to a slightly lower affinity than for the native peptide, H6A having a stronger affinity than the other two mutants. These results indicate that Aβ only needs two histidines for binding Cu(I), His13-His14 dyad being the major form. 4). They are necessary to maintain the homeostasis in cells and play an important role in signaling [74] but are also reactive oxidants, able to damage biomolecules. In cells, endogenous enzymes are in charge of the antioxidant defense to prevent the ROS mediated damages. [47] The superoxide (O2 • ) anion, the first ROS produced by the one-electron reduction of dioxygen, is capable of inactivating few enzymes, [47] but has a poor reactivity with most of the bio-inorganic substrates due to low rate constant (usually below 10 2 L mol -1 s -1 ). [START_REF] Gardès-Albert | Aspects physicochimiques des radicaux libres centrés sur l'oxygène[END_REF][START_REF] Bielski | [END_REF] To remove a potential excess of O2 a diffusion rate close to the limit (k around 10 9 L mol -1 s -1 ). [START_REF] Bielski | [END_REF][77]

). [78] The hydroxyl radical (HO • ) is the result of the third one-electron reduction of oxygen.

It can also be produced in the presence of metal ions from H2O2 or H2O2 and O2 • by the Fenton reaction or the Haber-Weiss reaction respectively (Figure I.C-6). HO • has a very short half-life (10 -9 s) compared with O2 • (10 -6 s) and is thus the more reactive and deleterious ROS, [74] being able to oxidize the biomolecules such as proteins, lipids, DNA [START_REF] Dorfman | Reactivity of the hydroxyl radical in aqueous solutions[END_REF] because of its very high redox potential (E°'=2.34 V [START_REF] Wardman | [END_REF] ). To control the quantity of pro-oxidants (ROS) and prevent the damages on the biomolecules, the body has protecting mechanisms including enzymatic and chemical antioxidants. However, in some diseases such as AD [81] , an imbalance may occur between prooxidants and antioxidants, due to a higher ROS production or a reduced activity of the enzymes responsible for the ROS degradation, leading to oxidative damages on biomolecules. [82] b. Metal-catalyzed ROS production Redox active metal ions such as copper and iron are involved in the ROS production via Fenton and Haber-Weiss reactions (Figure I.C-6). In the presence of a reducing agent, they can have a catalytic activity. [83] In AD, Cu and Fe can be coordinated to Aβ and the resulting complex could be directly involved in the ROS production. ROS production has been mostly studied with Cu-Aβ, as Fe-Aβ has a lower redox activity. [84] Iron is found in the amyloid plaques predominantly in a colloidal form (originating from ferritin), however histochemical studies indicate that it could also be bound to Aβ. [85] The coordination mode of Fe(II) with Aβ has been characterized [86] and Fe(III) does not form a stable complex with Aβ because it finally converts into Fe(III)(HO)3 and precipitates. Thus, the physiological stable formation of Fe(III)-Aβ is unlikely. However, ROS production by Fe-Aβ still might be relevant as Fe(II)-Aβ is stable and the Fe(III) complex formed during ROS production might not have time to precipitate. As the involvement of iron bound to Aβ in ROS production is still unclear, we focus here only on Cu-Aβ.

In the case of copper, the pro-oxidant role of the Cu-Aβ system is not clearly established as the complex is more active in ROS production than several biological relevant Cu-peptides or Cu-proteins [87] but less efficient than loosely-bound copper. [84,[87][88][89][90][91] However, in vitro studies have shown that Cu-Aβ is able to catalyze the formation of H2O2 and HO • in the presence of O2 and a reducing agent such as ascorbate (Figure I.C-7). [84,[87][88]92] Moreover, although it was generally proposed that H2O2 production by Cu-Aβ occurs via a two-electron process, a recent study has highlighted the formation of superoxide as an intermediate in the production of H2O2 by Cu-Aβ and O2. [93] The energy required for the rearrangement between the Cu(I) and Cu(II) geometries (linear and square-planar respectively) being very high, the electron transfer would rather proceed via a low-populated redox-active state in which Cu(I) and Cu(II) binding modes are highly similar, thus inducing a low reorganization energy. This transient state, called "inbetween" state, is in equilibrium with the resting states (Figure I.C-8, bottom section). It has been studied by calculations [96] and characterized with MS/MS by identifying the sites of oxidative damage on the peptide. [95] By comparing the non-specific oxidations detected on Aβ28 after the radiation-induced ROS production with the copper-mediated oxidations of Aβ28, Asp1, His 13 and His14 have been found to be the metal-specific targeted amino acid residues.

Furthermore, kinetic studies of the copper-mediated Aβ28 oxidation have shown that Asp1 would be the first amino acid residues damaged. Thus, in this study, the proposed ligands for both Cu(II) and Cu(I) coordination in the in-between state are Asp1, His 13 and His14. As they have been found to be the main targets for HO • , they are supposed to be the amino acid residues the closest from copper during the metal-catalyzed ROS production.

I.C.3. Metal-catalyzed oxidation of Aβ

During the metal-catalyzed ROS production, the Aβ peptide undergoes oxidative damages. This is in line with the detection of oxidized Aβ in amyloid plaques in vivo. [97] Studies on single amino acid residue oxidations could allow a prediction on the residues targeted during the metal-catalyzed oxidation (MCO) of Aβ. [98][99][100] The physiological main targets for HO • are the sulfur-containing amino acids (methionine, cysteine), the basic amino acids (arginine, histidine, lysine) and the aromatic amino acids (phenylalanine, tyrosine, tryptophan). [START_REF] Bonnefont-Rousselot | Radicaux libres et stress oxydant : aspects biologiques et pathologiques[END_REF] Table I.C-1 provides the main oxidation products of these amino acid residues.

Oxidation of Aβ28 by HO • produced by γ-radiolysis has shown that His and Phe residues are mainly targeted, [95] in line with the oxidations reported previously for free amino acid residues.

However, in the case of MCO of Aβ, the ROS are produced at the metal center. Thus, the oxidations are site-specific and can differ from the amino acid oxidations usually detected without metal.

Table I.C-1: Main oxidation products of the principal amino acid residues undergoing HO • attack. [START_REF] Bonnefont-Rousselot | Radicaux libres et stress oxydant : aspects biologiques et pathologiques[END_REF] Amino [88,95,[START_REF] Schöneich | [END_REF][103] or hydrogen peroxide. [104] found to be more sensitive to oxidation, His6 being not detected on its oxidized form [95,[103][104] or affected after longer oxidation time. [START_REF] Schöneich | [END_REF] This is in line with the predominant binding mode of Cu(I) to His13 and His14 (see section I.C.1.c). b. Aspartate

The Aβ peptide has 3 aspartates residues at positions 1, 7 and 23. In the literature, only Asp1 has been found to be oxidized. Actually, as Asp1 is involved in the coordination of Cu(II), [57][58] it is a preferential target for the hydroxyl radical produced at the metal center.

Different damages have been detected during MCO of Asp1 both in the presence of ascorbate [95,106] and of hydrogen peroxide [104] . The oxidative decarboxylation and deamination of Asp1 leads to the formation of a pyruvate function (Figure I.C-12, blue pathway). [95,104,106] Asp1 is also subject to a backbone cleavage on the α-position of the peptide, leading to an isocyanate function (Figure I.C-12, red pathway). [95,106] Another oxidation of Asp1 into 2-hydroxyaspartate corresponding to the formal addition of an oxygen atom has also been described (Figure I.C-12, green pathway). [95] c. Tyrosine Although the amino acid residues involved in copper coordination are more vulnerable to oxidation, non-coordinating amino acid residues can also be oxidized. It is the case for Tyr10 which is sensitive to oxidation and is responsible for the Aβ peptide cross-linking by dityrosine formation (Figure I.C-13). [98] This latter, induced by Cu(II), has been detected for Aβ in the presence of H2O2. [107] MCO of Tyr10 into dityrosine was found to have an impact on aggregation as Aβ cross-linking was correlated with the formation of covalent oligomers. [108- 109] Furthermore, a study has proposed that Tyr10 acts as a gate that promotes the electron transfer from Met35 to Cu(II) for its reduction in Cu(I). [110] Figure I.C-13: Tyrosine cross-linking mechanism leading to the formation of dityrosine [111] d. Phenylalanines Three phenylalanines are present in the Aβ sequence at positions 4, 19 and 20. None of them are involved in the Cu(II) or Cu(I) coordination, nevertheless Phe19 and Phe20 have been found oxidized during MCO of Aβ in the presence of Cu(II) and ascorbate. [95] Phe19 and Phe20 has been detected with the formal addition of an oxygen atom, likely oxidized into hydroxyphenylalanine (Figure I.C-14). [98] This oxidation seems to occur after the oxidation of Asp1 which is involved in Cu binding. [95] Figure I.C-14: Structural formula of phenylalanine and the three hydroxyphenylalanines.

e. Methionine

Methionine is an amino acid residue very sensitive to oxidation. In vivo, the enzyme methionine sulfoxide reductase is responsible for the reduction of the methionine sulfoxide (Figure I.C-15), a main oxidized form of the methionine. [112] Methionine can also be converted into sulfuranyl / hydroxysulfuranyl radical cation by a one-electron oxidation. [113]

Figure I.C-15: Structural formula of methionine (left) and methionine sulfoxide (right).

Reviews have reported about oxidation of the methionine of the Aβ peptide located at position 35 and its role in toxicity and oxidative stress. [114][115] Although methionine is very sensitive to oxidation, its conversion into methionine sulfoxide occurs only after the oxidation of His13 and His14 during the in vitro MCO of Aβ in the presence of Cu(II)/ascorbate. [START_REF] Schöneich | [END_REF] This highlights the site-specificity of the amino acid residue oxidation catalyzed by the bound copper.

Met35 has also been found to promote Tyr10 oxidation [116] and to interact with Gly33, inducing its peroxidation by promoting the formation of a carbon-centered radical, leading to a hydroperoxide. [90,117] f. Other cleavages Other oxidative cleavages have been reported for Aβ bound to Cu(II) in the presence of H2O2 such as the cleavage of the peptide bond of Asp1/Ala2, Ala2/Glu3, Val12/His13 or His13/His14. [104]

Chapter II: Methodologies

This chapter is a summary of the experimental conditions and techniques implemented for the studies presented throughout the manuscript. For each spectroscopic technique, the general principles are defined and the application of the technique to our study is described as well as the corresponding experimental conditions.

II.A. Preparation of the Aβ peptide II.A.1. Solubilisation and monomerization

All the peptides are commercially available, in the form of powder (purchased from Genecust, Luxembourg, purity grade >95%). The shorter Aβ16 and Aβ28 peptides are weighed and solubilized in water, resulting in an acidic solution due to the presence of trifluoroacetate as counterion in the powder (pH 2). For the full-length peptide Aβ40 which is subject to aggregation, the solution is prepared freshly. The powder is solubilized in NaOH 50 mM, resulting in a basic solution (pH 13) to remove preformed aggregates and slow down the aggregation, and further purified via Fast Protein Liquid Chromatography (FPLC) when used for aggregation studies (see Section II.A.4). [1] Usually, the Aβ peptide powder commercially available contains an unknown quantity of counterions (such as trifluoroacetate) and the dosage by weight is not precise. Thus, the dosage is performed by UV-Visible spectroscopy by using the absorption of Tyr10. The pH of each solution was taken into account as deprotonation of Tyr at high pH induces a change in the UV spectra obtained (Figure II.A-1).

II.A.2. Dosage

For the UV-Visible dosage, the Tyr10 is considered as a free tyrosine amino acid residue. As the peptide can aggregate, a little light scattering can occur and has to be taken into account. Thus, to avoid an overestimation of the concentration, a correction is made by subtracting the background absorbance to the maximum of absorbance of Tyr.

Figure II.A-1: pH titration of Tyr in Aβ16 (0.3 mM). UV-vis absorption spectrum from tyrosine band (red)

to tyrosinate band (blue). Picture from reference [1] .

At low pH (until pH 8.5 according to Figure II.A-1), Tyr10 in the Aβ peptide has a maximum of absorbance at 276 nm and is corrected with the absorbance at 296 nm with an extinction coefficient of ε276-ε296=1410 M -1 .cm -1 . [1] At higher pH, the dosage has to be realized around pH 13, where only the deprotonated form of Tyr is present. Tyrosinate has a maximum of absorbance at 293 nm and is corrected by the absorbance at 360 nm with an extinction coefficient of ε293-ε360=2400 M -1 .cm -1 . [1] For mutant or shorter peptides without Tyr (i.e. Y10F-Aβ or Aβ1-7), the dosage was performed via phenylalanine absorption (ε258-ε280=195 M -1 .cm -1 for one Phe). [2] More details about UV-visible spectroscopy are given in Section II.C.1.

II.A.3. Oxidation and purification of Aβ

The metal-catalyzed oxidation of Aβ is carried out at 60 µM of Aβ28 or Aβ40 with 50 µM of CuSO4 and 0.5 mM of ascorbate in 50 mM of phosphate buffer at pH 7.4, under air atmosphere. The reaction occurs during 30 min under stirring and is completed when the ascorbate has fully reacted. Then, the solution is concentrated by centrifugation with Amicon A mass spectrometer is composed of:

-An ion source which produces gas-phase ions from the molecules of the studied sample -A mass analyzer whose goal is to sort the ions based on their m/z value ratio.

-A detector that provides an electric signal related to the number of detected ions.

-A data processing system (IT) to obtain a mass spectrum with the intensity given as a function of m/z.

All the results presented in this manuscript were obtained on mass spectrometers equipped with electrospray ionization (ESI) and ion trap.

II.B.2. Electrospray Ionization (ESI)

The electrospray ionization (ESI) is an atmospheric pressure ionization process developed by J.B. Fenn (Nobel Prize in Chemistry 2002) in 1984. [3] ESI is considered as a soft ionization technique as little fragmentations are obtained in the source. Its main advantage is to directly generate ions in gaseous phase from solution, under atmospheric pressure. This The liquid sample is infused at low flow rate (usually 1 to 100 µL.min -1 ) through a capillary subjected to a high electric field, the latter being obtained by the application of a voltage difference (3.0 to 6.0 kV) between the capillary and a surrounding counter-electrode. [4] A constant flow of inert gas (nebulizer gas, usually N2) move along the tube containing the capillary. At the end of the capillary, a spray is formed via the Tailor cone originated from the high voltage applied on the capillary. The sample is turned into charged micro-droplets in suspension. Those droplets are moving towards the counter-electrode and the solvent of the droplets evaporate, leading to smaller droplets. Afterwards, the charge density of the droplet becomes too high and the gas-phase ions are released during the Coulomb-explosion. The ions produced are then transported to the analyzer.

In ESI, the ions obtained can be cations or anions, generated by protonation or deprotonation of the molecules, respectively. As explained above, for molecules with several ) and H. Steinwedel [5] described an ion trap which has been used for the mass spectrometry for the first time in 1984 by Stafford and his colleagues. [6] This analyzer selects ions in the ion flow arriving from the ion source and send The ion trap also allows to isolate specific ions, to fragment them and then to detect the fragment ions. This mode of operation is called tandem mass spectrometry (MS/MS or MS 2 ).

Because fragment ions are trapped prior to be detected, another possibility is to isolate one of them and to fragment it: in this case, the mode of operation is MS/MS/MS or MS 3 . This can be After its accumulation and isolation in the ion trap, the ion is excited: a kinetic energy of the order of a few tens of eV is provided to make it resonate. With this energy, the ion collides with neutral atoms (helium) present in the trap and acquires an internal energy, resulting in bond breakage and leading to its fragmentation in smaller fragment ions. This type of fragmentation is known as Collision Induced Dissociation (CID).

b. Orbitrap [7] The orbitrap, described in 2000 by Makarov [8] ), according to the nomenclature of Biemann. [9] Figure II.B-5: Schematic view of the possible fragmentations undergone by a polypeptide chain. [9] In the ion trap we used for our studies, the fragmentation occurs by CID leading to the

LC-MS and LC-MS/MS conditions

High Performance Liquid Chromatography / Mass Spectrometry (LC/MS) analysis was performed on an ion-trap mass spectrometer (LCQ DECA XP Max, ThermoFisher), equipped with an electrospray ionization source, coupled to an Ultimate 3000 LC System (Dionex, Voisins-le-Bretonneux, France). Samples (10 µL) at 60 µM of Aβ were injected onto the column (Acclaim 120 C18, 50 × 3 mm, 3 µm, ThermoScientific), at room temperature. The gradient elution was carried out with formic acid 0.1% (mobile phase A) and acetonitrile/water (80/20 v/v) formic acid 0.1% (mobile phase B) at a flow-rate of 0.5 mL.min -1 . The mobile phase gradient was programmed with the following time course: 5% mobile phase B at 0 min, held 3 minutes, linear increase to 55% B at 8 min, linear increase to 100% of B at 9 min, held 2 min, linear decrease to 5% B at 12 min and held 3 min. The mass spectrometer was used as a detector, working in the full scan positive mode between 150 and 2000 Da followed by data dependent scans of the two first most intense ions, with dynamic exclusion enabled. Isolation width was set at 1 Da and collision energy at 28% (units as given by the manufacturer), using wideband activation. The generated tandem MS data was searched using the SEQUEST algorithm against the human Aβ peptide sequence.

Conditions of tryptic digestion

The 100 µL solution of Aβ was filtered by using Amicon HRMS is a powerful tool to characterize molecules and biomolecules as it provides the exact mass of the ion related to them, thus allowing to differentiate two molecules of close masses. In our studies, HRMS was used: (i) to identify some of the oxidized amino acid residues of Aβ, (ii) to get the chromatographic traces of some specific ions of Aβ and associated peptides, thus allowing to perform relative quantification. HRMS was also used to check for digestion efficiency, systematically found close to 100 % (non-digested peptide not detected).

LC-HRMS conditions for the characterization of the oxidation sites of Aβ40

The same operating conditions than for LC-MS (column and mobile phase gradient)

were used to carry out high resolution mass spectrometry (LC/HRMS) experiments, by using a LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific, Les Ulis, France) coupled to an Ultimate 3000 LC System (Dionex, Voisins-le-Bretonneux, France). The Orbitrap cell was operated in the full-scan mode at a resolution power of 60 000. The UV-Visible spectroscopy (UV-Vis) is a technique applied to measure the light absorbance by a compound in the ultraviolet-visible spectral region. This region can be divided into three wavelength ranges: near UV (185-400 nm), visible (400-700 nm) and very-near infrared (700-1100 nm). Usually, the absorbance is not measured below 190 nm since dioxygen, water vapor and the quartz cuvettes used for the measurements absorb and thus disturb the measure.

LC-HRMS conditions for the kinetics study of Aβ40

The absorbance originates from the interaction of the photons of the light with the electrons of the bonds of the species present in the sample (such as molecules). Molecules with π-electrons or non-binding electrons (n-electrons) are susceptible to absorb in UV-Vis.

A spectrophotometer is composed of a light source (usually a combination of a deuterium lamp for the UV range (< 350 nm) and a tungsten filament for visible range (> 350 nm)), a dispersive system to select the wavelength, a space for the sample (usually in a cuvette) and a detector which converts the light detected into an electric signal. The selection of the wavelengths can be realized either (i) by a monochromator setup that isolates the different wavelengths of the light and introduce each monochromatic light into the sample or (ii) by a photodiode array (PDA) detector that detects the different wavelengths of the light passed through the sample after its dispersion by a spectrograph. As seen above, the detector converts the transmitted light into an electric signal. Two values can be extract from the data:

-the transmittance (T), defined as the transmitted intensity (It) over the initial intensity (I0) ratio (Equation. II.C-1).

𝑻 = 𝑰 𝒕 𝑰 𝟎

Equation. II.C-1

-the absorbance (A), given by Equation. II.C-2.

𝑨 = -𝒍𝒐𝒈 𝑻

Equation. II.C-2

UV-Vis can also be employed for quantitative analysis since the absorbance of the light can be directly related to the concentration of the compound in certain conditions defined by the Beer-Lambert law (Equation II.C-3).

