Axin-Mediated Regulation of Lifespan and Muscle Health in C. elegans Requires AMPK-FOXO Signaling.

Aging is a significant risk factor for several diseases. Studies have uncovered multiple signaling pathways that modulate aging, including insulin/insulin-like growth factor-1 signaling (IIS). In Caenorhabditis elegans, the key regulator of IIS is DAF-16/FOXO. One of the kinases that affects DAF-16 function is the AMPK catalytic subunit homolog AAK-2. In this study, we report that PRY-1/Axin plays an essential role in AAK-2 and DAF-16-mediated regulation of life span. The pry-1 mutant transcriptome contains many genes associated with aging and muscle function. Consistent with this, pry-1 is strongly expressed in muscles, and muscle-specific overexpression of pry-1 extends life span, delays muscle aging, and improves mitochondrial morphology in AAK-2-DAF-16-dependent manner. Furthermore, PRY-1 is necessary for AAK-2 phosphorylation. Taken together, our data demonstrate that PRY-1 functions in muscles to promote the life span of animals. This study establishes Axin as a major regulator of muscle health and aging.

Introduction

Aging is defined as a progressive functional decline in living organisms. It is characterized by hallmarks such as genomic instability, epigenetic alterations, mitochondrial dysfunction, and telomere attrition, and is thought to be regulated in part by genetic pathways (Lopez-Otin et al., 2013). Several genes and pathways have been identified that govern and modulate life span and are conserved in higher eukaryotes (Kenyon, 2010; Lapierre and Hansen, 2012; Uno and Nishida, 2016). Insulin/insulin-like growth factor-1 signaling (IIS) was the first pathway shown to be involved in the regulation of aging in Caenorhabditis elegans (Kenyon, 2011; Kenyon et al., 1993). Subsequent studies have demonstrated that the IIS pathway is conserved across eukaryotes (Uno and Nishida, 2016). In C. elegans, reduction in the activity of the IIS receptor homolog DAF-2 leads to a prolonged life span, which is dependent on DAF-16, a FOXO transcription factor homolog (Kenyon et al., 1993). This modulation of life span by DAF-16 involves translocation to the nucleus followed by either the activation or repression of genes involved in stress response, metabolism, and autophagy (Lee et al., 2003; Melendez et al., 2003; Murphy et al., 2003).

The activity of DAF-16 is regulated by phosphorylation (Kenyon, 2010). One of the kinases involved in this process is the α2 catalytic subunit homolog of AMPK, AAK-2 (Greer et al., 2007a), a phenomenon that is conserved in the mammalian system (Greer et al., 2007b). AAK-2 also plays a crucial role in aging. It is essential for DAF-2-mediated life span extension, and its overexpression extends the life span of animals (Apfeld et al., 2004; Mair et al., 2011). Interestingly, a truncated version of AAK-2 bearing only the catalytic domain was found to be more effective than the full-length wild-type form, suggesting that AAK-2 activity is regulated during the normal aging process (Mair et al., 2011). As in C. elegans, AMPK in Drosophila is also involved in life span regulation. Specifically, overexpression of the α2 subunit in muscles and fat bodies extends the life span of transgenic animals (Stenesen et al., 2013).

AMPK is an established energy sensor in eukaryotes that is phosphorylated by several kinases, including LKB1 (Burkewitz et al., 2014; Hardie et al., 2012). Studies in mouse and human cell culture models have shown that, under the condition of glucose starvation, AMPK forms a complex with LKB1 and the scaffolding protein Axin (Zhang et al., 2013b). The multimeric complex regulates AMPK activation, leading to phosphorylation of downstream targets (Hardie and Lin, 2017; Hardie et al., 2012). The involvement of Axin in AMPK complex formation is essential, since Axin knockdown drastically reduces AMPK activity, leading to fatty liver in starved mice (Zhang et al., 2013b). In addition to their role in AMPK regulation, Axin family members are also involved in multiple biological processes during development and post-development (Mallick et al., 2019b). Since its discovery as a negative regulator of WNT signaling, Axin has been demonstrated to participate in other, non-WNT, pathways as well. In all cases, a common thread is Axin's role as a scaffold protein in recruiting other factors to form complexes (Mallick et al., 2019b). However, whether the scaffolding role of Axin affects FOXO activity remains to be investigated.

In this study, we report that the C. elegans Axin homolog PRY-1, which is necessary for embryonic and larval processes, is also essential for normal life span maintenance. Previously, it was found that metformin-mediated life span extension depends on another C. elegans Axin-like gene, axl-1. However, axl-1 mutants do not show defects in aging and age-related processes (Chen et al., 2017). Our work has revealed that animals lacking pry-1 function during adulthood are short-lived and show increased deterioration in aging-associated processes. Consistent with this, pry-1 mutant transcriptome contains many aging-related protein-coding and miRNA genes. We found that pry-1 is broadly expressed in adults, with high levels in body wall muscles (BWMs). Moreover, muscle-specific knockdown of pry-1 caused an increase in the proportion of fragmented mitochondria and led to a reduction in life span. Conversely, overexpression of pry-1 in muscles improved both phenotypes. Thus, pry-1 appears to affect life span by regulating muscle mitochondria health. Interestingly, muscle-specific expression of mouse Axin (mAxin1) in C. elegans also extended life span, suggesting that Axin's role in aging may be conserved. It is worth mentioning that Axin is expressed in mouse and human skeletal muscles (Smith et al., 2019; Uhlen et al., 2015). To investigate PRY-1's mechanism of action, we performed a combination of molecular genetics and biochemical experiments. The results revealed that PRY-1's role in aging depends on AAK-2 and DAF-16. Our data suggest that PRY-1 presumably forms a complex with AAK-2 leading to its phosphorylation, thereby promoting nuclear localization of DAF-16 in the intestine and life span maintenance.

