IMPACT is a GCN2 inhibitor that limits lifespan in Caenorhabditis elegans
IMPACT is an ancient protein that exerts fundamental roles in the cellular adaptive response to nutritional stress [26, 28, 30, 32]. IMPACT is conserved across the metazoan phylum alongside its interacting partners GCN1and GCN2, through which it negatively influences eIF2? phosphorylation and limits ISR activation in response to amino acid restriction and other stress conditions [28, 30]. Despite its important functions, no previous work had addressed the role of IMPACT in multicellular organisms. Here, we identified the IMPACT homolog of C. elegans (IMPT-1) and demonstrated that its partial loss-of-function is sufficient to induce the ISR under the fed state. In turn, impt-1 knockdown extends lifespan and ameliorates stress response in GCN-2 and ISR dependent manners. These effects also depend on several genes required for lifespan extension promoted by DR in C. elegans such as skn-1, daf-16, and others. These genetic interactions occur in a timely fashion, in which knocking down skn-1 during larval stages is sufficient to block lifespan extension exerted by impt-1 knockdown. Impt-1 silencing induces SKN-1 function in L2 worms, and affects DAF-16 function later in adulthood. These results highlight the pleiotropic functions of IMPT-1 on longevity, where its knockdown during development leads to a chain of events first manifested by ISR up-regulation, followed by SKN-1 activation and later by DAF-16 induction (Fig. 7).
Working model of lifespan extension induced by impt-1 knockdown in C. elegans. IMPACT acts as an inhibitor of GCN-2. Impt-1 knockdown activates the GCN-2 branch of the ISR, increases the phosphorylation of eIF2? and induces ATF-5 expression. This pathway is further stimulated by an increase in uncharged tRNAs exerted by dietary restriction. In early larval stages, ATF-5 induces SKN-1 or acts in concert with it to activate a stress response program that leads to DAF-16 induction in adults and confers lifespan extension. Impt-1 also interacts with hsf-1 during larval stages and with aak-2 and let-363 during adulthood to control lifespan
A similar phenomenon is observed when worms are offered excess NAD+ starting at late embryonic development [40, 41]. These protocols prolong lifespan by a mechanism that requires activation of the mitochondrial unfolded protein response (UPRmt) first observed in day 1 adults and that consequentially leads to DAF-16 translocation and sod-3 transcription in day 3 adults. Lifespan extension induced by NAD+ donors also requires the sirtuin SIR-2.1 and SKN-1 [41, 42], similarly to impt-1 knockdown-induced longevity (Fig. 5 and data not shown). Interestingly, mitochondrial stress induced by inhibition of certain components of the electron transport chain (i.e., isp-1 and clk-1) promotes eIF2? phosphorylation during development, activates UPRmt and prolongs lifespan in C. elegans, and this requires GCN-2 [43]. Moreover, mild mitochondrial stress [44] or stimulation of UPRmt [42] during the larval stages are sufficient to render worms long lived. We thus suggest that multiple metabolic pathways can converge into GCN-2 and the ISR pathway during development to regulate lifespan and IMPT-1, as an inhibitor of the GCN-2 pathway, limits their effects.
How exactly the activation of GCN-2 and the ISR pathway initiates downstream events during development that culminate in lifespan extension is not absolutely clear. Since expressions of the ISR transcription factor ATF-5 and the SKN-1 target gene gst-4 are more prematurely up-regulated by impt-1 RNAi and both ATF-5 and SKN-1 are required during larval stages for the longevity phenotype of impt-1 knockdown models, we propose that these two transcription factors are acting in concert to trigger a longevity pathway. Indeed, in human cells, ATF4 (the mammalian ATF-5 homolog) has been shown to dimerize with NRF2 (the mammalian SKN-1 homolog) to activate the enhancer of the heme oxygenase-1 gene [45]. In C. elegans, SKN-1 and ATF-5 are mutually regulated in response to endoplasmic reticulum (ER) or oxidative stress [46]. Importantly, atf-5 is a SKN-1 target gene and atf-5 ablation prevents ER stress from inducing transcription of skn-1 and its target genes [46]. Thus, these studies and our observations provide evidence to suggest that SKN-1 function is controlled by the ISR. However, NRF2 is also a direct substrate of the ER stress-regulated eIF2? kinase PERK, and it has been proposed to mediate PERK-dependent cell survival independently of eIF2? phosphorylation in mouse cells [47]. Whether SKN-1 and ATF-5 act together or in parallel to control lifespan of C. elegans is a matter for future studies.
The importance of IMPT-1 and GCN-2 during larval stages is further supported by delayed development in the impt-1(ok3233) heterozygous mutants and mid-larval arrest in impt-1(ok3233) homozygous mutants and in N2 worms treated with krs-1 RNAi from eggs ([17] and confirmed by our own data). Given that both krs-1 and impt-1 ablations activate ISR when initiated in early development (Fig. 1 and [17]), these interventions could represent a stress signal that inhibits progression through the larval stages [48–50]. However, GCN-2 depletion by RNAi does not reverse the larval arrest phenotype of impt-1(ok3233) homozygous mutants (data not shown), indicating independence of GCN-2. Similarly, the knockdown of IMPACT in mouse neuronal cell lines partially affects neuritogenesis in a GCN2-independent manner [32]. In addition, it has been shown that Yih1 controls yeast cell cycle independently of Gcn2 or Gcn1 [51]. These phenotypes are in sharp contrast with the effects of impt-1 knockdown on longevity and stress resistance, which require both gcn-2 and gcn-1, evidencing the broad, yet poorly understood roles of IMPACT.
