Résumé confidentiel / Confidential abstract

La maladie d'Alzheimer est une pathologie neurodégénérative progressive liée à l'âge qui détériore premièrement les fonctions liées aux mémoires de travail et épisodiques, avant de s'étendre à l'ensemble des procédures mémorielles dans les stades plus avancés. L'ensemble des traitements existant à ce jour sont palliatifs. Au niveau histologique, la maladie d'Alzheimer est caractérisée par l'accumulation extra- et intracellulaire de différentes protéines agrégées (appelées amyloïde) dans les tissus cérébraux, entrainant des dysfonctions importantes du circuit neuronal. De fait, la majorité des approches thérapeutiques en développement consistent à tenter de réduire ou supprimer ces agrégats protéiques. Cependant, la maladie d'Alzheimer étant étroitement corrélée au vieillissement, certaines de ses caractéristiques biologiques sont parfois confondues avec celles du vieillissement non pathologique. L'une de ces caractéristiques est la diminution des différents mécanismes liés à l'homéostasie protéique (protéostasie). L'hypothèse réalisée au cours de mes travaux est que le rétablissement de ces mécanismes diminués par l'âge constituerait une approche thérapeutique crédible, complémentaire aux approches actuelles, à la pathologie complexe qu'est la maladie d'Alzheimer. C'est en suivant cette optique que je me suis intéressé au rôle et à la régulation de l'un des systèmes majeusr du contrôle de la protéostasie : l'UPR (unfolded protein response), et en particulier au facteur de transcription XBP-1s, considéré comme l'une des pièces maîtresses de ce réseau de signalisation cellulaire / Alzheimer's disease is a neurodegenerative pathology strongly correlated to aging. Its symptoms are characterized by an impaired short term memory process in the early stages of the disease and later on by a loss of all type of memory process. There is actually no cure for this pathology. At the histo-pathological levels, the disease show an accumulation of aggregated proteins in the brain (called amyloid protein) in the intra or extra cellular space, which act as a disruptor of the normal neuronal function and activity. Thus, most of the therapeutic approach to treat the disease aim at removing those proteins aggregates from the brain. However, some of the Alzheimer's disease characteristics could be melded with normal aging : One such case is the global decrease of the proteostasis mechanism in the cell which normally happen in normal brain. The assumption made during this work is that the recovery of these mechanisms impaired by age would constitute a credible therapeutic approach, complementary to the other existing approaches to the complex disease that is Alzheimer's disease. Following this hypothesis I was interested in the role and regulation of one of the major system controlling proteostasis: the UPR (unfolded protein response), and particulary to the XBP-1s transcription factor , considered one of the master regulator of this cellular network

Master of Science / Department of Animal Sciences and Industry / Barry Bradford / During the transition period, 3 wk before to 3 wk after calving, dairy cows experience a variety of sudden hormonal and metabolic shifts that could result in metabolic disorders or diseases, which can be detrimental to the productive life and longevity of the cow. Cows undergo a negative energy balance, where they cannot consume enough feed to meet their energy requirements. To make up this deficit, cows mobilize adipose tissue in the form of non-esterified fatty acids (NEFA) which are transported to the liver and are either used for fuel or stored as triglycerides. High levels of circulating NEFA can lead to endoplasmic reticulum (ER) stress, which is linked to inflammation. This low-grade inflammation can compromise cell function. To mitigate this inflammation, sodium salicylate, a non-steroidal anti-inflammatory drug (NSAID), was given to mature (3+ parity) cows for 7 d after parturition via their drinking water. Blood was collected daily and a glucose turnover assay was performed. Liver, muscle, and adipose tissue was collected on d 7. Overall, it appeared that SS increased insulin sensitivity and depressed gluconeogenesis post-transcriptionally. Multiple in vitro studies were performed on immortalized bovine mammary epithelium (MAC-T) cells to determine the action of SS when ER stress was induced with palmitate (PALM). Treatment with SS did not mitigate, and in some cases exacerbated, the ER stress response. The addition of bovine serum albumin (BSA), a common component of cell culture media, may alter reactive oxygen species (ROS) measurements due to its antioxidant property. Overall, SS seems to alter metabolic processes and the cellular response to stress.