𝑨 𝝀 = 𝜺 𝝀 . 𝒍. 𝑪

Equation II.C-3

Aλ is the absorbance (no dimension), ε the molar extinction coefficient (in L.mol -1 .cm - 1 ), l the optical path length (in cm) and C is the concentration of the compound in solution (in mol.L -1 ) for the wavelength λ where the measure is realized.

To be valid, the Beer-Lambert Law has to meet the following requirements [10] :

-A monochromatic light has to be used

Ascorbate consumption conditions

Ascorbate consumption is monitored by UV-Visible spectroscopy at 265 nm in a 1 cm length cuvette containing a phosphate buffered solution (50 mM, pH 7.4) of Aβ (12 µM), CuSO4 (10 µM) and ascorbate (100 µM). The ascorbate absorption is monitored every 10 s under shaking (800 rpm).

II.D. Fluorescence spectroscopy II.D.1. General principles [10] Fluorescence is an optical phenomenon that occurs usually on polyaromatic, plane or The fluorescence lifetime (τ) corresponds to the time the fluorophore stays in the excited state before emitting a photon by radiative deactivation. The lifetime is dependent on the environment of the molecule. Usually, the lifetime of a fluorophore is in the nanosecond range.

The intensity of fluorescence emission of very weakly absorbing and diluted sample is directly related with its absorbance and thus with its concentration (Equation II.D-2). [10] This relation allows to realized quantitative experiments, however it is important to establish the maximal concentration from which the fluorescence emission is not proportional to the concentration anymore. In our experimental conditions, the fluorescence intensity is proportional to the number of 7-OH-CCA molecules formed, which in turn is proportional to the HO • radicals trapped by CCA. Thus, monitoring 7-OH-CCA fluorescence gives information on the amount of HO • radical exiting the Aβ/copper system, when ROS are produced in the presence of ascorbate.

The hydroxyl group of 7-OH-CCA has a pKa of 7.4 [11] and only the deprotonated form emits light at 450 nm. Thus, the pH has to be controlled very precisely during the experiments, especially since the experiments are performed at pH 7.4 in our studies.

7-OH-CCA Fluorescence conditions

The fluorescence of 7-OH-CCA is monitored at 450 nm upon excitation at 395 nm with a microplate reader, at 25°C. The microplate contains a phosphate buffered (50 mM, pH 7.4) solution of Aβ, with CuSO4, and ascorbate or ascorbate and hydrogen peroxide (H2O2), depending on the studies.

II.D.3. Thioflavin T fluorescence

The Aβ peptide has the ability to aggregate into structured aggregates such as oligomers and fibrils (see Section I.B.4). The fibrils are known to be composed of β-sheet structures of the Aβ peptide. [12] To monitor the Aβ fibrillar formation, a fluorescent dye capable of interacting with β-sheet structures with a resulting enhanced fluorescence is commonly used:

Thioflavin T (ThT). [13][14] ThT is a benzothiazole salt (Figure II.D-5a) used first as histological marker of amyloid fibrils and, due to its water solubility and its good affinity for fibrils (in the low µM range), [15] it was then used to monitor the aggregation process during in vitro experiments. The above described increment of the ThT fluorescence yield is related to its spatial arrangement. The carbon-carbon bond between the benzothiazole and the aniline moieties is free for rotation when ThT is not bound (Figure II.D-5a, bottom panel). This rotation quenches the excited states created upon photon excitation, resulting in a low fluorescence. However, the preclusion of the free rotation is supposed to arise upon binding to β-sheet structures such as fibrils. The rotational blockage of the bond between the two moieties preserves the excited states, leading to an enhanced fluorescence yield. [15][16] Figure II.D-5b shows a proposed model of ThT binding to fibrils, named "Channel" model. In this model, ThT is proposed to bind to fibrils in channel-like motifs along the surface of the fibrils, in a parallel alignment to the long-axis of fibrils. As ThT interacts with fibrils made of the stacking of different amino acid sequences, the interaction seems to be independent of the amino acid chemical nature. Thus, the "Channel" model could be a good hypothesis for ThT binding to fibrils.

Aggregation conditions

For Aβ aggregation studies by fluorescence, the purified peptide (see Section II.A. In the absence of a magnetic field, the spin moments of the nuclei are uniformly distributed and the total magnetic moment resulting is zero. However, when a magnetic field B0 is applied, a Larmor precession occurs due to the interaction between the magnetic field and the magnetic moment of the nucleus. where h is the Planck constant (J.s -1 ), γ the gyromagnetic ratio (rad⋅s -1 ⋅T -1 ) and B0 the magnetic field (T). Thus, the higher the magnetic field strength, the higher the energy difference between the two spin states and the higher the difference of population between these two states, leading to a more intense magnetic moment. The frequency of precession of each nucleus is dependent on the chemical environment such as electron environment and neighboring nuclei. A proton close to an electron-attracting group will have a higher frequency, and its proximity with other protons nuclei leads to spinspin coupling. Thus, the frequency of each proton is informative on its chemical environment.

The NMR spectrum obtained by Fourier Transform contains the intensity of NMR absorption as a function of the chemical shift δ (in ppm) calculated from Equation II.E-2.

𝜹 = 𝝂 -𝝂 𝒓𝒆𝒇 𝝂 𝒓𝒆𝒇 . 𝟏𝟎 𝟔

Equation II.E-2

Where ν is the proton frequency, νref the frequency of the reference. where h is the Planck's constant (6.626 x 10 -34 J.s), ν the radiation frequency (in s -1 ), ge the Landé factor (2.00232 for a free electron), μB the Bohr magneton (9.2740 x 10 -28 J.G -1 ) and B0

the external magnetic field (in G).

The application of an electromagnetic wave perpendicular to B0 and with a frequency ν allows the transition between the two Zeeman levels, corresponding to an orientation change of the magnetic moment of the electron.

In EPR, an absorption spectrum is obtained by the detection of the residual electromagnetic field after it passed through the paramagnetic sample. The frequency is fixed and the magnetic field varies. When B0 value of the applied magnetic field is directly related to the frequency of the electromagnetic wave (via Equation II.F-1), the wave is absorbed by the sample, leading to the transition between the Zeeman levels. From the spectrum, the Landé factor (g) can be calculated, specific for each system studied. In the case of a free electron, g = ge = 2.00232. For anisotropic systems such as metal ions complexes, the response of the molecule is different depending to its orientation with respect to the magnetic field. Thus, 3 transitions occur in x, y and z directions. When the nucleus bearing the spin density has a nuclear spin I ≠ 0, a coupling of the electron magnetic moment occurs with the magnetic moment of the nucleus, called hyperfine coupling. The EPR spectrum is strongly impacted. The EPR spectrum will thus present a maximum of 2 I + 1 lines for each electronic transition, due to the electron / nuclear magnetic moment coupling. The hyperfine coupling constant of a nucleus (A) is directly related to the spectral line spacing 𝒜 by the Equation II.F-3.

𝑨 = 𝓐 . 𝒈 . 𝝁 𝑩 𝒉 𝒄

Equation II.F-3

𝒜 (in G) is the distance between two of the hyperfine lines, g the Landé factor, μB the Bohr magneton (9.2740 x 10 -28 J.G -1 ), h is the Planck's constant (6.626 x 10 -34 J.s) and c the light speed (9.9979.10 10 cm.s -1 ). A is expressed in 10 -4 cm -1 .

The electron magnetic moment can also interact with the nuclear magnetic moment of an another nucleus, resulting in a super-hyperfine coupling.

Thus, an EPR spectrum gives insights into the nature of the paramagnetic center and on its environment as well.

II.F.1. Application to Cu(II) coordination study

As Cu(II) ion is paramagnetic (d 9 ), EPR is a powerful technique for investigating binding modes of Cu(II) complexes. Cu(II)-peptide complexes have an axial symmetry, then 2

Landé factors g // (when the magnetic field is in the z axis direction, described by Equation II.F-4) and g  (when the magnetic field is in the xy plane, described by Equation II.F-5) Where 𝒜 // (in G) is the distance between two of the hyperfine signals, g // the Landé factor, μB the Bohr magneton (9.2740 x 10 -28 J.G -1 ), h is the Planck's constant (6.626 x 10 -34 J.s) and c the light speed (9.9979.10 10 cm.s -1 ). A // is expressed in 10 -4 cm -1 .

characterize
The hyperfine interaction in the xy plane is generally much lower than the one in the z axis and is resulting in a broadening of the signal related to g  thus not resolved rather (see

Figure II.F-4
). [17] Figure II.F-4: EPR spectrum of Cu(II) in water.

The determination of the A // , g // and g  values are essential to characterize the coordination mode of Cu(II)-Aβ. They give insights into the nature of the neighboring atoms in the equatorial plane (i.e. number of oxygens and nitrogens) according to Peisach and Blumberg empirical correlation. [18] Thus, these EPR parameters will be used for comparison between different Cu(II)-peptide complexes in our studies.

EPR conditions

EPR spectra were recorded on a Bruker Elexsys E500 spectrometer equipped with a continuous flow cryostat (Oxford). Analysis was performed with aqueous solution containing 10% of glycerol, 65 Cu (450 µM) and the Aβ peptide (500 µM). pH was adjusted with H2SO4 (1 M) and NaOH (1 M). Experimental parameters were set as follow: T = 120 K, ν = 9.5 GHz, microwave power = 20.5 mW, Amplitude modulation = 10.0 G, Modulation frequency = 100 kHz.

II.G. X-Ray Absorption Near Edge Structure (XANES) II.G.1. X-Ray absorption: general principles [19] X-rays are electromagnetic waves with wavelengths between 10 -12 m and 10 -8 m and an energy range of 0.1-100 keV, used in X-ray absorption spectroscopy (XAS) at synchrotron radiation facilities. This technique is based on the interaction between X-rays and matter. Xrays can be absorbed by an atom, resulting in an excitation of a core electron from its ground state to a high-energy electron unoccupied orbital or to the continuum. XAS is based on the application of X-rays on a sample and the detection of either the absorption or the fluorescence generated during relaxation. The starting energy of X-rays is chosen in order to be lower than the absorption edge of the atom, and is increased during the experiment. XAS spectra are presented as the absorption/fluorescence as a function of the incident X-ray energy (in eV).

As eV. Zn data were collected from 9510 to 9630 eV using 5 eV step and 3 s counting time, from 9630 to 9700 eV using 0.5 eV step and 3 s counting time, and from 9700 to 10000 eV with a kstep of 0.05 Å -1 and 3s counting time.

For each sample, three spectra were averaged and resulting XANES spectra were background-corrected by a linear regression through the pre-edge region and a polynomial through the post-edge region and normalized to the edge jump. All spectra were individually inspected prior to data averaging to ensure that beam damage was not occurring.

Zn samples were prepared by adding the solution of ZnSO4 (0.9 mM) and peptide (1 mM) in the presence of 10% glycerol as cryoprotectant (pH adjusted to 7.1) into the sample holder. The sample was immediately frozen in liquid nitrogen.

Chapter III: Oxidation of the Aβ peptide

This chapter focuses on the oxidation of the Aβ peptide by the Cu/ascorbate/O2 system and the characterization of its oxidation sites. First, the oxidation of Aβ40 is investigated by The exact nature of the oxidized amino acid residues cannot be determined here, even if, among the residues of the tryptic Aβ6-16 peptide, histidine and tyrosine residues are the most sensitive to oxidation by HO • . [1] Several peaks are detected on the chromatogram trace of HDGYEVHHQK+16, probably due to the presence of different oxidized species with a mass shift of +16 Da. For HDGYEVHHQK+32, only one peak is detected with a low intensity.

Only one oxidation has been detected for the tryptic Aβ17-28 peptide ( Table III.A-1 summarizes the detected non-oxidized and oxidized tryptic peptides along with the monoisotopic masses of their corresponding protonated ions. Specific mass shifts of -45 Da and -89 Da have been found on the N-terminal tryptic peptide Aβ1-5, corresponding respectively to the decarboxylation-deamination and to the oxidative cleavage of Asp1. [2][3] Usually, aromatic amino acid residues are more prone to oxidation than aliphatic ones, [4] in particular because of their high electron density which favors the hydroxyl radicals attack. Thus, Common oxidations (mass increase of 16 Da) were also detected on every tryptic peptide. As oxidation leading to the formal addition of an oxygen atom can occur on several amino acid residues, we cannot conclude on the nature of the damaged amino acid residue of each tryptic peptide, even if some of the amino acid residues are more sensitive than the other ones. The same tryptic peptides were thus sequenced by LC-MS/MS in order to identify the oxidized amino acid residues. After oxidation, purification and digestion, the Aβ40 peptide was analyzed by LC-MS/MS in order to determine the site of oxidation on each tryptic peptide. As explained in Section II.B.3.c, by using Collision Induced Dissociation (CID), the peptide is cleaved at the peptide bond, leading to the formation of b and y ions, whose differences in mass are equal to the mass of the residues. [5] These ions are detected and their masses compared to the theoretical masses obtained from the peptide sequence. The oxidized residues are identified when the b or y ions values are increased by +16, as compared to the non-oxidized peptide.

The N-terminal tryptic Aβ1-5 peptide was first analyzed by LC-MS/MS but the spectra obtained were not informative. This small peptide is usually fragmented as a mono-protonated ion, leading to a poor quality of the MS/MS spectrum. In addition, the terminal arginine residue (an amino acid residue which has the highest pKa value of the side-chain function) tends to preclude the proton mobility during fragmentation, resulting in the absence of b ions. [6] Figure III.A-5 shows two MS/MS spectra of the doubly protonated ion of the oxidized Aβ6-16 (+ 16 Da). For the first one (top), the oxidation is detected on His13, oxidized in 2oxohistidine while for the second one (bottom), His14 is detected oxidized in 2-oxohistidine. [7] For both MS/MS spectra, the peptide sequence is well covered by both the b and y ions, allowing a reliable characterization of the oxidation of His13 and His14. No other MS/MS spectrum was obtained, assuming that only those two amino acid residues are oxidized in the 6-16 moiety of Aβ40.With these results, we can reasonably assume that the mass shift of +32 Da previously detected in HRMS is related to the oxidation of both His13 and His14 into 2-oxohistidine on the same peptide. However, this hypothesis cannot be confirmed here as no Aβ6-16 ion with an increase of mass of 32 Da was fragmented and detected in MS/MS. This could be due to the fact that only a few part of the Aβ40 peptide undergoes this double oxidation, resulting in a too low concentration to allow fragmentation.

Two different oxidations were also found on the Aβ17-28 tryptic peptide, as shown in the two MS/MS spectra of the doubly protonated ion corresponding to Aβ17-28 with a mass shift of +16 Da in Figure III.A-6. The two oxidations are located respectively on Phe19 and Phe20. The +16 Da mass shift on phenylalanine is related to the formation of hydrophenylalanine (see Section I.C.3.d for chemical structure). [8] Although two amino acid residues are targeted by ROS, no double oxidation of Aβ17-28 was detected in LC-HRMS or in LC-MS/MS, as it was the case for Aβ6-16. The oxidations of Phe19 and Phe20 may occur on the same peptide, but this double oxidation is likely to occur in minority on the Aβ peptide and is thus not detected in MS. The Aβ29-40 tryptic peptide was finally sequenced and, as assumed above, the oxidation detected with a mass shift of +16 Da is located on Met35, as shown by Figure III.A-7. This was expected and previously described [9][10][11][12] as methionine residue is very sensitive to oxidation and can be damaged by both the hydrogen peroxide [13][14] and the hydroxyl radical [1,14] which are produced during metal-catalyzed ROS production. [15][16] The results obtained on Aβ40 corroborate the study previously realized in the team with the truncated Aβ28 peptide. [17] Similar oxidative damages and targeted residues were detected:

oxidative cleavage and decarboxylation-deamination of Asp1, [2][3] His13 and His14 oxidized into 2-oxohistidine [7] and Phe19 and 20 detected as hydroxyphenylalanine. [8] Furthermore, Met35 was also found oxidized into methionine sulfoxide, in good agreement with what is widely described in the literature. [18][19] Figure III.A-8 summarizes the oxidations detected on Aβ40 in the present study along with the chemical structures of the oxidized amino acid residues.

Among the identified oxidized residues, three of them (Asp1, His13 and His14) are proposed to be involved in the coordination of copper in the "in-between" state of Cu-Aβ, [17] i.e. in the redox competent state responsible for ROS production (see Section I.C.2.c). They are thus in close vicinity of copper and are preferential targets for the ROS attack. Their oxidation during MCO of Aβ was expected. This is not the case for Phe or Met residues, whose oxidation could appear as side damages resulting from an escaping of the ROS away from the catalytic center. At this point, we cannot conclude on the identity of the amino acid residues targeted first by HO • because the level of oxidation for each targeted residue identified above is not known. The following part tends to answer this question.

III.B. Kinetics of Aβ 40 oxidation

The characterization of the amino acid residues of Aβ targeted by ROS and the nature of the oxidations, as investigated above, is carried out at the end of the reaction. In order to understand in what order the oxidative attack of ROS happens during MCO and what are the preferential targets of the ROS attack (among those identified above), the kinetics of the Aβ40 peptide oxidation was monitored by LC-HRMS. Among the several amino acid residues oxidized, some of them could be preferentially targeted by ROS. Thus, the reaction started by mixing Aβ, copper and ascorbate, was stopped every minutes and the resulting reaction mixture was then analyzed by LC-HRMS.

A semi-quantitative approach was used to study the level of each oxidized residue as a function of the reaction time.

III.B.1. Experimental section

Copper(II)-catalyzed oxidation of the Aβ40 peptide was carried out by mixing Aβ40, Cu(II) and ascorbate in phosphate buffer (50 mM, pH 7.4) to reach final concentrations of 60, 50 and 500 μM, respectively (substoichiometry of copper to avoid free Cu(II) in solution) for a reaction mixture volume of 2 mL. Incubation was done at room temperature for a controlled reaction time. A volume of 100 μL of the reaction mixture was taken out every minutes between 0 and 14 min and the reaction was stopped by adding 400 μL of HCl 14.8 mM (final pH 2). At pH 2, copper is not bound to Aβ anymore and ascorbate is fully protonated into ascorbic acid (pKa 4.2 [20] ), thus the ROS production is stopped. The 400 µL solution of Aβ was filtered by using Amicon 3 kDa centrifugal device (Millipore) by centrifugation for 15 min at 13500 rpm.

The final volume is around 100 µL (volume before dilution in HCl). Trypsin digestion was then carried out and the four tryptic peptides described before were obtained (see Section II.B.3 for more details).

III.B.2. Results

The trace chromatograms were obtained for every oxidized tryptic peptide of Aβ40, by using two monoisotopic ions corresponding to two charge states of the peptide. The mass accuracy was systematically set below 10 ppm. The m/z ratio used for the detection are listed in Table III.A-1. 1c). The tryptic peptide with the oxidation on both Phe was not detected (i.e. LVFFAEDVGSNK+32). Phe oxidation seems to be slower than Asp1 or His oxidation, in particular at the beginning of the reaction where a lag phase is observed. The curve starts to increase after 2 min of reaction and does not seem to reach a plateau around 8 min.

For several reasons, the evolution of Met35 oxidation as a function of the reaction time does not provide interesting information. First, methionine is very sensitive to oxidation and peptide/protein handling commonly participate to its oxidation. This is probably the reason why Met35 is already oxidized before MCO of Aβ has started (Figure III.B-1d). Second, methionine sulfoxide formed by the ROS attack can be reduced in non-oxidized methionine by ascorbate. [21] As ascorbate is present in quite high concentration in the reaction mixture, this phenomenon would be responsible for an underestimation of the level of methionine sulfoxide generated during MCO. However, a slight increase of the gradient is observed during the reaction, thus Met35 seems to be oxidized during the course of the reaction.

