An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in chronic and acute neurodegenerative diseases. Indeed, markers of ER stress are common features of neurons destined to die in these conditions. In the present study we demonstrate that PUMA, a BH3-only member of the Bcl-2 family is essential for ER stress-induced cell death. PUMA is known to be a key transcriptional target of p53, however we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism involving instead the ER stress inducible transcription factor ATF4. Specifically, we demonstrate that ectopic expression of ATF4 sensitizes neurons to ER stress induced apoptosis, and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP, and that CHOP in turn directly activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER stress induced neuronal death involving ATF4-CHOP mediated transactivation of the pro-apoptotic Bcl-2 family member PUMA. Protein aggregates and markers of ER stress response have also been observed in dying neurons in several animal models of cerebral ischemia. Therefore, to decipher the significance of the ER stress apoptotic response, we investigate the role of ATF4-CHOP signaling pathway in ischemic neuronal injury. Ischemic stroke results from a transient or permanent reduction in cerebral blood flow in the brain. In spite of much research in trying to develop therapeutic strategies, most clinical trials have failed. These failures demonstrate that effective treatments require a more complete understanding of molecular signals that lead to neuronal death. However, stroke is a complex scenario since distinct mechanisms may involve in rapid and/or delayed neuronal death. The signaling pathways regulating these mechanisms however are not fully defined. Previous studies had suggested that ER stress playing a pivotal role in post-ischemic neuronal death. Yet, the relevance of ER stress signals was not fully known in ischemic neuronal injury. Accordingly, this thesis research attempts to explore the functional role of ER stress -inducible pathway, ATF4-CHOP axis, in different models of neuronal death (delayed and excitotoxic cell death) evoked by ischemia. The data indicates that ATF4 is essential in delayed type of death in vitro. In focal ischemia model (tMCAO) ATF4 also plays a role as a mediator of death signal in vivo. However, CHOP function looks more complex, and our data did not support the role of CHOP in ischemic neuronal death.

Mutations in the COL10A1 gene cause metaphyseal chondrodysplasia type Schmid (MCDS) by triggering ER stress and unfolded protein response (UPR). MCDS is characterised by a mild short-limb dwarfism accompanied by expansion of the cartilage growth plate hypertrophic zone (HZ) and altered differentiation of hypertrophic chondrocytes (HCs). ATF6 is one of the UPR mediators, which exists in two isoforms, ATF6α and ATF6β. Activation and up-regulation of ATF6α was a prominent biochemical sign of ER stress in a mouse model of MCDS, COL10a1 p.N617K. Although ATF6β is induced and activated in response to ER stress in a similar fashion to ATF6α, the role and significance of ATF6β in the pathology of many ER stress-associated diseases including MCDS is unknown. Here we utilized a combination of in vitro and in vivo approaches to define the precise role of each isoform of ATF6 in MCDS.To investigate the functions of ATF6α and ATF6β in vitro, we developed a MCDS cell model system (expressing either the wild type collagen X or one of the following MCDS-causing mutant forms of the protein: p.N617K, G618V, Y598D, and NC1del10) in which the expression of either ATF6α or ATF6β was efficiently silenced using siRNAs. ATF6α knockdown in HeLa cells expressing different MCDS-causing mutations suppressed the increased expression of UPR-associated genes such as BiP leading to an elevated ER stress, based on increased XBP1 splicing and/or ATF4 protein. In contrast, ATF6β knockdown did not significantly affect the mutant collagen X-induced increased expression of UPR-associated genes. Furthermore, the ER stress levels were significantly reduced in the ATF6β knockdown MCDS mutant cells based on the lower levels of XBP1 splicing and/or ATF4 protein detected. We then crossed the ATF6α/β knockout mice models with COL10a1 p.N617K mouse model of MCDS to investigate the function of ATF6α and ATF6β in vivo. Ablation of ATF6α in MCDS mice further- reduced the endochondral bone growth rate, further expanded the growth plate hypertrophic zone, and disrupted differentiation of HCs. Therefore, ATF6α appeared to play a chondroprotective role in MCDS as its deficiency caused an increase in the severity of the disease. Of particular note, the level of ER stress was further increased in the absence of ATF6α in MCDS, based on enhanced activities of