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Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder associated with aging. AD is by far the best understood and most studied neurodegenerative disease. Substantial advances have been made over the last decade, however it is debatable how much closer we are to a clinically useful therapy. A long standing goal in the AD field has been to improve the accuracy of early detection, with the assumption that the ability to intervene earlier in the disease process will lead to a better clinical outcome. Major facets of this effort have been the continued development and improvement of AD biomarkers, with a strong focus on developing imaging modalities. AD is accompanied by two pathological hallmarks in the brain: extracellular neuritic plaques composed of the beta-amyloid peptide (Aβ) and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein. Evidence of Aβ as the driving force behind the progression of AD (the amyloid cascade hypothesis) was first published by Hardy & Higgins in 1992, and this peptide has been the focus of therapeutic and diagnostic testing for decades. Significant technological advances in recent years now allow imaging of amyloid pathology in vivo. These methods evaluate Aβ burden in a living person, and could potentially serve as both a biomarker, and as a diagnostic tool to detect disease. Pittsburgh Compound B (PiB) is currently the best studied of these imaging agents, however, our current knowledge of the quantitative relationship between PiB binding and amyloid pathology in the brain is limited. A better understanding of how these variables relate to one another is essential for the continued development of reliable diagnostic biomarkers for AD. We analyzed increasingly insoluble pools of Aβ to quantify their relative contributions to the overall Aβ burden, and to determine if any of these measures could be used to predict disease status. We found that the amount of PiB binding in a cortical region of the brain could distinguish cases of mild cognitive impairment (MCI) when corrected to the amount of PiB binding in the cerebellum. As the Aβ peptide ages, the amino acid aspartate may spontaneously convert to an isoaspartate residue through a succinimide intermediary. The presence of iso-Asp Aβ has been used to indicate the presence of aged plaques in AD and Down syndrome cases. We sought to investigate the potential relationship between levels of ‘aged’ Aβ in the plasma as indicated by iso-Asp Aβ and disease state, as a potential biomarker for the presence of AD pathology. We found that AD cases had lower levels of all forms of Aβ in plasma when standardized to the group average, and that plasma levels of Aβ and iso-Asp Aβ were reversed between disease groups. A follow up study is required, however, these initial data are a promising step towards utilizing aged iso-Asp Aβ plasma levels as a potential biomarker to indicate disease state.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22689号 / 医博第4633号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 伊佐 正, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La Maladie d’Alzheimer (MA) est un des challenges sanitaires les plus importants du XXIème siècle. En effet, elle touche 35,6 millions de personnes en 2010, et en atteindra probablement quatre fois plus en 2050.Cependant, peu d’éléments sont à ce jour connus concernant les mécanismes de toxicité de cette maladie. Il semble néanmoins acquis que l’agrégation du peptide amyloïde Aβ est l’élément déclenchant une cascade d’événements cellulaires aboutissant à trois types de lésions : les dégénérescences neurofibrillaires, les plaques amyloïdes et une atrophie corticale, révélatrice d’une importante mort neuronale.Différents modèles transgéniques d’étude de la toxicité du peptide Aβ ont été créés au cours des vingt dernières années. Cependant, aucun modèle de levure n’a encore vu le jour, alors que cet organisme est utilisé depuis de nombreuses années pour l’étude des protéines amyloïdes.J’ai donc cherché à créer ce modèle de toxicité au cours de ma thèse. L’adressage d’une protéine de fusion Aβ-GFP à la voie de sécrétion permet de rendre son expression toxique chez la levure. J’ai démontré que le trafic intracellulaire était un élément important pour la génération d’espèces toxiques. Les orthologues de PICALM, facteur de prédisposition à la MA, sont impliqués dans la toxicité, montrant une conservation des mécanismes de toxicité avec l’Homme. Les constructions semblent avoir la capacité de traverser les membranes afin d’atteindre des cibles cellulaires comme la mitochondrie.Le modèle ainsi construit nous permettra de mettre en place une étude de la relation entre la structure et la toxicité du peptide Aβ et mieux comprendre les mécanismes cellulaires régissant la MA. / Alzheimer’s Disease (AD) is one of the most important sanitary challenges of the XXIst century. Indeed, 35.6 million people are affected in 2010, and there will be probably four times more in 2050. However, little is known about the mechanisms of toxicity of this disease. Nevertheless, it seems that aggregation of the Aβ peptide is the triggering factor of a cascade of cellular events that leads to three characteristic lesions: neurofibrillary tangles, amyloid plaques and a cortical atrophy, revealing an important neuronal death. Different transgenic models for the study of the Aβ peptide toxicity have been created during the past twenty years. However, no yeast model has yet seen the light of day, whereas this organism is used for several years to study amyloid proteins.Therefore I worked to create this model during my thesis. Addressing an Aβ-GFP fusion to the secretory pathway enable this construction to become toxic in yeast. I proved intracellular pathways are important for generation of toxic species. PICALM orthologs, an AD predisposing factor, are involved in toxicity, showing conservation in the mechanisms of toxicity between yeast and man. The constructions seem to be able to cross membranes and reach cytoplasmic targets as mitochondria.Thus, this model will allow us to set a study of the relationship between structure and toxicity of the Aβ peptide and better understand the cellular mechanisms governing AD

Les récepteurs AMPAR sont les principaux responsables de la transmission excitatrice rapide dans le système nerveux central, y compris dans les neurones d’hippocampe étudiés ici. Ils sont très dynamiques dans la membrane. Au sein des épines dendritiques, ils peuvent se déplacer par traffic membranaire entre les compartiments intracellulaires et la membrane plasmique. Une fois à la surface, ils se déplacent par diffusion latérale et peuvent s'ancrer réversiblement avec des protéines de la densité postsynaptique ou retourner dans des compartiments endocytaires. Les oligomères Aß augmentent l'endocytose des récepteurs AMPAR, diminuent la densité des épines dendritique et provoquent des défaillances globales dans la transmission synaptique excitatrice. Ces effets, sont englobés dans le terme "synaptotoxicité des oligomères Aß" et sont un domaine principal d'étude de l'étiologie de la maladie d'Alzheimer. Wnt5a un ligand Wnt endogène connu pour activer la voie non-canonique dans les neurones d'hippocampe, génère une augmentation des courants excitateurs et des aggrégats de PSD95 et protége les neurones contre la synaptotoxicité des oligomères Aβ. Compte tenu du fait que Wnt5a semble contrecarrer les effets nocifs causés par les oligomères Aß, nous avons procédé à l'étude du mécanisme par lequel Wnt5a protège de la synaptotoxicité des oligomères Aβ. Cela nous a conduit à évaluer l'effet de Wnt5a sur l'un des facteurs dans la transmission glutamatergique, la dynamique des récepteurs AMPAR. En utilisant la microscopie à super-résolution dans les neurones d'hippocampe vivants et fixés, nous avons trouvé que Wnt5a module la dynamique et la localisation des récepteurs AMPAR. Plus précisément, Wnt5a stabilise les récepteurs AMPAR dans les sites synaptiques et extrasynaptiques. Ceci est corrélé avec une augmentation de la co-localisation et de l'interaction entre GluA2 et PSD95. Ces effets ne sont exercés que par l'activation non-canonique de la signalisation Wnt, à travers le ligand Wnt5a et non par les effets canoniques de Wnt7a. De manière intéressante, la pré-incubation de Wnt5a prévient la toxicité des oligomères Aß et maintient la dynamique basale des récepteurs AMPAR. Nos données suggèrent que Wnt5a empêche les effets des oligomères Aβ en favorisant leur stabilisation dans les sites synaptiques. / AMPARs (AMPARs) are responsible for most fast excitatory synaptic transmission in the central nervous system, including hippocampal neurons studied here. AMPARs are highly dynamic in the plasma membrane. Within dendritic spines, they move by membrane trafficking between intracellular compartments and the plasma membrane. Once at the surface, they move through lateral brownian diffusion and can reversibly anchor to postsynaptic density proteins or return to endocytic compartments. Aβ oligomers increase endocytosis of AMPARs, diminish dendritic spine density and cause overall failures in excitatory transmission. These effects, among others, are englobed in the term “Aβ oligomers synaptotoxicity” and are a main focus on the study of Alzheimers disease ethiology. On the contrary, Wnt5a - an endogenous Wnt ligand known to activate the non-canonical pathway in hippocampal neurons - generates an increase in excitatory currents and in clusters of PSD95 and protects neurons against Aβ oligomers synaptotoxicity. Given the fact that Wnt5a seems to counteract the distresses caused by Aβ oligomers, we