The role of reactive oxygen species
Mild inhibition of the mitochondrial electron transport chain (ETC) increases life span in many species [1-10]. In C. elegans, mutations in ETC genes such as clk-1 (which encodes a mitochondrial hydroxylase involved in ubiquinone production), isp-1 (which encodes Rieske iron-sulfur protein in complex III), and nuo-6 (which encodes a subunit of NADH dehydrogenase complex) [1-3, 40] have been shown to extend life span. RNAi knock-down of mitochondrial ETC components also increases the life span of C. elegans, and that of Drosophila as well [6, 9]. In mice, mutants that exhibit decreased mitochondrial respiration such as Surf1−/− (mutation in cytochrome c oxidase) [8] and Mclk1+/− (heterozygous knockout of the mouse homolog of clk-1) have long life spans [7]. These findings support the notion that reduced mitochondrial respiration leads to longevity in mammals and suggest the existence of evolutionarily conserved underlying mechanisms.
Because mitochondria are the major source of ROS, one possible mechanism by which a reduction in mitochondrial respiration influences life span may involve changes in the level of ROS. Historically, ROS were believed to be the key causes of aging [41]. The free radical theory of aging proposed that ROS, which are natural byproducts of mitochondrial respiration, cause damage to macromolecules and organelles such as mitochondria. This mitochondrial damage in turn stimulates increased ROS generation, thus evoking a ‘vicious cycle’ that ultimately leads to deterioration of the cell and the organism [41]. However, several studies have shown that many conditions that increase ROS do not decrease life span, in worms and in mice [42, 43, 48]. Moreover, recent studies have highlighted the beneficial role of ROS in longevity [16, 31, 44-48]. For example, antimycin treatment that blocks mitochondria complex III and thus impairs ETC function, triggers excessive ROS production in the mitochondria [49]. Interestingly, antimycin treatment increases the life span of C. elegans [5]. Consistent with this, several studies have shown that long-lived clk-1, isp-1, and nuo-6 mutant worms have increased ROS levels [16, 31, 50]. Likewise, long-lived Mclk1+/− mice exhibit decreased respiration rates and elevated H2O2 levels [7, 51]. These data raise the intriguing possibility that increased ROS may play a causal role in the longevity conferred by the inhibition of mitochondrial respiration.
The Hekimi group recently addressed this issue directly and showed that increased ROS are required for the longevity of isp-1 and nuo-6 mutants [31]. They reported that isp-1 and nuo-6 mutants have increased superoxide levels, whereas clk-1 mutants have elevated overall ROS levels. N-acetylcysteine (NAC), a well-defined antioxidant, significantly decreased the life span of isp-1 and nuo-6 mutants but had little or no effect on the life span of wild-type animals. In case of clk-1 mutants, NAC treatment had marginal effects on life span [31]. In addition, three groups independently showed that wild-type worms treated with the ROS-generating chemicals juglone or paraquat are long lived [16, 31, 45]. These studies are consistent with the previous finding by Schulz et al. that restriction of glucose metabolism increases life span through elevating ROS and that antioxidant treatment suppresses this longevity [46]. Together, these data suggest that increased ROS generation in mitochondrial respiration-defective mutants promotes longevity in C. elegans.
In contrast to the long-lived mitochondrial respiration mutants described above, a mutant allele of mev-1, which encodes a cytochrome b large subunit in complex II [52, 53], cause a short life span [34, 52]. The mev-1 mutants were initially characterized in an EMS mutagenesis screen to identify mutants that are hypersensitive to paraquat treatment [52], and were subsequently shown that these mutants display increased ROS levels [54]. Studies in our laboratory and by Dingely et al. showed that mev-1 mutants have even higher ROS levels than the long-lived clk-1 or isp-1 mutants [16, 55]. Therefore, it is possible that mev-1 mutants are short lived because of excessive ROS, which is similar to the conditions that decrease life span by treatment of high concentrations of ROS-generating chemicals [16, 45].
