Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.

En esta tesis se estudian los efectos farmacológicos de derivados lipídicos frente el glioma y el Alzheimer. Los beneficios de este tipo de fármacos, basados en la terapia lipídica de membrana, están asociados con la modulación de la composición y las propiedades fisicoquímicas de membrana. En concreto, el ácido 2-hidroxioleico (2OHOA) es un potente fármaco antitumoral que fue diseñado para regular la composición y la estructura de la membrana lipídica así como la función de importantes proteínas de membrana. Por otro lado, el ácido 2-hidroxiaraquidónico (2OHARA), el ácido 2-hidroxieicosapentaenóico (2OHEPA), y el ácido 2-hidroxidocosahexanóico (2OHDHA) son derivados lipídicos hidroxilados que fueron diseñados en nuestro grupo de investigación para el tratamiento del Alzheimer. Este trabajo se ha basado en el estudio del funcionamiento de estos derivados de ácidos grasos hidroxilados en la modulación de las vías de señalización de la UPR (respuesta a las proteínas mal plegadas) y de la autofagia en células de glioma y células neuronales. / In this thesis, the pharmacological effects of lipid derivatives against glioma and Alzheimer's Disease are studied. The benefits of this type of drugs, which are based on the lipid membrane therapy, are associated with the modulation of the composition and physicochemical properties of membranes. 2-Hydroxyoleic acid (2OHOA) is a potent antitumor drug designed to regulate membrane lipid composition and structure and the function of important membrane proteins. In addition, 2- hydroxyarachidonic acid (2OHARA; LP204A1), 2-hydroxyeicosapentaenoic acid (2OHEPA; LP205A1), and 2-hydroxydocosahexanoic acid (2OHDHA; LP226A1) are new hydroxy derivated lipids designed in our group for the treatment of Alzheimer’s Disease. The main goal of this work was to study how these synthetic hydroxy derivates modulate the unfolded protein response and the autophagy pathways in glioma cells and neuron-like cells for Alzheimer’s Disease.

La mort cellulaire programmée (PCD pour Programmed Cell Death) est un processus essentiel aux cellules. Le PCD a d’abord été caractérisé dans le développement cellulaire et peut être divisé en plusieurs groupes selon les caractéristiques observées. L’apoptose, un sous-groupe du PCD, est caractérisé par plusieurs distinctions morphologiques et signalétiques attribué tout d’abord aux organismes complexes pour son rôle dans le développement et dans le maintien de l’intégrité tissulaire. Depuis la dernière décennie, de nombreuses études font état de l’existence d’un programme apoptotique dans des organismes unicellulaires comme les levures. Ce programme apoptotique a surtout été étudié chez les levures Saccharomyces cerevisiae et Schizosaccharomyces pombe et partage certaines caractéristiques avec l’apoptose des mammifères. Par contre, l’apoptose associé aux levures est distinct à certains égards entre autre par l’absence de certains homologues présents chez les mammifères. L’intérêt au niveau de l’étude du phénomène apoptotique chez les levures est sans cesse grandissant par la facilité avec laquelle les levures peuvent être utilisées comme système modèle. L’apoptose peut être induit dans les cellules de différentes façons en réponse à des stimuli internes ou externes. L’accumulation de protéines mal repliées au niveau du réticulum endoplasmique (RE) causant un stress est un inducteur bien caractérisé de la voie apoptotique. La signalisation de l’apoptose dans un cas de stress au RE fait appel aux transducteurs des signaux de la voie du UPR ( Unfolded Protein Response). Récemment, il a été montré que la calnexine, une chaperone transmembranaire du RE connue et caractérisée surtout pour ses fonctions d’aide au repliement des protéines et au contrôle de qualité, joue un rôle dans la transduction du signal apoptotique en réponse au stress du RE chez mammifères. Le rôle de la calnexine dans ce cas consiste principalement en l’échafaudage pour le clivage par la caspase 8 de la protéine apoptotique Bap31. Nous avons tout d’abord démontré que le stress du RE et que la déficience en inositol, un précurseur essentiel de nombreuses molécules signalétiques, sont deux inducteurs de l’apoptose chez la levure S. pombe. Ces deux voies semblent induire l’apoptose par deux voies distinctes puisque seule la voie de la déficience en inositol induit l’apoptose de façon dépendante à la métacaspase Pca1p. La calnexine, essentielle à la viabilité chez la levure S. pombe, est impliquée dans ces deux phénomènes apoptotiques. L’apoptose induit par le stress du RE nécessite une version de la calnexine ancrée à la membrane du RE pour être optimal. De façon opposée, l’apoptose induit par une déficience en inositol nécessite la présence de la queue cytosolique ancrée à la membrane de la calnexine pour être retardé. Ces deux actions différentes imputables à une même protéine laisse croire à une double fonction pro et anti-apoptotique de celle-ci. Suite