Dissertationen zum Thema „Protéine précurseur de l'amyloïde“
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Lessard, Christian. „Étude sur la régulation et de la voie moléculaire de la Béta-sécrétase et de préséniline“. Doctoral thesis, Université Laval, 2010. http://hdl.handle.net/20.500.11794/21906.
Der volle Inhalt der QuelleItkin, Anna. „Etude pluridisciplinaire de peptides liés à la maladie d'Alzheimer: de la protéine précurseur de l'amyloïde (APP) aux oligomères de beta-amyloïde et aux inhibiteurs de gamma-sécrétase“. Doctoral thesis, Universite Libre de Bruxelles, 2012. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209705.
Der volle Inhalt der QuelleLes propriétés conformationnelles du segment transmembranaire (TM) de l’APP peuvent affecter sa protéolyse par la γ-sécrétase. Ces propriétés ne sont pas encore clairement établies. Afin de comprendre le rôle des variations structurelles du TM dans le traitement de l'APP, des détails structurels des peptides APP_TM4K, chimiquement synthétisés, ont été étudiés dans la bicouche lipidique en utilisant la réflexion totale atténuée par spectroscopie infrarouge à transformée de Fourier (ATR-FTIR) et la résonance magnétique nucléaire à l’état solide (ssNMR). Tandis que la structure secondaire globale du peptide APP_TM4K est hélicoidale, une hétérogénéité conformationnelle et orientée a été observée pour le site de clivage γ et, dans une plus faible mesure, pour le site de clivage ζ. Ces variabilités conformationnelles autour des sites de clivage γ et ζ peuvent avoir des implications importantes dans le mécanisme de clivage et donc dans la production d’Aβ. Il a été aussi démontré que la dernière glycine dans le motif de dimérisation GxxxG est transmembranaire. Ceci peut impliquer que la dimérisation via ce motif pourrait servir d’ancrage et conférer une orientation transmembranaire stable au segment transmembranaire de l’APP.
Le peptide amyloïde β est directement lié à la maladie d’Alzheimer. Partant de sa forme monomérique, l’Aβ s'agrège pour produire en final des fibrilles et aussi de manière transitoire toute une gamme d'oligomères, ces derniers étant la plupart neurotoxiques. Une dérégulation de l’homéostasie du Ca2+ dans le cerveau vieillissant et dans des troubles neurodégénératifs joue un rôle crucial dans de nombreux processus et contribue au dysfonctionnement et à la mort cellulaire. Nous avons postulé que le calcium peut permettre ou accélérer l'accumulation d'Aβ. Le modèle d'accumulation d'Aβ (1-40) et celui d'Aβ (1-40) E22G, un peptide amyloïde portant la mutation arctique qui cause une apparition prématurée de la maladie, ont été comparé. Nous avons constaté qu'en présence de Ca2+, l’Aβ (1-40) forme de préférence des oligomères semblables à ceux formés par l’Aβ (1-40) E22G avec ou sans Ca2+, tandis qu'en absence de Ca2+ l'Aβ (1-40) s’agrège sous forme de fibrilles. Les ressemblances morphologiques entre oligomères ont été confirmées par microscopie de force atomique. La distribution des oligomères et des fibrilles dans des échantillons différents a été détectée par électrophorèse sur gel suivie d’une analyse par Western blot, dont les résultats ont été confirmés par des expériences de fluorescence à la thioflavine T. Dans les échantillons sans Ca2+, l’ATR-FTIR révèle la conversion des oligomères en feuillets β antiparallèles en la conformation caractéristique des fibrilles en feuillets β parallèles. En général, ces résultats nous ont ameré à conclure que les ions calcium stimulent la formation d'oligomères d'Aβ (1-40), qui sont impliqués dans la pathogénèse d'AD.
Malgré les progrès énormes obtenus dans la compréhension de la maladie (AD), il reste un défi majeur, celui du développement de médicaments nouveaux et efficaces. Afin d’obtenir des éclaircissements sur le mécanisme d'action de deux nouveaux puissants modulateurs de la γ-sécrétase - le benzyl-carprofen et le sulfonyl-carprofen dans la bicouche lipidique, la technique de RMN à l’état solide a été employée. Précédemment, les dérivés du carprofen ont été localisés dans des membranes de lipides par des expériences de diffusion (scattering) des neutrons. Les contraintes déterminées à partir des expériences de ssNMR ont permis d’affiner leurs positions et d’obtenir une orientation précise dans la double couche lipidique. Ces résultats combinés indiquent que le mécanisme probable de modulation du clivage par la γ-sécrétase est une interaction directe des carprofènes avec le domaine TM de l’APP. Une telle interaction, empêcherait à la formation de dimères d'APP, dimérisation nécessaire au clivage séquentiel par la γ-sécrétase, diminuant ou réduisant ainsi énormément la production d’Aβ, tout particulièrement d’Aβ42.
Les résultats de ce travail apporte de nouvelles informations sur les processus clés impliqués dans l'AD; Production de l'Aβ à partir de l'APP, formation des oligomères d'Aβ et mécanisme d'action potentiel de molécules thérapeutiques. Nous pensons que ces résultats pourront permettre une meilleure compréhension de la maladie et pourront aider dans la conception de nouveaux médicaments contre cette maladie.
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. There is no cure and the disease is fatal. One of the characteristic histopathological markers of AD is the presence of proteinaceous deposits, amyloid plaques, in the brain. These plaques are formed by the amyloid β-peptides (Aβ) 40- and 42-residue-long, which are protease cleavage products of the amyloid precursor protein (APP). Elucidation of some of the key processes in the cause and the development of AD is crucial for the development of new and efficient treatments.
Conformational properties of the transmembrane (TM) segment of APP may affect its proteolytic processing by γ-secretase. These properties have not been definitely established. In addressing the role of structural variations of the TM sequence in APP processing, structural details of the chemically synthesized APP_TM4K peptides within the membrane bilayers were studied using Attenuated total reflection Fourier transform spectroscopy (ATR-FTIR) and solid-state nuclear magnetic resonance (ssNMR) techniques. While the overall secondary structure of the APP_TM4K peptide is an α-helix, conformational and orientational heterogeneity was observed for the γ-cleavage site and, to a smaller extent, for the ζ-cleavage site. Evidence for the conformational variability around γ- and ζ-cleavage sites may have important implications for the cleavage mechanism and hence for the Aβ production. It was also found that the last glycine within the sequence of GxxxG motifs is in the transmembrane orientation, implying that dimerization via these motifs may act as an anchor, confining the TM dimer to the stable transmembrane orientation.
Amyloid β-peptide is directly linked to AD. Starting from its monomeric form, Aβ aggregates into fibrils and / or oligomers, the latter being the most neurotoxic. Dysregulation of Ca2+ homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. The aggregation pattern of Aβ(1-40) and of Aβ(1-40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease, were compared. We found that in the presence of Ca2+, Aβ(1-40) preferentially formed oligomers similar to those formed by Aβ(1-40)E22G with or without added Ca2+, whereas in the absence of added Ca2+ the Aβ(1-40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy (AFM) imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results which were further supported by thioflavin T fluorescence experiments. In the samples without Ca2+, Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1-40), that have been implicated in the pathogenesis of AD.
Despite the tremendous progress in understanding AD, there remains the challenge of the development of new and efficient drugs. In order to shed light onto the mechanism of action of two new potent γ-secretase modulators -- benzyl-carprofen and sulfonyl-carprofen within lipid bilayers, ssNMR technique was employed. Using neutron scattering experiments it was previously found that sulfonyl-carprofen and benzyl-carprofen partition into the headgroup region of the lipid bilayer. The orientational constraints derived from the ssNMR experiments refined their position into precise orientation. Combined, these results indicate that carprofen-derivatives can directly interact with the region of APP that mediates dimerization. Such interaction, would interfere with proper APP-dimer formation, which is necessary for the sequential cleavage by γ-secretase, diminishing or greatly reducing Aβ42 production.
Results obtained during this work shed new light onto some of the key processes in AD: Aβ production from APP, formation of Aβ oligomers and insights into the mechanism of action of potential therapeutics. We believe that these results will promote a better understanding of the disease and will help in future drug design.
Doctorat en Sciences
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Proulx, Marie-Claude. „Identification de facteurs régulant le complexe γ-sécrétase dans le contexte de la maladie d'alzheimer“. Master's thesis, Université Laval, 2007. http://hdl.handle.net/20.500.11794/19313.
