Thèses sur le sujet « IP3R3 »

Le cellule mieloidi del sistema immunitario innato sono in grado di riconoscere microbi o prodotti microbici attraverso recettori per i profili molecolari (PRRs, pattern recognition receptors). Tra questi, i recettori Toll-like (TLRs, Toll-like receptors) sono quelli più estensivamente caratterizzati. Il TLR4, insieme alle proteine CD14 e MD-2, forma il complesso multi-recettoriale che riconosce il Lipopolisaccaride (LPS), principale componente della membrana esterna dei batteri Gram-negativi. Il CD14 concentra il segnale dell’LPS e media la rilocalizzazione di TLR4 e MD-2 all’interno dell’endosoma. In seguito ad un’esposizione acuta ad LPS, il CD14, indipendentemente dal TLR4, attiva la via di trasduzione del segnale di NFAT attraverso chinasi della famiglia src e l’attivazione della PLCγ2 che portano ad un rapido ingresso di Ca2+ e all’attivazione della proteina calcineurina. Questa funzione del CD14 è cellula-specifica, essendo attiva nelle cellule dendritiche (DCs, dendritic cells) e non nei macrofagi. In questo lavoro abbiamo studiato il meccanismo di mobilizzazione del Ca2+ nelle DCs e abbiamo proposto una spiegazione per la mancata attivazione della via di trasduzione del segnale di CD14/NFAT nei macrofagi in risposta all’LPS. È stato dimostrato che, sia in DCs murine che in una sottopopolazione di DCs umane CD14+ recentemente descritta, il recettore di tipo 3 dell’IP3 (IP3R3) è espresso non soltanto in compartimenti intracellulari, quali il reticolo endoplasmatico (ER, endoplasmic reticulum) ma anche a livello della membrana plasmatica (PM, plasma membrane) e, in modo interessante, colocalizza con il CD14 nelle zattere lipidiche (lipid rafts). È stato riscontrato che, la mobilizzazione del Ca2+ nelle DCs stimolate con l’LPS è dovuto ad un flusso diretto di Ca2+ dallo spazio extracellulare che dipende dall’IP3R3 e richiede l’IP4, piuttosto che l’IP3, come secondo messaggero. Infatti, il silenziamento dell’IP3R3 o l’inibizione dell’ITPKB, la chinasi che converte l’IP3 in IP4, abolisce l’entrata di Ca2+ e l’attivazione di NFAT. In accordo con tali dati in vitro, l’inibizione dell’ITPKB in vivo, previene l’attivazione di NFAT, riducendo pertanto la permeabilità vascolare, che dipende dall’attivazione di NFAT nelle DCs. Diversamente dalle DCs, sebbene l’IP3R3 sia espresso a livello della PM dei macrofagi, presenta un minor livello di colocalizzazione con il CD14. Inoltre, in seguito ad una stimolazione con l’LPS, l’internalizzazione del CD14 avviene più rapidamente nei macrofagi rispetto a quanto avviene nelle DCs. Questi dati possono in parte spiegare il motivo della mancata attivazione della via di trasduzione del segnale di CD14/NFAT nei macrofagi in risposta all’LPS. Complessivamente, questi dati indicano che il meccanismo di mobilizzazione del Ca2+ innescato dal CD14 richiede l’attivazione dell’ITPKB e la produzione dell’IP4 come secondo messaggero, il quale, in seguito, apre l’IP3R3 a livello della PM, inducendo un flusso monofasico di Ca2+ che porta all’attivazione della cascata di trasduzione del segnale di NFAT. Infine, questi dati suggeriscono che l’ITPKB potrebbe essere presa in considerazione come nuovo bersaglio molecolare per terapie anti-infiammatorie volte a inibire funzioni specifiche delle DCs.<br>Innate immune myeloid cells sense the presence of microbes or microbial products through pattern recognition receptors. Among these, Toll-like receptors (TLRs) are the best-characterized. Toll-like receptor 4 (TLR4), together with CD14 and MD-2, forms the multi-receptor complex for the Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria. CD14 concentrates the LPS signal and mediates the relocation of TLR4 and MD-2 to the endosome. After acute LPS exposure, CD14, independently of TLR4, activates the NFAT signalling pathway through src family kinase and PLCγ2 activation that leads to a rapid Ca2+ influx and Calcineurin activation. This function of CD14 is cell type specific, being active in dendritic cells (DCs) and not in macrophages. In the present work, we investigated the mechanism of CD14-mediated Ca2+ mobilization in DCs and we proposed an explanation for the lack of CD14/NFAT pathway activation in macrophages in response to LPS. We revealed that, both in mouse DCs and in a newly discovered human CD14+ DC subpopulation, IP3 receptor 3 (IP3R3) is expressed not only in the intracellular compartments such as endoplasmic reticulum (ER) but also at the plasma membrane (PM) and interestingly, it colocalizes with CD14 in lipid rafts. We found that, Ca2+ mobilization in LPS-stimulated DCs is due to a direct Ca2+ influx from the extracellular space, that relies on IP3R3 and requires IP4 rather than IP3 as second messenger. Indeed, the silencing of IP3R3 or the inhibition of ITPKB, the kinase implicated in the IP3 to IP4 conversion, abolishes Ca2+ entry and NFAT activation. Conforming to our in vitro results, the inhibition of ITPKB in vivo, prevents the activation of NFAT thus reducing vascular permeability, which depends on NFAT activation in DCs. Differently from DCs, although IP3R3 is expressed at the PM of macrophages it shows a low level of colocalization with CD14. Besides, upon LPS stimulation, CD14 internalization occurs more rapidly in macrophages compared to DCs. These reasons explain at least in part the absence of CD14/NFAT pathway activation in macrophages in response to LPS. Taken together, our results indicate that the mechanism of Ca2+ mobilization triggered by CD14 requires the activation of ITPKB and the production of IP4 as second messenger, which, in turn, opens IP3R3 at the PM inducing a monophasic Ca2+ influx that leads to the activation of the NFAT pathway. Finally, our data suggest that ITPKB could be considered as a new target for anti-inflammatory therapies aimed at inhibiting specific DC functions.

