Les astrocytes sont des cellules gliales du système nerveux central, essentielles à la formation des synapses, à la barrière hémato-encéphalique ainsi qu’au maintien de l'homéostasie. Récemment, les astrocytes ont été identifiés comme éléments clés du traitement de l'information dans le système nerveux central. Les astrocytes peuvent communiquer avec les neurones au niveau des synapses et moduler la communication neuronale en libérant des gliotransmetteurs et en absorbant des neurotransmetteurs. L’utilisation de nouvelles techniques comme la microscopie à super-résolution et les indicateurs calciques encodés génétiquement a permis de révéler une grande diversité spatio-temporelle des signaux calciques astrocytaires. La majorité de ces signaux sont observés au sein de leurs prolongements cellulaires, qui sont le site de communication entre neurones et astrocytes. Ces prolongements sont trop fins pour être observés en microscopie optique conventionnelle, de sorte que la microscopie à super-résolution et la modélisation informatique sont les seules méthodes adaptées à leur étude. Les travaux présentés dans cette thèse ont pour but d’étudier l'effet des propriétés spatiales (telles que la géométrie cellulaire, les distributions moléculaires et la diffusion) sur les signaux calciques dans les prolongements astrocytaires. Historiquement, les signaux calciques ont été modélisés à l'aide d'approches déterministes non spatiales. Ces modèles ont permis l'étude des signaux calciques à l’échelle de la cellule entière voire à l’échelle du réseau de cellules. Ces méthodes ne prennent cependant pas en compte la stochasticité inhérente aux interactions moléculaires ainsi que les effets de diffusion, qui jouent un rôle important dans les petits volumes. Cette thèse présente un modèle stochastique et spatial qui a été développé dans le but d’étudier les signaux calciques dans les prolongements fins astrocytaires. Ce travail a été réalisé en collaboration avec des expérimentateurs, qui nous ont fourni des données de microscopie électronique et à super-résolution. Ces données ont permis de valider le modèle. Les simulations du modèle suggèrent que (1) la diffusion moléculaire, fortement influencée par la concentration et la cinétique des buffers calciques endogènes et exogènes, (2) l'organisation spatiale intracellulaire des molécules, notamment le co-clustering des canaux calciques, (3) la géométrie du reticulum endoplasmique et sa localisation dans la cellule, (4) la géométrie cellulaire influencent fortement les signaux calciques et pourraient être responsables de leur grande diversité spatio-temporelle. Ces travaux contribuent à une meilleure compréhension du traitement de l’information par les astrocytes, un prérequis pour une meilleure compréhension de la communication entre les neurones et les astrocytes ainsi que de son influence sur le fonctionnement du cerveau. / Astrocytes are predominant glial cells in the central nervous system, which are essential for the formation of synapses, participate to the blood-brain barrier and maintain the metabolic, ionic and neurotransmitter homeostasis. Recently, astrocytes have emerged as key elements of information processing in the central nervous system. Astrocytes can contact neurons at synapses and modulate neuronal communication via the release of gliotransmitters and the uptake of neurotransmitters. The use of super-resolution microscopy and highly sensitive genetically encoded Ca2+ indicators (GECIs) has revealed a striking spatiotemporal diversity of Ca2+ signals in astrocytes. Most astrocytic signals occur in processes, which are the sites of neuron-astrocyte communication. Those processes are too fine to be resolved by conventional light microscopy so that super-resolution microscopy and computational modeling remain the only methodologies to study those compartments. The work presented in this thesis aims at investigating the effect of spatial properties (as e.g cellular geometry, molecular distributions and diffusion) on Ca2+ signals in those processes, which are deemed essential in such small volumes. Historically, Ca2+ signals were modeled with deterministic well-mixed approaches, which enabled the study of Ca2+ signals in astrocytic networks or whole-cell events. Those methods however ignore the stochasticity inherent to molecular interactions as well as diffusion effects, which both play important roles in small volumes. In this thesis, we present the spatially-extended stochastic model that we have developed in order to investigate Ca2+ signals in fine astrocytic processes. This work was performed in collaboration with experimentalists that performed electron as well as super-resolution microscopy. The model was validated against experimental data. Simulations of the model suggest that (1) molecular diffusion, strongly influenced by the concentration and kinetics of endogenous and exogenous buffers, (2) intracellular spatial organization of molecules, notably the co-clustering of Ca2+ channels, (3) ER geometry and localization within the cell, (4) cellular geometry strongly influence Ca2+ dynamics and can be responsible for the striking diversity of astrocytic Ca2+ signals. This work contributes to a better understanding of astrocyte Ca2+ signals, a prerequisite for understanding neuron-astrocyte communication and its influence on brain function.