Der volle Inhalt der QuelleVaillant-Beuchot, Loan. „Étude des mécanismes liés aux dysfonctions mitochondriales, à l'altération de la mitophagie et aux défauts du transport mitochondrial dans la maladie d'Alzheimer“. Electronic Thesis or Diss., Université Côte d'Azur, 2022. http://www.theses.fr/2022COAZ6019.
Der volle Inhalt der QuelleMitochondria are essential organelles in cells, ensuring energy production with ATP synthesis, calcium buffering, apoptosis regulation. These functions are altered at early stages of Alzheimer's disease (AD) and are essentially induced by the Amyloid (Aβ), produced after the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretase. Aβ is a major actor of AD development but all the treatments targeting this peptide remain ineffective. C-terminal APP fragments (APP-CTFs: C83 and C99 (Aβ precursor) are other fragments presenting specific toxicity in AD and new potential therapeutic targets. My project is focus on the study of APP-CTFs toxicity, independently of Aβ, on the structure, function of mitochondria, their degradation by mitophagy and on mitochondrial transport proteins. They constitute the complex allowing mitochondrial transport in cells, especially in neurons, closely linked to mitochondrial renewal, particularly in neurons.First axe: APP-CTFs impact on mitochondrial structure, function and mitophagy. We described APP-CTFs accumulation in mitochondrial fraction in vitro (human neuroblastoma cells expressing APP Swedish double mutation (SH-SY5Y-APPswe) or C99 fragment (SH-SY5Y-C99)) and in vivo (3xTgAD mice expressing APPswe, TauP301L, PS1 (M146V) or C99 fragment after viral injection). We inhibit the cleavage of APP-CTFs and the production of Aβ by pharmacological approaches, to abolish γ-secretase activity. Ours results show for the first time in vitro and in vivo, that high concentration of APP-CTFs independently of Aβ, impact mitochondrial structure, function and alter mitophagy process, resulting in an accumulation of altered mitochondria producing high levels of toxic reactive oxygen species. In addition, our results in patient brains of sporadic AD (SAD) patients show altered mitophagic protein levels correlating with APP-CTFs accumulation (1-2).Second axe: study of the effects of APP, APP-CTFs and Aβ peptide on mitochondrial transport machinery. I reported the specific regulation of mitochondrial transport protein by endogenous APP (Mice fibroblasts APP WT and KO) and the overexpression of APPswe (and in SH-SY-5Y-APPswe cells). APP-CTFs and Aβ differentially regulate mitochondrial transport protein levels in treated SH-SY-5Y-APPswe cells with γ-secretase inhibitor. These results were validated in mice fibroblasts KO for presenilins (catalytic compounds of γ-secretase) avoiding APP-CTFs degradation. APP-CTFs and Aβ impair the recruitment of mitochondria to its transport machinery in differentiated SHSY-5Y. The progression of the disease deregulates the levels of mitochondrial transport protein in vivo (3xTgAD and WT mice brains, C99 injected mice brains) and in SAD patients brains. The analyses of young and old mice brains and of SAD patients samples at different stages of the disease, allowed us to demonstrate an impact of aging in the regulation of mitochondrial transport protein levels. This phenomenon occurs also in addition with AD progression (3).These studies highlight new molecular mechanisms impacting mitochondrial homeostasis during AD progression. Our findings will bring new therapeutic research to slow down mitochondrial dysfunctions and/or to stimulate their renewal in AD context.(1). Vaillant-Beuchot L.*, Mary A.* et al. Acta Neuropathologica 2020.(2). Mary A.*, Vaillant-Beuchot L.* et al. Médecine/sciences 2021.(3). Vaillant-Beuchot et al. En cours de soumission
Ndiaye, Séga. „Recherche des partenaires de l'amyloïde-bêta 1-42 par spectrométrie de masse“. Paris 6, 2011. http://www.theses.fr/2011PA066540.
Der volle Inhalt der QuelleJordà, Siquier Tomàs. „Rôles physiologiques et pathologiques de la protéine précurseur amyloïde à la présynapse“. Thesis, Bordeaux, 2021. http://www.theses.fr/2021BORD0054.
Der volle Inhalt der QuelleAlzheimer’s disease (AD) is a progressive neurodegenerative disease which affects 47 million people worldwide, being the most prominent type of dementia. The etiology of the disease is unknown but genetic evidence from the familial form of the disease indicates that the amyloid precursor protein (APP) plays a key role in the pathology. Importantly, APP is the substrate in the proteolytic reaction producing Aβ peptides which compose the amyloid plaques, one of the main pathological hallmarks in AD brain. In addition, APP is ubiquitously expressed by neurons where it interacts with multiple presynaptic proteins but the role of these interactions is elusive.The aim of my thesis was to study the physiological and pathological functions of APP related to its location at the presynapse. First, we studied the consequences on presynaptic mechanisms of the genetic deletion of presenilin, the catalytic subunit of γ-secretase, the intramembrane protease which cleaves APP. We observed that in absence of presenilin, APP accumulates in axons. By combining optogenetic to electrophysiology, we assessed synaptic transmission and plasticity in the CA3 region of the hippocampus. The presynaptic facilitation, the increase in synaptic vesicle release during repetitive stimulation, was altered whereas the basal neurotransmission was not. The impairment of presynaptic mechanisms was due to the accumulation of APP Cter, which decreases the abundancy of synaptotagmin-7, a calcium sensor essential for facilitation. Using a similar approach, we investigated the consequences of the genetic deletion of APP itself and observed again an impairment of presynaptic facilitation. Together, these results demonstrate the importance of APP homeostasis in presynaptic plasticity.I then investigated possible alterations of APP, other than the amyloid peptides, in the AD brain. I discovered that APP dramatically accumulates together with presynaptic proteins around dense-core amyloid plaques in human AD brain. In addition, the Nter domain, but not the Cter domain of APP is enriched in the core of amyloid plaques uncovering a potential pathological role of the secreted APP Nter in dense-core plaques. Ultrastructural analysis of APP accumulations reveals abundant multivesicular bodies containing presynaptic vesicle proteins and autophagosomal built-up of APP. Finally, we observed that outside the APP accumulations, presynaptic proteins were downregulated, in the neuropil area of the outer molecular layer of the dentate gyrus. Altogether, the data I collected during my thesis supports a role of presynaptic APP in physiology and in AD pathology and highlights APP accumulations as a pathological site where presynaptic proteins are mis-distributed
Itkin, Anna. „Multidisniplinary study of Alzheimer's disease-related peptides : from amyloid precursor protein (APP) to amyloid β-oligomers and γ-secretase modulators“. Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAF051/document.
Der volle Inhalt der QuelleA histopathological characteristic of Alzheimer’s disease (AD) is the presence of amyloid plaques formed by amyloid β(A) peptides of 40 and 42 residues-long, which are the cleavage products of APP by proteases. To understand the role of structural changes in the TM domain of APP, APP_TM4K peptides were studied in the lipid bilayer using ATR-FTIR and ssNMR. While the overall secondary structure of the APP_TM4K peptide is helical, conformational and orientational heterogeneity was observed for the y- and for the -cleavage sites, which may have implications for the cleavage mechanism and therefore the production of Aβ. Starting from its monomeric form, Aβ peptides aggregate into fibrils and / or oligomers, the latter being the most neurotoxic. We found that in the presence of Ca2 +, Aβ (1-40) preferably forms oligomers, whereas in the absence of a2 + Aβ (1-40) aggregates into fibrils. In samples without Ca2 +, ATR-FTIR shows conversion from antiparallel β sheet conformation of oligomers into parallel β sheets, characteristic of fibrils. These results led us to conclude that Ca2 +stimulates the formation of oligomers of Aβ (1-40), that have been implicated in the pathogenesis of AD. Position and precise orientation of two new drugs powerful modulators of γ-secretase benzyl-carprofen and carprofen sulfonyl in the lipid bilayer were obtained from neutron scattering and ssNMR experiments. These results indicate that carprofen-derivatives can directly interact with APP. Such interaction would interfere with proper APP-dimer formation, which is necessary for the sequential cleavage by β -secretase, diminishing or greatly reducing Aβ42 production
Lanoue, Edith. „Co-expression de la pro-protéine convertase SPC3 et du précurseur neuroendocrinien proSAAS“. Mémoire, Université de Sherbrooke, 2001. http://savoirs.usherbrooke.ca/handle/11143/3278.
Der volle Inhalt der QuelleBertrand, Eric. „Contribution à l'étude du précurseur de la protéine amyloïde : analyse fonctionnelle du domaine cytoplasmique“. Palaiseau, Ecole polytechnique, 2001. http://www.theses.fr/2001EPXX0016.
