A genetic model of methionine restriction extends Drosophila health- and lifespan

Abstract

Loss of metabolic homeostasis is a hallmark of aging and is characterized by dramatic metabolic reprogramming. To analyze how the fate of labeled methionine is altered during aging, we applied ¹³C5-Methionine labeling to Drosophila and demonstrated significant changes in the activity of different branches of the methionine metabolism as flies age. We further tested whether targeted degradation of methionine metabolism components would "reset" methionine metabolism flux and extend the fly lifespan. Specifically, we created transgenic flies with inducible expression of Methioninase, a bacterial enzyme capable of degrading methionine and revealed methionine requirements for normal maintenance of lifespan. We also demonstrated that microbiota-derived methionine is an alternative and important source in addition to food-derived methionine. In this genetic model of methionine restriction (MetR), we also demonstrate that either whole-body or tissue-specific Methioninase expression can dramatically extend Drosophila health- and lifespan and exerts physiological effects associated with MetR. Interestingly, while previous dietary MetR extended lifespan in flies only in low amino acid conditions, MetR from Methioninase expression extends lifespan independently of amino acid levels in the food. Finally, because impairment of the methionine metabolism has been previously associated with the development of Alzheimer's disease, we compared methionine metabolism reprogramming between aging flies and a Drosophila model relevant to Alzheimer's disease, and found that overexpression of human Tau caused methionine metabolism flux reprogramming similar to the changes found in aged flies. Altogether, our study highlights Methioninase as a potential agent for health- and lifespan extension.

Keywords: 13C-Methionine labeling; Alzheimer’s disease; Methioninase; aging; methionine restriction.

Fig. 1.

Age-dependent changes of the fate of labeled methionine. (A and B) Food consumption in 1-, 4-, and 6-wk-old male flies measured by qPCR using two different oligonucleotides. Twenty biological replicates per age. (C) Scheme of methionine metabolism. Blue color marks the labeled carbons. Relative labeling enrichment for methionine (D), SAM (E), SAH (F), and MTA (G) in whole male flies fed with 1 mM of labeled ¹³C5-methionine tracer in chemically defined food (lacking endogenous methionine) for 5 d. M0–M12 mark the number of labeled carbons. Labeling enrichment is the proportion of a particular labeled metabolite form among all measured isoforms. Note that although multiple isoforms of the same metabolite with different numbers of labeled carbons were measured, many of them are not present in Drosophila cells, and they are not displayed on the graph. Five biological replicates per condition. Means ± SD. Y and M symbols mark young (1 wk) and medium-age (4 wk) male flies *P < 0.05, **P < 0.01, ***P < 0.001. (H) The enrichment ratio (M1/M4+M5+M1) between methionine (M1) produced from the salvage pathway and total labeled methionine (M4+M5+M1) reflects the activity of the salvage (MTA cycle) pathway. Means ± SD; **P < 0.01. (I) Relative labeling enrichment for cystathionine. Means ± SD, ***P < 0.001.

Fig. 2.

Methioninase is a genetic tool for MetR. (A) Scheme of methionine degradation by Methioninase. (B) Immunoblot analysis of Methioninase and tubulin in tubulin-Gal80ts,tubulin-Gal4 flies > control flies or flies expressing Methioninase (in duplicates). (C) Relative mRNA levels of Methioninase in tubulin-Gal80ts,tubulin-Gal4 > control flies or flies expressing Methioninase. Four biological replicates per condition. Means ± SD, ***P < 0.001. (D) PCA of tubulin-Gal80ts,tubulin-Gal4 > control flies, or flies expressing Methioninase. (E) MSEA of the metabolites that changed significantly in either tubulin-Gal80ts,tubulin-Gal4 > control flies or flies expressing Methioninase.

Fig. 3.

Comparison of genetic (Methioninase) and dietary (food depletion) models of MetR. Box plots of relative levels of methionine (A), SAM (B), SAH (C), Cystathionine (D), MTA (E) in tubulin-Gal4,tubulin-Gal80ts > control flies or flies expressing Methioninase maintained on either standard laboratory food (marked as Lab food) or on the chemically defined food (CDF) supplemented with a range of concentrations of methionine (no methionine, 0.15 mM, 1 mM, or 4 mM). Four biological replicates per condition. Means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. (F) MSEA of the metabolites that changed significantly in tubulin-Gal4,tubulin-Gal80ts > control flies maintained on chemically defined food supplemented with either no methionine or 1 mM methionine. (G) Overlap of metabolites with more than a twofold change in the two models of methionine deprivation: 1) tubulin-Gal80ts tubulin-Gal4 flies > control flies (marked as Contr) or flies expressing Methioninase and 2) tubulin-Gal4,tubulin-Gal80ts > control flies maintained on chemically defined food supplemented with either no methionine (marked as no Met) or 1 mM methionine (marked as 1 mM Met).

Fig. 4.

Methionine requirements for normal maintenance of lifespan. Ubiquitous adult-onset expression of Methioninase suppresses lifespan in both male (A) and female (B) flies, which can be rescued by supplementation with a mixture of 3 mM of methionine, SAM, and cysteine. In males, mean lifespan for controls 51 d, and for Methioninase 20 d; P < 0.0001, log-rank test. In females, mean lifespan for controls 52 d, and for Methioninase 20 d; P < 0.0001, log-rank test. Survival of super survivor flies under ubiquitous adult-onset expression of Methioninase can be suppressed by treatment with a mixture of antibiotics in both male (C) and female (D) flies. Survival of male (E) and female (F) flies with ubiquitous adult-onset expression of control or Methioninase maintained on chemically defined food with either 1 mM methionine or with no methionine.

Fig. 5.

Low levels of Methioninase decrease levels of methionine and extend lifespan independently of the amino acid status in the food. Relative levels of methionine in male (A) and female (B) ActinGS > control or Methioninase flies fed with either EtOH or RU486. (C) Relative levels of methionine sulfoxide in male ActinGS > control or Methioninase flies fed with either EtOH or RU486. Relative levels of methionine in male (D) and female (E) TubulinGS > control or Methioninase flies fed with either EtOH or RU486. (F) Survival of female ActinGS > control or Methioninase flies fed with either EtOH or RU486. (G) Climbing of female ActinGS > control or Methioninase flies fed with either EtOH or RU486. (H) Proportion of smurf females at 50 d of age in female ActinGS > control or Methioninase flies fed with either EtOH or RU486. **P < 0.01, ***P < 0.001.

Fig. 6.

Tissue-specific Methioninase expression decreases levels of methionine and extends lifespan independently of the amino acid status in the food. (A) Relative levels of methionine in heads of tubulin-Gal80ts, elav-Gal4 > control or Methioninase female flies. Means ± SD. Relative levels of methionine in thoraxes of female (B) and male (C) tubulin-Gal80ts, Dmef-Gal4 > or tubulin-Gal80ts, elav-Gal4 > control or Methioninase flies. (D) Neuronal-specific leaky expression of Methioninase in ElavGS > Methioninase female flies extends lifespan. (E) Intestine-specific leaky expression of Methioninase in TIGS-2 > Methioninase male flies extends lifespan. (F) Fat body-specific leaky expression of Methioninase in WB-FB-GS > Methioninase male flies extends lifespan. **P < 0.01, ***P < 0.001.