L'activation chronique du stress du réticulum endoplasmique (RE) est une caractéristique commune à de nombreux cancers. De façon générale, toutes perturbations susceptibles d'induire une altération de l'homéostasie protéique activent un stress du RE. Afin de s'adapter, les cellules mettent en place une réponse nommée UPR pour Unfolded Protein Response. Ce programme d'adaptation est relayé par trois protéines localisées dans la membrane du RE (i) la protéine IRE1 (Inositol Requiring Enzyme 1) (ii) le facteur de transcription ATF6 (Activating Transcription Factor 6) (iii) la protéine kinase PERK (PKR-like Endoplasmic Reticulum Kinase). Parmi ces protéines, IRE1 est la seule à être conservée au cours de l'évolution. Elle possède deux activités enzymatiques, sérine/thréonine kinase et endoribonucléase. Sa cible la plus connue est l'ARNm de XBP1 (X-box Binding Protein 1) qui subit un épissage non conventionnel cytoplasmique conduisant à un décalage de phase. Ainsi, l'ARNm est traduit en un facteur de transcription actif, XBP1s (s,spliced). Le rôle de l'UPR a été particulièrement étudié dans les cancers solides. En revanche, les connaissances actuelles sur le rôle précis du stress du RE en général, et de la voie IRE1/XBP1 en particulier dans hémopathies malignes sont extrêmement parcellaires. Une étude clinique démontre que l'expression de XBP1s est corrélée à un meilleur pronostic chez les patients atteints de leucémies aiguës myéloïdes (LAMs). Cependant aucune étude fonctionnelle ne permet actuellement d'expliquer cette corrélation. Afin d'appréhender le rôle de XBP1, nous avons donc mis en place un modèle d'expression inductible dans des cellules leucémiques. L'étude de ce modèle a permis de mettre en évidence une chimiosensibilité accrue à l'aracytine, la doxorubicine et l'étoposide dans les cellules exprimant XBP1s. Nous avons pu démontrer que l'activation spécifique de la voie XBP1 active une réponse apoptotique et inhibe la croissance tumorale dans un modèle de xénogreffe. De façon à caractériser les mécanismes moléculaires sous-jacents et de mettre en évidence de nouvelles cibles, nous avons réalisé une expérience d'immunoprécipitation de la chromatine suivi d'un séquençage. Nous avons pu ainsi identifier le long ARN non codant MIR22HG, précurseur du microARN 22 comme étant une cible directe de XBP1. De nombreuses cibles caractérisées de ce microARN se classent dans la catégorie des oncogènes. Parmi ces cibles la Sirtuine 1 est surexprimée chez les patients et jouerait un rôle pro-survie dans la réponse aux dommages à l'ADN intervenant ainsi dans la résistance au traitement. Ces résultats suggèrent que le microARN 22 pourrait être un marqueur prédictif de la réponse au traitement dans les LAMs. Dans un second temps, nous avons étudié la régulation de XBP1 par un oncogène majeur, FLT3-ITD (Fms-Like Tyrosine kinase-3 receptor - Internal Tandem Duplication) et mis en évidence un mécanisme de rétrocontrôle inattendu entre ces deux acteurs, suggérant ainsi que l'expression de XBP1 peut être dérégulée dans les LAM. Ainsi l'ensemble de nos résultats permet d'appréhender le rôle et la régulation de XBP1 dans leucémies aiguës myéloïdes et pourrait, à terme, permettre de développer de nouveaux biomarqueurs utiles dans la prise en charge des patients atteints de LAM. / Endoplasmic reticulum stress activation is a common feature of cancer cells. Generally, endoplasmic reticulum (ER) stress is triggered by any situation inducing an accumulation of misfolded proteins in the ER. To cope with these perturbations, cells set off a conserved and adaptive intracellular signaling known as UPR or Unfolded Protein Response. UPR involves the activation of three sensors which are transmembrane proteins of ER (i) IRE1 (Inositol Requiring Enzyme 1), (ii) ATF6 (Activation Transcription Factor 6) and (iii) PERK (PKR-like Endoplasmic Reticulum Kinase). IRE1 is the most conserved branch of the three pathways and signals through two catalytic domains: a kinase and an RNAse L like endoribonucleolytic domains. Its most described target is the mRNA of the transcription factor XBP1 (X-box Binding Protein). IRE1 participates to the non-conventional splicing of XBP1 mRNA (XBP1s,spliced) leading to a frameshift and the expression of a potent transcription activator. In solid tumors, the implication of ER stress has been well characterized; however, the current knowledge on the precise role of the UPR in hematological malignancies, notably in leukemia, is extremely poor. A clinical study conducted by Schardt et al. highlighted a correlation between XPB1s activation and a favorable prognosis in acute myeloid leukemia (AML). Firstly, in order to decipher on the role of XBP1, we set up a model enabling the inducible expression of XBP1s in leukemic cells. In this model, XBP1s expression potentiates the effect of chemotherapeutic treatments, aracytine, doxorubicin and etoposide. We also report that XBP1s expression induces apoptosis and inhibits tumor growth in xenograft model. In order to characterize molecular mechanism and new targets, we perform a chromatin immunoprecipitation followed by sequencing. We thus identify the long non-coding MIR22HG, precursor of the microRNA 22 as a direct XBP1 target gene. Many miR-22 targets are classified as oncogenes. Among these targets, Sirtuin 1 is overexpressed in AML patients and could act as a pro-survival factor upon DNA damages, causing drug resistance. These results suggest that miR-22 could be a potent chemotherapeutic response biomarker in AML patients. Secondly, we study XBP1 regulation by the major AML oncogene FLT3-ITD (Fms-Like Tyrosine kinase-3 receptor - Internal Tandem Duplication) and highlight an unexpected feedback mechanism suggesting that XBP1 expression could be deregulated in AML. Taken together these results enable a better understanding of the role and regulation of XBP1 in acute myeloid leukemia and could, in the longer term, enable the development of new useful biomarkers in the management of patients.