Results

pry-1 Transcriptome Contains Genes Involved in Life Span Regulation

The involvement of PRY-1 in multiple signaling pathways and biological events is well documented (Mallick et al., 2019b). Earlier, we reported both mRNA and microRNA (miRNA) transcriptome profiles of pry-1 mutant that revealed 2,665 differentially expressed protein-coding genes and six miRNAs (Mallick et al., 2019a; Ranawade et al., 2018). The characterization of differentially expressed genes showed pry-1's crucial role in miRNA-mediated seam cell development (Mallick et al., 2019a) and lipid metabolism (Ranawade et al., 2018). In this study, we have specifically focused on the genes linked to aging. Of the differentially expressed miRNAs, mir-246 is involved in aging and stress response (de Lencastre et al., 2010). The mRNA transcriptome dataset was analyzed using Gene Ontology (GO) terms, which revealed that aging-related protein-coding genes and gene families are overrepresented (69 in total; relative frequency (RF): 2, p < 4.91 × 10−9; 33% upregulated and 67% downregulated) and that they are linked to biological activities such as cellular processes (26 genes), metabolic processes (24 genes), and biological regulation (13 genes) (Figure 1A; Table S1). Within cellular processes, candidates include genes linked to lipid metabolism (aap-1, hyl-1, elo-2, ctl-2, cat-1, and lipl-4), which further supports the essential role of lipids in pry-1-mediated signaling (Ranawade et al., 2018) and suggests that pry-1 may affect lipid metabolism to regulate aging.

Figure 1

pry-1 is Required for the Normal Life Span of Animals

(A) Sixty-nine differentially expressed genes in pry-1 mutant transcriptome are linked to aging.

(B) More than a quarter of DAF-16 direct targets is present in pry-1 mutant transcriptome.

(C) Life span of pry-1(mu38) animals.

(D) Life span of Adult-specific pry-1(RNAi) animals.

(E) Life span rescue experiments following two different treatments during adulthood, namely subjecting pry-1(mu38) to a 25°C upshift and 31°C 1hr heat-shock to pry-1(mu38); hs::pry-1 animals. The control worms consist of pry-1(mu38) alone, pry-1(mu38) subjected to 31°C 1hr heat shock, and pry-1(mu38); hs::pry-1 without heat shock.

(F) Life span analysis of Cbr-pry-1(RNAi) animals.

(E and F) See Transparent Methods and Table S2 for life span data and statistical analyses.

Further GO term analysis of protein-coding aging-related genes showed that they are linked to 32 distinct signaling pathways and include well-known factors such as AAP-1 (PI3K adapter subunit) and DAF-16, both belonging to the IIS pathway (Lapierre and Hansen, 2012; Uno and Nishida, 2016), and XBP-1, a human XBP1 ortholog that acts downstream of IRE-1 and PEK-1-mediated signaling (Ron and Walter, 2007). Thus, pry-1 appears to interact with multiple genetic networks. We also compared the pry-1 transcriptome with differentially expressed genes of the DAF-2-DAF-16 signaling pathway (Lin et al., 2018) and found a significant overlap (415 genes; RF: 1.7, p < 2.228 × 10−31, Table S3). Additionally, 29 of 109 DAF-16 direct targets (Li and Zhang, 2016) are present in the pry-1 dataset (27% overlap, p < 0.01; two-thirds downregulated), including four (dod-17, prdx-3, nnt-1, and daf-16) that are directly involved in aging (Figure 1B; Table S3). Taken together, these in silico analyses suggest that pry-1 acts in part via DAF-16 to regulate life span in C. elegans.

Mutations in pry-1 Reduce Life Span

In accordance with the above data suggesting pry-1's role in aging, pry-1 expression was found to be significantly higher in older adults (Figure S1A).We also found that the mean life span of pry-1(mu38) animals was 80% (p < 0.001) shorter compared with that of wild-type animals (Figure 1C; Table S2). A similar reduction in life span was also observed with a CRISPR allele, gk3682, that deletes a roughly 750-bp region, including the 5′ UTR and the first exon (Mallick et al., 2019a) (Figure 1C; Table S2). As pry-1 is also involved in developmental processes (Mallick et al., 2019b), we took an RNAi approach to knock down the gene function specifically during adulthood. As expected, pry-1(RNAi) animals were found to be short-lived, with 22–31% (p < 0.01) reduced mean life span (Figure 1D; Table S2).

To further investigate whether pry-1 affects aging, we performed two sets of rescue experiments. One of these involved making use of the cold-sensitive allele mu38. While the life span defect of pry-1(mu38) was severe at 20°C (mean life span 81% lower than N2, p < 0.001, Figure 1E; Table S2), the animals appeared healthier and showed an improved life span at 25°C (50% lower than N2, p < 0.01, Figure 1E; Table S2). When day-1 pry-1(mu38) adults were upshifted from 20°C to 25°C, life span was extended by 107% (6.4 ± 0.4 days mean life span compared with 3.2 ± 0.1 days for untreated mu38 control, p < 0.01). In the other experiment, transgenic animals were generated carrying a heat-shock promoter-driven pry-1. The hs::pry-1 transgene efficiently rescued the life span defect of pry-1(mu38) animals upon heat-shock during adulthood (58% longer mean life span compared to control animals, p < 0.001, Figure 1E; Table S2). Interestingly, no such effect was observed when the transgene was expressed in wild-type background (Figure S1B; Table S2).

Since our lab had previously reported a conserved role of C. briggsae pry-1 during development (Seetharaman et al., 2010), we investigated whether Cbr-pry-1 is also involved in aging. The results revealed both the sy5353 mutant allele and adult RNAi caused a shorter life span in animals (Figures 1F and S1C; Table S2). These data show that pry-1 function in life span maintenance is conserved in nematodes.

pry-1 Knockdown in Adults Causes Accelerated Aging and Increased Expression of Stress Response Markers

Several physiological and molecular changes occur in animals during the aging process. These include a decline in tissue function, oxidative stress, accumulation of mis/unfolded proteins, and altered lipid distributions (Huang et al., 2004; Lopez-Otin et al., 2013). To characterize such changes in pry-1(RNAi) animals, we analyzed the age-dependent decline in pharyngeal pumping and body bending. Adult-specific knockdown led to a significant reduction in rates of pharyngeal pumping and body bending starting on days 7 and 2, respectively (Figures 2A and 2B). Similar phenotypes were also observed in pry-1(mu38) mutants, although the defects were more severe (Figures S2A and S2B). Consistent with the adult-specific role of pry-1, we found that heat-shocked pry-1(mu38); hs::pry-1 adults showed significant improvements in both these aging-related markers (Figures S2C and S2D). Together, the results demonstrate that pry-1 is needed to delay aging-associated physical deterioration in animals.

