Les maladies neurodégénératives liées à l'âge, telle celle de Parkinson, sont un problème majeur de santé publique. Cependant, la maladie de Parkinson reste incurable et les traitements sont très limités. En effet, les causes de la maladie restent encore mal comprises et la recherche se concentre sur ses mécanismes moléculaires. Dans cette étude, nous nous sommes intéressés à deux phénomènes anormaux se produisant dans la maladie de Parkinson : l'agrégation de l'α-synucléine et l'activation des cellules microgliales. Pour étudier la polymérisation de l'α-synucléine, nous avons établi de nouvelles méthodes permettant la production in vitro de différents types d'oligomères d'α-synucléine. Grâce à des méthodes biophysiques de pointe, nous avons caractérisé ces différents oligomères à l'échelle moléculaire. Puis nous avons étudié leurs effets toxiques sur les neurones. Ensuite, nous nous sommes intéressés à l'activation des microglies et en particulier à leurs canaux potassiques et aux changements liés au vieillissement. Nous avons identifié les canaux Kv1.3 et Kir2.1 et montré qu'ils étaient impliqués dans l'activation des microglies. En parallèle, nous avons établi une méthode originale qui permet l'isolation et la culture de microglies primaires issues de cerveaux adultes. En comparaison à celles de nouveaux-nés, les microglies adultes montrent des différences subtiles mais cruciales qui soutiennent l'hypothèse de changements liés au vieillissement. Globalement, nos résultats suggèrent qu'il est possible de développer de nouvelles approches thérapeutiques contre la maladie de Parkinson en modulant l'action des microglies ou en bloquant l'oligomérisation de l' α-synucléine.

<p>Microglia are a population of specialized, tissue-resident immune cells that make up around 10% of total cells in our brain. They actively prune neuronal synapses, engulf cellular debris, and misfolded protein aggregates such as the Alzheimer’s Disease (AD)-associated amyloid-beta (Aβ) by the process of phagocytosis. During AD, microglia are unable to phagocytose Aβ, perhaps due to the several disease-associated changes affecting their normal function. Functional molecules such as lipids and metabolites also influence microglial behavior but have primarily remained uncharacterized to date. The overarching question of this work is, <i>How do microglia become dysfunctional in chronic inflammation</i>? To this end, we developed new chemical tools to better understand and investigate the microglial response to Aβ <i>in vitro</i> and <i>in vivo</i>. Specifically, we introduce three new tools. (1) Recombinant human Aβ was developed via a rapid, refined, and robust method for expressing, purifying, and characterizing the protein. (2) A pH-sensitive fluorophore conjugate of Aβ (called Aβ^pH) was developed to identify and separate Aβ-specific phagocytic and non-phagocytic glial cells <i>ex vivo</i> and <i>in vivo</i>. (3) New lysosomal, mitochondrial, and nuclei-targeting pH-activable fluorescent probes (called LysoShine, MitoShine, and NucShine, respectively) to visualize subcellular organelles in live microglia. Next, we asked, <i>What changes occur to the global lipid and metabolite profiles of microglia in the presence of Aβ in vitro and in vivo</i>? We screened 1500 lipids comprising 10 lipid classes and 700 metabolites in microglia exposed to Aβ. We found significant changes in specific lipid classes with acute and prolonged Aβ exposure. We also identified a lipid-related protein that was differentially regulated due to Aβ <i>in vivo</i>. This new lipid reprogramming mechanism “turned on” in the presence of cellular stress was also present in microglia in the brains of the 5xFAD mouse model, suggesting a generic response to inflammation and toxicity. It is well known that activated microglia induce reactive astrocytes during inflammation. Therefore, we asked, <i>What changes in proteins, lipids, and metabolites occur in astrocytes due to their reactive state? </i>We provide a comprehensive characterization of reactive astrocytes comprising 3660 proteins, 1500 lipids, and 700 metabolites. These microglia and astrocytes datasets will be available to the scientific community as a web application. We propose a final model wherein the molecules secreted by reactive astrocytes may also induce lipid-related changes to the microglial cell state in inflammation. In conclusion, this thesis highlights chemical neuroimmunology as the new frontier of neuroscience propelled by the development of new chemical tools and techniques to characterize glial cell states and function in neurodegeneration.</p>