The unfolded protein response (UPR) is induced by a variety of external and internal stimuli, including accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses such as Herpes Simplex Virus type 1 (HSV-1) induce host cells to produce viral proteins many of which undergo glycosylation and other modifications in the ER, causing stress to the ER and consequently UPR activation. I have tested the hypothesis that HSV-1 has evolved strategies to regulate the UPR in order to suppress aspects of the UPR that might interfere with viral replication and to promote pathways that aid its own survival and replication. The purpose of this study was to test the hypothesis that HSV-1 selectively modulates the three pathways (PERK, ATF6, and IRE-1) of the UPR in epithelial and neuronal cells and to examine the similarities and the differences between these two types of cells in their responses to ER stress. Vero and ONS-76 cells were used as models of epithelial and neuronal cells respectively and qRT PCR technique was used for analyzing RNA levels of transcripts of spliced Xbp1, HERP, CHOP and BIP, selected target genes for three pathways of the UPR. HSV-1 DNA synthesis and infectious virus production in infected cells showed that compared to the permissive Vero cells, ONS-76 cells seemed to be semi-permissive to HSV-1 infection with limited viral DNA synthesis and infectious virus production. The kinetics of transcript and protein synthesis for genes representing immediate early, early and late classes of viral genes was also monitored. Expression of the immediate early gene, ICP0, was similar in both cell types but the expression of the early gene, TK and late genes VP16 and VP 5 was different. My work reveals that HSV-1 infection in cells of epithelial and neuronal origins results in activation of the UPR, but through cell type selective regulation of the three signal transduction pathways of the UPR (PERK, ATF6, and IRE-1). While HSV-1 infection resulted in upregulation of Spliced Xbp1 and its target gene HERP (IRE1 pathway) and downregulation of BIP (ATF6 pathway) in both cell types, CHOP (PERK pathway) was upregulated only in ONS cells. My results suggest that some aspects of the UPR are regulated differently in cells representing the sites for HSV-1 lytic and latent infections. This may indicate the need for increasing the capacity for protein folding and degradation (Xbp1 and ATF6-induced) in both cells but a requirement for suppressing apoptosis (PERK-induced) only in epithelial cells. As well, I show that HSV-1 infection not only selectively activates the UPR pathways in different cell types, but also inactivates some components of the UPR pathways activated by the drug thapsigargin.

