Abstracts Category : Other

How cellular ATP/ADP ratios and reactive oxygen species affect AMPK signalling

by Elizabeth Hinchy

Mitochondria are key generators of cellular ATP, vital to complex life. Historically, mitochondrial generation of reactive oxygen species (ROS) was considered to be an unregulated process, produced by dysfunctional mitochondria. More recently, mitochondrial ROS generated by complex I, particularly by the process of reverse electron transfer (RET), has emerged as a potentially biologically relevant signal that is tightly-regulated and dependent on mitochondrial status. ROS production by RET is reported to play a role in the innate immune response and lifespan extension in fruit flies. One way in which mitochondrial ROS may behave as a signal is by altering the activity of AMP-activated protein kinase (AMPK), a key metabolic sensor and regulator of cell metabolism, which is activated when cellular ATP levels decrease during energy demand. Mitochondria can signal to AMPK via the magnitude of the cellular ATP/AMP and ATP/ADP ratios, which alter in response to mitochondrial function. Our view is mitochondria may also signal to AMPK via ROS. Important studies have helped to clarify the role of exogenous or cytosolic ROS in AMPK regulation. However, the effects of mitochondrial ROS on AMPK activity, specifically that generated by complex I, remain unclear and is the main focus of this thesis. I characterized the effects of exogenous H2O2 on cellular AMPK activity, ATP/ADP ratios and cellular redox state in a cell model. I then compounded this with selective mitochondria generated ROS by the mitochondria-targeted redox-cycler, MitoParaquat (MPQ). AMPK activity appeared to correlate with decreasing cell ATP/ADP ratios, indicating that both sources of ROS primarily activate AMPK in an AMP/ADP-dependent mechanism. In parallel, I developed an approach for analyzing the redox state of candidate proteins, an important step in determining if a protein is directly regulated by ROS. I also initiated development of a cell model for studying the downstream effects of mitochondrial ROS production by RET, by expressing alternative respiratory enzymes in a mammalian cell line.