In this thesis, we start with the description of the biophysical properties of the plasma membrane models upon signaling processess such as the increased cytoso- lic concentration of calcium ions, or posttranslational modifications of membrane proteins. Calcium signaling is characterized by a rapid increase of its cytosolic concentration. We identify calcium binding sites and characterize the binding in the plasma membrane models of increasing complexity from pure phospholipid bilayers, through cholesterol and peptide rich lipid membranes, to membranes ex- tracted from HEK293 cells. We use Time-Dependent Fluorescent Shift method, which provides direct information on hydration and mobility in defined regions of a lipid bilayer, accompanied with molecular dynamic (MD) simulations, which give molecular details of the studied interactions. The initial step of signaling mediated by PAG protein is its double palmi- toylation. We investigate changes of the biophysical properties of both the lipid membrane and the peptide itself upon the incorporation of the palmitoyls. Em- ploying all atom MD simulations, we study inter- and intramolecular interactions as well as changes in membrane hydration, thickness, or lipid ordering. The second part of the thesis, realized in a direct collaboration with a phar- macological...

Indiana University-Purdue University Indianapolis (IUPUI) / Glutamate clearance by astrocytes is an essential part of physiological excitatory neurotransmission. Failure to adapt or maintain low levels of glutamate in the central nervous system is associated with multiple acute and chronic neurodegenerative diseases. The primary excitatory amino acid transporters (EAATs) in human astrocytes are EAAT1 and EAAT2 (GLAST and GLT-1 respectively in rodents). While the inhibition of a ubiquitously-expressed serine/threonine protein kinase, the calcium/calmodulindependent kinase (CaMKII) results in diminished glutamate uptake in cultured primary rodent astrocytes, the molecular mechanism underlying this regulation is unknown. In order to delineate this mechanism, we use a heterologous expression model to explore CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of CaMKII and overexpression of a dominant-negative version of CaMKII (Asp136Asn) reduces [3H]-glutamate uptake by EAAT1, without altering EAAT2 mediated glutamate uptake. Surprisingly, overexpression of a constitutively active autophosphorylation mutant (Thr287Asp) to increase autonomous CaMKII activity and a mutant incapable of autophosphorylation (Thr287Val) had no effect on either EAAT1 or EAAT2 mediated glutamate uptake. Pulldown of FLAGtagged glutamate transporters suggests CaMKII does not interact with EAAT1 or EAAT2. SPOTS peptide arrays and recombinant GST-fusion proteins of the intracellular N- and C-termini of EAAT1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N-terminus. Introducing an Ala (a non-phospho mimetic) but not an Asp (phosphomimetic) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. In sum, this is the first report of a glutamate transporter being identified as a direct CaMKII substrate. These findings indicate that CaMKII signaling is a critical driver of homeostatic glutamate uptake by EAAT1. Aberrations in basal CaMKII activity disrupt glutamate uptake, which can perpetuate glutamate-mediated excitotoxicity and result in cellular death.

<p>    </p> <p>When mammalian eggs are fertilized, they undergo an egg-to-embryo transition during which different egg activation events take place. Egg activation events include the establishment of blocks to polyspermy, which prevent multiple sperm from fertilizing an egg. One of these blocks to polyspermy occurs at the level of the egg plasma membrane (the membrane block to polyspermy). Previous work in our lab provides evidence that the mammalian membrane block to polyspermy is mediated by sperm-induced calcium signaling and the egg’s actomyosin cytoskeleton (McAvey et al., 2002). This thesis research builds upon this foundation, testing hypotheses about two specific effector molecules, one involved in calcium signaling and one with the actin cytoskeleton, and also developing the use of an actin probe for live-cell imaging, with the goal of imaging actin dynamics in eggs undergoing fertilization. Specifically, we examined the calcium effector molecule Ca2+/Calmodulin-dependent-protein kinase IIg (<strong>CaMKII</strong>g), based on previous studies showing that CaMKII plays a role in the membrane block (Gardner et al., 2007) and that the g isoform of CaMKII is necessary and sufficient for eggs to complete meiosis (Backs et al., 2010). We tested the hypothesis that CaMKIIg would mediate the membrane block to polyspermy but found that egg activation driven by expression of a constitutively active form of CaMKIIg was not sufficient to establish the membrane block. Our studies of the actin cytoskeleton focused on the Arp2/3 complex as a candidate. We tested the hypothesis that Arp2/3, which mediates actin filament branching, was involved in membrane block establishment, building on the finding that disruption of actin with the drug cytochalasin D impairs the membrane block (McAvey et al., 2022). These studies used the Arp2/3 inhibitor CK666, predicting that we would see increased sperm incorporation in CK666-treated eggs. However, an assay of sperm incorporation over time indicated that Arp2/3 may not play a significant role in the membrane block to polyspermy, although follow-up studies will be beneficial. Lastly, the actin probe SiR- Actin was assessed for use on oocytes undergoing live-cell imaging during meiosis I and II. Oocytes were treated with differing concentrations of SiR-Actin and live cell imaged while maturing through meiosis I or completing meiosis II. Higher doses and longer exposure to SiR- Actin caused abnormalities in oocytes during meiosis I but not in eggs completing meiosis II. Together, this work sets the stage of a range of future studies into the mammalian membrane block to polyspermy. </p>

