La sénescence cellulaire, arrêt stable de la prolifération cellulaire, est accompagnée de la sécrétion de nombreux facteurs pro-inflammatoires (programme sécrétoire associé à la sénescence appelé SASP). La sénescence est induite par divers stimuli, et joue un rôle clé dans de multiples contextes physiopathologiques. Cependant, la régulation de la sénescence est encore mal comprise. Notre laboratoire a récemment identifié le récepteur inositol 1,4,5-trisphosphate de type 2 (ITPR2, canal calcique du ER) comme nouveau régulateur de la sénescence. L'expression du gène ITPR2 est réprimée dans la plupart des cancers, mais sa régulation transcriptionnelle est peu connue. Dans ce contexte, le premier objectif de ma thèse était de caractériser de nouveaux régulateurs de l’expression d’ITPR2. Par un criblage (siRNA) et une analyse Nanostring, nous avons identifié le récepteur nucléaire RXRA comme répresseur transcriptionnel d’ITPR2. Nous avons montré que dans les fibroblastes primaires humains, le knockdown de RXRA induit l’expression d’ITPR2 et de ce fait la signalisation calcique, la production d’espèces réactives de l’oxygène (ROS), le dommage de l’ADN et finalement la sénescence via l’activation de la voie p53-p21. Inversement, la surexpression constitutive de RXRA retarde la sénescence réplicative. Les molécules du SASP, induisant ou renforçant la sénescence, peuvent réguler la signalisation calcique. Le deuxième objectif de ma thèse était d’étudier le rôle du SASP et la participation de la signalisation calcique dans celui-ci. Nous avons observé que le SASP induit la sénescence cellulaire accompagnée d’une différenciation neuroendocrine (NED) dans des cellules de cancer du sein. Le SASP induit une accumulation de calcium dans le cytoplasme qui paraît être impliquée dans la régulation de la NED. Une analyse de données d’échantillons de tumeurs du sein humaines et observé que les échantillons positifs pour la NED présentent des marques de sénescence / Cellular senescence is a stable proliferation arrest accompanied with senescence-associated secretory phenotype (SASP). Senescence is induced by diverse stimuli such as telomere shortening and oncogene activation and plays key roles in many physiopathological contexts like embryonic development, cancer and aging. However the molecular mechanisms regulating senescence remain partially understood. Our laboratory recently identified a new senescence regulator: the inositol 1,4,5-trisphosphate receptor type 2 (ITPR2), an ER calcium release channel. ITPR2 is repressed in many cancers, but its transcriptional regulation is barely known. Therefore, the first aim of my thesis was to characterize new ITPR2 regulators. Through siRNA screen and Nanostring analysis, we identified the nuclear receptor RXRA as a transcriptional repressor of ITPR2. We found that in primary human fibroblasts, RXRA knockdown induces ITPR2 expression and thereby calcium signaling, reactive oxygen species (ROS) production, DNA damage and ultimately senescence through p53-p21 axis. Conversely, RXRA overexpression delays replicative senescence. SASP has been described to induce/reinforce senescence, and most of the SASP factors are able to regulate calcium signaling through their receptors. The second aim of my thesis was to investigate the role of the SASP and the participation of calcium signaling in it. We observed that the SASP induces senescence accompanied with a neuroendocrine differentiation (NED) in some breast cancer cells. Interestingly, SASP triggers calcium accumulation in the cytoplasm which seems to be involved in the regulation of NED. We then analyzed human breast tumor datasets and observed that NED-positive samples display some senescence marks: functional p53, low proliferation level and Sprouty 2 expression. Altogether, my work identified RXRA as a new senescence regulator and showed calcium signaling is involved in SASP-induced NED in breast cancer cells

Glia are non-excitable cells found in nervous tissue, and have an important role in synaptic plasticity and the maintenance of neuronal environment, as well as the activity, development, degeneration, and repair of neurons. Glial cells are interconnected via gap junctions to form a multicellular syncytium and utilize intercellular and intracellular Ca2+signals to regulate their functions. Glial Ca2+ signals regulate important cell functions that include gene expression, cell proliferation, metabolism, ion transport systems, release of cell products, and cell death. Consequently, significant alterations of glial Ca2+ signals are associated with pathological processes such as epilepsy, Alzheimer's disease and stroke. Two major forms of Ca2+ signals, intercellular Ca2+ waves and intracellular Ca2+ oscillations occur within glia. Intercellular Ca2+ waves consist of the propagation of elevations in intracellular calcium concentration ([Ca2+]i) between neighboring cells, while intracellular Ca2+ oscillations consist of repetitive elevations in [Ca2+]i that remain confined to single cells. Ca2+ signals are initiated by either a localized chemical, mechanical and electrical stimuli. However, the exact mechanism of their initiation, propagation and modulation is not fully understood. Previous studies have led to the hypothesis that mechanically-induced intercellular Ca2+ waves in glia are mediated by the diffusion of second messenger inositol (1,4,5)-trisphosphate (IP3) through the gap junctions (GJ). However, intracellular Ca2+ may also diffuse between cells during the