Introdução: A Doença de Alzheimer (DA) é uma desordem neurodegenerativa e a forma mais comum de demência. Processos inflamatórios parecem desempenhar importante papel na fisiopatologia da DA. A neuroinflamação é caracterizada pela ativação da microglia e a liberação de citocinas inflamatórias, tais como IL-1β, IL-6 e TNF-α. Porém, não se sabe qual é a real contribuição destes marcadores inflamatórios no desenvolvimento da DA. Objetivos: A proposta deste estudo é avaliar a possível relação entre marcadores inflamatórios no liquido cefalorraquidiano de indivíduos com comprometimento cognitivo leve amnéstico (CCL-a), com 60 anos ou mais, e comparar com controles saudáveis da mesma faixa etária. Métodos: Foram examinadas as concentrações de IL-1β, IL-6 e TNF-α no líquido cefalorraquidiano de sujeitos com CCL-a e em controles pelo método ELISA. Diagnósticos de CCL-a foram baseados na anamnese e nos critérios de Petersen, corroborados pela escala CDR. Para avaliar a função cognitiva o teste de memória e reconhecimento de palavras do CERAD e o Teste do Relógio foram aplicados aos participantes. Para a avaliação de sintomas depressivos usou-se o GDS. Resultados: Este estudo demonstrou diminuição significativa nos níveis de IL-1β (13.735 vs 22.932 pg/mL; p <0.001) e TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), mas não nos níveis da IL-6 (4.178 vs 5.689 pg/mL; p: 0.106), entre casos e controles. Indivíduos com IL-1β < 17 pg/mL possuem 7.2 (CI: 1.5-36; p: 0.016) mais chances de evoluírem à CCL-a. Além disso, houve correlação positiva entre IL-1β e a pontuação da lista de palavras do CERAD (rs: 0.299; p: 0.046). A análise de regressão linear mostrou que os níveis de IL-1β podem explicar 13.7% (β: 24.545; p: 0.012) da variância da pontuação do CERAD, o que sugere uma dependência linear direta. Conclusões: A neuroinflamação, mediada pela IL-1β e pelo TNF-α, provavelmente possui importante papel na prevenção de CCL-a. / Introduction: Alzheimer Disease (AD) is a neurodegenerative disorder and the most common form of dementia. Inflammatory processes may play a significant role at the pathophysiology of AD. Neuroinflammation is characterized by activation of microglia and the release of inflammatory cytokines, such as IL-1β, IL-6 and TNF-α. Although, it is unknown what is the real contribution of these inflammatory markers in the development of AD. Aims: The purpose of this study is to assess the possibly relationship between inflammatory markers in CSF of amnestic MCI subjects, with sixty years or older, and compare to aged healthy controls. Methods: We examined concentrations of IL-1β, IL-6 and TNF-α at CSF of amnestic MCI subjects and controls by ELISA. MCI diagnoses were based on anamnesis and Petersen criteria, corroborated by CDR. To assess the cognitive function the word list memory test and word recognition of CERAD and Clock Drawing Test were applied to subjects, and to evaluated depression symptoms the GDS was used. Results: This study demonstrated significant diminish in the levels of IL-1β (13.735 vs 22.932 pg/mL; p <0.001) and TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), but not IL-6 (1.913 vs 2.627 pg/mL; p: 0.002), between cases and controls. Individuals with IL-1β < 17 pg/mL were at a 7.2 (CI: 1.5-36; p: 0.016) increased odds of aMCI. Furthermore, there was a positive correlation between IL-1β and the CERAD word list score (rs: 0.299; p: 0.046). The linear regression analysis showed that IL-1β levels can explain 13.7% (β: 24.545; p: 0.012) of the variance on this CERAD subscore, suggesting a direct linear dependence. Conclusion: Neuroinflamation mediated by IL-1β and TNF-α may play an important role in preventing aMCI.

