Klotho interferes with a novel FGF-signalling pathway and insulin/Igf-like signalling to improve longevity and stress resistance in Caenorhabditis elegans

1- Genetic characterization of the two klotho genes in C. elegans

The C. elegans genome contains two sequences homologous to the mammals klotho gene : C50F7.10 and E02H9.5. Although these two paralogues differ in their genomic organization (Figure 1A), they encode ORFs of similar size and appear genetically redundant, since no obvious function emerged from previous mass RNA-mediated gene interference (RNAi) assays [29]. In addition, a sole putative klotho gene has been predicted in the C. briggsae genome [30]. Considering that the latter species diverged from C. elegans ~100 millions years ago, it is probable that a unique gene has been retained by evolution.

Figure 1. Characterization of Klotho in C. elegans. (A) Genomic organization of both C. elegans C50F7.10 (1,95 kb) and E02H9.5 (2,3 kb) genes, localized on chromosome IV and III, respectively. Coding regions are indicated by boxes, and introns are represented as lines. The corresponding ORFs share similar size (about 1,44 kb) and are organized in 8 and 7 exons for C50F7.10 and E02H9.5, respectively. (B) The predictive molecular organization of either C50F7.10 or E02H9.5 gene products essentially consists in a sole b-glucosidase-like KL1 domain. Note that a KL1 form of Klotho may be expressed either by differential splicing or post- translational cleavage, in mammals. (C) Alignment of alternatively spliced forms of human and mouse Klotho compared to both C. elegans C50F7.10 and E02H9.5 gene products, identified in the WormBase bank as WP: CE 04248 and WP: CE 09122, respectively. Identical amino acid residues are highlighted. The conserved KL1 domain is underlined. Alignment was performed using the ESPript program [60].

The predictive sequences of the C50F7.10 and E02H9.5 gene products (479 and 475 amino-acids, respectively) are 76% identical and share 32 to 34% amino acid identity with their mammalian counterparts. Interestingly, both C. elegans predicted gene products consist of a sole β-glucosidase-like domain that is homologous to the highly conserved KL1 domain of human and mouse Klotho proteins but that lacks a secretory signal peptide (Figure 1B,C). However, a potential secretion mechanism, via an endoplasmic reticulum/Golgi complex-independent pathway, cannot be excluded. Such a mechanism has been described in mammals for some secreted FGFs [31,32]. Furthermore, the membrane-bound Klotho could not be observed at the surface of kidney cells in mammals, but only as a diffuse expression in the cytoplasm. In addition, a combination of both conventional and a novel, Klotho-dependent, secretory pathways has also been reported [33,34].

Figure 2. A genetic interaction between Klotho and the EGL-17/EGL-15 (FGFR) signalling pathway can positively modulate lifespan. Adult worm lifespan analysis was performed at 20°C on RNAi plates as described in the text. Animals of indicated genotype were submitted to either klotho RNAi (solid symbol) or control L4440 vector RNAi (open symbol) throughout adulthood. See Table 1 for corresponding quantitative data and statistical analysis.

2- Klotho requires the EGL-17/EGL-15 (FGF-R) complex to regulate C. elegans longevity

To analyse the in vivo function of Klotho in C. elegans, we have chosen a genetic approach by using RNA-mediated gene interference (RNAi). To circumvent the genetic redundancy of C50F7.10 and E02H9.5, we designed RNAi capable of simultaneously reducing the expression of both paralogues.

As previously reported in mammals [1], we then assessed whether klotho gene knockdown can affect worm lifespan. As expected, N2 (wild type) worms showed a highly significant reduction (LogRank p < 0.0001) of longevity when submitted to klotho RNAi, in comparison with control (Figure 2A; Table 1). Similar results were observed when the E02H9.5 (ok1830) knockout strain was tested in place of the N2 (wild type) strain, supporting the genetic redundancy of the two paralogues (not shown).

In mammalian cells, the membrane-bound Klotho isoform has been reported to bind to multiple FGFRs, increasing their affinity to FGF23. Because of its weak affinity to FGF23, the secreted Klotho is not likely to function as a soluble co-receptor for FGF23. Whether this secreted Klotho product can modulate lifespan by interacting with FGF signalling in a FGF23 independent manner has not been investigated.