To summarize, Asp1, His13 and His14 seem to be the first targets of ROS attack. Similar tendencies were obtained with the truncated Aβ28 peptide, resulting in a proposition of Cu-Aβ binding mode in the "in-between" state. [17] As Asp1, His13 and His14 are the first targets for HO • during the ROS production, and because they are involved in Cu(II) or/and Cu(I) coordination in the resting states, [22][23][24][25][26][27] they were proposed to participate in Cu binding in this transient state. The results obtained with Aβ40 are consistent with this hypothesis. With this in mind, the stop of Asp and His oxidations after 8 min could be interpreted as a change of coordination during MCO. Indeed, if Asp1, His13 and His14 are involved in copper coordination during the ROS production, they are first targeted and are quickly oxidized. Thus, as they are damaged, they may not be good ligand under their oxidized form, leading to a change of coordination.

Phe oxidation starts after a 2-min lag phase and reach a much lower level of oxidation.

As Asp1, His13 and His14 residues are targeted first by HO • , the oxidation of phenylalanine residues is likely to be a side damage of the ROS production by the system, and would start when the amino acid residues targeted first by HO • are sufficiently oxidized to let escape the ROS. This is in line with the lag phase at the beginning of the reaction, indicating that Phe residues are not directly targeted by HO • , although they are very sensitive to HO • oxidation. [1] The same applies for Met35 which is not coordinated to copper.

III.C. NMR study of Aβox

The above results were obtained by MS based techniques. In order to corroborate them, we wanted to analyze Aβox by using a different technique. The chemical modification undergone by the amino acid residues of Aβ after MCO were analyzed by 1 H NMR. We have chosen to use the truncated Aβ28 instead of Aβ40, as the complete attribution of the 1 H NMR is available. [28] As the only damage observed on the 29-40 moiety of Aβ40 was attributed to Met35 oxidation and is well-described, [9][10][11][12] we keep the essential information by working on Aβ28 instead of Aβ40.

III.C.1. Experimental section

The methodology and conditions used for these experiments are detailed in Section II.E.2.

III.C.2. Results

1 H NMR of Aβ28 leads to a spectrum with numerous peaks. In order to be able to observe the impact of oxidation on the targeted amino acid residues (Asp1, His13, His14, Phe19 and Phe20), the present study was carried out at pH 10.5, where Hα of Asp1 and the aromatic protons of both His and Phe residues are well separated from other protons and thus easily detectable.

A complete NMR study of Aβ28 was previously realized in the team, [28] leading to the attribution of all the protons of Aβ28 (see Annex II for 1 H chemical shifts table and Aβ28 sequence along with the atom identifiers of each amino acid residue). Although the study was carried out at pH 7.4, it was useful to help us to attribute the protons of the amino acid residues of interest. Unlike the C-terminal moiety of Aβox (from Ala21 to Lys28) which is not disturbed by oxidation, the majority of the amino acid residues of the N-terminal moiety are strongly affected. For example, the apolar aliphatic amino acid residues such as Val and Leu residues have broaden protons signals (around 0.7-0.9 ppm) although they are weakly sensitive to oxidation. [1] Thus, the oxidation of some amino acid residues of the N-terminal moiety of Aβ28 Another signal is present only in the Aβ28ox spectrum around 3.6 ppm, with a signature corresponding to two diastereotopic protons. These protons are probably related to an oxidation product of Aβ, but the origin is unknown as its chemical shift does not correspond to an oxidized amino acid residue listed above (see Section I.C.3) or detected previously in MS (see Section III.A).

III.C.3. Summary

The interpretation of 1 H NMR spectrum of Aβ28ox was made difficult by the strong broadening of the peaks obtained. The origin of this broadening could tentatively be explained by the fact that (i) different oxidized species are generated during MCO of Aβ, thus leading to different chemical shifts for a given proton, (ii) the numerous chemical modifications undergone by the peptide may also change the environment of the protons owned by the amino acid residues not oxidized that are close to the oxidized one and (iii) the high level of concentration needed to perform NMR experiments requires for Aβox to be highly reconcentrated (from 60 µM to 400 µM), which might result in a modification of the sample (e.g. oligomerization for instance).

However, the presence of signals not affected have highlighted that the C-terminal moiety of the Aβ28 peptide (between Ala21 and Lys28) is not affected by oxidation. This is in line with identification of Asp1, His13, His14, Phe19 and Phe20 as the only oxidized amino acid residues detected in MS.

In the 1-20 part of Aβ28, the NMR spectrum of Aβ28ox does not provide information on the oxidized amino acid residues as the majority of the signals are broad. Thus, the identification of Asp1, His13 and His14 as the main oxidized amino acid residues in MS cannot be confirmed by NMR. However, the presence of a peak in Aβ28ox spectrum attributed to the methyl group of the pyruvate confirms the oxidation of Asp1 by decarboxylation and deamination.

III.D. Conclusion

In the present study, LC-HRMS, LC-MS/MS and 1 H NMR have been used to investigate the oxidation of the Aβ peptide during the ROS production. Both techniques have highlighted the chemical changes undergone by the peptide due to the ROS oxidative attack.

MS experiments enabled to determine the oxidized amino acid residues of the Aβ sequence and to characterize the nature of the oxidation undergone by each amino acid residue.

Asp1 is found oxidized either by decarboxylation-deamination or by oxidative cleavage, leading to the formation of a pyruvate or an isocyanate function respectively. His13 and 14 are found oxidized into 2-oxohistdines, Phe19 and Phe20 into hydroxyphenylalanine and Met35 into methionine sulfoxide. Furthermore, through a kinetic study of the amino acid residues oxidation, Asp1, His13 and His14 have been found to be the main targets of ROS, while Phe19, Phe20 and Met35 are secondarily targeted. The results are in line with the previous study realized on the truncated Aβ28 [17] and complete it by showing that (i) as expected, Met35 is also oxidized during the MCO of Aβ40 but as a secondary target and (ii) that Aβ28 is an appropriate model for oxidation studies as the same main oxidations are detected for both the truncated and the full-length peptides.

Although the 1 H NMR spectrum of Aβ28ox is not easily exploitable since the proton signals are broaden, the presence of signals not disturbed by oxidation has shown that no oxidation occurs in the 21 to 28 part of the Aβ28. Moreover, the decarboxylation and deamination undergone by Asp1, leading to the formation of a N-terminal pyruvate function, is confirmed by the presence of a signal attributed to the protons of the pyruvate methyl group.

Kinetic study of the amino acid residues oxidation has also shown that besides being the main targets, Asp1, His13 and His14 are strongly oxidized during the first minutes of the MCO and then are not targeted anymore. This phenomenon was also observed in the previous study [17] and has led to the proposition that these three amino acid residues would be the main ligands for Cu(II) and Cu(I) in the "in-between" state (see Section I.C.2.c) during the ROS production.

As they are targeted by the ROS, the chemical modifications they undergo may impact their aptitude to be a good ligand for copper. Hence, a change of coordination may occur and would explain the reason why they are not oxidized anymore after a few minutes of MCO.

As addressed above (see Section I.C.1) Asp1, His13 and His14 are also involved in Cu(II) or/and Cu(I) coordination in the resting states. Thus, the MCO of Aβ may have consequences on the copper coordination both in the resting and "in-between" states.

Chapter IV: Consequences of Aβ oxidation

This chapter focuses on the consequences of the Aβ peptide oxidation regarding metal coordination, as well as Reactive Oxygen Species (ROS) production and peptide aggregation, two key events of Alzheimer's Disease (AD). [1] The coordination of Cu(II), Cu(I) and Zn in 2016 [2] along with a summary of the article, written in French (requirement of the doctoral school). The supporting information related to the article are situated at the end of the chapter.

IV.A.1. Article

IV.A.2. French summary

Cet article, publié dans la revue Metallomics en 2016, [2] porte sur les conséquences de

IV.B. Zn coordination with Aβox

Zn ions are found in the amyloid plaques extracted from AD brains [3] and they can form complexes with the Aβ peptide. According to the proposed models (see Section I.C.1.a), Zn(II) would be bound by imidazole rings of His6 and either His13 or His14 residues in equilibrium, the carboxylate group of Glu11 and the carboxylate group of either Asp1, Glu3 or Asp7, with a preference for Asp1. [4] Asp1, His13 and His14 being found oxidized during MCO of Aβ, the

IV.B.2. Results

The main oxidized amino acid residues of Aβ28ox (Asp1, His 13 and His14, see Chapter III) are involved in the coordination of Zn(II). Thus, in order to investigate the impact of Aβ oxidation on Zn(II) coordination, mutated peptides are used to mimic Aβox. The D1N mutated Aβ16 peptide does not have any N-terminal carboxylate group and is employed to mimic the oxidation of Asp1. H13A mutated Aβ16 peptide bears only two His on its sequence and is used to mimic the oxidation of one His residue. In addition, H6A-H13A and H6A-H14A mutated Aβ16 peptides are used to mimic the oxidation of two His residues. In the above study (Section IV.A), Aβ7 (sequence DAEFRHD) was used to mimic the double His oxidation, but as Zn(II) also binds Glu11, its use would simulate a double His oxidation along with an unavailability of Glu11 for binding. Oxidation of Asp1 thus would not contribute that much to the Zn-Aβox signature, even if its carboxylate group, involved in Zn coordination, is strongly affected during Aβ oxidation. This could be explained by the dynamic exchange existing between the carboxylate groups of Asp1, Glu3 and Asp7 regarding Zn coordination. [4] The behavior of Zn-Aβ28ox seems to be close to that of Zn-H13A, Zn-(H6A-H13A) and

Zn-(H6A-H14A), since XANES signatures exhibit some similarities. A broaden peak is observed at 9665-9670 eV for Zn-Aβox and could result from the growth of a peak at 9668 eV, whose shape evolves to become well-defined, from Zn-H13A to Zn-(H6AH14A) and to Znbuffer. A similar evolution is observed for another growing and broaden peak detected at 9680 eV, which clearly appears in Zn-buffer spectrum. These elements would suggest that Zn affinity for Aβ would decrease upon Aβ oxidation, because the residues involved in zinc coordination are mainly targeted during MCO. In particular, the oxidation of one or two histidine residues would be responsible for this. This is in line with the affinity values of Zn for His mutants, lower than for Aβ. [4] Unfortunately, as the signatures in the white line region are broad, define precisely the several binding modes of Zn with oxidized species is precluded. For the same reason, it was not possible to obtain reliable linear combination fitting of the Zn-Aβox signature, as we found that different conclusions can be made according to the result of the fitting (see Annex III for examples of linear combination fittings).

It has been previously shown that the intensity of the Zn XANES white line was related to the number of ligands in the Zn(II) complex. [5] In the case of D1N, Aβ28 and H13A peptides, the white line intensity is 1.3 (or 1.4 for H13A), related to a four-coordination of the metal center. The same results were previously obtained for the other two His mutants (H6A and H14A). [4] Zn in buffer related white line intensity (around 2.3) allows to deduce that 6 ligands are coordinated to loosely bound Zn. In the case of H6A-H13 and H6A-H14A, both intensities being equal to 1.7, it is likely that Zn is bound by 5 ligands, probably including carboxylate groups instead of His residues. Zn-Aβ28ox related white line intensity being equal to 1.4, the number of ligands is the same than for Aβ28 or H13A.

In summary, the XANES signature of Zn-Aβ28ox shows that the Zn binding mode is affected when Aβ is oxidized, mainly because of the oxidation of His residue(s). However, the modification is not drastic as the number of ligands does not change. The oxidation of Asp1 would not have a substantial impact on Zn binding mode, in line with the existing dynamic exchange between Asp1, Glu3 and Asp7 in Zn coordination.

In the amyloid cascade hypothesis, the coordination of Zn ion to Aβ is proposed to modulate the aggregation process (see Section I.B.3) and hence to impact the morphology of the formed aggregates. [6][7] Thus, besides the fact that the chemical modifications undergone by Aβ during ROS attack could directly impact its ability to aggregate, the change of Zn binding mode due to Aβ oxidation could also have an effect on the aggregation process in the presence of Zn.

IV.C. Aggregation of Aβox

Aggregation of the Aβ peptide is one of the main events of AD, leading to the formation of amyloid plaques. [8] As addressed above (see Section I.B.4), Aβ is an unfolded peptide which is prone to aggregation and can form β-sheet rich structures. [9] In the previous chapter, the oxidized amino acid residues have been identified after MCO of the full-length Aβ40 peptide.

Asp1, His13 and His14 are the main amino acid residues targeted by ROS, but some minor oxidations may also occur on Phe19, Phe20 and Met35. As Aβ40 undergoes chemical modifications during MCO, its propensity to aggregate into β-sheet rich structures might be affected. In the present study, the aggregation capability of both Aβ40 and Aβ40ox is studied and compared. Aggregation into β-sheet structures is monitored by Thioflavin-T (ThT) fluorescence as a function of time, [10][11] and the presence (or absence) of fibrillar species at the end of the fluorescence experiment is investigated by Transmission Electron Microscopy (TEM). [12] This part presents the preliminary results we obtained on Aβ40 and Aβ40ox aggregation without metals.

IV.C.1. Experimental section

The preparation of Aβ40 and Aβ40ox solutions is detailed in Section II.A. The solutions of Aβ40 and Aβ40ox (20 µM) were incubated during 200 h at 37°C and the fibril formation was monitored by ThT fluorescence (the fluorescence conditions are presented in Section II.D.3).

The fluorescence curves obtained are an average of the fluorescence curves of 3 replicates.

Then, the solutions were collected and prepared for TEM using the conventional negative staining procedure. 20 μL of solution was adsorbed on Formvar-carbon-coated grids for 2 min, blotted, and negatively stained with uranyl acetate (1%) for 1 min. Grids were examined with a TEM (Jeol JEM-1400, JEOL Inc, Peabody, MA, USA) at 80 kV. Images were acquired using a digital camera (Gatan Orius, Gatan Inc, Pleasanton, CA, USA) at ×25 000 magnification. As Phosphate buffer reacts with uranyl acetate, HEPES buffer was preferred and employed for TEM experiments, and thus for ThT fluorescence experiments, as well.

IV.C.2. Results

a. Aggregation of Aβ and Aβox

ThT is a fluorescent dye whose fluorescence yield is enhanced when it interacts with βsheet rich structures such as Aβ fibrils (see Section II.D.3 for more details). [13][14] In addition, it displays a bathochromic shift of both excitation and emission maximum wavelengths. [15] Thus, The change of the overall charge of the peptide could also be the origin of the modification of aggregation process. [16][17] The oxidation of the protonated Asp1 residue can lead to the formation of the neutral pyruvate function. [18][19][20] Thus, although Asp1 is not located

ThT
200 nm 200 nm
on the peptide moiety responsible for aggregation, its alteration could disturb the aggregation process.

The aggregation study of mutated peptides (such as H13A-Aβ40, H14A-Aβ40 to mimic His oxidation or the N-terminal acetylated AcAβ to mimic Asp1 oxidation) could be interesting to determine if the Asp1 or the His oxidation is the origin of the Aβ fibrillization inhibition. [21] .

IV.C.3. Outlook

The preliminary results obtained by both fluorescence and TEM are very promising as they highlight a strong modification in the aggregation process of Aβ subjected to MCO.

Similar results were previously obtained after catalytic photo-oxygenation of Aβ. [22] However, as aggregation is a very sensitive process, the results have to be taken with caution. A series of controls should be carried out in order to ensure that the different aggregation behavior of Aβox effectively comes from the oxidative damages undergone by Aβ and not from the multistep purification procedure (described in Section II.A) of Aβox. The purification procedure undergone by the oxidized peptide will be carried out on the non-oxidized Aβ40 as well to ensure that it does aggregate after the procedure. Moreover, aggregation experiments will be realized with a mixture of oxidized and non-oxidized peptides at different ratios and compared with the samples containing the same quantity of non-oxidized peptide than in the mixture. The comparison of the fluorescence curves might be informative on the possible effect of Aβ40ox on Aβ40 aggregation process. The set of new experiments is being carried out at the laboratory and should complete the presently described results.

IV.D. Conclusion

In the present studies, the impact of MCO of Aβ has been investigated on metal ions coordination, ROS production and aggregation. and Cu ions can modulate the aggregation process by binding Aβ, [6][7] the impact of Aβ oxidation on the aggregation in the presence of Cu or Zn would also be studied.

The oxidation of the Aβ peptide occurring during ROS production induces strong modifications in ROS production and aggregation, two key events of AD. It seems to favor the amorphous-like aggregates such as oligomeric species which are proposed to be more toxic than fibers. [23][24] It also leads to an enhanced catalytic activity for ROS production. Thus, Aβ oxidation appears to be a detrimental event for AD, for both aggregation and ROS production processes.

Experimental

Titration of Aβ28, AcAβ28 and Aβ40

All the synthetic peptides were bought from GeneCust (Dudelange, Luxembourg), with purity grade > 95%. Stock solutions of the Aβ28 (sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNK) and Ac-Aβ28 (sequence AcDAEFRHDSGYEVHHQKLVFFAEDVGSNK), peptides were prepared by dissolving the powder in milliQ water (resulting pH ≈ 2). Peptide concentration was then determined by UV-visible absorption of Tyr10 considered as free tyrosine (at pH 2, (ε276-ε296) = 1410 M -1 cm -1 ). Stock solution of Aβ40 peptide (sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) was prepared by dissolving the powder in NaOH (50 mM) and purifying the solution in FPLC. The peptide concentration was then determined by UV-visible absorption of Tyr10, considered as free tyrosine ((ε293-ε360) = 2400 M -1 cm -1 ) in NaOH (50 mM, resulting pH ≈ 13).

Proteolytic digestion

The solution of Aβ was filtered using Amicon 3 kDa centrifugal device (Millipore) by centrifugation for 15 min at 13500 rpm, then washed and centrifuged twice with 200 μL sodium hydrogenocarbonate (100 mM, pH 8). The concentrated sample (approx. 50 μL) was recovered and transferred to an Eppendorf ProteinLoBind 1.5 mL vial. Trypsin (0.05 ng/μL in formic acid 0.1%) was added to obtain a Aβ/trypsin ratio of 20/1 (w/w) and digestion was carried out at 37°C for 3h in a Thermomixer (Eppendorf), 10 s mixing at 750 rpm every min.

Mass spectrometry

High Performance Liquid Chromatography / Mass Spectrometry (HPLC/MS) analysis was performed on an ion-trap mass spectrometer (LCQ DECA XP Max, ThermoFisher), equipped with an electrospray ionization source, coupled to a SpectraSystem HPLC system. Sample (10 µL of Aβ tryptic digest) was injected onto the column (Phenomenex, Synergi Fusion RP-C18, 250 × 1 mm, 4 µm), at room temperature. Gradient elution was carried out with formic acid 0.1% (mobile phase A) and acetonitrile/water (80/20 v/v) formic acid 0.1% (mobile phase B) at a flow-rate of 50 µL.min -1 . The mobile phase gradient was programmed with the following time course: 12% mobile phase B at 0 min, held 3 minutes, linear increase to 100% B at 15 min, held 4 min, linear decrease to 12% B at 20 min and held 5 min. The mass spectrometer was used as a detector, working in the full scan positive mode between 50 and 2000 Da followed by data dependent scans of the two first most intense ions, with dynamic exclusion enabled. Isolation width was set at 1 Da and collision energy at 28% (units as given by the manufacturer), using wideband activation. The generated tandem MS data was searched using the SEQUEST algorithm against the human Aβ peptide sequence. Dynamic modifications were specified according to the expected mass shift due to the Aβ peptide oxidation (Supporting Information, Table s1). The same operating conditions (column and mobile phase gradient) were used to carry out high resolution mass spectrometry (HPLC/HRMS) experiments, by using a LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific, Les Ulis, France) coupled to an Ultimate 3000 LC System (Dionex, Voisins-le-Bretonneux, France). The Orbitrap cell was operated in the full-scan mode at a resolution power of 60 000. HPLC/HRMS was also used to check for digestion efficiency, systematically found close to100 % (non-digested peptide not detected). HPLC High Performance Liquid Chromatography analysis was performed on an Agilent 1200 series device (Agilent technologies) equipped with a DAD detector. Sample (10 µL) was injected onto the column (Acclaim 120 C18, 50 × 3 mm, 3 µm, ThermoScientific), at room temperature. The gradient elution was carried out with formic acid 0.1% (mobile phase A) and acetonitrile/water (80/20 v/v) formic acid 0.1% (mobile phase B) at a flow-rate of 0.5 mL.min -1 . The mobile phase gradient was programmed with the following time course: 5% mobile phase B at 0 min, held 3 minutes, linear increase to 55% B at 8 min, linear increase to 100% of B at 9 min, held 2 min, linear decrease to 5% B at 12 min and held 3 min. Aβ28 was detected with the absorption of Tyr10 at 276 nm.