Figure 2

Adult-Specific Lowering of pry-1 Accelerates Aging-Associated Markers

(A and B) Pharyngeal pumping of RNAi-treated pry-1 in day-7 and day-8 adults (A) and body bending starting day-2 of adulthood (B). Data represent the mean of at least two replicates (n ≥ 30 animals) and error bars represent the standard deviation. Significance was calculated using Student's t-test ∗p < 0.05, ∗∗p < 0.01.

(C) Representative images of animals showing aging pigment (lipofuscin), ROS marker (sod-3::GFP), UPR-ER marker (hsp-4::GFP), and UPR-MT markers (hsp-6::GFP and hsp-60::GFP). Scale bar is 0.1mm.

(D) Quantification of fluorescence intensity shown in panel C.

(E) Oil red O staining of total lipid droplets in day-8 control and pry-1(RNAi) animals. Scale bar is 0.1mm. (D and E) Data represent the mean of two replicates (n ≥ 15 animals in each), and error bars represent the standard deviation. Significance was calculated using Student's t-test. ∗∗p < 0.01.

Next, we measured lipofuscin levels in adults. In C. elegans, lipofuscin, a product of oxidative damage and autophagy, is visible as auto fluorescent granules in the intestine and serves as a biomarker of aging (Davis et al., 1982). Quantification of the intestinal autofluorescence showed a 30% increase (p < 0.05) in pry-1(RNAi) adults compared with that in N2 control animals (Figures 2C and 2D). The expression of an oxidative stress marker, manganese superoxide dismutase (sod-3), was also investigated (Lopez-Otin et al., 2013). The RNAi-mediated knockdown of pry-1 caused no significant change in sod-3:GFP fluorescence (Figures 2C and 2D), suggesting that pry-1 function is not essential for the maintenance of oxidative stress.

Other indicators of premature aging include unfolded protein response (UPR) associated with mitochondria and ER (UPRMT and UPRER, respectively) (Lopez-Otin et al., 2013). Upon activation, these UPR pathways increase the expression of chaperones and heat shock proteins such as hsp-6 and hsp-60 (UPRMT) (Tran and Van Aken, 2020) and hsp-4/Bip (UPRER) (Ron and Walter, 2007). We found that GFP fluorescence of all three markers, namely hsp-6::GFP, hsp-60::GFP, and hsp-4::GFP, was significantly increased in pry-1(RNAi) day-8 adults compared with that in controls (70%, 40%, and 50% higher, respectively, p < 0.01) (Figures 2C and 2D). Moreover, it was observed that pry-1 transcriptome contains genes involved in IRE-1/IRE1 and PEK-1/PERK-mediated UPRER signaling (57 genes, 49% overlap, R.F. 3.2, p < 5.87 × 10−18; and 10 genes, 43% overlap, R.F. 2.9, p < 0.001, respectively) (Table S4), including the key downstream factor, XBP-1, which activates hsp-4 expression (Ron and Walter, 2007).

Collectively, the above data provide evidence that pry-1 plays an essential role in the maintenance of aging-associated processes and stress response in animals. One possibility may be that pry-1 affects aging by regulating lipid metabolism. This is supported by our previous results demonstrating that lipid synthesis is compromised in pry-1 mutant animals (Mallick and Gupta, 2020; Ranawade et al., 2018). More importantly, adult-specific knockdown of pry-1 caused a significant reduction in lipid content in day-8 adults (Figure 2E). Given that daf-16 is also necessary for lipid synthesis (Murphy et al., 2003; Ogg et al., 1997; Zhang et al., 2013a) and pry-1 and daf-16 transcriptomes contain a common set of lipid synthesis and transport genes (such as fat-5-7 and vit-1/3/4/5) (Table S3), it is conceivable that pry-1 and daf-16 interact to regulate lipid levels, leading to a normal life span of animals.

pry-1 Knockdown Suppresses Life Span Extension of mom-2/WNT Mutants

As PRY-1 is an established negative regulator of WNT signaling, we examined its genetic interactions with WNT ligands. Of the five known ligands, loss-of-function mutations in mom-2 and cwn-2 cause an extension of life span (Lezzerini and Budovskaya, 2014). When pry-1 was knocked down in mom-2(or42) and cwn-2(ok895) backgrounds, life span extension was significantly reduced in mom-2 mutants (13.6% reduction in mean life span, p < 0.05, Figure 3A; Table S2) but remained unchanged in the cwn-2 animals (Figure 3B; Table S2). We also analyzed the requirements of bar-1/β-catenin, a component of the canonical WNT signaling that plays a role in aging (Xu et al., 2019; Zhang et al., 2018), in the mom-2-pry-1 pathway. Since pry-1-mediated WNT signaling negatively regulates bar-1, removing bar-1 function is expected to suppress the phenotype of pry-1 mutants. However, we observed that the life span of bar-1 null mutants was further shortened by pry-1 RNAi (Figure 3C; Table S2), suggesting that bar-1 is unlikely to participate in the pry-1-mediated aging process. Further support for this model comes from a bar-1 RNAi experiment that failed to suppress the life span phenotype of pry-1(mu38); hs::pry-1 animals (Figure 3D; Table S2). These data suggest that PRY-1 may act downstream of MOM-2 in a pathway that is independent of BAR-1 and likely to utilize DAF-16-mediated signaling.

Figure 3

pry-1 Functions Downstream of WNT Ligand mom-2 and Independently of β-Catenin bar-1 to Regulate Life Span

(A–C) Life span analysis following RNAi knockdown of pry-1 in WNT pathway mutants, mom-2(or42) (A), cwn-2(ok895) (B), and bar-1(ga80) (C).