Cette thèse de doctorat englobe deux projets majeurs visant à étudier l'interaction entre les nanotubes à effet tunnel inter-péricytes (IP-TNT), le couplage neurovasculaire et la modulation des cellules gliales dans le contexte du glaucome. Le premier projet se concentre sur la caractérisation et l'importance fonctionnelle des IP-TNT dans la régulation du couplage neurovasculaire, tandis que le second projet explore le rôle des cellules gliales, en particulier S100Β, dans la modulation des réponses des péricytes pendant l'hypertension oculaire (HTO), un facteur de risque important pour le développement du glaucome. Dans le premier projet, nous avons étudié la présence et les implications fonctionnelles des IP-TNT dans l'unité neurovasculaire. Grâce à des techniques d'imagerie avancées et à des expériences d'imagerie en direct chez la souris, nous avons visualisé et caractérisé ces nanotubes à effet tunnel qui relient les péricytes voisins dans la rétine. Nous avons découvert que les IP-TNT jouent un rôle crucial en facilitant la communication intercellulaire et la signalisation calcique entre les péricytes. Ces nanotubes contribuent à la régulation du flux sanguin capillaire et au couplage neurovasculaire, assurant l'apport efficace d'oxygène et de nutriments aux neurones actifs. Nos résultats mettent en lumière les interactions cellulaires complexes au sein de l'unité neurovasculaire et élargissent notre compréhension des mécanismes qui sous-tendent le couplage neurovasculaire. Dans le second projet, nous nous sommes concentrés sur le rôle des cellules gliales, en particulier la protéine S100Β qui se lie au calcium, dans la modulation des réponses des péricytes au cours de l'HTO, une caractéristique pathologique clé du glaucome. Grâce à une combinaison d'expériences in vivo, d'analyses moléculaires et de techniques d'imagerie, nous avons étudié l'impact de la S100Β sur les niveaux de calcium des péricytes et sur le flux sanguin capillaire. Nous avons observé que la S100Β est régulée à la hausse dans les cellules gliales, y compris les cellules de Müller et les astrocytes, au cours de l'HTO. L'administration de la protéine recombinante exogène S100Β a exacerbé l'influx de calcium intra-péricyte et altéré le flux sanguin capillaire, tandis que le blocage de la fonction S100Β a amélioré les niveaux de calcium des péricytes et rétabli un flux sanguin basal. La neutralisation de la S100Β a également protégé les cellules ganglionnaires de la rétine de la mort induite par l'HTO. Ces résultats mettent en évidence le rôle critique des cellules gliales et de la S100Β dans les déficits du couplage neurovasculaire au cours du glaucome, et donnent un aperçu des cibles thérapeutiques potentielles pour préserver la santé et la fonction de la rétine. Collectivement, les résultats des deux projets contribuent à notre compréhension de l'interaction complexe entre les IP-TNT, le couplage neurovasculaire et la modulation des cellules gliales dans le contexte du glaucome. En élucidant le rôle des IP-TNT dans la régulation neurovasculaire et l'impact des cellules gliales, en particulier la S100Β, sur les réponses des péricytes, cette thèse fournit des informations précieuses sur les mécanismes sous-jacents de la pathogenèse du glaucome. Ces résultats peuvent ouvrir la voie au développement de stratégies thérapeutiques innovantes ciblant les IP-TNT et la modulation médiée par les cellules gliales afin de préserver la fonction rétinienne et de prévenir la perte de vision dans le glaucome et les maladies neurodégénératives associées / This PhD thesis encompasses two major projects aimed at investigating the interplay between interpericyte tunneling nanotubes (IP-TNTs), neurovascular coupling, and glial cell modulation in the context of glaucoma. The first project focuses on the characterization and functional significance of IP-TNTs in neurovascular coupling regulation, while the second project explores the role of glial cells, particularly S100Β, in modulating pericyte responses during ocular hypertension (OHT), an important risk factor for developing glaucoma. In the first project, we investigated the presence and functional implications of IP-TNTs in the neurovascular unit. Through advanced imaging techniques and live imaging experiments in mice, we visualized and characterized these tunneling nanotubes connecting neighboring pericytes in the retina. We found that IP-TNTs play a crucial role in facilitating intercellular communication and calcium signaling between pericytes. These nanotubes contribute to the regulation of capillary blood flow and neurovascular coupling, ensuring the efficient delivery of oxygen and nutrients to active neurons. Our findings shed light on the intricate cellular interactions within the neurovascular unit and expand our understanding of the mechanisms underlying neurovascular coupling. In the second project, we focused on the role of glial cells, specifically the calcium-binding protein S100Β, in modulating pericyte responses during OHT, a key pathological feature of glaucoma. Through a combination of in vivo experiments, molecular analyses, and imaging techniques, we investigated the impact of S100Β on pericyte calcium levels and capillary blood flow. We observed that S100Β is upregulated in glial cells, including Müller cells and astrocytes, during OHT. Administration of recombinant S100Β protein exacerbated intrapericyte calcium influx and impaired capillary blood flow, while blocking S100Β function improved pericyte calcium levels and restored normal blood flow. Notably, S100Β neutralization also protected retinal ganglion cells from OHT-induced death. These findings highlight the critical role of glial cells and S100Β in neurovascular coupling deficits during glaucoma, providing insights into potential therapeutic targets for preserving retinal health and function. Collectively, the results from both projects contribute to our understanding of the complex interplay between IP-TNTs, neurovascular coupling, and glial cell modulation in the context of glaucoma. By elucidating the role of IP-TNTs in neurovascular regulation and the impact of glial cells, particularly S100Β, on pericyte responses, this thesis provides valuable insights into the underlying mechanisms of glaucoma pathogenesis. These findings may pave the way for the development of innovative therapeutic strategies targeting IP-TNTs and glial cell-mediated modulation to preserve retinal function and prevent vision loss in glaucoma and related neurodegenerative diseases

