Les cellules murales du cerveau sont un groupe de cellules neurovasculaires qui présentent une hétérogénéité moléculaire, morphologique et fonctionnelle exceptionnelle. Celles en contact avec les plus petis vaisseaux du cerveau, les péricytes du lit capillaire moyen sont connues pour être essentielles à l'homéostasie cérébrale, bien que leur capacité contractile ait longtemps été débattue. Cependant, nombre de leurs propriétés physiologiques, telles que leurs mécanismes de signalisation calcique, n'ont pas encore été élucidées. Cette thèse vise donc à identifier les mécanismes cellulaires de la signalisation calcique des péricytes des capillaires cérébraux. Dans le chapitre 2, nous utilisons la pharmacologie et l'imagerie des péricytes cérébraux exprimant l'indicateur de calcium GCaMP6f (provenant de souris transgéniques PDGFRβ-Cre::GCaMP6f) pour découvrir ces mécanismes. Contrairement aux péricytes engainants dont la signalisation du calcique dépend des canaux calcique voltage-dépendants, nous constatons que les signaux calcique des péricytes capillaire moyen sont indépendants des canaux calcique voltage-dépendants. Au contraire, nous constatons que les signaux calciques transitoires des pericytes du lit capillaire moyen sont inhibés par l'élimination du Ca2+ extracellulaire, l'inhibition des canaux Orai opérés par les réserves, le blocage du remplissage des réserves du réticulum endoplasmique, ainsi que l'inhibition des récepteurs de la ryanodine (RyRs) et des récepteurs de l'inositol trisphosphate (IP3Rs). Nous constatons également que l'entrée de Ca2+ opérée par les réserves peut être induite par la déplétion des réserves du réticulum endoplasmique et inhibée par les bloqueurs d'Orai dans les pericytes du lit capillaire moyen, et que l'influx basal de Ca2+ est largement dépendant de la déplétion des réserves. Enfin, nous montrons que l'entrée de Ca2+ opérée par les réserves d'Orai amplifie les élévations de Ca2+ cytosolique en réponse au vasoconstricteur endothéline-1. Nous concluons que la signalisation calcique dans les pericytes du lit capillaire moyen, qu'elle soit spontanée ou induite de façon agoniste, est régulée par le couplage entre la libération des réserves du réticulum endoplasmique et les voies d'influx opérées par les réserves. / Brain mural cells are a grouping of neurovascular cells that display exceptional molecular, morphological, and functional heterogeneity. Mid-capillary pericytes, the mural cells which contact the smallest vessels of the brain, are known to be critical to brain homeostasis, and their contractile ability has long been debated. However, many of their physiological properties, such as their Ca2+ signaling mechanisms, have not been elucidated. This thesis aims to uncover the cellular mechanisms of brain mid-capillary pericyte Ca2+ signaling. In chapter 2, we harness pharmacology and imaging of brain pericytes expressing the calcium indicator GCaMP6f (from transgenic PDGFRβ-Cre::GCaMP6f mice) to uncover these mechanisms. In contrast to ensheathing pericytes whose Ca2+ signaling is dependent on voltage-gated Ca2+ channels (VGCCs), we find that mid-capillary pericyte Ca2+ signals are independent of VGCCs. Instead, we find that mid-capillary pericyte Ca2+ transients are inhibited by removal of extracellular Ca2+, inhibition of store-operated Orai channels, blockade of endoplasmic reticulum store filling, as well as inhibition of ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs). We further find that store-operated Ca2+ entry can be induced by endoplasmic reticulum store depletion and inhibited by Orai blockers in mid-capillary pericytes, and that basal Ca2+ influx is largely dependent on store depletion. Finally, we show that Orai store-operated Ca2+ entry amplifies cytosolic Ca2+ elevations in response to the vasoconstrictor endothelin-1. We conclude that both spontaneous and Gq-coupled protein receptor agonist-induced Ca2+ signaling in mid-capillary pericytes is regulated by coupling between endoplasmic reticulum store release and store-operated influx pathways.

Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of sphingosine 1-phosphate (S1-P) on miniature endplate potentials (MEPPs) were studied. Extracellular application of S1-P (1 and 30 μM) had no significant effects on the frequency and amplitude of MEPPs. Delivery into nerve terminals by liposomes containing 10-5, 10-4 or 10-3 M S1-P was associated with a concentration-dependent increase in MEPP frequency of 37, 63 and 86%. The per cent of median MEPP amplitude was not significantly changed, but there was an increase in the number of 'giant' MEPPs. Pre-exposure of the preparations to S1-P 10-5 but not 10-8 M entrapped in liposomes for 15 min blocked the effects of subsequent superfusion of S1-P (10-4 M)-filled liposomes on MEPP frequency. Thus, intracellular S1-P receptors seem to undergo 'desensitization' to higher concentrations of S1-P. The result provides the first evidence that S1-P acting intracellularly but not extracellularly enhances spontaneous transmitter release at the frog neuromuscular junction.

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Ca2+ is a universal intracellular messenger that regulates many cellular responses. Most cells express inositol 1,4,5-trisphosphate receptors (IP3R) that mediate Ca2+ release from the endoplasmic reticulum (ER) when they bind IP3 produced after activation of cell-surface receptors. Vertebrate genomes encode three closely related subtypes of IP3R (IP3R1-3). High-resolution optical analyses have revealed a hierarchy of IP3-evoked Ca2+ signals that are thought to arise from the co-regulation of IP3Rs by IP3 and Ca2+. The smallest events ('blips') report the opening of single IP3Rs, Ca2+ 'puffs' report the almost simultaneous opening of a few clustered IP3Rs, and as stimulus intensities increase further Ca2+ signals propagate regeneratively as Ca2+ waves. The aim of this study was to establish whether all three IP3R subtypes can generate Ca2+ puffs. I first used a haploid cell line (HAP1 cells) to generate, using CRISPR/Cas9, a line lacking all endogenous IP3Rs. However, for analyses of Ca2+ puffs, I used HEK cells that had been engineered, using CRISPR/Cas9 to disrupt endogenous genes, to express single IP3R subtypes. Local Ca2+ signals evoked by flash-photolysis of caged- IP3 were recorded using Cal520 and total internal reflection fluorescence (TIRF) microscopy in human embryonic kidney (HEK) cells. The Flika algorithm was used, and validated, for automated detection of Ca2+ puffs and to measure their properties. IP3 evoked Ca2+ puffs in wild-type HEK cells and in cells expressing single IP3R subtypes. In wild-type cells, the Ca2+ signals invariably propagated regeneratively to give global increases in cytosolic [Ca2+]. This occurred less frequently in cells expressing single IP3R subtypes, commensurate with their lower overall levels of IP3R expression. The properties of the Ca2+ puffs, including their rise and decay times, durations, the size of the unitary fluorescence steps as channels closed channel during the falling phase, and the estimated number of active IP3Rs in each Ca2+ puff, were broadly similar in each of the four cell lines. The latter observation suggests that despite lower overall levels of IP3R expression (~30%) in cells with single subtypes relative to WT cells, there is a mechanism that ensures formation of similarly sized IP3R clusters. The only significant differences between cell lines were the slower kinetics of the Ca2+ puffs evoked by IP3R2, which may suggest dissociation of IP3 from its receptor contributes to the termination of Ca2+ puffs. My results demonstrate, for the first time, that all three IP3R subtypes can generate Ca2+ puffs. I conclude that Ca2+ puffs are fundamental building blocks of all IP3-evoked Ca2+ signals.