(D) Effect of bar-1 RNAi in pry-1(mu38); hs::pry-1 animals.

(A–D) See Transparent Methods and Table S2 for life span data and statistical analyses.

Tissue-Specific Analysis Shows that pry-1 is Needed in Muscles and Hypodermis

To investigate the requirements of pry-1 in life span regulation, we examined its in vivo expression pattern. Previously, a 3.6-kb pry-1 proximal promoter was used to drive the coding sequence of pry-1 fused to a GFP reporter, which showed fluorescence throughout development, specifically in the vulval precursor cells, neurons, BWM, and some hypodermal cells (Korswagen et al., 2002). We further characterized pry-1 expression, which revealed expression in almost all tissues during development. Expression in seam cells, neuronal cells, muscles, hypodermis, and intestine was readily visible (Figure 4A). This pattern of localization matches well with tissue enrichment of differentially expressed genes in the pry-1 transcriptome using WormBase tissue ontology tool (Table S5, see Transparent Methods). The most enriched tissues include neurons and muscles.

Figure 4

Expression Pattern of pry-1 in Adults and Its Tissue-Specific Requirements for Life Span Maintenance

(A) Representative image of pry-1p::pry-1::GFP animals showing GFP fluorescence in muscles, intestine, seam cells, and neurons. Also see Figure S3. Scale bar is 0.1mm.

(B–E) Life span analysis after tissue-specific RNAi knockdown of pry-1. Also see Figures S4B and S4C pry-1 RNAi knockdown control (B) and pry-1 RNAi knockdown in muscle (C), hypodermis (D), and intestine (E). (B–E) See Transparent Methods and Table S2 for life span data and statistical analyses.

A closer examination of GFP localization in developing animals revealed bright fluorescence in the ventral cord region, which includes neuronal and non-neuronal cells. The expression was largely similar in adults, although the fluorescence was much higher in BWMs (Figures 4A, S3A, and S3B). The posterior end of the intestine, near the rectal opening, showed a strong signal in L4 and adult animals; however, the rest of the intestine lacked a detectable expression. In general, GFP was diffused and not localized to any specific subcellular structures except in the case of muscles and posterior intestine, where nuclei are visible (see arrows in Figures S3B and S3C). The fluorescence continued to persist in older adults, consistent with the role of pry-1 in aging. A similar pattern of expression for pry-1 was also observed in C. briggsae transgenic animals, with a marked increase in fluorescence in muscles throughout adulthood (Figure S4A). This pattern of pry-1 expression in both nematodes suggests that the gene may play a conserved role in maintaining muscle health during aging.

Given that pry-1 is expressed in muscles as well as other tissues, we examined its tissue-specific requirements for life span maintenance. To this end, RNAi experiments were performed in adults using strains that allow tissue-specific knockdowns in muscles, hypodermis, intestine, and neurons (see Transparent Methods). The results showed that pry-1 RNAi caused a significant reduction in mean life span when knocked down in the hypodermis and muscles (26% lower mean life span in hypodermis RNAi and 12% in muscle RNAi, p < 0.05) (Figures 4B–4D). No such effect was observed in other tissues (Figures 4E, S4B, and S4C). We conclude that pry-1 functions in muscles and hypodermis to maintain the life span of animals. Further support for this comes from the analysis of transgenic strains in which pry-1 expression was driven by hypodermal and muscle-specific promoters (lin-26p::pry-1 and unc-54p::pry-1, respectively). In both cases, the life span defect of pry-1(mu38) animals was significantly rescued (41% and 56% increases in mean life span by lin-26p::pry-1 and unc-54p::pry-1, respectively, p < 0.01) (Figures S5A and S5B; Table S2).

Having uncovered the role of pry-1 in hypodermis and muscles, we examined whether overexpression of the gene in these two tissues can extend the life span. Interestingly, while muscle-specific expression (unc-54p::pry-1) extended the life span significantly (13% increase in mean life span, p < 0.05), no such effect was observed in the case of hypodermis-specific expression (lin-26p::pry-1) (Figures 5A and 5B; Table S2). In fact, lin-26p::pry-1 animals were short-lived, suggesting that a lack of spatiotemporal control is detrimental (Figure 5B; Table S2). These data, together with RNAi and rescue experiments, firmly establish that pry-1 functions in both muscles and hypodermis for the maintenance of life span, and its hypodermal expression needs to be tightly regulated. Furthermore, the results have revealed a role of pry-1 in muscles that is beneficial to animals throughout the life span. Interestingly, muscle-specific expression of mAxin1 also caused animals to live longer (14% increase in mean life span, p < 0.05) (Figure S5C; Table S2).

Figure 5

pry-1 Overexpression in the Muscle Extends Life Span and Improves Muscle Physiology

(A and B) Effects of tissue-specific overexpression of pry-1. Overexpression in muscle (A), also see Figure S5B and Table S2, and in hypodermis (B). See Transparent Methods and Table S2 for life span data and statistical analyses.

(C and D) qPCR analysis of muscle genes tnt-4, mlc-1 and unc-96 in day-1 pry-1(mu38) (C) and unc-54p::pry-1 (D) adults. Data represent the mean of two replicates and error bars represent the SEM. Significance was calculated using Bio-Rad software (t test). ∗∗p < 0.01.

(E–G) Representative images of muscle mitochondrial morphologies revealed by myo-3p::GFP(mito) reporter in the control, pry-1(mu38), pry-1(RNAi), and unc-54p::pry-1 transgenic animals. The control for whole-animal RNAi experiment was N2 (E) and for muscle-specific RNAi was an RNAi-sensitive strain (F), each fed with bacteria carrying an empty vector (L4440) (see Transparent Methods for genotypes). Day-2 adults were used for pry-1(mu38), whereas day-8 adults for pry-1(RNAi) and unc-54p::pry-1 animals. Scale bar is 25μm.