Le cancer du sein est le cancer féminin le plus fréquent et le plus létal chez la femme dans le monde. Malgré l'amélioration du dépistage dans les phases précoces du développement tumoral, il demeure difficile de traiter les phases tardives lorsque les processus métastatiques sont engagés. Le développement métastatique dépend notamment de l'acquisition de capacités migratoires par les cellules épithéliales impliquant un remodelage du cytosquelette, hautement dépendant de la concentration calcique intracellulaire. Alors que les travaux se sont intéressés à l'implication des canaux ioniques membranaires dans les processus de migration, le rôle des récepteurs à l'inositol 1,4,5-trisphosphate (IP₃Rs) reste peu étudié et donc méconnu. Dans un premier temps nous avons montré une augmentation du niveau d'expression d'IP₃R3 avec le niveau du potentiel migratoire de trois lignées cancéreuses mammaires humaines : MCF-7 (les moins migrantes), MDA-MB-231 et MDA-MB-435s (les plus migrantes). D'autre part, nous montrons que la modulation de l'expression d'IP₃R3 module leurs capacités migratoires: elles sont diminuées par l'inhibition d'IP₃R3, alors qu'elles sont augmentées par la surexpression d'IP₃R3 dans les cellules mammaires peu migrantes. De plus, l'inhibition d'IP₃R3 révèle un signal calcique oscillant, alors que sa surexpression induit un signal calcique maintenu dans ces cellules. Dans un second temps, nous avons mis en évidence une corrélation inverse entre le niveau d'expression d'IP₃R3 et la morphologie arrondie de ces trois lignées cellulaires. En effet, plus la morphologie cellulaire est arrondie, plus l'expression d'IP₃R3 est importante. Par ailleurs, l'inhibition d'IP₃R3 induit un arrondissement des cellules migrantes et une diminution des protrusions membranaires accompagnés d’une diminution de leur adhésion. Ces résultats suggèrent fortement l'implication d'IP₃R3 dans la modulation des acteurs du cytosquelette. En effet, l'inhibition d'IP₃R3 induit une diminution de l'expression de l'ARHGAP18, de l'activité de RhoA et de l'expression de Cdc42. Ces Rho GTPases permettent à leur tour une diminution de la phosphorylation de FAK_⁸⁶¹ et la réorganisation du cytosquelette d'actine et de profiline. Enfin, dans un modèle migratoire, le profil oscillant s'établit dès réalisation d'une blessure et prédomine 3 h après dans les cellules du front de migration alors qu'il se met en place dans les cellules à l'arrière uniquement après 3 h. L'intervalle spatio-temporel de ce signal calcique oscillant reflète une dynamique calcique intracellulaire nécessaire aux processus migratoires et au remodelage du cytosquelette des cellules du cancer du sein. En conclusion, nos résultats révèlent un rôle clé de l'IP₃R3 dans les processus migratoires et dans le remodelage du cytosquelette de profilactine via la voie ARHGAP18/ RhoA/ FAK associé à une modulation du profil calcique intracellulaire<br>Breast cancer is the most common lethal cancer in women in worldwide. In spite of screening improvement in early stages of tumor development, it remains difficult to cure late stages when metastases are begun. Metastatic development depends on migratory capacities acquisition by epithelial cells, involving a cytoskeleton remodeling, highly depending of intracellular calcium concentration. While plasma membrane calcium channels have been highly studied in migration processes, the role of IP₃Rs remains misunderstood. Firstly, we highlighted a correlation between IP₃R3 expression level and migratory potential of three human breast cancer cell lines with different migratory potential. Indeed, the more the migratory profile increases, the more is the expression of IP₃R3. Moreover, IP₃R3 expression modulation regulates their migratory capacities. The migratory capacities decreased when silencing IP₃R3, whereas they increased by overexpressing IP₃R3 in the low migratory breast cancer cell line (MCF-7). Furthermore, IP₃R3 silencing reveals an oscillating calcium profile, while its overexpression induces a sustained calcium profile. Secondly, we demonstrated a reverse correlation between IP₃R3 expression level and rounded shape of these three cell lines. Indeed, the longer the cell has an elongated morphology, the greater the IP₃R3 expression is important. Moreover, IP₃R3 silencing induced a rounded shape of migrating cells and decreased of protrusions and adhesion. Our results suggest IP₃R3 implication in modulation of cytoskeleton actors. Indeed, knockdown of IP₃R3 induced a decrease of ARHGAP18 and Cdc42 expression, RhoA and FAK_⁸⁶¹ activity, and a reorganization of profilactin cytoskeleton. Furthermore, in a migratory model, oscillating profile is revealed at wound realization and predominates 3 h amer in cells of the front wound whereas it sets up in cells of back only amer 3 h. The spatio-temporal gap of this oscillating calcium signal reflects an intracellular calcium dynamic essential to migratory processes and cytoskeleton remodelling in breast cancer. In conclusion, our results reveal a key role of IP₃R3 in migratory processes by profilactin cytoskeleton remodeling through ARHGAP18/ RhoA/ FAK pathway thanks to intracellular calcium profile modulation

L'apoptose, ou « mort cellulaire programmée », joue un rôle clé dans de nombreux processus biologiques. Les protéines de la famille Bcl-2, dont l'expression est souvent altérée dans les cellules tumorales, sont les principaux régulateurs de l'apoptose. Parmi cette famille, la fonction exacte du répresseur apoptotique Nrh, aussi appelé BCL2L10 ou Bcl-B, reste à ce jour mal comprise. Bien que son expression ne soit pas détectable dans la plupart des tissus sains, on retrouve des niveaux élevés de Nrh corrélés à un mauvais pronostique dans les cancers du sein et de la prostate. Nous avons mis au jour un nouveau mécanisme selon lequel Nrz, l'orthologue de Nrh chez le poisson zèbre, interagit avec le domaine de liaison du ligand IP3 du canal calcique IP3R1. Il s'est avéré que la régulation négative des flux calciques par Nrz est critique lors du développement embryonnaire du poisson zèbre. Grâce à ces nouvelles données, nous avons cherché à comprendre la fonction de Nrh chez l'Homme, dans un contexte pathologique. Nous avons montré que Nrh interagit via son domaine BH4 avec le domaine de liaison du ligand du récepteur IP3R1 humain pour réguler l'homéostasie calcique et la mort cellulaire. Cette interaction définit Nrh comme la seule protéine de la famille Bcl-2 à réguler négativement la mort cellulaire exclusivement au niveau du réticulum endoplasmique. Pour aller plus loin, nous avons montré que la dissociation du complexe Nrh/IP3Rs sensibilise des cellules tumorales mammaires à l'action d'agents chimiothérapeutiques. Pour finir, nos résultats apportent une explication moléculaire sur la contribution de Nrh dans la résistance aux thérapies anti-tumorales<br>Apoptosis, also called “Programmed Cell Death”, plays a key role in many biological processes and pathologies. The B-cell lymphoma 2 (Bcl-2) proteins, whose expression is often altered in tumor cells, are the main regulators of apoptosis.Among this family, the actual physiological function of the human apoptosis inhibitor Nrh, also referred to as BCL2L10 or Bcl-B, remains elusive. Although in most healthy tissues the Nrh protein is nearly undetectable, clinical studies have shown that Nrh expression is correlated with poor prognosis in breast and prostate carcinomas. We have shed light on a novel mechanism by which Nrz, the zebrafish ortholog of Nrh, was found to interact with the Ligand Binding Domain (LBD) of the Inositol-1,4,5-triphosphate receptor (IP3R) type-I Ca2+ channel. Indeed, the regulation of IP3Rs-mediated Ca2+ signaling by Nrz was shown to be critical during zebrafish embryogenesis. We used the knowledge gained with the zebrafish model to investigate Nrh function in cancer. We showed that Nrh interacts with the LBD of IP3Rs via its BH4 (Bcl-2 Homology 4) domain, which is critical to regulate intracellular Ca2+ trafficking and cell death. Actually, this interaction seems to be unique among the Bcl-2 family, and sets Nrh as the only Bcl-2 homolog to negatively regulate apoptosis by acting exclusively at the Endoplasmic Reticulum. Furthermore, we showed that disruption of the Nrh/IP3Rs complex primes Nrh-dependent cells to apoptotic cell death and enhances chemotherapy efficiency in breast cancer cell lines.Lastly our results bring a new insight to the role of Nrh regarding chemotherapy resistance

