The roles of Hypoxia Inducible Factor-1, Glutamate and their interaction in Ischemic Tolerance

Abstract

Stroke is a debilitating disorder with no effective treatments. The clinical importance of promoting cerebral ischemic tolerance is apparent; however, our knowledge of the signaling mechanisms involved in the phenomenon of ischemic preconditioning remains insufficient to be able to translate to the clinical environment. This dissertation is aimed at an investigation of endogenous pathways, which are both innate and adaptive (in response to a preconditioning stimulus), and by which the brain might protect itself from ischemia. Defining key proteins and mechanisms involved in those pathways can potentially be used as tolerance-producing drugs. Hypoxia Inducible Factor-1α (HIF-1α) is the major regulator of oxygen homeostasis and functions to induce a wide array of transcripts. HIF-1's downstream targets mediate a number of cellular mechanisms that can facilitate cellular adaptation to hypoxia and promote survival. We have sought to understand the mechanisms of HIF-1 stability and degradation and to determine its use as a potential therapy for the treatment of stroke (Chapter 1). In the brain, HIF-1 is present in neurons as well as astrocytes. Considering their important role in maintaining glutamate homeostasis and providing support necessary for the survival of neurons, we examined the effects of glutamate toxicity on the viability of astrocytes exposed to hypoxia and the role that HIF-1 plays under these conditions (Chapter 2). Moreover, given that glutamate toxicity is responsible for the majority of the damage that occurs after ischemia, we aimed to better understand the mechanisms of tolerance induced by adaptation to chronic levels of glutamate (Chapter3). Overall, in this thesis we identified novel pathways that promote neuronal and astrocytic survival after ischemia. First, we demonstrated that HIF-1α oxidation by the formation of reactive oxygen species during ischemia increased its susceptibility to degradation by the 20S proteasome. Proteasome inhibition increased HIF-1 accumulation and reduced ischemia-induced neurotoxicity. Next, we observed that stabilization of HIF-1α under hypoxia has a protective effect on astrocytes in maintaining cell morphology and viability in response to glutamate toxicity. We found that the presence of the antioxidant, glutathione, was correlated with an increased expression of HIF-1α and its inhibition decreased HIF-1α protein levels and cell viability. Finally, for the first time we show that an in vivo model of glutamate preconditioning was also able to induce tolerance and decrease brain infarct volume in addition to edema volume in both young adult (9-month) and aged (22-month) mice. Taking advantage of these mechanisms may lead to the development of more feasible and less dangerous therapeutic agents and approaches to treat ischemic strokes.