Der volle Inhalt der QuelleBenoit, Matthieu. „Etudes biophysiques de l'interaction entre la protéine humaine TRBP et un précurseur de microARN oncogène“. Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-01038628.
Der volle Inhalt der QuelleBera, Alakesh. „Etude des changements structuraux de la protéine prion induite par les acides nucléiques conduisant à la formation d'oligomères de la protéine du prion et à des polymères de l'amyloïde“. Orléans, 2006. http://www.theses.fr/2006ORLE2015.
Der volle Inhalt der QuelleLongpré, Jean-Michel. „Identification de déterminants moléculaires impliqués dans la biosynthèse et l'activation des ADAMTS (a desintegrin and metalloprotease with thrombospondin type 1 repeat)“. Thèse, Université de Sherbrooke, 2007. http://savoirs.usherbrooke.ca/handle/11143/4260.
Der volle Inhalt der QuelleBoissonneault, Vincent. „Régulation de la protéine mBACE1 par les miARN miR-298 et miR-328“. Thesis, Université Laval, 2006. http://www.theses.ulaval.ca/2006/24104/24104.pdf.
Der volle Inhalt der QuelleLefranc-Jullien, Solveig. „Biologie cellulaire et pharmacologie des sécrétases impliquées dans la voie de maturation physiopathologique de la protéine précurseur du peptide ß amyloïde“. Nice, 2005. http://www.theses.fr/2005NICE4055.
Der volle Inhalt der QuelleAlzheimer's disease (AD) is characterized by the cortical and subcortical accumulation of proteinaceous deposits called senile plaques. The main constituent of these aggregates is referred to as amyloid b peptide (Ab). Ab is generated from the b amyloid precursor protein (bAPP) by the subsequent attacks by b- and g-secretases, which liberates the N- and C-terminal moieties respectively by a process called the amyloidogenic pathway. In the non-amyloidogenic pathway, a-secretase cleaves APP within the b-amyloid peptide, thereby preventing deposition of the intact amyloid peptide. Inhibition of b- and g-secretase, or stimulation of a-secretase, is a rational strategy for therapeutic intervention in AD. The work presented in this thesis was aimed to study the cellular biology and the pharmacology of these three secretases. Concerning b-secretase, I contributed to the design of a novel fluorimetric assay which confirmed the identity of BACE1 and BACE2 as b-secretase whereas cathepsin D proved to be a b-secretase " false positive " candidate. Our fluorimetric assay allowed us to identify two generations of new inhibitors. The first generation was a series of peptidomimetic statine-derived compounds. One of them, JMV1195, appeared potent and rather selective in vitro but was unable to block cellular b-secretase activity. Thus, we have designed a new permeable inhibitor, JMV2764 that corresponds to a derivative of JMV1195 to which a penetratin sequence had been added at its N-terminus. JMV2764 was able to affect BACE1 activity in vitro, and to modify Ab production in various cell systems. In a second part of my work, I developed an original cellular approach to study the g-secretase activity. BAPP undergoes two hydrolysis by g-secretase, at the C-terminus of Ab (g-site) and few residues C-terminal to the g-site (e-site). This e-cleavage resembles the S3 Notch cleavage. It appeared of most interest to discriminate the two g-secretase activities on bAPP to study them separately. I developed a cellular model based on the expression of the e-secretase-derived N-terminal fragment of bAPP (APPe) and I showed that APPe was a useful tool to study g-secretase activities and to design specific inhibitors without facing any rate-limiting effect of e-secretase-derived cleavage. Finally, I was interested in the a-secretase identity. Two disintegrin, ADAM10 and ADAM17 were identified as a-secretases. ADAM10 is involved in constitutive and regulated pathway and ADAM17 in the regulated pathway. Initially, I observed that in absence of ADAM10, constitutive secretion strongly decrease without totaly disappearing. I could show that the prohormone convertase 7 (PC7) acted upstream of a-secretase. Moreover, thanks to the development of a fluorimetric assay of a-secretase activity I demonstrated that ADAM9 participate in this pathway due its involvement in the shedding of ADAM10
Ben, Aissa Manel. „Développement de nouvelles stratégies anti-amyloïdes ciblant la protéine précurseur amyloïde pour le traitement de la maladie d'alzheimer“. Thesis, Université Laval, 2012. http://www.theses.ulaval.ca/2012/29049/29049.pdf.
Der volle Inhalt der QuelleMeckler, Xavier. „Caractérisation de la localisation neuronale de la protéine précurseur du peptide amyloïde : arguments en faveur d'un métabolisme présynaptique“. Bordeaux 2, 2006. http://www.theses.fr/2006BOR21330.
Der volle Inhalt der QuelleThe amyloid precursor protein APP is known for its implication in Alzheimer's disease. Indeed, amyloid peptide is the main component of senile plaque, one of the two characteristic lesions of the disease. Despite numerous studies made on APP ans its metabolism in the brain, the neuronal function of this protein is still unknown. The difficulty to define APP's function come probably from its complex metabolism made by several enzymes called secretases that cleave APP in many fragments, each potentially responsible for a particular function. The first part of this study is made during the early development of cultured rat hippocampal neurons and show the implication of APP cytosolic domain in the neuronal distribution of a GTPase involved in membrane morphogenesis, the dynamin 3. The second part of this study is dedicated to the potential synaptic metabolism and partners of APP. We show the synaptic enrichment of APP, APP metabolic derivatives and APP secretases. A synaptic membranes fractionation technique allowed us to show APP's enrichment in the presynaptic membrane. The analysis of several components of APP metabolism in the synaptic fractionations and the isolation of APP synaptic partners let suppose that APP can be metabolized into amyloid peptide in presynaptic terminals and that this metabolism could involve vesicles recycling
Samaan, Raghda. „Étude des interactions entre précurseur protéique amyloi͏̈de et lipides : analyses dans des systèmes membranaires biomimétiques“. Lyon 1, 2004. http://www.theses.fr/2004LYO10157.
Der volle Inhalt der QuelleGuyon, Antoine. „Insertion d’une mutation protectrice pour la maladie d’Alzheimer dans le gène de la protéine précurseur de l’amyloïde via le système CRISPR/Cas9“. Doctoral thesis, Université Laval, 2021. http://hdl.handle.net/20.500.11794/68776.
Der volle Inhalt der QuelleAlzheimer’s disease (AD) is the most common form of dementia in the world, withnearly fifty million people affected currently. The most common symptoms of this diseaseare memory loss, difficulties in task management, and temporal and spatial confusions. There is currently no treatment for this disease. The amyloid precursor protein (APP) is usually cut by the alpha-secretase enzyme; however, abnormal cleavage by the beta-site APP cleaving enzyme 1 (BACE1) leads to the formation of beta-amyloid peptides. These peptides in turn forms aggregates, which accumulate as plaques in the brains of Alzheimer patients. Many non-silent APP mutationscause changes to the amino acid composition of the protein and result in increased plaque accumulation. These mutations are called familial forms of Alzheimer’s disease (FAD).However, one of these mutations (Icelandic A673T mutation) has been shown to confer aprotection against the on set and development of AD. This mutation of a single mutation inexon 16 changes an alanine into a threonine and has been shown to reduce the cleavage ofthe APP protein by BACE1 by 40%.This kind of single point mutation is the perfect target for the newly discoveredCRISPR/Cas9 technology, which opens new perspectives for the development of preventiveor curative treatments for genetic diseases and in our case Alzheimer’s. The Cas9endonuclease is a powerful tool for the modification of genetic data. The protein has been shown to cut double-stranded DNA with the help of a guide RNA (gRNA) to target a specified sequence adjacent to a PAM (protospacer adjacent motif). The base CRISPRsystem has been coopted by many different research teams; one of which used the technology to develop a technique they called base editing. This technique allows researchers toexchange cytidine bases for thymine and guanine bases for adenine with a strong accuracy. The first article of this thesis aims to demonstrate that the addition of the A673Tmutation in codominance with another pathological form of AD may have beneficial effectson the reduction of beta-amyloid peptides in patients’ brains. To determine if the mutationwas protective, plasmids carrying the A673T mutation along with another random FADmutation were used. Ultimately, we confirmed the beneficial effect for many forms of FAD,in particular the London V717I mutation demonstrated the greatest reduction in beta amyloidproteins. The second article of this thesis deals with the insertion of the A673T mutation by theCRISPR/Cas9 derived system, base editing. Several base editor complexes were compared and optimized to achieve the most effective and accurate genome modification possible. A candidate was selected after testing on HEK293T cells and SH-SY5Y neuroblastoma. The third part of this manuscript presents the results obtained when using lentiviraland AAV vectors to infect induced human and mouse neurons with a base editor complex and harvested mouse neurons with FAD forms. This whole approach has opened up an avenue for a potential therapy for Alzheimer’sdisease.