Tables

Table S1: Monoisotopic masses used for high resolution mass spectrometry. Monoisotopic apparent masses (m/z in Th) of mono-, di-and triply-protonated ions of the tryptic peptides of oxidized Aβ16; +16 accounts for the formal addition of one oxygen atom during oxidation (conversion of Histidine into oxohistidine).

Position Peptide [M+H] + [M+2H] 2+

[M+3H] 3+ S1. S1. S1. bien plus faible que dans le cas des états au repos. Cet état fugace étant très peu peuplé (de l'ordre de 0.1 % [1] ), la caractérisation directe de son mode de coordination par les techniques spectroscopiques traditionnelles [2] n'est pas possible. La détection des principales oxydations encourues par le peptide Aβ durant la production de ROS par spectrométrie de masse (MS) a mené à la proposition de 3 principaux ligands du cuivre dans l'état IB. [3] L'aspartate situé en première position, et les histidines situées Comme le cuivre a un mode de coordination avec le peptide Aβ impliquant des acides aminés spécifiques, la mutation de ces derniers entraîne un changement du mode de coordination. Les modes de coordination de Cu(I) et Cu(II) dans les états au repos ont été étudiés avec de nombreux mutants de Aβ. Pour Cu(II), la non-disponibilité de l'amine terminale de l'Asp1 (via l'utilisation d'un peptide avec une acétylation de l'amine N-terminale) entraîne un profond changement de coordination. [4][5] Pour Cu(I), la présence d'une seule histidine dans la séquence (via la mutation de deux histidines par des alanines) a également un impact sur le mode de coordination. [6] De la même manière, la mutation d'un acide aminé impliqué dans la sphère de coordination du cuivre dans l'état IB devrait induire une variation dans la production des ROS, puisque c'est cet état qui est responsable du transfert électronique.

La production de ROS par le cuivre avec les différents peptides modifiés a été suivie par la fluorescence de l'acide 7-hydroxycoumarine-3-carboxylique (7-OH-CCA), produit par le piégeage des radicaux hydroxyles (HO • ) par l'acide coumarine-3-carboxylique (CCA). Le blocage de l'amine terminale ainsi que la mutation de l'Asp1 menant à un changement de la chaîne latérale contenant le groupe carboxylate de l'Asp1 ont fortement diminué la vitesse de production de HO

VI.B. French summary

Ce chapitre est composé d'une communication publiée dans Dalton Transactions en août 2016. [1] L'article détaille l'étude réalisée sur les propriétés pro-et anti-oxydantes de l'ascorbate dans le contexte de la maladie d'Alzheimer. L'ascorbate est un réducteur présent en grande concentration dans le cerveau humain (jusqu'à 10 mM dans les neurones). [2] Il est classé dans la catégorie des anti-oxydants puisqu'il a la capacité de réagir avec certaines espèces oxydantes telles que les espèces réactives de l'oxygène (ROS), en particulier O2 d'oxydo-réduction entre l'ascorbate et le dioxygène n'est pas efficace [3] mais le devient en présence d'un catalyseur tel que le cuivre. L'ascorbate réduit l'ion cuivrique en ion cuivreux, et ce dernier transfère à son tour un électron au dioxygène, formant ainsi l'anion superoxyde.

Le cuivre est ensuite à nouveau réduit par l'ascorbate, ce qui entretient le cycle catalytique.

Lorsque le cuivre est coordiné au peptide Aβ, il reste capable de cycler entre ses états d'oxydation Cu(II)-Aβ et Cu(I)-Aβ, le système catalysant ainsi la production de ROS. [4][5] Dans ce cas, l'ascorbate peut donc être considéré comme ayant des propriétés pro-oxydantes.

Les effets pro-et anti-oxydants de l'ascorbate ont été étudiés par spectroscopie de fluorescence, en présence de cuivre libre ou du complexe Cu-Aβ et de dioxygène. pKa (I/II) = 7.8 [6] Figure S2: Schematic view of the proposed Cu(I) coordination site in Aβ. [6] Fluorescent detection of HO • by CCA Fluorescence emission of 7-OH-CCA is very sensitive to pH as, the hydroxyl-group has a pKa of 7.5 (Figures S2 andS3), and only the deprotonated form emits at 450 nm after excitation at 390 nm. Since the experiment is performed at pH 7.4, i.e. very close to the pKa, the pH has to be strictly stable in order to have accurate results. Because the oxidation of ascorbate releases protons, it is not easy to keep the pH constant even with a high concentrated buffer, this issue being more important at higher ascorbate concentrations. Therefore, we Since similar results are obtained whatever the length of the peptide (see Figure S10), the MS experiments were performed with Aβ28 instead of Aβ16 for technical reason. S1. [1] H. Eury in Etude de l'intéraction de la thioflavine T et de complexes de ru (ii) avec le peptide amyloïde bêta dans le cadre de la maladie d'alzheimer, Vol. 2013. Nous avons également étudié la cinétique d'oxydation des acides aminés par spectrométrie de masse haute résolution, afin de déterminer s'il y avait des cibles privilégiées pour les ROS parmi ces 6 acides aminés retrouvés oxydés. La figure 3 montre les courbes d'évolution de l'oxydation au cours du temps pour les 4 peptides obtenus après la digestion trypsique du peptide Aβ40. L'oxydation de Asp1 et des histidines a lieu dès le début de la réaction de production de ROS et semble s'arrêter au bout de 8 min (figures 3a et 3b). Au contraire, l'oxydation des phénylalanines n'a lieu qu'au bout de quelques minutes, après la phase de latence observée sur la courbe (figure 3c). La quantité de phénylalanine oxydée ne cesse de s'accroitre pendant le reste de la réaction. Pour la Met35 (figure 3d), aucune conclusion ne peut être faite, l'ascorbate présent lors de la réaction ayant la capacité de réduire la méthionine oxydée en méthionine, ce qui a pour conséquence de biaiser les résultats.

LC-MS Experiments

Les résultats ainsi obtenus ont permis d'identifier Asp1, His13 et His14 comme cibles privilégiées des ROS car ils subissent des dommages oxydatifs dès le début de la réaction alors que les phénylalanines, cibles secondaires, sont oxydées après un temps de latence.

  OF ABBREVIATIONS ..................................................................................................................... GENERAL INTRODUCTION ................................................................................................................... CHAPTER I: CONTEXT OF THE PROJECT ......................................................................................... I.A. ALZHEIMER'S DISEASE .................................................................................................................... c. Tyrosine ....

  ....................................................................................................................................c. Tandem Mass Spectrometry (MS/MS) applied to proteomic analysis ........................................... II.B.4. Analysis of Aβ by MS and MS/MS ................................................................................... a. MS/MS ........................................................................................................................................... b. High-Resolution Mass Spectrometry (HRMS) ................................................................................. II.C. UV-VISIBLE SPECTROSCOPY ........................................................................................................ II.C.1. General principles ........................................................................................................... II.C.2. Ascorbate consumption .................................................................................................. II.D. FLUORESCENCE SPECTROSCOPY ....................................................................................................

  III.D. CONCLUSION ........................................................................................................................... REFERENCES ........................................................................................................................................... CHAPTER IV: CONSEQUENCES OF AΒ OXIDATION ........................................................................... IV.A. CU COORDINATION AND ROS PRODUCTION WITH AΒOX ................................................................... IV.A.

  Figure I.A-1 shows the repartition of the 46.8 million people living with dementia. In Europe, 10.5 million people are estimated to suffer from a neurodegenerative disease.

Figure I.A- 3 :

 3 Figure I.A-3: Neuropathological lesions revealed by immunohistochemistry. (a) Senile plaques observed by immunohistochemistry with antibodies against Aβ. (b) Neurofibrillary tangles observed by immunohistochemistry with antibodies against phosphorylated Tau (Pictures from reference [11])

  . Clinical diagnosis In 1984, the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) has

I.B. 1 .

 1 Aβ: Structure and formation Aβ is a 38 to 43 amino acid residue peptide (Figure I.B-1) derived from the enzymatic cleavage of APP. Depending on the exact location of the cleavage on the C-terminal part, several lengths can be formed, from Aβ1-38 to Aβ1-43. However, the most abundant species produced in the brain are Aβ1-40 and to a lesser extent Aβ1-42. Aβ is amphiphilic: the N-terminal moiety is hydrophilic while the C-terminal one is hydrophobic.

Figure I.B- 1 :

 1 Figure I.B-1: Amino acid sequence of Aβ1-43 (1-letter code)

  Figure I.B-2: A schematic view of APP proteolytic cleavage. In the non-amyloidogenic pathway, APP is first cleaved by α-secretase and then by γ-secretase to form truncated Aβ17-40/42 peptides or by β-secretase leading to the formation of the truncated Aβ1-16. In the amyloidogenic pathway, APP is cleaved consecutively by the β-and γ-secretases leading to the formation of full-length Aβ1-40/42 peptides.

  domain, APP proteolysis by both β-and γ-secretases leads to the formation of a mutated peptide. The possible mutations are shown in Figure I.B-3.

Figure I.B- 3 :

 3 Figure I.B-3: Familial AD mutations on Aβ1-43. The amino acid residues mutated and the names of the mutations are colored.(1-letter code).[13]

Figure I.B- 4 :

 4 Figure I.B-4: Schematic representation of the amyloid cascade hypothesis with the intervention of metal ions.

Figure I.B- 5 :

 5 Figure I.B-5: Schematic representation of amyloid aggregation (top section) and AFM images (2 × 2 µM) of the Aβ peptide at the oligomeric and fibrillary stages superimposed with the typical sigmoid curve of fibril formation (bottom section). Picture from reference[46] .

(

  TEM) give information about the size and shape of aggregates (see AFM images in Figure I.B-5). I.C. Aβ, metal ions and Reactive Oxygen Species I.C.1. Coordination of Aβ with metal ions

Figure I.C- 1 :

 1 Figure I.C-1:Proposed coordination sites of Zn(II) to the Aβ peptide at pH 7.4.[56]

  5 are shown in Figure I.C-2. For component I, it is now established that Cu(II) is bound to the NH2 terminus, the adjacent CO function from Asp1-Ala2 and to imidazole rings of His6 and either His13 or

Figure I.C- 2 :

 2 Figure I.C-2: Schematic representation of equatorial Cu(II)-Aβ binding sites depending on the pH. The pKa of the different components are indicated in the pH scale (Picture from reference [65]).

Figure I.C- 2 )

 2 Figure I.C-2) is the most accepted model and it will be used as the component II model thereafter.

  Cu(I) coordination to the Aβ peptide Copper is a redox-active ion which is present physiologically in two redox states: Cu(I) and Cu(II). Cu(I) coordination with Aβ has been investigated more recently than Cu(II) coordination and the involvement of histidine residues is now consensual. Several binding models are suggested, two of them being most populated (Figure I.C-3).

Figure I.C- 3 :

 3 Figure I.C-3: Schematic view of the two proposed models for Cu(I) coordination in the Aβ peptide.Adapted from reference[66] .

I.C. 2 .

 2 Reactive Oxygen Species a. ROS and oxidative stress Reactive oxygen species (ROS) are radicals and molecules deriving from the incomplete reduction of dioxygen. They are produced in small quantity during the in vivo metabolism of oxygen, through four successive 1-electron reductions of O2 leading to H2O formation (Figure I.C-

Figure I.C- 4 :

 4 Figure I.C-4: Schematic view of the ROS production during oxygen reduction (black pathway) and the enzymes involved in ROS detoxification (blue pathways)

Figure I.C- 5 : ( 1 )

 51 Figure I.C-5: (1) Dismutation of superoxide into dioxygen and hydrogen peroxide catalyzed in living systems by SOD. (2) Dismutation of hydrogen peroxide into dioxygen and water catalyzed in living systems by catalase. (3) Hydrogen peroxide reduction catalyzed by the glutathione peroxidase (GSH). GS-SH: Glutathione disulfide.

Figure I.C- 6 : ( 1 )

 61 Figure I.C-6: (1) Fenton reaction (2) Haber-Weiss reaction catalyzed by iron ions.

Figure I.C- 7 :Figure I.C- 8 :

 78 Figure I.C-7: Mechanism of ROS production from a reductant and dioxygen catalyzed by the Cu-Aβ complex. The ROS produced are the superoxide anion (O2 • ), hydrogen peroxide (H2O2) and the hydroxyl radical (HO • ).

acid residue 3

 3 reported the damages undergone by the Aβ peptide during the copper-mediated oxidation. The amino acid residues damaged are summarized in Figure I.C-9 and further described in the following paragraphs.

Figure I.C- 9 :

 9 Figure I.C-9: Schematic view of the different oxidative modifications (black circle), cleavages (blue arrows) and interactions (red arrows) undergone by the Aβ42 peptide during the copper-mediated oxidation (from reference [87]).

Figure I.C- 10 :

 10 Figure I.C-10: Structural formula of histidine (left) and 2-oxo-histidine (right).

Figure I.C- 11 : 2 -

 112 Figure I.C-11: 2-oxo-histidine formation from the oxidative attack of hydroxyl radical at the C-2 positionof the imidazole ring of Histidine[105]

  Figure I.C-12 shows an oxidative mechanism leading to the formation of either pyruvate (blue pathway), isocyanate (red pathway) or 2hydroxyaspartate (green pathway) function through the formation of an alkoxyl radical.

Figure I.C- 12 :

 12 Figure I.C-12: Mechanism of aspartate oxidation with three different pathways starting from alkoxyl radical and leading to the formation of 2-hydroxyaspartate (green), isocyanate (red) and pyruvate (blue).

Ultra 3

 3 kDa membrane (Millipore), washed with Ethylenediaminetetraacetic acid (EDTA) (10 equivalents) to remove copper, then with water and finally with a NaOH (50 mM, pH ≈ 13) solution. The oxidized peptide solution is recovered and the concentration is determined by UV-visible absorption of Tyr10, considered as free tyrosine ((ε293-ε360) = 2400 M -1 cm -1 ) in NaOH (50 mM, resulting pH ≈ 13). As the oxidized peptide solution has a background absorbance at 293 nm from an unknown origin, the curve is fitted to subtract the absorbance due to the tailing (Figure II.A-2). The Aβ28 peptide was used instead of Aβ16 for technical reasons (membrane cut-off of 3 kDa).

Figure II.A- 2 :

 2 Figure II.A-2: UV-Vis spectrum of oxidized Aβ28 (black curve) and fit (blue curve) to subtract the background absorbance from Tyr absorbance.

  technique was first dedicated to proteins studies as it may produce multiple charged ions from large molecules owning several ionizable sites, thus improving the sensitivity and extending the mass range of the analyzer for the study of high molecular weight molecules such as proteins and peptides. Nowadays, ESI is also used for the study of smaller molecules. The principle of electrospray ionization is illustrated in Figure II.B-1.

Figure II.B- 1 :

 1 Figure II.B-1: Principle of electrospray ionization.

  ionizable sites, the formation of multiple charged ions can occur, by addition/removal of several protons on the same molecule. Usually, the ions are either under the form [M + nH] n+ in positive mode or [M -nH] n-in negative mode, where n is the number of protons. They are observed at the mass M+1 or M-1 for monocharged ions or more generally at the mass (M+n)/n or (M-n)/n for n-times charged ions. Thus, the technique is not suitable for molecules without protonation sites such as apolar compounds. Other types of ions can be detected such as sodium ([M + Na] + ) or potassium ([M + K] + ) adducts and in some cases, ammonium adduct [M + NH4] + . Furthermore, it is likely to detect dimers that are due to the bridging of two monomers by a proton (or Na + /K + ). Those dimers are under the form [MHM] + or [MHM]  and observed at the mass 2M+1 or 2M-1 for monocharged ions. II.B.3. Ion trap a. Principle In 1960, W. Paul (Nobel Prize in Physics 1989

  them to the detector by deflecting them through an electric field. The ion trap consists of two hyperbolic electrodes called endcap electrodes and a hyperbolic ring electrode between them (Figure II.B-2).Ions come through the entrance endcap electrode and are trapped in the ion trap center by an oscillating electric field that plays on the ion trajectory, allowing to isolate the ion into the trap or to eject it towards the detector. Actually, an appropriate variation of the electric field allows to specifically destabilized the ion trajectory in order to eject the ions one by one. This oscillating electric field originates from alternating voltages applied on the endcap electrodes (negative voltage) and on the ring electrode (positive voltage).

Figure II.B- 2 :

 2 Figure II.B-2: Schematic representation of an ion trap.

  repeated several times (up to 10 on most of the commercially available spectrometers) and is called MS n process (Figure II.B-3). However, for each additional fragmentation, a little part of the ions is lost in the trap and the ions detected are less intense and the technique is less sensitive.

Figure II.B- 3 :

 3 Figure II.B-3: Schematic view of the ion trap operation for successive fragmentation mode (MS n ).

  and marketed since 2005, is an ion trap analyzer composed of an inner spindle-shaped electrode and an outer barrel-shaped electrode, both subjected to a direct voltage. The ions are injected tangentially into the field, trapped in the orbitrap and they adopt an orbital motion around the spindle-shaped electrode. The specific geometry of the trap and the electrostatic attraction towards the inner electrode, compensated by the ions inertia, force the ions to cycle around the inner electrode in complex spiral patterns (Figure II.B-4, red arrow). The axial component of the resulting oscillations is independent of initial parameters of ions such as initial kinetic energy of injected ions. However, its frequency is proportional to (m/z) -1/2 . Thus, the axial oscillations of the ion are detected as an image current induced in the outer electrode and transformed into mass spectra using Fourier Transform. The orbitrap delivers low-ppm mass accuracy with high resolution (up to 150 000 for ions produced by laser ablation) and is thus employed to do High-Resolution Mass Spectrometry (HRMS).

Figure II.B- 4 :

 4 Figure II.B-4: Schematic view of the orbitrap. The red arrow shows the complex spiral trajectory of the ion around the inner electrode.

  peptide bond breakage and thus mainly to the formation of b and y ions (Figure II.B-5, orange arrows). By detecting and combining the series of ions, the sequence of the protein/peptide can be determined. II.B.4. Analysis of Aβ by MS and MS/MS a. MS/MS Tandem Mass Spectrometry is a relevant tool for protein/peptide sequencing. It can also provide information and allow for identification of a modified amino acid residue in a polypeptide chain, since any modification will affect the masses of the corresponding b and y ions. Thus, the characterization of the oxidation sites of the Aβ peptide can be carried out by analyzing the oxidized peptide by MS/MS (usually coupled to liquid chromatography, LC-MS/MS), the b and y ions detected providing information about a mass shift on one amino acid residue of the sequence, related to its oxidation. However, CID fragmentation is usually less efficient as the mass of the molecule increases and the direct fragmentation of the whole Aβ40 peptide (top-down fragmentation) did not provide reliable results with the spectrometer we used. Thus, a proteolytic digestion was realized in order to cleave the Aβ peptide into smaller peptides, more easily fragmented (bottom-up fragmentation). Trypsin was chosen for digestion and cleaves peptides at the carboxyl side of the lysine or arginine residues, except when they are followed by a proline residue. The four tryptic peptides obtained from Aβ40 digestion by trypsin are shown in Figure II.B-6, and their monoisotopic masses (m/z) used for their detection are listed in Table II.B-1 Table II.B-1 along with the ones of the Aβ16, Aβ28 and Aβ40 peptides. A complete table listing the monoisotopic masses of the peptides studied as well as their oxidized counterparts is shown in Annex I.

