Background: The volvocine lineage, containing unicellular Chlamydomonas reinhardtii and differentiated multicellular Volvox carteri, is a powerful model for comparative studies aiming at understanding emergence of multicellularity. Tetrabaena socialis is the simplest multicellular volvocine alga and belongs to the family Tetrabaenaceae that is sister to more complex multicellular volvocine families, Goniaceae and Volvocaceae. Thus, T. socialis is a key species to elucidate the initial steps in the evolution of multicellularity. In the asexual life cycle of C. reinhardtii and multicellular volvocine species, reproductive cells form daughter cells/colonies by multiple fission. In embryogenesis of the multicellular species, daughter protoplasts are connected to one another by cytoplasmic bridges formed by incomplete cytokinesis during multiple fission. These bridges are important for arranging the daughter protoplasts in appropriate positions such that species-specific integrated multicellular individuals are shaped. Detailed comparative studies of cytokinesis between unicellular and simple multicellular volvocine species will help to elucidate the emergence of multicellularity from the unicellular ancestor. However, the cytokinesis-related genes between closely related unicellular and multicellular species have not been subjected to a comparative analysis. Results: Here we focused on dynamin-related protein 1 (DRP1), which is known for its role in cytokinesis in land plants. Immunofluorescence microscopy using an antibody against T. socialis DRP1 revealed that volvocine DRP1 was localized to division planes during cytokinesis in unicellular C. reinhardtii and two simple multicellular volvocine species T. socialis and Gonium pectorale. DRP1 signals were mainly observed in the newly formed division planes of unicellular C. reinhardtii during multiple fission, whereas in multicellular T. socialis and G. pectorale, DRP1 signals were observed in all division planes during embryogenesis. Conclusions: These results indicate that the molecular mechanisms of cytokinesis may be different in unicellular and multicellular volvocine algae. The localization of DRP1 during multiple fission might have been modified in the common ancestor of multicellular volvocine algae. This modification may have been essential for the re-orientation of cells and shaping colonies during the emergence of multicellularity in this lineage.

Eukaryotic cells are unique in their ability to form complex multicellular organisms giving rise to distinct physiological systems. However, the ability for such complexity to evolve likely stems from an early event in which endosymbiosis of an aerobic prokaryote by a eukaryotic precursor gave rise to the eukaryotic organelle we now know as mitochondria. Mitochondria are colloquially known as the “power house” of the cell due to their ability to produce ATP through oxidative phosphorylation, but perform numerous other vital functions within the cell including sequestration of cytosolic Ca2+, production and sequestration of reactive oxygen species (ROS), and initiation of various forms of cell death. Mitochondria are especially important in neurons given their high demand for ATP and the importance of Ca2+ signaling in neuron excitability and development. Neurons are highly compartmentalized and plastic cells requiring the ability to control energy supply and Ca2+ signaling locally within given specialized structures such as dendritic spines or synaptic boutons. Therefore, mitochondria must be able to localize to particular sub-cellular locales and respond functionally to signaling occurring in that environment. Mitochondrial transport and function are heavily dependent upon the ability of mitochondria to undergo opposing and reversible fission and fusion events. Mitochondrial fission and fusion are themselves regulated by GTPase enzymes which physically catalyze constriction and fusion of the mitochondrial membranes. Mutations in mitochondrial fission and fusion enzymes specifically cause neurological disease in humans and recent work has illustrated the necessity of a proper balance of mitochondrial fission in neuron development, survival, and plasticity. Despite recognizing the importance of mitochondrial fission and fusion in neuron survival, development, and function we lack a concrete understanding of how changes in the equilibrium of fission and fusion impact these processes in vivo. In this thesis we investigate how promoting or inhibiting mitochondrial fission, through phosphoregulation of the mitochondrial fission enzyme Dynamin related protein 1 (Drp1) at mitochondria, impacts neuron survival and memory in vivo. We find that inhibiting phosphorylation of Drp1 at Serine 656 (S656) at the mitochondria, through deletion of a mitochondrial targeted A kinase anchoring protein (AKAP) known as AKAP1 in mice, increases cerebral infarct volume following transient occlusion of the mid-cerebral artery. Oppositely, promoting phosphorylation of Drp1-S656 at the mitochondria, through deletion of the PP2A regulatory subunit Bβ2 which localizes the PP2A heterotrimer to mitochondria, decreases cerebral infarct volume following occlusion of the mid-cerebral artery. Mechanistic in vitro studies in primary neurons reveal these effects are dependent upon the phosphorylation state of Drp1-S656 and likely due to altered mitochondrial respiratory capacity, ROS production, and Ca2+ homeostasis. Interestingly, we also observe improved hippocampal dependent memory in mice in which AKAP1 has been deleted which also appears dependent upon the phosphorylation state of Drp1-S656 and Ca2+ homeostasis. Ultimately, these findings provide insight into how phosphoregulation of Drp1 at the mitochondria alters neuron survival and function through shifting the mitochondrial fission/fusion equilibrium and consequently mitochondrial function.

