The upregulation of apoptosis is a hallmark of several neurodegenerative disorders including ischemic stroke. In neurons, as in other cell types, Bax and tBid are critical regulators of the intrinsic pathway upstream of mitochondrial outer membrane permeabilization (MOMP) and caspase activation. The characterization of the molecular events that occur during the early stages is therefore extremely important from a therapeutic standpoint. Here I show that two independent genetic pilot screens looking for novel regulators of Bax activation identified a common hit in the E3 ubiquitin ligase Trim17. Knockdown of Trim17 was found to protect against tBid-induced death in primary cortical neurons and allowed for the maintenance of mitochondrial function and oxidative phosphorylation under this apoptotic stress. The RING-domain of Trim17 was found to interact with Opa1 in mouse brain extracts. Furthermore, Opa1 co-immunoprecipitated with exogenously expressed full-length Trim17 from HEK293 cells. Knockdown of Trim17 in neurons increased Opa1 protein levels under steady-state conditions. These results suggest that Trim17 regulates Bax-dependent apoptosis in neurons via the modulation of Opa1 levels.

The upregulation of apoptosis is a hallmark of several neurodegenerative disorders including ischemic stroke. In neurons, as in other cell types, Bax and tBid are critical regulators of the intrinsic pathway upstream of mitochondrial outer membrane permeabilization (MOMP) and caspase activation. The characterization of the molecular events that occur during the early stages is therefore extremely important from a therapeutic standpoint. Here I show that two independent genetic pilot screens looking for novel regulators of Bax activation identified a common hit in the E3 ubiquitin ligase Trim17. Knockdown of Trim17 was found to protect against tBid-induced death in primary cortical neurons and allowed for the maintenance of mitochondrial function and oxidative phosphorylation under this apoptotic stress. The RING-domain of Trim17 was found to interact with Opa1 in mouse brain extracts. Furthermore, Opa1 co-immunoprecipitated with exogenously expressed full-length Trim17 from HEK293 cells. Knockdown of Trim17 in neurons increased Opa1 protein levels under steady-state conditions. These results suggest that Trim17 regulates Bax-dependent apoptosis in neurons via the modulation of Opa1 levels.