proceeded to study the mechanism through which Wnt5a protects from Aβ oligomers synaptotoxicity. This led us to evaluate the effect of Wnt5a on one of the important factors in glutamatergic transmission, i.e. AMPAR receptor dynamics. By using super-resolution microscopy in live and fixed hippocampal neurons, we found that Wnt5a modulates the dynamic and localization of AMPARs. Specifically, Wnt5a stabilizes AMPARs in synaptic and extrasynaptic sites. This correlates with an increase in co-localization and interaction between GluA2 and PSD95. These effects are exerted only by non-canonical activation of Wnt signaling, through Wnt5a ligand and not by the canonical effects of Wnt7a. Interestingly, pre-incubation of Wnt5a prevents toxicity of Aβ oligomers and maintains basal AMPARs dynamics. Our data suggest that Wnt5a prevents Aβ oligomers effects by promoting their stabilization in synaptic sites. / Los receptores AMPA (AMPARs) son los principales responsables de la respuesta excitatoria rápida en el sistema nervioso central, incluyendo neuronas hipocampales, estudiadas en esta tesis. A diferencia de otros receptores glutamatérgicos, los AMPARs son altamente dinámicos. Dentro de las espinas dendríticas, se pueden mover hacia y desde compartimentos endocíticos y hacia la membrana plasmática. Una vez en la superficie, a través de difusión lateral, se pueden anclar a proteínas de la densidad postsináptica o regresar a compartimentos endocíticos. Por otro lado, los oligómeros Aβ (oAβ) aumentan la endocitosis de AMPARs, disminuyen la densidad de espinas dendríticas y causan una falla generalizada de la transmisión sináptica excitatoria. Estos efectos, entre otros, se engloban en el término “sinaptotoxicidad por oAβ” y es uno de los principales puntos de estudio en la etiología de la enfermedad de Alzheimer. Al contrario, Wnt5a un ligando endógeno conocido por activar la vía no canónica en neuronas hipocampales, genera un aumento en corrientes excitatorias y en los clusters de PSD95 y protege a las neuronas contra la sinaptotoxicidad causada por oAβ. Debido a esto, procedimos a estudiar el mecanismo por el cual Wnt5a protege de la sinaptotoxicidad causada por Aβ. Esto nos llevó a evaluar los efectos de Wnt5a en uno de los principales factores en la transmisión glutamatérgica, la dinámica de los AMPARs. Con el uso de microscopía de super-resolución en neuronas hipocampales vivas, encontramos que Wnt5a modula la dinámica y localización de los AMPARs. Específicamente, Wnt5a estabiliza los AMPARs en espinas y dendritas. Lo cual se correlaciona con un aumento en la co-localización e interacción entre GluA2 y PSD95. Estos efectos son causados únicamente por la activación no-canónica de la vía Wnt, a través del ligando Wnt5a y no por los efectos canónicos de Wnt7a. De manera interesante, la pre-incubación de Wnt5a previene la toxicicidad de los oligómeros Aβ y mantiene la dinámica basal de los AMPARs. Esta data sugiere que Wnt5a promueve la estabilización de AMPARs, previniendo los efectos synaptotóxicos de los oAβ .

Background Traumatic brain injury (TBI) is a complex disease with a spectrum of symptoms and disabilities. Over the past decade TBI has become the focus of research due to growing epidemiological and clinical evidences that TBI incidences are strong risk factors for Alzheimer’s disease (AD). Major pathological hallmarks of AD are massive accumulations of amyloid-β peptide (Aβ) toxic oligomers and plaques. Neuroinflammation is also considered as a common denominator in AD and aging. The epidemiological and experimental studies have supported that non-steroidal anti-inflammatory drugs markedly reduce the age-related prevalence of AD and can slow amyloid deposition by mechanisms that still remain elusive. S100A9 is a multifunctional cytokine with diverse roles in the cell signaling pathways associated with inflammation and cancers. A widespread expression of S100A9 was also reported in many other ailments involving inflammatory processes, such as AD, malaria, cerebral ischemia and TBI, implying that S100A9 may be a universal biomarker of inflammation. The distinctive feature of S100A9 compared to other pro-inflammatory cytokines is its ability to self-assemble into amyloids, which may lead to the loss of its signaling functions and acquired amyloid cytotoxicity, exceeding that of Aβ. Methods S100A9 properties was studied under various ex vivo and in vitro conditions. First, human and mouse tissues with TBI and AD were subjected to microscopic, immunohistochemical and immunofluorescent techniques. Then, aged mouse treated with native, oligomeric and fibrillary S100A9 was also studied by using behavioral and neurochemical analysis. Moreover, S100A9 was established as a biomarker of dementia progression and compared with others such as Aβ42 and tau proteins, by studying cerebrospinal