Xu, Zhou. „Etude des relations structure-activité de la protéine du prion“. Paris 7, 2011. http://www.theses.fr/2011PA077079.
Der volle Inhalt der QuellePrion diseases are fatal infectious neurodegenerative disorders related to the misfolding of the endogenous protein PrP. At least two conformations are accessible by this protein, an a-helical normal one, PrPc, and a P-sheet enriched amyloid one, PrPSc. Stanley Prusiner's « protein only » hypothesis proposes that the infectious agent of these diseases may be exclusively made of PrPSc protein. The latter has first been identified in infected brains as aggregated amyloid deposits, which were resistant to proteinase K degradation. Although breakthrough knowledge has been gained in the past decades about the physico-chemical properties of the infectious agent, the transmission phenomenon, the pathological spread of the disease in animals and the cellular mecanisms implicated, important issues remain unanswered, such as the understanding of the physiological and pathological functions of different regions of thee protein. In this thesis, we tried to bring forward some insights into these functions and their properties. On the one hand, we identified a protective role of PrPc in the maintenance of genomic stability through its unstructured N-terminus region. On the other hand, our data suggest that the two C-terminal helices H2 and H3 may be the sufficient and necessary domain for prion conformational change and replication. Based on these results, we propose a functional model for the different regions of the protein. In particular, m molecular mecanisms of H2H3 domain conversion stimulate our fundamental understanding of the conformational change of PrP relatively to the concept of prion in general
Ferland, Mélissa. „Préséniline 1 et voies de signalisation“. Thesis, Université Laval, 2003. http://www.theses.ulaval.ca/2003/21021/21021.pdf.
Der volle Inhalt der QuelleCachard-Chastel, Marthe. „Synergie d'effets neurochimique et comportemental entre un agoniste du récepteur 5-HT4 de la sérotonine et un inhibiteur de l'acétylcholinestérase chez la souris : hypothèse concernant le rôle du précurseur de la protéine β-amyloïde associé à la maladie d'Alzheimer et son traitement“. Université Paris XI, 2007. http://www.theses.fr/2007PA114834.
Der volle Inhalt der QuelleBourdet, Isabelle. „La Drosophile comme modèle pour l'étude de la maladie d'Alzheimer : rôle de la protéine précurseur Amyloïde dans la mémoire olfactive“. Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066256/document.
Der volle Inhalt der QuelleAlzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of memory. The amyloid peptide (Aβ), the principal component of senile plaques found in patients’ brains, has been considered as the main cause of memory dysfunction. However, the exact molecular mechanisms that underlie memory decline remain unknown. Aβ is produced by the proteolysis of a transmembrane protein named Amyloid Precursor Protein (APP). It has been suggested that in addition to the accumulation of Aβ, APP loss of function may play a crucial role in the cognitive dysfunction associated with AD, especially at the onset of the disease. Drosophila contains a single APP ortholog APP-like (APPL), that undergoes processing pathways similar to that of APP. We have previously highlighted in young flies the involvement of APPL in associative olfactory memory (Goguel et al., 2011). During my thesis, we sought firstly to identify which form of APPL, among its numerous metabolites, is critical for memory, and secondly, to analyze the effect of promoting the amyloidogenic pathway in the young adult brain. Our results suggest several types of functional interactions between APPL and its metabolites: a positive interaction between the full length membrane and the secreted form - which would underlie implementation of memory under physiological conditions - and a negative interaction between APPL and dAβ - which would rather participate to the progression of the memory decline observed during AD
Arnaud, Karen. „Sécrétion du précurseur de la protéine amyloïde par les plexus choroïdes : implications dans la neurogenèse adulte et la maladie d'Alzheimer“. Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066214/document.
Der volle Inhalt der QuelleAging and degeneration of the brain with cognitive decline and neurologic symptoms are major individual and societal problems. The major age-related brain degeneration disease is Alzheimer’s disease (AD) with about 40 million people affected in 2015.Physiologically, the Amyloid Precursor Protein (APP) is cleaved by an alpha-secretase, releasing soluble APP (sAPP) an important regulator of adult neurogenesis. This cleavage prevents two others in positions beta and gamma that generate the ßA4 toxic peptide, a hallmark of Alzheimer Disease.Next generation RNA-sequencing has revealed that APP is the 16th most expressed genes in the choroid plexus (CP), suggesting that it may be a major source of sAPP and ßA4 in the cerebrospinal fluid (CSF). If so, adult neurogenesis in the SVZ and hippocampus may be regulated by the choroid plexus and impeded in mutations favoring ßA4 production. My thesis project fell under the possibility to regulate App expression in the CP, and follow consequences on adult neurogenesis and plaques formation in AD. Using viral vectors to modulate App expression in the CP, we confirmed the importance of sAPP coming from CP in adult neurogenesis. With so, CP seems to be an important source of APPin the brain, and could have a key role in AD
Robert, Sylvain. „Influence des récepteurs 5-HT4 de la sérotonine sur la maturation du précurseur de la protéine ß-amyloi͏̈de associée à la maladie d'Alzheimer“. Paris 11, 2004. http://www.theses.fr/2004PA114819.
Der volle Inhalt der QuelleThe serotonin 5-HT4 receptors are heptahelical transmembrane receptors that are positively coupled to adenylyl cyclase. In the first part of this work, we showed that the human 5-HT4 receptor stimulated the secretion of the non-amyloi͏̈dogenic soluble form of the amyloid precursor protein (sAPPa), a key protein involved in Alzheimer's disease which has neuroprotective and memory enhancing effects. In the second part of this work we showed that this process which was cAMP dependent but PKA independent, involved a new signaling pathway of the 5-HT4 receptors that involved the small G proteins, Rac and Rap 1. Rac activation results from activation of Rap1 through the cAMP-activated guanine nucleotide exchange factor Epac. Futhermore, we showed that secretion of sAPPa induced by the human 5-HT4 receptor is due to its specific effect on a a-secretase activity. These findings may lead to the identification of new targets for the regulation of APP processing
Lopez-Perez, Elvira. „La voie α-sécrétase de maturation de la protéine précurseur du peptide β-amyloi͏̈de dans la maladie d'Alzheimer : activités protéolytiques et régulation“. Nice, 2000. http://www.theses.fr/2000NICE5459.
Der volle Inhalt der QuelleRayah, Amel. „Identification des voies biochimiques stimulées par le récepteur purinergique P2X7 qui sont impliquées dans le clivage protéolytique du précurseur de la protéine amyloïde (APP)“. Thesis, Paris 11, 2015. http://www.theses.fr/2015PA11T043.
Der volle Inhalt der QuelleThe amyloid protein precursor (APP) can be cleaved in neural cells by α-secretases to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic receptor P2X7 (P2X7R), a member of the P2X receptor family of ATP-gated cation channels, triggers sAPPα shedding from neural cells. Here, we demonstrate that theactivation of Ezrin/Radixin/Moesin proteins (ERM) is required for the P2X7R-dependent proteolyticprocessing of APP leading to sAPPα release. Indeed, the down regulation of ERM by siRNA blocksthe P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggers thephosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R.Using specific pharmacological inhibitors, we have established the order in which several enzymestrigger the P2X7R-dependent release of sAPPα. Thus, a Rho-kinase and the MAPK modules ERK1/2and JNK act upstream of ERM while a PI3Kinase activity is triggered downstream. This work for the first time identifies ERM as major partners in the regulated non-amyloidogenic processing of APP. Inaddition, we have recently established that the stimulation of P2X7R leads to the proteolytic cleavage of NrCAM by ADAM17 and the shedding of the soluble extracellular domain of NrCAM. Our results clearly show that the proteolytic cleavage of NrCAM is dependant of ERM activation and fixation tothe intracellular region of NrCAM. Thus, our results strongly suggest that ERM are required for the proteolytic cleavage of numerous substrates after P2X7R stimulation. Our findings suggest that ERM play a central role in the proteolytic cleavage of transmembrane proteins and act as molecular linkswhich aggregate ADAMs and substrates at the plasma membrane promoting the cleavage of substrates
Marquilly, Claire. „Rôle de la protéine APPL dans la croissance axonale des corps pédonculés chez Drosophila melanogaster“. Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT045/document.