à la découverte de l’existence d’un clivage endogène de la calnexine en situation normale de croissance, un modèle a été élaboré expliquant les rôles distincts de la calnexine dans ces deux voies apoptotiques. Ce modèle fait état d’un rôle associé au clivage de la calnexine dans l’apoptose. / Programmed Cell Death (PCD) is an essential process to the cells. PCD was first characterized in cell development and can be separated in sub-groups depending of cell death characteristics observed. Apoptosis is one of the PCD sub-groups that was first associated to complex organisms for its roles in cell development and in maintenance of tissues integrity. The apoptotic pathway is characterized by specific morphological and signalization characteristics. In the last ten years, numerous studies demonstrated the existence of apoptosis in unicellular organisms such as yeast. This apoptotic program was extensively studied in the two yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe and share characteristics with the mammalian one. However, yeast apoptosis is distinctive at many points as yeast do not encodes all the mammalian homologues of the apoptotic pathway. Although yeast and mammalian apoptosis seems to differs, the interest about yeast apoptosis is growing given that yeast is an excellent and easily tractable model system. External and internal stimuli can induce apoptosis by different ways. Accumulation of unfolded or incompletely folded proteins in the endoplasmic reticulum (ER) causing ER stress is a well-known inducer of the apoptotic pathway. Signalization of ER-stress induced apoptosis involves the same transducers than the UPR (Unfolded Protein Response) pathway. It was recently shown that calnexin, a transmembrane chaperone of the ER, is implicated in ER-stress apoptosis in mammalian cells. In this particular case, it was demonstrated that calnexin acts as a scaffold in the cleavage of the apoptotic protein Bap31 by caspase 8. We demonstrated that ER stress and inositol deficiency, a precursor of many important signalization molecules, are two situations leading to apoptosis in the yeast S. pombe. These two pathways leading to apoptosis seems to differ as only inositol deficiency is dependant of the yeast metacaspase Pca1p. We also demonstrated that S. pombe calnexin, essential for cell viability of this yeast, takes part in these two apoptotic process. ER stress induced apoptosis needs a calnexin anchors to the ER membrane to be efficient. However, apoptosis induced by inositol starvation needs the calnexin C-terminal tail with the transmembrane domain to be delayed. These two opposite actions from the same protein lead to the hypothesis that calnexin encodes both pro and anti-apoptotic functions. By the discovery that calnexin is cleaved under normal culture conditions, a model was elaborated explaining the distinctive roles of calnexin in these two apoptotic pathways. This model proposed a role of calnexin cleavage to apoptosis.

Plusieurs expériences et études cliniques ont démontré que l’activation du système rénine-angiotensine (RAS) peut induire l’hypertension, un facteur de risque majeur pour les maladies cardiovasculaires et rénales. L’angiotensinogène (Agt) est l’unique substrat du RAS. Cependant, il n’a pas encore été démontré si l’activation du RAS intrarénal peut à elle seule induire des dommages rénaux, indépendamment de l’hypertension systémique, et ainsi jouer un rôle prépondérant dans la progression de la néphropathie diabétique. Afin d’explorer le rôle du RAS intrarénal dans les dommages rénaux, un diabète a été induit par l’injection de streptozotocin chez des souris transgéniques (Tg) surexprimant l’Agt de rat dans les cellules des tubules proximaux du rein (RPTC). Les souris Tg diabétiques ont été traitées soit avec des inhibiteurs du RAS (perindopril et losartan), de l’insuline ou une combinaison des deux pour 4 semaines avant d’être euthanasiées. Pour une autre étude, des souris Tg non-diabétiques ont été traitées soit avec des inhibiteurs du RAS, l’hydralazine (vasodilatateur) ou l’apocynine (inhibiteur de la NADPH oxydase) pour une période de 8 semaines avant l’euthanasie. Des souris non-Tg ont été utilisées comme contrôles. Des cellules immortalisées de tubule proximal de rat (IRPTC) transfectées de manière stable avec un plasmide contenant l’Agt ou un plasmide contrôle ont été employées comme modèle in vitro. Nos résultats ont démontré que les souris Tg présentaient une augmentation significative de la pression systolique, l’albuminurie, l’apoptose des RPTC et l’expression de gènes pro-apoptotiques par rapport aux souris non-Tg. Les mêmes changements ont été observés chez les souris Tg diabétiques par rapport aux souris non-Tg diabétiques. L’insuline et/ou les inhibiteurs du RAS ont permis d’atténuer ces changements, sauf l’hypertension qui n’était réduite que par les inhibiteurs du RAS. Chez les IRPTC transfectées avec l’Agt in vitro, les hautes concentrations de glucose augmentent l’apoptose