The role of HIF-1 in longevity induced by impaired respiration and ROS
How do ROS extend the lifespan of respiration mutants? Consistent with experimental results using cultured mammalian cells, recent studies with model organisms show that HIF-1 is stabilized when ROS levels are increased. Mclk1+/− mice, which display reduced mitochondrial respiration, exhibit elevated ROS levels [51] and increased HIF-1α protein levels under normal oxygen conditions [56]. In addition, the increase in HIF-1 levels caused by RNAi knock-down of Mclk1 was abolished by a H2O2-specific antioxidant peptide targeted to mitochondria [56]. In C. elegans, we showed that defects in mitochondrial respiration elevated ROS levels, which lead to increased HIF-1 activity [16]. We found that clk-1 and isp-1 mutations induced several HIF-1 target genes and that the extended life span promoted by clk-1 and isp-1 mutations requires the HIF-1 transcription factor. Moreover, treating the worms with paraquat led to up-regulation of a HIF-1 target gene in a hif-1-dependent manner [16]. Together these data suggest that mutations affecting mitochondrial respiration lengthen life span by increasing ROS levels and HIF-1activity. A recent paper reported that an E. coli TCA cycle-related mutant that cannot provide reducing equivalents used in the ETC, and thus has reduced respiration, showed an extended chronological life span [57]. Interestingly, the longevity of these mutants required ArcA, which is the functional homolog of eukaryotic HIF [57], suggesting that this longevity regulation is functionally conserved between prokaryotes and eukaryotes.
Several issues regarding the role of HIF-1 in the longevity caused by increased ROS remain to be addressed. First, controversy remains regarding how HIF-1 is activated under low oxygen conditions. One unexpected observation regarding hypoxia is that cells exhibit increased ROS levels under hypoxia [58-60]. Although controversial, several recent studies proposed that mitochondrial complex III is the main source of ROS required for HIF-1 stabilization [61-63]. Under normoxic conditions, growth factors required for vasculogenesis and cytokines required for the maintenance of hematopoietic stem cells stabilize HIF-1, and this stabilization requires ROS [64, 65]. It has been suggested that ROS oxidize Fe2+, the coactivator of the HIF prolyl hydroxylase, to Fe3+ through Fenton's reaction in cultured mammalian cells [66]. Follow-up on our study showing that increased ROS lead to the activation of HIF-1 in C. elegans will be crucial to examine this issue in vivo. Second, what are the HIF-1 target genes that mediate the longevity induced by elevated ROS levels? In cultured mammalian cells, increased ROS levels under hypoxia prolong their replicative life span via stabilization of HIF-1, which leads to up-regulation of human telomerase reverse transcriptase (hTERT) and subsequent extension of telomeres [67, 68]. It will be important to determine which HIF-1 target genes are responsible for paraquat-induced longevity in C. elegans. Third, because the hif-1 mutation only partially suppresses the paraquat-induced longevity and because not all hif-1-dependent hypoxia genes were induced by inhibiting respiration [16], additional genes appear to be required in parallel to the conventional HIF-1 signaling pathway. Identification of these genes will increase our understanding of the mechanisms by which increased ROS extend life span.
Mitochondrial unfolded protein response
In contrast to the mitochondrial respiration mutants, we found that longevity caused by RNAi targeting ETC components showed only a partial dependency on hif-1[16]. Together with findings that the effect of respiratory-chain RNAi and respiration mutants may exert different outputs in gene expression and metabolism [11, 13], these studies suggest that there are additional mechanisms by which impaired ETC function (in particular that induced by RNAi) increases life span. Recently, several groups showed that inhibition of mitochondrial respiration triggers the mitochondrial unfolded protein response (UPRmito) [40, 69, 70] and UPRmito has been shown to be required for the longevity caused by impaired mitochondrial respiration [69]. An especially striking finding of Durieux et al. is that RNAi against an ETC component in a single tissue induced longevity of the whole animal, and this RNAi effect conveys a cell non-autonomous signal to neighboring tissues to elicit UPRmito [69]. Specifically, Durieux et al. showed that RNAi knock-down of the ETC component cco-1 (cytochrome c oxidase-1 subunit Vb/COX4) in the intestine or neurons is sufficient to extend the life span of C. elegans. In addition, they showed that cco-1 RNAi in neurons causes UPRmito in the intestine and coined the term “mitokine” for the unidentified signaling molecules that presumably relay the signal induced by reduced mitochondrial respiration in one tissue to other tissues [69]. Based on these results, and together with the suggestion that ROS and HIF-1 mediate the longevity conferred by a reduction in mitochondrial respiration, it is tempting to speculate that ROS such as hydrogen peroxide might travel locally and that HIF-1-target genes propagate a signaling cascade in various tissues to promote longevity.