PERK and IRE1 signalling pathways in compensation for the ATF6α loss. Paradoxically, ablation of ATF6β in MCDS mice reduced the intracellular retention of collagen X protein, and alleviated the ER stress as judged by the attenuated activities of PERK and IRE1 signalling pathways. The reduced ER stress resulting from deficiency for ATF6β in MCDS mice restored the expression of collagen X mRNA towards normal and improved the differentiation of HCs, causing a mark decrease in the expansion of HZ. The results presented within this thesis greatly increased our understanding of the function of ATF6α and ATF6β and their interplay in the pathogenesis of MCDS. We demonstrated an indispensable beneficiary role for ATF6α but a detrimental role for its closely related isoform, ATF6β, in pathology of MCDS. We also showed that the role of ATF6β should not be ignored. These findings may be used to develop a potential therapeutic strategy for MCDS through targeting and enhancing ATF6α-dependent and/or attenuating/blocking of ATF6β-dependent signalling pathways.

The prevalence of type 2 diabetes is increasing dramatically worldwide. Type 2 diabetes is a major health and socio-economic burden. Genetic predisposition and the obesity epidemic, due to sedentary life style and high caloric food intake, are associated with development of type 2 diabetes. Circulating free fatty acids (FFAs), in particular saturated FFAs, are linked with insulin resistance and β cell dysfunction. Following this background we performed RNA sequencing of human pancreatic islets treated with the saturated FFA palmitate to acquire a global image of the islet response to this insult. We identified several stress pathways induced by palmitate with a major induction of the endoplasmic reticulum (ER) stress response. The ER stress response, in particular the PKR-like ER kinase (PERK) branch, has been shown to be induced by saturated FFA. It leads to increased β cell apoptosis both in fluorescence activated cell sorter (FACS) purified rat β cells and human islets. We further clarified the role of this pathway by studying the involvement of the constitutive repressor of eIF2α phosphorylation (CReP) in a monogenic form of diabetes. CReP is a repressor of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation. A direct target of PERK, eIF2α is involved in translational attenuation and induction of apoptosis. We have shown that CReP loss-of-function leads to a new syndrome of young onset diabetes, intellectual disability and microcephaly. The identified R658C mutation abrogated CReP activity leading to increased eIF2α phosphorylation and β cell apoptosis. To further demonstrate the importance of eIF2α dysregulation in β cell demise, we used guanabenz, a chemical inhibitor of growth arrest DNA damage inducible 34 (GADD34). GADD34 is an ER stress-induced repressor of eIF2α phosphorylation. Guanabenz potentiated FFA-mediated ER stress and apoptosis in clonal and primary rat β cells and in human islets through the activation of CCAAT/enhancer binding protein homologous protein (CHOP), downstream of eIF2α. Guanabenz administration in mice impaired glucose tolerance and led to β cell dysfunction. In ex vivo experiments guanabenz also induced β cell dysfunction in mouse and rat islets.In conclusion our data demonstrate that the dysregulation of signaling in the PERK/eIF2α pathway is crucial for β cell demise. Together with previously reported monogenic diabetes caused by loss-of-function mutations in PERK in man and the eIF2αS51A mutation in mice, our findings suggest that a narrow regulation of PERK/eIF2α signaling is central for proper β cell function and survival. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

An increasing body of evidence points to a key role of endoplasmic reticulum (ER) stress in chronic and acute neurodegenerative diseases. Indeed, markers of ER stress are common features of neurons destined to die in these conditions. In the present study we demonstrate that PUMA, a BH3-only member of the Bcl-2 family is essential for ER stress-induced cell death. PUMA is known to be a key transcriptional target of p53, however we have found that ER stress triggers PUMA induction and cell death through a p53-independent mechanism involving instead the ER stress inducible transcription factor ATF4. Specifically, we demonstrate that ectopic expression of ATF4 sensitizes neurons to ER stress induced apoptosis, and that ATF4-deficient neurons exhibit markedly reduced levels of PUMA expression and cell death. However, chromatin immunoprecipitation experiments suggest that ATF4 does not directly regulate the PUMA promoter. Rather, we found that ATF4 induces expression of the transcription factor CHOP, and that CHOP in turn directly activates PUMA