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First published online October 12, 2006
doi: 10.1242/10.1242/jcs.03199
Research Article |
1 Molecular Biology Institute, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095-1489, USA
2 Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095-1489, USA
* Author for correspondence (e-mail: fuyut{at}microbio.ucla.edu)
Accepted 3 August 2006
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Summary |
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Key words: Rheb, TOR, S6K, Oxidative stress, Aging
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Introduction |
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). A variety of mechanisms exists to protect an organism from stress including antioxidant enzymes, protein re-folding or degradation, DNA repair and stress-sensing pathways that effect gene expression of stress-defense genes (Finkel and Holbrook, 2000).
In eukaryotes, the target-of-rapamycin (TOR) signaling pathway regulates several processes including cell growth and protein translation (Aspuria and Tamanoi, 2004; Inoki et al., 2005b; Pan et al., 2004). TOR kinase activity is thought to stimulate protein synthesis by activation of S6 kinase (S6K) and repression of the translational inhibitor, 4E-BP. TOR proteins exist in multi-protein complexes and bind the small GTPase Rheb; Rheb proteins have been shown to stimulate TOR kinase activity (Long et al., 2005; Smith et al., 2005; Urano et al., 2005). In Drosophila, we as well as others have shown that Rheb can stimulate cell growth through TOR (Patel et al., 2003; Saucedo et al., 2003; Stocker et al., 2003). Furthermore, Tsc2, which functions as a GTPase-activating protein (GAP) when in complex with Tsc1, can negatively regulate Rheb-TOR signaling by stimulating the hydrolysis of GTP bound to Rheb, to GDP (Castro et al., 2003; Garami et al., 2003; Inoki et al., 2003; Tee et al., 2003; Zhang et al., 2003) and inhibit cell growth (Gao and Pan, 2001; Inoki et al., 2002; Potter et al., 2001; Tapon et al., 2001). However, TOR proteins also play important roles in the regulation of ribosome biogenesis, lifespan and in a number of age-related diseases (such as cancer and neurodegenerative disorders) (Inoki et al., 2005a; Sarbassov dos et al., 2005). Since evidence for the contribution of stress to aging and age-related disease is growing, the influence of TOR signaling on the response of the whole organism to stress needs to be investigated.
Here we use the genetically tractable organism Drosophila to examine the effect of the Rheb-TOR-S6K signaling on the stress response. Drosophila has proven to be a useful system to assess the effects of various signaling pathways on stress (Clancy et al., 2001; Hwangbo et al., 2004; Simon et al., 2003; Wang et al., 2003), because flies can be readily exposed to stress and the consequences of the exposure can be tested with a large number of animals over a short period of time. We find that adult flies with increased Rheb-TOR signaling through S6K are sensitive to oxidative stress. Similarly, we find that alteration of Rheb-TOR and S6K signaling affects the starvation response, suggesting that increased TOR signaling may affect the response to various forms of stress. Further, we find that increasing Rheb-TOR signaling in muscle sensitizes flies to oxidative stress and increasing Rheb-TOR signaling through S6K in the whole organism results in early senescence of locomotor activity. These data support a role for Rheb-TOR signaling in aging and age-related disease. Since levels of Rheb-TOR signaling are increased by elevated insulin levels, nutrient availability as well as in several disease states, these situations may perturb the normal stress response.
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Results |
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; Saucedo et al., 2003; Stocker et al., 2003). We used the GAL4/UAS system (Brand and Perrimon, 1993) to ubiquitously overexpress Rheb in adult flies to examine the stress response of adult flies with increased Rheb-TOR activity. To generate adults overexpressing Rheb (hs>Rheb and da>Rheb), we crossed flies carrying the UAS-Rheb transgene (Patel et al., 2003) to flies carrying either hs- or da-GAL4, which allow overexpression at low and high levels, respectively. Northern analysis of total RNA from 5-day-old adults reveals that Rheb is overexpressed at least fourfold with hs-GAL4 and 19-fold with da-GAL4 at 25°C (Fig. 1A). Overexpression of Rheb with hs-GAL4 results in phosphorylation of T398 of S6K (Fig. 1B) suggesting that low levels of Rheb overexpression can promote TOR signaling in adults. Flies overexpressing Rheb with hs-GAL4 seem fit overall, as they show normal body size and weight (Fig. 1C) and display normal developmental times (Fig. 1D), thus we used these flies for the stress experiments described below.