et l’activité de la caspase-3 par rapport aux cellules contrôles et l’insuline et/ou les inhibiteurs du RAS empêchent ces augmentations. En plus des changements physiologiques, les RPTC des souris Tg présentent aussi une augmentation significative de la production des espèces réactive de l’oxygène (ROS) et de l’activité de la NADPH oxydase, ainsi qu’une augmentation de l’expression du facteur de croissance transformant-beta 1 (TGF-β1), de l’inhibiteur activateur du plasminogène de type 1 (PAI-1), des protéines de la matrice extracellulaire, du collagène de type IV et de la sousunité p47 de la NADPH oxydase. Le traitement des souris Tg avec l’apocynine et le perindopril a permis d’améliorer tous ces changements, sauf l’hypertension qui n’était pas corrigée par l’apocynine. D’autre part, l’hydralazine a prévenu l’hypertension, sans modifier l’albuminurie, l’apoptose des RPTC ou l’expression des gènes pro-apoptotiques. Ces résultats montrent bien que l’activation du RAS intrarénal et l’hyperglycémie agissent de concert pour induire l’albuminurie et l’apoptose des RPTC, indépendamment de l’hypertension systémique. La génération des ROS via l’activation de la NADPH oxydase induit en partie l’action du RAS intrarénal sur l’apoptose des RPTC, la fibrose tubulo-interstitielle et l’albuminurie chez les souris Tg. D’autre part, une expérience en cours a tenté d’encore mieux délimiter les effets de l’activation du RAS intrarénal, tout en éliminant la néphrotoxicité du STZ. Pour cette étude, les souris Tg surexprimant l’Agt de rat dans leurs RPTC ont été croisées aux souris Ins2Akita, un modèle spontané de diabète de type I, afin de générer des souris Akita-rAgt-Tg. Les résultats préliminaires indiquent que le RAS intrarénal est activé dans les souris Akita et que la combinaison avec l’hyperglycémie induit du stress du réticulum endoplasmique (ER) dans les RPTC in vivo. Le stress du ER contribue à l’apoptose des RPTC observée dans le diabète, à tout le moins dans le modèle Akita. Le traitement avec des inhibiteurs du RAS permet d’atténuer certains des dommanges rénaux observés dans les souris Akita-rAgt-Tg. / Experimental and clinical studies have shown that renin-angiotensin system (RAS)activation may lead to hypertension, a major cardiovascular and renal risk factor. Angiotensinogen (Agt) is the sole substrate of the RAS. However, it is unclear whether intrarenal RAS activation alone could induce kidney injury independently of systemic hypertension and play an important role in the progression of diabetic nephropathy (DN). To explore the role of intrarenal RAS in kidney injury, transgenic (Tg) mice overexpressing rat Agt in their renal proximal tubular cells (RPTCs) were rendered diabetic by streptozotocin (STZ). Diabetic Tg mice were treated with RAS blockers (perindopril and losartan), insulin or a combination of both and then euthanized after 4 weeks of treatment. In a separate study, non-diabetic Tg mice were treated with RAS blockers or hydralazine (a vasodilator) or apocynin (an NADPH oxidase inhibitor) and then euthanized after 8 weeks of treatment. Non-Tg littermates served as controls in both studies. Immortalized rat proximal tubule cells (IRPTCs) stably transfected with Agt cDNA or control plasmid were used in the experiments as an in vitro model. Our results showed that non-diabetic Tg mice displayed a significant increase in systolic blood pressure (SBP), albuminuria, RPTC apoptosis, and proapoptotic gene expression. Diabetic Tg mice had a further increase of albuminuria, RPTC apoptosis, and proapoptotic gene expression, though the SBP of the diabetic Tg mice was similar to that of non-diabetic Tg mice. RAS blockers and/or insulin treatments markedly attenuated these changes, except that insulin had no impact on hypertension. In vitro, high-glucose melieu significantly increased apoptosis and caspase-3 activity in Agt stable transfectants compared to control cells, and these changes were attenuated by insulin and/or RAS blockers. Furthermore, non-diabetic Tg mice showed significantly elevated reactive oxygen species (ROS) production and NADPH oxidase activity, as well as enhanced expression of transforming growth factor-beta 1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), extracellular matrix proteins, collagen type IV, and NADPH oxidase subunit p47 in their RPTC. Treatment with apocynin and perindopril ameliorated these changes, but apocynin had no effect on SBP. In contrast, hydralazine prevented hypertension but not albuminuria, RPTC apoptosis, or proapoptotic gene expression. These data indicate that intrarenal RAS activation and hyperglycemia act in concert to induce albuminuria and RPTC apoptosis independent of systemic hypertension. ROS generation via NADPH oxidase activation mediates, at least in part, intrarenal RAS action on RPTC apoptosis, tubulointerstitial fibrosis and albuminuria in Tg mice. On the other hand, in an on-going experiment, to avoid the nephro-toxic effects of STZ and further delineate the effects of intrarenal RAS activation, Tg mice overexpressing rat Agt in their RPTCs were crossbred with Ins2Akita mice, a spontaneous type I diabetes model, to generate Akita-rAgt-Tg mice. Preliminary data indicated that hyperglycaemia and intrarenal RAS activation induced endoplasmic reticulum (ER) stress in RPTC in vivo, and the ER stress pathway contributed to RPTC apoptosis in diabetes, at least in the Akita model. RAS blockade was effective in attenuating some parameters of renal injury in AkitarAgt-Tg mice.

