On the benefits of the tryptophan metabolite 3-hydroxyanthranilic acid in Caenorhabditis elegans and mouse aging

Abstract

Tryptophan metabolism through the kynurenine pathway influences molecular processes critical to healthy aging including immune signaling, redox homeostasis, and energy production. Aberrant kynurenine metabolism occurs during normal aging and is implicated in many age-associated pathologies including chronic inflammation, atherosclerosis, neurodegeneration, and cancer. We and others previously identified three kynurenine pathway genes-tdo-2, kynu-1, and acsd-1-for which decreasing expression extends lifespan in invertebrates. Here we report that knockdown of haao-1, a fourth gene encoding the enzyme 3-hydroxyanthranilic acid (3HAA) dioxygenase (HAAO), extends lifespan by ~30% and delays age-associated health decline in Caenorhabditis elegans. Lifespan extension is mediated by increased physiological levels of the HAAO substrate 3HAA. 3HAA increases oxidative stress resistance and activates the Nrf2/SKN-1 oxidative stress response. In pilot studies, female Haao knockout mice or aging wild type male mice fed 3HAA supplemented diet were also long-lived. HAAO and 3HAA represent potential therapeutic targets for aging and age-associated disease.

Fig. 1. Knockdown of haao-1 extends healthy lifespan in C. elegans.

a Kynurenine pathway schematic (created with BioRender). Metabolites are shown in bubbles. C. elegans gene names for enzymes are shown in italics. RNAi knockdown of kynu-1, haao-1, or tdo-2: b extends lifespan at 15 °C, 20 °C, and 25 °C (colored text indicates change in mean lifespan relative to empty vector (EV) RNAi); c slows growth but allows worms to maintain body size (i.e., worm area) later in life; and d slows motivated and unmotivated crawl speed early in life but allows worms to maintain speed and thrashing frequency in liquid later in life. For box-and-whisker plots, center line represents median, boxes indicate first and third quartiles, and whiskers represent 5th and 95th percentiles. e tdo-2(RNAi) reduces brood size at 15 °C while kynu-1(RNAi) increased brood size at 25 °C. Lifespan extension from kynu-1(RNAi), haao-1(RNAi), or tdo-2(RNAi) displays different patterns of dependence on (f) tissue and (g) genes in established aging pathways. Error bars indicate standard error of mean. *p < 0.05, **p < 0.01, ***p < 0.001 (log rank test, panels b, f, g; two-sided Welch’s t test, panels c–e). Abbreviations: TRP tryptophan, NFK N-formylkynurenine, KYN kynurenine, KA kynurenic acid, AA anthranilic acid, 3HK 3-hydroxykynurenine, XA xanthurenic acid, 3HAA 3-hydroxyanthranilic acid, CA cinnabarinic acid, ACMSA aminocarboxymuconate semialdehyde, AMSA aminomuconic semialdehyde, QA quinolinic acid, Acetyl-CoA acetyl coenzyme A, NAD⁺ nicotinamide adenine dinucleotide. Summary statistics are provided in Supplementary Data 1 and source data are provided in Source Data.

Fig. 2. 3HAA mediates lifespan extension from reduced haao-1 activity.

a C. elegans lacking haao-1 (haao-1(tm4637)) accumulate a red coloration with age relative to wild type. Animals shown are age 21 days from egg. Cutout provides an enlarged view of the head region. Arrows highlight areas of distinct coloration difference between wild type and haao-1 animals. b NGM plates supplemented with 3HAA are red in color. c 3HAA levels measured by LC-MS/MS are elevated ~10-fold in 8-day-old worms subjected to haao-1(RNAi), but not kynu-1(RNAi) or tdo-2(RNAi), relative to EV(RNAi) (N = 9 biologically independent samples per condition). d Supplementing C. elegans media with 0.01 to 1 mM 3HAA extends lifespan. e 1 mM 3HAA mimics lifespan extension from haao-1 knockdown or deletion and similarly f does not affect brood size and g elevates thrashing capacity in liquid across lifespan. For box-and-whisker plots, center line represents median, boxes indicate first and third quartiles, and whiskers represent 5th and 95th percentiles. h 1 mM 3HAA increases lifespan in animals subjected to tdo-2(RNAi), kynu-1(tm4924) single deletion, or kynu-1(tm4924) haao-1(tm4637) double deletion to a degree similar to wild type animals; lifespan extension is largely eliminated in haao-1(tm4637) animals. Colored text indicates change in mean lifespan relative to control. Error bars indicate standard error of mean. *p < 0.05, **p < 0.01, ***p < 0.001 (two-sided Welch’s t test, panels c, f, g; log rank test, panels d, e, h). Summary statistics are provided in Supplementary Data 1 and source data are provided in Source Data.