Introdução: A Doença de Alzheimer (DA) é uma desordem neurodegenerativa e a forma mais comum de demência. Processos inflamatórios parecem desempenhar importante papel na fisiopatologia da DA. A neuroinflamação é caracterizada pela ativação da microglia e a liberação de citocinas inflamatórias, tais como IL-1β, IL-6 e TNF-α. Porém, não se sabe qual é a real contribuição destes marcadores inflamatórios no desenvolvimento da DA. Objetivos: A proposta deste estudo é avaliar a possível relação entre marcadores inflamatórios no liquido cefalorraquidiano de indivíduos com comprometimento cognitivo leve amnéstico (CCL-a), com 60 anos ou mais, e comparar com controles saudáveis da mesma faixa etária. Métodos: Foram examinadas as concentrações de IL-1β, IL-6 e TNF-α no líquido cefalorraquidiano de sujeitos com CCL-a e em controles pelo método ELISA. Diagnósticos de CCL-a foram baseados na anamnese e nos critérios de Petersen, corroborados pela escala CDR. Para avaliar a função cognitiva o teste de memória e reconhecimento de palavras do CERAD e o Teste do Relógio foram aplicados aos participantes. Para a avaliação de sintomas depressivos usou-se o GDS. Resultados: Este estudo demonstrou diminuição significativa nos níveis de IL-1β (13.735 vs 22.932 pg/mL; p <0.001) e TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), mas não nos níveis da IL-6 (4.178 vs 5.689 pg/mL; p: 0.106), entre casos e controles. Indivíduos com IL-1β < 17 pg/mL possuem 7.2 (CI: 1.5-36; p: 0.016) mais chances de evoluírem à CCL-a. Além disso, houve correlação positiva entre IL-1β e a pontuação da lista de palavras do CERAD (rs: 0.299; p: 0.046). A análise de regressão linear mostrou que os níveis de IL-1β podem explicar 13.7% (β: 24.545; p: 0.012) da variância da pontuação do CERAD, o que sugere uma dependência linear direta. Conclusões: A neuroinflamação, mediada pela IL-1β e pelo TNF-α, provavelmente possui importante papel na prevenção de CCL-a. / Introduction: Alzheimer Disease (AD) is a neurodegenerative disorder and the most common form of dementia. Inflammatory processes may play a significant role at the pathophysiology of AD. Neuroinflammation is characterized by activation of microglia and the release of inflammatory cytokines, such as IL-1β, IL-6 and TNF-α. Although, it is unknown what is the real contribution of these inflammatory markers in the development of AD. Aims: The purpose of this study is to assess the possibly relationship between inflammatory markers in CSF of amnestic MCI subjects, with sixty years or older, and compare to aged healthy controls. Methods: We examined concentrations of IL-1β, IL-6 and TNF-α at CSF of amnestic MCI subjects and controls by ELISA. MCI diagnoses were based on anamnesis and Petersen criteria, corroborated by CDR. To assess the cognitive function the word list memory test and word recognition of CERAD and Clock Drawing Test were applied to subjects, and to evaluated depression symptoms the GDS was used. Results: This study demonstrated significant diminish in the levels of IL-1β (13.735 vs 22.932 pg/mL; p <0.001) and TNF-α (1.913 vs 2.627 pg/mL; p: 0.002), but not IL-6 (1.913 vs 2.627 pg/mL; p: 0.002), between cases and controls. Individuals with IL-1β < 17 pg/mL were at a 7.2 (CI: 1.5-36; p: 0.016) increased odds of aMCI. Furthermore, there was a positive correlation between IL-1β and the CERAD word list score (rs: 0.299; p: 0.046). The linear regression analysis showed that IL-1β levels can explain 13.7% (β: 24.545; p: 0.012) of the variance on this CERAD subscore, suggesting a direct linear dependence. Conclusion: Neuroinflamation mediated by IL-1β and TNF-α may play an important role in preventing aMCI.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-10-18T19:17:10Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao -Gilderlanio Santana de Araujo.pdf: 9533988 bytes, checksum: 951b1cf090729a87ebf3a8741ff00ad4 (MD5) / Made available in DSpace on 2016-10-18T19:17:10Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao -Gilderlanio Santana de Araujo.pdf: 9533988 bytes, checksum: 951b1cf090729a87ebf3a8741ff00ad4 (MD5) Previous issue date: 2013-03-07 / FACEPE / O desenvolvimento de técnicas de genotipagem de baixo custo (SNP arrays) e as anotações de milhares de polimorfismos de nucleotídeo único (SNPs) em bancos de dados públicos têm originado um crescente número de estudos de associação em escala genômica (do inglês, Genome-Wide Associations Studies - GWAS). Nesses estudos, um enorme número de SNPs (centenas de milhares) são avaliados com métodos estatísticos univariados de forma a encontrar SNPs associados a um determinado fenótipo. Testes univariados são incapazes de capturar relações de alta ordem entre os SNPs, algo comum em doenças genéticas complexas e são afetados pela alta correlação entre SNPs na mesma região genômica. Métodos de aprendizado de máquina, como o Random Forest (RF), têm sido aplicados em dados de GWAS para realizar a previsão de riscos de doenças e capturar os SNPs associados às mesmas. Apesar de RF ser um método com reconhecido desempenho em dados de alta dimensionalidade e na captura de relações não-lineares, o uso de todos os SNPs presentes em um estudo GWAS é computacionalmente inviável. Neste estudo propomos o uso de redes biológicas para a seleção inicial de SNPs candidatos a serem usados pela RF. A partir de um conjunto inicial de genes já relacionados à doença na literatura, usamos ferramentas de redes de interação gene-gene, para encontrar novos