No description available.

Plasmodium falciparum is responsible for the most virulent form of human malaria. The biology of the intra-erythrocytic stage of P. falciparum is the most well studied as it is this stage that marks the clinical manifestation of malaria. To establish a successful infection, P. falciparum brings about extensive remodeling of erythrocytes, its host compartment. The infected erythrocytes harbor several parasite induced membranous structures. Most importantly, pathogenesis related structures termed knobs, which impart cytoadherence, appear on the cell surface of the infected erythrocytes. For bringing about such eccentric renovations in its host compartment, the parasite exports 8% of its genome (~400 proteins) to various destinations in the host cell. Studies from our lab have shown that proteins belonging to heat shock protein40 (Hsp40) and heat shock protein70 (Hsp70) group of chaperones are also exported to the host compartment. We and others have implicated these chaperones in important processes such as protein trafficking and chaperoning assembly of parasitic proteins into the cytoadherent knobs. As detailed above, malaria parasite invests a lot of energy in exporting a large number of proteins including chaperones in the red blood cell to meet its pathogenic demands. In order to do so, it heavily relies on its secretory pathway. However, it is known that the parasite experiences a significant amount of oxidative stress on account of heme detoxification, its own metabolism and the immune system of the host. The parasite also effluxes large quantities of reduced thiols such as glutathione and homocysteine into the extracellular milieu indicative of redox perturbation. Additionally, the parasite lacks Peroxiredoxin IV, which otherwise localizes in the ER and carries out detoxification of peroxide generated as a result of oxidative protein folding. Together, these factors indicate that maintaining redox homeostasis is a challenging task for the parasite. It also implies that the ER, where the redox balance is even more critical as it requires oxidising environment for protein folding, is predisposed to stress. In light of this fact and the importance of secretory pathway in malaria pathogenesis, we decided to address the ways and mechanisms used by the parasite to tackle perturbations in its secretory pathway. Examination of a canonical unfolded protein response pathway in P. falciparum ER-stress is a condition arising whenever the load of unfolded proteins increases the folding capacity of the ER. However, eukaryotes have evolved a fairly well conserved homeostatic response pathway known as unfolded protein response (UPR) to tackle ER-stress. This signal transduction pathway is composed of three arms involving three ER-transmembrane signal transducers namely; IRE1, ATF6 and PERK. IRE1 brings about splicing of a bZIP transcription factor, XBP1/Hac1 and ATF6 becomes activated upon getting proteolytically cleaved in the Golgi. These transcription factors then migrate to the nucleus where they bind onto the ER-stress elements thereby, leading to the transcriptional up-regulation of the UPR targets such as ER chaperones and components of ER associated degradation (ERAD) pathway which rescue the function of the ER. PERK on the other hand brings about translational attenuation by phosphorylating eIF2α, thereby providing parasite the benefit of time to recover. We started our examination on UPR in Plasmodium by carrying out in silico analysis of the major components of UPR in the parasite by using Homo sapiens protein sequences as the query. We found that the parasite lacks the homologues of all the transcriptional regulators of canonical UPR. Only PERK component of the UPR was found to be present in the parasite. To rule out the existence of the canonical UPR in P. falciparum, we examined the status of UPR targets by subjecting the parasites to treatment with DTT. DTT perturbs the disulfide oxidation in the ER and thereby inhibits protein folding leading to ER-stress. Owing to the missing components of a canonical UPR, we did not find up-regulation of known UPR targets such as ER-chaperones including PfBiP, PfGrp94, PfPDI and ERAD marker Derlin1 at transcript as well as protein level. Owing to the presence of a PERK homologue, phosphorylation of eIF2α followed by attenuation of protein synthesis was observed upon subjecting the parasites to DTT mediated ER-stress. In the absence of