We then asked whether FGF signalling is a determinant in the regulation of worm lifespan by Klotho. To avoid possible redundant effects of the two EGL-15 ligands, we first tested whether klotho gene deficiency alters lifespan in an egl-15 gene-dependent manner.

As shown in Figure 2B and Table 1, the mean life-span of the egl-15 (n484) reduction-of-function allele, fed with either control or klotho RNAi, was clearly identical to N2 (wild-type) worms fed with klotho RNAi only (LogRank p = 0.037; p = 0.54, respectively).

Downregulation of both egl-15 and klotho genes reduces lifespan by a similar order of magnitude, strongly suggesting that the entire modulation of lifespan by klotho gene in worms, is mediated by the FGFR EGL-15. Interestingly, egl-15 (n484) has been reported to mimic the phenotype of an egl-17 reduction-of-function allele [35]. On the basis of this mutant allele effect, we then assessed whether klotho gene deficiency might alter lifespan in an egl-17 gene-dependent mechanism. As expected (Figure 2C; Table 1), the mean lifespan of the egl-17 (n1377) reduction-of-function allele, fed with either control or klotho RNAi, was comparable to that of N2 (wild-type) worms fed with Klotho RNAi only (LogRank p = 0.235; p = 0.261, respectively). Since either reduction-of-function of egl-17 or klotho gene knock down can impair worm lifespan to a similar degree, our data indicate that EGL-17 alone (of the EGL-15 ligands) is necessary and sufficient to modulate lifespan in a Klotho-dependent manner. Moreover, we demonstrate, for the first time, a specific involvement of EGL-17 for normal post-developmental events in worm. Taken together, our results support genetic evidence that Klotho can signal via the EGL-17/EGL-15 pathway to regulate worm lifespan in physiological conditions.

Of the two known FGFR ligands in worm, LET-756 has been primarily implicated in fluid balance regulation [24], and more recently in muscle protein degradation [28]. However, because of the essential function accomplished by LET-756, we were not able to assess its implication in the regulation of lifespan, but we cannot exclude that it does not also play a role. Moreover, according to our results, such a potential role should be independent of Klotho.

3- Klotho is not involved in the worm fluid balance homeostasis

In C. elegans, the vital maintenance of fluid homeostasis is under the control of the LET-756/EGL-15 (B) complex which activates the LET-60 (RAS) /MPK-1 (MAPK) pathway [36]. Such a regulatory pathway is reminiscent of the role of FGF23 in the kidney, where it regulates ionic homeostasis and, therefore, helps in the maintenance of global fluid balance. In fact, the mammalian FGF-23/Klotho /FGFR-1 complex has been reported to activate the MAP kinase pathway [8].

We then assessed for a possible link between Klotho and functions implicating LET-756/EGL-15. In C. elegans, the existence of a negative regulator of FGFR signalling allows genetic activation of EGL-15. EGL-15 (FGFR) signalling is normally attenuated by a receptor tyrosine phosphatase, encoded by the clr-1 gene. Reduced CLR-1 receptor activity provokes an over-activation of EGL-15 (FGFR) signalling leading to a Clr (clear) phenotype, characterized by fluid accumulation within the pseudo-coelom [37]. Thus, clr-1 mutants have proven useful for screening Soc (suppressor of Clr) regulators, which can reverse the Clr phenotype by decreasing EGL-15 signalling [35,36].

We took advantage of the Clr model phenotype described above to assess whether klotho gene deficiency might diminish EGL-15 signalling in a clr-1 genetic background and thus suppress, at least partially, the Clr phenotype.

When temperature-sensitive allele clr-1(e1745) worms are shifted to restrictive temperature (25°C), they develop a Clr phenotype. We then pre-induced clr-1(e1745) worms to either klotho or control RNAi at permissive temperature (15°C) before shifting them to the restrictive temperature.

Figure 3. Klotho gene knockdown cannot suppress the Clr phenotype induced by genetic activation of EGL-15. Adult worms were pre-induced with either klotho or control RNAi at permissive temperature (15°C), prior to being either maintained at 15°C or shifted to restrictive temperature (25°C) which allows the development of a Clr phenotype. (A) Experiments were conducted in a clr-1(e1745) genetic background. The induced Clr phenotype is characterized by both intestine and gonad floating in an enlarged fluid-filled pseudocoelomic cavity. (B) Controls, performed in a clr-1(e1745); let-756(s2613) reduction-of-function double mutant show a partial suppression of Clr phenotype, independent of the klotho gene status.

