Mitochondrial stress communication in mammalian cells

Abstract

Mitochondria are crucial organelles in energy transduction, with an emerging role in cell signaling beginning to be appreciated. Due to the endosymbiotic nature of mitochondria and as a by-product of energy transduction via the electron transport chain (ETC), mitochondria are inherently exposed to many stresses (e.g. reactive oxygen species, genomic damage and proteotoxic stress). We postulated that there would be active intracellular and extracellular pathways of mitochondrial communication engaged to maintain cellular fitness, however this has not been well studied in mammalian cells. The overarching aim of this thesis was to explore how mammalian cells communicate mitochondrial stress. More specifically, to characterise intracellular signaling adaptations to mitochondrial stress and to investigate cell non-autonomous communication of mitochondrial stress through secreted proteins. To address the first aim, a xeno-cybrid model was employed where cells had mismatch between nuclear and mitochondrial genomes resulting in an impairment of ETC function. Compared with controls, cells with ETC dysfunction had substantial oxidative defects, but were viable and maintained cellular ATP. This ‘rescue’ of cell survival appeared to be driven by upregulation of glycolysis and pro-survival Akt and AMPK signaling. To interrogate a potential upstream mediator, ATM kinase was explored, and it was found that a number of adaptations were reversed by a selective ATM kinase inhibitor. These results therefore support a role for ATM kinase as an important adaptation promoting survival of mammalian cells in response to mitochondrial stress. To address cell non-autonomous communication, HEK293 cells, and primary murine hepatocytes and adipocytes were incubated with ETC inhibitors (rotenone or antimycin A) or an oxidative phosphorylation uncoupler (Dinitrophenol), and secreted proteins were analysed by LC-MS/MS to map the mitochondrial stress-sensitive secretome. Significant alterations in secreted proteins were observed for HEK293 cells and adipocytes with ETC complex I and III inhibition and in hepatocytes treated with DNP. Collectively, the work presented in this thesis sheds new light on the pathways employed by mammalian mitochondria to signal and respond to stress.