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Mild overexpression of Rheb sensitizes adult flies to oxidative stress
To test the effects of oxidative stress on flies with increased Rheb-TOR activity, we administered daily oxidative agents in 5% sucrose/PBS for 6 hours hs>Rheb to adult flies and then returned these flies to normal growth medium. As a control, each genotype was similarly fed 5% sucrose/PBS alone for 6 hours before returning them to normal growth medium. While all genotypes survive daily 6-hour feedings of the 5% sucrose/PBS diet (Fig. 2A,B), we found hs>Rheb flies to be sensitive to 5% sucrose/PBS containing 5% H2O2 (Fig. 2A). This increased sensitivity of hs>Rheb flies to H2O2 was also observed with continuous exposure to 5% H2O2 in 5% sucrose/PBS (data not shown). Post-mortem examination of hs>Rheb flies fed 5% H2O2 revealed necrotic gut tissue probably owing to activity of ingested H2O2 suggesting that mortality is due to toxicity of the oxidative agent and not due to starvation by avoidance of 5% sucrose/PBS containing 5% H2O2. Thus mild overexpression of Rheb appears to sensitize adult flies to oxidative stress. A similar sensitivity to oxidative stress can be observed in hs>Rheb flies fed yet another oxidative agent, 20 mM paraquat (methyl viologen) (Fig. 2B). By contrast, hs>Rheb flies were found to be as sensitive to 25 mg/ml G418 as control flies, suggesting that the effects of increased Rheb-TOR activity increases sensitivity to oxidative stress rather than to all toxic compounds in general (Fig. 2C).
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; Zhang et al., 2000).
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) with da-GAL4 (da>S6KSTDETE) were sensitive to oxidative stress (Fig. 3A). The S6KSTDETE variant mimics an active, hyperphosphorylated form of S6K (Barcelo and Stewart, 2002). Since strong sensitivity to oxidative stress was observed in flies overexpressing Rheb or S6KSTDETE, increased TOR activity might sensitize flies to oxidative stress by increasing S6K activity.
Reduced Rheb-TOR-S6K signaling confers resistance to oxidative stress
Since increasing systemic Rheb-TOR-S6K signaling sensitizes flies to oxidative stress, we tested whether reducing Rheb-TOR-S6K signaling in adult flies provides resistance to oxidative stress. We reduced Rheb-TOR-S6K signaling in adults by expressing the Tsc1/2 complex, Tsc2, or dominant-negative forms of TOR (FRB fragment) or S6K (KQ, kinase-dead form).
Co-overexpression of Tsc1 and Tsc2 during larval development can severely reduce cell growth by antagonizing TOR activity (Gao and Pan, 2001; Potter et al., 2001; Tapon et al., 2001). However, we were able to obtain viable adults co-overexpressing both Tsc1 and Tsc2 with hs-GAL4; co-overexpression of Tsc1 and Tsc2 with da- or tub-GAL4 is early larval lethal (data not shown) similarly to Rheb mutants (Patel et al., 2003; Saucedo et al., 2003). We found that weak co-overexpression of Tsc1 and Tsc2 with hs-GAL4 (hs>Tsc1/2) provides resistance to oxidative stress (Fig. 3B). Unlike Tsc1/2 co-overexpression, overexpression of Tsc1 or Tsc2 alone does not suppress growth in larval tissues (presumably as a result of low GAP activity of Tsc2 without Tsc1 in vivo), thus we were able to overexpress Tsc2 in adults using da-GAL4 or tub-GAL4. Strong overexpression of Tsc2 with da- or tub-GAL4 (da>Tsc2 and tub>Tsc2) flies provides resistance to oxidative stress similarly to hs>Tsc1/2 flies (Fig. 3B). This is consistent with the observation that Tsc2 can exhibit GAP activity towards Rheb (Zhang et al., 2003). Similarly, we found that overexpression of a dominant-negative form of TOR (TORFRB) with da-GAL4 (da>TORFRB) or a dominant-negative form of S6K (S6KKQ) with hs-(hs>S6KKQ) and da-GAL4 (da>S6KKQ) also conferred resistance to oxidative stress (Fig. 3B).
