Here, researchers use genetic engineering try to pin down specific sources of oxidizing molecules within a cell and identify their different effects on health and longevity. Cells are liquid bags of chemical machinery, and the presence of too many reactive oxygen species (ROS) - also known as free radicals - can produce a level of damage to that machinery that significantly impacts cell function, or even kills cells. This is known as oxidative stress, and levels of oxidative stress are seen to rise alongside the other manifestations of degenerative aging. However, ROS molecules are also used as signals: produced by mitochondria and triggering increased cell maintenance, among other activities. They are critical in the beneficial response to exercise, for example. Many methods of modestly extending life span in laboratory species involve reductions in levels of ROS, and many others involve increases - either can under the right circumstances extend healthy life by reducing net levels of cell damage or triggering other mechanisms relevant to health. A cell is a complex, reactive system and few aspects of cell state have straightforward relationships with one another. Oxidative stress features prominently in many theories of aging, but many of these theories are older and too simplified to be useful given the present state of knowledge and explorations of oxidative stress and ROS signaling.
Historically, mitochondrial ROS (mtROS) production and oxidative damage have been associated with aging and age-related diseases such as Parkinson's disease. 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 Caenorhabditis 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. 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 complex III and complex IV. NDI1 is a rotenone-insensitive 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 complex I. 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 complex I 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 report that ROS increase with age as mitochondrial function deteriorates. However, we also demonstrate that increasing ROS production specifically through respiratory complex I reverse electron transport extends Drosophila lifespan. We show that NDI1 over-reduces the CoQ pool and increases ROS via reverse electron transport (RET) through complex I. Importantly, restoration of CoQ redox state via NDI1 expression rescued mitochondrial function and longevity in two distinct models of mitochondrial dysfunction. 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.