Dans de nombreuses maladies neurodégénératives dont la Maladie de Parkinson, mais également dans le processus de vieillissement normal, il a été observé une accumulation excessive des niveaux de fer associée à une production accrue d‘espèces réactives de l’oxygène (ROS).Il est bien connu que le fer (i) participe à la réaction de Fenton pour produire le ROS le plus toxique, le radical hydroxyle, (ii) accélère l’auto-oxidation de la dopamine, augmentant le stress oxydant et (iii) augmente la peroxydation lipidique qui va déclencher l'agrégation des protéines, dont l’α-synucléine, une caractéristique de la maladie de Parkinson. Tous ces phénomènes rendent les neurones dopaminergiques très sensible au stress oxydant.Récemment, une nouvelle forme de mort cellulaire a été découverte sur des cellules cancéreuses. Cette mort cellulaire est appelée « ferroptose » car elle dépend essentiellement du fer intracellulaire. De plus, elle est liée à une très grande augmentation du stress lipidique.Le principal objectif de ce travail de thèse a donc porté sur la possible implication de la ferroptose dans la mort des cellules dopaminergiques.Dans un premier temps, nous avons montré que la lignée de neurones dopaminergiques (LUHMES) est un modèle particulièrement adapté à l’étude de mort cellulaire induite par le fer comparativement à d’autres modèles plus classique.La seconde partie du travail a donc été consacrée à l'étude de l’implication de la ferroptose dans les neurones dopaminergiques. D’abord par une approche cellulaire avec les cellules LUHMES, nous avons pu observer (i) que l’inducteur spécifique de cette mort, l’érastine, est très efficace pour induire la mort des cellules dopaminergiques, (ii) que l'inhibition de la ferroptose protégeait de la mort induite par des agents oxydants classiquement utilisés dans les études sur la maladie de Parkinson comme le MPP+ et (iii) que la PKC joue un rôle majeur dans la mort par ferroptose. Tous ces résultats ont ensuite été confirmés dans un modèle de culture organotypique, à l’interface entre la culture cellulaire et le modèle animal.Enfin, nous avons montré que l’utilisation des inhibiteurs de la ferroptose ainsi que celui de la PKC, confère une protection des neurones dopaminergiques dans un modèle de souris MPTP, modèle d’étude de la maladie de Parkinson.En conclusion ce travail de thèse montre pour la première fois l'implication de la ferroptose dans une maladie neurodégénérative comme la maladie de Parkinson et suggèrent que le développement d'inhibiteurs spécifiques de cette mort cellulaire pourrait être des futures cibles thérapeutiques possibles. / In many neurodegenerative diseases including Parkinson's disease, as well as in the regular aging process, an excessive accumulation of iron levels associated with increased production of oxygen species (ROS) have been observed.It is well known that iron (i) participates in the Fenton reaction to produce the most toxic ROS, the hydroxyl radical, (ii) accelerates auto-oxidation of dopamine, increasing the oxidative stress and (iii) increases lipid peroxidation that will trigger the aggregation of proteins, including α-synuclein, a hallmark of Parkinson's disease. All theses factors make dopamine neurons very sensitive to oxidative stress.Recently, a new form of cell death was found on cancer cells. This cell death is called ferroptosis because it essentially depends on the intracellular iron. In addition, it is linked to a very large increase in lipid stress.The main objective of this work has focused on the possible involvement of ferroptosis in the death of dopamine cells.First, we showed that the line of dopaminergic neurons (LUHMES) is a model particularly suited to the study of cell death induced by iron compared to other more conventional models.The second part of the work has been devoted to the study of the involvement of ferroptose in dopaminergic neurons. First by a cellular approach with LUHMES cells, we observed that (i) the specific inducer of this death, erastin, is very effective to induce the death of dopaminergic cells, (ii) inhibition of ferroptosis protected from death induced by oxidizing agents conventionally used in studies of Parkinson's disease like MPP+ and (iii) that PKC plays a major role in death by ferroptosis. These results were then confirmed in an organotypic culture model, at the interface between cell culture and animal models.Finally, we have shown that the use of ferroptosis inhibitors and PKC inhibitor, provides protection of dopaminergic neurons in MPTP mice model, animal model for studying of Parkinson's disease.In conclusion this study demonstrates for the first time the involvement of ferroptosis in a neurodegenerative disease such as Parkinson's disease and suggest that the development of specific inhibitors of this cell death could be possible future therapeutic targets.