As shown in Figure 3A, klotho knockdown failed to suppress the Clr phenotype, characterized by both intestine and gonad floating in an enlarged fluid-filled pseudocoelomic cavity. As controls, similar experiments were done using a clr-1(e1745); let-756(s2613) reduction-of-function double mutant in place of clr-1(e1745) mutant allele. In accordance with Borland's data [36], our results show that let-756 gene activity reduction leads to a partial suppression of the Clr phenotype (Figure 3B). However, this latter observation appears to be independent of the klotho gene status.

Taken together, our data suggest that Klotho is not involved in the maintenance of fluid balance in the nematode. Furthermore, our observations again argue for a lack of interaction between Klotho and the LET-756 ligand.

4- Klotho targets the C. elegans Insulin/Igf-like/Daf-2 receptor and requires Daf-2/Daf-16 signalling pathway for lifespan modulation

Overexpression of Klotho in mice has been reported to inhibit the activation of the Insulin/Igf-like receptor [2]. Furthermore, loss-of-function of the worm daf-2 gene, that encodes for an Insulin/Igf-like receptor, can double worm lifespan [38]. We therefore asked whether Klotho behaves in the same way, by acting as a constitutive Insulin/Igf-likesignalling pathway repressor, in worm. With this in mind, we assessed whether klotho gene deficiency can modulate worm lifespan in a daf-2 gene-dependent manner. Statistical analysis (Table 2) showed that klotho knockdown failed to induce detectable lifespan modulation (LogRank p = 0.027) in a daf-2 (e1370) genetic background (Figure 4B). However, we detected (Table 2) a slightly significant lifespan modulation (LogRank p = 0.002) when klotho knockdown was assessed in a daf-2 (m577) genetic background (Figure 4C). One possible explanation for such a Klotho RNAi effect discrepancy between the two daf-2 reduction-of-function alleles assessed for lifespan could be differences in their daf-2 mutant class. Thus, while daf-2(e1370) belongs to the daf-2 class 2-allele and behaves as a kinase-dead-like mutant, the daf-2 (m577) reduction-of-function allele, which belongs to the daf-2 class 1-allele, is modified by a point-mutation in the ligand-binding site and therefore may exhibit residual, ligand-independent, catalytic activity [39,40]. Taken together, our data argue for in vivo lifespan modulation by Klotho mediated by the Insulin/Igf-like/Daf-2 receptor activity by either a ligand-dependent or -independent effect.

In mammalian cell cultures, Klotho overexpression can diminish the recruitment of phosphatidyl-inositol 3-kinase (PI3-kinase) regulatory subunits that normally leads to Insulin/Igf-like receptor activation [2]. However, to date, the direct inhibition of PI3-kinase catalytic activity by Klotho has not been demonstrated. We investigated the involvement of PI3-kinase/AGE-1 in worms by submitting the strong loss-of-function allele age-1(mg44) mutant to klotho knockdown. As shown (Figure 4D; Table 2) we cannot detect any significant (LogRank p = 0.077) lifespan variation when compared to control. Taken together, our results demonstrate a fully functional Insulin/Igf-like/PI3-kinase signalling pathway for worm lifespan modulation by Klotho.

Figure 4. Klotho targets daf-2 gene activity and requires a functional daf-2/daf-16 genetic pathway for lifespan modulation. See Figure 2 for legend and Table 2 for corresponding quantitative and statistical analysis.

The worm lifespan extension induced by different daf-2 class-mutants is believed to occur by de-repression of the FOXO transcription factor family member DAF-16 [41]. Daf-16 gene products are thought to be permanently under negative regulation by the daf-2/age-1 genetic pathway. Therefore, strong daf-16 loss-of-function alleles can suppress the long-lived phenotype of daf-2 mutants. We thus submitted the daf-16 (mgDf50) deletion mutant to klotho RNAi. As expected, worm lifespan was not affected (LogRank p = 0.996) in this genetic background (Figure 4E; Table 2). This result makes the daf-16 gene a major output for klotho gene effect on worm lifespan modulation.