Les mitochondries sont des organites essentiels qui fusionnent et fissionnent en permanence. Cette dynamique est régulée par différentes GTPase, notamment OPA1 qui est impliquée dans la fusion mitochondriale. OPA1 possède également une fonction anti-apoptotique, régulée par BNIP3, un membre pro-apoptotique de la famille Bcl-2. En conditions de stress, comme l'hypoxie, la protéine BNIP3 est induite et inhibe OPA1, ce qui conduit à une fragmentation des mitochondries et à l'apoptose. En plus de sa fonction pro-apoptotique, BNIP3 a récemment été impliquée dans l'autophagie et la mitophagie, une forme d'autophagie sélective des mitochondries qui assure le contrôle qualité de l'organite. Mon travail de thèse a visé à étudier l'implication des protéines BNIP3 et OPA1 dans la balance entre survie par autophagie-mitophagie et mort par apoptose dans les neurones. La protéine BNIP3 a été induite ou surexprimée en utilisant, respectivement, un mimétique de l'hypoxie ou des lentivirus, dans des neurones en culture primaire. Par des analyses biochimiques et de microscopie nous avons étudié les effets de cette induction/surexpression sur la morphologie mitochondriale, l'autophagie-mitophagie et l'apoptose, ainsi que l'impact d'OPA1 sur ces processus. Nous avons montré que l'induction de BNIP3 provoque une séquence d'évènements qui débute par la fragmentation du réseau mitochondrial, est suivie par un processus d'autophagie-mitophagie de survie et se termine par la mort des neurones. Alors que la surexpression d'OPA1 diminue la fragmentation des mitochondries et la mort des neurones, elle n'affecte pas l'autophagie-mitophagie dans nos conditions. Les mutations du gène d'OPA1 sont responsables d'une maladie neurodégénérative, l'Atrophie Optique Dominante Autosomale de type 1 (ADOA1), qui se traduit par une atrophie du nerf optique, pouvant conduire à la cécité, et des atteintes neurologiques extra-oculaires variées. Nous avons démontré dans des modèles in vitro et in vivo de l'ADOA1 que le taux de protéine BNIP3 basal est réduit. Ceci s'accompagne in vitro d'une diminution de l'autophagie-mitophagie qui pourrait contribuer à sensibiliser les neurones haploinsuffisants en protéine OPA1 à différents stress. BNIP3 participerait donc, de concert avec son partenaire OPA1, au contrôle du destin des neurones, favorisant la survie cellulaire grâce à son activité pro-autophagique et mitophagique lors de dommages modérés, mais entraînant la mort quand les dommages sont trop conséquents. La découverte d'une modulation de l'expression de BNIP3 dans des modèles d'ADOA1 permet de proposer un rôle de la protéine dans l'étiologie de la maladie. / Mitochondria are essential organelles that constantly fuse and divide. These dynamic processes are controlled by various GTPases, such as OPA1, which is involved in mitochondrial fusion. OPA1 has also an anti-apoptotic function, which is regulated by BNIP3, a pro-apoptotic member of the Bcl-2 family. Upon stresses, such as hypoxia, BNIP3 is induced and inactivates OPA1, leading to mitochondrial fragmentation and apoptosis. Besides its pro-apoptotic function, BNIP3 was recently shown to be involved in autophagy and mitophagy, a selective autophagy of mitochondria that ensures their quality control. This study aims to decipher the role of BNIP3 and its partner OPA1 in the balance between survival by autophagy-mitophagy and death by apoptosis in neurons. BNIP3 induction or overexpression was achieved, respectively, using a mimetic of hypoxia or lentiviruses, in primarily cultured neurons. Various microscopy and biochemistry approaches were used to analyse the impact of this induction/overexpression on mitochondrial morphology, autophagy-mitophagy and apoptosis. We showed that BNIP3 induction led to a sequence of events that started with mitochondrial network fragmentation, followed by pro-survival autophagic and mitophagic processes, and ended by cell death. OPA1 mutations lead to a neurodegenerative condition: Autosomal Dominant Optic Atrophy type 1 (ADOA1). ADOA1 patients suffer from optic nerve atrophy leading to blindness and various extra-ocular neurological defects. We evidenced in in vitro and in vivo ADOA1 models a lowered BNIP3 basal level. This decrease is associated in vitro with a reduction of autophagy-mitophagy that could lead to increased susceptibility to various stresses. BNIP3 thus controls, together with its partner OPA1, the fate of neurons, favoring cell survival upon moderate damages thanks to its pro-autophagic and mitophagic activity, or leading to cell death upon extensive damages. Having demonstrated that BNIP3 expression is affected in ADOA1 models, we propose a role of the protein in the etiology of the disease.

The cell possesses a number of vital mechanisms to respond to different stressors. Mitochondria are dynamic organelles which undergo constant changes in length, transport and inner membrane structure and curvature. Invaginations of this inner membrane, cristae, have been known to respond to the energetic state of mitochondria, but the regulation of these changes as well as the consequences thereof remain undetermined. We find that Optic Atrophy 1 (OPA1), a protein involved in inner membrane fusion and cristae maintenance during cell death, can respond to the energetic state of mitochondria and the cell. Moreover, OPA1-dependent changes in cristae structure are required for resistance to starvation induced cell death, proper functioning of the electron transport chain, for growth in galactose media and for maintenance of ATP synthase assembly. Interestingly, we demonstrate that select members of the mitochondrial solute carriers (SLC25A) interact with OPA1 and affect the response of OPA1 to substrate levels. Taken together, we propose an SLC25A-dependent role for OPA1 in sensing energy substrate availability and responding to alter cristae, bioenergetics and cellular survival. We also identified KIAA0664 as a novel OPA1-interacting protein, describe its subcellular localization and investigate its role in mitochondrial fusion and in mitochondrial localization. Finally, since both known carriers of mitochondrial glutathione were demonstrated to interact with OPA1, we investigated the role of OPA1 in cellular glutathione redox. OPA1 depleted cells demonstrated both increased total cellular glutathione and a shift in redox to its reduced form. The role of OPA1 in glutathione levels and redox ratios required GTPase activity, but surprisingly not fusion. Since glutathione is a master regulator of reactive oxygen species detoxification, these findings may shed light on the role of OPA1 in ROS-induced cell death pathways.