a canonical UPR, the parasites were found to be hypersensitive to ER-stress in comparison to the mammalian counterpart. In the presence of DTT, the parasites showed perturbation in the redox homeostasis as indicated by increase in the levels of ROS. Next, we sought to examine if the parasites resorted to any alternate means of increasing the availability of chaperones in the ER. For this, we analysed the involvement of another Hsp70 family member, Hsp70-x which is homologous to BiP and which is known to traverse the ER while getting exported to the erythrocyte compartment. Interestingly, we found that upon exposure to ER-stress, the export of this protein is partially blocked and around 30% of the protein is retained in the ER. On the other hand, there was no effect on the trafficking of another exported chaperone KAHsp40. This indicates that the parasite possibly recruits this pool of retained Hsp70-x for the chaperoning of unfolded proteins in the ER. Global response to ER-stress in P. falciparum To dig deeper into the parasite specific strategies employed for dealing with ER-stress at a global level, we carried out high throughput transcriptomic and proteomic analysis upon subjecting the parasites to DTT mediated ER-stress. Microarray based gene expression profiling was carried out upon subjecting the parasites to DTT mediated ER-stress. We found that the parasite mounts a transcriptional response as indicated by up-regulation of 155 transcripts. In congruence with our biochemical analysis, we did not find up-regulation of ER chaperones as well as ERAD proteins. Functional grouping of the up-regulated genes revealed large number of hypothetical proteins in our list of differentially expressed genes. The genes encoding exported proteins represent yet another abundant class. In the course of examining the involvement of Plasmodium specific transcriptional regulators mediating response to DTT induced ER-stress, we identified 4 genes belonging to the family of AP2 transcription factors. AP2 (Apetela-2) are specific transcription factors which are possessed by apicomplexa and bring about regulation of developmental processes and stress response in plants. On comparing our list of up-regulated genes with the previously known targets of AP2 factors, we found that an entire cascade of AP2 factors is up-regulated upon DTT-mediated ER stress. Thus, AP2 factors appear to be the major stress response mediators as they are together responsible for the up-regulation of 60% of genes identified in this study. In addition, another striking observation made, was the up-regulation of a few sexual stage specific transcripts. 2D Gel electrophoresis and 2D-DIGE based Proteomic analysis indicated an up-regulation of secretory proteins and some components of vesicular trafficking and secretory machinery possibly to overcome the block in the functions of the secretory pathway. ER-stress triggers stage transition in P. falciparum Intrigued by the up-regulation of a few sexual stage specific genes, we were curious to examine if there was a functional significance of this observation. To this end, we decided to investigate the effect of ER-stress on induction of gametocytes, the only sexual stage found in humans. Indeed, we found a two fold induction in the numbers of gametocytes formed upon challenging the parasite with DTT mediated ER-stress. The induction of gametocytogenesis was also observed by using a clinical isolate of P. falciparum for the assay. The DTT treated cultures progressed through the gametocytogenesis pathway normally forming all the five morphologically distinct stages. Then we sought to examine if this phenomenon could be simulated in the physiological scenario as well. For this, we made use of a rodent model of malaria, P. berghei. Two different treatment regimes involving 1) direct injection of increasing concentration of DTT into P. berghei infected mice and 2) injection of DTT pretreated P. berghei infected erythrocytes into healthy mice were followed. In both cases, a significant increase in the gametocyte induction was observed. Having seen that Plasmodium undergoes gametocytogenesis upon exposure to ER-stress not only in in vitro cultures but also in in vivo scenario, we wanted to identify the players involved in the commitment to sexual stage. Recently, a transcription factor belonging to AP2 class of transcription factors, referred to as AP2-G has been implicated in committing the asexual parasites for transition to gametocyte stage. To examine the role of this factor in the phenotype observed by us, we looked at the effect of DTT on AP2-G. Interestingly, we found around 6 folds up-regulation in the expression of AP2-G levels under ER-stress. The downstream targets of AP2-G, many of which are the markers of gametocyte were also found to be up-regulated upon being exposed to DTT mediated ER-stress indicating the launch of a transcriptional program which together works in the direction of transition to gametocytes. Having