fluid (CSF) samples from different stages of dementia. Finally, in vitro experiments on S100A9 amyloidogenesis, co-aggregation with Aβ40 and Aβ42, digestion and cytotoxicity were also performed by using spectroscopic, atomic force microscopy and cell biology methods. Results S100A9-driven amyloid-neuroinflammatory cascade serves as a link between TBI and AD. We have found that S100A9 contributes to the plaque formation and intraneuronal responses in AD, being a part of the amyloid-neuroinflammatory cascade. In TBI we have found that extensive S100A9 neuronal production and amyloid self-assembly is triggered immediately after injury, leading to apoptotic pathways and neuronal loss. S100A9 is an integral component of both TBI precursor-plaques, formed prior to Aβ deposition, and AD plaques, characterized by different degree of amyloid maturation, indicating that all plaques are associated with inflammation. Both intra- and extracellular amyloid-neuroinflammatory cascades are intertwined and showed similar tendencies in human and mouse tissues in TBI and AD. Ex vivo findings are further supported by in vitro experiments on S100A9 amyloidogenesis, digestion and cytotoxicity. Importantly, being highly amyloidogenic itself, S100A9 can trigger and aggravate Aβ amyloid self-assembly and significantly contribute to amyloid cytotoxicity. Moreover, the CSF dynamics of S100A9 levels matches very closely the content of Aβ42 in AD, vascular dementia and mild cognitive impairment due to AD, emphasizing the involvement of S100A9 together with Aβ in the amyloid-neuroinflammatory cascade in these ailments. Conclusions The conclusions of this thesis is that the inflammatory pathways and S100A9 specifically represent a potential target for the therapeutic interventions during various post-TBI stages and far prior AD development to halt and reverse these damaging processes. / Role of pro-inflammatory S100A9 protein in amyloid-neuroinflammatory cascade in Alzheimer’s disease and traumatic brain injury

Depuis le début du XXIème siècle, de nombreuses études ont souligné les relations étroites existant entre la voie des récepteurs nucléaires Liver X Receptors (LXRs), leurs gènes cibles et la maladie d'Alzheimer (MA). La stimulation de cette voie est considérée par conséquent comme une approche thérapeutique prometteuse. Cette voie régule de manière complexe et précise l'homéostasie cérébrale du cholestérol et le métabolisme des peptides β-amyloïde (Aβ). Les effets des oxystérols (produits de l'autoxydation du cholestérol) tels que le 24S-hydroxycholestérol (24S-PH-chol) et le 27-hydroxycholestérol (27-OH-chol), tous deux agonistes naturels endogènes des LXRs, sont très largement étudiés à l'échelle neuronale et gliale. Toutefois, peu d'études ont été effectuées sur ces effets au niveau des cellules endothéliales des capillaires cérébraux (CECCs) et des péricytes cérébraux, qui constitue la barrière hémato-encéphalique (BHE), et qui sont d'une part exposées à des flux quotidiens d'oxystérols, et d'autre part impliquées dans les échanges de peptides Aβ entre les compartiments sanguin et cérébral. En utilisant un modèle in vitro de BHE, ces travaux de thèse tendent à mettre en évidence sur l'influence de ces deux oxystérols sur les CECCs et sur les péricytes cérébraux, en se focalisant sur l'expression des gènes cibles des récepteurs LXRs et sur le transport des peptides Aβ. Nos résultats démontrent que le 24S-OH-chol induisent l'expression des transporteurs ATP Binding Cassette sub-familly A member 1 (ABCA1, pour les CECCs et les péricytes) et ABCG1 (seulement au niveau des CECCs), qui corrèle avec une augmentation de l'efflux cellulaire de cholestérol vers les (apo)liprotéines. De plus, ces oxysérols n'exercent aucune influence sur l'accumulation des peptides Aβ dans les péricytes cérébraux, mais diminuent le transport apical-à-basolatéral (influx) des peptides Aβ à travers les CECCs. Cette restriction d'influx est indépendante de la fonction du transporteur ABCA1, et semble être sous l'influence de la P-gp (P-glycoprotéine), pompe d'efflux de la BHE. Ces résultats soulignet l'importance des récepteurs LXRs dans le métabolisme cérébral du cholestérol et les échanges de peptides Aβ, et par conséquent dans la MA. / As several studies have demonstrated a close relationship between Liver X Receptors (LXRs) signalling pathway, their target genes and Alzheimer's disease (AD), stimulation of these nuclear receptors is now considered as a promising therapeutical approach in AD. This pathway strictly regulates brain cholesterol homeostasis and amyloid-β (Aβ) peptide metabolism. Actions of oxysterols such as 24S-hydroxycholesterol (24S-OH-chol) or 27-hydroxycholesterol (27-OH-chol), both known as LXR endogenous agonists, are therefore under intense scrutiny in neurons and glial cells. But little is