Mitochondrial dysfunction has been identified as a key factor in the progression of Parkinson's disease. Mitochondrial dysfunction has been shown to induce stress pathways, leading to neuronal dysfunction and cell death. Our lab has previously identified that, in neurons, reconfiguring the mitochondria using supercomplex assembly factors is protective against excitotoxic stress. For this thesis, we sought to characterize the stress pathways and synaptic impairment in an in vitro mitochondrial dysfunction model. Then, to determine if we can rescue the deficits shown, we manipulated mitochondrial integrity using the inner mitochondrial membrane targeted isoform of MCL1, which has previously been shown to regulate cristae structure and mitochondrial supercomplex assembly. We demonstrate that the integrated stress response is activated upon mitochondrial dysfunction. Next, we show mitochondrial dysfunction leads to a downregulation of synaptic genes involved in neurotransmission. Finally, our results show that both the antiapoptotic outer mitochondrial membrane-targeted isoform, and MCL1-Matrix are able to prevent cell death in response to mitochondrial dysfunction; however, MCL1-Matrix confers greater reduction in ISR activation and reactive oxygen species production. These data suggest that manipulating mitochondrial integrity, using MCL1-Matrix, confers a broad protective effect against neuronal stressors and may be used as a novel approach to preventing Parkinson's disease.

La morphologie mitochondriale résulte d’un équilibre dynamique entre les processus de fusion et de fission, impactant la physiologie cellulaire. Plusieurs données montrent une relation entre la fonction mitochondriale, des maladies cardiovasculaires et le vieillissement. OPA1 (optic atrophy 1) est une protéine qui contrôle la fusion de la membrane interne de la mitochondrie, et dont la mutation induit la maladie ADOA (autosomal dominant optic atrophy). Les travaux menés récemment indiquent que la mutation OPA1 est impliquée dans le dysfonctionnement cardiaque mais son impact sur la fonction vasculaire est encore inconnu. Notre étude a pour ambition d’examiner le rôle d’OPA1 sur la fonction vasculaire, notamment dans le développement de l’hypertension artérielle et le vieillissement vasculaire. Avec un modèle de souris hétérozygotes Opa1+/-, nous montrons dans cette étude que la protéine OPA1 joue un rôle protecteur dans le système vasculaire. En effet, les souris déficientes en OPA1 développent une hypertension-L-NAME dépendante plus grave qui est associée avec une dysfonction endothéliale plus importante et une altération de remodelage vasculaire. D’autre part, présentant une fonction vasculaire normale à 6 mois, les souris Opa1+/- commencent à développer un dysfonctionnement vasculaire à 12 mois qui pourrait induire le développement de pathologies vasculaires. En conclusion, ces résultats suggèrent pour la première fois que la dynamique mitochondriale peut jouer un rôle important sur la fonction et l’adaptation des vaisseaux dans les conditions pathologiques et dans le vieillissement vasculaire. Des études complémentaires seront nécessaires afin de clarifier le rôle de la protéine OPA1 dans l’hypertension. Ces données peuvent contribuer à la recherche de nouvelles cibles thérapeutiques pour prévenir les complications de l’hypertension et les maladies vasculaires liées à l’âge. / Defects in mitochondrial dynamics have been associated with various disorders, including cardiovascular diseases. OPA1 is essential for mitochondrial inner membrane fusion. Mutation in Opa1 is associated with the autosomal dominant optic atrophy (ADOA). Since then, OPA1 has been reported to be associated with cell apoptosis, cell proliferation, mitochondrial ATP synthesis, calcium homeostasis and ROS production. These data suggest that OPA1 has a potential role in vascular cells and subsequently affects vascular function. On the other hand,OPA1 is also associated with age-related changes of mitochondria and simultaneously contribute to the development of many dysfunctions in different organs. In this study, we investigated impacts of OPA1 mutation on vascular function in physiological and pathological condition like hypertension and vascular aging. By using an Opa1+/- heterozygote mouse model, we show that the OPA1 protein plays a protective role in the vascular system. Indeed, Opa1+/- mice developed a hypertension more severe than WT mice which was associated with more important endothelial dysfunction and altered vascular remodeling. In addition, although initial vascular function was normal, at 12 months, Opa1+/- mice displayed vascular dysfunction which might predict onset of vascular diseases at a later time. These results suggest for the first time that mitochondrial dynamics might play an important role in vascular function and adaptation in pathological conditions and in vascular aging. More studies are needed to clarify the role of the protein OPA1 in hypertension. These data may help to identify novel therapeutic targets to prevent complications of hypertension and vascular age-related diseases.