Diabetes mellitus and its complications stands as arguably the most formidable pandemic of the 21st century. While rodent models of diabetes mellitus have been extensively explored, none have managed to faithfully replicate the full spectrum of pathological hallmarks and secondary complications witnessed in diabetic patients. Among the commonly affected organs is the kidney, manifesting in the form of diabetic kidney disease (DKD). Recently, our clinical understanding of incretins as critical regulators of disease progression in diabetic patients including DKD has undergone significant expansion. In particular, the incretin hormone gastric inhibitory polypeptide (GIP) axis has taken central stage. A ground-breaking development in this realm was the creation of a GIP receptor dominant negative (GIPRdn) mouse, exhibiting all the characteristic features observed in DKD patients. This study sheds light on the heightened susceptibility of these mice to lethal acute kidney injury (AKI) induced by ischemia-reperfusion injury (IRI). Notably, isolated renal GIPRdn-tubules displayed accelerated cell death propagation and increased tubular necrosis. Expanding on previous cell culture experiments involving hyperglycemia, it became apparent that tubules of GIPRdn mice express elevated levels of the intracellular thioredoxin interacting protein (TXNIP), previously reported to be responsible for the degradation of glucose transporter 1 (GLUT1). This phenomenon is crucial in maintaining intracellular glucose homeostasis. The study further indicates an association between TXNIP and the downregulation of thioredoxin reductase 1 (TXNRD1), a selenoenzyme playing a pivotal role in protecting renal tubules from ferroptosis in a glutathione-independent manner. Intriguingly, the inhibition of TXNRD1 with the small molecule ferroptocide (FTC) in GIPRdn tubules resulted in severe tubular necrosis, a condition effectively reversed by the ferroptosis inhibitor ferrostatin 1 (Fer-1). This nuanced exploration establishes a connection between DKD and a heightened sensitivity to kidney tubular ferroptosis, thereby presenting a potential avenue for intervention with ferrostatins. Importantly, the administration of a single dose of Fer-1 significantly prolonged the survival of GIPRdn mice following IRI. In conclusion, this study illuminates the intricate dynamics of DKD, highlighting a pronounced sensitization to kidney tubular ferroptosis. The findings suggest that ferrostatins, particularly exemplified by Fer-1, hold promise as potential therapeutic agents in mitigating the severity of this condition, offering hope for improved outcomes in individuals struggling with diabetes-related kidney complications.:Acknowledgments Abstract Zusammenfassung List of abbreviations List of tables List of Figures 1. Introduction 1.1. Diabetes mellitus 1.1.1. Definition and description 1.1.2. Epidemiology 1.1.3. Classification of diabetes mellitus 1.1.4. Diagnosis of diabetes mellitus 1.1.5. Type 2 Diabetes Mellitus 1.1.6. Long-term complications of T2DM 1.1.6.1. Diabetic Nephropathy 1.1.6.2. Therapies for diabetic nephropathy 1.1.7. Animal models for diabetic kidney disease 1.1.7.1. Diabetic eNOS knockout mouse 1.1.7.2. Bradykinin B2 Receptor (B2R) deficient Ins2Akita/+ mouse 1.1.7.3. Decorin-deficient streptozotocin diabetic mouse 1.1.7.4. NONcNZO mouse 1.1.7.5. OVE26 mouse 1.1.7.6. Black and tan, brachyuric (BTBR) ob/ob mouse 1.1.8. Incretin hormones and GIPRdn diabetic mouse model 1.1.8.1. Generation of GIPRdn diabetic mouse model 1.2. Regulated cell death 1.3. Ferroptosis 1.3.1 Mechanism of ferroptosis 1.3.1.1 Sensitization to ferroptosis by ether phospholipids 1.3.1.2 Hydropersulfides and ferroptosis 1.3.2 Ferroptosis inducers (FINs) and inhibitors 1.3.3 Ferroptosis in the kidney 1.4 Aims 2. Materials and Methods 2.1. Reagents 2.2. Experimental models: cell lines and mouse strains 2.2.1. Cell culture conditions 2.2.2. Mice 2.2.2.1. Genotyping 2.2.2.1.1. DNA isolation 2.2.2.1.2. Polymerase Chain Reaction (PCR) 2.2.2.1.3. Gel electrophoresis 2.2.2.2. Body weight 2.2.2.3. Blood glucose 2.2.2.4. Blood collection and serum parameters 2.2.3. Isolation of primary murine renal tubules 2.2.4. Generation of a 3D-printed double chamber 2.3. Experimental procedures 2.3.1. Plating and treatment of cells 2.3.2. Fluorescence activated cell sorting (FACS) 2.3.3. Western Blotting (WB) 2.3.4. Induction of cell death on isolated murine tubules 2.3.5. LDH release assay 2.3.6. Evaluation of speed of cell death propagation (exponential plateau – growth equation) 2.3.7. Time lapse imaging and processing of the time lapse data 2.3.8. Fluorescence Lifetime Imaging Microscopy (FLIM) 2.3.8.1. Time domain data analysis 2.3.8.2. FLIM time lapse video generation 2.3.9. Thioredoxin Reductase Activity assay 2.3.10. Bilateral kidney Ischemia and Reperfusion injury (IRI) 2.3.11. Immunohistology and semi-quantitative scoring 2.3.12. Measurements of sulfur-containing metabolites by ultra-performance liquid chromatography-mass spectroscopy (LC-MS) 2.4. Statistical analysis 3. Results 3.1. Characterization of diabetic kidney disease in GIPRdn mice 3.1.1. Blood glucose viii 3.1.2. Body weight 3.1.3. Serum parameters 3.1.4. Histological analysis of the kidneys 3.2. The spontaneous death of GIPRdn tubules is characterized by a non-random pattern of necrotic cell death 3.3. GIPRdn tubules are more prone to undergo spontaneous death compared to WT tubules 3.4. Spontaneous necrosis of GIPRdn and WT tubules is partially mediated by ferroptosis 3.5. GIPRdn tubules show downregulation of the PRX pathway compared to the non-diabetic tubules 3.6. GIPRdn tubules show altered hydropersulfides pathway 3.7. GIPRdn tubules show altered etherglycerophospholipids (etherPLs) pathway. 