induction. Specifically, we demonstrate that CHOP binds to the PUMA promoter during ER stress and that CHOP knockdown attenuates PUMA induction and neuronal apoptosis. In summary, we have identified a key signaling pathway in ER stress induced neuronal death involving ATF4-CHOP mediated transactivation of the pro-apoptotic Bcl-2 family member PUMA. Protein aggregates and markers of ER stress response have also been observed in dying neurons in several animal models of cerebral ischemia. Therefore, to decipher the significance of the ER stress apoptotic response, we investigate the role of ATF4-CHOP signaling pathway in ischemic neuronal injury. Ischemic stroke results from a transient or permanent reduction in cerebral blood flow in the brain. In spite of much research in trying to develop therapeutic strategies, most clinical trials have failed. These failures demonstrate that effective treatments require a more complete understanding of molecular signals that lead to neuronal death. However, stroke is a complex scenario since distinct mechanisms may involve in rapid and/or delayed neuronal death. The signaling pathways regulating these mechanisms however are not fully defined. Previous studies had suggested that ER stress playing a pivotal role in post-ischemic neuronal death. Yet, the relevance of ER stress signals was not fully known in ischemic neuronal injury. Accordingly, this thesis research attempts to explore the functional role of ER stress -inducible pathway, ATF4-CHOP axis, in different models of neuronal death (delayed and excitotoxic cell death) evoked by ischemia. The data indicates that ATF4 is essential in delayed type of death in vitro. In focal ischemia model (tMCAO) ATF4 also plays a role as a mediator of death signal in vivo. However, CHOP function looks more complex, and our data did not support the role of CHOP in ischemic neuronal death.

The endoplasmic reticulum (ER) facilitates the synthesis, assembly and quality control of all secretory, transmembrane, and resident proteins of the endomembrane system. An accumulation of unfolded proteins or a disruption in the specialized folding environment within the organelle causes ER stress, thus impairing the folding capacity of the ER. In response to this stress, the ER initiates a signaling cascade called the unfolded protein response (UPR) in an attempt to restore ER homeostasis. The vertebrate UPR is propagated by three ER-resident transmembrane proteins (i.e., PERK, IRE1α, and ATF6α), each initiating a signaling cascade that ultimately culminates in production of a transcriptional activator. The UPR was originally characterized as a pathway for the upregulation of ER chaperones, and a comprehensive body of subsequent work has shown that protein synthesis, folding, oxidation, trafficking, and degradation are all transcriptionally enhanced by the UPR. However, UPR activation is also accompanied by extensive mRNA suppression. The mechanisms responsible for this suppression and its consequences for physiological processes beyond the realm of ER protein folding and processing are only now beginning to be described. The overall goal of my thesis work was to explore this process of UPR-mediated gene suppression by identifying the mechanisms involved and the cellular processes affected. As a result, I characterized a novel mechanism of UPR-mediated transcriptional repression involving the translational regulation of the transcription factor C/EBPβ resulting in the suppression of the gene Il4ra, encoding an essential subunit of the IL-4/IL-13 receptor. As a consequence of this suppression, a novel effect of ER stress was identified in the impairment of IL-4/IL-13 signaling, a finding of potential significance in the study of inflammatory disease. In addition to this mechanism, I validated a novel approach to the identification of UPR-regulated transcription factors using publically available bioinformatic software. Through this analysis, I identified the transcription factor HNF4α as a novel post-translational UPR-regulated transcription factor, the regulation of which, resulted in the suppression of a number of lipid metabolic genes. This analysis not only identified a novel UPR-regulated transcription factor, but also presented a new tool for the characterization of UPR-mediated gene suppression. My work represents an independent and original investigation into the process of UPR-mediated gene suppression; and reveals that the UPR facilitates transcriptional suppression through the transcriptional, translational, and post-translational regulation of multiple transcription factors, resulting in the coordinated attenuation of physiological pathways. This function of the UPR is likely to contribute to metabolic, inflammatory, and other chronic disease states.

