Tissue-specific down-regulation of S-adenosyl-homocysteine via suppression of dAhcyL1/dAhcyL2 extends health span and life span in Drosophila

Abstract

Aging is a risk factor for many human pathologies and is characterized by extensive metabolic changes. Using targeted high-throughput metabolite profiling in Drosophila melanogaster at different ages, we demonstrate that methionine metabolism changes strikingly during aging. Methionine generates the methyl donor S-adenosyl-methionine (SAM), which is converted via methylation to S-adenosyl-homocysteine (SAH), which accumulates during aging. A targeted RNAi screen against methionine pathway components revealed significant life span extension in response to down-regulation of two noncanonical Drosophila homologs of the SAH hydrolase Ahcy (S-adenosyl-L-homocysteine hydrolase [SAHH[), CG9977/dAhcyL1 and Ahcy89E/CG8956/dAhcyL2, which act as dominant-negative regulators of canonical AHCY. Importantly, tissue-specific down-regulation of dAhcyL1/L2 in the brain and intestine extends health and life span. Furthermore, metabolomic analysis of dAhcyL1-deficient flies revealed its effect on age-dependent metabolic reprogramming and H3K4 methylation. Altogether, reprogramming of methionine metabolism in young flies and suppression of age-dependent SAH accumulation lead to increased life span. These studies highlight the role of noncanonical Ahcy enzymes as determinants of healthy aging and longevity.

Keywords: CG8956/Ahcy89E/AhcyL2; CG9977/AhcyL1; S-adenosyl-homocysteine (SAH); aging; life span; methionine restriction.

Figure 1.

Age-dependent metabolism reprogramming. (A) Life spans of male yw (blue) and OreR (red) flies. Samples were collected at 1, 4, and 7 wk of age for metabolomics analyses by LC-MS/MS. (B,C) Heat maps showing the metabolites significantly affected in both yw and OreR flies between 1 and 7 wk of age. (D) Metabolic set enrichment analysis of the metabolites that changed significantly in both yw and OreR flies between 1 and 7 wk of age. (E) Box plots of individual methionine metabolism pathway metabolites significantly affected in both yw and OreR flies between 1 and 7 wk of age. (F) Methionine metabolism pathway. Arrows indicate the metabolites significantly affected in yw and OreR flies.

Figure 2.

Methionine metabolism reprogramming in naturally selected long-lived flies. (A) Life spans of control (B3) and long-lived (O1 and O3) males. (B) PCA of control (B3) and long-lived (O1 and O3) males. (C) Heat map showing the metabolites of the methionine metabolism pathway. (D) The methionine metabolism pathway. Arrows point to the metabolites that are significantly changed between control (B3) and long-lived (O1 and O3) flies at 1 wk of age (red) and 4 wk of age (blue).

Figure 3.

Screen of methionine metabolism components for life span regulation. (A) RNAi screen results ordered by ascending life span. Each bar represents the difference ([black bars] median; [gray bars] mean) in life span between ActinGS>RNAi flies and ActinGS>RNAi flies fed with RU486 starting at day 7. (*) P < 0.05; (**) P < 0.01. (B) Ubiquitous adult-onset expression of Rheb RNAi increases life span in males. P < 0.0001. (C) Ubiquitous adult-onset expression of dAhcyL1 RNAi increases life span in males. P < 0.0001 (for both 1× and 2× concentrations of RU486). (D) Ubiquitous adult-onset expression of dAhcyL1 RNAi-2 increases life span in females. P < 0.0001 (for both 1× and 2× concentrations of RU486). (E) Ubiquitous adult-onset expression of dAhcyL1 RNAi-1 (HM05009) increases life span in males. P < 0.0001. (F) Ubiquitous adult-onset expression of dAhcyL2 RNAi-1 increases life span in males. P < 0.0001. (G) Ubiquitous adult-onset expression of dAhcyL2 RNAi-2 increases life span in males. P < 0.0001.

Figure 4.

