EAK proteins: novel conserved regulators of C. elegans lifespan.

FoxO transcription factors (TFs) extend lifespan in invertebrates and may participate in the control of human longevity. The role of FoxO TFs in lifespan regulation has been studied most extensively in C. elegans, where a conserved insulin/insulin-like growth factor signaling (IIS) pathway and the germline both control lifespan by regulating the subcellular localization of the FoxO transcription factor DAF-16. Although the control of FoxO activity through modulation of its subcellular localization is well established, nuclear translocation of FoxO is not sufficient for full FoxO activation, suggesting that undiscovered inputs regulate FoxO activity after its translocation to the nucleus. We have recently discovered a new conserved pathway, the EAK (enhancer-of-akt-1) pathway, which acts in parallel to the Akt/PKB family of serine-threonine kinases to regulate DAF-16/FoxO activity. Whereas mutation of Akt/PKB promotes the nuclear accumulation of DAF-16/FoxO, mutation of eak genes increases nuclear DAF-16/FoxO activity without influencing DAF-16/FoxO subcellular localization. Thus, EAK proteins regulate the activity of nuclear DAF-16/FoxO. Two EAK proteins, EAK-2/HSD-1 and EAK-7, influence C. elegans lifespan and are conserved in mammals. The discovery of the EAK pathway defines a new conserved FoxO regulatory input and may have implications relevant to aging and the pathogenesis of aging-associated diseases.

dysregulation similar to that observed in Type 2 diabetes, as FoxO1 haploinsufficiency protects mice against insulin resistance induced by a high-fat diet [17], and both liver-specific and osteoblast-specific FoxO1 deletion ameliorate glucose intolerance in mouse models of insulin resistance [18,19,20]. Thus, in mammals, FoxO TFs have context-dependent effects on the development of phenotypes associated with agerelated disease. Elucidating the regulatory mechanisms that maintain the balance of FoxO TF activity may prove to be crucial for understanding and combating the progression of age-related disease.

DAF-16/FoxO regulation in the control of C. elegans lifespan
DAF-16/FoxO is required for C. elegans lifespan modulation by IIS and the germline [1,2], as well as in some contexts of dietary restriction [21]. Reduction of IIS and ablation of the germline both extend lifespan by increasing DAF-16/FoxO activity. Neither intervention increases lifespan in daf-16/FoxO null mutants [1,2], indicating that DAF-16/FoxO is the critical target of IIS and the germline in lifespan control.
How the germline promotes the cytoplasmic sequestration of DAF-16/FoxO is not entirely understood. Notably, germline ablation extends lifespan in animals with reduced IIS [2]. In animals lacking a germline, translocation of cytoplasmic DAF-16/FoxO to the nucleus requires the nuclear receptor DAF-12 [29] and its steroid hormone ligands [29,30], which are known as dafachronic acids (DAs) [31]. The conserved protein KRI-1 is required for DAF-16/FoxO nuclear translocation in animals lacking a germline but largely dispensable for DAF-16/FoxO nuclear localization in animals with reduced IIS [29]. In aggregate, these observations suggest that IIS and the germline control the subcellular localization of DAF-16/FoxO via distinct mechanisms.

Nuclear translocation is not sufficient for full DAF-16/FoxO activation
Although nuclear localization of DAF-16/FoxO is clearly necessary for DAF-16/FoxO-dependent lifespan extension, multiple lines of evidence indicate that it is not sufficient for full DAF-16/FoxO activation. For example, a DAF-16/FoxO mutant lacking all four canonical Akt/PKB phosphorylation sites localizes to the nucleus but fails to fully extend lifespan [24,29]. This indicates that a second pathway acts in parallel to Akt/PKB and the germline to inhibit the activity of nuclear DAF-16/FoxO.

