Reactive oxygen species (ROS) are largely generated in the mitochondria of the cell, a side-effect of the energetic processes taking place there to power cellular operations. ROS cause damage that must be repaired by reacting with molecular machinery in the cell, and that stress on the cell increases with age, and features prominently in most discussions of aging. ROS also play an important role as signals, however, triggering important processes related as cellular maintenance. That exercise is beneficial, for example, depends upon an increase in ROS production, and a number of ways of increasing life span in laboratory species incorporate some degree of increased ROS generation.
Historically, mitochondrial ROS (mtROS) production and oxidative damage have been associated with aging and age-related diseases. In fact, the age-related increase in ROS has been viewed as a cause of the aging process while mitochondrial dysfunction is considered a hallmark of aging, as a consequence of ROS accumulation. However, pioneering work in Caenorhabiditis elegans has shown that mutations in genes encoding subunits of the electron transport chain (ETC) or genes required for biosynthesis of ubiquinone extend lifespan despite reducing mitochondrial function. The lifespan extension conferred by many of these alterations is ROS dependent, as reduction of ROS abolishes this effect. Moreover, chemical inhibition of glycolysis or exposure to metabolic poisons that block respiratory complex I (CI) or complex III (CIII) also prolong lifespan in C. elegans in a ROS-dependent manner. Various studies have shown that ROS act as secondary messengers in many cellular pathways, including those which protect against or repair damage. ROS-dependent activation of these protective pathways may explain their positive effect on lifespan. The confusion over the apparent dual nature of ROS may, in part, be due to a lack of resolution as without focused genetic or biochemical models it is impossible to determine the site from which ROS originate.
A promising path to resolving ROS production in vivo is the use of alternative respiratory enzymes, absent from mammals and flies, to modulate ROS generation at specific sites of the ETC. The alternative oxidase (AOX) of Ciona intestinalis is a cyanide-resistant terminal oxidase able to reduce oxygen to water with electrons from reduced ubiquinone (CoQ), thus bypassing CIII and complex IV (CIV). NDI1 is an alternative NADH dehydrogenase found in plants and fungi, which is present on the matrix-face of the mitochondrial inner membrane where it is able to oxidize NADH and reduce ubiquinone, effectively bypassing CI. Our group and others have demonstrated that allotopic expression of NDI1 in Drosophila melanogaster can extend lifespan under a variety of conditions and rescue developmental lethality in flies with an RNAi-mediated decrease in CI levels.
To determine the role of increased ROS production in regulating longevity, we utilized allotopic expression of NDI1 and AOX, along with Drosophila genetic tools to regulate ROS production from specific sites in the ETC. We show that NDI1 over-reduces the CoQ pool and increases ROS via reverse electron transport (RET) through CI. Importantly, restoration of CoQ redox state via NDI1 expression rescued mitochondrial function and longevity in two distinct models of mitochondrial dysfunction. We show that mitochondrial ROS production increases with age and that un-detoxified ROS can be detrimental to Drosophila lifespan, while increasing ROS production specifically from reduced CoQ, possibly via RET, acts as a signal to maintain mitochondrial function (notably CI) and extend lifespan. It is possible that an intact CI is required for lifespan extension in fruit flies, as metformin, which increases lifespan by blocking CI and increasing ROS in worms, fails to do so in fruit flies. If the mechanism we describe here is conserved in mammals, manipulation of the redox state of CoQ may be a strategy for the extension of both mean and maximum lifespan and the road to new therapeutic interventions for aging and age-related diseases.