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

Abstract

Klotho exerts anti-aging properties in mammals in two different ways. While membrane-bound Klotho, which is primarily expressed in the kidney, acts as an obligate co-receptor of FGF23 to regulate phosphate homeostasis, secreted Klotho, resulting from the shedding of the KL1-KL2 ectodomain into the bloodstream, inhibits Insulin/IGF1 signalling. However, the underlying molecular mechanisms are not fully understood. Here, we investigated the biological role of Klotho in Caenorhabditis elegans. Two redundant homologues of the klotho gene exist in C. elegans and encode predicted proteins homologous to the  glucosidase-like KL1 domain of mammalian Klotho. We have used a genetic approach to investigate the functional activity of Klotho in C. elegans. Here, we report that whereas Klotho requires EGL-15 (FGFR) and EGL-17 to promote longevity and oxidative stress resistance, it is not involved in the regulation of fluid homeostasis, controlled by LET-756. Besides revealing a new post-developmental role for EGL-17, our data suggest that the KL1 form of Klotho is involved in FGF23-independent FGF signalling. We also report a genetic interaction between Klotho and the DAF-2 (Ins/IGF1R)/DAF-16 (FOXO) pathway. While the regulation of longevity requires functional DAF-2/DAF-16 signalling, the control of oxidative stress resistance involves a DAF-2- independent, DAF-16-dependent pathway, suggesting that Klotho may target either DAF-2 or DAF-16, depending of environmental conditions. Thus, the predictive KL1 form of Klotho appears to crosstalk with both FGF and Insulin/IGF1/FOXO pathways to exert anti-aging properties in C. elegans.

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].

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.

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.

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.

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.

Figure 6.

In adult worms the FGFR EGL-15(5A) targeted for activation by the Klotho KL1 isoform can allow EGL-17 ligand binding. Under physiological conditions, the Klotho/EGL-15/EGL-17 complex constitutively represses the DAF-2 (Insulin/Igf-like) receptors by a still unknown pathway. Such complexes may induce DAF-16 (FOXO) de-repression and subsequent overexpression of longevity factors, such as antioxidant enzymes. When worms have to cope with a potent stress, the Klotho/EGL-15/EGL-17 complex may directly activate DAF-16 by a DAF-2-independent pathway (dashed line). Such activation mechanism remains to be elucidated.