Mitochondria are important cellular organelles whose functions include generation of ATP, sequestration and release of pro-apoptotic molecules and calcium buffering. Mitochondria function is tightly linked to organelle morphology, which exits in a dynamic spectrum between a highly interconnected/fused mitochondria network to a punctate/fragmented scattering of individual mitochondrion. A family of large GTPase enzymes modulates this spectrum, with fusion catalyzed through the actions of mitofusin 1 and 2 (Mfn1/2) on the outer mitochondria membrane (OMM) and optic atrophy 1 (Opa1) causing fusion of the inner mitochondria membrane (IMM). On the other end of the spectrum, fragmentation is catalyzed through the actions of dynamin-related protein 1 (Drp1). Drp1 is recruited from the cytosol to binding partners at the OMM, organizes into concentric spiral rings, undergoes GTP hydrolysis to constrict the ring and pinches mitochondrion in two. While fragmentation is achieved through the action of only one GTPase enzyme, the mechanisms behind the complex regulation of Drp1 remain relatively obscure. In order to expand upon known Drp1 regulatory mechanisms, an examination of how both Drp1 splicing and Drp1 recruitment to the OMM contributes to protein regulation is necessary. Drp1 contains three alternatively spliced exons, resulting in the potential generation of eight protein isoforms. Each of these isoforms is capable of inducing mitochondrial fragmentation, however one exon arrangement (termed Drp1-x01) can also bind to microtubules within the cell. Characterization of the Drp1-x01 isoform at both the RNA and protein level indicate an important, yet incompletely characterized, role in immune system biology. Drp1 is capable of interacting with several proteins localized at the OMM. Among these, mitochondria fission factor (Mff) has been implicated in the formation of Drp1 spirals and the eventual fragmentation process. Mff contains four alternatively spliced exons as well as several phosphorylation sites identified through nonbiased phosphoproteomic screens. Inclusion of alternative exons to the Mff structure decreases its ability to recruit Drp1 from the cytosol, while phosphomimetic substitutions to conserved serine residues enhances the Drp1::Mff interaction. Taken together, this suggests that regulation of mitochondrial fragmentation occurs at the pretranslational (alternative splicing) and the posttranslational (phosphorylation) level is critical for maintaining the complex, yet essential, balance between mitochondrial fission and fragmentation.