genes que possam estar associados a doença. Logo, é possível extrair um número reduzido de SNPs tornando a aplicação do método RF viável. Os experimentos realizados nesse estudo concentram-se em investigar quais polimorfismos podem influenciar na suscetibilidade à doença de Alzheimer (DA) e ao comprometimento cognitivo leve (MCI). O resultado final das análises é a delineação de uma metodologia para o uso de RF, para a análise de dados de GWAS, assim como a caracterização de potenciais fatores de riscos da DA. / The development of low cost genotyping techniques (SNP arrays) and annotations of thousands of single nucleotide polymorphisms (SNPs) in public databases has led to an increasing number of Genome-Wide Associations Studies (GWAS). In these studies, a large number of SNPs (hundreds of thousands) are evaluated with univariate statistical methods in order to find SNPs associated with a particular phenotype. Univariate tests are unable to capture high-order relationships among SNPs, which are common in complex genetic diseases, and are affected by the high correlation between SNPs at the same genomic region. Machine learning methods, such as the Random Forest (RF), have been applied to GWAS data to perform the prediction of the risk of diseases and capture a set of SNPs associated with them. Although, RF is a method with recognized performance in high dimensional data and capacity to capture non-linear relationships, the use of all SNPs present in GWAS data is computationally intractable. In this study we propose the use of biological networks for the initial selection of candidate SNPs to be used by RF. From an initial set of genes already related to a disease based on the literature, we use tools for construct gene-gene interaction networks, to find novel genes that might be associated with disease. Therefore, it is possible to extract a small number of SNPs making the method RF feasible. The experiments conducted in this study focus on investigating which polymorphisms may influence the susceptibility of Alzheimer’s disease (AD) and mild cognitive impairment (MCI). This work presents a delineation of a methodology on using RF for analysis of GWAS data, and characterization of potential risk factors for AD.

Le développement de nouveaux médicaments, la validation et la standardisation des marqueurs de neuroimagerie et biochimie de la Maladie d'Alzheimer (MA) seront les principaux objectifs de la recherche de cette maladie dans les années à venir. La présente thèse vise à aborder ces questions cruciales. La première partie de la thèse fait le point sur l’application correcte des Procédures Opérationelles Standards (SOPs) pour l'acquisition de protocoles de neuroimagerie structurelle et l’application des marqueurs de neuroimagerie cérébrale dans 10 cliniques italiennes spécialisées dans les troubles de la mémoire. La deuxième partie traite de l'application de plusieurs marqueurs de neuroimagerie structurelle et fonctionnelle dans le cadre des études cliniques sur des patients ayant des troubles précoces de la maladie d'Alzheimer. Les résultats ont révélé que la mise en oeuvre des SOPs pour l’acquisition des séquences d’imagerie par résonance magnétique (IRM), au niveau multicentrique, réduit la variance des mesures des marqueurs de neuroimagerie détectée par différents scanners. En outre, les résultats de la deuxième partie de la thèse ont montré un impact significatif des thérapies anticholinestérasiques pour réduire l'atrophie de l'hippocampe, l'amincissement cortical ainsi que une augmentation de l'activation de zones du cerveau liées à l'IRM fonctionnelle chez des patients ayant des troubles précoces de la MA. Ces résultats prometteurs confirment l'hypothèse que les marqueurs de neuroimagerie structurelle et fonctionnelle appliqués avec SOPs pourraient être utilisés comme critère d'évaluation substitutif dans les études cliniques pour les patients ayant des troubles précoces de la maladie d'Alzheimer. / The development of new drugs and the validation and standardization of neuroimaging and biological markers for Alzheimer’s Disease (AD) clinical treatment trials is expected to be one of the major goals of AD research in the upcoming years. The present thesis aims to adress these critical issues. The first part of the thesis is focused on the proper application of Standard Operating Procedures (SOPs) for the structural neuroimaging protocols of acquisition and the implementation of neuroimaging markers in 10 Italian Memory Clinics. The second part of the thesis deals with the application of several structural and functional neuroimaging markers in the context of clinical trials investigation in mild cognitive impairment individuals. Results revealed that the implementation of SOPs at multicentre level reduces the variance of neuroimaging markers measurement detected by different scanners. Moreover, results from the employment of neuroimaging markers in pre-dementia trials in mild cognitive impairment individuals showed a significant impact of anticholinesterase therapies in reducing the hippocampal rate of atrophy, the cortical thinning as well as in increasing the activation of brain areas related to functional Magnetic Resonance Imaging (fMRI) face and location macthing tasks. These promising results support the hypothesis that structural and functional neuroimaging markers applied in a standardized manner migh be utilized as candidate surrogate outcomes in future pre-dementia trials for AD.