In summary our data demonstrate that Klotho can significantly convert an N2 (wild type) worm lifespan (Figure 4A; Table 2) to a shorter one. However, since this negative lifespan modulation vanished when daf-2, age-1 or daf-16 mutants were submitted to klotho knockdown, we propose that Klotho de-represses DAF-16 by constitutively down-regulating DAF-2. Therefore we provide here strong evidence for a direct link between the Insulin/Igf-like/PI3-kinase/FOXO signal-ling pathway and klotho gene action on lifespan modulation in a whole organism, which then argues for an evolutionary conserved mechanism.

Figure 5. Klotho requires a functional EGL-17/EGL-15 signalling pathway to improve oxidative stress resistance by a daf-16-dependent but daf-2-independent genetic pathway. Adult worms of the indicated genotype were pre-induced to either klotho or control RNAi at 20°C, then subjected to oxidative treatment by 25mM Menadione during 72 h and their viability scored. Controls were performed in the absence of Menadione. Results are mean values +/- SD of at least four independent experiments. Statistical analysis was done by a Student t-test at *p < 0.05 signification level. At least 100 worms were scored for each test condition. All experiments were performed at 20°C.

5- Klotho requires functional EGL-17/EGL-15 signalling to improve oxidative stress resistance in a DAF-16-dependent and DAF-2-independent mechanisms

It has been reported that Klotho can increase oxidative stress resistance at the cellular and organism levels in mammals [42,43]. It is also believed that FOXO transcription factor de-repression by Klotho-induced downregulation of Insulin/Igf-like signalling should induce antioxidant enzymes overexpression.

Two antioxidant enzymes: manganese superoxide dismutase and catalase are known to facilitate removal of reactive oxygen species towards a permanent molecular scavenger action. Since increased resistance to oxidative stress has been associated with increased longevity in various species, including C. elegans [44], we then asked if Klotho may help protecting worms when subjected to a sub-lethal oxidative stress induced by Menadione [45].

As shown in Figure 5, both the egl-15 (n484) reduction-of-function allele and the daf-16 (mgDf50) deletion mutant, fed with either control or klotho RNAi, displayed an increased sensitivity to oxidative stress in similar fashion to the N2 (wild-type) worms fed with klotho RNAi only. As expected, similar results were obtained when the egl-17(n1377) reduction-of-function allele was used in place of the egl-15(n484) mutant (not shown).

These data suggest that Klotho requires functional EGL-17/EGL-15 signalling to improve oxidative stress resistance in a DAF-16-dependent manner. We further checked whether Klotho requires a fully functional DAF-2 tyrosine kinase domain to improve oxidative stress resistance. Surprisingly, the daf-2(e1370) loss-of-function allele showed an increased susceptibility to oxidation when fed with klotho RNAi with respect to control RNAi. Since the functional activity of the tyrosine kinase domain is impaired in this class mutant allele, our results strongly suggest that Klotho may activate DAF-16 by a different pathway from the canonical worm DAF-2/DAF-16 signalling cascade. Interestingly, some reports using mammalian models have shown possible FOXO activation by alternative pathways that involve inhibition of the serum- and glucocorticoid-inducible kinase SGK [46] or activation of either the c-Jun NH2-terminal kinase (JNK) [47] or β-catenin [48].

Strains and culture conditions

The following strains were used: Wild type Bristol (N2); E02H9.5 (ok1830); MT1079 egl-15(n484); MT3188 egl-17(n1377); CB3241 clr-1(e1745); PJ1153 clr-1(e1745) [let-756(s2613) unc-32(e189) ccls55]; CB1370 daf-2(e1370); GR1032 age-1(mg44)/mnC1[dpy-10(e128) unc-52(e444)]; GR1307 daf-16(mgDf50) which were all provided by the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resources). DR1567 daf-2(m577) was kindly provided by David Gems, University College London, UK. Worms were maintained at 15°C and, unless specified, cultured at 20°C as described previously [57].