Alzheimer’s disease (AD) is characterized pathologically by proteinaceous aggregates composed primarily of amyloid \(\beta (A \beta)\) and tau. Diseases characterized by abnormal deposition of tau are collectively termed “tauopathies.” \(A \beta\) acts upstream of tau in the AD pathogenesis pathway, but tau expression is required for the neurodegenerative effects of \(A \beta\). Mitochondrial abnormalities have been documented in Alzheimer’s disease and related tauopathies, but the causal relationship between mitochondrial changes and neurodegeneration, as well as specific mechanisms promoting mitochondrial dysfunction, are unclear. Mitochondrial morphology is regulated by fission and fusion events within and between individual mitochondria, and misregulation of this process has been observed in several neurodegenerative diseases. The contribution of mitochondrial dynamics to the pathogenesis of Alzheimer’s disease and tauopathy has not yet been determined. We have found that expression of tau promotes elongation of mitochondria in Drosophila and vertebrate neurons. Elongation is followed by mitochondrial dysfunction, aberrant cell cycle reactivation, and cell death, which can be rescued in vivo by genetically restoring the proper balance of mitochondrial fission and fusion. Tau induces mitochondrial elongation by inhibiting mitochondrial localization of DRP1, the primary effector of fission. We have previously demonstrated that direct tau-mediated stabilization of filamentous (F)-actin is critical for neurotoxicity. Here we show that actin stabilization is responsible for the mislocalization of DRP1 following tau expression. Additionally, we identify regulatory roles for F-actin and myosin II in DRP1 localization. Similarly to overexpression of human tau, loss of endogenous Drosophila tau (dtau) induces mitochondrial elongation, but through distinct mechanisms. Expression of human \(A \beta\)in Drosophila induces mitochondrial fragmentation and neuronal toxicity, which are reversed by depletion of dtau. Together, we demonstrate that human disease-associated tau induces neurotoxicity through disruption of mitochondrial dynamics, which can be mediated by enhanced actin stabilization. We also observe a novel role for dtau in the regulation of mitochondrial dynamics, a function critical to the ability of endogenous tau to mediate the effects of \(A \beta\). These findings offer new insights into the contribution of mitochondrial dysfunction to AD and tauopathy, and highlight the emerging role of mitochondrial dynamics in the pathogenesis of neurodegenerative disease.

Chapter 1: Molecular Mechanism of Mitochondrial Fragmentation and Glucose Metabolism under Disturbed Flow in Endothelial Cells: Focus on the Role of Dynamin-Related Protein 1. The luminal surface of the endothelium is continually exposed to dynamic blood flow patterns that is known to alter immunometabolic phenotypes of the endothelial cells (ECs). Recent literature reported that inhibition of the metabolic reprogramming to glycolysis or enhancement of oxidative phosphorylation (OXPHOS) is considered as an effective strategy to prevent EC proinflammatory activation and eventually the progression of vascular diseases. Endothelial mitochondria are highly dynamic organelles playing versatile roles in maintaining endothelial cell homeostasis working as bioenergetic, biosynthetic, and signaling organelles. The balance between fusion and fission processes modulates mitochondrial network, which is essential for maintaining mitochondrial homeostasis. Disruption of the orchestrated balance, especially toward excessive fission resulting in fragmented and dysfunctional mitochondria, has been shown to be associated with atheroprone phenotypes of ECs. However, there is a key knowledge gap with respect to morphology of EC mitochondria under different flow conditions and its role on EC immunometabolic phenotypes.In chapter 1, the purpose of this study was to investigate the effect of different flow patterns on mitochondrial morphology in ECs and its implication in immunometabolic endothelial phenotype. The overarching hypothesis of the Chapter 1 was that disturbed flow (DF) will increase mitochondrial fragmentation, which will facilitate glycolysis and inflammatory activation in ECs. In the study, mitochondrial morphology was analyzed in ECs at multiple segments of the aorta and arteries in EC-specific photo-activatable mitochondria (EC-PhAM) mice. Increased mitochondrial fragmentation was observed at atheroprone regions (e.g., lesser curvature of the aortic arch, LC) with increased dynamin-related protein 1 (Drp1) activity, compared with the atheroprotective regions (e.g., thoracic aorta, TA). The atheroprone regions also showed a higher level of endothelial activation and glycolysis. Carotid artery partial ligation surgery, as a surgical model of DF, significantly induced mitochondrial fragmentation with elevated Drp1 activity and increased EC activation. in vitro experiments recapitulated in vivo