Der volle Inhalt der QuelleIn the drosophila brain, mushroom bodies are involved in olfactory memory and learning. This structure is composed of different types of / neurons. These neurons form an orthogonal structure, with the branch projecting dorsally and the branch projecting medially. The aim of this study is to understand mechanisms and pathways involved during the development of these neurons.The drosophila APPL protein (Amyloïd Precursor Protein-Like) is the homologue of the human APP, known to be involved in Alzheimer’s disease. This pathology is characterized by neuronal degeneration inducing cognitive and memory defects. In spite of the numerous studies focused on the pathological function of APP during the last decades, few things are known on the physiological functions of this protein and more particularly during the development. This is from this perspective that we studied the APPL function and its interaction with proteins during the mushroom bodies development.The APPL protein was identified as a co-receptor of the PCP pathway (Planar Cell Polarity), involved in the axonal growth regulation. During the development, APPL allows the recruitment and the activation of the ABL protein (Abelson Tyrosine Kinase), which phosphorylates DSH (dishevelled) and so activates the axonal growth pathway.The first part investigates the regulation of ABL activity during the / neuron development. If it’s already established that APPL regulates positively the kinase activity of ABL, I show here that the HTT protein (Huntingtin) allows a negative regulation of ABL activity. In human, HTT is involved in the Huntington’s disease, another neurodegenerative disorder. This thesis work shows that HTT regulates the phosphorylation level of ABL, and therefore its activity.The second part investigates the interaction between APPL and ARM (armadillo), the homologue of the human -catenin, during the development of the / neurons. I show that this interaction is independent of the APPL function in the PCP pathway. Moreover, this interaction between APPL and ARM involves the actin cytoskeleton dynamic function of ARM, and not its Wnt pathway function.The third and last part presents new mutant alleles of APPL obtained with the CRISPR-CAS9 technique. The creation and analysis of these new alleles lead us to propose that vnd (ventral nervous system defective), neighbor gene of Appl, is also involved in / neurons development, and can interact genetically with Appl
Paumier, Jean-Michel. „Métalloprotéases matricielles et maladie d'Alzheimer : étude des rôles de MT1- et MT5-MMP dans l'amyloïdogenèse et la neuro-inflammation“. Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0709.
Der volle Inhalt der QuelleDuring my Ph.D., I studied the role of metalloproteinases MT1- and MT5-MMP in Alzheimer's disease (AD). In vivo, our work demonstrates a beneficial effect of the MT5-MMP deletion in the 5xFAD mouse model of AD, which is characterized by: i) a decrease in the levels of toxic metabolites of APP, including C99 and Aβ ; ii) a reduction in the inflammatory response with decreased levels of pro-inflammatory cytokines; iii) preservation of the integrity of neural networks, synaptic activities and cognitive performance. In vitro, we show that MT5-MMP interacts with APP, increases its localization in early endosomes - one of the important sites of BACE-1 (beta-site amyloid precursor protein cleaving enzyme 1) γ-secretase - and increases the release of Aβ. After overexpression in HEK (human embryonic kidney) cells stably expressing the Swedish APP mutation (HEKSwe), we show that MT1-MMP stimulates the production of C99 and Aβ according to a process dependent on BACE-1. Our data indicates that MT1-MMP interacts with the APP and cleaves it in partnership with MMP-2 to generate two fragments with unknown properties. MT1-MMP, like MT5-MMP, favors the trafficking of APP to early endosomes, which may explain its pro-amyloidogenic potential. Finally, overexpression of a catalytically inactive form of MT1-MMP induces none of the effects observed with the active form of the protease. All of this work makes it possible to consider MT1- and MT5-MMP as new therapeutic targets to target both pro-amyloidogenic and pro-inflammatory processes characteristic of AD
Hoareau, Céline. „Rôle de la reelin du neurodéveloppement à la neurodégénérescence“. Paris 6, 2006. http://www.theses.fr/2006PA066277.
Der volle Inhalt der QuelleGalioot, Amandine. „Contribution à l'étude du rôle des SER/THR protéine-phosphatases PP1/PP2A dans les processus de mort cellulaire et de maturation du précurseur du peptide Beta-Amyloïde“. Paris 7, 2013. http://www.theses.fr/2013PA077219.
Der volle Inhalt der QuelleOuTlabhas previousry proposed a concept denominated "Drug Phosphatase Technology" (DFT) based on the use of cationic penetrating sequences capable of interacting with PP1/PP2A phosphatases in order to deregulate specific intracellular signais Ser/Thr proteins-phosphatases of PP1 and PP2A family are key factors of the cellular signalization and deregulation of their activity through interaction with cellular or viral protein often leads to severe dysfunctions. For exemple, E4orf4 protein of adenoviruses interacts with PP2A1 and leads to apoptotic death in infected or transformed cells while healthy cells remain unaffected. In a first part of this work, we have identified an interacting sequence between PP2A1 and E4orf4 from which we - have characterized the peptide DFT-F4orf44, which provokes the apoptosis of a subset of human tumoral cellswithout affecting healthy cells of fibroblastic type. Protein-phosphatases of PP1 and PP2A family also play a crucial role in the physiological regulation of neuronal substracts Tau and APP (Amylold Peptid Precursor). A diminution of the phosphatase activity in nerve cells leads to a drastically increasjng of phosphorylation state of these proteins for patients affected by Alzheimer's disease. I The second part of this work has allowed the identification of the phosphatase proteins responsible for the regulation of phosphorylation of APP T668 residue, which is an essential modification for APP maturation and processing
Vingtdeux-Didier, Valérie. „Aspects moléculaires et cellulaires impliqués dans le clivage ou la dégradation des fragments carboxy-terminaux et du domaine intracellulaire du Précurseur du Peptide Amyloïde (APP-CTFs et AICD)“. Lille 2, 2006. http://www.theses.fr/2006LIL2S036.
Der volle Inhalt der QuelleAlzheimer's disease (AD) is characterized by two distinct pathologies: neurofibrillary tangles (NFT) and extracellular amyloid plaques composed of beta-amyloid peptide (Abeta). Abeta derive from the catabolism of the Amyloid Precursor Protein (APP). A relationship between APP metabolism and NFT is observed in AD. This relation is illustrated by a significant decrease of APP-CTFs and AIDD, which correlated with the progression of NFT. APP-CTFs phosphorylation is also modified in AD. The main objectives of this thesis were to identify the degradations pathways of APP-CTFs and AICD. Our results demonstrated that increase in the phosphorylation of APP-CTFs facilitates their processing by the gamma-secretase. Moreover, our data demonstrate for the first time that the endosome/lysosome pathway mediates the degradation of AICD and we describe a novel secretion pathway of APP catabolic derivatives
Petit-Paitel, Agnès. „Étude du rôle des présénilines dans la maturation de la protéine précurseur du peptide amyloi͏̈de dans la maladie d'Alzheimer et dans la maturation du récepteur Notch-1 : une fonction controversée“. Paris 7, 2002. http://www.theses.fr/2002PA077149.
Der volle Inhalt der QuelleBour, Alexandra. „Implication d'un dérivé sécrété du précurseur de la protéine β-amyloi͏̈de (sAPP695) et de différentes isoformes de l'apolipoprotéine E humaine (apoE3 et apoE4 ) dans les processus mnésiques chez la Souris“. Université Louis Pasteur (Strasbourg) (1971-2008), 2004. https://publication-theses.unistra.fr/public/theses_doctorat/2004/BOUR_Alexandra_2004.pdf.
Der volle Inhalt der QuelleAn alteration of the beta-amyloid precursor protein (APP) metabolism is associated with Alzheimer's disease (AD) as well as age-related memory impairments. This alteration seems to affect cerebral level of sAPP, a promnestic and soluble form of APP. On the other hand, the epsilon 4 allele, but not the epsilon 3 allele, of apolipoprotéine E (apoE) gene is a genetic risk factor for AD and for age-related memory impairments. Moreover, APP metabolism seems to be influenced by apoE. First, the effects of exogenous sAPP administration in mice were evaluated in object recognition, spatial recognition and bar-press learning tasks. According to the results, sAPP seems to promote early memory processes. The post-training blockade of endogenous sAPPs with specific antibodies induced memory deficits suggesting a central role of sAPPs in memory consolidation mechanisms. Besides this study, we showed the deleterious effect of the new ventilation system of some animal housing rooms on mice memory performance. Then, the memory performance of "aged" (14-15 months) apoE3 and apoE4 transgenic mice (homozygous carriers of the human apoE allele epsilon 3 and epsilon 4, respectively) were evaluated in spatial memory tasks (spatial recognition and Morris water maze) and active and passive avoidance memory tasks. Compared to apoE3 mice, apoE4 mice, especially females, showed more severe memory deficits. Thus, apoE4 female mice appear as a good model for the study of age-related memory deficits. In a last experiment using the Morris water maze task, the administration of sAPP restored a good level of spatial memory performance in "aged" female apoE4 mice. Altogether, our results suggest that the modulation of cerebral sAPP levels must be considered as a potential therapeutic approach in the treatment of age-related memory deficits in general, or those associated with AD in peculiar
Ouellet, Stéphane. „Études des mécanismes de régulation de la transcription des gènes humains P21CIP1/WAF1, GPC3 (GLYPICAN 3) et A-beta-PP (AMYLOID beta-PRECURSOR PROTEIN)“. Thesis, Université Laval, 2010. http://www.theses.ulaval.ca/2010/27537/27537.pdf.