Doctor en Bioquímica<br>La macroautofagia, comúnmente referida como “autofagia” es la principal vía de degradación de proteínas, organelos y material citoplasmático, permitiendo de este modo el reciclaje del material intracelular. Este proceso consiste en el englobamiento de fracciones citosólicas por una estructura multimembranar llamada “autofagosoma”, el cual posteriormente se fusiona con el lisosoma para formar el “autofagolisosoma”. Luego el material comprendido en el autofagolisosoma es degradado por enzimas hidrolíticas. Un estudio mostró que la inhibición de la enzima inositolmonofosfatasa (IMPasa) usando litio y L690.330, inducía una disminución de los niveles basales del IP3 y en consecuencia la generación de autofagia. Nuestros resultados confirmaron estos datos previos, demostrando que el pre tratamiento con mio-inositol revierte la autofagia inducida por litio y L-690.330. Además se demuestra que el pre tratamiento con mio-inositol también revertía la autofagia inducida por privación de nutrientes. IP3 es ligando de su receptor de IP3 (IP3R), el cual es el principal canal de Ca2+ a nivel del retículo endoplásmico. El principal objetivo de esta tesis es evaluar el rol del IP3R en la regulación de la autofagia. Los resultados mostraron que la disminución de los niveles proteicos del IP3R usando siRNA específicos, así como el tratamiento con antagonistas químicos del IP3R, tales como xestosponginas B y C, estimulaban significativamente el aumento en los niveles de autofagia. Además, xestospongina B, así como también la privación de nutrientes, indujo una pérdida en la interacción entre Bcl-2 y Beclin-1, los cuales interactúan en condiciones basales. El tratamiento con xestospongina B no perturbó los niveles de Ca2+, tanto en retículo endoplásmico como en el citosol, concluyendo que la autofagia inducida por xestospongina B es independiente de una fluctuación del Ca2+. Los experimentos de inmunoprecipitación mostraron que Beclin-1 (regulador clave en la inducción de la autofagia) interactúa tanto con IP3R así como con Bcl-2 en condiciones basales, y la interacción de este complejo es atenuado bajo condiciones de privación de nutrientes o por tratamiento con ABT737, el cual es un mimetizador de dominios BH3. Este resultado sugiere la presencia de un complejo proteico en la regulación de la autofagia. El papel del retículo endoplásmico en el desarrollo de la autofagia toma gran significancia debido al reclutamiento de proteínas clave (IP3R, Beclin-1 and Bcl-2). La relación entre autofagia y estrés de retículo no es clara y por lo tanto se evaluó el efecto de agentes inductores de estrés de retículo en la inducción de la autofagia. Los resultados mostraron que tunicamicina, tapsigargina y brefeldina-A (agentes inductores de estrés de retículo) activaron el UPR (respuesta a proteínas mal plegadas) e indujeron autofagia. La disminución de los niveles de proteínas claves en el desarrollo de la autofagia (Atg5, Atg10, Atg12, Vps34 y Beclin-1) usando específicos RNAs interferentes atenuaron la autofagia inducida por agentes inductores de estrés de retículo y xestospongina B. Además, la sobreexpresión de Bcl-2 y Bcl-XL con destinación a retículo endoplásmico atenuó la autofagia inducida por xestospongina B e inhibidores de la IMPasa. Esta tesis muestra novedosos resultados, los cuales dan cuenta de un complejo proteico IP3R/Beclin-1/Bcl-2 en la regulación de la autofagia.<br>Macroautophagy (herein referred to as “autophagy”) is the major catabolic pathway for entire organelles, long-lived/ aberrant proteins and superfluous portions of the cytosol. It consists of the stepwise engulfment of substrate elements into distinctive multimembraned “autophagosomes”, which after fusion with lysosomes form singlemembraned autophagolysosomes. Into the autophagolysosome, the engulfed material is degradated by lisosomal hidrolytic enzymes, leading the recyclage of intracellular material. A study has suggested that myo-inositol-1,4,5-trisphosphate (IP3) could regulate autophagy because inhibition of inositol monophosphatase (IMPasa) by lithium or L-690.330 stimulates autophagy through the depletion of IP3. Our results have confirmed that the reduction of intracellular IP3 levels by IMPasa inhibitors (lithium and L.690.330) stimulates autophagy, whereas the enhancement of IP3 levels by pre treatment whit mio-inositol inhibits the lithium and L.690.330 effect. Moreover we have demostred that autophagy induced by nutrient privation was also inhibited by treatment with mio-inositol, but the effect of nutrient privation in the intracellular IP3 basal levels was not evaluated. IP3 acts on the IP3 receptor (IP3R), an IP3‑activated Ca2+ channel of the endoplasmic reticulum membrane and consequently we wanted to evaluate de roll of IP3R in the regulation of autophagy. The results obtained in this thesis show that knockdown of the IP3 receptor (IP3R) with specifics small interfering RNAs and pharmacological IP3R antagonist (xestospongin B and C) are a strong stimulus for the induction of autophagy, in addition, xestospongin B (like nutrient starvation) induced loss in the interaction between Beclin-1 and Bcl-2. Moreover, the autophagy promoted by xestospongin B not produced alterations in the steady-state Ca2+ levels in the ER or in the cytosol, therefore the autophagy induced by xestospongin B was Ca2+-independent. Immunoprecipitation assays shown that Beclin- 1 (key protein in the regulation of autophagy) interacts with IP3R and Bcl-2 in basal conditions, and this interaction may be attenuated both by nutrient starvation or ABT737 treatment, which is a mimetic compound of BH3. These results suggest the presence of a protein complex in the regulation of autophagy. The treatment whit ER stressors such as tunicamycin, thapsigargin and brepheldine A induced Unfolded Protein Responses (UPR) and autophagy. The autophagy induced by these agents showed to be IRE1α dependent, but the inhibition of autophagy showed an increase in the cell death, indicating a pro survival function of the autophagy upon endoplasmic reticumum stress conditions. The autophagy induced by treatment with xestospongin B and ER stressors was inhibited by knockdown of Atg5, Atg10, Atg12, Vps34 and Beclin-1, which are keys proteins in the autophagic process. We have also evaluated the roll of Bcl-2 and Bcl-XL in the inhibition of autophgy, and the results showed that Autophagy triggered by IMPasa inhibitors and xestospongin B was inhibited by Bcl-2 and Bcl-XL over expression specifically targeted to ER but not Bcl-2 or Bcl-XL proteins targeted to mitocondria. Altogether, these results suggest that IP3R form a regulator complex with Bcl-2 and Beclin-1, which exerts a major role in the physiological control of autophagy

Orientador: Prof. Dr. Alexandre Hiroaki Kihara<br>Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2016.<br>Anóxia é uma das maiores causas de morbidade e mortalidade neonatal, especialmente em neonatos pré-maturos, constituindo um importante problema de saúde pública devido às sequelas neurológicas permanentes em pacientes. A privação de oxigênio dispara uma série de cascatas, culminando em morte celular em regiões cerebrais mais vulneráveis, como o hipocampo. Neste processo de morte celular causada pela privação de oxigênio, o cálcio citosólico possui um papel crucial. Receptores intracelulares de inositol 1,4,5-trifosfato (IP3Rs) são importantes reguladores de níveis de deste cálcio, no entanto, não se sabe sobre sua função na anóxia. O objetivo deste estudo é analisar se os IP3Rs do tipo 1 (IP3R1) participam no processo de morte no hipocampo de ratos após a anóxia neonatal. A análise quantitativa de real-time PCR revelou uma diminuição da expressão gênica de IP3R1 24 horas após a anóxia neonatal. Na análise da distribuição de células IP3R1-positivas foi observada uma densidade de IP3R1 na região de CA1 em ambos os grupos, porém, não se observou diferença entre os grupos controle e anóxia. Interessantemente os animais anóxia apresentaram uma alta colocalização de IP3R1 e marcador de núcleo (DAPI), sugerindo que a anóxia causa uma translocação de IP3R1 para o núcleo nas células hipocampais. Além disso, o padrão de marcação mostrou diferentes tamanhos de clusters dos receptores, indicando uma organização diferente entre os grupos. Foi injetado 2-APB, um bloqueador de IP3R1, ou veículo, no hipocampo de forma bilateral após a anóxia. Foi utilizado metodologias de marcação de células degeneradas e foi visto que no grupo 2APB houve uma diminuição do número de células FJC-positivas e TUNEL-positivas em comparação ao grupo veículo anóxia. Porém, não foi observado nenhuma diferença de marcação entre os grupos na imunofluorescência de caspase-3 ativada. Não foi detectada nenhuma diferença entre os grupos no teste de labirinto de Barnes. No teste de campo aberto, observou-se que o grupo 2APB apresentam maiores níveis de ansiedade. Desta forma, este estudo pode contribuir com novas perspectivas na investigação de mecanismos de neurodegeneração ativadas pela privação de oxigênio.<br>Anoxia is one of the most prevalent causes of neonatal morbidity and mortality, especially in preterm neonates, constituting an important public health problem due to permanent neurological sequelae observed in patients. Oxygen deprivation triggers a series of simultaneous cascades, culminating in cell death mainly located in more vulnerable metabolic brain regions, such as the hippocampus. In the process of cell death by oxygen deprivation, cytosolic calcium plays crucial roles. Intracellular inositol 1,4,5-trisphosphate receptors (IP3Rs) are important regulators of cytosolic calcium levels, although the role of these receptors in neonatal anoxia is completely unknown. This study focused on the functional role of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in rat hippocampus after neonatal anoxia. Quantitative real-time PCR analysis revealed a decrease of IP3R1 gene expression 24 hours after neonatal anoxia. Distribution analysis of IP3R-positive cells was performed and we observed higher IP3R1 pixels quantity in CA1 of both groups; however, we were not able to observe alterations between control and anoxia animals. Interestingly, we observed that anoxia animals present a higher colocalization of IP3R1 and nucleus marker (DAPI), suggesting that neonatal anoxia may cause IP3R1 translocation to the nucleus in hippocampal cells. Furthermore, puncta-labelling pattern showed different cluster sizes, larger in control group, indicating different organization between groups. We injected 2-APB, an IP3R1 blocker, or vehicle in hippocampus bilaterally after anoxia. Labelling techniques of degenerate cells was performed and we observed that 2APB group decrease the number of FJC-positive cells compared to vehicle anoxia group. In contrast, TUNEL labelling and active caspase-3 immunofluorescence showed no difference between groups. Barnes maze test showed no differences between 2APB group and anoxia vehicle group. On the other hand, the open field test showed that 2APB group presents higher anxiety levels than vehicle group. In this way, this study may contribute to new perspectives in the investigation of neurodegenerative mechanisms triggered by oxygen deprivation.