(H) Quantification of phenotypes in panels E, F, and G. Data represent the mean of two replicates (n = 20 animals in each) and error bars standard deviations. Statistical analyses were carried out using the two-tailed Fisher's exact test by comparing mitochondrial morphology between normal (tubular) and defective (intermediate and fragmented) categories and indicated by stars (∗). ∗p < 0.05, ∗∗∗p < 0.0001. See Transparent Methods for details.

(I) Body bending analysis of unc-54p::pry-1 adults between day-8 and day-11. Also see Figure S5D.

(J) Number of autophagic vesicles per muscle cell at day-2 of adulthood revealed by dyc-1S::lgg-1::GFP, a GFP marker of autophagic vesicles in body-wall muscles. (I-J) Data represent the mean of two replicates (n ≥ 15 animals in each) and error bars standard deviations. Significance was calculated using Student's t-test. ∗p < 0.05, ∗∗p < 0.01.

(K) lgg-1 transcript levels in pry-1(mu38) and unc-54p::pry-1 animals. Data represent the means of two replicates and error bars the SEM. Significance was calculated using Bio-Rad software (t test). ∗p < 0.05.

Overexpression of pry-1 in Muscles Improves Muscle Health and Mitochondrial Morphology

The life span extension observed in unc-54p::pry-1 animals led us to investigate the cellular and molecular basis of pry-1's role in muscle health. Based on GO analysis, we found that pry-1 transcriptome contains a significant number of muscle-associated genes (31 of 123, 25.2%, R.F. 1.7, p < 0.002) (Table S5). A majority of these genes are downregulated (90.3%, 28 of 31 genes), suggesting that pry-1 is needed to maintain their expression. Further investigation identified two broad categories, namely muscle structure development (21 genes) and muscle contraction (15 genes) (Table S5), both of which include core components of the sarcomere, such as the subunits of troponin complex (tnt-3, tnt-4), twitchin/titin (unc-22), myosin complex (mlc-1, unc-15, unc-54), and voltage-gated potassium channels (unc-58, unc-103, slo-1) (Table S5). We chose three genes at random to validate changes in their expression by quantitative Polymerase Chain Reaction (qPCR): mlc-1 and tnt-4 (involved in muscle contraction and structure development), and unc-96 (involved in muscle structure development). The results confirmed that tnt-4 and unc-96 were indeed downregulated in pry-1(mu38) mutants, whereas mlc-1 expression was unchanged (Figure 5C). As expected, all three genes were upregulated in unc-54p::pry-1 animals (Figure 5D).

Since muscle health is linked to mitochondrial homeostasis (Gouspillou and Hepple, 2016; Hood et al., 2019; Mergoud Dit Lamarche et al., 2018; Regmi et al., 2014), we speculated that pry-1 is necessary to maintain the expression of mitochondrial genes. Indeed, genes associated with mitochondrial structure and function are overrepresented in the pry-1 transcriptome (173 genes, 27%, R.F. 1.8, p < 1.691 × 10−15) (Table S6). These include genes that function in the mitochondrial membrane (52 of 220, 24% overlap, R.F. 1.6, p < 5.567 × 10−4), mitochondrial outer membrane (10 of 30, 33% overlap, R.F. 2.2, p < 0.01), mitochondrial matrix (37 of 137, 27% overlap, R.F. 1.8, p < 2.298 × 10−4), and mitochondrial gene expression (18 of 53, 34% overlap, R.F. 2.2, p < 5.146 × 10−4) (See also Table S6).

Further support of pry-1's role in mitochondrial health comes from examination of BWMs using an organelle-specific GFP reporter, mitoGFP (Benedetti et al., 2006). The mitoGFP was used earlier to demonstrate age-dependent fragmentation of muscle mitochondria and, consequently, the loss of muscle function, since significantly fewer adults exhibited a tubular mitochondrial morphology (Mergoud Dit Lamarche et al., 2018; Regmi et al., 2014). We found that while muscle-specific, but not whole animal, pry-1 RNAi caused a subtle but statistically significant defect in mitochondria in older adults, pry-1(mu38) animals exhibited a drastic increase in fragmented mitochondria (Figures 5E, 5F, and 5H). In contrast, the morphology was better preserved in unc-54p::pry-1 adults compared with wild-type controls (Figures 5G and 5H), demonstrating that pry-1 is needed to maintain muscle mitochondrial homeostasis.

The above results led us to investigate whether the mitochondrial network architecture mirrors the functional state of muscles. Studies have shown that the loss of locomotion and pharyngeal pumping are associated with fragmented mitochondrial structure in older worms (Mergoud Dit Lamarche et al., 2018; Regmi et al., 2014). Since a similar correlation is also seen in pry-1(mu38) day-1 adults, we wondered whether unc-54p::pry-1 animals will appear healthier with respect to these aging-related markers. The experiments revealed that, while overexpression of pry-1 in muscles led to a significantly improved body bending rate in adults, pharyngeal pumping and thrashing were comparable to that of controls (Figures 5I and S5D–S5F). These results are consistent with pry-1's role in maintaining the mitochondrial network, which may contribute to the improvement of muscle health.

Another process that affects muscle aging is autophagy, in which damaged mitochondria are selectively removed (Madeo et al., 2015; Twig and Shirihai, 2011). While autophagy is beneficial for longevity, its effect is detrimental in the presence of increased mitochondrial permeability, which triggers mitochondrial fragmentation (Zhou et al., 2019). Since muscle autophagy increases with age (Mergoud Dit Lamarche et al., 2018), we investigated whether the process is affected in pry-1 mutants that are short-lived. The analysis of autophagic vesicles, using dyc-1S::lgg-1::GFP marker (Mergoud Dit Lamarche et al., 2018), revealed that vesicle number per muscle cell was significantly higher in pry-1(mu38) animals compared with that in controls (Figure 5J). Similar results were also obtained by the analysis of lgg-1 transcripts (Figure 5K). As expected, no such effect was found in the unc-54p::pry-1 genetic background (Figures 5J and 5K). Overall, our data demonstrate that pry-1 regulates muscle mitochondrial morphology to maintain muscle structure and function.