Fig. 3. Elevating 3HAA protects C. elegans against oxidative stress and activates the Nrf2/SKN-1 oxidative stress response.

a Knockdown or deletion of haao-1 or 1 mM 3HAA increases survival of C. elegans challenged with 20 mM paraquat. b 500 μM H₂O₂ reduces visible red color of 1 mM 3HAA in water. c 3HAA reduces H₂O₂ in water in a dose-dependent manner. d Deletion of haao-1 or 1 mM 3HAA represses the age-dependent increase in H₂O₂ secretion in C. elegans. e C. elegans lacking haao-1 have elevated endogenous H₂O₂ (HyPer). haao-1(RNAi) or 1 mM 3HAA increases fluorescence in C. elegans strains transgenically expressing f the skn-1::GFP fusion protein or g the gcs-1p::GFP or gst-4p::GFP promoter activity reporters. Hypomorphic skn-1 loss-of-function largely prevents lifespan extension from h haao-1(RNAi) or (i) 1 mM 3HAA. Colored text indicates change in mean lifespan relative to control. Error bars indicate standard error of mean. *p < 0.05, **p < 0.01, ***p < 0.001 (log rank test, panels a, h, i; ANOVA; panels c, d; Welch’s t test, panels f, g). Summary statistics are provided in Supplementary Data 1 and source data are provided in Source Data.

Fig. 4. Dietary 3HAA extends lifespan, improves health metrics, and promotes a less inflammatory immune cell profile in mice.

a C57BL/6J mice fed chow supplemented with 312.5 ppm (low dose, L; N = 6) or 3125 ppm (high dose, H; N = 7) 3HAA starting at 27 months of age live longer than mice fed control diet (C; N = 8). Kaplan-Meier survival curves show remaining lifespan following introduction of the 3HAA diet. Colored text indicates change in mean remaining lifespan relative to control. b Neither 3HAA supplemented diet affected body weight. c Dietary 3HAA supplementation increases 3HAA concentration in serum and urine. d Mice on low dose 3HAA diet trended toward increased all paw grip strength after 13 weeks. Dietary 3HAA alters circulating immune cell profiles after 10 weeks: e shifting myeloid cells away from neutrophils/granulocytes and toward monocytes, f decreasing or preventing an increase in natural killer (NK) within several sub-lineages, g preventing an increase in double negative (DN) T cells, and h creating a trend toward reducing B cell counts. ^#p < 0.1, ^†, *p < 0^.05, ^‡, **p < 0.01, ***p < 0.001 vs. control diet (Wald test in Cox regression with weighted estimation, panel a; two-sided Welch’s t test, panels b–h). For panel d, *^, **^, *** indicate significance vs. Week 0; ^{†, ‡} indicate significance vs. control diet. Error bars indicates standard error of mean. Summary statistics are provided in Supplementary Data 1 and source data are provided in Source Data.

Fig. 5. Knocking out Haao extends lifespan and improves health metrics in mice.

a Deletion of Haao (Haao^-/-, KO; N = 45) extends lifespan relative to wild type (N = 37) in C57BL/6N mice. Kaplan-Meier survival curves show lifespan from birth. Gray text indicates change in mean lifespan. b Haao^-/- mice have lower body weight in early- to mid-adulthood. c Haao deletion increases 3HAA concentration in plasma and urine. d Haao^-/- mice had significantly increased grip strength at 12 and 18 months of age. ^#p < 0.1, ^†, *p < 0^.05, ^‡, **p < 0.01, ***p < 0.001 vs. control diet (log rank test, panel a; two-sided Welch’s t test, panels b–d). Error bars indicate standard error of mean. Summary statistics are provided in Supplementary Data 1 and source data are provided in Source Data.