observations. Inhibition of Drp1 activity by mdivi-1 attenuated the DF-induced atheroprone EC phenotypes, showing the close relationship between mitochondrial morphology and atheroprone phenotypes of ECs. As for the molecular mechanism, hypoxia-inducible factor 1 α (HIF-1α) stabilization and its nuclear translocation was significantly increased under DF, which was attenuated by mdivi-1 treatment. Mitochondrial reactive oxygen species (mtROS) and succinate, which are known to reduce prolyl hydroxylase domain 2 (PHD2) activity thereby increasing HIF-1α stabilization, were significantly elevated under DF, but those were attenuated by mdivi-1 treatment. Finally, a 7-week voluntary wheel-running exercise training significantly decreased mitochondrial fragmentation with a down-regulation of VCAM-1 expression at the LC. In conclusion, our data suggest that DF induces mitochondrial fragmentation with increased Drp1 activity, which is associated with an atheroprone EC phenotype. In addition, regular practice of aerobic exercise reduces mitochondrial fragmentation and prevents ECs from an atheroprone endothelial phenotype at the atheroprone regions. Chapter 2: Molecular Mechanisms for Unidirectional Flow (UF)/Exercise-Induced improvement of Mitochondrial Integrity: Focus on phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) /PARKIN-Dependent Mitochondrial Autophagy (Mitophagy) Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) is an essential molecule in the mitophagy process and known to act as a cytoprotective protein involved in several cellular mechanisms in mammalian cells. It has been documented that the loss of PINK1 expression in mice and various cell types enhance susceptibility to stress-induced cell damage, while the overexpression of PINK1 significantly attenuates stress-induced mitochondrial and cellular dysfunction.In chapter 2, the purpose of this study was to investigate PINK1 expression and its subcellular localization under an exercise-mimic laminar shear stress (LSS) condition in human primary endothelial cells and in exercizing mice, and its implications on endothelial homeostasis and cardiovascular disease (CVD) prevention. The overarching hypothesis of the Chapter 2 was that unidirectional flow (UF) will increase cytosolic PINK1 expression through which UF-preconditioned ECs will be more protective against an accumulation of dysfunctional mitochondria via a greater mitophagy induction. In this study, we measured the full-length PINK1 (FL-PINK1) mRNA and protein expression levels in ECs under unidirectional laminar shear stress (LSS). LSS significantly elevated both FL-PINK1 mRNA and protein expressions in ECs. Mitochondrial fractionation assays showed a decrease in FL-PINK1 accumulation in the mitochondria with an increase in the cytosolic FL-PINK1 level under LSS. Confocal microscopic analysis confirmed these subcellular localization patterns suggesting downregulation of mitophagy induction. Indeed, mitophagy flux was decreased under LSS, determined by a mtKeima probe. Mitochondrial morphometric analysis and mitochondrial membrane potential determined by tetraethylbenzimidazolylcarbocyanine iodide (JC-1) showed mitochondrial elongation and increased mitochondrial membrane potential under LSS respectively, suggesting that an elevation of cytosolic PINK1 is not related to an immediate induction of mitophagy. However, increased cytosolic PINK1 elevated mitophagic sensitivity toward dysfunctional mitochondria in pathological conditions. Preconditioned ECs with LSS showed lower mtDNA lesions under angiotensin II stimulation. Moreover, LSS-preconditioned ECs showed rapid Parkin recruitment and mitophagy induction in response to mitochondrial toxin (i.e., carbonyl cyanide chlorophenylhydrazone, CCCP) treatment compared to the control. We measured PINK1 expression at ECs of the thoracic aorta in exercised mice, a physiological LSS-enhanced model, which was significantly elevated compared to sedentary animals. In addition, exercise-preconditioned mice were more protective to angiotensin II-induced mtDNA lesion formation in the mouse abdominal aorta than sedentary mice, suggesting a potential protective mechanism of exercise in a PINK1-dependent manner. In conclusion, LSS increases a cytosolic pool of FL-PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria or activate other cytoprotective mechanisms in ECs. Our data suggest that exercise may support mitochondrial homeostasis in vascular ECs by enhancing PINK1-dependent cell protection mechanisms. / Kinesiology