Impt-1 knockdown by RNAi or by its heterozygous null mutation render worms with very similar phenotypes but also with some fundamentally different ones. Both interventions decrease impt-1, reduce pharyngeal pumping, increase lifespan, and promote stress resistance at similar levels. On the other hand, while impt-1
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reduces brood size, delays development and decreases triglyceride content, impt-1 RNAi does not affect these parameters, or in the case of developmental timing even accelerates it. Impt-1 RNAi also does not promote eIF2? phosphorylation as impt-1
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does, at least not in the time point that we analyzed, but it increases ATF-5 expression, which is directly downstream of eIF2?. These distinctions may be explained by different hypotheses, including maternally inherited traits, RNAi delivery constraints or timing. More importantly, they highlight the fact that these phenotypes are not completely associated, and that impt-1 knockdown can increase lifespan without continuous hyperphosphorylation of eIF2? or affecting fertility, development, and fat accumulation.
In a broad spectrum, the phenotypes of impt-1 knockdown models can resemble the characteristics of dietary restricted animals, i.e., increased lifespan, improved stress response, delayed development, reduced fertility, decreased fat accumulation, and diminished food intake. Additionally, the levels of IMPT-1 and uncharged tRNAs seem to limit each other’s effects. For example, when impt-1 is decreased (e.g., in impt-1
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mutants or impt-1 RNAi) but uncharged tRNAs levels are low (e.g., during the fed state), lifespan extension is limited, as observed when uncharged tRNA levels increase (e.g., during DR) in the presence of high levels of IMPT-1 (e.g., in N2 worms or control RNAi). However, when DR and impt-1 knockdown are combined, lifespan is extended to its maximum under these circumstances. This could be interpreted through the perspective that DR and impt-1 knockdown act in parallel pathways. However, the genetic interaction of impt-1 with essentially all downstream components of the DR pathway suggests otherwise, that DR and impt-1 indeed share the same route to control lifespan. One alternative explanation is that impt-1 knockdown releases more GCN-1 to interact with GCN-2 and respond more efficiently to uncharged tRNAs that are more abundant during DR. Therefore, loss of impt-1 would have an effect under the fed state, and this effect would be exacerbated under DR. Considering that impt-1 knockdown (Fig. 2), eat-2 [17] and krs-1 RNAi (Additional file 6: Figure S6) all increase lifespan in GCN-2 or GCN-1 dependent manners, we suggest that these interventions converge into activating GCN-2 to exert their effects.
However, how can impt-1 knockdown increase GCN-2 activation under the fed state? A similar effect was observed in mouse neuronal cells, which express high levels of IMPACT. The knockdown of IMPACT in these cells resulted in increased levels of phosphorylated GCN2, the active form of the kinase, and of phosphorylated eIF2? [32]. It is possible that the resulting increase in GCN-1 available for interacting with GCN-2 will render GCN-2 more sensitive to the basal levels of uncharged tRNAs that occur naturally in the cells, as a result of each cycle of translation elongation as they exit the ribosomes.
And why would DR and krs-1 RNAi increase lifespan when applied to adult worms while impt-1 knockdown requires activation of SKN-1 during development? One possible explanation is a model where stoichiometric balance determines the effects. In such a model, uncharged tRNA levels are sufficiently high during development (perhaps due to increased protein synthesis) and IMPT-1 becomes rate limiting, exerting an important inhibitory pressure on GCN-2. On the other hand, uncharged tRNAs are less abundant in adults and therefore IMPT-1 is dispensable for GCN-2 regulation in the absence of a proper stimulus. This model is supported by the important functions of IMPT-1 during development and by the fact that krs-1 RNAi during adulthood adds to impt-1
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to prolong lifespan. Curiously, while krs-1 RNAi applied to adult N2 worms increases lifespan in our hands, another study reported decreased lifespan of N2 worms under the same RNAi clone [17]. These discrepancies might be related to slightly different protocols that could sensitize or desensitize the GCN-2 pathway. One such difference in our study is the use of FUdR, which inhibits progeny.
Reduced food intake is usually the primary cause of DR. The fact that impt-1 knockdown causes the pharyngeal pumping rate to be decreased indicates that this phenotype may also explain or at least partially contribute to longevity. Interestingly, brain-specific activation of GCN2 and phosphorylation of eIF2? leads to an aversive behavior against amino acid deficient diets [52–54]. Hyperactivation of the GCN2 pathway may therefore represent an evolutionarily conserved hub to control food intake and metabolic adaptation in response to changes in nutrient availability.