<p dir="ltr">Membrane-dependent enzymes play crucial roles in cellular signaling by transducing extracellular signals into intracellular responses. Phospholipase Cepsilon (PLCe) and diacylglycerol kinase alpha (DGKa) are membrane-associated enzymes regulated by G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), controlling signaling pathways essential for numerous cellular processes. PLCe catalyzes the hydrolysis of phosphatidylinositol phosphates into inositol phosphates (IPX) and diacylglycerol (DAG), triggering calcium release from intracellular stores and activating protein kinase C (PKC)-dependent pathways. While PLCe is crucial for normal cardiovascular function, hyperactivation or sustained activation can lead to hypertrophy. Due to structural heterogeneity, previous studies focused on isolated regulatory domains or the catalytic core. In this work, I present the first cryo-EM reconstruction of the largest PLCe fragment to date in complex with an antigen-binding fragment (Fab). This structure reveals the domain architecture of the N-terminal regions of the lipase and defines an extended membrane-binding surface critical for maximal basal and G protein-dependent activity. These findings lay the groundwork for high-resolution structures of the full-length enzyme and its complexes with the small GTPase Rap1A. Additionally, I explored the role of mAKAP in the Rap1A–PLCe pathway, alongside the guanine nucleotide exchange factor (GEF) function of PLCe toward Rap1A. In parallel, cryo-EM studies of DGKa bound to a covalent inhibitor were initiated. DGKa reduces DAG, thereby limiting PKC activity, and its inhibition is emerging as a promising cancer immunotherapy target. We have established a protocol for structural studies of full-length DGKa, which will elucidate its structures in basal and inhibited states.</p>

<p dir="ltr">Wound healing is a complex biological process orchestrated by intricate cellular and biochemical interactions. This study leverages a multiscale modeling approach, integrating agent-based and ordinary differential equation (ODE) methods within CompuCell3D, to investigate wound detection and calcium signaling in juvenile zebrafish. Calcium as a ubiquitous secondary messenger plays a crucial role in translating wound stimuli into cellular responses. We focus on the initial phase of wound detection, a multi-step process beginning at the subcellular level with the release of Damage-Associated Molecular Patterns (DAMPs) and subsequent calcium signaling. We hypothesize that an ATP diffusion wave acts as the primary trigger, initiating a downstream calcium signaling cascade mediated by inositol triphosphate (IP3). Calcium and IP3 production and movement from the injured cells to healthy ones would then coordinate a tightly regulated wound response. To investigate this hypothesis, we adapted existing equations from a Drosophila wing disc injury model. We carefully modified them to accurately represent the zebrafish system in our in-silico setup, specifically focusing on relevant agonists. Model predictions were rigorously compared to the zebrafish’s experimental data to validate the computational approach. Our findings provide preliminary evidence suggesting that ATP diffusion through the interstitial spaces of injured tissue may be a potent agonist, triggering localized calcium release closely resembling experimental observations. This multiscale modeling framework offers a promising avenue for significant advancements in wound healing research. It has the potential to facilitate the development of novel therapeutic strategies and discoveries by enabling the integration of cell signaling pathways and tissue engineering.</p>

Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development. The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function. First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta. The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.

Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development. The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function. First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta. The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.

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