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La maladie de Parkinson (MP) est une affection neurodégénérative fréquente d’étiologie inconnue, touchant environ 5% de la population mondiale après 80 ans. Environ 10% des cas correspondent à des formes familiales à transmission mendélienne. Pendant longtemps, un dysfonctionnement mitochondrial a été soupçonné jouer un rôle dans la physiopathologie de la MP. Cette possibilité a été récemment corroborée par des découvertes majeures réalisées dans le cadre des formes autosomiques récessives. Parkine et PINK1, les produits de deux gènes associés à ces formes familiales, participent au sein d’une même voie moléculaire au contrôle de la qualité mitochondriale, par la régulation du transport, de la dynamique, de la biogenèse et de la clairance de ces organites.L’objectif de ce travail a été d’élucider certains des mécanismes moléculaires sous-jacents à la régulation de l’homéostasie mitochondriale par Parkine et PINK1. Nous avons utilisé un ensemble d’approches de biologie moléculaire et cellulaire, de biochimie et de microscopie confocale, afin d’identifier et de caractériser des interacteurs moléculaires de Parkine et PINK1 à la membrane mitochondriale externe (MME).Dans la première partie de ce travail, nous avons découvert que la Parkine et PINK1 s’associent sur la MME de mitochondries dysfonctionnelles à proximité de la translocase de la MME (TOM), un complexe dédié à l’import de la grande majorité des protéines mitochondriales. Nous avons montré que ces interactions protéiques jouent un rôle clé dans l’activation du programme de dégradation mitochondriale régulé par la voie PINK1/Parkine. Nous avons également observé que la GTPase de type dynamine Drp1, impliquée dans la fission mitochondriale, est recrutée au niveau de mitochondries endommagées à proximité de Parkine et PINK1 ; ainsi, les processus de fission et de dégradation mitochondriales pourraient être spatialement coordonnés. Dans la deuxième partie de ce projet, nous avons caractérisé l’interaction fonctionnelle entre la Parkine et l’enzyme neuroprotectrice multifonctionnelle de la matrice mitochondriale, 17B-hydroxystéroïde déshydrogénase de type 10 (HSD17B10), dont les taux s’étaient révélés être diminués chez la souris déficiente en Parkine. Nous avons mis en évidence un effet protecteur d’HSD17B10 vis-à-vis de la mitochondrie qui était indépendant de son activité catalytique. Nous avons de plus montré que la Parkine interagit directement avec HSD17B10 à proximité de la machinerie TOM et qu’elle régule positivement l’abondance mitochondriale de cette protéine ; cela suggère qu’elle pourrait promouvoir son import.Dans l’ensemble, ces résultats approfondissent notre connaissance des mécanismes moléculaires mis en jeu par la Parkine et PINK1 dans le contrôle de la qualité mitochondriale, élargissant ainsi notre compréhension de leur rôle dans la physiopathologie des formes autosomiques récessive de MP. / Parkinson’s disease (PD) is a common neurodegenerative disorder of unknown etiology, affecting nearly 5% of the world population over the age of 80. Nearly 10% of PD cases are familial forms with Mendelian inheritance pattern. Mitochondrial dysfunction has long been suspected to play a role in the physiopathology of sporadic PD. This possibility has been recently corroborated by major discoveries in the field of autosomal recessive PD. Parkin and PINK1, the products of two genes associated with these forms, participate in a common molecular pathway focused on maintenance of mitochondrial quality, with roles in mitochondrial transport, dynamics, biogenesis and clearance.The aim of this work was to elucidate some of the molecular mechanisms underlying the regulation of mitochondrial homeostasis by Parkin and PINK1. We used a combination of approaches in molecular and cell biology, biochemistry and confocal microscopy to identify and characterize molecular interactors of Parkin and PINK1 on the outer mitochondrial membrane (OMM).In the first part of my project, we discovered that Parkin and PINK1 associate on dysfunctional mitochondria in proximity of the translocase of the OMM (TOM), a complex devoted to the mitochondrial import of the vast majority of the mitochondrial proteins. We provided evidence that these associations play a key role in activation of the mitochondrial degradation program mediated by the PINK1/Parkin pathway. We also observed that the dynamin-related GTPase Drp1, involved in mitochondrial fission is recruited to defective mitochondria in proximity of Parkin and PINK1, suggesting that mitochondrial fission occurs at sites where mitochondrial clearance is initiated.In the second part of my project, we characterized the functional interaction between Parkin and the multifunctional neuroprotective mitochondrial matrix enzyme 17B-hydroxysteroid dehydrogenase type 10 (HSD17B10), previously found by the team to be altered in abundance in Parkin-deficient mice. We demonstrated that HSD17B10 exerts a mitochondrion-protective function independent of its enzymatic activity. In addition, we provided evidence that Parkin directly interacts with HSD17B10 at the TOM machinery and that it positively regulates its mitochondrial levels, possibly through the regulation of its mitochondrial import.Altogether, these results provide novel insights into the molecular mechanisms by which Parkin and PINK1 control mitochondrial quality, and deepen our understanding of the role of these proteins in the physiopathology of autosomal recessive PD.

