SummaryIncreased production of reactive oxygen species (ROS) has long been considered a cause of aging. However, recent studies have implicated ROS as essential secondary messengers. Here we show that the site of ROS production significantly contributes to their apparent dual nature. 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. Reverse electron transport rescued pathogenesis induced by severe oxidative stress, highlighting the importance of the site of ROS production in signaling. Furthermore, preventing ubiquinone reduction, through knockdown of PINK1, shortens lifespan and accelerates aging; phenotypes that are rescued by increasing reverse electron transport. These results illustrate that the source of a ROS signal is vital in determining its effects on cellular physiology and establish that manipulation of ubiquinone redox state is a valid strategy to delay aging.

Reactive Oxygen Species (ROS) can cause oxidative damage and have been proposed to be the main cause of aging and age-related diseases including cancer, diabetes and Parkinson's disease. Accordingly, mitochondria from old individuals have higher levels of ROS. However, ROS also participate in cellular signaling, are instrumental for several physiological processes and boosting ROS levels in model organisms extends lifespan. The current consensus is that low levels of ROS are beneficial, facilitating adaptation to stress via signaling, whereas high levels of ROS are deleterious because they trigger oxidative stress. Based on this model the amount of ROS should determine the physiological effect. However, recent data suggests that the site at which ROS are generated is also instrumental in determining effects on cellular homeostasis. The best example of site-specific ROS signaling is reverse electron transport (RET). RET is produced when electrons from ubiquinol are transferred back to respiratory complex I, reducing NAD+ to NADH. This process generates a significant amount of ROS. RET has been shown to be instrumental for the activation of macrophages in response to bacterial infection, re-organization of the electron transport chain in response to changes in energy supply and adaptation of the carotid body to changes in oxygen levels. In Drosophila melanogaster, stimulating RET extends lifespan. Here, we review what is known about RET, as an example of site-specific ROS signaling, and its implications for the field of redox biology.

Defects in mitochondrial OXPHOS are associated with diverse and mostly intractable human disorders. The single-subunit alternative oxidase (AOX) found in many eukaryotes, but not in arthropods or vertebrates, offers a potential bypass of the OXPHOS cytochrome chain under conditions of pathological OXPHOS inhibition. We have engineered Ciona intestinalis AOX for conditional expression in Drosophila melanogaster. Ubiquitous AOX expression produced no detrimental phenotype in wild-type flies. However, mitochondrial suspensions from AOX-expressing flies exhibited a significant cyanide-resistant substrate oxidation, and the flies were partially resistant to both cyanide and antimycin. AOX expression was able to complement the semilethality of partial knockdown of both cyclope (COXVIc) and the complex IV assembly factor Surf1. It also rescued the locomotor defect and excess mitochondrial ROS production of flies mutated in dj-1beta, a Drosophila homolog of the human Parkinson's disease gene DJ1. AOX appears to offer promise as a wide-spectrum therapeutic tool in OXPHOS disorders.

BackgroundAging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.Methodology/Principal FindingsWe investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψm). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.Conclusions/SignificanceThese findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

Previous studies have consistently shown that caloric restriction (CR) decreases mitochondrial reactive oxygen species (ROS) (mitROS) generation and oxidative damage to mtDNA and mitochondrial proteins, and increases maximum longevity, although the mechanisms responsible for this are unknown. We recently found that protein restriction (PR) also produces these changes independent of energy restriction. Various facts link methionine to aging, and methionine restriction (MetR) without energy restriction increases, like CR, maximum longevity. We have thus hypothesized that MetR is responsible for the decrease in mitROS generation and oxidative stress in PR and CR. In this investigation we subjected male rats to exactly the same dietary protocol of MetR that is known to increase their longevity. We have found, for the first time, that MetR profoundly decreases mitROS production, decreases oxidative damage to mtDNA, lowers membrane unsaturation, and decreases all five markers of protein oxidation measured in rat heart and liver mitochondria. The concentration of complexes I and IV also decreases in MetR. The decrease in mitROS generation occurs in complexes I and III in liver and in complex I in heart mitochondria, and is due to an increase in efficiency of the respiratory chain in avoiding electron leak to oxygen. These changes are strikingly similar to those observed in CR and PR, suggesting that the decrease in methionine ingestion is responsible for the decrease in mitochondrial ROS production and oxidative stress, and possibly part of the decrease in aging rate, occurring during caloric restriction.

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Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.aging | mitochondria | respiratory chain | free radicals M itochondria are key metabolic organelles whose oxidative phosphorylation (OXPHOS) system is considered to be one of the most efficient producers of bioenergy. When OX-PHOS function is compromized (e.g., by mutations or toxins), bioenergy supply and cellular homeostasis are seriously disrupted, which can be lethal.OXPHOS complex I plays a central role in the regulation of ATP production, intermediary metabolism, and apoptosis (1, 2), and mutations affecting it cause many human pathologies (3). It has also been proposed as a pacemaker of the aging process (4). Treatments inferred to decrease the production of reactive oxygen species (ROS) at the level of complex I can prolong lifespan in Drosophila (5). All these characteristics make it essential to understand better the role of complex I in vivo and its involvement in aging.Many organisms possess alternative enzymes that can bypass or replace the proton-translocating complexes of the mitochondrial respiratory chain. These include the alternative oxidases (AOX) and the NADH dehydrogenases of the Ndi and Nde families. Together these enzymes provide an alternative respiratory chain that potentially allows the maintenance of redox homeostasis and intermediary metabolism under conditions where flux through the "standard" respiratory chain is limited by high ATP levels, the action of toxins or other physiological restraints (6, 7). AOX acts as a bypass of complexes III and IV, whereas Nde or Ndi can bypass complex I.In previous studies these bypass enzymes were shown to be active when introduced into the mitochondria of higher metazoans such as mammals (8-12), arthropods (13), or nematodes (14), all of which lack endogenous alternative enzymes. Fu...

The brain is the most complex human organ, consuming more energy than any other tissue in proportion to its size. It relies heavily on mitochondria to produce energy and is made up of mitotic and postmitotic cells that need to closely coordinate their metabolism to maintain essential bodily functions. During aging, damaged mitochondria that produce less ATP and more reactive oxygen species (ROS) accumulate. The current consensus is that ROS cause oxidative stress, damaging mitochondria and resulting in an energetic crisis that triggers neurodegenerative diseases and accelerates aging. However, in model organisms, increasing mitochondrial ROS (mtROS) in the brain extends lifespan, suggesting that ROS may participate in signaling that protects the brain. Here, we summarize the mechanisms by which mtROS are produced at the molecular level, how different brain cells and regions produce different amounts of mtROS, and how mtROS levels change during aging. Finally, we critically discuss the possible roles of ROS in aging as signaling molecules and damaging agents, addressing whether age-associated increases in mtROS are a cause or a consequence of aging.