La maladie d’Alzheimer (MA) est la maladie neurodégénérative touchant le plus de personnes dans le monde. Jusqu’à présent, aucun traitement curatif n’existe pour soigner cette maladie, d’où la nécessité d’identifier et d’étudier de nouvelles cibles thérapeutiques.La découverte des effets bénéfiques de la caféine, antagoniste du récepteur à adénosine A2A (A2AR), conjuguée à une surexpression de ce dernier chez les patients atteints de la MA, font de ce récepteur une cible d’intérêt. En effet, des antagonistes des A2ARs ont montré leur capacité à améliorer les performances cognitives de par une diminution de la charge amyloïde associée à une diminution la phosphorylation de la protéine Tau.Bien que plusieurs antagonistes aient été développés pour le traitement de maladies neurodégénératives, ceux-ci présentent un manque d’efficacité corrélée à de faibles propriétés pharmacocinétiques. Ainsi, à partir d’études de modélisation moléculaire, deux nouvelles familles d’antagonistes présentant un noyau central benzoxazole ou quinazoline ont été conçus, synthétisés et évalués pharmacologiquement. Trois composés ont été sélectionnés et font actuellement l’objet d’études pharmacologiques complémentaires sur modèles animaux. / Alzheimer’s disease (AD) is the most prevalent form of dementia in the aged population. So far, there is no way to halt or slow-down AD. Therefore, there is a constant need of developing novel therapeutic strategies.In recent years, adenosine A2A receptor (A2AR) has attracted a growing interest since it has been proved that this receptor is over-expressed during AD. Also, epidemiological studies showed that people consuming regularly caffeine-based beverages over a lifetime are substantially less likely to develop this disease. Indeed, A2AR antagonists improve memory performance as it reduces β-amyloid deposits and Tau-phosphorylation.Though several antagonists have been developed for the treatment of neurodegenerative diseases, current research efforts are focus on developing new antagonists with relevant ADME properties and a better efficacy. Based on a molecular modeling-guided design, we synthesised new A2AR antagonists with benzoxazole and quinazoline as central scaffold. Three molecules were selected and will be subject to evaluation on animal’s model.

Lors du screening de composés du ‘human chemical exposome’, les herbicides triazines et les insecticides pyrazoles, notamment le fipronil, ont révélé leur capacité à induire la production de peptides amyloïde β -42 et -43. Ayant tendance à s’agréger en oligomères puis en plaques, ils sont une caractéristique de la maladie d’Alzheimer (MA). Cette découverte informe sur le danger potentiel de ces produits et apporte des outils pour décrypter les mécanismes menant à l’altération du rapport des différentes formes d’amyloïdes. Par ailleurs, la dérégulation de DYRK1A, impliquée dans la trisomie 21, affecte aussi ces peptides amyloïdes, ainsi que la protéine Tau qui est alors hyperphosphorylée et a tendance à s’agréger en enchevêtrements neurofibillaires, une autre caractéristique de la MA. Ce même effet sur Tau est observé avec CDK5 dans la MA, les AVC et les traumatismes crâniens. Le développement d’inhibiteurs pharmacologiques spécifiques de ces deux kinases est donc un enjeu pour ManRos Therapeutics. / In the screening of ‘human chemical exposome’ compounds, triazine herbicides and pyrazole insecticides, especially fipronil, have shown their ability to induce β-amyloid -42 and -43 peptide production. As they tend to aggregate into oligomers then into plaques, they are a characteristic of Alzheimer's disease (AD). This discovery informs about the potential danger of these products and provides tools to decipher the mechanisms leading to the alteration of the ratio of the different forms of amyloid.Moreover, dysregulation of DYRK1A, involved in trisomy 21, also affects these amyloid peptides, as well as the Tau protein which is then hyperphosphorylated and tends to aggregate into neurofibillar tangles, another characteristic of AD. This same effect on Tau is observed with CDK5 in AD, stroke and brain injury. The development of specific pharmacological inhibitors of these two kinases is therefore an issue for ManRos Therapeutics.