daf-16/FOXO Functions Downstream of pry-1 to Maintain Life Span

As described above, we found that daf-16 is downregulated in the pry-1 transcriptome. daf-16 encodes several isoforms, three of which, R13H8.1b, d, and f (WormBase WS261 release), influence the rate of the aging process (Chen et al., 2015; Kwon et al., 2010). To examine whether pry-1 affects these isoforms, we performed qPCR analysis. In the case of pry-1(mu38), transcripts for R13H8.1b/c (daf-16a) and R13H8.1d/f/h/i/k (daf-16d/f/h/i/k) were significantly downregulated (Figure S6A). An opposite trend was observed in unc-54p::pry-1 animals (Figure S6B). How might pry-1 regulate transcription of daf-16? Previously, two intestinal GATA transcription factors, elt-2 and elt-4, were shown to promote daf-16 transcription, leading to longevity (Bansal et al., 2014). Using qPCR, we found that the expression of both elt-2 and elt-4 was significantly upregulated in the muscle-specific line (unc-54p::pry-1) (Figure S6C). Thus, pry-1 may use these GATA factors directly or indirectly to affect daf-16 transcription.

To investigate whether the interaction of pry-1 with daf-16 is affected by daf-2 signaling (IIS), we knocked down pry-1 in both daf-2 and daf-16 mutant backgrounds. While the knockdown caused a reduction in daf-2(e1370ts) life span (Figure S6D), no change was observed in daf-16(mu86) animals (Figure 6A), suggesting that pry-1 may act genetically downstream of daf-2 but upstream of daf-16. The results of the following two experiments are most consistent with the possibility of daf-16 acting downstream of pry-1: One, daf-16 RNAi suppressed the life span extension observed in pry-1(mu38); hsp::pry-1 animals (Figure S6E), and, two, life span defect of pry-1(mu38) animals is significantly rescued by daf-16 overexpression (Figure 6B).

Figure 6

Life Span Regulation by pry-1 Depends on daf-16 Function in the Intestine

(A) pry-1 RNAi knockdown in daf-16(mu86) animals.

(B) daf-16 overexpression in pry-1(mu38) animals.

(C) Localization of GFP fluorescence in unc-54p::pry-1 intestinal nuclei. Scale bar is 50μm. 100% of the unc-54p::pry-1 animals (n = 30) showed nuclear localization for DAF-16:GFP.

(D and E) Intestine-specific daf-16 RNAi knockdown (D) and muscle-specific knockdown (E) in unc-54p::pry-1 animals.

(A, B, D, and E) See Transparent Methods and Table S2 for life span data and statistical analyses.

(F) Western blot analysis of AAK-2 phosphorylation in control, pry-1 mutant, and unc-54p::pry-1 animals. Data represent the means of two replicates and error bars the standard deviation. Significance was calculated using Student's t-test. ∗∗p < 0.01.

Since DAF-16's function depends on its nuclear localization (Kenyon, 2010), we investigated whether PRY-1 plays a role in this process. The fluorescence of DAF-16:GFP in unc-54p::pry-1 animals was localized frequently to intestinal nuclei (Figure 6C). Consistent with this, sod-3, a direct target of daf-16, was overexpressed (Figure S6F). The transgenic worms also exhibited a higher level of lipids (Figures S7A and S7B), which supports pry-1's role in lipid synthesis (Mallick and Gupta, 2020; Ranawade et al., 2018). Moreover, there was a significant up-regulation of fatty acid desaturases (fat-5, fat-6, and fat-7) and the SREBP homolog sbp-1 (Figure S7C). These findings, along with the known role of daf-16 in promoting lipid synthesis (Papsdorf and Brunet, 2019), lead us to propose that pry-1 interacts with daf-16 to regulate lipids.

To examine whether daf-16 acts locally in the intestine or via a long-range signal by functioning in the muscle, we performed tissue-specific RNAi experiments. Life span extension of unc-54p::pry-1 was completely abolished by daf-16 knockdown in the intestine (Figure 6D), the tissue where it acts primarily to regulate life span (Libina et al., 2003). No such effect was observed following muscle-specific knockdown (Figure 6E). Overall, the results show that daf-16 is involved in pry-1-mediated life span regulation and that life span extension observed in muscle-overexpressed pry-1 animals depends on daf-16 function in the intestine.

DAF-16-Mediated PRY-1 Signaling Depends on AAK-2 Function

Next, we determined the nature of interaction between pry-1 and daf-16. In the mammalian system, Axin forms a complex with AMPK and LKB1 upon glucose starvation, resulting in phosphorylation of AMPK (Zhang et al., 2013b). The activated AMPK, in turn, phosphorylates a number of targets, including FOXO family members, preferentially FOXO3 (Greer et al., 2007b; Mihaylova and Shaw, 2011). Since the AMPK-FOXO interaction also occurs in C. elegans where AAK-2 phosphorylates DAF-16 and plays a role in DAF-16-dependent life span extension (Greer et al., 2007a; Mair et al., 2011), we investigated whether PRY-1 is involved in activating AAK-2. For this, AAK-2 phosphorylation was quantified in worm protein extracts. The results showed that, while the phosphorylated AAK-2 level was drastically reduced in pry-1 mutants when probed with phospho-AMPK (T172) antibody, it was significantly increased in unc-54p::pry-1 animals (Figure 6F). To determine whether a reduced AAK-2 signal in pry-1 mutants is due to a lower abundance of protein, we examined GFP fluorescence in aak-2p::aak-2::GFP transgenic animals and found no change in fluorescence intensity in pry-1(mu38) mutants compared with that in the control (Figure S7D). Thus, PRY-1 is necessary for AAK-2 phosphorylation.

