Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB

Abstract

Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity.

Figure 1. CRTC-1 regulates longevity

a, CRTC-1 is the sole C. elegans CRTC family member, with conserved CREB-, calcineurin- and 14-3-3-binding domains and nuclear export signals (NES). Conserved AMPK phosphorylation sites are found at serines 76 and 179. b, Pcrtc-1::RFP is expressed throughout the intestine of the worm and in neurons in the head (inset) and tail. c, RNAi inhibition of crtc-1 and tax-6 increases C. elegans wild-type median lifespan by 53% and 60% respectively (log rank, P < 0.0001 in each case). d, Overexpressing aak-2 (aa 1–321) increases lifespan by 37.5% (log rank, P < 0.0001). e–g, Coexpression of the NUP-160::GFP nuclear pore complex subunit to mark the nuclear envelope (green), CRTC-1::RFP (red) and merge (×20 magnification). e, Under well-fed conditions CRTC-1::RFP is found throughout intestinal cells. f, g, Overnight starvation (f) or 33 °C overnight (g) induce cytosolic translocation of CRTC-1::RFP. h, 4′,6-diamidino-2-phenylindole (DAPI) staining of C. elegans intestinal nuclei (×20). White arrow indicates nucleolus. i, Confocal image of NUP-160::GFP (green) marking nuclear membrane and CRTC-1::RFP (Red) after 33 °C overnight showing nuclear exclusion of CRTC-1::RFP after heat stress (×100).

Figure 2. CRTC-1 is a target of AAK-2 and TAX-6

a, CRTC-1::RFP (red) is excluded from the nucleus of intestinal cells when coexpressed with a constitutively active AMPK catalytic subunit AAK-2 (aa 1–321, T181D)::GFP but localized throughout the cell in the presence of kinase-dead AAK-2 (aa 1–321, T181A)::GFP (×63 magnification). b, tax-6 RNAi results in nuclear exclusion of CRTC-1::RFP (red). White arrows represent intestinal nuclei (×20). DIC, differential interference contrast microscopy. c, Combined RNAi for the two C. elegans 14-3-3 proteins results in nuclear localization of CRTC-1::RFP under basal conditions. White arrows represent intestinal nuclei (×20). d, In vitro kinase assay showing that AMPK directly phosphorylates CRTC-1. Purified rat AMPK holoenzyme was incubated with GST or GST::CRTC-1 in the presence of AMP. Phosphorylation was detected with phospho-Ser (p-Ser) 14-3-3 binding motif antibody. Coomassie stained gel shows protein loaded.

Figure 3. Calcineurin and AMPK regulate lifespan through phosphorylation of CRTC-1

a, RNAi for crtc-1 or tax-6 has no effect in a tax-6 (ok2065) mutant background (log rank, P = 0.28 and P = 0.73, respectively). b, Mutations to the conserved AMPK target sites serines 76 and 179 together are sufficient to retain CRTC-1::RFP in the nucleus (×10). Insets are magnification of intestinal region. c, CRTC-1(S76A,S179A) is refractory to both tax-6 RNAi and AAK-2 overexpressor (o/e) in C. elegans intestinal cells (×40). White dashed lines and arrows indicate nuclei. d, RNAi of tax-6 extends the lifespan of C. elegans expressing wild-type CRTC-1::RFP (log rank, P < 0.0001), but has no effect on the S76A, S179A double mutant (log rank, P = 0.57). There is no significant difference between the lifespans of CRTC-1::RFP and CRTC-1(S76A,S179A) fed empty vector (log rank, P = 0.48). d, AAK-2 (aa 1–321) overexpression increases wild-type lifespan (log rank, P < 0.0001) but has no effect on worms expressing CRTC-1 (S6A, S179A) (log rank, P = 0.65).

Figure 4. CREB activity regulates lifespan

a, Co-immunoprecipitation using lysate from a C. elegans transgenic strain (AGD744) coexpressing 3×Flag::CRTC-1 and HA::CRH-1 showing CRTC-1 and CRH-1 interact in vivo. b, c, CREB transcriptional reporter (pCRE::GFP) is significantly affected by RNAi of crh-1, crtc-1 or tax-6 (two-way ANOVA, F = 18.54, P < 0.0001). Error bars represent data ± s.e.m. Control worms express GFP in head neurons and in some body tissues including muscle, hypodermis and intestine. d, RNAi of crh-1 from larvae or adulthood extends wild-type lifespan (log rank, P < 0.001). e, RNAi of crtc-1 extends wild-type lifespan (log rank, P < 0.001) but has no effect on crh-1 (nn3315) nulls (log rank, P = 0.53). f, Heat maps (clustered display of expression relative to wild-type) for genes differentially expressed in crh-1 (nn3315) nulls, tax-6 (ok2065) nulls, and aak-2 overexpressors (adjusted P value < 0.05 and fold change larger than twofold). The 22 most upregulated genes in all mutants are expanded (full gene list available in Supplementary Material). g, Venn diagram of all differentially expressed genes compared to wild-type in tax-6 (ok2065) nulls, aak-2 overexpressors and crh-1 (nn3315) nulls. Transcriptional profiles of aak-2 overexpressors and tax-6 nulls are more similar to crh-1 nulls than expected by chance (one-tailed Fisher’s exact test, P = 2.3× 10⁻¹⁹ and P = 1.1× 10⁻²²⁵, respectively). WT, wild type. h, Promoters of differentially expressed genes are significantly enriched for CRE motifs (full bar) and TATA boxes (darker bar) (P values for binomial proportion test compared to CRE background shown above each mutant, asterisks indicate significant P-values). ‘Common’ denotes genes differentially expressed in all 3 backgrounds. TSS, transcription start site.