3.8. Ferrostatin-1 but not Empagliflozin reverses ferroptosis induction in different cell lines as well as in isolated kidney tubules 3.9. GIPRdn mice are more sensitive to IRI-induced acute kidney injury compared to their WT littermates 3.10. Ferrostatin-1 ameliorates the sensitivity of GIPRdn to ischemia reperfusion injury-induced acute kidney injury 4. Discussion 4.1. The GIPRdn mouse model 4.2 Ferroptosis in diabetic nephropathy 4.2.1. Ferroptotic cell death is involved in the spontaneous death of diabetic tubules 4.2.2. Possible mechanisms behind the enhanced sensitivity of the GIPRdn kidney tubules to ferroptosis 4.3. Therapeutic consequences of the study 4.3.1. SGLT2 inhibitor empagliflozin does not have a protective effect on diabetic tubules undergoing spontaneous death 4.4. Outlook and limitations of the study References

No description available.

Regulated cell death (RCD) is indispensable for homeostasis and plays a crucial role in the pathophysiology of numerous diseases. Adrenocortical carcinomas (ACCs) represent a rare and highly malignant type of cancer. Currently, the most common therapeutic options include the complete surgical removal of the adrenal gland and/or the administration of mitotane, a derivative of the pesticide DDT. Yet patient survival remains poor and the mechanism of action of mitotane remains elusive. In this thesis it is demonstrated that the human ACC cell line NCI-H295R is sensitive to mitotane-induced cell death. In the first part, the involvement of three different RCD pathways, namely apoptosis, necroptosis and ferroptosis, in mitotane induced necrosis was investigated. To this end, different inhibitors were used, which were not able to block mitotane-induced cell death. When the medium was supplemented with insulin, transferrin, sodium selenite and linoleic acid (ITS+1) no cell death of the ACC cells was observed. This phenomenon was attributed to the presence of linoleic acid, since ITS supplementation lacking this component was not able to reverse mitotane-induced necrosis. Identification of new drug targets for alternative options of ACC treatment led to the investigation of key molecules involved in the pathways of necroptosis and ferroptosis. The receptor-interacting protein kinase 1 and 3 (RIPK1 and 3) and the mixed lineage kinase domain-like protein (MLKL) were considered as interesting targets given their crucial role in the execution of necroptosis. A western blot analysis of those molecules revealed the presence only of RIPK1, suggesting that the necroptosis machinery is not present in the NCI-H295R cells. Of interest, evaluation of the expression levels of glutathione peroxidase four (GPX4), one of the main inhibitory molecules of ferroptosis, showed a much higher expression in the ACC cells compared to the standard cell line used for studying ferroptosis, the human fibrosarcoma HT1080 cells. A hypothesis that the NCI-H295R cells are susceptible to ferroptosis induction was formed based on this finding. Compounds representative of all the four classes of ferroptosis inducers (FINs) were tested. Direct inhibition of GPX4 using the small compound RSL3, a type II FIN, led to high necrotic populations. Co-treatment with the ferroptosis inhibitor ferrostatin-1 (Fer-1) completely reversed RSL3-induced ferroptosis. Type IV FIN FINO2, that causes indirect loss of the enzymatic activity of GPX4, lead also to high necrotic populations, while Fer-1 prevented FINO2-induced ferroptosis. Data from public databases concerning gene methylation or mutation status of ACC tissues and normal human adrenal tissues was used to investigate potential key players of ferroptosis that might be either mutated or silenced in ACCs. Of note, glutathione peroxidases 3 and 5 (GPX3 and 5) were highly methylated, while the enzyme cystathionine gamma-lyase (CSE) involved in the transsulfuration pathway via the break down of cystathionine into cysteine and α-ketobutyrate and ammonia was found to be highly mutated. Collectively, these data point towards a high sensitivity of ACCs to ferroptosis induction. This could provide a new chapter for the therapeutic approaches of ACCs. Additionally, these findings provide a better understanding of the biology of this type of cancer that highly mutates or silences ferroptosis-related genes. The second part of this thesis focuses on the involvement of RCD in spontaneous cell death in isolated murine tubules. Existing literature points towards an involvement of necroptosis and ferroptosis pathways in the kidney in models of acute kidney injury (AKI). Acute tubular necrosis (ATN) represents a hallmark of AKI. While the work in the Linkermann lab has shown that isolated tubules perfused with type I FIN erastin undergo cell death in a “wave-of-death” manner, no deeper insights into the propagation of tubular necrotic injury exist. A protocol for isolation of murine kidney tubules was established, providing an ex-vivo model for investigation of tubular death. The absence of potentially confounding blood cells as well as immune cells was ensured by extensive washing steps as well as the use of collagenase. Visual observation and staining of isolated tubules with the nucleic acid stain SYTOX green revealed a spontaneous cell death in a “wave-of-death” manner. This wave was running in parallel with a calcium concentration change, indicating its involvement in the spontaneous necrosis. To investigate the potential involvement of mitochondria in this process, electron microscopy images were obtained from parts of the tubules with different levels of damage which revealed highly damaged and ballooned mitochondria. These data provided with a phenotypic characterisation of the spontaneous tubular necrosis. Aiming to approach this type of death genetically, necroptosis and pyroptosis deficient mice (MLKL/GSDMDDKO) were used. Comparison of the LDH release, used as a measure of necrosis, from isolated kidney tubules of the MLKL/GSDMDDKO mice and wild type (WT) mice showed no difference. This indicated that neither necroptosis nor pyroptosis are involved in the tubular necrosis. Therefore, the next step was to investigate the effects of Fer-1 at the levels of LDH of isolated tubules from WT mice. A significantly lower LDH release was observed in tubules treated with Fer-1 compared to the ones treated with vehicle. However, this reduction in the LDH release was not complete, suggesting that ferroptosis is only partially responsible for the spontaneous death of isolated tubules. The difference of male and female mice towards AKI sensitivity has been noted in the literature in that female mice are less susceptible compared to the male mice. Therefore, the next step was to investigate whether this protection of females can be observed at the level of isolated tubules. Indeed, the LDH release from tubules isolated from female mice was significantly less compared to the LDH release of tubules isolated from male mice. Based on the data obtained from isolated tubules from WT male mice treated with Fer-1, a similar experiment was performed with tubules isolated from WT female mice. No difference in the LDH release was observed between the Fer-1-treated tubules and the vehicle-treated ones, indicating that another cell death pathway might be involved. The most obvious difference between male and female organisms is the sex hormones. Whether testosterone or β-estradiol are responsible for the higher susceptibility or protection against cell death has been a debate over the last years. To test this hypothesis, three different cell lines were utilised. A pre-treatment of 16 h with either testosterone or β-estradiol was performed. Treatment with either type I FIN erastin or type II FIN RSL3 followed, and cells were analysed via flow cytometry. Data revealed protective effects of β-estradiol against ferroptosis induction. Next, the effects of β-estradiol in a simultaneous treatment with RSL3 were investigated. Interestingly the protective effects of the hormone were still observed. Among the metabolites of β-estradiol, 2-hydroxyestradiol (2-OHE2) has been reported to exert antioxidant effects. Therefore, 2-OHE2 was used in a simultaneous treatment with RSL3, and the obtained data showed that it was a much more potent inhibitor of necrotic cell death than β-estradiol even at lower concentrations. Collectively these data indicate that the lower susceptibility of female organisms towards cell death might be explained by the presence of β-estradiol and its more potent antioxidant metabolites. Such findings could change the way the two sexes are approached scientifically, while providing new insights on different therapeutic strategies between male and female organisms.

Renal tubules represent an intercellular unit and function as a syncytium. When acute tubular necrosis was first visualized to occur through a process of synchronized regulated necrosis (SRN) in handpicked primary renal tubules, it became obvious that SRN actually promotes nephron loss. This realization adds to our current understanding of acute kidney injury (AKI)-chronic kidney disease (CKD) transition and argues for the prevention of AKI episodes to prevent CKD progression. Because SRN is triggered by necroptosis and executed by ferroptosis, 2 recently identified signaling pathways of regulated necrosis, a combination therapy employing necrostatins and ferrostatins may be beneficial for protection against nephron loss. Clinical trials in AKI and during the process of kidney transplantation are now required to prevent SRN. Additionally, necrotic cell death drives autoimmunity and necroinflammation and therefore represents a therapeutic target even for the prevention of antibody-mediated rejection of allografts years after the transplantation process.