Le réticulum endoplasmique rugueux (RE), assure la synthèse, le repliement et la maturation des protéines de la voie de sécrétion. Les altérations des fonctions physiologiques du RE, entrainent l’accumulation de protéines mal repliées dans la lumière du RE, une condition appelée stress RE. En réponse au stress RE, un mécanisme compensatoire adaptatif appelé Unfolded Protein Response (UPR) est activé afin de restaurer l’homéostasie du RE et de permettre la survie de la cellule. Dans le cas d’un stress RE sévère ou prolongé, les altérations ne pouvant plus être compensées, la cellule est éliminée par apoptose contribuant ainsi au développement de pathologies cardiaques. Le but des recherches actuelles sur le stress RE en physiopathologie cardiaque n’est pas d’inhiber la réponse au stress RE, mais plutôt de la moduler afin de limiter l’apoptose des cardiomyocytes et de protéger le cœur. Dans ce contexte, nous avons mis en évidence que le stress RE induit une modification importante de l’architecture des cardiomyocytes associée à une altération de la fonction mitochondriale. De plus, nous avons montré que SIRT1, une désacétylase dépendante du NAD+, inhibe l’apoptose mitochondriale induite par un stress RE en limitant spécifiquement l’activation de la voie PERK de la réponse UPR via la désacétylation du facteur eIF2á sur la lysine K143. Enfin, nos résultats indiquent que SIRT1 protège les cardiomyocytes de l’apoptose induite par le stress RE en favorisant la mitophagie, via une activation de la voie de signalisation eEF2K/eEF2. Ces résultats montrent que SIRT1 est impliquée dans la régulation de la réponse autophagique et apoptotique des cardiomyocytes au stress RE et suggèrent que cette désacétylase serait une cible thérapeutique intéressante pour limiter le développement des pathologies cardiaques liées au stress RE. / The endoplasmic reticulum (ER) functions to properly synthesize, fold and process secreted and transmembrane proteins. Impairment of ER function induces an accumulation of misfolded proteins in the ER lumen, a condition termed ER stress. In response to ER stress, an adaptive compensatory mechanism called Unfolded Protein Response (UPR) is activated to restore ER homeostasis and promote cell survival. In the case of severe or prolonged ER stress, homeostasis cannot be restored and the cell is eliminated by apoptosis contributing to the development of cardiac pathologies. Currently, cardiac therapy based on ER stress modulation to conserve beneficial adaptations and to avoid cardiomyocyte apoptosis is viewed as a promising avenue towards effective therapies of ER stress-associated cardiac diseases.In this context, we demonstrated that ER stress induces architectural modifications and alterations of the mitochondrial function in cardiomyocytes. Furthermore, we showed that SIRT1, a NAD+-dependent deacetylase, inhibits mitochondrial apoptosis by modulating the activation of the PERK pathway of the UPR through deacetylation of the translation initiation factor eIF2á on lysine K143. Our results also indicate that SIRT1 protects cardiomyocyte from ER stress-induced apoptosis by activating mitophagy through eEF2K/eEF2 pathway. Collectively, these data demonstrate that SIRT1 regulates ER stress-induced autophagy and apoptosis in the heart and suggest that this deacetylase may be a therapeutic target to protect the heart against ER stress-induced injury.