L'encodage de la mémoire dépend de changements durables dans l'activité des circuits synaptiques dans un ensemble de neurones interconnectés. La région CA3 de l'hippocampe reçoit des informations directement ou indirectement (à travers le gyrus denté - GD) en provenance des structures corticales. Des données théoriques et comportementales ont montré que la région CA3 est importante pour l'encodage de la mémoire épisodique, en particulier au stade initial de l'acquisition, en développant vraisemblablement une représentation instantanée d'un contexte. Les neurones pyramidaux CA3 reçoivent une variété de connections afférentes, parmi lesquelles les fibres moussues (FM), les axones des cellules du gyrus denté. Ces connections synaptiqes ont attiré une attention par leurs propriétés morphologiques et fonctionnelles uniques. Malgré les nombreuses études comportementales et computationnelle, les liens entre plasticité des circuits CA3 et encodage de la mémoire ne sont pas bien compris.Le cadre général de ce projet de thèse se situe dans l'étude des mécanismes synaptiques de l'encodage de la mémoire épisodique dans des conditions physiologiques ainsi que dans un modèle de souris de la maladie d'Alzheimer (MA). En effet, la MA se caractérise à un stade précoce par une mémoire épisodique altérée, qui peut être associée à une dysrégulation de la plasticité des circuits CA3.À l'aide de techniques d'enregistrement électrophysiologique, nous avons d'abord exploré les modifications dans les circuits CA3 peu de temps (quelques heures) après conditionnement de la peur contextuelle chez les souris adultes C57Bl6j. Nous avons observé une augmentation de la fréquence des IPSC spontanés accompagnée de changements mineurs dans le nombre de filopodia issus des boutons synaptiques des FM, tandis que les EPSCs et les plasticités à court terme de ces synapses ne sont pas modifiés. Cependant, cette augmentation n'est peut observée 24 heures après l'apprentissage contextuel. Nous avons également tenté de modéliser de manière simplifiée les réseaux neuronaux GD-CA3, afin d'étudier si et dans quelle mesure les interneurones locaux dans la région CA3 contribuent à la précision de l'encodage de la mémoire. [...]Dans l'ensemble ce travail a révélé que la transmission inhibitrice des circuits locaux CA3 de l'hippocampe pourrait être importante dans l'encodage de la mémoire épisodique. Dans le modèle murin de la MA avec déficit de mémoire, il y a une réduction de la transmission GABAergique et des courants médiés par les KAR réduits cellules pyramidales de CA3. Finalement, avons observé une modification transcriptionnelle d'un certain nombre de gènes dans CA3, à des stades précoces de développement de la pathologie dans notre modèle de MA. Notre étude pourrait contribuer à la compréhension des mécanismes pathologiques précoces de la MA, au niveau synaptique ainsi qu'au niveau transcriptionnel, et fournir des idées nouvelles sur les mécanismes sous-jacents au codage rapide de la mémoire contextuelle. / Memory encoding is thought to proceed from durable changes in the activity of synaptic circuits to the storage of patterns of electrical events in a sparsely distributed ensemble of neurons. Located at the entry level of hippocampal circuitry, the CA3 region of hippocampus is thought to be important for episodic memory encoding, especially at the initial stage of acquisition, by presumably developing an instant representation of a context. CA3 pyramidal neurons receive a variety of diverse inputs, among which the mossy fiber (MF) inputs draw special attention for its peculiar structure and unique synaptic properties. However, the links between the plasticity of CA3 circuits and memory encoding are not well understood.This thesis project aimed to address the synaptic mechanisms of episodic memory encoding in physiological conditions as well as in a mouse model of Alzheimer's disease (AD).Using electrophysiological recording techniques, we first explored the changes in CA3 circuits shortly after one-trial contextual fear conditioning in adult C57Bl6j mice. We show that despite hardly any changes in filopodia number of MF terminals, an increase in spontaneous IPSC frequency can be registered, while the EPSCs and short-term plasticities of theses synapses are unaltered. However, this increase cannot be seen anymore 24 hours after the contextual learning. We also tried to do simplified computational modeling of the DG-CA3 neuronal networks, to investigate if and to what extent the local interneurons in CA3 region contribute to memory encoding precision.AD is characterized at an early stage by impaired episodic memory, which may involve dysregulation of the plasticity of CA3 circuits.In the next step, we searched for synaptic deficits in CA3 local circuit in the early stage of AD pathology, taking advantage of a familial AD mouse model: 6-month male APP/PS1 mice. We report that there is a reduction in spontaneous IPSC frequency in CA3 neurons together with decreased inhibitory charges of evoked events at MF-CA3 synapses, whereas the short-term plasticity of these synapses and intrinsic properties of CA3 neurons remain unaffected. Furthermore, there is a robust reduction in Kainate receptor (KAR) mediated currents at MF-CA3 synapses, and the same results can be obtained from PSKO mice too, suggesting that disturbed function of γ-secretase and NCad processing pathways might underlie the dysfunction of KARs at MF-CA3 synapses.Finally, to screen for changes on a transcriptome level, we performed RNA-seq with dissected CA3 tissue from APP/PS1 mice and found a list of up- and down-regulated genes at this early stage of AD. Moreover, we carried out ChIP-seq for a histone modification marker: H3K4me3, which has been shown to be directly related to one-trial contextual memory, and here we report that there is a concrete decrease in H3K4me3 levels at the promoter areas of various genes in CA3 neurons. However, these genes are not overlapping much with the down-regulated genes from RNA-seq result, suggesting that other epigenetic mechanisms might play more important roles in expressing early deficits in this AD mouse model.Taken together, we show that inhibitory innervations of hippocampal CA3 local circuits might be important for episodic memory encoding, and in early AD mouse model with memory deficits, there is reduced GABAergic transmission and reduced KAR-mediated currents in CA3 neurons, together with many active transcriptional regulations across the genome. Our study might contribute to the understanding of early AD pathologies at synaptic level as well as transcriptional level, and provide novel insights into the mechanisms underlying rapid encoding of contextual memory.