Le calcium est un second messager impliqué dans la plupart des fonctions neuronales et possède un rôle particulièrement important dans la régulation de l'expression génique. Pour activer l'expression de certains gènes, une augmentation de la concentration calcique au sein du noyau est essentielle. L'augmentation du calcium intracellulaire dépend entre autre, de la mobilisation du calcium contenu dans les stocks intracellulaires par des seconds messagers comme l'IP3, le cADPR et le NAADP, plus récemment découvert. Au cours de ma thèse, j'ai montré que dans les neurones d'aplysies, l'Aplysia ADP ribosyl cyclase, l'enzyme de synthèse du cADPR et du NAADP migrait dans le noyau suite à la dépolarisation et à l'entrée de calcium par les canaux voltage dépendants de type L. De plus, grâce à la microscopie confocale et l'utilisation de sondes calciques fluorescentes, nous avons observé sur des noyaux de neurones isolés, que les seconds messagers étaient capables de mobiliser le calcium contenu dans l'enveloppe nucléaire pour générer des oscillations calciques nucléoplasmiques. Nos données pharmacologiques montrent que le NAADP active un récepteur qui lui est propre et que ce dernier coopère avec les RyRs et IP3R pour générer ces signaux. Finalement, afin d'explorer les rôles du NAADP dans la physiologie du neurone entier, j'ai mis au point un modèle de neurones spinaux embryonnaire de souris. Les résultats préliminaires nous permettent de poser de nouvelles hypothèses concernant l'implication du NAADP dans certaines grandes fonctions neuronales régulées par les neurotrophines.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13429号 / 論医博第2233号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 妹尾 浩, 教授 川口 義弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Nur77 is a member of the orphan nuclear receptor family, where it is known to play an important role in apoptosis in both negative selection in T cells and in cancer cell lines. In the development of T cells, it is critical for the immune system to discriminate self from non-self by eliminating auto-reactive cells. It was originally thought that Nur77 initiated apoptosis by activating downstream gene targets. However, it is now clear that Nur77 has its own distinct role outside of the nucleus and the precise mechanisms by which Nur77 induces apoptosis in T cells still needs to be clarified. Calcium plays an important role as a second messenger in various cellular responses, one of which includes apoptosis. The IP3 receptor controls efflux of calcium from the ER and can be activated through TCR activation. This signal induces a rise in cytoplasmic calcium levels ultimately causing cell death through mechanisms that remain unclear. Here, we use a double positive DO11.10 T cell line with tetracycline responsive Nur77, to examine the effects of cytosolic Nur77. Through co-immunoprecipitation experiments we suggest, that the presence of Nur77 disrupts the IP3R/Bcl-2 interaction. In this study, we also investigated the effect of Nur77 on intracellular calcium levels. We show that Nur77 increases baseline calcium levels and causes emptying of ER calcium stores. We suggest a model where cytosolic Nur77 disrupts the IP3R/Bcl-2 interaction by binding Bcl-2 at the mitochondria or ER, causing calcium release through the IP3R and apoptosis of the cell.

La ICSI porcina es una herramienta con gran potencial aplicativo en diversos campos, entre los que destacan la producción de animales transgénicos, y la recuperación de razas en peligro de extinción. Aunque en la actualidad existen referencias de obtención de descendencia viva, el rendimiento es inferior al de otras especies, posiblemente debido al desconocimiento de las condiciones idóneas, y la dificultad de los cigotos para alcanzar el estadío de blastocisto in vitro. El presente trabajo se llevó a cabo para determinar diferentes factores que podrían afectar al rendimiento de la técnica, estudiando el efecto de 1) la secuencia de cultivo de los zigotos recién inyectados; 2) modificaciones en el sistema de MIV tradicional, y por último 3) la activación exógena del ovocito mediante la inyección de inositol trifosfato con el espermatozoide. El objetivo global de este estudio fue el de incrementar el rendimiento final de la ICSI en la especie porcina. / ICSI in pigs is a tool with an important applicable potential in diverse fields. One of this is the production of transgenic animals, and the conservation of endangered species. Even though there are some cases of living offspring, its output is still quite low comparing to other species, possibly due to unknown factors referring to ideal conditions for the development, and to the difficulty of the zygotes to reach the blastocyst stage in vitro. The goal of this study was to evaluate different factors affecting the ICSI performance. This was done by studying 1) the sequence of culture of the injected oocytes; 2) In vitro maturation (IVM) modifications, through meiotic inhibitors, such as roscovitine, and changes in IVM duration time, and finally 3) the exogenous oocyte activation through inositol triphosphate (InsP3) injection together with the sperm. The main objective of this study was to increase the final performance of ICSI in pigs.