Les lymphomes non hodgkiniens (LNH) regroupent un panel hétérogène de pathologies originaires de cellules lymphatiques. Parmi les LNH à cellules B matures, la leucémie lymphoïde chronique (LLC) constitue la forme de leucémie de l’adulte la plus fréquente en Occident. La physiopathologie des LNH à cellules B matures est marquée par l’inhibition des mécanismes de la mort cellulaire, notamment via la surexpression de la protéine MCL-1. Une première partie de ce travail de thèse a été de déterminer quelles pouvaient être les enzymes de déubiquitination (DUBs) impliquées dans la survie des LNH à cellules B matures et la stabilisation de MCL-1. Notre étude a permis d’identifier la DUB USP14, qui est liée au système ubiquitine-protéasome, comme capable de réguler MCL-1 et la survie cellulaire. Nos travaux montrent également pour la première fois que l’activité DUB des cellules, ainsi que l’activité d’USP14, sont directement régulées par la signalisation du BCR via l'activité de la tyrosine kinase SYK. Le FTY720, un analogue de la sphingosine utilisé comme immunosuppresseur dans la sclérose en plaques, a montré un effet cytotoxique dans des hémopathies malignes sans toutefois que son mécanisme d’action soit clairement expliqué. Une deuxième partie de ce travail de thèse a été de caractériser la mort induite par le FTY720. Notre étude montre que la caractérisation de la morphologie cellulaire et des marqueurs induits par la mort due au FTY720 dans les LLC correspond en fait à une nécrose cellulaire programmée indépendante de RIPK1, mais dépendante d'une enzyme régulatrice de la fission mitochondriale, DRP1. / Non-Hodgkin lymphoma (NHL) include a diverse range of pathologies originate from the lymphatic cells. Among the mature B-cell NHL, chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The pathophysiology of mature B-cell NHL is marked by the inhibition of cell death mechanisms, particularly through the overexpression of MCL-1 protein. The first part of this thesis was to determine which deubiquitinating enzymes (DUBs) are involved in the survival of mature B-cell NHL and in the stabilization of MCL-1. Our study identified the DUB USP14, which is linked to the ubiquitin-proteasome system, as able to regulate MCL-1 and cell survival. Our work also shows for the first time that the DUB activity of the cells and the activity of USP14 are directly regulated by BCR signaling through the activity of the SYK tyrosine kinase. FTY720, a sphingosine analog used as an immunosuppressive drug in multiple sclerosis, showed a cytotoxic effect in hematological malignancies but its mechanism of action is not well understood. A second part of this thesis was to characterize the death induced by FTY720. Our study shows that the characterization of the cellular morphology and markers induced by death due to FTY720 in the LLC corresponds in fact to a programmed RIPK1-independent necrosis cell death, but dependent on DRP1, a regulatory enzyme of the mitochondrial fission.

Les neuropathies à anticorps anti-MAG sont secondaires à une gammapathie monoclonale IgM dirigée contre la MAG des gaines de myéline des nerfs périphériques. Le traitement est celui de l’hémopathie sous‐jacente. Même si les thérapeutiques sont de plus en plus efficaces, les hémopathies restent le plus souvent incurables. Le rituximab est couramment utilisé dans le traitement des neuropathies à anticorps anti‐MAG, mais son efficacité n’a pas pu être clairement démontrée dans deux études contrôlées. Le FTY720 ou fingolimod est un sphingolipide, analogue de la sphingosine, qui inhibe les récepteurs de la sphingosine-1-phosphate (S1P). Il est utilisé comme immunosuppresseur dans la Sclérose en Plaques. Des études ont également rapporté un effet cytotoxique du FTY720 dans des hémopathies sans toutefois clairement expliquer son mécanisme d’action. L’objectif de ce travail est d’élucider les mécanismes moléculaires de l’effet cytotoxique du FTY720 dans un modèle d’hémopathie B, la leucémie lymphoïde chronique (LLC). Des cellules leucémiques primaires de LLC et une lignée cellulaire MEC1 ont été utilisées comme modèle expérimental in vitro. Le FTY720, comme la sphingosine, entraîne une cytotoxicité dose‐dépendante dans la LLC. Cet effet, médié par la forme non phosphorylée de FTY720, est indépendant des récepteurs au S1P. Le FTY720 induit l’expression de