O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and ubiquitous post-translational modification that has been shown to play a role in regulating a variety of intracellular processes. The pathway responsible for generating the O-GlcNAc modification, the hexosamine biosynthetic pathway (HBP), has also been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. To investigate if the effects of HBP flux on PKC translocation observed by others is related to the O-GlcNAc modification, O-GlcNAc levels in human astroglial cells were elevated using four separate O-GlcNAc modulating agents followed by analysis of cytosol and membrane concentrations of PKC-epsilon, -alpha, -betaII, and -iota. Of the four PKC isoforms analyzed, PKC-epsilon showed a significant reduction in its membrane associated levels in response to all agents tested whereas PKC-alpha showed reductions in response to only two agents. Investigation of the mechanism for the reductions in membrane associated PKC-epsilon and -alpha indicate that the increased O-GlcNAc levels did not disrupt the activation of these isoforms or their ability to translocate to the plasma membrane. Furthermore, results indicate that these reductions are not due to a disruption in the Hsp70 mediated recycling of the isoforms. It was found; however, that increased O-GlcNAc levels resulted in increased degradation of PKC-epsilon suggesting that the decreases in membrane associated PKC-epsilon may be a result of increased phosphatase or protease activity. Additional studies revealed that decreases in membrane bound PKC-epsilon and PKC-alpha, both of which act as anti-apoptotic enzymes, correlated with an increase in poly-(ADP-ribose) polymerase (PARP) cleavage -- a well characterized hallmark of apoptosis. In addition to PKC, the effects of increased O-GlcNAc levels on a related kinase, Akt, were also examined. Initial investigation of the effects of increased O-GlcNAc modification of Akt activation using glucosamine or streptozotocin revealed a relatively large, short-term increase in Akt phosphorylation in response to these treatments. However, further analysis with other O-GlcNAc modulators indicated that this activation was not related to O-GlcNAc protein modification. Furthermore, this activation does not appear to be related to any hyperosmotic effects associated with the treatment conditions, nor does it appear to be related to oxidative stress. Therefore, further investigation is needed to characterize the novel pathway responsible for Akt activation following glucosamine or streptozotocin treatment.

Le stress oxydatif joue un rôle majeur dans la mort des cellules neuronales dans diverses conditions neuropathologiques. Cependant, les astrocytes réactifs, en produisant des facteurs neuroprotecteurs et antioxydants, sont capables de protéger les neurones contre le stress oxydatif. De ce fait, la protection des cellules gliales contre les facteurs nocifs, s’avère indispensable pour prévenir les dommages des cellules nerveuses. Les globines du cerveau, en particulier, la neuroglobine (Ngb) et l’hémoglobine (Hb), exprimées dans les cellules nerveuses, jouent un rôle important dans le métabolisme de l’oxygène. Récemment, il a été démontré, que ces protéines exercent des effets neuroprotecteurs dans les modèles expérimentaux de maladies neurodégénératives. Cependant, aucun effet glioprotecteur n’a été rapporté. Les objectifs de ce travail de thèse sont, de mettre en évidence les effets protecteurs de l’Hb et la Ngb dans les astrocytes en culture en présence d’un stress oxydant, et d’élucider les mécanismes intracellulaires mis en jeu. Nous avons démontré que l’Hb et la Ngb sont capables de promouvoir la survie des astrocytes en condition de stress oxydatif, et ce en réduisant significativement la surproduction des ROS, la surexpression des gènes pro-inflammatoires (IL-6, IL-33, iNOS), le dysfonctionnement mitochondrial et la stimulation de l’activité de la caspase-3/7. Nous avons montré aussi que les effets anti-apoptotiques impliquent l’activation des voies de signalisation ERK-MAPK. En outre, nous avons vérifié les effets glioprotecteurs sur un modèle animal de stress oxydatif chronique, les souris KO TP53INP1, ainsi que sur un modèle animal d’hypoxie. / Oxidative stress plays a major role in the death of neuronal cells under various neuropathological conditions. However, reactive astrocytes, by producing neuroprotective and antioxidant factors, are able to protect neurons against oxidative stress. Therefore, protecting glial cells from harmful factors is essential to prevent nerve cell damage. Brain globins, in particular, neuroglobin (Ngb) and hemoglobin (Hb), expressed in neurons and glial cells, play an important role in the metabolism of oxygen. Recently, it has been demonstrated that these proteins exert neuroprotective effects in experimental models of neurodegenerative diseases. However, no glioprotective effect has been reported. The objectives of this thesis work are to demonstrate the protective effects of Hb and Ngb in cultured astrocytes in the presence of oxidative stress and to elucidate the intracellular mechanisms involved. We have demonstrated That Hb and Ngb are able to promote the survival of astrocytes under oxidative stress conditions by significantly reducing over-production of ROS, overexpression of pro-inflammatory genes (IL-6, IL-33, iNOS) Mitochondrial dysfunction and stimulation of caspase-3/7 activity. We have also shown that anti-apoptotic effects involve the activation of ERK-MAPK signaling pathways. In addition, we verified the glioprotective effects on an animal model of chronic oxidative stress, KO mice TP53INP1, as well as on an animal model of hypoxia.