Expression of RNAi constructs

E02H9.5 and C50F7.10 entire ORFs were cloned from C. elegans cDNA into a pBluescript II KS (+/-) vector (Stratagene, La Jolla, CA, USA). RNAi constructs were obtained after E02H9.5 and C50F7.10 ORFs were amplified from pBluescript II KS (+/-) and 1425 and 1464 bp respective fragments were inserted at the SmaI site of a pPD129.36 (L4440) vector (a generous gift of Johnathan Ewbank, CNRS, Marseille, France). RNAi producing bacteria were obtained as follows: after transformation with either pPD129.36, E02H9.5 or C50F7.10 RNAi construct, HT115 (DE3) strain bacteria were grown in LB medium in presence of both Ampicillin (50 μg/ml) and Tetracyclin (12,5 μg/ml) during 8 h, then seeded on NGM plates containing FUdR (98,5 μg/ml), Ampicillin (50 μg/ml) and IPTG (1mM) and induced overnight.

RNAi feeding and lifespan assays

Synchronized L4 larvae fed with RNAi producing bacteria were allowed to grow at 20°C until death on supplemented NGM plates, including FUdr to avoid offspring [58]. Worms were examined every day for touch-provoked movement, dead worms were scored and worms that crawled off the plate or displayed extruded internal organs were censored. All lifespan assays were repeated at least three times. XLSTAT-life statistical software (Addinsoft, New York, NY, USA) was used to plot survival data by the Kaplan-Meier method and differences between survival curves were calculated using the LogRank test with 95% confidence.

Real-time PCR

Synchronized N2 adult worms were fed with RNAi producing bacteria during 4 days at 20°C, total RNA was isolated and cDNAs were synthesized as previously described [45]. Real-time PCRs were performed in capillaries, using LightCycler Fast Start DNA Master^plus Sybr Green I kit (Roche Diagnostics, Meylan, France). Primers for the reference gene TBA2 are: Forward primer: 5’-CCTCCTCCGAATGAATGA AA-3’; Reverse primer: 5’-TCCGATACTGGAAACG GAAG-3’. Primers for E02H9.5 are: Forward primer: 5’-ATAGGCAATATTTTGTTAGT-3’; Reverse primer: 5’-GCTGAAGCCGCGGCCATTCA-3’. Primers for C50F7.10 are: Forward primer : 5’-GATATTCTGACC TCTTACAG-3’; Reverse primer : : 5’-ATCCCAAGT ACTGAATCCGC -3’. To avoid cross-amplification of RNA sequence present in RNAi-fed worms, both E02H9.5 and C50F7.10 specific forward primers were designed to hybridize the 5’-UTR region not included in the RNAi-targeted sequences. The corresponding reverse primers were designed to hybridize the boundary of the first and the second exons in order to prevent cross-amplification of possible contaminating genomic DNA. The RNA level of each gene of interest was normalized to the tba-2 reference gene level for comparison and results of three independent experiments were treated as described in (Pfaffl, 2001). As shown in Figure S1, each construct was able to induce a similar knockdown of both C50F7.10 and E02H9.5 gene expression. Thus, in this study, the knockdown of both paralogues, indifferently induced by either construct, will be referred as klotho RNAi and the product of either C. elegans gene will be named Klotho.

Clr phenotype suppression assay.Clr-1-suppressing activity was assayed in a temperature-sensitive clr-1(e1745) background. After 4 days of RNAi treatment at 15°C, adult worms were either maintained at the permissive temperature (15°C) or shifted to the restrictive temperature (25°C), then examined 24h later for the suppression of the Clr phenotype. At least 50 worms were examined per plate and results are representative of three independent experiments. Worms were photographed under bright-field illumination.

Oxidative stress resistance assays

Menadione was used as a potent oxidative stress inducer, as previously described [45]. After 4 days of RNAi treatment at 20°C, adult worms from different genetic backgrounds were transferred onto 24-well plates (about 30 worms per well) containing S complete medium supplemented with 25 mM of Menadione (Sigma-Aldrich) and transformed HT115 bacteria to maintain the RNAi treatment during the assay. Controls were performed in the absence of Menadione. At least four independent experiments were carried out for 72h at 20°C and the viability of worms was scored, using 1 μM SYTOX Green Nucleic Acid Stain (Molecular probes) to discriminate fluorescent dead worms. Survival histograms were analyzed with XLSTAT software (Addinsoft, New York, NY, USA), using the Student's-t-test at *p < 0.05 signification level.