Etude de la dualité d AIF, dans la mort cellulaire programmée indépendante des caspases via son partenaire yH2AX et dans la phosphorylation oxydative via les complexes et supercomplexes mitochondriaux

L apoptosis inducing factor (AIF) est une flavoprotéine mitochondriale à deux visages, à travers sa fonction vitale dans la phoshorylation oxydative et sa fonction apoptotique lors de la mort cellulaire programmée (MCP). Suite à un stimulus de mort cellulaire, AIF est relarguée de la mitochondrie vers le noyau, et forme un complexe de dégradation de l ADN avec l histone H2AX et la DNase Cyclophiline A. Ce dégradosome est crucial pour l exécution de la MCP indépendante des caspases aussi appelée nécroptose. Ce travail de thèse détermine l implication de la forme activée d H2AX par phosphorylation sur la sérine 139, gH2AX dans le dégradosome lors de la nécroptose induite par l agent alkylant de l ADN, MNNG. La transduction de fibroblastes embryonnaires murins (MEF) H2AX-/- avec la forme non-phosphorylable ou phosphomimétique d H2AX pour la sérine 139, a permis de démontrer que gH2AX joue un rôle conformationnel dans l activation du dégradosome et l exécution de la nécroptose. De plus, nous avons démontré que les kinases, activées suite aux dommages de l ADN, ATM et DNA-PK contrôlent la nécroptose via l activation d H2AX. D autre part, nous avons étudié la fonction d AIF dans la mitochondrie en utilisant un modèle de MEF inductible pour la délétion d AIF. L analyse de l activité de la phosphorylation oxydative dans ce système in vitro inédit nous a permis de suivre les conséquences progressives de la perte d AIF. Ainsi, nous avons confirmé qu AIF est essentiel pour le fonctionnement de ce mécanisme énergétique mitochondrial. Et pour la première fois, le rôle vital de cette protéine mitochondriale n est pas seulement associé au complexe I mais aussi aux supercomplexes.Apoptosis inducing factor (AIF) is a mitochondrial protein with two faces: a vital role in mitochondrial oxidative phosphorylation and a death function in programmed cell death (PCD). On one hand, after cell death triggering, AIF is released from mitochondria and involved in a DNA degradation complex (degradosome) with histone H2AX and DNAse cyclophilin A. This complex is crucial in caspase-independent PCD mediated by DNA damage, named necroptosis. H2AX and its activated form gH2AX, phosphorylated on serine 139, had often been described in DNA repair. In this thesis work, we characterized this activated form in necroptosis induced by alkylating agent MNNG (N-methyl-N'-nitro-N-nitrosoguanidine). Transduction of H2AX KO embryonic murine fibrobasts (MEF), with mutant forms of H2AX for serine 139 - non phosphorylable or phosphomimetic - demonstrated that gH2AX have a conformational role for degradosome activation and MNNG-induced necroptosis execution. With pharmacological and cellular approaches, we also demonstrated that kinases usually involved in DNA damage response also controlled necroptosis via H2AX activation by serine 139 phosphorylation. On the other hand, by using an innovative in vitro model of inducible MEF for AIF deletion, and many techniques as native electrophoresis, in-gel activity, oxygraphy, we confirmed the implication of AIF in mitochondrial oxidative phosphorylation. For the first time, we also described the role of this mitochondrial flavoprotein, not only for complex I but also for mitochondrial supercomplexes formation.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Modulation of Protein Quality Control and Proteasome to Autophagy Switch in Immortalized Myoblasts from Duchenne Muscular Dystrophy Patients

The maintenance of proteome integrity is of primary importance in post-mitotic tissues such as muscle cells; thus, protein quality control mechanisms must be carefully regulated to ensure their optimal efficiency, a failure of these processes being associated with various muscular disorders. Duchenne muscular dystrophy (DMD) is one of the most common and severe forms of muscular dystrophies and is caused by mutations in the dystrophin gene. Protein quality control modulations have been diversely observed in degenerating muscles of patients suffering from DMD or in animal models of the disease. In this study, we investigated whether modulations of protein quality control mechanisms already pre-exist in undifferentiated myoblasts originating from DMD patients. We report for the first time that the absence of dystrophin in human myoblasts is associated with protein aggregation stress characterized by an increase of protein aggregates. This stress is combined with BAG1 to BAG3 switch, NFκB activation and up-regulation of BAG3/HSPB8 complexes that ensure preferential routing of misfolded/aggregated proteins to autophagy rather than to deficient 26S proteasome. In this context, restoration of pre-existing alterations of protein quality control processes might represent an alternative strategy for DMD therapies

Mitochondrial AIF Loss Causes Metabolic Reprogramming, Caspase-Independent Cell Death Blockade, Embryonic Lethality, and Perinatal Hydrocephalus