spread of intercellular Ca2+ wave. Alternatively, Ca2+ waves may be mediated by the release of extracellular messengers, e.g. ATP, that act via phospholipase C (PLC) -linked receptors, e.g. P2y receptors. It is also unknown if the propagation of Ca2+waves requires the regeneration of the signaling message by each cell. An interesting consequence of the propagation of an intercellular Ca2+ wave in glia is that they induce intracellular Ca2+ oscillations in cells that participate in its propagation. These intracellular Ca2+ oscillations may serve to resolve information contained in the position and strength of a local stimulus that induces intercellular Ca2+ wave propagation. Although the mechanism by which Ca2+ waves initiate Ca2+ oscillations is unknown it would seem likely that the mechanism of wave propagation is linked to the mechanism of initiation of Ca2+ oscillations. Guided by previous findings, I hypothesized that intercellular Ca2+ waves propagate by the diffusion of IP3 via gap junctions between neighboring cells to establish an intercellular gradient of IP3 concentration ([IP3]i that within individual cells initiates distinct intracellular Ca2+ oscillations. Two specific aims were investigated to test this hypothesis. The First Specific Aim was to determine if intercellular Ca2+ waves in glia are initiated by the generation of IP3 within a stimulated cell, and propagated by diffusion of IP3 molecules between neighboring cells via gap junctions. The Second Specific Aim, was to determine if intercellular Ca2+ waves induce distinct intracellular Ca2+ oscillations by establishing a specific gradient of oscillation-promoting [IP3]i within the glial syncytium. The initiation and propagation of intercellular Ca2+ waves and intracellular Ca2+ oscillations were examined in primary cultures of rat neonatal cortical glia, utilizing the techniques of a) the intracellular measurement of [Ca2+]i by fluorescence videomicroscopy, b) the photorelease of second messengers IP3 and Ca2+from their photolabile carriers, c) the loading of specific drugs by electroporation into defined zones of glial cultures, and d) identification of cell types by immunocytochemistry. The results of the Specific Aim 1 demonstrated the following: Mechanically-induced intercellular Ca2+ waves reequired PLC activation, the subsequent production of IP3 within the stimulated cell, and release of Ca2+ from intracellular calcium stores. Propagation of Ca2+waves depended on the presence of gap junctions. The release of Ca2+ via IP3 receptor/channels (IP3Rs) was necessary for Ca2+ wave propagation. In contrast, release of Ca2+ from ryanodine receptor/channels (RyRs) occurred in the mechanically-stimulated cell as well as in cells propagating a Ca2+ wave, but was not required for Ca2+ wave initiation and propagation. The propagation of Ca2+ waves through cells that contained heparin to block IP3Rs, or additional [Ca2+]i buffers, demonstrated that the regeneration of IP3 in the non-stimulated cells was not necessary for the propagation of the Ca2+ wave. Ca2+ waves were not mediated by extracellular signals, since Ca2+ waves were not affected by the extracellular perfusion or the inhibition of G proteins. Ca2+ was found to be a poor propagating signal of Ca2+ waves, since intercellular Ca2+ diffusion was not detected during Ca2+ wave propagation. These results are consistent with the hypothesis that Ca2+ waves propagate by diffusion of IP3molecules between neighboring cells via GIs. The [Ca2+]i increase in the stimulated cell occurred due to a Ca2+ influx from extracellular environment, and a release of Ca2+ from intracellular Ca2+ stores, and appeared to contribute to the activation of PLC and the generation of IP3. Ca2+ influx however, was not a necessary event in Ca2+ wave initiation or propagation, because Ca2+ waves occurred in the absence of extracellular Ca2+. By contrast, a [Ca2+]i increase in the absence of [IP3]i increase did not generate intercellular Ca2+waves. The results of the Specific Aim 2 demonstrated the following: An intercellular Ca2+ wave induced intracellular Ca2+ oscillations in a zone of cells at a specific distance from the stimulated cell. The initiation, frequency and duration of Ca2+ oscillations depended on the cells' distance from the Ca2+ wave origin, and not on the cell type or the magnitude of the Ca2+ wave. Modulation of the [IP3]i achieved by acetylcholine (ACh), a neurotransmitter that initiates IP3 production, or by intracellular photorelease of IP3 altered the oscillatory activity of individual cells and shifted the zone of oscillating cells away from the stimulated cell. Ca2+ oscillations spread through individual cells as an intracellular Ca2+ wave that was initiated from a specific site within the cell, independent of the orientation of the initial intercellular Ca2+ wave. These results are consistent with the hypothesis that an intercellular Ca2+ wave initiates Ca2+ oscillations by establishing a specific gradient of oscillation-promoting [IP3]i within the glial syncytium. The findings of this study support the hypothesis that intercellular diffusion of IP3 is the dominant mechanism of Ca2+ wave propagation and initiation of Ca2+ wave-induced Ca2+ oscillations. The significance of these results is that the glial syncytium may utilize specific intracellular Ca2+ oscillations to decode the position and strength of stimuli that induce intercellular Ca2+ waves, and thus integrate and coordinate multicellular functions of glia in the CNS.