dAhcyL1 and dAhcyL2 regulate health span. (A) Ubiquitous adult-onset expression of dAhcyL1 RNAi-1 improves climbing ability in 40-d-old males. (*) P < 0.05. Means ± SD. (B) Ubiquitous adult-onset expression of dAhcyL1 RNAi-2 improves climbing ability in 40-d-old males. (*) P < 0.05. Means ± SD. (C) Ubiquitous adult-onset expression of dAhcyL2 RNAi-1 improves climbing ability in 40-d-old males. (**) P < 0.01. Means ± SD. (D) Ubiquitous adult-onset expression of dAhcyL1 RNAi-1 improves egg laying in 40-d-old flies. (*) P < 0.05. Means ± SD. (E) Ubiquitous adult-onset expression of dAhcyL1 RNAi-2 suppresses the number of “Smurf” females at 40 d of age. (***) P < 0.001. Means ± SD. (F) Ubiquitous adult-onset expression of dAhcyL2 RNAi-1 suppresses the number of “Smurf” females at 40 d of age. (*) P < 0.05. Means ± SD. (G) Fat body-specific adult-onset expression of dAhcyL1 RNAi-1 does not affect life span in males. (H) Fat body-specific adult-onset expression of dAhcyL2 RNAi-1 does not affect life span in males. (I) Brain-specific adult-onset expression of dAhcyL1 RNAi-1 increases life span in males. P < 0.0001. (J) Brain-specific adult-onset expression of dAhcyL2 RNAi-1 does not affect life span in males. (K) Intestine-specific adult-onset expression of dAhcyL1 RNAi-1 increases life span in females. P < 0.0001. (L) Intestine-specific adult-onset expression of dAhcyL1 RNAi-2 increases life span in females. P < 0.001

Figure 5.

Noncanonical functions of dAhcy are not involved in life span regulation. (A) Scheme of IP₃R-dAhcyL1 function. (B) Ubiquitous adult-onset expression of Itp-r83A RNAi does not affect life span in females. (C) Ubiquitous adult-onset expression of Itp-r83A.v does not affect life span in females. (D) Scheme of ribonucleotide reductase (RNR) regulation by dAhcyL1. (E) Relative levels of dCDP in tubulinGal80ts, tubulinGal4 flies expressing either control (gfp) or dAhcyL1 RNAi-1 for 2 wk at 29.5°C. Means ± SD. (F) Relative levels of dTDP in tubulinGal80ts, tubulinGal4 flies expressing either control (gfp) or dAhcyL1 RNAi-1 for 2 wk at 29.5°C. Means ± SD. (G) Relative levels of dGTP in tubulinGal80ts, tubulinGal4 flies expressing either control (gfp) or dAhcyL1 RNAi-1 for 2 wk at 29.5°C. (H) Ubiquitous adult-onset expression of RnrL RNAi does not affect life span in males.

Figure 6.

dAhcyL1 and dAhcyL2 are involved in the regulation of methionine metabolism. (A) Scheme of methionine metabolism regulation by dAhcyL1 and dAhcyL2. (B) Relative levels of SAH in tubulinGal80ts, tubulinGal4 flies expressing either control (gfp) or Ahcy13 RNAi-1 for 2 wk at 29.5°C. (***) P < 0.0001. Means ± SD. (C) Relative levels of SAH in tubulinGal80ts, tubulinGal4 flies expressing either control (gfp) or dAhcyL1 RNAi-1 for 2 wk at 29.5°C. (*) P < 0.05. Means ± SD. (D) Relative levels of homocysteine in tubulinGal80ts, tubulinGal4 flies expressing either control (gfp) or dAhcyL1 RNAi-1 for 2 wk at 29.5°C. (*) P < 0.05. Means ± SD. (E) Ubiquitous adult-onset expression of Ahcy13 RNAi-2 significantly suppresses life span in males. P < 0.001. (F) Ubiquitous adult-onset expression of Ahcy13 RNAi-1 significantly suppresses life span in males. P < 0.001. (G) Brain-specific adult-onset expression of Ahcy13 RNAi-1 significantly suppresses life span in males. P < 0.001. (H) Feeding adult flies with SAM significantly suppresses life span in males. P < 0.001. (I) Feeding adult flies with 100 µM cycloleucine significantly increases life span in males. P < 0.001.

Figure 7.

dAhcyL1 and dAhcyL2 are involved in the regulation of H3K4me3 levels. (A) Immunoblot analysis of H3K4me3 and total histone 3 in ActinGS-Gal4>dAhcyL1 RNAi-1 or control RNAi flies fed with RU486 from 7 to 40 d of age. (B) Relative mRNA levels of dAhcyL1, ENOPH1, SMS, and MTAP from 40-d-old ActinGS-Gal4>dAhcyL1 RNAi-1 flies fed with RU486 from 7 to 40 d of age. (*) P < 0.05; (**) P < 0.01. Means ± SD. (C) Ubiquitous adult-onset expression of dAhcyL1 RNAi-1 increases life span in males under a standard sugar/yeast diet (1× SY) P < 0.001 but not with a diet where amounts of sugar and yeast were reduced (0.5× SY). (D) Ubiquitous adult-onset expression of dAhcyL1 RNAi-1 increases life span in males maintained on a high-methionine diet (4.5 g/L). (E) Working model.