Figure II.B- 6 :

 6 Figure II.B-6: Sequence of the four tryptic peptides obtained after the Aβ40 digestion by trypsin.

Figure

  Figure II.C-1 is a schematic representation of an UV-Vis experiment. The UV-Vis light (or beam) with an initial intensity I0 is created at the light source and goes through the sample. Upon light excitation, the energy given by the absorbed photon allows the transition of an electron from its ground state to an excited state. This energy has to correspond to the energy of permitted transitions (Figure II.C-1, green arrow in energy level diagram). Then, the light exiting from the sample with a transmitted intensity It is analyzed at the detector. An UV-Vis spectrum is obtained with absorption or transmission of the light (see definitions beyond) as a function of the wavelength.

Figure II.C- 1 :

 1 Figure II.C-1: A schematic view of an UV-Vis spectroscopy experiment.

-

  Low concentration of the compounds in the solution (A < 1, i.e. at least 10% of light is transmitted) -Solution studied not fluorescent or heterogeneous -No photochemical transformations of the compounds studied -No interaction between the compounds studied and the solvent The absorbance is an additive value. If for a given wavelength, two compounds 1 and 2 absorb, the total absorbance is equal to the sum of the absorbance of each compound, as given by Equation II.C-4. 𝑨 = 𝑨 𝟏 + 𝑨 𝟐 = 𝒍 (𝜺 𝟏 . 𝒄 𝟏 + 𝜺 𝟐 . 𝒄 𝟐 ) Equation II.C-4 II.C.2. Ascorbate consumption The Reactive Oxygen Species (ROS) produced by the Aβ/copper/ascorbate system can indirectly be monitored by ascorbate consumption. As discussed above (see Section I.C.2.b), ascorbate (Figure II.C-2a) gives an electron to reduce Cu(II)-Aβ and thus produce ROS. The decrease of ascorbate concentration can be monitored by UV-Visible spectroscopy as ascorbate has a maximal absorption in UV at 265 nm (Figure II.C-2b).

Figure

  Figure II.C-2: (a) Structure of L-ascorbate anion and (b) UV-Visible spectrum of ascorbate (0.1 mM) in phosphate buffer (50 mM, pH 7.4) (right panel).

  cyclic molecules named fluorophores. Upon a specific light excitation, they can re-emit light at a higher wavelength. The Jablonski diagram illustrates the fluorescence process (Figure II.D-1).

Figure II.D- 1 :

 1 Figure II.D-1: Jablonski diagram.

Figure II.D- 2 :

 2 Figure II.D-2: Schematic spectrum of excitation (blue curve) and emission (red curve) of a fluorophore, highlighting the Stokes shift (difference between maximum wavelengths of emission and excitation).

A

  fluorophore is characterized by its excitation and emission wavelengths (Figure II.D-2) and by the quantum yield (Φf), the lifetime (τ) and the fluorescence intensity (If). The quantum yield characterizes the efficiency of fluorescence compared with the other deactivation pathways for a given fluorophore. It is directly related to the ratio between the number of photons emitted and number of photons absorbed which is turned into the ratio of the fluorescence intensity (If) and the absorption intensity (Ia) (Equation II.D-1). The value of quantum yield is between 0 and 1, the best fluorophores having a quantum yield close to 1.Moreover, the quantum yield varies with the environment of the fluorophore such as concentration, pH and the nature of the solvent.

Figure

  Figure II.D-3: Coumarin-3-carboxylic acid (left) and 7-hydroxycoumarin-3-carboxylic acid (right) structures.

Figure II.D- 4 :

 4 Figure II.D-4: Excitation spectrum (black curve, λemission = 450 nm) and emission spectrum (blue curve, λexcitation = 395 nm) of the 7-hydroxycoumarin-3-carboxylic acid (50 µM) in phosphate buffer at pH 7.4.

Figure II.D- 5 :

 5 Figure II.D-5: (a) Structure of ThT (top) and representation of the rotation of the bond between the benzothiazole moiety (pink) and aniline moiety (blue) (bottom). (b) "Channel" model of ThT binding to fibril-like β-sheets. ThT is proposed to bind along surface side-chain grooves running parallel to the long axis of the β-sheet. Pictures from Reference[16]

  The 1 H nucleus can occupy 2 energy levels (2I+1) related to two spin alignments to 0 B : parallel (magnetic quantum number m = ½) or anti-parallel (m = -½) (Figure II.E-1). The level of lower energy is slightly more populated by nuclei than the other one, resulting in a non-zero magnetic moment vector sum called magnetization ( M ) parallel to 0 B (Figure II.E-2a).

Figure II.E- 1 :

 1 Figure II.E-1: Energy level of spin states for a nucleus with a spin I = ½ (such as 1 H) subjected to a magnetic field 0 B .

Figure II.E- 2

 2 Figure II.E-2 Schematic view of the different phenomena during a 1 H NMR experiment.

Figure II.E- 3 :

 3 Figure II.E-3: Fourier transform of the Free Induction Decay (FID) into NMR spectrum.

For 1 H

 1 NMR experiments, the tetramethylsilane (TMS) is used as reference with its 12 protons that have the same chemical shift, far from the one of the protons usually studied.II.E.2. Application to Aβ chemical structure study a. Samples preparationAβ28 peptideA stock solution of Aβ28 2 mM was diluted with D2O to reach a peptide concentration of 0.4 mM, and the pH was adjusted to 10.5 with NaOD 1 mM.Aβ28ox peptideA stock solution of Aβ28ox 2.7 mM (for preparation, see Section II.A.3) was diluted with D2O to reach a peptide concentration of 0.4 mM, and the pH was adjusted to 10.5 with NaOD 1 mM.A volume of 600 µL is required to run the NMR experiment.b. NMR conditionsThe 1 H NMR experiments were recorded on a Bruker Avance 500 spectrometer equipped with a 5 mm triple resonance inverse Z-gradient probe (TBI 1 H,31 P, BB). The presaturation of the water signal was achieved with a zqpr sequence (Bruker).1 H NMR experiments are performed at 298K. The parameters for the acquisition are d1 = 30 and p1 = 6.6 µs. II.F. Electron Paramagnetic Resonance II.F.1. General principles Electron Paramagnetic Resonance (EPR) is an absorption spectroscopy which has similarities with NMR. The electromagnetic waves interact with the magnetic moment associated with electrons and not with nuclei. Yevgeny Zavoisky and Brebis Bleaney have developed independently EPR at the same time in 1944. EPR is mainly employed for studying compounds with an impaired number of electrons such as radicals and paramagnetic metal complexes.An electron is characterized by the spin quantum number S = ½, the magnetic moment μ and the two spin states ms = -½. and ms = + ½. In the absence of a magnetic field, the electron spin states are degenerated. However, when subjected to a magnetic field (B0), the magnetic moment of the electron aligns itself parallel (ms = -½) or antiparralel (ms = + ½) to B0. It results in a removal of degeneracy, the spin states having thus two different energies, ms = -½ having the lower one (Figure II.F-1). Those energy levels are called Zeeman levels.

Figure II.F- 1 :

 1 Figure II.F-1: Energy level of spin states for an electron subjected to a magnetic field 0 B .

Figure

  Figure II.F-2 shows a schematic view of an EPR equipment. A microwave source (usually a Gunn diode) provides the electromagnetic wave of fixed frequency ν, which goes through an attenuator, and is sent to the sample cavity by passing into the circulator. The sample cavity is surrounded by a magnet which generates the static magnetic field 0 B . The resulting

Figure II.F- 2 :

 2 Figure II.F-2: Schematic representation of an EPR equipment.

Figure II.F- 3 :

 3 Figure II.F-3: Example of EPR spectrum. (Top) absorption and (bottom) first derivative as a function of the magnetic field strength.

  3 different Landé factors (gx, gy, gz, described by the Equation II.F-2) characterize the system, resulting in 3 signals at respectively Bx,res, By,res and Bz,res values of magnetic field on the EPR spectrum. 𝒈 𝒙/𝒚/𝒛 = 𝒉𝝂 𝝁 𝑩 . 𝑩 𝒙/𝒚/𝒛 𝒓𝒆𝒔 Equation II.F-2

  the system, resulting in two different signals centred at B // res and B  res values of magnetic field. 𝒈 // = 𝒉𝝂 𝝁 𝑩 . 𝑩 // 𝒓𝒆𝒔 Equation II.F-4 𝒈  = 𝒉𝝂 𝝁 𝑩 . 𝑩  𝒓𝒆𝒔 Equation II.F-5 As Cu(II) has an electron spin of S = ½ and a nuclear spin of I = 3/2, the hyperfine coupling will result in four signals (2I+1) for each Zeeman transition. The signal related to g // is subdivided into 4 distinct signals, as shown in Figure II.F-4. The hyperfine coupling constant A // can be calculated from the spectrum, with the Equation II.F-6. 𝑨 // = 𝓐 // 𝒈 // 𝝁 𝑩 𝒉 𝒄 Equation II.F-6

  Figure II.G-1 shows theBohr model which illustrates the phenomenon. When the electron is excited, a core hole is created, filled in approximately a femto-second after the relaxation of an electron from a highenergy electron orbital, with a release of energy, leading in some cases to fluorescence light emission. Depending on the X-ray energy, the excited electron can come from K-shell, L-shells or M-shells. The closer the electron from the nucleus, the higher the energy required to excite it.

Figure II.G- 1 :

 1 Figure II.G-1: Bohr model of an atom with the different electron shells.

  an example, Figure II.G-2 shows a spectrum of the X-ray absorption of NiO as a function of the energy, as along with the X-ray Absorption Near Edge Structure (XANES) and the Extended X-ray Absorption Fine Structure (EXAFS) regions of the spectrum.

Figure II.G- 2 :

 2 Figure II.G-2: NiO X-ray absorption spectrum highlighting the XANES and EXAFS regions.

using

  Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) and to High Resolution Mass Spectrometry (LC-HRMS), in order to characterize the oxidative damages on Aβ and to identify the oxidized amino acid residues. The study is complemented by proton Nuclear Magnetic Resonance ( 1 H NMR) analysis of the oxidized and non-oxidized Aβ peptide. The kinetics of the amino acid residues oxidation of Aβ40 is finally monitored by LC-HRMS. All these studies may provide information on the chemical modifications undergone by the amino acid residues of Aβ during oxidation, and will be further useful for studying the consequence of Aβ oxidation regarding Reactive Oxygen Species (ROS) production and aggregation, two events implicated in Alzheimer's Disease. III.A. Characterization of the oxidation sites During the ROS production catalyzed by the Cu-Aβ complex, the Aβ peptide is targeted by ROS. The amino acid residues damaged by ROS undergo chemical modifications, leading to a change of their mass. Thus, the characterization of Aβ oxidation can be investigated by MS. After Metal Catalyzed Oxidation (MCO) of Aβ by the copper/ascorbate/O2 system, purification and tryptic digestion, the sample is analyzed by LC-HRMS in order to measure the exact mass of every tryptic peptides and thus deduce the nature of the chemical modification undergone. Then, LC-MS/MS analysis of the sample is carried out in order to sequence the peptide and identify the amino acid residues targeted by ROS. The combination of HRMS and MS/MS is a great source of information for the investigation of the oxidation sites on the Aβ peptide. III.A.1. Experimental section Copper(II)-catalyzed oxidation of the Aβ40 peptide was carried out by mixing Aβ40, Cu(II) and ascorbate in phosphate buffer (50 mM, pH 7.4) to reach final concentrations of respectively 60, 50 and 500 μM (sub-stoichiometry of copper to avoid free Cu(II) in solution) in a reaction mixture volume of 100 μL. Incubation was done at room temperature for at least 15 min. Trypsin digestion was then realized in order to obtain the four tryptic peptides described above (see Section II.B.3). LC-HRMS and LC-MS/MS conditions are detailed in Section II.B.4. III.A.2. Results a. Detection of the oxidized tryptic peptides by LC-HRMS The digested oxidized Aβ40 was analyzed by LC-HRMS and the specific modifications due to amino acid residues oxidation were searched. As a reminder, the most common products obtained upon ROS oxidation of Aβ result from: (i) formal addition of one oxygen atom (mass shift of +16 Da), (ii) formal addition of molecular oxygen (+32 Da), (iii) carbonylation (+14 Da), (iv) decarboxylation/deamination of Asp1 (-45 Da), (v) oxidative cleavage of Asp1 (-89 Da), (vi) dityrosine cross-linking (formally -1 Da) (see Section I.C.3 for more information).From the total ion current (TIC) chromatogram obtained, chromatograms were traced from the theoretical monoisotopic masses of each tryptic peptide of Aβ40 and for their oxidized counterparts. Mass spectra were extracted at the retention time where the maximal intensity of the chromatographic peak is detected, showing the experimental monoisotopic masses. Thus, the theoretical and experimental monoisotopic masses were compared to evaluate the accuracy of the detection. All the masses were measured with a mass accuracy of 5 ppm. For the tryptic Aβ1-5 peptide that contains the N-terminal part of the peptide, four different ions are found (Figure III.A-1). First, the ion at m/z 637.2925 corresponds to the mono-protonated non-oxidized Aβ1-5. Ions at m/z 592.2715 and 548.2444, related to mass shifts of -45 Da and -89 Da, are specific mass modifications assigned respectively to the decarboxylation and deamination of Asp1 (DAEFRdd) and to the oxidative cleavage of Asp1 (DAEFRox).

7 Figure

 7 Figure III.A-1: (Top, left panel) Trace chromatograms of DAEFR and its oxidation products DAEFRdd, DAEFRox and DAEFR+16 obtained by LC-HRMS for Aβ40 submitted to MCO for 15 min. (Top, right panel) Mass spectra extracted from the corresponding trace chromatograms at the retention time of maximal intensity of the peak. (Bottom) Theoretical (th) and experimental (exp) monoisotopic masses of native and oxidized Aβ1-5 tryptic peptides and mass difference between experimental and theoretical masses. NL: normalized intensity, RT: retention time (min).

Figure

  Figure III.A-2 shows the mass spectra obtained for the Aβ6-16 tryptic peptide and its two oxidized homologs. The doubly protonated ion at m/z 668.8032 is the major ion of Aβ6-16 detected in the sample. Two other doubly protonated ions are detected at m/z 676.7997 and 684.7979, corresponding to the Aβ6-16 peptide with the formal addition of one and two oxygen atoms, respectively (+ 16 Da and +32 Da).

Figure

  Figure III.A-2: (Top, left panel) Trace chromatograms of HDSGYEVHHQK and its oxidation products HDSGYEVHHQK+16 and HDSGYEVHHQK+32 obtained by LC-HRMS for Aβ40 submitted to MCO for 15 min. (Top, right panel) Mass spectra extracted from the corresponding trace chromatograms at the retention time of maximal intensity of the peak. (Bottom) Theoretical (th) and experimental (exp) monoisotopic masses of native and oxidized Aβ6-16 tryptic peptides and mass difference between experimental and theoretical masses. NL: normalized intensity, RT: retention time (min).

4 Figure

 4 Figure III.A-3: (Top, left panel) Trace chromatograms of LVFFAEDVGSNK and its oxidation product LVFFAEDVGSNK+16 obtained by LC-HRMS for Aβ40 submitted to MCO for 15 min. (Top, right panel) Mass spectra extracted from the corresponding trace chromatograms at the retention time of maximal intensity of the peak. (Bottom) Theoretical (th) and experimental (exp) monoisotopic masses of native and oxidized Aβ17-28 tryptic peptides and mass difference between experimental and theoretical masses. NL: normalized intensity, RT: retention time (min).

9 Figure

 9 Figure III.A-4: : (Top, left panel) Trace chromatograms of GAIIGLMVGGVV and its oxidation product GAIIGLMVGGVV+16 obtained by LC-HRMS for Aβ40 submitted to MCO for 15 min. (Top, right panel) Mass spectra extracted from the corresponding trace chromatograms at the retention time of maximal intensity of the peak.(Bottom) Theoretical (th) and experimental (exp) monoisotopic masses of native and oxidized Aβ29-40 tryptic peptides and mass difference between experimental and theoretical masses. NL: normalized intensity, RT: retention time (min).

  the results obtained highlight the particular sensitivity of Asp1 during Aβ oxidation. The two oxidative damages detected on Asp1 strongly affect the chemical structure of the N-terminal part of the peptide (see Section I.C.3.b for chemical structures). Such a modification would probably have a significant impact on the behavior of the oxidized Aβ peptide regarding copper coordination, ROS production and aggregation.

Figure III.A- 5 :

 5 Figure III.A-5: Fragmentation spectra of doubly protonated ion (m/z 676.9) of the tryptic Aβ6-16 peptide allowing for identification of His13 (top) or His14 (bottom) oxidation. b and y ions series (charge 1+, 2+ and 3+) detected are summarized in the peptide sequence. * indicates an increase of mass of +16 Da on the colored amino acid residue.

Figure III.A- 6 :

 6 Figure III.A-6: Fragmentation spectra of doubly protonated ion (m/z 671.4) of tryptic Aβ17-28 with oxidation on Phe19 (top) or Phe20 (bottom). b and y ions series (charge 1+ and 2+) detected are summarized in the peptide sequence. * indicates an increase of mass of +16 Da on the colored amino acid residue.

Figure III.A- 7 :

 7 Figure III.A-7: Fragmentation spectra of doubly protonated ion (m/z 551.3) of tryptic Aβ29-40 with oxidation on Met35. b and y ions series (charge 1+ and 2+) detected are summarized in the peptide sequence. * indicates an increase of mass of +16 Da on the colored amino acid residue.

Figure III.A- 8 :

 8 Figure III.A-8: (Top) Peptide sequence of Aβ40 with the oxidized amino acid residues (black circle) detected along with the nature of oxidation and the change of mass and (bottom) chemical structure of the oxidized amino acid residues. The 3-hydroxyphenylalanine is shown as an example of phenylalanine oxidation product, but the 2-and 4-hydroxyphenylalanines can also be formed (see Section I.C.3.d).

  Figure III.B-1 shows the area ratio between the oxidized peptide and the equivalent non-oxidized peptide (control at t=0) as a function of reaction time. Such a calculated ratio gives a general tendency of the evolution of the oxidized species throughout the completion of the reaction. It cannot be considered as an accurate quantification because each peptide has a different propensity to get ionized in the ESI source and cannot be formally compared to another one. Decarboxylation-deamination of Asp1 (DAEFRdd) seems to be the major oxidative reaction since the level of DAEFRdd strongly increases with the reaction time. DAEFRox (oxidative cleavage of Asp1) also increases with time, but to a much lesser extent (Figure III.B-1a). The level of DAEFRdd is increasing with time and seem to stop after 8 min. For DAEFRox, the tendency is less clear, but the formation rate of DAEFRox seems also to slow down after 8 min.

Figure III.B- 1 : 1 . 16 (

 1116 Figure III.B-1: Oxidation of the Aβ40 tryptic peptides as a function of the time. Area ratio between the oxidized and the equivalent non-oxidized (control, t=0) tryptic peptide of Aβ40 as a function of the reaction time. (a) Decarboxylation and deamination of Asp1 (DAEFRdd) and oxidative cleavage of Asp1 (DAEFRox); (b) oxidation of His13/His14 (HDSGYEVHHQK+16 and +32); (c) oxidation of Phe19/20 (LVFFAEDVGSNK+16); (d) oxidation of Met35 (GAIIGLMVGGVV+16). Mass tolerance set at 10 ppm.m/z ratios used for detection are specified in TableIII.A-1.