Der volle Inhalt der QuelleGene transcription is the first step to the production of any given protein. Understanding of the molecular mechanisms regulating gene expression, such as the binding of transcription factors to genes promoters, is essential to the understanding of biological functions and to develop new powerful therapies against many clinically documented pathologies. We investigated the transcriptional regulatory mechanisms of three human genes very differently expressed and involved in diverse pathologies in an attempt to reveal structurals and functionals differencies between these mechanisms. AβPP and p21 genes are both expressed in adult while GPC3 is only transcribed in a tissus specific manner before birth. The expression of the AβPP gene is also specific to tissue and its over-expression may be involved in Alzheimer disease and Down syndrome. P21 gene is expressed in many types of cells and is strongly induced by DNA damage. Finally, we demonstrated that GPC3 is differently expressed in neuroblastoma and Wilms' tumor. P21 : The characterization of the proximal promoter from the p21 gene in normal human proliferating fibroblasts revealed seven DNA-protein footprints of which one bears a perfect consensus sequence for the NFI family of transcription factors. EMSA, CHIP, anti-RNA and transient transfection of recombinant constructs analyses clearly demonstrated that NFI interact with the most proximal LMPCR footprint on the p21 promoter and functions as a repressor. Upon serum starvation, a change in the electrophoretic mobility of the NFI DNA-protein complex was observed that may contribute to the activation mechanistic of the p21 gene throughout cell senescence and differentiation. AβPP : We demonstrated that Sp1, like USF, recognizes an element in the human AβPP gene that is necessary for full promoter activity. In cellulo footprinting analysis revealed at least eight DNA-protein interactions including CTCF, USF and many Sp1 target sites. These results were further supported by EMSA and transient transfection analysis. GPC3 : The characterisation of the entire GPC3 gene promoter revealed 1) a particular DNA structure in the promoter and 2) eight large protected regions. The use of competitor oligos in EMSA experiments and super-shift assays showed that an NFY-type transcription factor (TF) may explain the GPC3 aberrant expression in SJNB-7.
Verlaeten, Olivier Jacques. „Altérations de l'expression génique dans l'hypothalamus de souris infectées par un Morbillivirus : recherche de gènes candidats de l'obésité murine viro-induite“. Lyon 1, 2002. http://www.theses.fr/2002LYO1T210.
Der volle Inhalt der QuelleDesbene, Cédric. „Implication de la phospholipase A2 cytoplamique dans la pathogénèse de la maladie d'Alzheimer“. Thesis, Université de Lorraine, 2012. http://www.theses.fr/2012LORR0235/document.
Der volle Inhalt der QuelleSoluble beta-amyloid (A-beta) oligomers putatively play a critical role in the early synapse loss and cognitive impairment observed in Alzheimer's disease. We previously demonstrated that A-beta oligomers activate cytosolic phospholipase A2 (cPLA2) which specifically releases arachidonic acid from membrane phospholipids. By using a single A-beta oligomers intra cerebro ventricular injection, we observed that cPLA2 gene suppression prevents both the alterations of cognitive abilities and the reduction of hippocampal synaptic markers levels which are observed in wild type mice. We further demonstrated that the A-beta oligomers-induced sphingomyelinase activation is suppressed and that the phosphorylation of Akt/PKB is preserved in neuronal cells isolated from KO mice. Interestingly, expression of the A-beta precursor protein (APP) is reduced in hippocampus homogenates and neuronal cells from KO mice, but the relationship with the resistance of these mice to the A-beta oligomers toxicity requires further investigation. These results therefore show that cPLA2 plays a key role in the A-beta oligomers-associated neurodegenerative effects, and as such represents a potential therapeutic target for the treatment of Alzheimer's disease
Gay, Bernard. „Analyse ultra-microscopique de la morphologie et des interactions des protéines de capside de l'Adénovirus et du rétrovirus HIV-1 : I. Protéines de l'apex de la capside d'Adénovirus humain, penton base et fibre : oligomérisation et assemblage en cellules d'insecte. II. Précurseur de la protéine de capside du HIV-1(Pr55Gag) : expression et assemblage sous forme de protéine recombinante en système baculovirus-cellule d'insecte“. Montpellier 1, 1994. http://www.theses.fr/1994MON1T025.
Der volle Inhalt der QuellePavoni, Serena. „Mise au point d’un nouveau modèle d’organoïde cérébral humain pour l’étude des mécanismes d’interaction de la protéine prion et de l’amyloïde β“. Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS427.
Der volle Inhalt der QuellePrion-like mechanisms are known to underlie most of human neurodegenerative diseases including Alzheimer’s disease (AD), which is characterized by two important pathological markers, β amyloid (or Aβ at the origin of the etiopathogenic amyloid cascade hypothesis) and phosphorylated tau protein. Furthermore, the prion protein (PrPC) interacts at multiple levels with the metabolism of Aβ, by mechanisms which are not well understood. To overcome the current limits in the development of efficient strategies to treat AD, the pharmaceutical industry requires innovative experimental models. However, even if a lot of progress has been achieved by using transgenic mouse models, to date no in vivo model can reflect the complexity of human brain or reproduce a clinical context. 2D in vitro cell culture models are unable to allow the aggregation and accumulation of pathological proteins as observed in vivo. The aim of this study consists in taking advantage of the research prospects offered by induced pluripotent stem cell (iPSCs) in the field of neurosciences. iPSCs can be used to generate 3D models of differentiation also called human cerebral organoids or mini-brains (MBs). Their ability to self-organise in 3D neuroectodermic tissue leds to a complex system that mimics different human cerebral structures in which we were able to characterize the expected markers. The study of the two proteins of interest (APP and PrPC) during neural differentiation has allowed us to follow the modulation of protein expression level occurring during the in vitro development of the human MBs. In order to use this model to reproduce the protein accumulation mechanisms seen in AD, we have tested chemical inductors such as Aftin-5 in order to modulate the APP post-transcriptional pathway towards a pathological outcome. Many strategies of treatment are adopted to lead APP cleavage and Aβ generation. The production of soluble fragments Aβ38, Aβ40, Aβ42 in the supernatant of organoids has been showed using ELISA technique. The levels generated are reproducible and the increase of Aβ42/Aβ40 ratio is consistent with extrapolated data from mouse and human models thus validating our model. Analysis at the gene and protein level has been assessed in order to understand the interaction between PrPC and APP after treatment. The long-term goal consists in improving this model which is notably hampered by the absence of vascularization and the low level of maturation of the neural tissue. The main challenge in MB culture thus consists in the integration of the vascular system, and also in increasing the speed of ageing process in vitro for the study of neurodegenerative diseases. In the long term, the prospect of automating the culture of MBs would allow the use of the system for cytotoxicity testing and/or high throughput screening for the discovery of new drugs for AD
Poirier, Véronique. „Manipulation expérimentale des progéniteurs neuronaux de la crête neurale de mammifère“. Cachan, Ecole normale supérieure, 1994. http://www.theses.fr/1994DENS0011.
Der volle Inhalt der QuelleEvrard, Caroline. „Implication des systèmes de dégradation cellulaire, de la protéine VCP et de nouvelles molécules sur le métabolisme de l’APP : aspects fondamentaux et appliqués à la maladie d’Alzheimer“. Thesis, Lille 2, 2018. http://www.theses.fr/2018LIL2S022/document.