marqueurs d’apoptose: exposition de la phosphaJdylsérine, clivage de PARP et de caspase 3. Cependant sa toxicité apparaît indépendante des caspases. La lipidation accrue de LC3 et la formation d’autophagolysosomes indiquent que le FTY720 augmente également le flux autophagique. Cependant, des inhibiteurs de l’autophagie ne permettent pas de bloquer la mort cellulaire induite par le FTY720, suggérant que l’autophagie a ici un rôle protecteur vis à vis de la toxicité du FTY720. Plusieurs éléments permettent de conclure que le FTY720 est responsable d’une nécrose cellulaire : aspect morphologique de nécrose en microscopie électronique, perméabilisation membranaire précoce avec relocalisation cytoplasmique de HMGB1, libération extracellulaire de LDH, perméabilisation de la membrane lysosomale associée à une activation des cathepsines. Au niveau moléculaire, l’action du FTY720 n’est pas bloquée par la nécrostatine 1, indiquant que la nécrose induite par le FTY720 est indépendante de RIPK1 (receptor interacJng protein 1), une kinase clef des voies extrinsèques de nécrose cellulaire programmée. Par contre, nos travaux ont établi l’implication de DRP1 (dynamin related protein), une enzyme régulatrice de la fission mitochondriale, dans le processus de nécrose induite par le FTY720. En plus d’une relocalisation précoce de DRP1 à la mitochondrie accompagnée d’une augmentation de sa phosphorylation sur des sites régulateurs de son activité, nos expériences montrent que la suppression de son expression par interférence à ARN dans les cellules leucémiques réduit fortement la mort cellulaire induite par le FTY720. Le FTY720 est donc responsable dans la LLC d’une nécrose cellulaire programmée dépendante de DRP1. Nos résultats illustrent l’implication des sphingolipides dans la régulation de la survie cellulaire et dans les voies de nécrose programmée. Le FTY720 a un mode d’action original différent de l’apoptose induite par les chimiothérapies classiques. Le FTY720 pourrait donc être une alternative thérapeutique dans les néoplasies B résistantes aux chimiothérapies usuelles et dans certaines manifestations auto‐immunes des hémopathies comme les neuropathies à anticorps anti‐MAG. / Fingolimod (FTY720) is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of haematological malignancies. Previous studies have indicated a role for FTY720 in inducing autophagy and caspase-independent cell death in cancer cells through incompletely characterized molecular mechanisms. Our study thus aims at a beeer understanding of the way of action of FTY720. In chronic lymphocytic leukaemia (CLL) cells, FTY720 induced cell death with typical features of apoptosis, including phosphatidylserine exposure and caspase-3 activation, and features of autophagy, including LC3 conversion, autophagolysosome formation and lysosomal cathepsins activation. However, neither caspase nor autophagy blockade prevented the cytotoxic effect of FTY720, suggesting another mechanism of cell death. Using electron and fluorescence microscopy, flow cytometry and biochemical analyses, we found that FTY720 treatment increased a fraction of annexin V-/7-AAD+ cells both in primary and transformed leukemic cells and induced morphological changes representative of necrosis, including oncosis, mitochondrial and plasma membrane alteration. FTY720 treatment resulted in increased plasma membrane permeability as shown by the extracellular translocation of the nuclear high mobility group box 1 (HMGB1) protein and by the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH). Interestingly, cell death induced by FTY720 was not prevented by pharmacological inhibition of RIPK1 and PP2A. In contrast, FTY720--‐induced necrosis was accompanied by an early relocation to the mitochondria of Dynamin Related Protein 1, DRP1. Importantly, FTY720 stimulation led to ma tior changes in the phosphorylation of serine residues associated with the mitochondrial fission activity of DRP1. Finally, siRNA--‐mediated knockdown of DRP1 significantly reduced necrotic cell death induced by FTY720. In this study, we thus demonstrate that in leukemic cells the cytotoxic effect of the immunosuppressive drug Fingolimod involves a DRP1--‐dependent regulated necrosis. These observations are important in line of the future development of Fingolimod as a new therapeutic agent in haematological malignancies.