A genetic screen for molecules that regulate nuclear DAF-16/FoxO activity
To identify components of this parallel DAF-16/FoxO regulatory pathway, we exploited the fact that in larvae, DAF-16/FoxO promotes developmental arrest in an alternative larval stage called dauer that is morphologically distinct from reproductively developing larvae [32]. We performed a genetic screen for mutants that enhance the weak dauer-constitutive phenotype of an akt-1 null mutant (eak screen). We identified 21 independent mutants that define seven eak genes, six of which have been cloned (Table 1) [33,34,35]. Strikingly, five of the six cloned eak genes are expressed specifically in the two endocrine XXX cells [34,35,36]; in contrast, eak-7 is expressed in the XXX cells as well as multiple other tissues [33]. The phenotypic similarity of all eak single mutants and the observation that no eak;eak double mutant combination tested to date exhibits phenotypic enhancement compared to eak single mutants indicate that the EAK proteins are components of a single pathway.
EAK proteins and AKT-1 both control DAF-16/FoxO target gene expression. However, in contrast to AKT-1, which inhibits DAF-16/FoxO activity by promoting its translocation from the nucleus to the cytoplasm, EAK proteins inhibit nuclear DAF-16/FoxO activity without influencing its subcellular localization [33,34,35]. Collectively, the data indicate that EAK proteins define a new conserved endocrine pathway that acts in parallel to Akt/PKB to regulate the activity of nuclear DAF-16/FoxO. Two eak genes, eak-2/hsd-1 and eak-7, have putative mammalian orthologs based on comparative sequence analysis [33,34].
The role of HSD-1 in lifespan control is complex. In wild-type animals, HSD-1 does not have a major impact on adult lifespan. However, in animals with reduced IIS, HSD-1 is required for full lifespan extension [34]. This is reminiscent of the requirement for DAF-36 and DAF-9 in lifespan extension induced by germline ablation [29,40]. Surprisingly, in contrast to daf-36 and daf-9 mutations, which substantially shorten the lifespan of animals lacking a germline [29,40], hsd-1 mutation does not affect lifespan extension induced by germline ablation [34]. This may be a consequence of the relative potencies of Δ 4 -and Δ 7 -DA and/or the anatomical source of distinct DAs (XXX cells vs. hypodermis and somatic gonad). Alternatively, HSD-1 may participate in the biosynthesis of a different steroid hormone that is required for full lifespan extension in the context of reduced IIS but dispensable for lifespan extension induced by germline ablation.
Notably, although eak-7 mutation strongly enhances the dauer-constitutive phenotype of akt-1 mutants, its enhancement of lifespan extension in akt-1 mutants is relatively modest [33]. This is likely a consequence of the influence of the germline on DAF-16/FoxO subcellular localization; since the germline promotes the cytoplasmic sequestration of DAF-16/FoxO [24], akt-1 mutation may not increase nuclear concentrations of DAF-16/FoxO to the extent that it does in early larval stages, before the germline has proliferated. In accordance with this, eak-7 mutation strongly enhances lifespan extension (by ~50-80%) in animals lacking a germline [33].

Potential role of the EAK pathway in diseases associated with aging
Although studies in mammalian cells have also implicated a regulatory input that controls the activity of FoxO TFs that are localized to the nucleus [41], the nature of this input is obscure. Our data suggest that the putative HSD-1 and EAK-7 mammalian orthologs Sdr42e1 and KIAA1609 may be components of this input. Recent work on mice harboring conditional knockout alleles of FoxO1 point to a potential role for FoxO TFs in the pathogenesis of Type 2 diabetes [17,18,19,20], cancer [14], and osteoporosis [16,42]; thus, it is conceivable that the EAK pathway may influence the pathogenesis of these aging-associated diseases.
The function of the putative mammalian HSD-1 ortholog Sdr42e1 is not known. Sdr42e1 mRNA is expressed in a limited number of mouse tissues, with strikingly high expression in liver [43], where FoxO1 plays an important role in metabolic homeostasis [19,20]. In contexts of insulin resistance, reduction of hepatic FoxO1 activity substantially ameliorates dysregulation of glucose metabolism in mice [19,20]. Since FoxO1 is nuclear in these circumstances [44], Sdr42e1 could play an important role in controlling hepatic FoxO1 activity and glucose homeostasis. The putative EAK-7 ortholog KIAA1609 is widely expressed in mouse tissues and is also present in mouse liver [43], indicating that KIAA1609 may also influence glucose metabolism in the context of insulin resistance. Both Sdr42e1 and KIAA1609 are expressed in mouse osteoblasts [43], where FoxO TFs promote the maintenance of normal bone mass and control metabolism nonautonomously through regulation of osteocalcin activity [16,42]; thus, EAK pathway activity in osteoblasts could influence the development of both osteoporosis and Type 2 diabetes. Intriguingly, KIAA1609 is highly expressed in mouse retinal pigment epithelium (RPE), where FoxO1 and FoxO3 are also expressed [43]. Although nothing is known about the function of FoxO TFs in RPE, the extreme www.impactaging.com levels of KIAA1609 expression in this tissue suggest that the EAK pathway could be important in regulating FoxO activity in RPE.
It is noteworthy that the genes encoding human Sdr42e1 and KIAA1609 lie within 2.5 megabases of each other at chromosome 16q23. Copy number variation in this genomic region could affect both genes coordinately, changing baseline EAK pathway activity (and, in turn, FoxO TF activity) accordingly.

SUMMARY
The EAK pathway is a new conserved FoxO regulatory input that acts in parallel to Akt/PKB and the germline to control C. elegans lifespan. Continued investigation of the C. elegans EAK pathway promises to reveal new insights into mechanisms of FoxO regulation. In light of the potential role of FoxO TFs in mammalian aging and the pathogenesis of aging-associated diseases, EAK proteins could emerge as promising targets for the development of new drugs to treat Type 2 diabetes, cancer, and osteoporosis. Studies of Sdr42e1 and KIAA1609 function in mouse models of aging and disease will be especially revealing in this regard.