seen that P. falciparum undergoes ametocytogenesis in response to DTT treatment both under in vitro and in vivo conditions, we sought to look for probable physiological analogue of DTT. Since glutathione is the major cellular redox buffer, critical for redox homeostasis, we quantitated the levels of both oxidized and reduced forms of this non protein thiol using Mass Spectrometric approach. We found that the levels of reduced forms of glutathione significantly increased upon treating the parasites with DTT. This indicates that the levels of glutathione could be one of the physiological triggers of gametocytogenesis. Conclusion In conclusion, our study analyses the ways and mechanisms employed by malaria parasite to cope with perturbations to its secretory pathway. We have established the absence of a canonical UPR in this parasite and our results suggest that Plasmodium has developed a three stage response to cope with ER stress: 1) an early adaptation to increase the local concentration of chaperones in the ER by partially blocking the export of a Hsp70 family member, 2) activation of gene expression cascade involving AP2 transcription factors and 3) a consequent switch to the transmissible sexual stage. Hence, our study throws light on a novel physiological adaptation utilised by malaria parasite to tackle stress to its secretory pathway. Gametocytogenesis, which can be transmitted to the mosquito vector, could hence serve as an effective means to escape ER-stress altogether. Importantly, while it is widely known that stress brings about switch towards sexual stages in P. falciparum, the molecular triggers involved in this process remain obscure in the field of malaria biology. Therefore, our findings also address this long standing question by providing the evidence of ER-stress being one such trigger required for switching to the transmissible sexual stages.

Les acides gras saturés (AGS) altèrent la fonctionnalité des organites dans de nombreux types cellulaires. Il a été proposé que ce processus, également nommé lipointoxication, puisse être responsable de plusieurs pathologies humaines telles que le diabète de Type 2.Au niveau cellulaire, l'accumulation d'AGS est associée à une augmentation du taux de saturation des phospholipides (PL) membranaires, les composants majoritaires des membranes des organites, mais également du taux de céramides, impliqués dans l'induction de l'apoptose.Dans une première partie de ce travail, nous avons étudié, chez le modèle cellulaire simple Saccharomyces cerevisiae, la contribution relative des PL saturés et des céramides à la cytotoxicité des AGS. Nous avons pu démontrer que les céramides agissaient à des étapes précoces de la voie de sécrétion, alors que les PL saturés impactaient des étapes plus tardives en altérant en particulier la formation de vésicules de sécrétion.Parallèlement, nous avons également constaté que le taux d'AGS était significativement augmenté dans les PL membranaires des patients atteints d'une maladie génétique, la mucoviscidose. La mutation la plus fréquente responsable de cette maladie, résulte en la rétention de la protéine correspondante dans le réticulum endoplasmique. Des molécules pharmacologiques, capables de corriger le trafic de la protéine à sa destination finale ont été isolées in vitro, mais des limitations importantes ont pu être observées lors des tests cliniques. Nous proposons dans le présent manuscrit que la lipointoxication liée aux AGS pourrait être un écueil important à l'utilisation des correcteurs actuels pour le traitement de la mucoviscidose. / Saturated fatty acids (SFA) have been reported to alter organelle integrity in many cell types. This process, also known as lipotoxicity, has been proposed to be responsible for several human pathologies such as type 2 diabetes.At the cellular level, SFA accumulation is associated with an increase of the saturation rate of membrane phospholipids (PL), the major components of organelle membranes, and an increase of ceramides levels, implicated in apoptosis induction.In the first part of this work, we took advantage of a simple yeast-based model to study the relative contributions of saturated PL and ceramides to SFA cytotoxicity. We demonstrated that ceramides act early in the secretory pathway, while saturated PL impact the later steps, and particularly the formation of secretory vesicles.In parallel, we observed that SFA amounts were significantly increased in the membrane PL of cystic fibrosis (CF) patient cells. The most common mutation responsible for this genetic disease results in the retention of the corresponding protein in the endoplasmic reticulum. Pharmacological agents, which correct the mistrafficking of the protein, have been isolated in vitro, but they did not show significant improvements in clinical trials. We propose in the present manuscript, that SFA-related lipointoxication could be an important bottleneck for the use of these pharmacological agents in clinical trials.

