Extended Data Fig. 1: acsd-1 LOF improves NAD+ levels, mitochondrial function, and lifespan through de novo synthesis in C. elegans.
From: De novo NAD+ synthesis enhances mitochondrial function and improves health
a, De novo synthesis of NAD+ from tryptophan. Names of the worm’s orthologues are in blue. b, acsd-1 expression pattern across different developmental stages in wild-type worms expressing extrachromosomal array of acsd-1::GFP transgene. Scale bar, 100 μm. c, acsd-1 expression pattern in adult wild-type worms expressing extrachromosomal array of acsd-1::GFP transgene. i, intestine; m, muscle; p, pharynx; v, vulva. d, acsd-1 mRNA levels in wild-type and rrf-3(pk1426) mutants (n = 6, each n represents a pool of ~600 worms). e, ACSD-1 activity in control (empty vector) versus acsd-1 RNAi-fed worms quantified in both wild-type and rrf-3 mutants (n = 3, where each n represents a pool of ~3,600 worms) with compensation for negative controls. f, QPRT-like activity can be detected in both wild-type worms and rrf-3 mutants (n = 3, each n represents a pool of ~3,600 worms). g, h, Effects of acsd-1 knockdown throughout the entire life on N2 (g) and rrf-3 mutant (h) worm lifespan. i, Lifespan of rrf-3(pk1426) mutants exposed to control or acsd-1 RNAi upon tryptophan supplementation. P*, ctrl versus ctrl + Trp 50 μM; P^, ctrl versus acsd-1 RNAi; P°, ctrl + Trp 50 μM versus acsd-1 RNAi + Trp 50 μM. j, Quantification of GFP signal in ges-1::mito::GFP reporter strain, expressing mitochondria-targeted GFP in the intestine at day 1 and 3 of adulthood (n = 4, each n represents a pool of 20 worms). k, Blue native PAGE on mitochondria extracted from rrf-3 mutant worms fed with either empty vector or acsd-1 RNAi bacteria at day 2 of adulthood (n = 3, each n represents mitochondria extracted from a pool of ~10,000 worms). l, Mitochondrial morphology in the Pmyo-3::mito::GFP reporter strain fed with control or acsd-1 RNAi. Stars represent nuclei. Scoring includes the total perimeter of the mitochondrial network, its total area, the area occupied by the mitochondria within the cell and the circularity assessment, in which 1 is a perfect circle and 0 is a line (n = 6 worms). m, Epistasis between RNAi for acsd-1 and the UPRmt regulator, ubl-5. P*, ctrl versus ctrl/acsd-1 RNAi; P°, ctrl/ubl-5 RNAi versus ubl-5/acsd-1 RNAi. n, Quantification of the GFP signal in hsp-4::GFP reporter strain (n = 4, each n represents a pool of 20 worms) at day 1 and 3 of adulthood. o, Quantification of the GFP signal in hsp-16.2::GFP reporter strain (n = 4, each n represents a pool of 20 worms). After the first time point sampled at 20 °C, worms were exposed to 37 °C, and the measurement was repeated every hour for 6 h. p, Expression of UPRmt genes in worms at day 2 of adulthood fed with control or acsd-1 RNAi (n = 6, each n represents a pool of ~600 worms). q, Expression of sod-3 mRNA at day 1 of adulthood in control or acsd-1 RNAi-fed worms (n = 3, each n represents a pool of ~600 worms). r, Survival of wild-type (N2) worms exposed to 4 mM paraquat starting at the L4 stage, in which the knockdown of acsd-1 was performed at different life stages. P*, ctrl versus acsd-1 RNAi whole life; P^, ctrl versus acsd-1 RNAi development; P°, ctrl versus acsd-1 RNAi adulthood. s, Epistasis between RNAi for acsd-1 and daf-16 in wild-type (N2) worms exposed to 4 mM paraquat. P*, ctrl versus ctrl/acsd-1 RNAi; P^, ctrl/daf-16 RNAi versus daf-16/acsd-1 RNAi. All worm assays, except for hsp-16.2::GFP reporter strain, were performed at 20 °C and repeated at least once. Data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. P values calculated using two-tailed t-test (d, e, j, l, n–q) or log-rank test (g–i, m, r, s). For individual P values, see Source Data. For lifespan values, see Extended Data Table 1.
