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Vésicules Extracellulaires : Biomarqueurs Et Véhicules De Propagation De Protéinopathies

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Vésicules Extracellulaires : Biomarqueurs Et Véhicules De Propagation De Protéinopathies Vésicules extracellulaires : Biomarqueurs et véhicules de propagation de protéinopathies Mémoire Jérôme Lamontagne-Proulx Maîtrise en Neurobiologie Maître ès sciences (M.Sc.) Québec, Canada © Jérôme Lamontagne-Proulx, 2018 Vésicules extracellulaires : Biomarqueurs et véhicules de propagation de protéinopathies Mémoire Jérôme Lamontagne-Proulx Sous la direction de : Francesca Cicchetti, directrice de recherche Résumé La maladie de Parkinson (MP) est une maladie neurodégénérative invalidante pour laquelle le diagnostic ne peut être donné qu’une fois la dégénérescence neuronale bien entamée, rendant impérative la découverte d’un biomarqueur ; un outil biologique permettant de prédire l’apparition de la pathologie ou d’évaluer sa progression. Mon projet de maîtrise visait donc l’étude des vésicules extracellulaires (VE) issues du sang comme test diagnostique ou comme marqueur de la progression de la MP. La quantification des VE effectuée par cytométrie de flux à haute sensibilité a révélé une augmentation spécifique des VE dérivées d’érythrocytes (VEE) chez les parkinsoniens comparés à leurs contrôles, ainsi qu’une forte corrélation avec la progression de la maladie. L’analyse quantitative de l’alpha- synucléine (α-Syn), principale protéine impliquée dans la pathologie de la maladie, a montré un niveau similaire entre les individus. Cependant, l’analyse protéomique des VEE a révélé une modulation de certaines protéines entre les patients et les donneurs sains. Nos résultats suggèrent que les VEE pourraient conduire au développement d’un marqueur pour suivre l’évolution de la maladie ainsi que l’effet de nouvelles thérapies. iii Abstract Parkinson’s disease (PD) is a debilitating neurodegenerative disease for which the diagnosis can only be confirmed once the degeneration state is very advanced, making imperative the discovery of a biomarker: a biological tool to predict the onset of pathology or its progression. My master’s project was designed to study extracellular vesicles (EV) from the blood in order to discover if they could be used as a diagnostic test or as a marker of disease progression. Quantification of EV performed by high-sensitivity flow cytometry demonstrated an increase in PD patients compared to their controls and a strong correlation with the progression of the disease only in EV derived from erythrocytes (EEV). Quantitative analysis of α-Syn, the main protein involved into PD pathogenesis, showed a similar level between individuals. However, analysis of the EEV proteome reveals a modulation of some proteins between patients and healthy donors. Our results suggest that EEV have the potential to lead to the development of a marker abled to track disease course as well as measuring the effect of new therapies. iv Table des matières Résumé .................................................................................................................................. iii Abstract ................................................................................................................................ iv Table des matières ................................................................................................................ v Liste des tableaux .............................................................................................................. viii Liste des figures ................................................................................................................... ix Abréviations .......................................................................................................................... x Remerciements ................................................................................................................... xiv Avant-propos ....................................................................................................................... xv CHAPITRE 1: INTRODUCTION ...................................................................................... 1 1.1 La maladie de Parkinson ............................................................................................... 2 1.1.1 Épidémiologie ........................................................................................................... 2 1.1.2 Étiologie .................................................................................................................... 3 1.1.2.1 Facteurs environnementaux................................................................................. 3 1.1.2.2 Facteurs génétiques ............................................................................................. 5 1.1.3 Neuropathologie ....................................................................................................... 7 1.1.3.1 Dégénérescence du système dopaminergique ..................................................... 7 1.1.3.2 L’alpha-synucléine et les corps de Lewy ............................................................ 8 1.1.3.3 Stress oxydatif et facteurs de mort cellulaire .................................................... 10 1.1.4 Diagnostic et test clinique ...................................................................................... 11 1.1.5 Biomarqueur .......................................................................................................... 16 1.1.5.1 Introduction ....................................................................................................... 16 1.1.5.2 Biomarqueurs génétiques .................................................................................. 18 1.1.5.3 Biomarqueurs par imagerie ............................................................................... 18 1.1.5.4 Biomarqueurs cliniques ..................................................................................... 19 1.1.5.5 Biomarqueurs biochimiques .............................................................................. 19 1.1.5.6 L’avenir des biomarqueurs ................................................................................ 22 1.2 Biomarqueur sanguin ................................................................................................... 22 1.2.1 Érythrocytes ........................................................................................................... 23 1.2.2 Plaquettes ................................................................................................................ 24 1.2.3 Leucocytes ............................................................................................................... 24 v 1.3 Vésicules extracellulaires ............................................................................................. 25 1.3.1 Exosomes ................................................................................................................. 25 1.3.2 Microvésicules ........................................................................................................ 26 1.3.3 Corps apoptotiques ................................................................................................ 27 1.3.4 Rôles des VE dans la maladie de Parkinson ............................................................ 29 1.3.4.1 Études des VE dans le système nerveux central ( in vitro ) ................................ 30 1.3.4.2 Études des VE dans le liquide céphalo-rachidien ............................................. 30 1.3.4.3 Études des VE dans l’urine ............................................................................... 31 1.3.4.4 Études des VE dans le système circulatoire ...................................................... 31 1.4 Les objectifs de recherche ............................................................................................ 32 CHAPITRE 2: ERYTHROCYTE-DERIVED EXTRACELLULAR VESICLES: A NOVEL, ROBUST AND SPECIFIC BIOMARKER THAT MAPS TO PARKINSON'S DISEASE STAGES ............................................................................................................ 34 2.1 Résumé ........................................................................................................................... 35 2.2 Abstract ......................................................................................................................... 37 2.3 Introduction .................................................................................................................. 38 2.4 Materials and methods ................................................................................................. 39 2.4.1 Ethics statement and participant recruitment ........................................................... 39 2.4.2 Preparation of platelet-free plasma and EV labeling ............................................... 39 2.4.3 Flow cytometry quantification ................................................................................. 40 2.4.4 Production and purification of EEV ........................................................................ 40 2.4.5 C-reactive protein, free hemoglobin and α-synuclein quantification ...................... 41 2.4.6 Scanning electron microscopy ................................................................................. 41 2.4.7 Transmission electron microscopy .......................................................................... 41 2.4.8 Mass spectrometry analysis and label free protein quantification ........................... 41 2.4.9 Statistical analyses ................................................................................................. 42 2.5 Results ...........................................................................................................................
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