The unfolded protein response (UPR) is induced by a variety of external and internal stimuli, including accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses such as Herpes Simplex Virus type 1 (HSV-1) induce host cells to produce viral proteins many of which undergo glycosylation and other modifications in the ER, causing stress to the ER and consequently UPR activation. I have tested the hypothesis that HSV-1 has evolved strategies to regulate the UPR in order to suppress aspects of the UPR that might interfere with viral replication and to promote pathways that aid its own survival and replication. The purpose of this study was to test the hypothesis that HSV-1 selectively modulates the three pathways (PERK, ATF6, and IRE-1) of the UPR in epithelial and neuronal cells and to examine the similarities and the differences between these two types of cells in their responses to ER stress. Vero and ONS-76 cells were used as models of epithelial and neuronal cells respectively and qRT PCR technique was used for analyzing RNA levels of transcripts of spliced Xbp1, HERP, CHOP and BIP, selected target genes for three pathways of the UPR. HSV-1 DNA synthesis and infectious virus production in infected cells showed that compared to the permissive Vero cells, ONS-76 cells seemed to be semi-permissive to HSV-1 infection with limited viral DNA synthesis and infectious virus production. The kinetics of transcript and protein synthesis for genes representing immediate early, early and late classes of viral genes was also monitored. Expression of the immediate early gene, ICP0, was similar in both cell types but the expression of the early gene, TK and late genes VP16 and VP 5 was different. My work reveals that HSV-1 infection in cells of epithelial and neuronal origins results in activation of the UPR, but through cell type selective regulation of the three signal transduction pathways of the UPR (PERK, ATF6, and IRE-1). While HSV-1 infection resulted in upregulation of Spliced Xbp1 and its target gene HERP (IRE1 pathway) and downregulation of BIP (ATF6 pathway) in both cell types, CHOP (PERK pathway) was upregulated only in ONS cells. My results suggest that some aspects of the UPR are regulated differently in cells representing the sites for HSV-1 lytic and latent infections. This may indicate the need for increasing the capacity for protein folding and degradation (Xbp1 and ATF6-induced) in both cells but a requirement for suppressing apoptosis (PERK-induced) only in epithelial cells. As well, I show that HSV-1 infection not only selectively activates the UPR pathways in different cell types, but also inactivates some components of the UPR pathways activated by the drug thapsigargin.

The unfolded protein response (UPR) is induced by a variety of external and internal stimuli, including accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses such as Herpes Simplex Virus type 1 (HSV-1) induce host cells to produce viral proteins many of which undergo glycosylation and other modifications in the ER, causing stress to the ER and consequently UPR activation. I have tested the hypothesis that HSV-1 has evolved strategies to regulate the UPR in order to suppress aspects of the UPR that might interfere with viral replication and to promote pathways that aid its own survival and replication. The purpose of this study was to test the hypothesis that HSV-1 selectively modulates the three pathways (PERK, ATF6, and IRE-1) of the UPR in epithelial and neuronal cells and to examine the similarities and the differences between these two types of cells in their responses to ER stress. Vero and ONS-76 cells were used as models of epithelial and neuronal cells respectively and qRT PCR technique was used for analyzing RNA levels of transcripts of spliced Xbp1, HERP, CHOP and BIP, selected target genes for three pathways of the UPR. HSV-1 DNA synthesis and infectious virus production in infected cells showed that compared to the permissive Vero cells, ONS-76 cells seemed to be semi-permissive to HSV-1 infection with limited viral DNA synthesis and infectious virus production. The kinetics of transcript and protein synthesis for genes representing immediate early, early and late classes of viral genes was also monitored. Expression of the immediate early gene, ICP0, was similar in both cell types but the expression of the early gene, TK and late genes VP16 and VP 5 was different. My work reveals that HSV-1 infection in cells of epithelial and neuronal origins results in activation of the UPR, but through cell type selective regulation of the three signal transduction pathways of the UPR (PERK, ATF6, and IRE-1). While HSV-1 infection resulted in upregulation of Spliced Xbp1 and its target gene HERP (IRE1 pathway) and downregulation of BIP (ATF6 pathway) in both cell types, CHOP (PERK pathway) was upregulated only in ONS cells. My results suggest that some aspects of the UPR are regulated differently in cells representing the sites for HSV-1 lytic and latent infections. This may indicate the need for increasing the capacity for protein folding and degradation (Xbp1 and ATF6-induced) in both cells but a requirement for suppressing apoptosis (PERK-induced) only in epithelial cells. As well, I show that HSV-1 infection not only selectively activates the UPR pathways in different cell types, but also inactivates some components of the UPR pathways activated by the drug thapsigargin.