International audienceObjectives: Apoptosis-Inducing Factor (AIF) is a protein involved in mitochondrial electron transport chain assembly/stability and in programmed cell death. The relevant role of this protein is underlined by the fact that mutations altering mitochondrial AIF properties result in acute pediatric mitochondriopathies and tumor metastasis. By generating an original AIF-deficient mouse strain, the present study sought to analyze, in a single paradigm, the cellular and developmental metabolic consequences of AIF loss and the subsequent oxidative phosphorylation (OXPHOS) dysfunction.Methods: We developed a novel AIF-deficient mouse strain and assessed, by molecular and cell biology approaches, the cellular, embryonic, and adult mice phenotypic alterations. Additionally, we carried out ex vivo assays with primary and immortalized AIF knockout mouse embryonic fibroblasts (MEFs) to establish the cell death characteristics and the metabolic adaptive responses provoked by the mitochondrial electron transport chain (ETC) breakdown.Results: AIF deficiency destabilized mitochondrial ETC and provoked supercomplex disorganization, mitochondrial transmembrane potential loss, and high generation of mitochondrial reactive oxygen species (ROS). AIF-/Y MEFs counterbalanced these OXPHOS alterations by mitochondrial network reorganization and a metabolic reprogramming towards anaerobic glycolysis illustrated by the AMPK phosphorylation at Thr172, the overexpression of the glucose assimilation transporter GLUT-4, the subsequent enhancement of glucose uptake, and the anaerobic lactate generation. A late phenotype was characterized by the activation of P53/P21-mediated senescence. Interestingly, about 2% of AIF-/Y MEFs diminished both mitochondrial mass and ROS levels and spontaneously proliferated. These cycling AIF-/Y MEFs were resistant to caspase-independent cell death inducers. The AIF-deficient mouse strain was embryonic lethal between E11.5 and E13.5 with energy loss, proliferation arrest, and increased apoptotic levels. Contrary to AIF-/Y MEFs, the AIF KO embryos were unable to reprogram their metabolism towards anaerobic glycolysis. Heterozygous AIF-/+ females displayed a progressive bone marrow, thymus, and spleen cellular loss. In addition, about 10% of AIF-/+ females developed perinatal hydrocephaly characterized by brain development impairment, meningeal fibrosis, and medullar hemorrhages; those mice died around 5 weeks of age. AIF-/+ with hydrocephaly exhibited loss of ciliated epithelium in the ependymal layer. This phenotype seemed triggered by the ROS excess. Accordingly, it was possible to diminish the occurrence of hydrocephalus AIF-/+ females by supplying dams and newborns with an antioxidant in drinking water.Conclusion: In a single knockout model and at three different levels (cell, embryo, and adult mice) we demonstrated that, by controlling the mitochondrial OXPHOS/metabolism, AIF is a key factor regulating cell differentiation and fate. Additionally, by shedding new light on the pathological consequences of mitochondrial OXPHOS dysfunction, our new findings pave the way for novel pharmacological strategies

AIF loss deregulates hematopoiesis and reveals different adaptive metabolic responses in bone marrow cells and thymocytes

International audienceMitochondrial metabolism is a tightly regulated process that plays a central role throughout the lifespan of hematopoietic cells. Herein, we analyze the consequences of the mitochondrial oxidative phosphorylation (OXPHOS)/metabolism disorder associated with the cell-specific hematopoietic ablation of apoptosis-inducing factor (AIF). AIF-null (AIF-/Y ) mice developed pancytopenia that was associated with hypocellular bone marrow (BM) and thymus atrophy. Although myeloid cells were relatively spared, the B-cell and erythroid lineages were altered with increased frequencies of precursor B cells, pro-erythroblasts I, and basophilic erythroblasts II. T-cell populations were dramatically reduced with a thymopoiesis blockade at a double negative (DN) immature state, with DN1 accumulation and delayed DN2/DN3 and DN3/DN4 transitions. In BM cells, the OXPHOS/metabolism dysfunction provoked by the loss of AIF was counterbalanced by the augmentation of the mitochondrial biogenesis and a shift towards anaerobic glycolysis. Nevertheless, in a caspase-independent process, the resulting excess of reactive oxygen species compromised the viability of the hematopoietic stem cells (HSC) and progenitors. This led to the progressive exhaustion of the HSC pool, a reduced capacity of the BM progenitors to differentiate into colonies in methylcellulose assays, and the absence of cell-autonomous HSC repopulating potential in vivo. In contrast to BM cells, AIF-/Y thymocytes compensated for the OXPHOS breakdown by enhancing fatty acid β-oxidation. By over-expressing CPT1, ACADL and PDK4, three key enzymes facilitating fatty acid β-oxidation (e.g., palmitic acid assimilation), the AIF-/Y thymocytes retrieved the ATP levels of the AIF +/Y cells. As a consequence, it was possible to significantly reestablish AIF-/Y thymopoiesis in vivo by feeding the animals with a high-fat diet complemented with an antioxidant. Overall, our data reveal that the mitochondrial signals regulated by AIF are critical to hematopoietic decision-making. Emerging as a link between mitochondrial metabolism and hematopoietic cell fate, AIF-mediated OXPHOS regulation represents a target for the development of new immunomodulatory therapeutics