L’homéostasie calcique est régulée par de nombreux canaux ioniques, parmi lesquels des canaux intracellulaires perméables au Ca2+, comme l’IP3R. Récemment, la protéine Bcl-2 a été montré comme régulant l’activité de ce canal ionique. Cependant, les acteurs moléculaires précis de cette interaction ne sont pas très bien établis. Ici nous montrons grâce à une nouvelle technique que l’IP3R est inhibé par le domaine BH4 de Bcl-2 et que ce domaine est nécessaire et suffisant pour inhiber son activité. De plus, la liaison de l’ABT-199 dans la poche hydrophobe de Bcl-2 conduit à un changement de structure du domaine BH4. Le niveau d’expression des différentes isoformes d’IP3R ainsi que des protéines Bcl-2 et Bcl-xL ont été étudié dans différentes lignées cancéreuses prostatiques. De manière intéressante, l’expression du récepteur à l’IP3 de type 3 (IP3R3) est augmentée en fonction de l’agressivité des lignées cancéreuses prostatiques. De plus, nous pouvons observer un effet important de l’IP3R3 sur la migration et l’invasion des lignées humaines de cancer de la prostate. Globalement, ces données montrent que l’IP3R3 participe à l’augmentation du potentiel métastatique des cellules cancéreuses prostatiques. Par conséquent, l’IP3R3 peut être un marqueur diagnostic intéressant ainsi qu’une cible thérapeutique, notamment pour les stades avancés de cancer de la prostate<br>Calcium homeostasis is regulated by various ion channels, among which intracellular Ca2+-permeable channels, such as IP3R. Lately, Bcl-2 protein have been shown to regulate this ion channel activity. However, the study of the functional properties of IP3R in interaction with Bcl-2 is not a straightforward procedure and the molecular players implicated in that interaction are still not well established. Here, we show with the use of a new electrophysiological method, that the IP3R is inhibited by Bcl-2 via its BH4 domain and that the BH4 domain of Bcl-2 can inhibit by itself the single channel activity of the IP3R. Moreover, the binding of the ABT-199 in the hydrophobic groove of Bcl-2 leads to a tail-flip structural change in BH4 domain. We also studied the expression level of different IP3R isoforms as well as Bcl-2 and Bcl-xL protein in different prostate cancer cell lines. Interestingly, IP3R type 3 (IP3R3) expression is increased according the aggressiveness of prostate cancer cell lines. Indeed, IP3R3 was expressed preferentially in highly aggressive prostate cancer cell lines. Moreover, we can observe an significantly important effect of the IP3R3 on migration and invasion properties of human prostate cancer cell lines. Our study also revealed that IP3R3 was not involved in viability, proliferation. Overall, these data provide evidence on IP3R3 contribution to the increased metastatic potential of human prostate cancer cells. Therefore, IP3R3 could provide new perspective molecular target for the disease suppression, in particular at its advances stages

Au cours du vieillissement, les artères cérébrales subissent des modifications structurelles et fonctionnelles, notamment au niveau des cellules musculaires lisses (CML). La CML a pour rôle de maintenir la réactivité vasculaire via une signalisation calcique qui fait intervenir différents acteurs pouvant ainsi réguler deux phénomènes : la contraction et la relaxation. Ces acteurs rassemblent, au sein d’une même cellule, des canaux (CCVD, RYR, IP3R), des pompes calciques (SERCA, PMCA, NCX, STIM/ORAI) et leurs régulateurs (PLB, FKBP12.6, TRPP2, SARAF, TRIC). La restriction calorique (RC), apparaît comme étant un facteur retardant le vieillissement et ses pathologies. Notre travail s’est donc fortement impliqué dans l’étude de la signalisation calcique de la CML, en se focalisant sur les altérations génomiques et fonctionnelles au cours du vieillissement des artères cérébrales chez la souris C57Bl6/j. Nous avons ainsi pu mettre en évidence une altération de la signalisation calcique qui passe en partie par une modulation des niveaux d’expressions génique et protéique des canaux et pompes calciques impliqués dans ce phénomène, et par une modification fonctionnelle en termes de signaux calciques et de contraction. Après 5 mois de régime RC, il a été mis en évidence un ralentissement des altérations de la signalisation calcique liées au vieillissement et une diminution de l’oxydation des CML<br>During aging, cerebral arteries undergo structural and functional changes, particularly in smooth muscle cells (SMC). SMC is responsible for maintaining vascular reactivity via calcium signaling involving different actors and can regulate two phenomena: contraction and relaxation. These actors regroup channels (CCVD, RYR, IP3R) calcium pumps (SERCA, PMCA, NCX, STIM / ORAI) and their regulators (PLB, FKBP12.6, TRPP2, SARAF, TRIC). Caloric restriction (CR) appears as a factor in delaying aging and its pathologies. Our work is strongly involved in the study of calcium signaling in SMC, focusing on genomic and functional alterations during aging of cerebral arteries in mice C57BL6/J. We were able to demonstrate an altered calcium signaling, which is partly through modulation of gene and protein expression levels of calcium channels and pumps involved in this phenomenon, and a functional change in terms of calcium signals and contraction. After 5 months under RC, it was highlighted a slow calcium signaling alterations associated with aging and a decrease of SMC oxidation by SAMP8

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.

L'apoptose est un processus cellulaire fondamental pour l'homéostasie tissulaire. Ce type de mort cellulaire est sous le contrôle des protéines de la famille Bcl-2 qui régulent la perméabilité de la membrane externe de la mitochondrie. Cependant, au-delà de leur rôle dans le contrôle de l'apoptose, les protéines de la famille Bcl-2 peuvent intervenir dans d'autres processus tels que le cycle cellulaire ou le métabolisme. Au sein du laboratoire, nous nous intéressons tout particulièrement aux rôles non-apoptotiques des protéines Bcl-2 au cours du développement embryonnaire. Grâce à l'utilisation du poisson zèbre, nous avons pu montrer que les protéines de la famille Bcl-2 contrôlent différents processus au cours du développement grâce à leur capacité à réguler l'homéostasie calcique. En effet, nous avons montré que la protéine anti-apoptotique Nrz participe au remodelage du cytosquelette d'actine au cours de l'épibolie en régulant la concentration de calcium cytosolique par son interaction avec le récepteur à l'IP3 (IP3R). Nous avons de plus pu montrer que Nrz diminue la sortie de calcium du réticulum endoplasmique en inhibant la fixation de l'IP3 sur son récepteur. Nous avons également identifié un nouveau membre pro-apoptotique de la famille Bcl-2, Bclwav, spécifiquement exprimée chez les poissons et le xénope. Cette protéine participe à la régulation de l'homéostasie calcique mitochondriale en interagissant avec VDAC. Nous avons de plus montré que cette activité est essentielle pour les mouvements de convergence et d'extension au cours du développement embryonnaire précoce du poisson zèbre<br>Apoptosis is a key cellular process for tissue homeostasis. Apoptotic cell death is under control of Bcl-2 family proteins which regulate outer mitochondrial membrane permeability. However, beyond their role in apoptosis, Bcl-2 family proteins are also involved in other cellular processes such as cell cycle or metabolism. In our laboratory we are interested in non-apoptotic functions of Bcl-2 family proteins in embryonic development. Using zebrafish model we have shown that Bcl-2 proteins control different processes during early development thanks to their ability to regulate calcium homeostasis. Indeed, we have shown that the anti-apoptotic protein Nrz participates in actin cytoskeleton remodeling during epiboly by regulating cytosolic calcium concentration via an interaction with the IP3 receptor (IP3R). We have also demonstrated that Nrz decreases calcium release from the endoplasmic reticulum by inhibiting IP3 fixation on its receptor. We have furthermore identified a new pro-apoptotic member of Bcl-2 family, Bcl-wav which is expressed only in fish and frogs. This protein regulates mitochondrial calcium homeostasis by interacting with VDAC. We have moreover shown that this activity is essential for convergence and extension movements during early zebrafish development

