Cell Culture, RNAi knockdown, and mitochondrial parameters

The primary fibroblast cell lines HT01 (#AG09599) and the WS01 (#AG03141) cells were acquired from Coriell Institute. The WRN-KD cells were siRNA knockdown in HT01 cells using WRN human siRNA ologo duplex (CAT#: SR322215, Origene). Briefly, siRNAs were incubated in Optimem with 4 ml RNA Interferin (siRNA transfection reagent, Polyplus) per 1 ng RNA for 15 min and added to complete media for a final concentration of 30 nM siRNA. After 3-day incubation, cells were applied for further experiments. Knockdown efficiency was examined by western blot. All other primary human fibroblasts (detailed in Supplementary Table 1) and blood samples (detailed in Supplementary Table 1) were from Chiba University. The protocols (SEI973 and SEI974) were approved by the Bioethics committee of the Chiba University Graduate School of Medicine, and was conducted in full compliance with the Declaration of Helsinki. Participants provided written informed consents. The mouse embryonic fibroblasts (MEFs) were generated from Wrn−/− embryos with MEFS from the wild type littermates as controls. All cells were maintained in GIBCO MEM medium supplemented with 15% FBS, 1% P&S, 1x Vitamin C, 1x Glutamine, and 1x NEAA, and grown in 20% O 2 /5% CO 2 at 37 °C. Flow cytometry was used mitochondrial parameters, electron microscopy for mitochondrial morphology, an XFe96 for mitochondrial oxygen consumption rate (OCR), and a luminescent assay for ATP quantification. First, human primary cells (HT01, WRN-KD, WS01) or MEFs (WT and Wrn−/− cells) were stained with designated dyes followed by flow cytometry to quantify relative mitochondrial ROS (using mitoSOX dye 3 μM for 30 min), mitochondrial membrane potential/MMP (TMRM dye, 10 nM for 15 min), or relative mitochondrial content (MitoTracker Green, 50 nm for 30 min)54. All reagents were from Life Technologies. Data was analyzed using FCS Express 4 software (De Novo Software). ATP levels in the primary human fibroblasts were examined using a commercial kit (abcam #ab113849). Cells of 6–10 passages were used for the experiment.

Mitophagy detection in human primary cells

A commercial kit (Dojindo Laboratories) was used to detect mitophagy in cultured primary fibroblasts and WRN-KD cells23,75. In brief, Mtphagy dye (Dojindo Laboratories) was added to the cells, where after it accumulates in the intact mitochondria. A decrease in pH (when mitochondria are localized to lysosomes) results in a high fluorescence signal from the dye, which was used to sort the cells using flow cytometry as previously described23,75. For microscopic observation, in brief, human cells were treated with Mtphagy dye for 30 min, then after the supernatant was discarded, cells were treated with NR (1 mM) for 24 h, and then the lysosomal dye was incubated for 30 min. Mitophagy events were imaged under the microscope. For the ULK1 and AMPK knockdown in human cells, we use ULK1 siRNA (Origene, #SR322391) and AMPK siRNA (Origene, #SR321409) transient transfection in WS01 fibroblasts.

Mice and mitochondrial functions

Mice carrying Wrn−/− allele30 were maintained under standard laboratory conditions at the NIA with free access to water and standard diets. All procedures were approved by the Animal Care and Use Committee of the Intramural Research Program of the National Institute on Aging (NIA), in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Our protocol 361-LMG received an ethical approval as a part of the review process. High resolution respirometry was performed on the Oxygraph-2k (O2k, OROBOROS Instruments, Innsbruck, Austria) according to previous report76. Mice were sacrificed by cervical dislocation and brain, heart and liver tissues were rapidly dissected and homogenized by 10–15 strokes of a 15 mL dounce homogenizer in Mir05 respiration buffer (110 mM sucrose, 60 mM K-lactobionate, 0.5 mM EGTA, 3 mM MgCl2, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, and 0.1% BSA essentially fatty acid free, pH 7.1 at 37 °C). Protein was quantified by BCA assay and 1 mg/mL homogenate loaded for respiration measurements at 37 °C. Malate (2.5 mM), pyruvate (5 mM) and ADP (2.5 mM) addition results in complex I mediated state 3 respiration. Subsequently, complex I inhibitor rotenone (0.5 μM) was added and then complex II substrate succinate (10 mM) injection initiates complex II mediated state 3 respiration. Complex III inhibitor antimycin A (2 μM) was then added followed by the artificial complex IV substrate ascorbate + TMPD (4 mM + 1 mM, respectively). Fatty acid metabolism of the heart homogenate was also performed using malate plus palmitoyl-carnitine (5 mM each) followed by state 3 respiration initiate with 2.5 mM ADP.