Damage to the endothelium is an important contributor to the initiation and progression of atherosclerosis. GRP78 is an endoplasmic reticulum (ER)-resident molecular chaperone in normal healthy endothelium that functions to assist in the correct folding of newly synthesized proteins and to prevent the aggregation of folding intermediates. In addition, GRP78 is present as a transmembrane protein on the surface of lesion-resident endothelial cells. Surface GRP78 is known to act as a surface signaling receptor in cancer cells and is activated by anti-GRP78 autoantibodies (GRP78a-Abs) isolated from the serum of cancer patients. However, the role of cell surface GRP78 on endothelial cells and the influence of GRP78a-Abs in atherosclerosis is unknown. The objectives of this study were to investigate the effects of GRP78a-Abs on lesion development, examine whether engagement of cell surface GRP78 by GRP78a-Abs modulates endothelial cell function, and determine whether GRP78a-Abs were associated with cardiovascular disease (CVD) in humans. This research showed that ApoE-/- mice with advanced atherosclerotic lesions have elevated serum levels of GRP78a-Abs and ApoE-/- mice immunized against recombinant GRP78 demonstrated a significant increase in GRP78a-Abs titers as well as accelerated lesion growth. Furthermore, this work demonstrated that activation of surface GRP78 on endothelial cells by GRP78a-Abs significantly increases gene expression of adhesion molecules ICAM-1 and VCAM-1 as well as leukocyte adhesion through the NFκB pathway. Additionally, middle-aged to elderly adults at risk for CVD showed a tendency toward elevated circulating GRP78a-Ab levels. Our results suggest that signaling through cell surface GRP78 can activate intracellular pathways that contribute to endothelial cell activation and augment atherosclerotic lesion development. These findings demonstrate a novel role for GRP78a-Abs and surface GRP78 receptor activity in endothelial cell function and the early stages of lesion development, as well as establish an initial framework for future work involving circulating GRP78a-Abs and atherosclerotic disease in humans. Furthermore, this work indicates inhibiting the interaction of GRP78a-Abs with cell surface GRP78 could present a novel therapeutic strategy to modulate lesion growth, thereby reducing the risk for atherosclerosis and cardiovascular disease. / Thesis / Doctor of Philosophy (PhD)

No description available.

In this project I sought to analyse the effects of different free fatty acids (FFAs) on INS-1E β-cells. The saturated fatty acid palmitate is considered toxic whereas the monounsaturated fatty acid oleate is harmless. In my working hypothesis I assumed an additional protective effect of oleate when used in combination with palmitate. Furthermore I aimed to explore in detail the possible causes and signalling pathways responsible for apoptosis or sustained cell survival. I examined the Endoplasmic Reticulum (ER) stress response, called unfolded protein response (UPR), as one essential criterion deciding about cell death or life. Analysis of viability and apoptosis confirmed the deleterious effect of palmitate on INS-1E β-cells after 24h of incubation. Oleate proved not to be harmful and even reversed the toxicity of palmitate. When the main components of the UPR were assessed using Western blot analyses and quantitative PCR was performed I found positive proof that palmitate activated the UPR and ultimately led to apoptosis. By contrast, oleate completely prevented UPR signalling. I conclude that oleate rescues INS-1E β-cells by inhibiting ER stress and its signalling.