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Increased Rheb-TOR signaling in muscle sensitizes flies to oxidative stress
To determine whether particular tissues overexpressing Rheb contribute to the stress sensitivity observed in hs>Rheb flies, we used both constitutive and inducible GAL4 drivers to express Rheb in neurons, fat bodies and muscle tissue. We tested the effects of Rheb overexpression in neurons and fat bodies on the oxidative stress response, because expression of JNK (in neurons) and foxo (in fat bodies), can increase Drosophila adult lifespan and provide stress resistance (Giannakou et al., 2004; Hwangbo et al., 2004; Wang et al., 2003). We also included muscle in our analysis as a result of our observation of early senescence of locomotor activity in flies overexpressing Rheb.
To examine the stress response of flies overexpressing Rheb in adult neural tissue, we used the pan-neural elav-GAL4 and elav GS-GAL4 drivers. We found that constitutive expression of Rheb in all neurons did not affect development to adulthood nor did it sensitize adult flies to oxidative stress (Fig. 4A). For inducible expression, we used the GeneSwitch system that uses an inducible progesterone-receptor–GAL4 fusion. This allows expression from UAS-transgenes (Osterwalder et al., 2001; Roman et al., 2001) after feeding of the antiprogestin, RU486, and allowed us to bypass the embryonic lethality resulting from Rheb overexpression with many constitutive tissue-specific GAL4 drivers. We found that elav GS>Rheb flies (± RU) are also insensitive to oxidative stress (Fig. 4A); together these data suggest that expression of Rheb in neurons does not sensitize the whole organism to stress. To examine the stress response in flies overexpressing Rheb in the adult fat bodies, we used lsp2-GAL4 as used by Teleman et al. (Teleman et al., 2005) to overexpress Dp110 (the catalytic subunit of PI3K) in adult fat bodies. We found that lsp2>Rheb flies are insensitive to oxidative stress (Fig. 4B). We also used DJ634-GAL4, which allows expression primarily in the adult fat body and muscle (Kapahi et al., 2003). Adult flies expressing Tsc2, TORFRB or S6KKQ with DJ634-GAL4 lived longer (Kapahi et al., 2004); however, we found that DJ634>Rheb flies were also insensitive to oxidative stress (Fig. 4B). To further examine the effects of overexpressing Rheb in adult fat bodies, we used the S106 GS-GAL4 (Giannakou et al., 2004; Hwangbo et al., 2004) to express Rheb in the abdominal fat body but also found these flies to be insensitive to oxidative stress (Fig. 4B). These data together imply that expression of Rheb in the fat bodies probably does not sensitize the whole organism to stress. In contrast to neurons and fat bodies, expression of Rheb in muscle showed a significant increase in sensitivity to oxidative stress. Using the pan-muscle driver, MHC (myosin heavy chain) GS-GAL4 (Wang et al., 2003); we found that MHC GS>Rheb flies (+RU) are sensitive to oxidative stress (compared with MHC GS>Rheb, –RU flies) (Fig. 4C,D). We also used 24B-GAL4 (Brand and Perrimon, 1993; Kapahi et al., 2004), which is expressed in muscle and fat body; however, we did not obtain many viable adults. These data together suggest that muscle might be one of the tissues affected in hs>Rheb flies when exposed to oxidative stress.
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Increased Rheb-TOR signaling through S6K promotes early senescence of locomotor activity
Fly strains that exhibit an increased resistance to stress or increased lifespan often perform well in negative geotaxis assays (Gargano et al., 2005; Mockett et al., 2001). These assays test the ability of flies to travel against gravity, an innate escape behavior. In our assays, flies were tapped down to the bottom of their vials; we then scored the number of flies that travel 5 cm against gravity in 10 seconds. We found that flies overexpressing Rheb with hs-GAL4 (hs>Rheb) perform as well as control flies in these assays 5 days after eclosion (Fig. 6). However, 30 days after eclosion, hs>Rheb flies perform poorly compared with control flies (Fig. 6). We also found that co-overexpression of S6KKQ can rescue this defect (Fig. 6), suggesting that increased TOR signaling through S6K is responsible for the observed early senescence of locomotor behavior.
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Discussion |
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The importance of the Rheb-TOR signaling to the stress response is not limited to oxidative stress. We found that alteration of this pathway similarly affects the starvation stress response indicating that increased TOR signaling could also influence the response to other forms of stress. In addition, we found that increasing Rheb-TOR signaling in the whole organism results in early senescence of locomotor activity. Further, we found that co-overexpression of dominant-negative S6K can rescue the early senescence of locomotor activity in flies overexpressing Rheb, suggesting the importance of S6K as an effector of TOR in aging and age-related disease. A recent study in yeast also points to the importance of the TOR signaling in oxidative stress response and aging (Powers et al., 2006).