Ca2+ is an important messenger that affects almost all cellular processes. Ca2+ signaling involves events that happen at various time-scales such as Ca2+ diffusion, trans-membrane Ca2+ transport and Ca2+-mediated protein-protein interactions. In this work, we utilized multi-scale computational methods to quantitatively characterize Ca2+ diffusion efficiency, Ca2+ binding thermodynamics and molecular bases of Ca2+-dependent protein-protein interaction. Specifically, we studied 1) the electrokinetic transport of Ca2+ in confined sub-µm geometry with complicated surfacial properties. We characterized the effective diffusion constant of Ca2+ in a cell-like environment, which helps to understand the spacial distribution of cytoplasmic Ca2+. 2) the association kinetics and activation mechanism of the protein phosphatase calcineurin (CaN) by its activator calmodulin (CaM) in the presence of Ca2+. We found that the association between CaM and CaN peptide is diffusion-limited and the rate could be tuned by charge density/distribution of CaN peptite. Moreover, we proposed an updated CaM/CaN interaction model in which a secondary interaction between CaN’s distal helix motif and CaM was highlighted. 3) the roles of Mg2+ and K+ in the active transport of Ca2+ by sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. We found that Mg2+ most likely act as inhibitor while K+ as agonist in SERCA’s transport process of Ca2+. Results reported in this work shed insights into various aspects of Ca2+ signaling from molecular to cellular level.

S’il est bien établi que le flux sanguin cérébral est distribué en fonction de la demandemétabolique des neurones, aucune étude n’a exploré la contribution du réseauvasculaire au processus de consolidation mnésique qui requiert un dialoguehippocampo-cortical permettant le remodelage progressif des réseaux neuronauxcorticaux sous-tendant la trace mnésique ancienne stabilisée.Utilisant un test comportemental induisant une mémoire olfactive associative chez lerat couplé à des techniques d’imagerie cellulaire ex vivo, nous montrons pour lapremière fois, chez le rat adulte sain, une dissociation fonctionnelle entre réactivité etarchitecture du réseau vasculaire cérébral. Nous mettons en évidence des modificationsde signalisation calcique des artères cérébrales qui suggèrent que leur dynamiques’adapte pour permettre l’expression du souvenir. De plus, suivant une cinétiquedifférente, le réseau vasculaire se densifie par angiogenèse dès le lendemain del’apprentissage, y compris dans les régions du cortex ne prenant en charge le souvenirque plusieurs semaines plus tard. En stimulant spécifiquement cette angiogenèse parinjection d’agents pharmacologiques dans le cortex, nous améliorons les performancesdes rats lors du rappel de mémoire ancienne.Pris dans leur ensemble, nos résultats soulignent l’importance de la plasticitévasculaire dans la modulation de la plasticité neuronale et des fonctions cognitives. Ilssuggèrent en outre que les changements structuraux précoces du réseau vasculairepourraient constituer un mécanisme permissif à l’origine de la régulation des épinesdendritiques corticales impliquées dans la formation et le stockage à long terme dessouvenirs.Mots / While there is consensus that cerebral blood flow is distributed according to themetabolic demand of neurons, the contribution of vascular networks to memoryconsolidation, the process by which memories acquire stability over time, remainsunknown. This process requires a transitory hippocampal-cortical interaction allowingthe progressive remodeling of cortical neuronal networks supporting the remotememory trace.By using a behavioral task requiring an associative olfactory memory coupled to cellularimaging techniques, we first reveal, in adult healthy rats, a functional dissociationbetween the reactivity and the architecture of cerebral vascular networks. We identifycalcium signaling changes that occur in specific cerebral arteries, pointing to theirability to adapt their dynamics upon retrieval to enable the successful expression ofeither recent or remote memories. Moreover, we show that vascular networks undergo atime-dependent densification via an angiogenesis mechanism as early as one day afterlearning, including in cortical regions which will only support memory storage andretrieval weeks later. By specifically stimulating this early cortical angiogenesis, we wereable to improve the performance of rats tested for remote memory.Taken together, our results highlight the importance of vascular plasticity inmodulating neuronal plasticity and cognitive functions. They also suggest that the earlystructural changes within vascular networks could constitute a permissive mechanismwhich regulates the development of cortical dendritic spines thought to support theprogressive formation and storage of enduring memories.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20099号 / 生博第359号 / 新制||生||47(附属図書館) / 33215 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 福澤 秀哉, 教授 佐藤 文彦, 教授 河内 孝之 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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