Modulation of the endocannabinoid system (ECS) is of great interest for its therapeutic relevance in several pathophysiological processes. The CB2 subtype is largely localized to immune effectors, including microglia within the central nervous system, where it promotes anti-inflammation. Recently, a rational drug design toward precise modulation of the CB2 active site revealed the novelty of Pyrrolo[2,1-c][1,4]benzodiazepines tricyclic chemotype with a high conformational similarity in comparison to the existing leads. These compounds are structurally unique, confirming their chemotype novelty. In our continuing search for new chemotypes as selective CB2 regulatory molecules, following SAR approaches, a total of 17 selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs were synthesized and tested for their ability to bind to the CB1 and CB2 receptor orthosteric sites. A competitive [H]CP-55,940 binding screen revealed five compounds that exhibited >60% displacement at 10 μM concentration. Further concentration-response analysis revealed two compounds, and , as potent and selective CB2 ligands with sub-micromolar activities ( = 146 nM and 137 nM, respectively). In order to support the potential efficacy and safety of the analogs, the oral and intravenous pharmacokinetic properties of compound were sought. Compound was orally bioavailable, reaching maximum brain concentrations of 602 ± 162 ng/g (p.o.) with an elimination half-life of 22.9 ± 3.73 h. Whether administered via the oral or intravenous route, the elimination half-lives ranged between 9.3 and 16.7 h in the liver and kidneys. These compounds represent novel chemotypes, which can be further optimized for improved affinity and selectivity toward the CB2 receptor.

La perturbation de la barrière hémo-encéphalique et la migration des lymphocytes de la périphérie vers le système nerveux central (SNC) sont des événements précoces dans la formation des lésions cérébrales de sclérose en plaques (SEP). Dans ce contexte, les lymphocytes passent au travers des barrières hémo-encéphalique ou hémo-méningée pour atteindre le SNC et sont des contributeurs importants dans l’inflammation et les dommages tissulaires. Pour migrer à travers les barrières du SNC, les lymphocytes pathogéniques expriment des molécules d’adhérence. Identifier les acteurs clés à la migration des lymphocytes pathogéniques en estimant la contribution des molécules d’adhérence dans ce processus est la prochaine étape pour le développement de thérapies pour traiter la SEP. L’objectif de ce projet est d’explorer le rôle de deux molécules d’adhérence que sont ALCAM (de l’anglais : activated leukocytes cell adhesion molecule) et DICAM (de l’anglais : dual immunoglobulin domain containing cell adhesion molecule) dans la migration des lymphocytes pathogéniques vers le SNC pendant la SEP. Notre objectif principal se subdivise en deux sous-objectifs. En premier, notre but est de caractériser le rôle d’ALCAM dans le passage des lymphocytes B à travers les barrières du SNC dans un contexte neuroinflammatoire. En second, nous explorons le rôle de DICAM dans la migration des lymphocytes T auxiliaires 17 (TH17) vers le SNC en neuroinflammation. Nous faisons l’hypothèse qu’ALCAM contribue à la migration des lymphocytes B vers le SNC et que DICAM est impliqué dans la migration des lymphocytes TH17 à travers la barrière hémo-encéphalique pendant la SEP. Ces molécules d’adhérence seraient alors impliquées dans la pathogenèse de la SEP et seraient de potentielles cibles thérapeutiques pour traiter cette maladie. Nous avons d’abord utilisé une combinaison de spectrométrie de masse, PCR quantitative, cytométrie de flux et microscopie afin d’explorer l’expression de chacune de ses deux molécules d’adhérence sur les lymphocytes d’intérêt périphériques ex vivo ou différenciés in vitro. Des analyses en cytométrie en flux et microscopie nous ont permis de caractériser leur expression dans le sang périphérique et dans les lésions cérébrales de personnes atteintes de SEP. Ensuite, les expériences d’adhérence en flux et de migration in vitro effectuées en déplétant la molécule d’adhérence d’intérêt ont permis de mettre en évidence leur rôle dans différentes étapes de la migration des lymphocytes à travers les cellules endothéliales des barrières du SNC. Pour finir, le traitement de plusieurs modèles murins de SEP, appelés EAE (de l’anglais : experimental autoimmune