Grado de magister en fisiología<br>La plasticidad muscular es la capacidad que presentan las células musculares esqueléticas de adaptarse a diferentes estímulos externos, modificando su fenotipo. La frecuencia de estimulación eléctrica (EE) (in vitro como in vivo) induce la transición fenotípica de un músculo. De modo interesante, el EE de baja frecuencia induce marcadores de transición fenotípica rápido/lenta por una vía dependiente de la activación del IP3R. Además, se ha establecido al uniportador de Ca2+ mitocondrial como un regulador del trofismo muscular. Hipotetizamos que la regulación de los niveles de RNAm del uniportador de Ca2+ mitocondrial y de sus proteínas reguladoras en respuesta a EE de baja frecuencia es dependiente de la activación del IP3R. Fibras musculares adultas aisladas desde el músculo flexor digitorium brevis (fdb) de ratones C57BL/6J de 8 a 10 semanas de edad fueron expuestas a EE de baja frecuencia en presencia o ausencia de inhibidores de la vía propuesta. Se observó que el estímulo eléctrico de baja frecuencia resulta en una disminución de los niveles de RNAm de MCU, MICU1, MICU2 y EMRE, mientras que el EE de alta frecuencia no genera modificaciones. Las fibras musculares esqueléticas pre-incubadas con apirasa (enzima que degrada ATP) y xestospongina B (inhibidor de los receptores de IP3) previenen la disminución de los niveles de RNAm de MCU, MICU1, MICU2 y EMRE mediada por un EE de baja frecuencia. El ATP extracelular exógeno (agregado al medio de incubación) resulta en una disminución de los niveles de RNAm de MCU, MICU1, MICU2 y EMRE). Además, este efecto de EE no es prevenido por actinomicina D (inhibidor de la transcripción). Este trabajo contribuye a la comprensión de los mecanismos moleculares involucrados en la plasticidad muscular en respuesta a ejercicio físico y en particular, al rol de la mitocondria en estos mecanismos.<br>Muscular plasticity is the ability of the skeletal muscle cells of different external stimuli, modifying their phenotype. The frequency of electrical stimulation (ES) (in vitro as in vivo) induces the phenotypic transition of a muscle. Interestingly, low frequency ES induces rapid / slow phenotypic transition markers by a pathway dependent on the activation of IP3R. In addition, the mitochondrial Ca2 + uniporter has been established as a regulator of muscle trophism. We hypothesized that the regulation of mRNA levels of the mitochondrial Ca2 + uniporter and its regulatory proteins in response to low frequency EE is dependent on the activation of IP3R. Adult muscle fibers isolated from the flexor digitorium brevis muscle (fdb) of C57BL / 6J mice from 8 to 10 weeks of age were exposed to low frequency ES in the presence or absence of inhibitors of the proposed pathway. It was observed that the low frequency electrical stimulus results in a decrease in mRNA levels of MCU, MICU1, MICU2 and EMRE, while high frequency ES does not generate modifications. Skeletal muscle fibers pre-incubated with apirasa (enzyme that degrades ATP) and xestospongin B (inhibitor of IP3 receptors) prevent the decrease of mRNA levels of MCU, MICU1, MICU2 and EMRE mediated by a low frequency ES. Exogenous extracellular ATP (added to the incubation medium) results in a decrease in mRNA levels of MCU, MICU1, MICU2 and EMRE). In addition, this effect of ES is not prevented by actinomycin D (transcription inhibitor). This work contributes to the understanding of the molecular mechanisms involved in muscle plasticity in response to physical exercise and, in particular, to the role of mitochondria in these mechanisms.<br>30/08/2019

Breast cancer is the most prevalent type of cancer and second leading cause of death in women. Among others, the triple negative breast cancer (TNBC) is the most invasive as it has the highest recurrence and death rates with no targeted therapeutic available thus far. Epidermal Growth Factor Receptor (EGFR) is one of the important targets as more than fifty percent of the TNBC overexpress it but all the therapies designed against it have failed to show significant results. The juxtamembrane domain of EGFR has been explored comparatively recently and has been used to design a decoy peptide with the anticipation to affect the EGFR downstream functions. Previous research has shown it to cause cell death in cancer cells. This study is aimed towards deciphering the mechanism of action of the stapled form of this decoy peptide-SAH5. It presents evidence that the peptide leads to an immediate intracellular calcium release from the Inositol 1,4,5 triphosphate on the endoplasmic reticulum, an inhibition of which can rescue SAH5 induced cell death. The study also demonstrate that the peptide is able to increase the production of Reactive Oxygen Species (ROS) in mitochondria, part of which is triggered by the peptide-induced calcium release.