Three additional experiments support PRY-1 playing a role in AAK-2 activation: First, pry-1 RNAi did not exacerbate the life span defect of aak-2(ok524) animals (Figure 7A; Table S2). Second, a constitutively active form of AMPKα2 (due to increased T172 phosphorylation), which causes a long-lived phenotype in worms (Mair et al., 2011), was unable to rescue the life span defect of pry-1(mu38) (Figure 7B; Table S2). And, three, aak-2 is expressed in BWMs and neurons during adulthood in a pattern that resembles pry-1 (Lee et al., 2008; Mair et al., 2011) (Figure S7E). Moreover, similar to aak-2 mutants, pry-1 mutant animals exhibited significantly reduced life span of dauers (55-70% reduction in mean life span in two different alleles compared to the control, p < 0.01) (Figure S7F; Table S2) (Narbonne and Roy, 2009). Taken together, these data support a model of PRY-1 promoting AAK-2 activation, likely through protein-protein interaction. The LKB1 homolog, PAR-4, required for AAK-2 activation (Lee et al., 2008) may also be involved in this process, since the life span defect of par-4 mutant was not enhanced by pry-1(RNAi) (Figure S7G). The activated AAK-2 may, in turn, act in a cell non-autonomous manner to affect DAF-16 function in the intestine.

Figure 7

PRY-1 Interacts with AAK-2 to Regulate DAF-16 Localization and Life Span Extension

(A) pry-1 RNAi knockdown in aak-2(ok524) animals.

(B) Constitutive activation of aak-2 in pry-1(mu38) animals.

(C and D) aak-2 RNAi knockdown in the muscle and intestine in unc-54p::pry-1 animals.

(A–D) See Transparent Methods and Table S2 for life span data and statistical analyses.

(E) aak-2 RNAi effect on DAF-16:GFP localization in unc-54p::pry-1 animals. Scale bar is 50 μm.

Data represent the means of two replicates (15 animals each) and error bars the standard deviation. Significance was calculated using Student's t-test. ∗∗p < 0.01.

(F) Venn diagram showing an overlapping set of genes between pry-1(mu38) and aak-2(gt33) transcriptomes.

The model above suggests that PRY-1 and AAK-2 affect the expression of a common set of genes. Indeed, the transcriptome data sets of pry-1 and aak-2 mutants (Ranawade et al., 2018; Shin et al., 2011) showed a significant overlap (192 shared genes, 132 upregulated and 60 downregulated; RF: 1.2, hyp.geo p < 0.006) (Figure 7F; Table S7). Of these, 60 (45%) are mutually upregulated and 28 (47%) mutually downregulated in both mutants. The overlapping set of differentially expressed genes are linked to GO processes such as muscle structure development (act-1, mel-26, unc-52, emb-9, unc-15, and unc-54), muscle contraction (unc-54), aging (daf-16, prmt-1, mpk-1, chc-1, cgh-1, dao-5, and glp-4), lipid metabolic processes (tat-4, ldp-1, sptl-3, pmt-1, lipin-1, and cgt-3), and regulation of lipid localization (daf-16, prmt-1, sams-1, tat-4, lea-1, vit-1, vit-3, vit-4, and vit-6). Moreover, a significant number of genes are associated with stress response (27) and catabolic process (25) (Table S7).

To further investigate the interaction of aak-2 with pry-1, tissue-specific knockdown experiments were performed. Both muscle and intestine-specific aak-2 RNAi abolished life span extension in unc-54p::pry-1 animals (Figures 7C and 7D; Table S2). Additionally, RNAi caused significantly fewer animals to show nuclear-localized DAF-16:GFP (Figures 7E and 7F). As with aak-2, RNAi knock-down of par-4 in the muscle suppressed the life span extension of unc-54p::pry-1 (Figure S7H), providing further evidence for PAR-4's involvement in PRY-1 and AAK-2 interaction. Collectively, the results described in this section lead us to conclude that PRY-1 interacts with PAR-4 and AAK-2 in the muscle, thereby affecting DAF-16 localization in the intestine and, ultimately, the life span of animals.

Discussion

Our results demonstrate the role of C. elegans Axin family member PRY-1 in life span maintenance, which involves AAK-2/AMPK-mediated DAF-16/FOXO signaling. We found that the pry-1 mutant transcriptome contains a significant number of aging-associated genes, including IIS and UPRER pathway components as well as those linked to lipid maintenance. Moreover, a significant number of DAF-16 direct targets are altered in pry-1 mutants, and a majority of these are downregulated. Consistent with these findings, previous studies have shown that both DAF-16 and XBP-1-mediated UPRER signaling regulate stress response, lipid metabolism, and longevity (Imanikia et al., 2019; Lee et al., 2003; Lin et al., 2018; Murphy et al., 2003; Taylor and Dillin, 2013). As expected from misregulation of aging-related genes, a partial or complete loss of PRY-1 activity resulted in a shorter life span. The aging phenotype was associated with physiological changes such as slower rates of body bending and pharyngeal pumping, an increase in aging pigment (lipofuscin), and higher expression of UPRER and UPRMT chaperones. Altogether, these data suggest that pry-1 affects multiple conserved pathways involved in stress maintenance and aging.

The characterization of pry-1 expression uncovered muscles as a major tissue for gene action. Other tissues showing a relatively lower abundance of pry-1 include neurons, hypodermis, and intestine. Since the WNT ligands, mom-2 and cwn-2, are localized in some of these tissues (Song et al., 2010) and both ligands affect life span (Lezzerini and Budovskaya, 2014), we investigated the possibility of pry-1 acting in a WNT-dependent manner. The results of genetic interaction experiments suggest that mom-2-mediated signaling may affect pry-1 function to maintain life span. However, such a mechanism may not involve the canonical WNT effector protein β-catenin. It is worth noting that WNT signaling has been shown to play roles in cellular senescence, aging, and age-related diseases (Brack et al., 2007; Gruber et al., 2016; Naito et al., 2010; Zhang et al., 2019). However, the regulation and function of Axin in the pathway is poorly understood.