Several defense mechanisms exist to protect cells from oxidative stress such as anti-oxidant enzymes (Finkel and Holbrook, 2000). We did not find differences in the expression of anti-oxidant enzyme genes (Cu/Zn SOD, MnSOD, catalase or thioredoxin reductase-1) nor in anti-oxidant activities of catalase, Cu/Zn SOD or MnSOD in Rheb-overexpressing flies under normal conditions (data not shown). We also did not observe a difference in the gene expression of anti-oxidant enzymes (catalase and MnSOD) nor in catalase activity in flies overexpressing Rheb after exposure to 5% H2O2. Upon oxidative or starvation stress, a key transcription factor in metazoans, FOXO, promotes the expression of several stress-defense genes, including the gene encoding Drosophila 4E-BP (Thor) and those encoding anti-oxidant enzymes and chaperones (Essers et al., 2004; Greer and Brunet, 2005; Murphy et al., 2003; Wang et al., 2005). We found strong 4E-BP expression after exposure to oxidative stress in flies overexpressing Rheb (data not shown), suggesting that Rheb-TOR signaling does not sensitize the whole organism to stress by antagonizing FOXO activity.
Several recent studies suggest a need for deceleration of protein translation during stress (Holcik and Sonenberg, 2005; Tettweiler et al., 2005). Increased Rheb-TOR-S6K signaling may stimulate 5'-cap-dependent translation resulting in increased sensitivity of the organism to stress. Increased Rheb-TOR signaling could stimulate 5'-cap-dependent translation by (1) mediating assembly of the pre-translation initiation complex by increasing S6K activity (Holz et al., 2005) or by (2) enabling 5'-cap dependent translation by inhibiting 4E-BP. However, flies mutant for 4E-BP or S6K display only mild growth defects during development (Miron et al., 2001; Montagne et al., 1999). Although their importance in translation control in adults needs to be investigated further, increasing Rheb-TOR signaling may not sensitize the whole organism merely by promoting 5' cap dependent translation through inhibition of 4E-BP or by stimulation of S6K. Furthermore, these data allow for the possibility that TOR or S6K can influence the stress response through unknown protein and gene targets unrelated to protein translation such as relieving the cytoplasmic retention by TOR of important stress response transcription factors (Beck and Hall, 1999; Powers et al., 2006).
The failure of flies with increased Rheb-TOR-S6K signaling to elicit a stress response could result in the accumulation of oxidative damage resulting in premature aging and degeneration. One sign of early aging that we observe in flies overexpressing Rheb is the early senescence of locomotor activity (in particular, negative geotaxis). The senescence of negative geotaxis is observed in older flies as well as in short-lived strains that exhibit sensitivity to oxidative stress (Gargano et al., 2005; Mockett et al., 2001). Thus, the early senescence of locomotor activity in Rheb-overexpressing flies suggests the possibility for early aging and degeneration of these flies. The overexpression of either dominant-negative TOR or S6K can rescue the effects of Rheb overexpression on negative geotaxis implying that increased TOR signaling through S6K may be responsible for the early senescence or degeneration of locomotor activity.
Decreasing TOR activity results in increased lifespan in several organisms (Kapahi et al., 2004; Powers, 3rd et al., 2006; Vellai et al., 2003) and has recently been shown to play an important role in age-related neurodegenerative disease (Inoki et al., 2005a; Nelson et al., 2005; Ravikumar et al., 2004). Although we cannot exclude the possibility that the early senescence of locomotor activity in Rheb-overexpressing flies is due to long-term neural degeneration in these flies, it is possible that muscle degeneration could explain this behavior, because the overexpression of Rheb in muscle can sensitize flies to oxidative stress. Increased apoptosis is observed in leg and thoracic (which includes flight muscle) muscles in aging flies (Zheng et al., 2005) providing an attractive mechanism to explain the decreased locomotor activity of aged flies. Increased Rheb-TOR signaling in skeletal muscle may accelerate the process by inhibiting the stress response. Alternatively, levels of Rheb-TOR signaling in myocardial tissue could be an important determinant of lifespan because decreasing insulin signaling in Drosophila adults can prevent the decline of cardiac performance with age (Wessells et al., 2004). Further work using Drosophila may provide valuable insights into efforts to control TOR and S6K activity to treat several age-related diseases and/or their progression.