known in brain capillary endothelial cells (BCECs) and brain pericytes (BPs) which compose the blood-brain barrier (BBB), which are continuously exposed to oxysterols fluxes and are involved in Aβ peptide exchanges between blood and brain. Using an in vitro BBB models, this study aims to highlight the influence of both oxysterols on BCECs and BPs, focusing on their involvement on LXRs target genes expression and on Aβ peptide transport. Our results demonstrate that 24S-OH-chol and 27-OH-chol induced the expression of ATP Binding Cassette sub-family A member 1 (ABCA1, in BCECs and BPs) and ABCG1 (only in BCECs), correlating with an increase of cellular cholesterol efflux to (apo)lipoproteins. Furthermore, these oxysterols do not modify Aβ peptide accumulation in BPs but decrease their influx across the BCECs. This latter process is ABCA1-independent and seems to be mediated by the P-gp (P-glycoprotein) efflux pump. These results highlight the importance of the BBB in the LXR-mediated effects in brain cholesterol metabolism and Aβ peptide clearance and, thus, in AD.

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The work detailed in this dissertation has an overarching theme of modulating microglia activation in both in vivo and in vitro models relevant to AD. The premise is that understanding microglia function in this context may lead to a better understanding of AD pathogenesis and thus to effective therapeutic interventions. In chapter 3, we employ a well-defined model of microglia activation whereby the intraperitoneal delivery of LPS results in CNS microglia activation and TNF-α production. Having previously identified that CD45 signaling pathways antagonized microglia TNF-α production in vitro and given that immunotherapy with anti-CD45 antibodies are already in clinical trials for both the treatment of malignant disorders as well as for tolerance induction following organ transplants, we investigated whether microglial CD45 could be a relevant molecular target in the opposition of microglia activation in vivo. Given that a number of epidemiological studies have shown an inverse correlation between the use of statins (a class of drugs that were initially described as specific inhibitors of cholesterol biosynthesis) and the incidence of Alzheimer's disease (Wolozin, 2000; Simon 2002; Zamrini, 2004); in chapter 4, we employed one of the most prescribed statins, lovastatin in our microglia activation paradigm. Interestingly, we show that statins only inhibit the enzyme HMG-CoA (the rate limiting step of cholesterol biosynthesis) but that they also display pleiotrophic effects including the inhibition of CD40 expression. Given the role of CD40 in microglia activation and its potential role in AD pathogenesis, we investigated whether lovastatin's protection in AD might be derived from effects on microglia function as governed by the CD40 pathway. In chapter 5, we provide evident for a model of microglia activation that addresses some of the current controversies concerning the role of microglia cells in AD pathogenesis. The model suggests that microglia cells exist in a number of distinct activation states, in one such state that we denote the "phagocytic state"; microglia function to clear cellular debris or foreign invaders or in the case of AD to remove β-amyloid/Aβ peptides. However, in response to certain co-stimuli (i.e. CD40 activation) these microglia take the form of an "antigen presenting cell" (APC) whereby they lose their phagocytic capacity in lieu of cytokine production and thus potential contributing to AD pathogenesis.

Alzheimer’s disease is neurodegenerative with amyloid plaque and neurofibrillary tangles as pathological hallmarks. The most abundant component in the amyloid plaque is the amyloid-β (Aβ) peptide, with presence of both isoform Aβ40 and Aβ42. In immunological methods studying the Aβ peptide a specific monoclonal antibody, 6E10, is routinly being used. In this master thesis work unspecific binding of 6E10 antibody to the blood coagulating protein factor X has been investigated. Factor X is a protein in the blood coagulation cascade where it forms protein complex that activates thrombin. Non-hemostatic functions with connections to nerves and Aβ peptide are also known. Studies with Western blot show clear binding of 6E10 to denatured factor X. Interaction studies with ELISA gives uncertain results, where binding is found but no clear binding curve is obtained. Studies with native factor X in real time measurements with SPR gave no binding at all. These results suggest binding to denatured factor X. Immunohistochemistry studies of colocalisation of factor X and Aβ peptide gave clear evidence that factor X and Aβ are found near each other in mouse brain tissue. Factor X is located outside the blood vessels and Aβ is located at the inside.