Dysfunction of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis is a common finding in advanced dementia. In this study, the function of the LHPA axis at different levels was investigated in patients with dementia and in healthy elderly. A subtle disturbance in the feedback regulation of the LHPA axis was found in patients with early (i.e., mild to moderate) Alzheimer’s disease (AD). After 0.5 mg dexamethasone, serum cortisol levels were less suppressed in AD patients and plasma adrenocorticotropin (ACTH) levels were lower as compared with healthy elderly. After stimulation with human corticotropin-releasing hormone a blunted ACTH response was found in AD patients while relative serum cortisol, dehydroepiandrosterone, and androstenedione responses were increased. Significant correlations were found between low plasma ACTH levels and temporal lobe atrophy and between low peak plasma ACTH levels and hippocampal atrophy measured with computer tomography. Patients with advanced AD and multi-infarct dementia had lower basal levels of dehydroepiandrosterone sulphate in combination with no difference in cortisol levels, resulting in a high cortisol/DHAS ratio. The difference persisted after adjustments for age and sex in a multivariate analysis. In patients with early AD, basal serum levels of dehydroepiandrosterone and androstenedione were increased, and this increase was accentuated after stimulation with ACTH. Peripheral glucocorticoid sensitivity was examined by skin vasoconstrictor blanching tests. Patients with AD and patients treated with glucocorticoids showed skin blanching at higher clobetasol concentrations than healthy elderly. These findings justify further investigations on the role of LHPA axis dysfunction in Alzheimer’s disease and its possible importance for the pathophysiology of the disease. / digitalisering@umu

Les tauopathies, dont la maladie d’Alzheimer (MA) est l’exemple le plus connu, sont des maladies neurodégénératives caractérisées par une agrégation intra-neuronale progressive de protéine Tau hyperphosphorylée conduisant inéluctablement à la mort du neurone. De nombreuses études convergent vers une implication du stress oxydant comme l’un des mécanismes précoces à l’origine de la MA. En effet, une accumulation de dommages oxydatifs aux acides nucléiques est observée aux stades précoces de la MA. Cependant, les mécanismes impliqués dans cette altération de l’intégrité des acides nucléiques au cours de la MA restent obscurs. Outre son rôle connu dans la stabilisation des microtubules, Tau est un acteur essentiel de la protection de l’intégrité des acides nucléiques neuronaux. En effet, au sein du laboratoire, il a été récemment montré in vivo que Tau protège l’ADN et l’ARN en condition physiologique ainsi qu’au cours d’un stress hyperthermique. L’hyperthermie est un outil inducteur de stress oxydant. Aucune étude n’a été menée pour examiner l’effet de la pathologie Tau sur la fonction protectrice de Tau vis-à-vis des acides nucléiques (AN).Cette problématique constitue le premier objectif de ma thèse. Pour cela, nous avons utilisé un modèle murin qui développe une agrégation et une hyperphosphorylation progressive de la protéine Tau : les souris THY-Tau22. Nous avons démontré, dans ce modèle, que l’hyperthermie induit des dommages aux AN uniquement dans les neurones présentant une pathologie précoce, à des stades qui précèdent la formation d’agrégats insolubles de Tau. Au sein de ces neurones, ces dommages aux AN induits par l’hyperthermie sont strictement corrélés à la formation d’oligomères de protéines Tau, formes précoces de l’agrégation de Tau. Une association similaire entre la présence d’oligomères de Tau et des dommages oxydatifs a également été mis en évidence dans des cerveaux de patients atteints de la MA. Le pré-traitement de ces souris avec du bleu de méthylène, un inhibiteur de l’agrégation de Tau, prévient la formation de ces oligomères ainsi que les dommages aux acides nucléiques. Ces résultats suggèrent que l’oligomérisation de Tau prévient la fonction protectrice de Tau vis-à-vis des acides nucléiques. Ce travail met également en lumière l’existence d’une fenêtre critique de vulnérabilité de l’ADN et l’ARN au cours de la progression de la pathologie.Dans le deuxième volet de ce manuscrit, nous nous sommes concentrés sur la relation entre la protéine Tau et l’ARN. Bien que l’interaction de Tau avec l’ARN soit connue depuis une vingtaine d’années, la fonction de