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Materials and Methods |
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), UAS-Tsc2 (Tapon et al., 2001), UAS-Rheb (Patel et al., 2003), UAS-TOR (Hennig and Neufeld, 2002), UAS-TORTED (Hennig and Neufeld, 2002), UAS-TORFRB (Hennig and Neufeld, 2002), UAS-S6KSTEDTE (Barcelo and Stewart, 2002) and UAS-S6KKQ (Barcelo and Stewart, 2002). Rheb was overexpressed in adult neurons using elav-GAL4 and elav GS-GAL4 (Osterwalder et al., 2001; Roman et al., 2001; Wang et al., 2003), in fat bodies using lsp2-GAL4 (Teleman et al., 2005), DJ634-GAL4 (Kapahi et al., 2004) and S106 GS-GAL4 (Giannakou et al., 2004; Hwangbo et al., 2004; Roman et al., 2001) and in muscle using 24B-GAL4 (Brand and Perrimon, 1993; Kapahi et al., 2004) and MHC GS-GAL4 (Osterwalder et al., 2001; Wang et al., 2003).
Northern analysis
Total RNA was isolated using TRIzol reagent (Invitrogen). 5 µg of Drosophila adult total RNA was separated on a denaturing 1% agarose-1.5% formaldehyde gel, transferred to nylon membrane and probed with [
-32P]dATP-labeled probes for Rheb and actin5C genes.
Western analysis
80 µg protein were separated on an 8% SDS-polyacrylamide gel. Phosphorylated S6K was detected using anti-phospho-S6K T398 antibody from Cell Signaling Technology.
Weight analysis
The mean weight of individual males and females of each genotype was determined with a Mettler ME30 precision scale (0.001-10 mg). Flies were grown and maintained at low density and weighed (n=50) 5 days post eclosion. Weight measurements were repeated three times.
Developmental rate
Embryos for each cohort were collected for 2 hours at 25°C. 25 embryos were immediately transferred to each food vial and allowed to develop at low density. 200 embryos were used for each genotype. Emerging adults were scored every 12 hours.
Stress tests
Embryos of each genotype were collected for 2 hours and then separated into new food vials (25 embryos per vial) to allow development to progress at low density. Newly eclosed adults were removed from their vials, maintained at low density (ten flies per vial) and allowed to mate for 5 days before exposure to each stress. Each stress test consisted of three to five vials, ten adult males per vial, repeated in three different experiments. Two different protocols were used to test sensitivity to oxidative stress. In one protocol (Fig. 3A,B), each genotype was placed into a vial containing a 3 mm Whatman filter wetted with a solution of 5% H2O2 in 5% sucrose/PBS. Vials were scored every 12 hours for the number of dead flies. Because Rheb-overexpressing adults were found to be sensitive to a continuous diet of 5% sucrose/PBS, a second type of oxidative stress test was performed by placing adults daily for 6 hours into a vial containing a 3 mm Whatmann filter wetted with a solution of 5% H2O2 or 20 mM paraquat (methyl viologen) in 5% sucrose/PBS (Fig. 2A,B and Fig. 3C). Adults were then transferred to normal growth medium. This treatment was repeated daily until the end of the experiment. Vials were scored every 24 hours for the number of dead flies. For starvation stress tests, flies were placed in a vial containing a 3 mm Whatmann filter wetted with PBS alone (Fig. 5A,B). Vials were scored every 12 hours for the number of dead flies. All stress tests were performed at 25°C.
Conditional expression of Rheb
Conditional expression of Rheb was effected by using the GeneSwitch (GS) system (Osterwalder et al., 2001; Roman et al., 2001; Wang et al., 2003). Tissue-specific conditional expression was obtained using elav GS-GAL4 (pan-neural), S106 GS-GAL4 (abdominal fat body) and MHC GS-GAL4 (pan-muscle). GS-GAL4 activity was induced by feeding flies 400 µg/ml RU486 for 24 hours; flies were then transferred to new vials for stress tests performed as described above.
Negative geotaxis assay
Ten adult males per empty vial were tapped down to the bottom of the vial and the number of flies that could travel 5 cm towards the top of each vial in 10 seconds was scored. Approximately 30-50 flies were used for each cohort; each experiment was repeated three times.
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Acknowledgments |
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