A metabolic signature for long life in the Caenorhabditis elegans Mit mutants

Abstract

Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial-derived α-ketoacids and α-hydroxyacids that are produced by long-lived Mit mutants but not by other long-lived mutants or by short-lived mitochondrial mutants. We show that accumulation of these compounds is dependent on concerted inhibition of three α-ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer's disease. When the expression of DLD in wild-type animals was reduced using RNA interference we observed an unprecedented effect on lifespan - as RNAi dosage was increased lifespan was significantly shortened, but, at higher doses, it was significantly lengthened, suggesting that DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype.

Figure 1. A metabolic signature for long-life in the C. elegans Mit mutants

(A) Exometabolome analysis of wild type worms (N2), isp-1(qm150) and mev-1(kn1) mutants: 120,000 worms were transferred to minimal media and their exometabolome sampled over a 20 hr period (at +0, 0.5, 1, 2, 5, 20 hr). GC-MS was used to identify and quantify metabolites within each sample. Data is presented using hierarchical clustering (Pearson’s correlation coefficient): left panel, metabolite variation across row; right panel, metabolite variation relative to the entire array. Asterisks mark several α-ketoacids and α-hydroxyacids that are significantly overproduced by isp-1 mutants (statistical analyses summarized in Supplementary Figure 1). (B, C) Exometabolome analysis of various Mit, Age and short-lived mutants. Data was collected as described in (A) following an 18 hr metabolite capture period. Results are presented as in the left panel of A. In (C) only metabolites that differed significantly (p<0.0062) between long-lived and short-lived ETC mutants are shown (full results are provided in Supplementary Figures 6 & 7). Strains are marked at the top of each panel. (D) Many of the compounds detected in the exometabolome of worms strains are related by redox reactions. Metabolites that are specifically enriched in the exometabolome of Mit mutants are highlighted. (E) Quantification of select α-ketoacids and α-hydroxyacids in the +18 hr exometabolome of wild type (N2), isp-1(qm150) and nuo-6(qm200) animals. Ordinate is plotted on a log scale.

Figure 2. Inhibition of dihydrolipoamide dehydrogenase activity leads to metabolic and phenotypic recapitulation of the Mit phenotype

(A, B) Cross-reactivity of α-DLD polyclonal antibody with human (HEK293T) and mouse (3T3-L1 fibroblast) whole-cell extracts, and with whole-worm extracts of N2, isp-1(qm150) and mev-1(kn1). Also tested in (B) were whole-worm extracts from a feeding RNAi dilution series (Rea et al. 2007) targeting DLD (RNAi to empty vector ratio - 0:1, 1:200, 1:20, 1:5, and 1:0). Animals were fed DLD RNAi from the time of hatching. All lanes contain 25 μg of protein; β-actin served as loading control in (B). (C) DLD activity in whole-worm extracts from two independently-collected DLD feeding RNAi dilution series. One unit of DLD activity is defined as the rate of production of 1 μmol of NADH in 1 min at 25°C (Bhaskaran et al. 2011) (error bars: +/− SD). (D) Temporal changes in the exometabolome of N2 worms following treatment with increasing amounts of DLD RNAi (RNAi to empty vector ratio - 0:1, 1:200, 1:20, 1:5, and 1:0). Excreted metabolites were collected at +0, 0.5, 2, 5 and 18 hr. Data was analyzed by GC-MS and is presented using hierarchical clustering – left panel, metabolite variation across row; right panel, metabolite variation relative to the entire array. (See also Supplementary Figures 10-15). (E, F) Increasing doses of RNAi targeting DLD (RNAi to empty vector ratio - 0:1, 1:1000, 1:500, 1:200, 1:100, 1:50, 1:20, 1:10, 1:5, 1:2 and 1:0) were fed to wild type C. elegans from the time of hatching and the effects on both adult size (E, Scale bar: 200 μm) and lifespan (F) measured. Lifespan data is the mean of four replicates (+/-) S.E.M. (n = 60 worms/condition/replicate; asterisks indicates significantly different from vector, p<0.005 (Bonferroni corrected), summary statistics are tabulated in Supplementary Figure 17). (G) DLD activity in whole-worm extracts from N2 and isp-1(qm150) animals (n = 3 replicates, error bars: +/− SD).

Figure 3. Exometabolome analysis of tpk-1(qm162) mutants reveals concerted inhibition of α-ketoacid dehydrogenase activity is sufficient to recapitulate the Mit metabolic phenotype

(A) The +18 hr exometabolome of the following strains were collected and analyzed by GC-MS: wild type worms (N2), isp-1(qm150) and nuo-6(qm200) Mit mutants, short-lived mev-1(kn1) mutants, N2 exposed to DLD RNAi from the time of hatching (RNAi to vector ratios of 0:1, 1:200, 1:20, 1:5, and 1:0), and tpk-1(qm162) mutants exposed for 18 hours to both normoxia or anoxia. Columns represent independent experimental replicates. Data is presented using hierarchical clustering – left panel, metabolite variation across row; right panel, metabolite variation relative to the entire array. Statistical analyses are summarized in Supplementary Figures 5. See also Supplementary Figure 16 for global correlation analysis. (B) Correlation matrix showing metabolic similarity between +18 hr exometabolome of long-lived Mit mutants, long-lived dld-1 disrupted animals, and long-lived tpk-1(qm162) mutants. Details of distance measure calculations are described in Supplementary Figure 16. (C) Model for the genesis of the Mit phenotype (BCAA - branched chain amino acids; Mito. ETC – mitochondrial electron transport chain).