The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells. This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR). The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6. The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK. <p>Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the immune cells infiltrating the pancreatic islets. Our group has previously shown that the pro-inflamatory cytokines interleukin-1beta (IL1-beta and interferon-gamma (IFN-gamma), via nitric oxide (NO) formation, downregulate expression and function of the ER Ca2+ pump SERCA2. This depletes beta-cell ER Ca2+ stores, leading to ER stress and apoptosis. Of note, IL1-beta alone triggers ER stress but does not induce beta-cell death, while IFN-gamma neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown.<p>Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis.<p>In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (<25%).<p>The first goal of the work was to understand the synergistic effects of IL1-beta and IFN-gamma leading to pancreatic beta-cell apoptosis. Our group previously observed, by microarray analysis of primary beta-cells, that IFN-gamma down-regulates mRNAs encoding for some ER chaperones. Against this background, our hypothesis was that IFN-gamma aggravates beta-cell ER stress by decreasing the ability of these cells to mount an adequate UPR. To test this hypothesis, we investigated whether IFN-gamma pre-treatment augments CPA-induced ER stress and beta cell death. The results obtained indicated that IFN-gamma pre-treatment potentiates CPA-induced apoptosis in INS-1E and primary beta-cells. This effect was specific for IFN-gamma since neither IL1-beta nor a low dose CPA pre-treatment potentiated CPA-induced apoptosis in INS-1E cells. These effects of IFN-gamma were mediated via the down regulation of genes involved in beta cell defense against ER stress, including the ER chaperones BiP, Orp150 and Grp94 as well as Sec61, a component of the ERAD pathway. This had functional consequences as evidenced by a decreased basal and CPA-induced activity of a reporter construct for the unfolded protein response element (UPRE) and augmented expression of the pro-apoptotic transcription factor Chop. <p>We next investigated the molecular regulation of the Chop gene in INS-1E cells in response to several pro-apoptotic and ER stress inducing agents, namely cytokines (IL1-beta and IFN-gamma), palmitate, or CPA. Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by these compounds. While cytokines (via NO production)- and palmitate-induced Chop expression was mediated via a C/EBP-ATF composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the ER stress response element (ERSE). Cytokines, palmitate and CPA induced ATF4 protein expression and further binding to the C/EBP-ATF composite site, as shown by Western blot and EMSA experiments. There was also formation of distinct AP-1 dimers and binding to the AP-1 site after exposure to cytokines or palmitate. <p>\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

L'obésité est un facteur de risque majeur de cancer. Il est connu qu’une adiposité élevée prédispose à un stress inflammatoire accru et potentialise la croissance tumorale. Néanmoins, les mécanismes restent mal définis. De façon intéressante, la sénescence cellulaire, ou le programme moléculaire causant l’arrêt du cycle cellulaire suite à un stress insurmontable, favorise l'inflammation chronique et délétère pendant l'obésité. Nous avons donc émis l’hypothèse que l'obésité puisse être un inducteur de sénescence protumoral qu’il est possible d’exploiter, via une stratégie sénolytique, pour ralentir ou même bloquer le développement de tumeurs. Grâce à des marquages de coupes histologiques de tumeurs métastatiques, nous avons montré que les masses malignes de patients ayant un indice de masse corporelle (IMC)>35 sont associées à des marqueurs de sénescence. Cette découverte suggère une charge élevée de cellules sénescentes chez ses patients. Alors que la sénolyse, ou l’élimination thérapeutique des cellules sénescentes, s'est révélée très prometteuse dans le traitement de plusieurs maladies liées à l'âge, son efficacité en tant que traitement du cancer est souvent mitigé et dépend des antécédents du patient. Dans notre étude, nous avons utilisé un modèle murin d'obésité induit par la diète combinée avec un modèle d’injections syngéniques de différentes lignées cancéreuses occasionnant des réponses immunogéniques faibles, légères ou hautes. Chez les souris sur une diète riche en gras, nous avons identifié des cellules cancéreuses sénescentes spécifiquement dans les tumeurs faiblement immunogènes, soit faiblement reconnue par le système immunitaire et donc difficile à traiter. Un traitement sénolytique avec l'inhibiteur de la famille BCL-2 ABT-263 abolit la réponse protumorale observée via l'ablation des cellules cancéreuses sénescentes. Ainsi, nous proposons que les thérapies combinatoires avec des agents sénolytiques devraient être envisagées pour traiter les patients cancéreux présentant une adiposité accrue. De plus, dans la même cohorte de patients où nous avons rapporté des marqueurs de sénescence dans les