encephalomyelitis), avec des anticorps bloquant anti-ALCAM ou anti-DICAM ont permis d’explorer le potentiel effet de tels traitements sur la sévérité de la maladie. Dans la première étude, nos résultats montrent qu’ALCAM est préférentiellement exprimée par les lymphocytes B pro-inflammatoires, mémoires et effecteurs au potentiel pathogénique. En tant que molécule d’adhérence, ALCAM contribue à leur migration à travers les cellules endothéliales des barrières hémo-encéphalique et hémo-méningée chez la souris et l’humain. De plus, nos expériences ont permis de montrer que la fréquence de lymphocytes B ALCAM+ est augmentée dans le sang périphérique des personnes atteintes de SEP et ces cellules sont aussi présentes dans les lésions et les infiltrats méningées en SEP. Finalement, bloquer ALCAM in vivo réduit la sévérité de la maladie EAE en diminuant l’infiltration des lymphocytes B au SNC. Dans la seconde étude, nous avons montré que parmi les sous-types de lymphocytes TH, DICAM est préférentiellement exprimée par les lymphocytes TH17. Dans les lésions de SEP, DICAM et son ligand αVβ3 co-localisent avec des marqueurs de cellules endothéliales suggérant que ces deux molécules pourraient être présentées à la lumière des vaisseaux aux lymphocytes TH17 circulants. Dans le sang périphérique, la fréquence de lymphocytes T CD4+ exprimant DICAM est augmentée chez les personnes atteintes de SEP et cette augmentation corrèle avec l’activité de la maladie. Nos expériences ont montré que DICAM est impliquée dans l’adhérence, l’arrêt et la diapédèse des lymphocytes TH17 à travers les cellules endothéliales de la barrière hémo-encéphalique in vitro et in vivo. Finalement, le traitement de souris EAE avec un anticorps bloquant DICAM permet de réduire la sévérité de la maladie et diminue la migration des lymphocytes TH17 vers le SNC. Nos résultats indiquent un rôle d’ALCAM dans la migration des lymphocytes B et que DICAM, préférentiellement exprimé par les TH17, médie leur migration vers le SNC. Bloquer ALCAM ou DICAM sont deux stratégies permettant de réduire l’accès au SNC de différents sous-types de cellules pathogéniques pendant la neuroinflammation. Ainsi, elles sont toutes deux de potentielles cibles thérapeutiques pour réduire la sévérité et la progression de la SEP. / Disruption of the blood-brain barrier and migration of lymphocytes from the periphery to the central nervous system (CNS) are early events in lesion formation during multiple sclerosis (MS). Lymphocytes readily cross the blood-brain barrier (BBB) and the blood-meningeal barrier (BMB) to infiltrate the CNS and are important contributors to inflammation and tissue damage. To migrate through the brain barriers, pathogenic lymphocytes express adhesion molecules. Identifying key players in lymphocyte migration by understanding the role of adhesion molecules is the next step to develop novel therapies to treat MS. The objective of this project is to explore the role of two distinct adhesion molecules ALCAM (activated leukocytes cell adhesion molecule) and DICAM (dual immunoglobulin domain containing cell adhesion molecule) in pathogenic lymphocytes migration to the CNS during MS. This thesis subdivides in two main objectives. First, we aim to characterize ALCAM role in B lymphocyte migration to the CNS during neuroinflammation. Second, we aim to explore DICAM role in T helper 17 (TH17) lymphocytes migration to the CNS in neuroinflammation. We hypothesized that ALCAM plays a role in B lymphocytes migration to the CNS during MS and that DICAM is involved in TH17 lymphocytes migration through the blood-brain barrier during MS. Those adhesion molecules might be involved in MS pathogenesis and therefore could become new therapeutic targets to treat MS. We first used mass spectrometry, quantitative PCR, flow cytometry and confocal microscopy to explore expression profiles of ALCAM and DICAM by peripheral lymphocytes subpopulations ex vivo and differentiated in vitro. Flow cytometry and confocal microscopy analysis also revealed how those adhesion molecules are expressed by lymphocytes in peripheral blood and brain lesions of people living with MS. Then, we performed flow adhesion and migration assay of lymphocytes depleted for the adhesion molecule of interest allowing us to address their role in multitstep migration process through brain barriers endothelial cells. Finally, using five distinct murine experimental autoimmune encephalomyelitis models (EAE), we explored how blocking ALCAM or DICAM in vivo could affect