Heavy ethanol use causes neurodegeneration manifested by neuronal loss and dysfunction. It is becoming imperative to delineate the underlying mechanism to promote the treatment of ethanol-induced neurodegeneration. Endoplasmic reticulum (ER) stress is a hallmark and an underlying mechanism of many neurodegenerative diseases. This study aims to investigate the role of ER stress in ethanol-induced neurodegeneration. In experimental design, adult mice were exposed to binge ethanol drinking by daily gavage for 1, 5, or 10 days and the response of ER stress was examined. We found the induction of ER stress appeared at 5 days and remained at 10 days. Moreover, the induction of ER stress was accompanied by an increase in neurodegeneration. With cell culture, we demonstrated that ethanol exposure resulted in neuronal apoptosis and that blocking ER stress by sodium phenylbutyrate (4-PBA) abolished ethanol-induced neuronal apoptosis, suggesting that ER stress contributes to ethanol-induced neurodegeneration. Mesencephalic astrocyte-derived neurotrophic factor (MANF) responds to ER stress and has been identified as a protein upregulated in ethanol-exposed developmental mouse brains. To investigate its implication in ethanol-induced neurodegeneration, we established a central nervous system (CNS)-specific Manf knockout mouse model and examined the effects of MANF deficiency on ethanol-induced neuronal apoptosis and ER stress using a third-trimester equivalent mouse model. We found MANF deficiency worsened ethanol-induced neuronal apoptosis and ER stress and that blocking ER stress abrogated the harmful effects of MANF deficiency on ethanol-induced neuronal apoptosis. Moreover, a whole transcriptome RNA sequencing supported the involvement of MANF in ER stress modulation and revealed candidates that may mediate the ER stress-buffering capacity of MANF. Collectively, these data suggest that MANF is neuroprotective against ethanol-induced neurodegeneration via ameliorating ER stress. Because MANF is a neurotrophic factor, we also examined the effects of MANF deficiency on neurogenesis. We observed that MANF deficiency increased neurogenesis in the subgranular zone of the hippocampal dentate gyrus and subventricular zone of the lateral ventricles in the mouse brain. Mechanistically, this finding was supported by a decrease of cell cycle inhibitors (p15 and p27), an increase of G2/M marker (phospho-histone H3), and an increase of neural progenitor markers (Sox2 and NeuroD1) in the brain of conditional Manf knockout mice. Our in vitro studies demonstrated that the gain-of-function of MANF inhibited cell cycle progression, whereas the loss-of-function of MANF promoted cell cycle progression. Taken together, these data suggest that MANF may affect the process of neurogenesis through altering cell cycle progression.

Das PCSK1-Gen kodiert für die Prohormonkonvertase 1/3 (PC1/3), einem Enzym, welches eine wichtige Rolle bei der Aktivierung von Prohormonen in ihre aktive Form spielt. Unter den Substraten der PC1/3 befinden sich Proinsulin, Proglukagon, Pro-POMC und andere Schlüsselmetabolite des Energiestoffwechsels. In genomweiten Assoziationsstudien wurde eine Korrelation zwischen Varianten in PCSK1 und Übergewicht gefunden. In Einzelfällen führte eine genetische bedingte PC1/3-Defizienz zu einem multiendokrinologischen Krankheitsbild mit schwerer Adipositas. Für einige der Varianten in PCSK1 wurde eine Retention im endoplasmatischen Retikulum (ER) gefunden. Retention im ER, ausgelöst durch fehlgefaltete Proteine, verursacht ER-Stress, welcher ggf. zu Apoptose führen kann. In dieser Arbeit wurde untersucht, ob 5 ausgewählte PCSK1-Varianten die Enzymreifung/-sekretion beeinträchtigen, ob ER-Stress induziert wird und letztlich, ob Apoptose induziert wird. Es fand sich für 2 Varianten eine Retention im ER und eine Induktion von ER-Stress. Apoptose wurde durch keine der untersuchten Varianten induziert.:Abkürzungsverzeichnis 1. Einführung und Hintergrund 1.1 Prohormonkonvertase 1/3 1.1.1. Entstehung und Reifung der Prohormonkonvertase 1/3 1.1.2. Substrate der Prohormonkonvertase 1/3 1.1.3. Prohormonkonvertase 1/3-Defizienz und Krankheitsbilder 1.1.4. Die untersuchten PCSK1-Varianten 1.2. Stress im endoplasmatischen Retikulum und Unfolded protein response 1.3. Ableitung der Rationalen 2. Publikation 3. Zusammenfassung Literaturverzeichnis A. Anhang A.1. Supplemental Information A.2. Methoden A.3. Darstellung des eigenen wissenschaftlichen Beitrags A.4. Erklärung über die eigenständige Abfassung der Arbeit A.5. Lebenslauf A.6. Liste der Veröffentlichungen A.7. Danksagung