tissus malins, les patients obèses ont aussi montré une expression importante de Toll-like receptor 4 (TLR4). Nous avons donc émis l’hypothèse que le récepteur TLR4 joue un rôle important dans l’établissement d’un microenvironnement tumoral qui favorise la sénescence cellulaire et la croissance tumorale de souris en surplus de poids. Dans notre étude, nous rapportons que l'expression systémique de TLR4 est importante pour la croissance tumorale induite par l'obésité. Nous montrons également que l’induction d’un stress du réticulum endoplasmique médié par Inositol requiring enzyme 1a (IRE1ɑ) dans les cellules myéloïdes associées à une tumeur, favorise la sénescence des cellules cancéreuses, dans un contexte de faible immunogénicité, via TLR4. Ce travail établit les fondements d’une compréhension moléculaire du lien entre les régimes à forte teneur calorique et l'immunité protumorale. / Obesity is a major risk factor for cancer. High adiposity predisposes to increased inflammatory stress, which potentiates tumor growth. However, the mechanisms remain poorly defined. Interestingly, cellular senescence, or the molecular program causing cell cycle arrest following insurmountable stress, is known to promote chronic and deleterious inflammation during obesity. We therefore hypothesized that obesity could be an inducer of a protumoral senescence that can be exploited, via a senolytic strategy, to slow down or even block tumor development. Through histological sections of metastatic tumor, we show that malignant masses from patients with a body mass index (BMI)>35 are associated with markers of senescence, suggesting a high burden of senescent cells in these patients. While senolysis, or the therapeutic elimination of senescent cells, has shown great promises in the treatment of several age-related diseases, its efficacy as a treatment for cancer is often elusive and depends on patients’ history. In our study, we used a mouse model of diet-induced obesity (DIO) combined with a model of syngeneic injections of different cancer cell lines causing low, mild, or high immunogenic responses. In mice under a DIO, we have identified senescent cancer cells specifically in weakly immunogenic tumors, or tumors poorly recognized by the immune system, and therefore difficult to treat. Moreover, a senolytic treatment with the BCL-2 family inhibitor ABT-263 abolishes the protumor response seen in these mice via the ablation of senescent cancer cells. Thus, combination therapies using senolytic agents should fall into consideration to treat cancer patients with increased adiposity. In addition, in the same cohort of patients where we reported markers of senescence in malignant tissues, obese patients also showed significant expression of TLR4. We therefore hypothesized that the TLR4 receptor plays an important role in establishing a tumor microenvironment that promotes cellular senescence and tumor growth in mice subjected to experimental obesity. In our second study, we report that systemic expression of TLR4 is important for obesity-induced tumor growth. Moreover, we show that the induction of an IRE1ɑ-mediated endoplasmic reticulum stress, in tumor-associated myeloid cells, promotes the senescence of cancer cells, in a context of low immunogenicity, via TLR4. This work lays the foundation for a molecular understanding of the link between high-calorie diets and protumoral immunity.

<p>The ß-carbolines norharman and harman structurally resemble the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that is known for its ability to damage neuromelanin-containing dopaminergic neurons of the substantia nigra and thereby induce parkinsonism. MPTP is, however, not normally present in the environment whereas the ß-carbolines are present in cooked food and tobacco smoke. </p><p>In this thesis it was demonstrated that norharman and harman had affinity to melanin and were retained in neuromelanin-containing neurons of frogs up to 30 days post-injection (the longest survival time examined). It was also demonstrated that norharman induced neurodegeneration, activation of glia cells and motor impairment in mice. Furthermore, this compound induced ER stress and cell death in PC12 cells. An in vitro model of dopamine melanin-loaded PC12 cells was developed in order to study the effect of melanin on norharman-induced toxicity. In this model, melanin seemed to attenuate toxicity induced by low concentrations of norharman. After exposure to the highest concentration of norharman, melanin clusters were disaggregated and there was an increased expression of stress proteins and caspases-3, known to be involved in apoptosis.</p><p>The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[<i>a</i>]anthracene was demonstrated to have a CYP1A1-dependent localization in endothelial cells in the choroid plexus, in the veins in the leptomeninges and in the cerebral veins of mice pre-treated with CYP1-inducers. </p><p>These results demonstrate that the distribution of environmental compounds could be influenced by the presence of neuromelanin and expression of CYP enzymes in the brain and that norharman may induce neurotoxic effects in vivo and in vitro.</p>