Familial Alzheimer's disease (FAD) -linked mutations in presenilin 1 and 2 (PS1, PS2) have been causally implicated in neurodegeneration and eventually neuronal cell death by amyloid toxicity and perturbation of cellular Ca2+ homeostasis. The mechanism governing this latter phenomenon remains unclear. In the cytosol, upon stimulation, both exaggerated and reduced Ca2+ release have been reported in different cell lines and neurons expressing PS1 and PS2 mutants. Despite the contradicting data yet available, it is undisputable that FAD-linked PS mutants cause imbalances of cellular Ca2+ homeostasis. Recent independent reports have strongly suggested that the FAD-linked PS1 and PS2 mutants interact directly or indirectly with both the ER Ca2+ uptake and release machinery by modulating SERCA pump, IP3R- and RyR channel activity. In the study described here, we took advantage of two available lines of transgenic (tg) mice expressing a mutant PS2 alone or together with a mutant amyloid precursor protein (APP) - both linked to FAD - in order to investigate its effects on Ca2+ homeostasis in a physiological environment more relevant to the pathology under study. We particularly focused our attention on Ca2+ dysregulation in cortical neurons from tg mice either single homozygous for human PS2-N141I or double homozygous for human PS2-N141I and human APP swedish mutations. Ca2+ measurements were carried by the fura-2/AM technique. This study highlights the role of PS2-N141I in modulating Ca2+ homeostasis of cortical neurons. We have demonstrated that, irrespective of the presence of mutant APP, modest expression of PS2-N141I altered the Ca2+ dynamics of intracellular stores. The total Ca2+ content of intracellular stores is partially depleted, as demonstrated by reduced Ca2+ release upon ionomycin stimulation. Consequently, the tg neurons have reduced Ca2+ release in response to IP3-generating agonists. However, the PS2 mutant does not affect the protein expression levels for both SERCA pump and IP3Rs. Our results suggest that the PS2-N141I FAD mutant causes a functional defect in ER Ca2+ entry/exit pathways. We also show that both tg neurons express very low levels of the mutant protein but show Ca2+ dysregulation, similar quantitatively and qualitatively to that previously reported in cell lines upon transient over-expression of the same mutant protein. Conversely, measurements of Ca2+ release via RyRs revealed a novel and unexpected finding in PS2 mutant tg mice i.e. increased Ca2+ release in response to caffeine. In addition, RyR2 protein expression level was elevated in tg neurons. Upregulation of RyRs function and protein levels could save as an adaptation phenomenon to compensate the reduction in Ca2+ release via IP3Rs or a direct effect of mutant PS2 on RyR or a secondary effect. Furthermore, PS2-N141I causes alteration in neuronal Ca2+ excitability. The tg neurons had significantly elevated number of picrotoxin-evoked synchronous Ca2+ oscillations, which required extracellular Ca2+ influx but not Ca2+ release from intracellular stores. Interestingly, while Ca2+ dysregulation appeared to be similar qualitatively and quantitatively in both single and double tg mouse models, the total amount of brain Ab42 and Ab40 peptides (ELISA) as well as their ratios were strikingly different between the two tg lines. These results strongly suggest that in tg mice the expression of mutant APP and/or Ab levels have no primary effect on the store Ca2+ content at this early stage and provide evidence that the quite similar effects on Ca2+ dynamics observed in both tg mice are due to the mutant PS2. Finally, results presented in this work suggest that although Ca2+ dysregulation is an early event in FAD, it does not affect neuronal response and vulnerability to cytotoxic stimuli at this early stage. The second part of this study focused on the role of Ab42 oligomers in cellular Ca2+ dynamics. Synthetic Ab42 oligomers reduced Ca2+ release in response to IP3 generating agonists in wt neurons but did not affect the total Ca2+ content as monitored by ionomycin. Conversely, Ab42 oligomers increased the Ca2+ release induced by caffeine. It is likely that Ab42 oligomers exert their effect on the activation pathway from IP3 generating agonists to IP3Rs. However, the mechanisms through which Ab42 deranges intracellular Ca2+ homeostasis require further investigation. Nonetheless, it is conceivable that in addition to Ab42 oligomers, also intracellular Ca2+ stores could become likely therapeutic targets in FAD and AD in general<br>E stato dimostrato che mutazioni in presenilina 1 e 2 (PS1, PS2) legate alle forme familiari della malattia di Alzheimer (FAD) sono implicate nella neurodegenerazione e in ultima istanza nella morte cellulare per effetto della tossicità del peptide amiloide e della perturbazione dell'omeostasi del Ca2+ cellulare. Il meccanismo alla base di quest'ultimo fenomeno non è ancora stato chiarito. Si è visto che in linee cellulari e in neuroni esprimenti mutazioni in PS1 e PS2, il rilascio di Ca2+ nel citosol in seguito a stimolazione cellulare può essere sia aumentato che ridotto. Nonostante i dati contraddittori attualmente disponibili, è innegabile che i mutanti di PS legati a FAD provocano uno squilibrio nell'omeostasi del Ca2+ cellulare. Recentemente studi indipendenti hanno dimostrato che i mutanti FAD in PS1 o PS2 interagiscono sia con i meccanismi di rilascio che di accumulo di Ca2+ nel reticolo endoplasmatico (RE), modulando sia l'attività  della pompa SERCA che i canali di tipo IP3R e RyR. In questo studio abbiamo impiegato due linee già  disponibili di topi transgenici (tg) esprimenti una PS2 mutata, da sola o assieme alla Proteina Precursore dell'Amiloide (APP) (entrambe con mutazioni legate a FAD) al fine di investigarne gli effetti sull'omeostasi del Ca2+ in una condizione fisiologica più rilevante per la patologia oggetto dello studio. Abbiamo focalizzato in particolare la nostra attenzione sull'alterazione del Ca2+ in neuroni corticali ottenuti da topi tg omozigoti per il solo mutante PS2-N141I o doppi omozigoti per le proteine mutate PS2-N141I e APPswe. Le misure di Ca2+ sono state effettuate con la tecnica del Fura-2/AM. Questo studio mette in evidenza il ruolo della PS2-N141I nel modulare l'omeostasi del Ca2+ in neuroni corticali murini. Abbiamo dimostrato che, indipendentemente dalla presenza del mutante di APP, l'espressione della PS2-N141I, in quantità  moderata, inficia le dinamiche del Ca2+ dei depositi intracellulari. In particolare,i depositi del Ca2+ sono parzialmente svuotati, come dimostrato dal ridotto rilascio di Ca2+ in seguito a stimolazione con ionomicina. Conseguentemente i neuroni tg hanno un ridotto rilascio di Ca2+in risposta ad agonisti legati alla generazione di IP3. Occorre notare che la PS2 mutata non altera i livelli di espressione delle proteine SERCA e IP3R. I nostri risultati suggeriscono che il mutante FAD PS2-N141I causa un difetto funzionale nelle vie di entrata e di uscita del Ca2+ dal RE. Abbiamo inoltre dimostrato che i neuroni di entrambe le linee tg esprimono bassi livelli di proteina mutante ma mostrano un'alterazione del segnale Ca2+ qualitativamente e quantitativamente simile a quella precedentemente riportata per le linee cellulari sovra-esprimenti la stessa proteina. Al contrario, misure del rilascio di Ca2+ via RyR hanno rivelato un effetto inatteso, ovvero un aumentato rilascio di Ca2+ in risposta a caffeina nei neuroni tg. Inoltre, i livelli proteici di RyR2 sono aumentati nei neuroni tg. Un aumento della funzionalità  e dei livelli proteici di RyR2 potrebbero essere un fenomeno adattativo per compensare la riduzione del rilascio di Ca2+ via IP3R oppure essere un effetto diretto dei mutanti PS2 su RyR2 o ancora un effetto secondario. Infine abbiamo dimostrato che la PS2-N141I causa un'alterazione nell'eccitabilità  neuronale. I neuroni tg hanno un numero significativamente più elevato di oscillazioni di Ca2+ sincronizzate, evocate da picrotossina, che dipendono dall' ingresso di Ca2+ extracellulare e non dal rilascio di Ca2+ dai depositi. E interessante notare che, mentre l'alterazione del Ca2+ è qualitativamente e quantitativamente simile nei singoli e doppi tg, sia il livello totale di peptidi Ab42 e Ab40 nel tessuto cerebrale (misurati mediante ELISA) che il loro rapporto sono notevolmente diversi tra le due linee tg. Questi risultati suggeriscono che nei topi tg l'espressione di APP mutata o i livelli di Ab non hanno alcun effetto primario sul contenuto dei depositi di Ca2+ in questo stadio precoce, e suggeriscono che gli effetti sulle dinamiche del segnale Ca2+, così simili nelle due linee tg, siano dovuti alla PS2 mutata. Infine, i risultati qui presentati suggeriscono che nonostante l'alterazione del segnale Ca2+ sia un evento precoce nei neuroni a questo stadio, non altera la vulnerabilità  neuronale a stimoli citotossici che agiscono atrraverso i depositi del Ca2+ . La seconda parte di questo studio si è concentrata sul ruolo degli oligomeri di Ab42 sulle dinamiche del Ca2+ cellulare. Nei neuroni di topi wt, oligomeri di Ab42 sintetico riducono il rilascio di Ca2+ in risposta ad agonisti legati alla produzione di IP3, ma non riducono il contenuto totale di Ca2+ dei depositi misurato mediante applicazione di ionomicina. D'altra parte, gli oligomeri di Ab42 aumentano il rilascio di Ca2+ indotto da caffeina. E' probabile che gli oligomeri di Ab42 agiscano sulla via attivata dagli agonisti legati alla produzione di IP3 e sull' IP3R. I meccanismi attraverso cui Ab42 altera l'omeostasi intracellulare del Ca2+ richiedono tuttavia ulteriori indagini. In conclusione, oltre agli oligomeri di Ab42 anche i depositi intracellulari di Ca2+ possono diventare un importante bersaglio terapeutico per intervenire sulla patologia di Alzheimer familiare ma anche sulla forma sporadica

Ca2+ is a universal and versatile intracellular messenger, regulating a vast array of biological processes due to variations in the frequency, amplitude, spatial and temporal dynamics of Ca2+ signals. Increases in cytosolic free Ca2+ concentration ([Ca2+]c) are due to influx from either an infinite extracellular Ca2+ pool or from the more limited intracellular Ca2+ stores. Stimulation of the endogenous muscarinic (M3) receptors of human embryonic kidney (HEK) cells with carbachol results in the activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3Rs), release of Ca2+ from the endoplasmic reticulum (ER), and activation of store-operated Ca2+ entry (SOCE). Lysosomes are the core digestive compartments of the cell, but their importance as signalling organelles is also now widely appreciated. Accumulating evidence indicates that lysosomal Ca2+ is important for their physiological functions. Lysosomal Ca2+ release triggers fusion during membrane trafficking and, through calmodulin, it regulates lysosome size. Luminal Ca2+ is critical for regulation of lysosomal biogenesis and autophagy during starvation through the transcription factor, TFEB. Furthermore, aberrant lysosomal Ca2+ is associated with some lysosomal storage diseases. Lysosomes in mammalian cells have long been suggested to accumulate Ca2+ via a low-affinity Ca2+-H+ exchanger (CAX). This is consistent with evidence that dissipating the lysosomal H+ gradient increased [Ca2+]c and decreased lysosomal free [Ca2+], and with the observation that lysosomal Ca2+ uptake was followed by an increase in pHly. Furthermore, heterologous expression of Xenopus CAX in mammalian cells attenuated carbachol-evoked Ca2+ signals. However, there is no known CAX in mammalian cells, and so the identity of the lysosomal Ca2+ uptake pathway in mammalian cells is unresolved. Using mammalian cells loaded with a fluorescent Ca2+ indicator, I show that dissipating the pHly gradient pharmacologically or by siRNA-mediated knockdown of an essential subunit of the H+ pump, increases the amplitude of IP3-evoked cytosolic Ca2+ signals without affecting those evoked by SOCE. A genetically encoded low-affinity Ca2+ sensor expressed on the lysosome surface reports larger increases in [Ca2+]c than the cytosolic sensor, but only when the Ca2+ signals are evoked by IP3R rather than SOCE. Using cells expressing single IP3R subtypes, I demonstrate that each of the three IP3R subtypes can deliver Ca2+ to lysosomes. I conclude that IP3Rs release Ca2+ within near-lysosome microdomains that fuel a low-affinity lysosomal Ca2+ uptake system. The temporal relationship between the increase in pHly and reduced Ca2+ sequestration suggests that pHly affects the organization of the microdomain rather than the Ca2+ uptake mechanism. I show that abrogation of the lysosome H+ gradient does not acutely prevent uptake of Ca2+ into lysosomes, but disrupts junctions with the ER where the exchange of Ca2+ occurs. The dipeptide, glycyl-L-phenylalanine 2-naphthylamide (L-GPN), is much used to disrupt lysosomes and release Ca2+ from them. The mechanism is widely assumed to require cleavage of GPN by cathepsin C, causing accumulation of amino acid residues, and osmotic lysis of lysosomal membranes. I show, using LysoTracker Red and Oregon Green-dextran to report pHly, that L-GPN is effective in HEK cells lacking functional cathepsin C, following CRISPR-Cas9-mediated gene disruption. Furthermore, D-GPN, which is resistant to cleavage by cathepsin C, is as effective as L-GPN at increasing pHly, and it is similarly effective in cells with and without cathepsin C. L-GPN and D-GPN increase cytosolic pH, and the effect is similar when the lysosomal V-ATPase is inhibited with bafilomycin A1. This is not consistent with GPN releasing the acidic contents of lysosomes. I conclude that the effects of GPN on lysosomes are not mediated by cathepsin C. Both L-GPN and D-GPN evoke Ca2+ release, the response is unaffected by inhibition or knock-out of cathepsin C, but it requires Ca2+ within the ER. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. I conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes. I conclude that all three IP3R subtypes selectively deliver Ca2+ to lysosomes, and that the low pH within lysosomes is required to maintain the junctions between ER and lysosomes, but not for lysosomal Ca2+ uptake. I suggest that GPN lacks the specificity required to allow selective release of Ca2+ from lysosomes.