Figure III.C-1 shows the 1 H

 1 Figure III.C-1 shows the 1 H NMR spectra obtained for Aβ28 (black line) and Aβ28ox (blue line) in the four shift ranges of interest: 0.5-2.0, 2.0-3.3, 3.5-4.7 and 6.7-7.9 ppm. Oxidation strongly affects the 1 H NMR signal, as the peaks of Aβ28ox are very broad compared with the one of Aβ28. Thus, only a qualitative comparison is made between Aβ28 and Aβ28ox. The amino acid residues of the C-terminal moiety are not affected by oxidation: Asp23, Val24, Gly25, Ser26 Asn27 and possibly Ala21 and Lys28. For the latter two, their proton signals have similar chemical shifts than Ala2 and Lys16 (around 1.4 ppm). The two doublets of Ala2 and Ala21 form a triplet-like signal and the two doublets of triplets of Lys16 and Lys28 are hidden under the Ala signals. After oxidation, a doublet corresponding to Hβ protons of an Ala residue in observed, hiding a part of another signal, probably a doublet of triplets of the Hγ protons of a Lys residue. Thus, as the amino acid residues close to Ala21 and Lys28 are not affected by oxidation, it is likely that Ala2 and Lys16 are affected by oxidation and Ala21 and that Lys28 are not.

Figure III.C- 1 : 1 H

 11 Figure III.C-1: 1 H NMR spectra of Aβ28 (a) and Aβ28ox (b) at pH 10.5.

  seems to affect the proton signals of the neighboring amino acid residues. As Val and Leu residues (except Val24 which is not affected by oxidation) are close to His13, His14, Phe19 or Phe20, which are found oxidized in MS experiments (see Section III.A), this could explain their broad NMR signals. However, some signals seem to be wider than the ones of Val and Leu residues. In particular, the signals of Hα and Hβ protons of Asp1 (at 3.68 and 2.45 ppm) are almost erased. Moreover, a signal is observed at 2.6 ppm only in Aβ28ox (Figure III.C-1b, red oval). This is likely to be the signal of the three equivalent protons of the methyl group in the pyruvate function formed by the decarboxylation-deamination of Asp1 (see Section I.C.3.b). Thus, the results are in line with the oxidation of Asp1 by decarboxylation-deamination detected in MS (see Section III.A). Signals assigned to Hδ and Hε of His6, His13 and His14 are observed at chemical shifts around 7.6 and 6.9 ppm respectively. Hα and Hβ peaks of His are not noticeable since they have chemical shifts around 3 and 4.5 ppm, as numerous other protons of the peptide (Figure III.C-1) and are thus difficult to isolate. 1 H NMR peaks of His are also strongly affected by oxidation. The same tendency is observed for the other aromatic residues (Phe and Tyr residues).

  (II) coordination to the oxidized Aβ peptide (Aβox) has been investigated by Electron Paramagnetic Resonance (EPR) and X-ray absorption (XANES). The ROS production during metal-catalyzed oxidation (MCO) of Aβ has been studied at different steps of Aβ oxidation. Finally, the impact of Aβ oxidation on fibrils formation has been studied by Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM). IV.A. Cu coordination and ROS production with Aβox This section focuses on the impact of Aβ oxidation on Cu(I) and Cu(II) binding modes and on the ROS production. It is composed of an article published in the journal Metallomics

  l'oxydation du peptide Aβ sur la production des espèces réactives de l'oxygène (ROS) et la coordination avec les ions Cu(I) et Cu(II). En présence d'oxygène et d'un agent réducteur comme l'ascorbate, le complexe cuivre-peptide a la capacité de produire des ROS en cyclant entre les états redox Cu(I) et Cu(II). Dans le chapitre précédent, nous avons montré que le peptide Aβ est une cible privilégiée pour les radicaux hydroxyles ainsi générés et nous avons identifié les acides aminés Asp1, His13 et His14 comme cibles préférentielles du radical hydroxyle. Ils sont tous trois connus pour être impliqués dans la sphère de coordination de Cu(II) et /ou Cu(I) (voir Section I.C.1), comme le montre la Figure IV.A-1.

Figure IV.A- 1 :[ 2 ]

 12 Figure IV.A-1: Modes de coordination du peptide Aβ avec les ions Cu(II) (panel a) et Cu(I) (panel b) à pHphysiologique.[2]

Figure IV.A- 2 :

 2 Figure IV.A-2: Vue schématique de l'attaque oxydative du radical hydroxyle sur le peptide Aβ lors de la production d'espèces réactives de l'oxygène.[2]

  binding mode of Zn(II) might be impacted by Aβ oxidation, as previously observed for Cu(II) and Cu(I) (Section IV.A). In the present study, the impact of Aβ oxidation on Zn(II) coordination is investigated by XANES. Several mutated peptides are employed to mimic the oxidation of each amino acid residue. As Zn(II) has a d 10 electronic configuration, it is "silent" is most of the classical spectroscopic techniques such as UV-Vis and EPR. Hence, XANES belongs to the few techniques suitable for Zn(II) coordination mode investigation. IV.B.1. Experimental section Stock solutions of Aβ28 (sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNK), D1N (sequence NAEFRHDSGYEVHHQK), H13A (sequence DAEFRHDSGYEVAHQK), H6A-H13A (sequence DAEFRADSGYEVAHQK) and H6A-H14A (sequence DAEFRADSG-YEVHAQK) were prepared and titrated (see Section II.A for more details). The preparation, purification and dosage of Aβ28ox is detailed in Section II.A.3. ZnSO4.H2O was purchased from Strem Chemicals. A Zn(II) stock solution (10 mM) was prepared in ultrapure water. HEPES buffer was bought from Sigma and dissolved in ultrapure water to reach a 0.5 M concentration and a pH 5.2 adjusted with NaOH (10 M) to obtain a final pH of 7.1. The methodology and the conditions of the Zn XANES experiments are detailed in Section II.G.2.b.

Figure IV.B- 1 :

 1 Figure IV.B-1: Zn XANES K-edge X-ray absorption spectra of Aβ28-Zn(II) (red curve), AcAβ16-Zn(II) (orange curve), Aβ28ox-Zn(II) (black curve), H13A-Zn(II) (blue curve), (H6A-H13A)-Zn(II) (purple curve), (H6A-H14A)-Zn(II) (green curve) and Zn(II) in buffer (grey curve). The conditions were as follow: 0.9 mM Zn(II), 1 mM peptide in a HEPES buffer (0.1 M, pH 7.1) solution.

Figure

  Figure IV.B-1 shows the Zn K-edge XANES spectra of Zn-Aβ28 (red curve), Zn-Aβ28ox (black curve), Zn-D1N (orange curve), Zn-H13A (blue curve), Zn-(H6A-H14A) (green curve)and Zn in buffer (grey curve). The XANES signatures of several Zn-peptide complexes are very different around 9670 eV, i.e. in the white line region which is very sensitive to the site geometry. In particular, the XANES signatures of Zn-Aβ28ox and Zn-Aβ28 are different between 9660 and 9680 eV, meaning that Aβ oxidation has an impact on Zn coordination, as expected.First, the XANES signature of Zn-D1N is very close to that of Zn-Aβ, suggesting that a modification of the N-terminal part of Aβ does not induce a strong change in Zn coordination.

  fluorescence monitoring is well-suited for kinetic study of Aβ aggregation into fibrils. Both Aβ40ox and Aβ40 solutions incubated at 37°C in the presence of ThT were monitored by fluorescence. Figure IV.C-1 shows the ThT fluorescence as a function of the reaction time for Aβ40 (grey curve) and Aβ40ox (blue curve). For Aβ40, fluorescence starts to increase after around 90 hours, meaning that the peptide aggregates and forms β-sheet structures (fibrils). The fluorescence half time is observed at t ½ = 134 hours. For Aβ40ox, fluorescence remains at the background level all along the 200 hours of the reaction time. This absence of fluorescence increase suggests that oxidation has a strong impact on the aggregation capability of Aβ because Aβ40ox does not form any β-sheet structures.

Figure IV.C- 1 :

 1 Figure IV.C-1: β-sheet formation during Aβ40 (grey curve) and Aβ40ox (blue curve) aggregation. ThT fluorescence as a function of the time of HEPES (50 mM, pH 7.4) buffered solution containing Aβ40 or Aβ40ox (20 µM) and ThT (10 µM) incubated at 37 °C.

Figure IV.C- 2 :

 2 Figure IV.C-2: TEM pictures of Aβ40 (left) and Aβ40ox (right) after 200 h of aggregation at 37°C

Figure IV.C- 3 :

 3 Figure IV.C-3: Structural model for Aβ40 fibrils, obtained from solid state NMR constraints. (a) Representation of a single molecular layer constituted by a double-layered structure, with a parallel βsheets formed by residues 12-24 (orange) and 30-40 (blue). The arrow shows the direction of the long axis of the fibril. (b) Aβ40 viewed down the long axis of the fibril. Green colors correspond to hydrophobic sidechains, magenta to polar, blue to positive and red to negative ones. Picture from Ref[21] .

  Zn(II), Cu(I) and Cu(II) binding modes with Aβox have been probed by XANES for the former two and by EPR for the latter. Aβ oxidation leads to a change in the coordination of the three metal ions. In addition, Cu(I) XANES and Cu(II) EPR results allowed to get relative quantification of the oxidative damages: around 40 % of Asp1 would be damaged and not available anymore for binding while around 40 % of the oxidized species would have two oxidized His. ROS production catalyzed by Cu-Aβox was also investigated. Cu-Aβox has a higher catalytic activity and released more HO • than Cu-Aβ. The change in ROS production occurred at a specific step of Aβ oxidation, when the peptide is sufficiently oxidized to induce a change in Cu coordination. Both aggregation monitoring by ThT fluorescence and sample imaging by TEM highlight a strong modification of the Aβ aggregation process, leading to amorphous-like aggregates instead of fibrillar aggregates. These preliminary results have to be validated by further controls and future experiments could be carried out with mutated peptides in order to determine if one specific amino acid residue oxidation in particular affects the aggregation process. Zn and Cu binding modes with Aβ were also strongly affected by Aβ oxidation. As Zn

FigureFiguresFigure S1 :Figure S2 :

 S1S2 Figure S8 and S9.

Figure S3 :

 S3 Figure S3: Remaining Aβ28 peptide. Trace chromatograms of Aβ28 (60 µM) after 30 min in the presence of Cu (50µM) (left panel) or Cu (50 µM) and ascorbate (0.5 mM) (right panel) in phosphate buffer pH 7.4 (50 mM). Aβ28 is detected with the [M+3H] 3+ , [M+4H] 4+ and [M+5H] 5+ m/z ratio ions: 1087.8503, 816.1397 and 653.1133. Mass tolerance set at 5 ppm. Ratio between the chromatogram area of Aβ28-Cu(II) with ascorbate to chromatogram area of Aβ28-Cu(II) without ascorbate is around 0.2, meaning that around 80% of Aβ28 is oxidized (at least one amino acid residue is oxidized).

Figure S4 :

 S4 Figure S4: Linear Combination Fitting of A28-Cu(I) and Aβ7-Cu(I) to reproduce Aβox-Cu(I) spectrum. The fitting allows deducing that around 40% of Cu(I) is bound the same way than Aβ7 (bearing only one His), and that no Cu(I) is released in buffer (0% of Cu(I)-HEPES contribution). Quantitative results of linear combination fitting (Right panel) and the resulting XANES spectra with 42% Cu(I)-Aβ7 and 58% Cu(I)-Aβ28 XANES signatures (blue curve) compared to Cu(I)-Aβox XANES spectrum (grey curve). Cu(I)-Aβ7 (green curve) and Cu(I)-Aβ28 (red curve) XANES signatures, weighted according to the result found in the linear combination fitting, are also shown (Left panel).

Figure S5 :

 S5 Figure S5: EPR spectra of Cu(II)-Aβox (black line) and Cu(II)-Aβ28 (grey line) at pH 7.4. Aqueous solution with 10% of glycerol containing 65 Cu (450 µM) and peptide (500 µM). T = 120 K, ν = 9.5 GHz, microwave power = 20.5 mW, Amplitude modulation = 10.0 G, Modulation frequency = 100 kHz.

Figure S6 :

 S6 Figure S6: Linear combination fitting of 60% Cu(II)-Aβ28 and 40% Cu(II)-AcAβ28 (red line) compared to Cu(II)-Aβox signature (black line).

Figure S7 :

 S7 Figure S7: Oxidation of Asp1 (DAEFRdd) in the presence or absence of H2O2. Trace chromatograms of the Aβ oxidized tryptic peptide DAEFRdd (decarboxylation and deamination of Asp1), after successive additions of ascorbate (4 nmol, final conc. 20 µM; left panel) or ascorbate/H2O2 (4/10 nmol, final conc. 20/50 µM; right panel). A28 25 µM and Cu(II) 20 µM, phosphate buffered pH 7.4 (50 mM). Mass tolerance set at 5 ppm; m/z ratios used for detection are specified in TableS1.

Figure S8 :

 S8 Figure S8: Oxidation of Asp1 (DAEFRox) in the presence or absence of H2O2Trace chromatograms of the oxidized Aβ tryptic peptide DAEFRox (oxidative cleavage of Asp1), after successive additions of ascorbate (4 nmol, final conc. 20 µM; left panel) or ascorbate/H2O2 (4/10 nmol, final conc. 20/50 µM; right panel). A28 25 µM and Cu(II) 20 µM, phosphate buffered pH 7.4 (50 mM). Mass tolerance set at 5 ppm; m/z ratios used for detection are specified in TableS1.

Figure S9 :

 S9 Figure S9: Oxidation of His (HDSGYEVHHQK+16) in the presence or absence of H2O2.Trace chromatograms of the oxidized Aβ tryptic peptide HDSGYEVHHQK+16 (formal addition of an oxygen atom), after successive additions of ascorbate (4 nmol, final conc. 20 µM; left panel) or ascorbate/H2O2 (4/10 nmol, final conc. 20/50 µM; right panel). A28 25 µM and Cu(II) 20 µM, phosphate buffered pH 7.4 (50 mM). Mass tolerance set at 5 ppm; m/z ratios used for detection are specified in TableS1.

Figure S10 :

 S10 Figure S10: MCO of Aβ in the presence of H2O2, identification of oxidized His13. Series of b and y ions (charge 1+ and 2+) used for the identification of the oxidation of His13, and corresponding MS/MS spectrum of the doubly protonated ion of HDSGYEVHHQK+16. Same experimental conditions as the ones of Figure S8.

Figure S11 :

 S11 Figure S11: MCO of Aβ in the presence of H2O2, identification of oxidized His14. Series of b and y ions (charge 1+ and 2+) used for the identification of the oxidation of His14, and corresponding MS/MS spectrum of the doubly protonated ion of HDSGYEVHHQK+16. Same experimental conditions as the ones of Figure S8.

Figure S12 :

 S12 Figure S12: Fluorescence curves of phosphate buffered solutions (50 mM) containing Cu (20 µM), Aβ peptide (25 µM, except the green curve: 50 µM), CCA (0,5 mM) with additions of ascorbate (20 µM) and hydrogen peroxide (50 µM) realized every 15 min during 2 hours.

Figure S13 :

 S13 Figure S13: Fluorescence at the plateau of phosphate buffered solution (50 mM) containing Cu (20 µM), Aβ peptide (25 µM), CCA (0,5 mM) as a function of the number of ascorbate and H2O2 additions. A total of 8 additions of 2 µL ascorbate (2 mM) and 2 µL hydrogen peroxide (5 mM) are realized, i.e. respectively 4 and 10 nmol for each addition (initial concentrations reaching 20 and 50 µM for the first addition, respectively).

Figure V.B- 1 :

 1 Figure V.B-1: Vue schématique de l'équilibre entre les états "resting" (au repos) et "in-between" du complexe Cu-Aβ pendant la production de ROS.

en position 13 et 14 ,

 14 qui sont impliqués dans la coordination de Cu(I) ou/et de Cu(II) au repos (voir Section I.C.1), sont les principaux acides aminés retrouvés oxydés après la production de ROS. Il est donc proposé que ces 3 acides aminés sont également les ligands du cuivre dans l'état IB qui est responsable de la production de ROS. La Figure V.B-2 schématise les réarrangements entre les états au repos, appelés états « resting » et les états IB, ainsi que les transferts électroniques entre Cu(II)-Aβ et l'ascorbate et entre Cu(I)-Aβ et le peroxyde d'hydrogène.

Figure V.B- 2 :

 2 Figure V.B-2: Représentation schématique des modes de coordination de Cu(II)-Aβ et Cu(I)-Aβ au repos (resting states) et la proposition de mode de coordination de Cu(I/II)-Aβ dans l'état « in-between ».[3]

Figure V.B- 3 :

 3 Figure V.B-3: Représentation schématique du mode de coordination proposé de Cu(I/II)-Aβ dans l'état « in-between » lors de la production de ROS en présence d'un substrat.

Figure V.B- 4 :

 4 Figure V.B-4: Mécanisme proposé des transferts électroniques entre l'ascorbate et le complexe Cu(II)-Aβ et entre le peroxyde d'hydrogène et le complexe Cu(I)-Aβ, réalisés par le passage vers des modes de coordination du complexe Cu-Aβ transitoires appelés états « in-between ». L'amine terminale et le groupe carboxylate de l'Asp1 sont indiqués en bleu et le motif imidazole de l'His impliquée dans l'état IB est indiqué en violet.

Figure VI.B- 1 :

 1 Figure VI.B-1: Effets pro-oxydants de l'ascorbate menant à la production de ROS catalysée par le cuivre (voie rouge) et effets anti-oxydants de l'ascorbate menant à la formation d'espèces non radicalaires (voie verte).

Figure VI.B- 2 :

 2 Figure VI.B-2: Structures chimique de l'acide coumarine-3-carboxylique (nommé CCA, panel a) et l'acide 7-hydroxycoumarine-3-carboxylique (nommé 7-OH-CCA, panel b).

Figure VI.B- 3 :

 3 Figure VI.B-3: Représentation schématique des effets pro-et anti-oxydants de l'ascorbate et conséquences sur les cibles biologiques environnantes et sur le peptide Aβ.AscH -est l'abréviation de l'ascorbate.[1]

Figure S1 :

 S1 Figure S1: Schematic view of the proposed Cu(II) binding site in Aβ in component I (left) and II (right).pKa (I/II) = 7.8[6]

  established a new methodology to take into account this possible pH drift: (i) For the gradient measurement (Figure2in Full Text), we have verified that the pH drift is negligible even at high ascorbate concentration. This is due to the weak ascorbate consumption during the first minutes of the experiment. (ii) For the plateau measurement (FigureS5and figure 3 in the Full Text), we have established a new protocol in which the pH was raised at the end of the reaction to pH 8.5, a region where 7-OH-CCA fluorescence is weakly sensitive to pH changes.Additionally, we have also verified that the rate of HO • trapped by CCA was independent of pH in the range of pH 7.0 to 7.4, where 7.0 is the minimal value of final pH obtained after experiment with high ascorbate concentration (FigureS4). Thus, the value of the measured fluorescence is proportional to the HO • trapped at pH 7.4. -3-carboxylate / 7-oxidocoumarin-3-carboxylate (pKa 7.5)

Figure S3 :

 S3 Figure S3: Fluorescence at 450 nm as a function of 7-OH-CCA concentration, after excitation at 390 nm. Phosphate-buffered solution (50 mM, pH 7.4) of 7-OH-CCA (concentration range from 0 to 0.5 mM).

Figure S4 :

 S4 Figure S4: UV-Visible spectra of the 7-OH-CCA (50 µM) in a phosphate buffered solution (50 mM) with pH increased from 5.9 to 9.4 at 25°C.

Figure S5 :

 S5 Figure S5: Determination of the pKa of the hydroxyl functional group of the 7-OH-CCA. Absorbance is plotted as a function of the pH of the acidic form absorbing at λmax = 338 nm (grey crosses) and the basic form absorbing at λmax = 388 nm (black crosses).

