The dynamin-related protein DRP-1 and the insulin signaling pathway cooperate to modulate Caenorhabditis elegans longevity.

Mitochondria are dynamic organelles able to undergo frequent morphological and numeral changes. A delicate balance between mitochondrial fusion and fission is critical for broad aspects of animal physiology, including apoptosis and control of mitochondrial inheritance and quality (Seo et al. 2010). From yeast to human, deregulations of mitochondrial network equilibrium as evident by disrupted mitochondrial morphology and accumulation of abnormally shaped mitochondria have been associated with senescence, aging and aging-related diseases (Sohal 1975; Yasuda et al. 2006; Lee et al. 2007). However, no direct evidence implicates mitochondrial dynamics in longevity determination in animals. The only evidence that mitochondrial plasticity positively impacts lifespan was shown in fungal models, in which reduced mitochondrial fission led to increased lifespan (Scheckhuber et al. 2007; Palermo et al. 2010). Mitochondrial dynamics are governed by molecular machineries that are highly conserved (Okamoto & Shaw 2005). The dynamin-related protein DRP-1 is the only profission protein identified in C. elegans and is demonstrated to control the scission of the mitochondrial outer membrane (Labrousse et al. 1999; Westermann 2010). To explore how mitochondrial fission can impact animal lifespan, we monitored the lifespan of worms either treated with drp-1 RNAi or bearing a putative null-mutation in the drp-1 gene (Breckenridge et al. 2008). The mean lifespan of wild-type worms treated with drp-1 RNAi or drp-1 mutant worms was indistinguishable from that of control animals (Fig.1A-B, Table S1), even though their mitochondrial morphology are greatly disrupted (Labrousse et al. 1999, Fig.2B-C and S1), indicating that reduced mitochondrial fission does not affect C. elegans lifespan under normal culturing condition Figure 1 Loss of drp-1 enhances the longevity phenotype of IIS mutant worms in a daf-16-dependent manner Figure 2 Loss of drp-1 affects mitochondrial morphology and stress resistance without affecting DAF-16 sub-cellular localization The insulin/IGF-1 signaling (IIS) pathway is a key longevity pathway and C. elegans mutants with reduced IIS, such as the phosphatidyl-inositol 3-kinase age-1 mutant (Friedman & Johnson 1988; Morris et al. 1996) and the tyrosine kinase insulin/IGF receptor daf-2 mutant (Kenyon et al. 1993), are long-lived (Fig.1A,C-D). Given that mitochondrial fission plays a critical role in insulin secretion in mammals (Yoon et al. 2010), we next tested how inactivating drp-1 might affect the longevity of IIS mutants. Strikingly, both age-1 and daf-2 mutants treated with drp-1 RNAi showed substantial further increase in mean lifespan (>65% compared to age-1 and daf-2 single mutant on control RNAi; Fig.1A, Table S1). Similarly, age-1;drp-1 and daf-2;drp-1 mutants exhibited a >75% increase in mean lifespan and an extension by up to 30 days in maximum lifespan when compared to their single mutant counterparts (Fig.1C-D, Table S1). Importantly, the enhancement of both the mean and maximum lifespans of age-1 mutants by the drp-1 mutation could be rescued by a transgene expressing drp-1 (Fig.S2). We also tested whether drp-1 inactivation will impact the lifespan of other long-lived strains, including worms undergoing the bacteria deprivation paradigm of dietary restriction (Kaeberlein et al. 2006), and worms with mild impairment of the mitochondrial respiratory chain (Felkai et al. 1999; Feng et al. 2001). We detected no or marginal effects (Table S2). Altogether, we demonstrated that inactivating drp-1 specifically and robustly synergizes with reduced IIS to prolong longevity. The longevity effect of reduced IIS is often associated with increased resistance to stress (Johnson et al. 2001). Surprisingly, wild-type and insulin mutant worms with drp-1 deletion were more sensitive to the oxidizing agent paraquat and to heat stress than their counterparts with wild-type drp-1 (Fig.2A, Table S3). The vulnerability of the age-1;drp-1 mutant to heat stress is due to drp-1 loss as it can be rescued by a transgene overexpressing drp-1 (Fig.S2). Therefore, inactivating drp-1 specifically prolongs longevity, but