Contexte: La physiopathologie de la fibrillation auriculaire (FA) a été caractérisée par des changements de concentration cellulaire de Ca2+ et des processus connexes menant à l'apparition et au maintien de la maladie. Les récepteurs de trisphosphate d'inositol (IP3R) sont des canaux calciques ligand-dépendants pour lesquels la surexpression dans la FA a été liée à un remodelage cardiaque. Les microARN (miR, miARN), petits ARN non codants, sont d'une longueur d'environ 22 nucléotides et régulent l'expression des gènes par déstabilisation de l'ARN ou inhibition de sa traduction. De plus en plus de preuves ont été apportées sur le rôle des miARN dans la physiopathologie des troubles cardiaques, y compris le remodelage défavorable induit par la FA. Objectif: Notre laboratoire a montré que le niveau nucléaire IP3R1 est régulé à la hausse dans le modèle canin de FA, ce qui produit une augmentation de la charge nucléaire en calcium. Cette étude vise donc à étudier le rôle des miARN dans la régulation d'IP3R1 qui initie et/ou perpétue la FA dans les cardiomyocytes auriculaires du modèle de FA chez le chien. Méthodes: Nous avons utilisé un modèle canin de AF établi par méta-cardiographie auriculaire pendant 600 bpm × une semaine; des cœurs perfusés par Langendorff pour isoler les cardiomyocytes auriculaires pour des expériences moléculaires; le criblage des miRs qui ciblent le gène ITPR1, codant IP3R1, en utilisant des bases de données en ligne; RT-qPCR pour mesurer l'expression de l'ARNm de ITPR1 et confirmer le niveau d'expression des miARN criblés; l'analyse Western Blot pour évaluer le niveau de protéine d’IP3R1; le test de la double luciférase reporter, la surexpression et l'abattement des miARN en culture primaire de cardiomyocytes isolées ou de lignées cellulaires appropriées; et l'imagerie par fluorescence calcique Fluo-4 AM pour évaluer le rôle potentiel des miARN sur la manipulation du Ca2+. Pour les expériences de manipulation des miARN, les cellules ont été transfectées avec 1) un miARN non codant (miR-NC, groupe témoin), 2) un miARN mimétique et 3) un inhibiteur du miARN (AMO). La signification statistique est calculée avec le test t de Student ou l'analyse unidirectionnelle de variance (ANOVA) suivie par le test de Tukey à comparaisons multiples en utilisant le logiciel GraphPad Prism version 6.00. Résultats: Nos données indiquent une augmentation du niveau de la protéine IP3R1 sans changement apparent de l'expression du gène ITPR1 dans les cardiomyocytes de l'oreillette gauche par rapport à notre modèle canin de FA. Sur la base de l'analyse informatique, il a été prédit que miR-26a ciblerait l'ARNm de l'ITPR1. La FA a considérablement réduit la régulation du miR-26a dans les cardiomyocytes de l'oreillette gauche. Le dosage de la double luciférase reporté dans les cellules H9C2 a montré que le miR-26a agissait directement sur la région non traduite 3′ (3′UTR) de l'ARNm ITPR1. De plus, la surexpression de miR-26a a réduit le niveau de la protéine IP3R1 et a diminué le taux diastolique [Ca2+] dans le noyau et le cytosol des cardiomyocytes de chien, des transistors de Ca2+ stimulés électriquement; tandis que le knockdown de miR-26a a inversé ces effets. L'expression de l'ARNm de l'ITPR1 est restée inchangée dans les cardiomyocytes de chien isolées après la transfection avec l'imitateur et l'inhibiteur de l'ARNm. Conclusion: La régulation à la hausse d'IP3R1 dans la FA est due à l'inhibition de la traduction par le miR-26a, qui est régulé à la baisse dans les cardiomyocytes auriculaires du modèle canin de FA. Ce changement est associé à une altération de la manipulation du Ca2+, qui se traduit par une augmentation des taux de Ca2+ diastolique nucléaire. Nos résultats suggèrent que la régulation à la baisse de miR-26a augmente l'expression de l’IP3R1, contribuant au remodelage pro-arythmique dans la FA.<br>Background: The pathophysiology of atrial fibrillation (AF) has been characterized by changes in the cellular concentration of Ca2+ and related processes leading to the initiation and maintenance of the condition. Inositol trisphosphate-receptors (IP3Rs) are ligand-gated calcium channels for which overexpression in AF has been linked to cardiac remodeling. microRNA (miR, miRNA)s, small non-coding RNAs, are around 22 nucleotides in length and regulate gene expression by mRNA destabilization or inhibition of its translation. A growing body of evidence has emerged about miRNA's role in the pathophysiology of cardiac disorders, including AF-induced adverse remodeling. Objective: Our laboratory has shown that nuclear IP3R1 level is upregulated in the dog AF model, producing increased nuclear calcium loading. Hence, this study aims to investigate the role of miRNAs in the regulation of IP3R1 initiating and/or perpetuating AF in atrial cardiomyocytes of the dog AF model. Methods: We used AF dog model established by atrial-tachypacing for 600 bpm × one week; Langendorff-perfused hearts to isolate atrial cardiomyocytes for molecular experiments; screening miRs that target ITPR1 gene, encoding IP3R1, using online databases; RT-qPCR to measure ITPR1 mRNA expression and confirm the expression level of the screened miRNAs; western blot analysis to evaluate the protein level of IP3R1; dual-luciferase reporter assay, overexpression and knockdown of miRNAs in primary culture of isolated cardiomyocytes or appropriate cell lines; and Fluo-4 AM calcium fluorescence imaging to assess the potential role of the miRNA on Ca2+ handling. For miRNA manipulation experiments, cells were transfected with 1) non-coding miRNA (miR-NC, control group), 2) miRNA mimic, and 3) inhibitor of the miRNA (AMO). Statistical significance is calculated with Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test using GraphPad Prism software version 6.00. Results: Our data indicated a rise in IP3R1 protein level with no apparent change in ITPR1 gene expression in left atrial cardiomyocytes from our dog AF model. Based on the computational analysis, miR-26a was predicted to target the ITPR1 mRNA. AF significantly downregulated miR-26a in left atrial cardiomyocytes. The dual-luciferase reporter assay in H9C2 cells showed that miR-26a directly acted on the 3′ untranslated region (3′UTR) of ITPR1 mRNA. In addition, miR-26a overexpression reduced the IP3R1 protein level and decreased the diastolic [Ca2+] in both nucleus and cytosol of the electrically-stimulated Ca2+ -transients, dog cardiomyocytes, while miR-26a knockdown reversed these effects. ITPR1 mRNA expression remained unaltered in isolated dog cardiomyocytes after transfection with the miRNA mimic and inhibitor. Conclusion: IP3R1 upregulation in AF is due to translation inhibition by miR-26a, which is downregulated in the atrial cardiomyocytes of the dog AF model. This change is associated with altered Ca2+ handling, reflected as enhanced nuclear diastolic Ca2+ levels. Our results suggest that miR-26a downregulation enhances the IP3R1 expression, contributing to pro-arrhythmic remodeling in AF.