La neurogenèse adulte est cruciale pour certaines fonctions mnésiques dépendantes de l'hippocampe. La mise en évidence d'une altération de la neurogenèse dans le cerveau de souris transgéniques modèles de la maladie d'Alzheimer (MA), en parallèle d'une réduction du contenu mitochondrial de leurs nouveaux neurones ouvre une nouvelle piste de recherche ciblant les mitochondries. Aujourd'hui, l'hypothèse d'un rôle causal des dysfonctionnements mitochondriaux dans l'étiologie des pathologies neurodégénératives est particulièrement pertinente dans la MA. Les mitochondries, " centrales électriques " et régulateurs du métabolisme oxydatif, forment un réseau dynamique qui s'adapte aux différents types et contextes cellulaires, via des événements antagonistes de fusion et de fission de leurs membranes. Les protéines clés ont été identifiées, dont OPA1 qui permet la fusion. Les dysfonctionnements de cette dynamique influent non seulement sur la forme et la distribution des mitochondries dans les neurones, mais affectent aussi leurs principales activités que sont respiration, régulation calcique, production de ROS et apoptose. Dans les neurones, cellules excitables à l'architecture complexe, les dysfonctionnements mitochondriaux ont des conséquences particulièrement cruciales pour la transmission synaptique. Au cours de cette thèse, nous avons étudié parallèlement des souris modèles de la MA, les souris Tg2576 (mutation d'APP) et des souris OPA1+/-, porteuses d'une mutation d'OPA1, modèles de l'Atrophie Optique Dominante. Nous avons observé chez ces deux lignées de souris une altération précoce des performances dans des tests comportementaux mettant en jeu le gyrus denté et les nouveaux neurones (tests de localisation d'objet et de séparation de patron). Nous avons démontré chez les souris Tg2576 et OPA1+/- que ces déficits cognitifs sont associés à des perturbations de la neurogenèse hippocampique adulte.[...] / Adult neurogenesis is crucial for some hippocampus-dependent memory functions. Both the demonstration of an alteration of neurogenesis in the brain of transgenic mouse models of Alzheimer's disease (AD), in parallel with a reduction in the mitochondrial content of their new neurons, open a new research avenue targeting the mitochondria. Today, the hypothesis of a causal role of mitochondrial dysfunctions in the etiology of neurodegenerative pathologies is particularly relevant in AD. Mitochondria, "power plants" and regulators of oxidative metabolism, form a dynamic network that adapts to different cell types and contexts, via antagonistic events of fusion and fission of their membranes. Key proteins have been identified, including OPA1 that allows fusion. Dysfunctions of this dynamics affect not only the shape and distribution of mitochondria in neurons, but also alter their main activities: respiration, calcium regulation, ROS production and apoptosis. In neurons, excitable cells with complex architecture, mitochondrial dysfunctions have particularly crucial consequences for synaptic transmission. In this thesis, we studied in parallel an AD mouse model, the Tg2576 mice (APP mutation) and the OPA1 +/- mice, carrying a mutation of OPA1, a Dominant Optic Atrophy model. In both mouse lines, we observed precocious performance alterations in behavioral tests involving the dentate gyrus and new neurons (object location, pattern separation tests). We demonstrated in Tg2576 and OPA1 +/- mice that these cognitive deficits are associated with disturbances of adult hippocampal neurogenesis. [...]

OPA1 regulates cristae structure and mitochondrial DNA (mtDNA) maintenance. Recently, our lab identified ATAD3A and SLC25 proteins as OPA1 interactors. After validating these interactions by co-immunoprecipitation, the role of these proteins in OPA1 function was examined. Previously, ATAD3A was implicated in mtDNA maintenance. However, no change in mtDNA content or nucleoid number was observed in my studies following long-term and short-term ATAD3A knockdown suggesting that OPA1 maintains mtDNA independently of ATAD3A. Previous data from our lab demonstrates that OPA1 oligomerization and cristae structure is altered by nutrients. SLC25 proteins transport nutrients into mitochondria. Therefore, OPA1 oligomerization and cristae structure was analyzed following SLC25 protein inhibition and knockdown. Decreased OPA1 oligomerization and cristae remodeling was observed following SLC25 protein inhibition and OGC knockdown. In addition these changes correlate with decreased ATP synthase monomers and oligomers suggesting that cristae remodeling may affect metabolism. Overall, these studies enhance our understanding of OPA1 function.

