Dissecting the role of Complex I ROS production via forward and reverse electron transport in the brain of Drosophila melanogaster

Abstract

The dual nature of reactive oxygen species (ROS) is a widely recognised paradox. For many years, mitochondrial ROS were characterised as the main cause of ageing and chronic ROS production was documented in many age-related diseases. In contrast, recent studies have described ROS as signalling molecules essential for maintaining cellular homeostasis. Further experimentation has revealed that the extent of ROS production, as well as the location, can be important in determining the behaviour of ROS. The best example is the process known as Reverse Electron Transport (RET). RET is associated with a large increase in site-specific ROS production at CI and is responsible for stimulating key signalling pathways that control stress adaptation and cellular fate. Additionally, the expression of the alternative NADH dehydrogenase, Ndi1, which promotes ROS-RET, leads to extension of lifespan in Drosophila melanogaster. Exploring ROS-RET signalling may be instrumental in understanding the role of ROS in health and disease. In the following chapters, I investigate the mechanisms behind ROS-RET. I use ex vivo ROS measurements in the brain of Drosophila melanogaster to demonstrate that ROS-RET occurs under physiological conditions when flies are exposed to heat stress. I describe in detail how manipulation of the electron transport chain affects the occurrence of ROS-RET. My results reveal that the entry of electrons through CI and CII is essential for ROS-RET to occur. However, blocking the exit of electrons (CIII-CIV) increases ROS production but not via ROS-RET. Finally I have performed a genome-wide RNAi screen, taking advantage of the alternative oxidase (AOX) that supresses ROS-RET, where I have found new candidate genes responsible for regulating mitochondrial ROS levels. In summary, I provide evidence of how ROS-RET can be regulated in vivo including its physiological stimulation, factors essential for its generation and new genes involved in its regulation.