cette interaction reste obscure. Notre hypothèse est que Tau pourrait être impliquée dans le métabolisme des ARN. Par stratégie TAP-tag, des partenaires protéiques de Tau ont été purifiés. Parmi eux, la protéine DEAD box protein 6 (DDX6), un acteur du métabolisme des ARN, a été identifiée comme un nouveau partenaire de Tau. DDX6 est une hélicase à ARN impliquée notamment dans la voie de régulation de l’expression génique par les microARN. Ce second projet vise à caractériser, comprendre la fonction de ce complexe ainsi que son impact dans la pathogénèse des tauopathies. Nous avons confirmé l’interaction entre Tau et DDX6 puis identifié les séquences de Tau impliquées dans ce complexe. Nos résultats suggèrent que l’interaction de Tau avec DDX6 stimule l’activité du microARN Let-7a. De manière particulièrement intéressante, des mutations de Tau responsables de formes familiales de tauopathies altèrent l’interaction Tau/DDX6 et abolissent l’effet de Tau sur l’activité du microARN Let-7a. L’ensemble de ces résultats met en évidence un rôle atypique de Tau, non décrit à ce jour, dans la voie de régulation du microARN Let-7a. Cette nouvelle fonction ouvre des perspectives sur le rôle de Tau dans le métabolisme des ARN et suggère un impact de la pathologie Tau sur la régulation de la voie des microARN. / Tauopathies are neurodegenerative disorders characterized by a progressive intraneuronal accumulation of hyperphosphorylated Tau leading to neuronal death. The most well-known tauopathy is Alzheimer Disease (AD). Numerous studies suggest that oxidative stress is one of the early mechanisms involved in AD. An increase in oxidative DNA and RNA damage occurs at early stages of AD. The mechanisms underlying the alteration of nucleic acid integrity during the course of AD are unclear. In addition to its well-described role in microtubule stabilization, Tau is an essential player in the protection of neuronal nucleic acid integrity. Indeed, we recently reported that Tau protect DNA and RNA integrity in vivo under physiological and hyperthermic conditions, which is known to be a strong inducer of oxidative stress. However, no study has been conducted to test the effects of Tau pathology on the protective function of Tau. This is the first objective of my thesis.To do that, we used a transgenic Tau pathology mouse model. With age, these mice develop a progressive Tau hyperphosphorylation and aggregation. We demonstrate, in this model, that hyperthermia selectively induces nucleic acid damage in neurons that display early Tau pathology without Tau fibrils. In these neurons, nucleic acid damage is strictly correlated with prefibrillar Tau oligomers. A similar association between prefibrillar Tau oligomers and nucleic acid oxidative damage was observed in AD brains. Pretreatment with Methylene Blue (MB), a Tau aggregation inhibitor reduced hyperthermia-induced Tau oligomerization as well as nucleic acid damage. These results suggest that Tau oligomerization triggers the loss of the nucleic acid protective function of Tau. This study highlights the existence of a critical window of DNA and RNA vulnerability during the progression of Tau pathology.In the second part of this manuscript, we have focused on the relationship between Tau and RNA. It has been reported that Tau bind to RNA. Although this interaction has been known for 20 years, the function of this interaction is still unclear. Based on this observation, we hypothesize that Tau is involved in RNA metabolism. Proteins interacting with Tau have been purified using the tandem affinity purification methodology. Thus, the DEAD box protein 6 (DDX6), known to be an actor of RNA metabolism, has been identified as a new Tau partner. DDX6 is a RNA helicase implicated in miRNA gene silencing mechanism. This project aims to understand Tau-DDX6 complex function in physiological conditions and its impact on tauopathies. We validate the interaction between Tau and DDX6, and identify Tau sequences involved in the interaction. Our results suggest that Tau-DDX6 complex enhance Let-7a activity. Interestingly, Tau mutations involved in inherited tauopathies impair Tau-DDX6 interaction and abolish the effect of Tau on Let-7a activity. All these results highlight a new and atypical function of Tau in microRNA pathway. This undescribed function offers promising prospects for the role of Tau in RNA metabolism and suggest a potential impact of Tau pathology on regulation of microRNA pathway.