La protéine Klotho (KL) possède des propriétés anti-vieillissement et anti-cancer. Les données cliniques montrent que l’expression de KL est associée à une meilleure survie des patients atteints de liposarcome. De plus, elle est réduite par rapport au tissu sain dans les liposarcomes dédifférenciés (DDLPS), un type de tumeur maligne rare mais de mauvais pronostic. Nos résultats montrent que KL sensibilise les DDLPS aux chimiothérapies (gemcitabine, navitoclax). L’abondance de KL dans les tumeurs pourrait donc servir de biomarqueur pour prédire l’efficacité des chimiothérapies et mettre en place une médecine plus personnalisée. De plus, des médicaments utilisés pour d’autres pathologies et connus pour stimuler l’expression de KL (Cozaar) pourraient être testés en association avec la chimiothérapie. Enfin, inspirés par le mode d’action de KL, nous avons testé la combinaison de la gemcitabine avec le navitoclax, qui s’est révélée très efficace sur les DDLPS. / Klotho (KL) is both an anti-ageing and anti-cancer protein. Analysis of clinical data highlights that high expression of KL is associated with a better overall survival of liposarcoma patients. Moreover, its expression in downregulated in dedifferentiated liposarcomas (DDLPS), a rare type of tumor associated with a poor prognosis due to high chemoresistance. Our results show that KL sensitizes DDLPS cells to chemotherapeutic agents (gemcitabine, navitoclax). So, abundance of KL in tumoral tissues could serve as a biomarker for predicting gemcitabine efficacy and so, could help for establishing personalized therapy. Moreover, drugs increasing KL expression could be tested in combination with chemotherapy. Based on KL mechanism of action, we also highlight that the combination between gemcitabine and navitoclax is very effective for killing DDLPS cells.

Sphingolipids are a class of lipid molecules that function both as structural membrane components and as bioactive signaling molecules. Sphingolipids can be produced de novo or salvaged and recycled. Despite the established roles of sphingolipids such as sphingosine 1-phosphate and ceramides in regulating signaling involved in pro- and anti-tumorigenic cellular processes, the role of the de novo sphingolipid biosynthesis pathway in cancer is unclear. The main objective of this thesis study was to determine whether there is an essential role for this pathway in cancer and whether its disruption can be a cancer-specific metabolic vulnerability. Here, we find that de novo sphingolipid synthesis through the rate-limiting enzyme serine palmitoyltransferase (SPT) is not required in cancer cells due to their salvage capacity. However, upregulation of SPT in cancer cells creates a requirement to detoxify its product, 3-ketodihydrosphingosine (3KDS), via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). We demonstrate that KDSR is essential in cancer cells both in vitro and in vivo to restrain the levels of its substrate 3KDS, the accumulation of which can disrupt ER structure and function, resulting in proteotoxic stress and cell death. Our findings also reveal that KDSR is essential specifically in cancer cells and not normal cells and that upregulation of SPT in cancer may act as a biomarker for sensitivity to targeting KDSR. Altogether, this thesis study provides new insights into the role of KDSR in the de novo sphingolipid biosynthesis pathway in both cancer and ER homeostasis and demonstrates the potential to exploit this for therapeutic purposes in a cancer-specific manner.