not stress resistance, of worms with reduced IIS, suggesting that manipulation of mitochondrial fission uncouples the function of IIS in aging and stress resistance. However, we cannot exclude that inactivating drp-1 causes an early stress in young worms but will confer stress resistance later in life. We next examined how inactivation of drp-1 affects mitochondrial morphology by comparing the indices of circularity (a mathematical estimation of circular shapes) of individual mitochondrion (see Supplementary Materials and Methods). We found that mitochondrial morphology was similarly disrupted in single drp-1 mutant and in IIS;drp-1 mutants (Fig.2C), suggesting DRP-1 does not cooperate with IIS to regulate mitochondrial fission. Interestingly, our data also revealed that mitochondria of daf-2 and age-1 single mutants presented lower indices of circularity than those of wild-type animals (Fig.2B-C). Signaling from DAF-2 and AGE-1 results in cytoplasmic retention of the FOXO transcription factor DAF-16, and DAF-16 translocation into the nucleus in age-1 and daf-2 mutants enables DAF-16-mediated regulation of target genes that contribute to diverse functional outcomes (Hekimi et al. 2001; Lee et al. 2001). Because the prolonged lifespan of age-1 and daf-2 mutants are fully dependent on daf-16 (Dorman et al. 1995), we next tested whether the extraordinary longevity of the IIS;drp-1 mutants also requires daf-16. We found that daf-16 RNAi completely abolished the synergistic effect of drp-1 and daf-2/age-1 mutations (Fig.1C-D, Table S1). Interestingly, we did not detect any changes in DAF-16∷GFP sub-cellular localization upon drp-1 inactivation (Fig.2D). Thus, the mechanism allowing DRP-1 to cooperate with IIS in modulating lifespan fully depends on DAF-16 but is unlikely to be mediated via DAF-16’s nuclear translocation. In considering a mechanism that enables drp-1 inactivation to specifically synergize with IIS mutants to extend lifespan, several possibilities come to mind. In mammals, mitochondrial fission is critical for insulin secretion (Zorzano et al. 2009; Yoon et al. 2010). Thus, drp-1 inactivation in worms may similarly interfere with insulin secretion and further reduce IIS in age-1/daf-2 mutants. This possibility is unlikely however as DAF-16 nuclear translocation was not enhanced in IIS;drp-1 double mutants. Although we found that age-1 and daf-2 single mutants show slight alterations in mitochondrial morphology, our data do not support the possibility that mitochondrial dynamics was further perturbed in IIS;drp-1 double mutants, as mitochondrial morphology in those mutants were similar to drp-1 single mutant. On the other hand, because the drp-1 single mutant has a brood size defect (Breckenridge et al. 2008), it is possible that germline proliferation is inhibited when drp-1 is inactivated, and defective germline proliferation is known to synergize with IIS mutant to increase lifespan (Hsin & Kenyon 1999; Spanier et al.). Alternatively, similar to what was shown with mitochondrial prohibitins (Artal-Sanz & Tavernarakis 2009), inactivation of drp-1 may perturb lipid metabolism, a process highly dependent on mitochondrial activity, to synergize with IIS mutants to extend lifespan. Lastly, C. elegans DRP-1 is shown to regulate apoptosis under sensitized conditions (Breckenridge et al. 2008). Intriguingly, daf-2 mutants exhibit enhanced germline apoptosis (Pinkston et al. 2006). It is possible that the apoptotic function of DRP-1 is induced in IIS mutants, and loss of drp-1 may block apoptosis in IIS mutants and further promote longevity. Interestingly, in fungal models, mitochondrial fission modulates wild-type lifespan by interfering with apoptosis (Scheckhuber et al. 2007; Palermo et al. 2010). It is possible that fungal aging is more closely connected to apoptosis (Rockenfeller & Madeo 2008) than C. elegans aging, providing an explanation for why reduced mitochondrial fission affects wild-type lifespan in fungi but not in worms. This is the first report of a manipulation of mitochondrial dynamics that positively impacts lifespan in an animal. As components of the mitochondrial fission/fusion machinery and IIS are highly conserved, our observations are likely relevant to mitochondrial biology and longevity in mammals.