It is widely accepted that activating mutations of genes encoding the Notch family of transmembrane receptors, specifically Notch1, are associated with oncogenic transformation. Previous data from our lab has shown that an active form of Notch1 (NICD) provides protection against apoptosis in D011.10 T cells; and that this effect may be attributed to NICD binding the pro-apoptotic protein Nur77. Nur77 is an immediate early gene that is upregulated during both negative selection of thymocytes and activation-induced apoptosis in D011.10 T cells. Nur77 upregulation is tightly regulated and requires MEF2D, NFAT, and the transcriptional co-activator, p300, to effectively respond to apoptotic stimuli. Here, we show that NICD has the ability to interfere with the transcription of Nur77, and that this interference is directly related to the inability of p300 to bind the Nur77 promoter in the presence of NICD. We also show that blocking Notch activation, through inhibition of gamma secretase or shRNA directed against Notch1, in T cell acute lymphoblastic leukemia (T-ALL) cell lines restores Nur77 expression in response to apoptotic stimuli. These observations support a mechanism by which NICD over-expression can suppress the activation of a known pro-apoptotic molecule, and further suggests this mechanism may operate in T-ALL.

Tese de doutoramento em Ciências da Saúde<br>Os astrócitos desempenham múltiplas funções desde a homeostasia cerebral ao controlo e processamento da atividade sináptica. Eles integram sinais neuronais por elevações complexas de cálcio (Ca2+) com impacto na comunicação neurónio-astrócito. As elevações de Ca2+ nos astrócitos podem ser divididas em dois tipos: globais (presentes no soma e principais processos) e/ou focais (presentes nos microdomínios). Apesar de estar descrito que as elevações globais de Ca2+ nos astrócitos podem modular a comunicação sináptica, continuam por esclarecer quais os mecanismos moleculares envolvidos. Desta forma, urge uma caracterização comportamental, estrutural e molecular detalhada para compreender esses mecanismos. Nesta tese, utilizámos o modelo de murganho que apresenta a deleção constitutiva do receptor 2 do inositol 1,4,5-trifosfato (IP3R2 KO), no qual as elevações globais de Ca2+ nos astrócitos estão ausentes. Na primeira parte deste trabalho (Capítulo 2) demonstramos que os murganhos IP3R2 KO têm um desenvolvimento somático e neurológico normal. Em seguida, a caracterização comportamental deste modelo transgénico (Capítulo 3) revelou que os murganhos IP3R2 KO apresentam uma melhoria do desempenho cognitivo em tarefas dependentes do hipocampo. Identificámos o factor de transcrição Foxo1 como modulador da expressão de genes específicos de astrócitos, responsáveis pela regulação do citoesqueleto e de espinhas dendríticas. A sobre-expressão do FOXO1 em astrócitos do hipocampo de murganho C57BL/6J foi suficiente para mimetizar a melhoria cognitiva verificada no modelo IP3R2 KO. Este resultado levou-nos a avaliar o papel da sinalização global de Ca2+ no contexto da depressão, uma doença que afeta comportamento dependente das regiões cortico-límbicas (Capítulo 4). Os murganhos IP3R2 KO apresentam uma surpreendente resiliência ao efeito ansiogénico do stress crónico. Por fim, explorámos o papel da sinalização de Ca2+ nos astrócitos no envelhecimento cognitivo (Capítulo 5). Os nossos resultados demonstram uma preservação do desempenho cognitivo em murganhos IP3R2 KO envelhecidos, caracterizado por alteração do rácio neurónio/astrócito e por refinamento dendrítico dos neurónios da camada V do córtex pré-frontal. Em suma, este trabalho contribuiu para uma melhor compreensão do papel da sinalização global de Ca2+ nos astrócitos desde o desenvolvimento até ao envelhecimento, num contexto de saúde e doença. Os resultados revelaram um alvo terapêutico específico em astrócitos com potencial aplicação em contextos de depressão e envelhecimento cognitivo.<br>Astrocytes are responsible for distinct functions ranging from brain homeostasis to the modulation of synaptic functioning. They integrate neuronal signals by complex calcium (Ca2+) elevations that control intracellular mechanisms that in turn drive the neuron-astrocyte dialogue, modulating the activity of cells and networks. It is now recognized that Ca2+ elevations in astrocytes appear spatially distributed in global (soma and main processes) and/or focal regions (microdomains). Although it is observed that global astrocytic Ca2+ signaling contributes to synaptic communication, its role in circuit computation and behavioral performance is still poorly understood. A detailed behavioral, structural and molecular characterization should provide us with putative mechanisms underlying the roles of astrocytic Ca2+. In this thesis, we took advantage of the inositol 1,4,5-trisphosphate receptor type 2 knockout (IP3R2 KO) mouse model, which lacks global Ca2+ signaling in astrocytes. In the first part of this work (Chapter 2), we demonstrate that IP3R2 KO mice retain a normal developmental maturation, as compared with WT littermates. Next, a detailed behavioral characterization of this mouse model (Chapter 3) showed that IP3R2 KO mice display enhanced cognitive performance in hippocampal-dependent tasks. We found Foxo1 as the most active transcription factor controlling the increased expression of astrocyte-specific genes related with fine cytoskeleton modulation and spinogenesis, which could underlie the cognitive enhancement observed. Moreover, specific overexpression of FOXO1 in hippocampal astrocytes of C57BL/6J mice was enough to recapitulate the enhanced fear memory observed in IP3R2 KO mice. This striking observation prompted us to test the role of global Ca2+ signaling in the context of depression, which affects cortico-limbic regions (Chapter 4). IP3R2 KO mice present an unexpected resilience to the installation of stress effects, namely translated into an increased self-care and a reduced anxious-like phenotype. Finally, we explored the role of astrocytic Ca2+ signaling in cortico-limbic performance in aged mice that display cognitive decline (Chapter 5). We observed a preserved cognitive performance in aged IP3R2 KO mice, an altered neuron/astrocyte ratio and a dendritic refinement of mPFC neurons. Overall, this work contributed to a better understanding on the role of global astrocytic Ca2+ signaling from development to aging, both in a health and disease context. We found a putative astrocyte-specific therapeutic target that could be used to prevent depression- and aging-related deficits.<br>The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), at the School of Medicine, University of Minho. Financial support was provided by a PhD grant (SFRH/BD/101298/2014 to SGG), FCT Investigator grants (IF/00328/2015 to JO, IF/01079/2014 to LP) and PTDC/MED-NEU/31417/2017 from the FCT – Foundation for Science and Technology, by BIAL Foundation grants (207/14 to JO and 427/14 to LP), by Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013); FEDER Funds, through the Competitiveness Factors Operational Programme (COMPETE), and The National Fund, through the FCT (POCI-01-0145-FEDER-007038).

La réaction acrosomique de Mammifère nécessite l'ouverture successive de trois canaux calciques : un canal calcique activé par de faibles dépolarisations (LVA), le récepteur à l'IP3 et un canal activé par la vidange des stocks (SOC) TRPC2. Nos données montrent une intéressante interaction fonctionnelle entre le canal LVA et les protéines dont l'activité est liée au niveau de remplissage du réticulum (vraisemblablement les canaux TRPCs et l'IP3R). Toute la signalisation calcique de la RA est sous le contrôle du canal LVA qui est le premier à s'activer. En utilisant les souris déficientes pour Cav3.1, Cav3.2, nous avons montré que la sous-unité α1H est la sous-unité majoritaire dans les cellules spermatogéniques sauvages. Nos données fournissent de nouvelles hypothèses concernant l'activation de TRPC2 : celle-ci pourrait être due à des modifications de son interaction avec la junctate et l'IP3R, induite par la vidange des stocks.