Figure S6 :

 S6 Figure S6: Impact of the pH on the formation of 7-OH-CCA during the metal-catalyzed production of the HO • . Reaction of Cu (50 µM), ascorbate (0.5 mM for grey panel and 1 mM for dark panel) in phosphate buffered solution (50 mM, pH 6.5, 7.0, 7.4, 8.0 or 8.5) containing CCA (0.5 mM). At the end of the reaction, the pH is ajusted to 8.5 with POPSO buffer (0.4 M, pH 9.0).

Figure S7 :

 S7 Figure S7: Reaction of Cu (50 µM, black curves) or Cu-Aβ16 (50-60 µM, grey curves) with ascorbate (0.5 mM, in phosphate buffered solution, pH 7.4). Left: Ascorbate consumption followed using UV (absorption of ascorbate at λmax = 264 nm) as a function of the time. Right: Fluorescence of the 7-OH-CCA produced by HO • trapping by CCA (0.5 mM) as a function of the time. When ascorbate is fully consumed (see left figure), POPSO buffer is added to increase the pH to 8.5. Arrow indicates the addition of POPSO on dotted curve.

Figure S8 :Figure S9 :

 S8S9 Figure S8: Trace chromatograms of the non-oxidized Aβ28 peptide (60 µM) in the presence of Cu II (50 µM) and ascorbate (0, 0.1, 0.2, 0.3, 0.4, 0.5 or 3.5 mM) at the end of the reaction. Mass tolerance set at 5 ppm; m/z ratios used for detection are specified in blue in table S1.

Figure S10 :Figure S11 :

 S10S11 Figure S10: Initial rates of 7-OH-CCA fluorescence at 450 nm, reflecting the scavenging of HO • by CCA. Phosphate-buffered solution (50 mM, pH 7.4) of CCA (0.5 mM), Cu (50 µM), ), Aβ16, Aβ28 or Aβ40 (60 µM)and ascorbate (concentration between 0 and 5 mM).

Figure 1 :

 1 Figure 1: Chemical structure of Aβ28 sequence along with the atom identifiers of each amino acid residue.

Figure 2 : 5 .Figure 5 :

 255 Figure 2: 1H NMR spectra of Aβ28 (top) and Aβ28ox (bottom) at 400 µM, pH 10.5.

Figure 1 :

 1 Figure 1 : Production d'espèces réactives de l'oxygène (ROS) catalysée par le complexe Cu-Aβ en présence d'ascorbate (Asc). L'anion superoxyde (O2 •-), le peroxyde d'hydrogène (H2O2) et le radical hydroxyle (HO • ) sont formés à partir du dioxygène.

Figure 2 :

 2 Figure 2 : Séquence peptidique de Aβ40 avec les acides aminés oxydés (cercles noirs) détectés ainsi que la nature de l'oxydation et le changement de masse (en Da) et structure chimique des acides aminés oxydés. La 3-hydroxyphenylalanine est montrée comme exemple de produit d'oxydation de la phénylalanine, mais les 2-et 4-hydroxyphenylalanines pourraient également être formés.

Figure 3 :

 3 Figure 3 : Oxydation des peptides obtenus après digestion trypsique de Aβ40 en fonction du temps. Ratio des aires entre le peptide trypsique de Aβ40 oxydé et son equivalent non-oxydé (contrôle, t=0) en fonction du temps de réaction. (a) Décarboxylation et déamination de Asp1 (DAEFRdd) et clivage oxydatif de Asp1 (DAEFRox); (b) oxydation de His13/His14 (HDSGYEVHHQK+16 et +32); (c) oxydation de Phe19/20 (LVFFAEDVGSNK+16); (d) oxydation de Met35 (GAIIGLMVGGVV+16).

Figure 4 :

 4 Figure 4 : Modes de coordination du peptide Aβ avec les ions Cu(II) (panel a) et Cu(I) (panel b) à pH physiologique.

Figure 5 :

 5 Figure 5 : Spectre XANES au seuil du Cu de Aβ-Cu(I) (courbe noire), Aβox-Cu(I) (courbe bleue) et Aβ7-Cu(I) (courbe grise). Les flèches indiquent l'évolution de la signature de Aβox-Cu(I) comparée à celle de Aβ-Cu(I).

Figure 6 : 5 .

 65 Figure 6 : Spectres RPE de Cu(II)-Aβox (courbe bleue), Cu(II)-Aβ (courbe noire) et Cu(II)-AcAβ (courbe grise) à pH 12,5.

Figure 7 :

 7 Figure 7 : Vue schématique de l'attaque oxydante du radical hydroxyle sur le peptide Aβ lors de la production d'espèces réactives de l'oxygène.

Figure 9 :

 9 Figure 9 : Vue schématique de l'équilibre entre les états "resting" (au repos) et "in-between" du complexe Cu-Aβ pendant la production de ROS.

Figure 10 :

 10 Figure 10 : Représentation schématique des modes de coordination de Cu(II)-Aβ et Cu(I)-Aβ au repos (resting states) et la proposition de mode de coordination de Cu(I/II)-Aβ dans l'état « in-between ».

Figure 11 :

 11 Figure 11 : Représentation schématique du mode de coordination proposé de Cu(I/II)-Aβ dans l'état « inbetween » lors de la production de ROS en présence d'un substrat.

Figure 12 :

 12 Figure 12 : Mécanisme proposé des transferts électroniques entre l'ascorbate (Asc -) et le complexe Cu(II)-Aβ et entre le dioxygène et le complexe Cu(I)-Aβ, réalisés par le passage vers des modes de coordination du complexe Cu-Aβ transitoires appelés états « in-between ». L'amine terminale et le groupe carboxylate de l'Asp1 sont indiqués en bleu et le motif imidazole de l'His impliquée dans l'état in-between est indiqué en violet. Asc • représente le radical ascorbyl.

Figure 14 :

 14 Figure 14 : Structures chimique de l'acide coumarine-3-carboxylique (nommé CCA, panel a) et l'acide 7hydroxycoumarine-3-carboxylique (nommé 7-OH-CCA, panel b).

Figure 15 :

 15 Figure 15 : Représentation schématique des effets pro-et anti-oxydants de l'ascorbate et conséquences sur les cibles biologiques environnantes et sur le peptide Aβ. AscH -est l'abréviation de l'ascorbate.

  ...................................................................................................................................... d. Phenylalanines ............................................................................................................................... e. Methionine ..................................................................................................................................... f. Other cleavages .............................................................................................................................. REFERENCES ........................................................................................................................................... II.A. PREPARATION OF THE AΒ PEPTIDE ................................................................................................ II.A.1. Solubilisation and monomerization ................................................................................ II.A.2. Dosage [1] ......................................................................................................................... II.A.3. Oxidation and purification of Aβ ..................................................................................... II.A.4. Preparation for aggregation ........................................................................................... II.B. MASS SPECTROMETRY ...............................................................................................................

CHAPTER II: METHODOLOGIES ...................................................................................................... II.B.

1. General principles ........................................................................................................... II.B.2. Electrospray Ionization (ESI) ............................................................................................ II.B.3. Ion trap ............................................................................................................................ a. Principle ......................................................................................................................................... b.

  Samples preparation ...................................................................................................................... b. NMR conditions .............................................................................................................................. II.F. ELECTRON PARAMAGNETIC RESONANCE ........................................................................................

...................................................................................................... II.D.2. Fluorescence of 7-hydroxycoumarin-3-carboxylic acid ................................................... II.D.3. Thioflavin T fluorescence ................................................................................................. II.E. PROTON NUCLEAR MAGNETIC RESONANCE ................................................................................... II.E.1. General principles ........................................................................................................... II.E.2. Application to Aβ chemical structure study .................................................................... a. II.F.1. General principles ........................................................................................................... II.F.1. Application to Cu(II) coordination study ......................................................................... II.G. X-RAY ABSORPTION NEAR EDGE STRUCTURE (XANES) ...................................................................

  . Cu K-edge XANES conditions .......................................................................................................... b. Zn K-edge XANES conditions .......................................................................................................... REFERENCES ........................................................................................................................................... III.A. CHARACTERIZATION OF THE OXIDATION SITES ................................................................................. III.A.1. Experimental section ....................................................................................................... III.A.2. Results ............................................................................................................................. a. Detection of the oxidized tryptic peptides by LC-HRMS ................................................................ b. Characterization of the oxidation sites by LC-MS/MS .................................................................... III.B. KINETICS OF AΒ40 OXIDATION ...................................................................................................... III.B.1. Experimental section ....................................................................................................... III.B.2. Results .............................................................................................................................

......................................................................... II.G.2. Application to Cu(I) and Zn(II) coordination .................................................................... aCHAPTER III: OXIDATION OF THE AΒ PEPTIDE ................................................................................. III.C. NMR STUDY OF AΒOX ............................................................................................................... III.C.1. Experimental section ....................................................................................................... III.C.2. Results ............................................................................................................................. III.C.3. Summary .........................................................................................................................

  IV.D. CONCLUSION ......................................................................................................................... REFERENCES ......................................................................................................................................... SUPPORTING INFORMATION .................................................................................................................... .B. FRENCH SUMMARY.................................................................................................................. V.C. SUPPORTING INFORMATION ...................................................................................................... REFERENCES ......................................................................................................................................... VI.A. COMMUNICATION................................................................................................................... VI.B. FRENCH SUMMARY.................................................................................................................. VI.C. SUPPORTING INFORMATION ...................................................................................................... REFERENCES ......................................................................................................................................... ANNEX I .............................................................................................................................................. ANNEX II .............................................................................................................................................

1. Article .............................................................................................................................. IV.A.2. French summary ...........................................................................................................

IV.B. ZN COORDINATION WITH AΒOX ................................................................................................. IV.B.1. Experimental section..................................................................................................... IV.B.2. Results ........................................................................................................................... IV.C. AGGREGATION OF AΒOX .......................................................................................................... IV.C.1. Experimental section ..................................................................................................... IV.C.2. Results ........................................................................................................................... a. Aggregation of Aβ and Aβox ........................................................................................................ b. Morphology of Aβ aggregates ...................................................................................................... IV.C.3. Outlook ......................................................................................................................... CHAPTER V: CHARACTERIZATION OF A REDOX COMPETENT CU BINDING MODE IN THE « IN-BETWEEN » STATE……………………………………………………………………………………………………………………………………. .127 V.A. ARTICLE ................................................................................................................................ VCHAPTER VI: PRO VERSUS ANTIOXIDANT PROPERTIES OF ASCORBATE ........................................ GENERAL CONCLUSION ................................................................................................................... ANNEX III ............................................................................................................................................ RESUME ..............................................................................................................................................

  Le cerveau humain est un organe très surprenant. Alors qu'il ne pèse que 2 % du poids total du corps et qu'il est constitué majoritairement d'eau (75%), il assure les fonctions cognitives et motrices du corps et traite les informations provenant de la vue, de l'ouïe, de l'odorat, du toucher et du goût. Il est l'une des parties du corps dont l'activité métabolique est la plus intense, et utilise au repos, à lui tout seul, 20% de l'oxygène consommé par l'organisme entier. Il est constitué de 100 milliards de neurones qui communiquent entre eux grâce aux neurotransmetteurs, messagers chimiques qui traversent les synapses. Le cerveau étant un organe extrêmement complexe et multitâche, les origines biologiques et chimiques de ses nombreuses fonctions ne sont pas encore toutes parfaitement connues.

De nombreuses pathologies sont liées à un dysfonctionnement métabolique du cerveau, mais pour la plupart d'entre elles, leur étiologie est inconnue. C'est le cas des maladies neurodégénératives, ou démences, qui touchent actuellement plus de 45 millions de personnes à travers le monde. En raison de l'augmentation de l'espérance de vie liée aux avancées de la médecine, le nombre de personnes atteintes de démence ne fait que s'accroître. Parmi les différentes maladies neurodégénératives, la maladie d'Alzheimer, découverte il y a plus d'un siècle, est la plus répandue. Bien que la cause de son développement soit encore inconnue à l'heure actuelle, deux types de lésions cérébrales sont observés chez les patients : (i) les enchevêtrements neurofibrillaires, ayant pour origine l'hyperphosphorylation de la protéine Tau et (ii) la formation de plaques amyloïdes extracellulaires. Les recherches scientifiques sont donc principalement dirigées vers l'étude de ces deux caractéristiques de la maladie, que ce soit d'un point de vue mécanistique, pour comprendre l'étiologie de la maladie, ou d'un point de vue thérapeutique, pour tenter de trouver un médicament efficace. Dans ce cadre global, le travail présenté ici s'est focalisé sur la problématique des plaques amyloïdes -appelées aussi plaques séniles -et plus précisément sur son composant principal, le peptide amyloïde-bêta (Aβ). Aβ est un peptide composé de 40 à 42 acides aminés, naturellement présent sous forme monomérique dans le cerveau. Dans le cas de la maladie d'Alzheimer, il est retrouvé sous forme agrégée dans les plaques amyloïdes. Ces dernières peuvent être formées dans l'espace inter-synaptique et empêcher le bon fonctionnement des neurones en obstruant le passage des neurotransmetteurs entre les synapses de deux cellules nerveuses. Un lien entre maladie d'Alzheimer et stress oxydant a également été démontré. En plus de sa capacité d'agrégation dans des conditions spécifiques à la maladie d'Alzheimer, Aβ est également à l'origine de la production d'espèces réactives de l'oxygène (ROS) car il est capable de chélater des ions métalliques ayant des propriétés oxydo-réductrices, ions cuivre ou fer par exemple. En présence d'un agent réducteur tel que l'ascorbate, naturellement présent dans le cerveau à des concentrations pouvant être localement importantes, le complexe Cu-Aβ formé peut catalyser la production de l'anion superoxyde (O2

  •-), du peroxyde d'hydrogène (H2O2) et du radical hydroxyle (HO • ) à partir du dioxygène. Ces ROS, et plus spécifiquement le radical hydroxyle, sont des espèces oxydantes réactives qui peuvent endommager les biomolécules environnantes (lipides, protéines, ADN). Lors de la production de ROS, le peptide Le chapitre IV se concentre sur l'étude des conséquences de l'oxydation de Aβ. Le mode de coordination du peptide oxydé (Aβox) avec les ions métalliques Cu(I) et Cu(II), catalyseurs de la production de ROS, est étudié par XANES et RPE respectivement, et l'effet de l'oxydation de Aβ sur la production de ROS est étudié par spectroscopie de fluorescence. La coordination du peptide Aβox avec l'ion Zn(II) est également étudiée par XANES et les conséquences de l'oxydation de Aβ sur le phénomène d'agrégation sont investiguées par spectroscopie de fluorescence et par microscopie électronique à transmission (TEM).

	Les principes généraux de chaque technique sont rappelés, et les conditions d'utilisation
	(matériel et méthodes) pour notre sujet d'étude sont présentées.

Aβ, lié au cuivre, subit également des attaques oxydantes. Dans ce contexte général, le projet de thèse a consisté à caractériser l'oxydation du peptide Aβ lors de la production de ROS, catalysée par le cuivre, et en l'étude des conséquences des dommages d'oxydation subis par Aβ sur la coordination d'ions métalliques, la production de ROS et l'agrégation du peptide oxydé. Le premier chapitre situe le contexte du projet de thèse. La maladie d'Alzheimer y est décrite avec les différentes étapes connues de son développement, ainsi que quelques approches diagnostiques et thérapeutiques. Les lésions cérébrales associées à la maladie sont présentées et une attention particulière est portée aux plaques amyloïdes et à la caractérisation du peptide Aβ qui fait l'objet de notre étude. Les différents événements impliquant le peptide Aβ y sont décrits : la formation des plaques amyloïdes, la production d'espèces réactives de l'oxygène (ROS) en présence d'ions métalliques pouvant entrainer l'oxydation des biomolécules et du peptide Aβ. Enfin, les sites d'oxydation du peptide Aβ décrits dans la littérature sont présentés. Le second chapitre expose la méthodologie du projet de thèse. La préparation d'échantillons, étape essentielle pour l'étude des peptides, y est présentée. Différentes techniques spectroscopies ont été utilisées : la Spectrométrie de Masse (MS), les spectroscopies UV-Visible et de fluorescence, la Résonance Magnétique Nucléaire du proton ( 1 H RMN) ainsi que la Résonance Paramagnétique Electronique (RPE) et l'absorption des rayons X (XANES). L'oxydation du peptide Aβ40 catalysée par le cuivre est étudiée dans le chapitre III par MS haute résolution et MS en tandem. Les acides aminés oxydés sont identifiés et la cinétique d'oxydation de chacun d'eux est présentée. Une étude du peptide oxydé est également réalisée par 1 H RMN. Dans le chapitre V, la production de ROS catalysée par le cuivre est étudiée par spectroscopie de fluorescence, avec une série de peptides Aβ ayant subi des modifications (troncation, mutation d'un acide aminé, blocage de l'amine N-terminale), afin de déterminer quels acides aminés sont liés au cuivre pendant la production de ROS. Enfin, le chapitre VI se focalise sur l'ascorbate, molécule connue pour ses propriétés antioxydantes, mais qui participe également à la production de ROS catalysée par un métal. Afin d'évaluer ses effets pro-et anti-oxydants dans le cadre de la maladie d'Alzheimer, les oxydations des molécules environnantes et du peptide Aβ lors de la production de ROS catalysée par le cuivre sont évaluées par spectroscopie de fluorescence et MS respectivement, pour différentes concentrations d'ascorbate.

Chapter I Chapter I: Context of the project I.A. Alzheimer's Disease In 1907, Aloïs Alzheimer related in the article "Über eine eigenartige Erkankung der

  oxidation

	High Performance Liquid Chromatography / High Resolution Mass Spectrometry
	(LC/HRMS) analysis was performed on a LTQ-Orbitrap XL mass spectrometer (ThermoFisher
	Scientific, Les Ulis, France) coupled to an Ultimate 3000 LC System (Dionex, Voisins-le-
	Bretonneux, France). Sample (10 µL of Aβ tryptic digest) was injected onto the column
	(Phenomenex, Synergi Fusion RP-C18, 250 × 1 mm, 4 µm), at room temperature. Gradient
	elution was carried out with formic acid 0.1% (mobile phase A) and acetonitrile/water (80/20
	v/v) formic acid 0.1% (mobile phase B) at a flow-rate of 50 µL.min -1 . The mobile phase gradient
	was programmed with the following time course: 12% mobile phase B at 0 min, held 3 minutes,
	linear increase to 100% B at 15 min, held 4 min, linear decrease to 12% B at 20 min and held
	5 min. The mass spectrometer was used as a detector, the Orbitrap cell operating in the full-
	scan mode at a resolution power of 60 000.
	II.C. UV-Visible spectroscopy

II.C.1. General principles

Table III .A-1: Theoretical monoisotopic masses (m/z) of mono-and doubly-protonated ions of the tryptic peptides of non-oxidized and oxidized Aβ40 detected. +16 and +32 account for the formal addition of one and two oxygen atoms during oxidation.

 III

	Position	Peptide	[M+H] + [M+2H] 2+
	1-5	DAEFR	637.2945 319.1512
	1-5	DAEFR+16	653.2895 327.1486
	1-5	DAEFRdd*	592.2731 296.6405
	1-5	DAEFRox**	548.2469 274.6273
	6-16	HDSGYEVHHQK	1336.6034 668.8056
	6-16	HDSGYEVHHQK+16 1352.5983 676.8031
	6-16	HDSGYEVHHQK+32 1368.5932 684.8005
	17-28	LVFFAEDVGSNK	1325.6741 663.3410
	17-28 LVFFAEDVGSNK+16 1341.6690 671.3384
	29-40	GAIIGLMVGGVV 1085.6392 543.3235
	29-40 GAIIGLMVGGVV+16 1101.6342 551.3210
	* DAEFRdd: oxidative decarboxylation and deamination of Asp1
	** DAEFRox: oxidative cleavage of Asp1
	b. Characterization of the oxidation sites by LC-MS/MS

Table S2 : Identification of the oxidized residues of Aβ, when oxidized in the presence of H2O2.