The finding that pry-1 is expressed in multiple tissues led us to investigate its tissue-specific function. The results of RNAi-mediated knockdowns and rescue experiments revealed that the gene is needed in the muscle and hypodermis to maintain life span. Interestingly, forced expression of pry-1 in muscles, but not in hypodermis, allowed animals to live longer. Considering that Axin homologs are expressed in muscles (Smith et al., 2019; Uhlen et al., 2015) and mouse Axin (mAxin1) extended the life span of C. elegans when ectopically expressed in the muscle tissue, we propose that the beneficial role of Axin in the muscle is evolutionarily conserved.

pry-1's involvement in muscle health was further investigated using the transcriptome data, which uncovered a significant number of genes involved in muscle structure development and function. Almost all of these were downregulated. Another group of genes regulated by pry-1 are associated with mitochondria and include those that function in the mitochondrial membrane, mitochondrial matrix, and mitochondrial ATP synthesis, suggesting that pry-1 plays a major role in maintaining the health of this vital organelle. As expected, mutant animals showed increased fragmentation of mitochondria, which may contribute to muscle aging and a shorter life span (Gouspillou and Hepple, 2016; Hood et al., 2019; Mergoud Dit Lamarche et al., 2018). In contrast, muscle-specific overexpression of pry-1 resulted in marked improvements in mitochondrial morphology and locomotion. The relationship between aging and muscle mitochondrial function is well described. For example, daf-2 mutants that have a longer life span show preservation of mitochondrial morphology and delayed muscle aging (Mergoud Dit Lamarche et al., 2018; Wang et al., 2019). Additionally, daf-16 is essential for the maintenance of muscle mitochondrial health (Wang et al., 2019). We found that both transcription and subcellular localization of DAF-16 is regulated by PRY-1. Moreover, genetic experiments revealed that the pry-1-mediated life span depends on daf-16. Interestingly, DAF-16 was nuclear localized in the intestine of unc-54p::pry-1 worms. This localization appears to be important, since the intestine-specific knockdown of daf-16 abolished the life span extension of transgenic animals.

We investigated the mechanism of PRY-1-mediated DAF-16 regulation and uncovered the role of AMPK homolog AAK-2 in this process. Specifically, PRY-1 is essential for the activation of AAK-2, which, in turn, promotes DAF-16 nuclear localization and life span extension of unc-54p::pry-1 animals. Previous work has reported the involvement of AAK-2 in regulating DAF-16 function (Chen et al., 2013; Greer et al., 2007a). Hence, these data, along with genetic interactions, aak-2::GFP expression, and pry-1 and aak-2 transcriptome analysis, support the following model: PRY-1, PAR-4, and AAK-2 form a complex in the muscle, leading to AAK-2 phosphorylation. Activated AAK-2 initiates cell non-autonomous signaling to regulate DAF-16 activity in the intestine to maintain the life span. This model is consistent with the previously reported role of AAK-2 (Burkewitz et al., 2014).

One of the outcomes of pry-1 interaction with daf-16 could be to affect lipid metabolism, since lipids are implicated in aging (Papsdorf and Brunet, 2019) and both genes promote monounsaturated fatty acid synthesis by transcriptionally regulating fatty acid desaturases such as fat-7 (Murphy et al., 2003; Ranawade et al., 2018; Zhang et al., 2013a). Other possibilities are also likely because DAF-16 interacts with multiple factors to regulate life span (Lapierre and Hansen, 2012; Uno and Nishida, 2016). We should point out that AXL-1, another Axin homolog in C. elegans, was reported previously to be necessary for metformin-induced life span extension, although axl-1 mutants have no aging-related phenotypes of their own (Chen et al., 2017). Thus, our work on PRY-1 provides the first evidence of an Axin family member regulating muscle health as well as life span.

Interactions between Axin and AMPK have been reported previously in mammalian systems. Specifically, the Axin-AMPK complex formation was enhanced in cultured cells when subjected to glucose deprivation, and Axin knockdown in the mouse liver impaired AMPK activation (Zhang et al., 2013b). AMPK is known to promote mitochondrial biogenesis and mitochondrial function in human umbilical vein cells and mice aorta (Marin et al., 2017). Moreover, AMPK phosphorylates all four human FOXO family members (Greer et al., 2007b). Similar to that with AMPK, AAK-2-mediated life span extension depends on mitochondrial network maintenance and DAF-16 regulation (Greer et al., 2007a; Uno and Nishida, 2016; Weir et al., 2017). Thus, it is plausible that Axin-AMPK-FOXO interact in a conserved manner to regulate disparate biological processes in eukaryotes. Studies in humans and other higher systems have established a connection between aging, muscle health, mitochondrial dysfunction, and diseases (Gouspillou and Hepple, 2016; Hood et al., 2019). Furthermore, Axin is essential for muscle maintenance, since myogenesis is abrogated in mutant animals (Huraskin et al., 2016) and Axin2 upregulation is associated with increased muscle fibrosis in aging mice (Arthur and Cooley, 2012; Brack et al., 2007). Since muscle mass and function progressively decline with age, understanding the mechanism of Axin's function in this tissue promises to uncover potential interventions for aging-associated muscle deterioration.

Limitations of the Study

We have shown that pry-1 is necessary to maintain muscle health and life span in C. elegans. However, it remains to be determined whether Axin homologs in other systems also regulate similar processes. Our conclusion that muscle-specific expression of pry-1 extends life span is based on the analysis of transgenic animals that constitutively express the gene throughout developmental and post-developmental periods. In the future, it will be worthwhile to investigate pry-1's role by activating its expression specifically during adulthood. The analysis of the pry-1 role in muscles led us to investigate its interactions with aak-2/AMPK and daf-16/FOXO. While our data demonstrates that the muscle-specific expression of pry-1 causes an increase in AAK-2 phosphorylation, whether PRY-1 physically interacts with AAK-2 is yet to be examined. Finally, we found that PRY-1-AAK-2-mediated signaling acts cell non-autonomously to promote nuclear localization of DAF-16 in the intestine, which is necessary for life span extension. However, the factors that facilitate communication between PRY-1 and DAF-16 remain to be identified.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Bhagwati P Gupta (guptab@mcmaster.ca).

Materials Availability

All data generated or analyzed in this study are included in this published article and its supplemental information.

Data and Code Availability

The published article includes all data generated or analyzed during this study.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.