lymphocytes migration to the SNC and disease severity. In the first manuscript, we described that ALCAM is preferentially expressed by B lymphocytes with memory, pro-inflammatory and effector phenotypes. Functionally, ALCAM is involved in B lymphocyte migration through both the BBB and the BMB in mouse and human. Interestingly, we showed that ALCAM expressing B lymphocytes are increased in peripheral blood of people living with MS and they are recovered in meningeal and parenchymal MS lesions. Last, blocking ALCAM in vivo alleviates EAE severity by reducing B lymphocyte infiltration to the CNS. In the second manuscript, we showed that TH17 lymphocytes preferentially express DICAM and can adhere both to DICAM and its ligand αVβ3. Moreover, DICAM and αVβ3 are both overexpressed by inflamed brain endothelial cells. In MS lesions, we described that both molecules colocalize with endothelial cell markers suggesting that it could be presented to the vessel lumen to the circulating TH17 lymphocytes. In peripheral blood, we showed that DICAM+ memory CD4+ T lymphocytes frequency is increased in people living with MS and it correlates with active form of the disease. Then, we described DICAM as a player in TH17 lymphocyte adhesion, arrest and migration through BBB endothelial cells in vitro and in vivo. Last, we showed that treating mice with a neutralizing DICAM antibody in several distinct models of EAE, reduced disease severity and TH17 cell migration to the SNC. Our data provide evidence of the role of ALCAM in memory B lymphocyte migration and that DICAM is preferentially expressed by TH17 cells and mediate their migration to the CNS during neuroinflammation. Collectively, our findings indicate that blocking ALCAM or DICAM are two ways to restrict different pathogenic cells access to the CNS during neuroinflammation and thus potentially to reduce the severity and worsening of a disease like MS.

Chronic pain affects 20 % of the global population, causes suffering, is difficult to treat, and constitutes a large economic burden for society. So far, the characterization of molecular mechanisms of chronic pain-like behaviors in animal models has not translated into effective treatments. In this thesis, consisting of five studies, pain patient biofluids were analyzed with modern proteomic methods to identify biomarker candidates that can be used to improve our understanding of the pathophysiology chronic pain and lead to more effective treatments. Paper I is a proof of concept study, where a multiplex solid phase-proximity ligation assay (SP-PLA) was applied to cerebrospinal fluid (CSF) for the first time. CSF reference protein levels and four biomarker candidates for ALS were presented. The investigated proteins were not altered by spinal cord stimulation (SCS) treatment for neuropathic pain. In Paper II, patient CSF was explored by dimethyl and label-free mass spectrometric (MS) proteomic methods. Twelve proteins, known for their roles in neuroprotection, nociceptive signaling, immune regulation, and synaptic plasticity, were identified to be associated with SCS treatment of neuropathic pain. In Paper III, proximity extension assay (PEA) was used to analyze levels of 92 proteins in serum from patients one year after painful disc herniation. Patients with residual pain had significantly higher serum levels of 41 inflammatory proteins. In Paper IV, levels of 55 proteins were analyzed by a 100-plex antibody suspension bead array (ASBA) in CSF samples from two neuropathic pain patient cohorts, one cohort of fibromyalgia patients and two control cohorts. CSF protein profiles consisting of levels of apolipoprotein C1, ectonucleotide pyrophosphatase/phosphodiesterase family member 2, angiotensinogen, prostaglandin-H2 D-isomerase, neurexin-1, superoxide dismutases 1 and 3 were found to be associated with neuropathic pain and fibromyalgia. In Paper V, higher CSF levels of five chemokines and LAPTGF-beta-1were detected in two patient cohorts with neuropathic pain compared with healthy controls. In conclusion, we demonstrate that combining MS proteomic and multiplex antibody-based methods for analysis of patient biofluid samples is a viable approach for discovery of biomarker candidates for the pathophysiology and treatment of chronic pain. Several biomarker candidates possibly reflecting systemic inflammation, lipid metabolism, and neuroinflammation in different pain conditions were identified for further investigation. / Uppsala Berzelii Technology Centre for Neurodiagnostics