Les démences regroupent un ensemble de pathologies cérébrales affectant progressivement les fonctions cognitives et survenant plus fréquemment chez les personnes âgées. L’augmentation du nombre de cas liée au vieillissement de la population et la lourdeur de la prise en charge font des démences un problème de santé publique important.La maladie d’Alzheimer (MA) est la plus fréquente des démences. Elle apparaît généralement après 65 ans et possède une forte composante génétique. En dehors de certaines formes familiales précoces liées à des mutations dans les gènes du précurseur de la protéineamyloïde, et des présénilines 1 et 2, la grande majorité des cas résulte de l’interaction de facteurs environnementaux avec divers gènes de susceptibilité.L’approche gène candidat a permis l’identification de nombreux gènes associés au risquede MA. Cependant, en raison de problèmes techniques et méthodologiques, seul le gène de l’apoprotéine E (APOE) a pu être identifié de manière robuste par cette approche. Les études d’association pangénomiques permettent d’identifier sans a priori des variations génétiques fréquentes associées à une maladie sur l’ensemble du génome. À partir de 2009, plusieurs consortia ayant pour objectif de réaliser ce type d’étude dans le champs de la MA ont identifiéquatre nouveaux gènes d’intérêt pour la MA, CLU, PICALM, CR1 et BIN1. Cependant,ces gènes n’expliquent qu’une petite partie de la variabilité génétique de la maladie et denombreux autres variants restent à découvrir.Durant cette thèse, nous avons d’abord chercher à répliquer les résultats des principauxgènes identifiés par approche gène candidat en utilisant les données du consortium EuropeanAlzheimer’s Disease Initiative (EADI). Nous avons pu montrer qu’une grande partie deces gènes présentait un faible niveau d’association avec la MA. En utilisant l’approche pangénomique,nous avons ensuite pu identifier 19 gènes associés au risque de MA en dehorsd’APOE, dont 11 n’ayant pas été identifiés par les précédentes études, via la mise en placed’une collaboration informelle entre consortia puis au sein du International Genomics ofAlzheimer’s Disease Project (IGAP).Nous nous sommes également interessés à plusieurs phénotypes intermédiaires associésà la MA, et en particulier aux taux plasmatiques des peptides amyloïde b (Ab), en partantde l’hypothèse qu’ils pourraient permettre la recherche de variants impliqués dans des mécanismesphysiopathologiques pré-symptomatiques. Ce travail a permis l’identification d’uneassociation potentielle entre le gène CTXN3 et les taux plasmatiques d’Ab1−42.En conclusion, l’utilisation des études d’association pangénomiques a permis d’identifierde nombreux nouveaux gènes associés au risque de MA. Ces gènes ouvrent des voies derecherche intéressantes pour mieux comprendre la physiopathologie de la MA et permettrele développement de traitements efficaces qui font actuellement défaut. / Dementia is a syndrom caused by several brain diseases progressively deteriorating cognitivefunctions and occurs more frequently in the elderly. The increased number of patients withdementia due to the ageing of the general population and the high cost of care add up tomake dementia a concerning public health issue.Alzheimer’s disease (AD) is the most common form of dementia. It is often diagnosedafter 65 years old and has a strong genetic component. Familial forms exist and are mainlycaused by mutations in the amyloid-b protein precursor, presenilin 1 and presenilin 2 genes.However, the vast majority of cases result from the complex interaction of environmental factors with susceptibility genes.Using a candidate gene approach, numerous genes associated with AD risk were identified,but due to technical and methodological problems, only the apoliprotein E (APOE) genewas replicated. Genome-wide association studies (GWAS) aim to identify frequent geneticvariants associated with disease risk in a hypothesis-free manner. Starting 2009, severalconsortia aiming to perform this type of analyses in the field of AD robustly identified fournew genes associated with AD risk, CLU, PICALM, CR1 and BIN1. However, these genes puttogether only explain a small proportion of the total genetic variance of AD and the searchfor new susceptibility genes remains an important goal for AD research.In this work, we first tried to replicate the results of the top genes reported using thecandidate gene approach, using GWAS data from the European Alzheimer’s Disease Initiative(EADI). Most of these genes showed weak levels of association. Using GWAS, we were ableto identify 19 new genes associated with AD risk besides APOE, including 11 that had notbeen reported by previous studies, first through an informal collaboration between consortia,then under the name of International Genomics of Alzheimer’s Disease Project (IGAP).Assuming that use of endophenotypes related to AD would be relevant for the discoveryof genetic variants involved in the early pathophysiology of AD, we then performed aGWAS of plasma amyloid-b (Ab) concentrations. This study showed suggestive asssociationsbetween the CTXN3 gene on chromosome 5 and Ab1−42 plasma levels.To sum up, using GWAS enabled us to identify new genes associated with AD risk. Thesegenes point to interesting new research hypotheses and hopefully, to a better understandingof AD pathophysiology and development of effective drugs.