 S2

	1-5	DAEFRdd*	592.2731	296.6405	198.0963
		DAEFRox**	548.2469	274.6274	183.4208
	6-16	HDSGYEVHHQK+16	1352.5983	676.8031	451.5380
	* DAEFRdd: oxidative decarboxylation and deamination of Asp1
	** DAEFRox: oxidative cleavage of Asp1

  • . Il a donc été déduit qu'à la fois l'amine terminale et l'oxygène du groupe carboxylate de l'Asp1 sont engagés dans la coordination du cuivre dans l'état IB. Il a également été montré que le nombre d'histidines présents dans la séquence peptidique a un impact sur la vitesse de production des radicaux hydroxyles. Moins il y a d'histidines, plus les HO • sont produits rapidement. De plus, cet effet est accru lorsque la séquence peptidique ne contient qu'une seule histidine. Le mode de coordination du Cu(I) avec les peptides ne possédant qu'une histidine, a été étudié par RMN du proton. Il a été montré que Cu(I) est coordiné par l'amine N-terminale de l'Asp1, le cycle imidazole de l'His et un groupe carboxylate. Toutes ces informations ont permis la proposition d'un mode de coordination unique des ions Cu(I) et Cu(II) avec le peptide Aβ dans l'état « in-between », responsable de la formation de ROS. L'ion métallique serait lié à l'amine N-terminale et au groupe carboxylate de l'Asp1, ainsi qu'à une histidine (voir Figure V.B-3). La séquence de Aβ comprenant 3 histidines, celles-ci sont supposées être en échange dynamique, comme cela est le cas dans les états au repos.

  • et HO • Figure VI.B-1,

	voie verte), limitant ainsi de possibles dommages qui résulteraient de l'oxydation de
	biomolécules environnantes.

Table S1 : monoisotopic apparent masses (m/z) of mono-and multi-protonated ions of non-oxidized Aβ28. m/z values in blue are used for Aβ28 detection in HRMS.

 S1

Table S2 : Remaining non-oxidized Aβ28 peptide (%) after the addition of Cu II (50 µM) and ascorbate (0 mM, 1 mM or 10 mM with only 1 mM consumed). Percentages are calculated with the peak areas of the chromatograms traces (see Figure S11).

 S2 His13 et His14) sont suffisamment endommagées. Ils sont donc plutôt les victimes d'une fuite lors de la production de HO • par le système Aβ-Cu, les radicaux ciblant avant tout les ligands du cuivre. Concernant l'influence de l'oxydation sur l'agrégation du peptide Aβ, les résultats préliminaires ont montré que le peptide oxydé a une faible tendance à agréger et à privilégier la formation de fibres. Cela marque une différence importante avec le peptide non-oxydé qui forme des fibres dans lesquelles il s'arrange en feuillets β. D'autres expériences sont en cours au laboratoire, elles visent à confirmer les premiers résultats obtenus et à mieux comprendre la manière dont le peptide oxydé se comporte en termes d'agrégation. Par l'étude de la production de ROS catalysée par le cuivre en utilisant différents peptides Aβ modifiés, il a été possible de proposer une sphère de coordination du cuivre avec Aβ dans l'état transitoire « in-between », responsable de la production de ROS. Ainsi, une histidine, l'amine N-terminale et le groupe carboxylate de l'aspartate 1 seraient liés au cuivre dans l'état « in-between ». Le peptide Aβ non-modifié possédant 3 résidus histidine, il est probable qu'ils soient tous trois impliquées dans la sphère de coordination du cuivre en échange dynamique, comme c'est déjà le cas pour les complexes Cu(I)-Aβ et Cu(II)-Aβ dans les états dits « au repos ». 'intérêt de cette étude et des résultats qu'elle a permis d'obtenir réside dans une meilleure connaissance des mécanismes et conséquences de l'activité redox du système Cu-Aβ, à rapprocher du lien existant entre stress oxydant et maladie d'Alzheimer. Des perspectives directes peuvent être envisagées, à plus ou moins long terme. Une meilleure connaissance de l'état redox responsable de la production de ROS peut permettre le développement de stratégies thérapeutiques ciblées et efficaces, pouvant conduire à une réduction des dommages oxydatifs qui seraient en cause dans la neurodégénérescence liée à la maladie. D'autre part, et étant donné sa spécificité, l'oxydation du peptide Aβ peut représenter une piste intéressante dans la recherche de biomarqueurs pour le diagnostic précoce de la maladie d'Alzheimer. Il serait ainsi intéressant de pouvoir purifier le peptide Aβ à partir de matériel biologique (sérum, liquide

	participe donc à sa propre destruction en contribuant à la production des ROS qui
	General conclusion l'endommagent irréversiblement.	Annex I
	Les travaux présentés dans ce manuscrit concernent l'oxydation du peptide amyloïde-High-Resolution Mass Spectrometry data
	bêta (Aβ) lors de la production d'espèces réactives de l'oxygène (ROS) catalysées par les ions
	cuivre. Différents aspects ont été étudiés autour de l'oxydation du peptide Aβ : (i)
	l'identification des résidus oxydés et les conséquences que peut entraîner cette oxydation quant
	à la coordination des ions métalliques, l'agrégation du peptide oxydé et la production de ROS
	par le peptide oxydé en présence de cuivre, (ii) la caractérisation du mode de coordination du
	complexe Cu-Aβ dans l'état transitoire, appelé état « in-between », responsable de la
	production de ROS et donc de l'oxydation de Aβ et (iii) les effets à la fois pro-et anti-oxydants
	de l'ascorbate envers les biomolécules et le peptide Aβ lors de la production de ROS catalysée
	par le cuivre.
	Starting ascorbate (mM) Consumed ascorbate (mM) Remaining Aβ28 (%)
	0	0	100%
	1 10	1 1 Annexes	1% 4%
	Nous avons pu montrer que l'oxydation du peptide Aβ a entraîné un changement de Ainsi, il ressort de cette étude que l'Aspartate 1 et les Histidines du peptide Aβ sont des
	coordination des deux ions métalliques Cu(I) et Cu(II) ainsi que de Zn(II). Lorsque le peptide acides aminés clés dans la production de ROS. Impliqués dans la coordination des ions Cu(I)
	est suffisamment oxydé, ce changement est à l'origine d'une production de ROS plus rapide et et Cu(II) dans les états au repos, ils le sont également dans l'état transitoire, seul état redox
	d'une quantité plus importante de HO • quittant le système Cu-Aβ. L'oxydation de Aβ peut donc activement impliqué dans la production de ROS. Ils sont les premiers ciblés et leur dégradation

L'étude du peptide après oxydation catalysée par le cuivre a permis d'identifier les principaux acides aminés oxydés : l'aspartate 1 ainsi que les histidines

13

et 14. Ces acides aminés étant impliqués dans les coordinations de Cu(I) et/ou Cu(II), il a été proposé qu'ils soient aussi engagés dans la coordination du cuivre pendant la production de ROS. Etant proches du site de production, cela expliquerait leur oxydation ciblée. Des oxydations collatérales ont aussi été détectées sur les phénylalanines 19 et 20 et sur la méthionine 35. Ces acides aminés ne sont pas engagés dans la sphère de coordination des ions métalliques (partie N-terminale), mais sont très sensibles aux attaques du radical hydroxyle. Il a été proposé qu'ils soient ciblés par les ROS quittant le système Cu-Aβ, lorsque les premières cibles des radicaux (Asp1, être considéré comme un événement délétère pour le peptide lui-même, mais également pour les biomolécules environnantes pour lesquelles on observe une augmentation des oxydations. Enfin, les propriétés pro-oxydantes et anti-oxydantes de l'ascorbate ont été étudiées. Antioxydant bien connu du grand public (notamment sous le nom de Vitamine C), l'ascorbate présente un double visage puisqu'il participe également à la production de ROS en transférant un électron au centre métallique, qui peut à son tour réduire l'oxygène moléculaire ou les espèces intermédiaires que constituent les ROS. L'étude a permis de déterminer que son effet dépend de sa concentration. Quelle qu'elle soit, l'ascorbate participe à la production de ROS, mais à forte concentration, il réagit aussi avec les radicaux hydroxyles formés et joue son rôle d'antioxydant vis-à-vis des biomolécules environnantes. Cependant, il n'est pas efficace dans la protection du peptide Aβ. Même à forte concentration dans le milieu, le peptide n'est pas protégé contre les attaques du radical hydroxyle. entraîne des changements importants quant à la coordination des métaux, l'agrégation et de la production de ROS. L'ascorbate, même présent en grande concentration, ne peut faire bénéficier Aβ de ses propriétés antioxydantes. Le peptide Aβ, en se liant à un ion cuivre, Lcéphalo-rachidien, …) pour étudier spécifiquement de possibles dégâts dus à l'oxydation. La production d'anticorps spécifiquement dirigés contre le peptide oxydé représenterait alors une avancée intéressante pour une telle étude. Enfin, une meilleure connaissance du système Cu-Aβ devrait permettre aussi de mieux comprendre le fonctionnement d'autres systèmes impliquant des peptides amyloïdogéniques mis en cause dans d'autres pathologies, tels que l'amyline, pour le diabète de type II, et l'alpha-synucléine, pour la maladie de Parkinson.

Table 1 : Monoisotopic apparent masses (m/z) of mono-and multi-protonated ions of Aβ40, Aβ28, Aβ16 and the tryptic peptides (Aβ1-5, Aβ6-16, Aβ17-28 and Aβ29-40) as well as their oxidized counterparts.

 1

	Name Sequence						[M+H] +	[M+2H] 2+	[M+3H] 3+	[M+4H] 4+	[M+5H] 5+
	A16 DAEFRHDSGYEVHHQK		1954.8796	977.9437	652.2984	489.4758	391.7822
	D	dd AEFRHDSGYEVHHQK		1909.8581	955.4330	637.2913	478.2204	382.7779
	DAEFRHDSGYEVH	+16 HQK		1970.8745	985.9412	657.6300	493.4745	394.9812
	DAEFRHDSGYEVH	+16 H	+16 QK		1986.8694	993.9386	662.9617	497.4732	398.1801
	D	dd AEFRHDSGYEVH	+16 HQK		1925.8530	963.4304	642.6229	482.2191	385.9769
	D	ox AEFRHDSGYEVHHQK		1865.8319	933.4199	622.6159	467.2138	373.9726
	D	ox AEFRHDSGYEVH	+16 HQK		1881.8268	941.4173	627.9475	471.2126	377.1716
	A28 DAEFRHDSGYEVHHQKLVFFAEDVGSNK		3261.5353	1631.2716	1087.8503	816.1397	653.1133
	D	dd AEFRHDSGYEVHHQKLVFFAEDVGSNK	3216.5138	1608.7608	1072.8432	804.8843	644.1090
	DAEFRHDSGYEVH	+16 HQKLVFFAEDVGSNK	3277.5302	1639.2690	1093.1820	820.1384	656.3123
	DAEFRHDSGYEVHHQKLVFFAEDVGSNK	+32	3293.5251	1647.2665	1098.5136	824.1372	659.5113
	D	dd AEFRHDSGYEVH	+16 HQKLVFFAEDVGSNK	3232.5088	1616.7583	1078.1748	808.8831	647.3080
	D	ox AEFRHDSGYEVHHQKLVFFAEDVGSNK	3172.4876	1586.7477	1058.1678	793.8778	635.3038
	D	ox AEFRHDSGYEVH	+16 HQKLVFFAEDVGSNK	3188.4825	1594.7452	1063.4994	797.8765	638.5028
	A40 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV	4328.1561	2164.5820	1443.3906	1082.7949	866.4375
	D	dd AEFRHDSGYEVHHQKLVFF…		4283.1347	2142.0713	1428.3834	1071.5395	857.4332
	DAEFRHDSGYEVH	+16 HQKLVFF…		4344.1511	2172.5794	1448.7222	1086.7936	869.6365
	DAEFRHDSGYEVH	+16 H	+16 QKLVFF…		4360.1460	2180.5769	1454.0539	1090.7924	872.8355
	D	dd AEFRHDSGYEVH	+16 HQKLVFF…		4299.1296	2150.0687	1433.7151	1075.5383	860.6322
	D	ox AEFRHDSGYEVHHQKLVFF…		4239.1085	2120.0581	1413.7080	1060.5330	848.6280
	D	ox AEFRHDSGYEVH	+16 HQKLVFF…		4255.1034	2128.0556	1419.0397	1064.5317	851.8269
	DAEFR

p HDSGYEVHHQK p Annex II 1 H NMR data

Table 2 : 1 H NMR chemical shifts of Aβ28 at pH 7.4. [1]

 2

  RésuméRésuméLe cerveau humain est un organe très surprenant. Alors qu'il ne pèse que 2 % du poids total du corps et qu'il est constitué majoritairement d'eau (75%), il assure les fonctions cognitives et motrices du corps et traite les informations provenant de la vue, de l'ouïe, de l'odorat, du toucher et du goût. Il est l'une des parties du corps dont l'activité métabolique est la plus intense, et utilise au repos, à lui tout seul, 20% de l'oxygène consommé par l'organisme entier. Il est constitué de 100 milliards de neurones qui communiquent entre eux grâce aux neurotransmetteurs, messagers chimiques qui traversent les synapses. Le cerveau étant un organe extrêmement complexe et multitâche, les origines biologiques et chimiques de ses nombreuses fonctions ne sont pas encore toutes parfaitement connues. Un lien entre maladie d'Alzheimer et stress oxydant a également été démontré. En plus de sa capacité d'agrégation dans des conditions spécifiques à la maladie d'Alzheimer, Aβ est également à l'origine de la production d'espèces réactives de l'oxygène (ROS) car il est capable de chélater des ions métalliques ayant des propriétés oxydo-réductrices, ions cuivre ou fer par exemple. En présence d'un agent réducteur tel que l'ascorbate, naturellement présent dans le cerveau à des concentrations pouvant être localement importantes, le complexe Cu-Aβ formé peut catalyser la production de l'anion superoxyde (O2 •-), du peroxyde d'hydrogène (H2O2) et du radical hydroxyle (HO • ) à partir du dioxygène (Figure1). Ces ROS, et plus spécifiquement le radical hydroxyle, sont des espèces oxydantes réactives qui peuvent endommager les biomolécules environnantes (lipides, protéines, ADN). Lors de la production de ROS, le peptide Aβ, lié au cuivre, subit également des attaques oxydantes.

	Dans ce cadre global, le travail présenté ici s'est focalisé sur la problématique des
	plaques amyloïdes -appelées aussi plaques séniles -et plus précisément sur son composant
	principal, le peptide amyloïde-bêta (Aβ). Aβ est un peptide composé de 40 à 42 acides aminés,
	naturellement présent sous forme monomérique dans le cerveau. Dans le cas de la maladie

De nombreuses pathologies sont liées à un dysfonctionnement métabolique du cerveau, mais pour la plupart, leur étiologie est inconnue. C'est le cas des maladies neurodégénératives, ou démences, qui touchent actuellement plus de 45 millions de personnes à travers le monde.

En raison de l'augmentation de l'espérance de vie liée aux avancées de la médecine, le nombre de personnes atteintes de démence ne fait que s'accroître. Parmi les différentes maladies neurodégénératives, la maladie d'Alzheimer, découverte il y a plus d'un siècle, est la plus répandue. Bien que la cause de son développement soit encore inconnue à l'heure actuelle, deux types de lésions cérébrales sont observés chez les patients : (i) les enchevêtrements neurofibrillaires, ayant pour origine l'hyperphosphorylation de la protéine Tau et (ii) la formation de plaques amyloïdes extracellulaires. Les recherches scientifiques sont donc principalement dirigées vers l'étude de ces deux caractéristiques de la maladie, que ce soit d'un point de vue mécanistique, pour comprendre l'étiologie de la maladie, ou d'un point de vue thérapeutique, pour tenter de trouver un médicament efficace. d'Alzheimer, il est retrouvé sous forme agrégée dans les plaques amyloïdes. Ces dernières peuvent être formées dans l'espace inter-synaptique et empêcher le bon fonctionnement des neurones en obstruant le passage des neurotransmetteurs entre les synapses de deux cellules nerveuses.

Remerciements

Supporting information

Metal-catalyzed oxidation of Aβ and the resulting reorganization of the Cu binding sites promote ROS production

Clémence Cheignon*, Peter Faller, Denis Testemale, Christelle Hureau and Fabrice Collin*

Supporting Information

Table S3: Linear fitting of HO production curves. Gradient and determination coefficients of fits for each curve of Figures 7 and8, for the two linear parts.

First linear part

Chapter VI: Pro versus antioxidant properties of Ascorbate

The chapter focuses on the study of the prooxidant and antioxidant properties of the ascorbate anion, in the context of Alzheimer's Disease (AD). The chapter is composed of a communication published in the journal Dalton Transactions in August 2016 [1] along with a summary of the article, written in French (requirement of the doctoral school). The supporting information related to the communication is situated at the end of the chapter (Section VI.C).

VI.A. Communication

Material and methods

Chemicals

Cu(II) used was from CuSO4. dissolving the powder in NaOH (50mM) and passing the solution through FPLC to obtain the monomeric fraction. The peptide concentration was then determined in NaOH (50mM) by UVvisible absorption of Tyr10, considered as free tyrosine ((ε293-ε360)=2400 M -1 cm -1 ).

Mass spectrometry

High Performance Liquid Chromatography / High Resolution Mass Spectrometry (HPLC/HRMS) analysis was performed on a LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific, Les Ulis, France) coupled to an Ultimate 3000 LC System (Dionex, Voisins-le-Bretonneux, France). The Orbitrap cell was operated in the full-scan mode at a resolution power of 60 000. Samples were washed three times with water prior analysis,by using Amicon 3 kDa centrifugal device (Millipore). Samples (10 µL) were then injected onto

Abstract

Alzheimer's Disease (AD) is the most frequent for of dementia in the elderly. A hallmark of AD is the extracellular formation of senile plaques in the brain of AD subjects, composed of the Amyloid-β peptide (Aβ) under aggregated form with metal ions such as copper ions. Aβ can form a complex with copper ions, able to catalyze reactive oxygen species (ROS) formation in the presence of a reducing agent such as ascorbate. These oxidative species can oxidize the surrounding molecules and the Aβ peptide itself. Being close to the production site of ROS, Aβ is the preferential target, especially for the hydroxyl radical HO • . The aim of this work was to study the ROS production by the Aβ/Cu/ascorbate system, to characterize the oxidation undergone by Aβ and to evaluate the consequences of Aβ oxidation on ROS production, metal ions coordination and aggregation. Several spectroscopic techniques have been used, in particular mass spectrometry (MS), fluorescence spectroscopy, electron paramagnetic resonance (EPR) and X-Ray absorption spectroscopy (XANES).

The oxidation sites of Aβ have been studied by mass spectrometry (MS and MS/MS). Thanks to the use of proteomic tools and high-resolution mass spectrometry (HRMS), the oxidized amino acid residues have been identified. Asp1, His 13 and His14 have been found to be the preferential targets for HO • on Aβ. This result was expected as these residues are involved in copper coordination, from which the ROS are generated.

The impact of Aβ oxidation on Cu(II), Cu(I) and Zn(II) on metal ions coordination, on ROS production and on Aβ aggregation has been studied. Results have shown that Aβ oxidation induces a change of coordination of Zn(II) as well as Cu(II) and Cu(I), leading to an increase of ROS production. Moreover, Aβ oxidation has also an impact on aggregation, as it does not favor fibrils formation.

The Cu-Aβ binding mode during ROS production has been deduced from the study of a series of mutated Aβ peptides. The hypothesis, in which the amino acid residues bound to Cu during the ROS production are the oxidized one (Asp1, His 13 and His14) has been corroborated by the results of this study, the mutation of Asp1 or the two His having an impact on ROS production.

Finally, the pro-and antioxidants effects of ascorbate have been investigated, showing that, on the Cu-Aβ system, ascorbate only has antioxidant properties at high concentration for surrounding molecules, but does not exhibit any protecting effect on Aβ itself.