OPA1 regulates cristae structure and mitochondrial DNA (mtDNA) maintenance. Recently, our lab identified ATAD3A and SLC25 proteins as OPA1 interactors. After validating these interactions by co-immunoprecipitation, the role of these proteins in OPA1 function was examined. Previously, ATAD3A was implicated in mtDNA maintenance. However, no change in mtDNA content or nucleoid number was observed in my studies following long-term and short-term ATAD3A knockdown suggesting that OPA1 maintains mtDNA independently of ATAD3A. Previous data from our lab demonstrates that OPA1 oligomerization and cristae structure is altered by nutrients. SLC25 proteins transport nutrients into mitochondria. Therefore, OPA1 oligomerization and cristae structure was analyzed following SLC25 protein inhibition and knockdown. Decreased OPA1 oligomerization and cristae remodeling was observed following SLC25 protein inhibition and OGC knockdown. In addition these changes correlate with decreased ATP synthase monomers and oligomers suggesting that cristae remodeling may affect metabolism. Overall, these studies enhance our understanding of OPA1 function.

L’atrophie optique dominante (AOD, MIM#165500) est une pathologie héréditaire affectant un individu sur 30 000 environ. Elle touche principalement les cellules ganglionnaires de la rétine qui composent le nerf optique, entraînant une baisse d’acuité visuelle. Cette pathologie, génétiquement et cliniquement hétérogène, est majoritairement due aux mutations du gène OPA1. La protéine mitochondriale OPA1 est impliquée dans de multiples fonctions telles que la fusion des mitochondries, le métabolisme énergétique, l’apoptose et la maintenance de l’ADN mitochondrial. Afin d’appréhender les effets globaux des dysfonctions d’OPA1, nous avons développé des approches métabolomiques et lipidomiques non-targeted sur des plasmas et des fibroblastes de patients ainsi que sur des modèles murins. En dépit des spécificités de chaque modèle et matrice, nos études ont clairement révélé des altérations métaboliques communes dont une déficience en aspartate. Ce déficit est impliqué dans le métabolisme des nucléotides et est en relation directe avec le défaut énergétique révélé dans nos différents modèles. Avec l’approche lipidomique, nous avons montré dans les fibroblastes de souris invalidés pour le gène OPA1 une augmentation importante des triglycérides qui est en lien avec le défaut énergétique. De plus, nous avons mis en évidence un remaniement majeur des phospholipides qui témoigne d’un profond remodelage des structures membranaires mitochondriales. Ces approches nous ont ainsi permis d’avoir de nouvelles données sur les implications physiopathologiques d’OPA1. L’ensemble de ce travail ouvre de nouvelles perspectives pour une meilleure prise en charge de la pathologie. / Dominant Optic Atrophy (DOA, MIM #165500) is an inherited disease affecting one of 30,000 individuals. It mostly affects the retinal ganglion cells that make up the optic nerve, leading to the decrease invisual acuity. This genetically and clinically heterogeneous pathology is mainly related to the mutations on OPA1 gene. The mitochondrial protein OPA1 has been implicated in many functions including mitochondrial fusion, energy metabolism, apoptosis and maintenance of mitochondrial DNA. In order to investigate the overall effects of OPA1 dysfunctions, we developed non-targeted metabolomic and lipidomic approaches on patients' plasmas and fibroblasts as well as on OPA1 knock-out mouse fibroblast model. Despite the specificities of each model and matrix, we clearly revealed a common metabolic alteration including an aspartate deficiency due to the energy defect observed in all our models and responsible for the alteration of nucleotide metabolism. With a lipidomic approach, we revealed in the knock-out cell model a huge increase of triglycerides which is related to the energetic deficiency. Moreover, we highlighted a major alteration on phospholipids, testifying a deep remodeling of mitochondrial membrane structures. Taken together, our analysis revealed new pathophysiological roles of OPA1. Finally, our work opens new perspectives to improve the diagnosis and the patient care.