Der volle Inhalt der QuelleAlzheimer's disease (AD) is a slow, progressive and irreversible neurodegenerative disease. There are two histopathological hallmarks found in AD brains: neurofibrillary tangles, caused by the intraneuronal accumulation of Tau protein aggregates in a hyper- and abnormally phosphorylated form; and amyloid deposits in the brain parenchyma which are mainly composed of amyloid peptides (Aβ) aggregates derived from the cleavage of the amyloid precursor protein (APP). AD physiopathology also includes a deregulation of protein homeostasis and degradation systems: proteasome, autophagy and the endosome/lysosome pathway, which are the main processes involved in the elimination of protein aggregates. Many evidences suggest that overproduction and aggregation of Aβ peptides are the main causes of AD and that strategies aiming to reduce their production and/or improve their clearance represent attractive approaches for AD therapeutics. Thus, this thesis aimed to study the respective contribution of g-secretase, proteasome and lysosomes in APP degradation; to determine the biological mode of action of chloroquine derivatives on APP metabolism in relationship with the Valosin-Containing Protein (VCP), supposed to be the target of these molecules, and at the same time, to study the relationship between APP and VCP. First, we have demonstrated that APP and its carboxy-terminal fragments (APP-CTFs) were mainly degraded by two pathways: g-secretase and the endosome/lysosome pathway. Next, we showed that VCP was involved in APP trafficking and processing. Finally, we have discovered that the action of our molecules does not depend on VCP but that they are indirect modulators of the β-secretase activity, reducing Aβ peptides secretion. In conclusion, this work contributed to a better understanding of APP metabolism and its degradation processes but also to characterize the biological effects of new indirect β-secretase inhibitors
Auboyer, Laura. „Génération de cellules souches pluripotentes induites de patients Alzheimer et production d'un modèle de culture en trois dimensions de neurones pour les recherches diagnostiques et thérapeutiques de la maladie d’Alzheimer“. Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT004/document.
Der volle Inhalt der QuelleAmyloid precursor protein (APP) and Tau protein are two main molecular actors of the Alzheimer’s disease (AD), which is of prime importance in Human Health. Intensive research is ongoing to understand these proteins’ metabolism, action and implication in the pathological mechanism of these affections. They are the target of most therapeutic approaches and are used for biological diagnosis. In the present PhD project, our objective was to investigate neuronal APP and Tau protein processing and metabolism using biochemical tools and innovative multiplex immunodetection system (MSD®) in diverse cell culture models of AD. The goal was to get a comprehensive view oh physiopathological processes based on the analysis of samples generated in neuronal differentiated human embryonic stem cell and induced pluripotent stem cells derived from AD-patients. We generated several cell lines from an healthy control individual, and AD patients showing sporadic and familial forms of the disease. This project offer the unique opportunity to combine state-to-the-art approaches to understand how the APP fragments and peptides are generated as well as the modifications of the Tau protein in normal and pathological situation
Laumet, Geoffroy. „Rôle des ADAM dans le processus physiopathologique de la maladie d'Alzheimer“. Phd thesis, Université du Droit et de la Santé - Lille II, 2010. http://tel.archives-ouvertes.fr/tel-00576504.
Der volle Inhalt der QuelleEysert, Fanny. „Etude des mécanismes impliquant le facteur de risque génétique FERMT2 dans le métabolisme de l’APP et ses conséquences dans le processus physiopathologique de la maladie d’Alzheimer“. Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S040.
Der volle Inhalt der QuelleThe establishment of genome-wide association study (GWAS) constitutes a major advance for the identification of new genetic susceptibility factors of Alzheimer’s Disease (AD). In contrast with the target gene approach, these analyses are done sans a priori and do not allow us to determine the role of the identified genes in the pathophysiological process of AD. In this context, only performing "post-GWAS analyses" can explain the molecular processes involving these genes. Our laboratory therefore aimed to identify the genetic risk factors identified by GWAS whose expression levels impact the APP metabolism. Moreover, our model allowed us to study the potential involvement of micro-RNAs (miRs) in the dysregulation of the expression of these genes.In this context, we showed that miR-4504, which is overexpressed in the brains of AD patients compared with controls, decreases the expression of FERMT2, a genetic susceptibility factor of AD. Our results show that FERMT2 under-expression is dependent on the presence of a variant (rs7143400) localized in the 3'UTR of FERMT2, which then leads to the modulation of the APP metabolism and the subsequent increase in Aβ peptide secretion.In this project, I was able to show that the effects of FERMT2 on APP metabolism require its direct inter-action with APP. In addition, using a model of primary neurons cultured in microfluidic devices enabled me to study neuronal functions of the FERMT2/APP complex. I was able to determine that FERMT2/APP interaction contributes to the regulation of axonal growth and synaptogenesis. Finally, by analyzing the long-term potentiation in the brains of mice in which FERMT2 under-expression was induced, we show a decrease in synaptic plasticity – potentially the underlying mechanism of the deleterious effect of decreased FERMT2 expression in neurons and of the pathophysiological process of AD.In conclusion, these results suggest that the genetic risk factor FERMT2, regulated by the presence of the functional variant rs7143400 and miR-4504, participates in the pathophysiological process of AD via synaptic alterations in an APP-dependent manner. This work would ultimately lead to a better understanding of the pathophysiological process leading to AD and help characterize new mechanisms involved in synaptic functions
Py, Nathalie. „Métalloprotéases matricielles et maladie d'Alzheimer : étude du rôle de MT1-MMP dans le métabolisme de l'APP/Aß“. Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM5073.
Der volle Inhalt der QuelleWe investigate the role of matrix metalloproteinases in the metabolism of beta amyloid peptide (Abeta) and its amyloid precursor protein (APP) in Alzheimer's disease (AD). Our results in the 5xFAD mouse model of AD indicate a cell-type and age-dependent upregulation of MMP-2 -and MT1-MMP active forms. This is concomitant with the increase of toxic forms of Abeta, but also of cytotoxic C99, a membrane fragment of APP generated by beta-secretase and that gives rise to Abeta after gamma-secretase cleavage. We show in HEK cells overproducing Abeta that while MT1-MMP interacts with APP and boosts C99 and Abeta production, MMP-2 does not interact with APP and degrades Abeta. These results uncover a MMP-specific regulatory crosstalk with amyloid and also MT1-MMP as a new pro-amyloidogenic proteinase. We want now to gain further insight into the mechanisms that support MT1-MMP effects, namely the possible modulation by MT1-MMP of beta- and gamma-secretase activities and/or APP trafficking
Letronne, Florent. „ADAM30 et métabolisme de l'APP : implication dans le développement physiopathologique de la maladie d'Alzheimer“. Thesis, Lille 2, 2014. http://www.theses.fr/2014LIL2S062/document.
Der volle Inhalt der QuelleProgressive intra-cerebral accumulation of amyloid peptides formed after sequential cleavage of the amyloid peptide precursor (APP) by secretases , is a central mecanism for Alzheimer’s disease. Therefore, a better understanding of APP regulation and homeostasy is now crucial. With this background, we postulate that the characterization of new actors in the APP metabolism could provide a more subtle understanding of this APP metabolism and trafficking. From their obvious implication in brain (development, plasticity and repair) and in APP metabolism (α-secretases), ADAMs (A Disintegrin And Metalloprotease) are an important protein proteins family which still have some undetermined function or role. Previously, a transcriptomic approach targeting ADAMs family bas been done at the laboratory on Alzheimer’s patient or control brains and found ADAM30 as under-expressed in Alzheimer’s patient brains. On cellular models, we confirmed that ADAM30 under-expression was associate with an increase in production/secretion of all the APP metabolim byproducts. Opposite results were found with ADAM30 over-expression. To replicate those results in another model closest to human pathophysiology, we have developed a triple transgenic mice model over-expressing APPSweInd and conditionally over-expressing ADAM30. In this model, we have observed and measured a decrease in amyloid deposits in mice brains over-expressing ADAM30. Secondly, because ADAM30 did not modulate secretase activities and did not cleave APP directly, we decided to determine ADAM30 substrats in the APP metabolism context. With a systematic approach, we have determined that Cathepsin D (CTSD) and Insulin Receptor Substrat 4 (IRS4) are two ADAM30 potential substrats. In our cellular models, we have found that ADAM30 is able to cleave and activate CTSD. This CTSD activity is required for ADAM30 action on APP metabolism. We have determined that ADAM30 specific action for CTSD is dependent on lysosome adressing sequence localised in APP C-terminal part. CTSD is a lysosomal protein and so ADAM30 would make CTSD specific activation easier. This mecanism would be able to increase APP degradation in endosome/lysosome pathway and reduce APP entry in its metabolism. To better understand ADAM30 specific action on CTSD and APP, we begin to investigate the potential role of IRS4 and the relation between insulin signaling pathway ans APP metabolism. Combined together, those data suggest that ADAM30 is a new APP metabolism actor, involved in an early APP regulation and degradation pathway dependent on lysosome activation. This study participate in a better understanding of the fine mecanism regulations involved in Alzheimer’s disease pathophysiological process
Desjardins, Alexandre. „Caractérisation de l’interaction entre la protéine Lin28 et le précurseur du microARN let-7g“. Thèse, 2013. http://hdl.handle.net/1866/11068.
Der volle Inhalt der QuelleThe regulation of gene expression is what allows our cells to adapt to their environment, to fight infections or, more generally, to express the appropriate level of proteins to meet a specific need. The microRNAs (miRNAs) are among the most important players in the regulation of gene expression. These small RNAs of 22 nucleotides are present in most multicellular species and are responsible for the direct control of more than 30% of protein-expressing genes in vertebrates. The miRNA lethal-7 (let-7) family consist of some of the most studied miRNAs and plays crucial roles in the cell. The appropriate regulation of the let-7 miRNAs level is essential for proper cellular development. The biogenesis of these miRNAs, from the primary transcript to their mature form is mainly regulated by Lin28, a highly-conserved pluripotent protein. This protein is composed of a cold shock domain (CSD) and two zinc-binding domains. These RNA-binding domains allow Lin28 to bind and inhibit the maturation of the let-7 miRNA. The objective of this thesis is to characterize the interaction between the Lin28 protein and the let-7g miRNA precursor to better understand the role of this protein in the inhibition of miARN biogenesis. Using biochemical and biophysical techniques, we first identified the main determinants of the interaction between Lin28 and the terminal loop of the precursor miRNA let-7g (TL-let-7g). We concluded that the C-terminal domain of Lin28, composed of a lysine-rich and arginine-rich motif in addition to two zinc-binding motifs, is sufficient to bind with high affinity a conserved guanine-rich bulge located on the TL-let-7g. In addition, because the sequence and RNA-binding specificity of this C-terminal domain are similar to those of the HIV protein NCp7, we defined this region as the NCp7-like domain of Lin28. Subsequently, we characterized the multimerization of three Lin28 proteins on the terminal loop of pre-let-7g. This study helped to reconcile apparent contradictions found in the current literature regarding the Lin28-binding sites on miRNA precursors. We identified three high-affinity binding sites on TL-let-7g that are bound in a stepwise manner by the three Lin28 proteins. As part of the formation of the multimeric complex, both RNA-binding domains of Lin28 play an important role. The CSD destabilizes the RNA and this exposes several binding sites, whereas the NCp7-like domain allows an orderly protein assembly and facilitates the initial binding of the protein. These results lead us to propose a new model for the interaction between Lin28 and pre-let-7g. In conclusion, these studies provide a better understanding of the molecular mechanisms involved in the post-transcriptional regulation of an important family of miRNAs and will help guide future projects in the expanding research area of miRNA biogenesis.
Trillaud-Doppia, Émilie. „Implication du domaine intracellulaire du précurseur de la protéine amyloïde dans la modulation de la plasticité synaptique“. Thèse, 2016. http://hdl.handle.net/1866/15997.
Der volle Inhalt der QuelleLa maladie d’Alzheimer est la forme la plus commune de démence liée au vieillissement ; elle est caractérisée par des déficits précoces d’apprentissage et de mémorisation et entraîne à terme une perte généralisée des fonctions cognitives supérieures. Alors que l’amyloïde-bêta (Aβ) et la protéine tau sont traditionnellement associées au développement de la maladie d’Alzheimer, des études récentes suggèrent que d’autres facteurs, tels que le domaine intracellulaire (APP-ICD) du précurseur de la protéine amyloïde (APP), pourraient jouer un rôle. Dans notre étude, nous avons testé si l’APP-ICD pourrait affecter les mécanismes de transmission ou de plasticité synaptique dans l’hippocampe, qui sous-tendent les processus d’apprentissage et de mémorisation. Nos résultats ont indiqué que la surexpression de l’APP-ICD dans des neurones du CA1 de l’hippocampe entraînait une diminution de la transmission synaptique dépendante des récepteurs AMPA et NMDA. Notre étude a montré que cet effet était spécifique de l’APP-ICD puisque son plus proche homologue l’APLP2-ICD n’a pas eu cet effet. De plus, l’APP-ICD entraînait un blocage de la potentialisation à long terme (LTP), une augmentation de l’expression et une facilitation de l’induction de la dépression à long terme (LTD), mais l’APLP2-ICD n’a eu aucun de ces effets. Notre étude a montré que cette différence observée en transmission et en plasticité synaptique entre les deux peptides réside dans le changement d’une seule alanine dans l’APP-ICD pour une proline dans l’APLP2-ICD, et que l’APP(PAV)-ICD n’avait pas d’effet sur la plasticité synaptique. Nous avons aussi démontré que l’APLP2(AAV)-ICD mimait l’effet de l’APP-ICD pour la facilitation de la LTD. Ensuite nous avons montré que la longue forme APP-APLP2-APP (APP avec un changement de la séquence de l’Aβ pour celle homologue de l’APLP2) ne montrait pas d’effet en comparaison avec l’APP-ICD, ni sur la transmission synaptique ni sur la plasticité synaptique. Cependant, en activant le clivage par les caspases préalablement à l’induction de la LTD ou la LTP, nous avons observé une facilitation de la LTD et un iii blocage de la LTP avec l’APP-APLP2-APP, des effets que nous n’avons pas reproduit avec la longue forme APLP2. La thréonine 668 phosphorylable se trouve immédiatement après l’alanine et le site de clivage par les caspases dans l’APP-APLP2-APP. La mutation de la Thr668 pour une alanine a aboli son effet sur la LTD et restauré la LTP. Finalement, nous avons montré que la facilitation de la LTD par l’APP-APLP2-APP dépendait de la libération de calcium intracellulaire par les récepteurs ryanodines. En conséquence, nous proposons l’émergence d’un nouveau domaine de l’APP jouant un rôle critique, en plus de l’Aβ, dans les processus à la base de l’apprentissage et qui en conséquence pourrait jouer un rôle dans le développement de la maladie d’Alzheimer.
Licea, Sara. „Rôle du jeûne et de la perturbation de la cascade de signalisation de l'insuline sur le clivage du précurseur de la protéine amyloïde (APP)“. Thèse, 2016. http://hdl.handle.net/1866/18578.
Der volle Inhalt der QuelleLittle is known about the mechanisms that trigger the onset of Alzheimer’s disease (AD), a primarily sporadic disease. Studies on the familial form of AD attributed a particular importance to Amyloid beta (Aβ), a cleavage product of the Amyloid precursor protein (APP). Many risk factors have been identified as potential triggers of the development of AD including Type 2 diabetes (T2D). Indeed, the impairment of insulin signaling by the desensitization of insulin receptors (IR) in the brain seems to be a common hallmark of both diseases. The long term effects of IR resistance on the accumulation of Aβ and Tau hyperphosphorylation have been studied, but the acute effects of IR perturbation is less characterized. Also, both diseases show metabolic defects. Our research aimed to determine whether acute perturbation of IR signaling affects APP processing and if starving (energy deprivation) could sensitize this processing to acute perturbation of IR. To assess this, we used small doses of Tyrphostin AG1024 to simulate IR perturbations rather than a complete blocakade of the receptors. To quantify APP processing, we measured the change of total full- lenght APP by Western blot. To ensure that this change reflected APP processing we developed a luciferase based readout system. This system allowed us to monitor the occurrence of APP cleavage via GAL4-UAS Firefly luciferase driven expression because we linked GAL4 transcription factor to the C-terminal region of APP. First, we showed that IR perturbation with Tyrphostin leads to APP processing and that starving increased IR susceptibility to a smaller doses of Tyrphostin. This APP processing occurs via the amyloidogenic pathway because inhibition of β- and γ-secretase inhibited APP processing. We showed that this processing absolutely requires IR perturbation, while IGF-1R perturbation alone is insufficient. Furthermore, neither autophagy, caspases nor proteasome seemed to be implicated in APP processing following starvation and small IR perturbation. The activity of mTOR is also not required. On the contrary, GSK3 activation is necessary for the processing and seems to affect γ-secretase cleavage. We then confirmed in primary cultured neurons that the combination of acute starvation and small IR perturbation causes APP cleavage and GSK3 is again strongly activated. Taken together, our results suggest that the impairment of IR signalling seen in T2D increases the processing of APP via the amyloidogenic pathway and thereby contributes to the pathology of AD.