Fly lines, lifespan and stem cell proliferation assays

To temporally control WRN knockdown, we used GeneSwitch lines expressing an inactivated form of Gal4 that is activated in the presence of the drug RU48677. We used w;DaGeneSwitch-Gal4 for ubiquitous knockdown and w;5961GeneSwitch-Gal4, UAS-nlsGFP/CyO (a gift from Benjamin Ohlstein78) for intestinal stem cell-specific knockdown. We used y,v,sc;WrnexoRNAi; (Bloomington line #38297) for knockdown, and w;LacZ RNAi (a gift from Masayuki Miura) as controls for non-specific RNAi effects. Knockdown of Wrnexo was confirmed by qPCR analysis. RNA was purified from single flies (3 flies/genotype) treated with EtOH, RU486 or RU486 and 5 mM NMN for 7 days with Nucleospin RNA purification kit following manufacturer’s protocol (Macherey-Nagel). cDNA was synthesized using iScript cDNA Synthesis kit (BioRad) and qPCR analysis was done with power SYBR Green PCR master mix (Thermo Fisher). To confirm knockdown two different WRNexo primer sets were used: WRNexo F1: 5′-GAAAAGAACGGAGATGCTGCC-3′, WRNexo R1: 5′-AGTCACCTCGTTGATCTTGGTC-3′. WRNexo F2: 5′-AGAACGGAGATGCTGCCTTTA-3′, WRNexo R2: 5′-AGTCACCTCGTTGATCTTGGT-3′, Actin was used as household gene with the following primers: Actin F: 5′-TTCCCCTCCATCGTCGGTC-3′ and Actin R: 5′-GATACCACGCTTGGACTGGG-3′. Reaction efficacy was optimized to 90–100% and the relative difference was calculated by the ddCt method79.

Flies were maintained at 25 °C on a 12-h light/dark cycle, using the following food recipe: 1 L distilled water, 13 g agar, 22 g molasses, 65 g malt extract, 18 g brewer’s yeast, 80 g corn flour, 10 g soy flour, 6.2 ml propionic acid, and 2 g methyl-p-benzoate in 7.3 ml of EtOH. For treatment food, RU486 was dissolved in the EtOH for 200 μM final concentration. Lifespan experiments were performed as previously described80. Briefly, female offspring of DaGeneSwitch and WrnexoRNAi lines were allowed to mate for 2–3 days, then transferred to vials with food ± RU486 and/or NMN at 5 mM final concentration. Up to 70 flies per vial were flipped thrice weekly, with dead flies counted visually. The Kaplan–Meier lifespan curves were generated using Prism. For statistics, multiple condition experiments were evaluated by one-way ANOVA with statistics using Peto’s log-rank test (Cox-Mantel test).

The 5961GeneSwitch-Gal4, UAS-nlsGFP line was crossed to Wrnexo or LacZ RNAi to evaluate stem cell proliferation in response to pathogenic insult. After allowed to mate for 2 days after eclosion female offspring were transferred to food with/without RU486 (200 μM), ±NR supplementation (0.78 mM) or NMN supplementation (5 mM). At six days of age (after four days of RNAi), they were transferred to 5% sucrose ± Erwinia carotovora carotovora 15 from 15 mL of overnight culture. After 24 h, guts were dissected in 1x phosphate-buffered saline (PBS), fixed for 45 min at room temperature (100 mM glutamic acid, 25 mM KCl, 20 mM MgSO 4 , 4 mM sodium phosphate, 1 mM MgCl 2 , and 4% formaldehyde), washed for 1 h at 4 °C (1x PBS, 0.5% bovine serum albumin and 0.1% Triton X-100), and then incubated with rabbit anti-phospho-Histone H3 Ser 10 (Upstate, 1:1000) overnight at 4 °C. Secondary antibody staining was done at room temperature for 2 h, using fluorescent antibody from Jackson Immunoresearch at 1:500. DAPI was used to stain DNA (1:1000). Guts were washed 3 × 10 min after each antibody. pH3 positive cells were counted manually on a Zeiss dissecting fluorescent microscope, with representative images captured on a Zeiss LSM 700 confocal microscope and processed using Adobe Photoshop/Illustrator. The UAS-nlsGFP construct was used to evaluate the total number of intestinal stem cells, to rule out cell death as a factor in the proliferative response. Comparisons between proliferation counts were done by one-way ANOVA, using Sidak’s post-hoc test.

C. elegans strains, and lifespan/healthspan studies

Standard C. elegans strain maintenance procedures were followed in all experiments81. Nematode rearing temperature was kept at 25 °C, unless noted otherwise. N2: wild type Bristol isolate was from Caenorhabditis Genetics Center and the wrn-1(gk99) was a gift from Dr. Hyeon-Sook Koo (Yonsei University, Korea)27. RNAi knockdown of designated genes was performed using standard protocol and verified by PCR21. Lifespan examination was performed at 25 °C on NGM plates containing 100 μM 5-FudR and seeded with 100 μL E. coli OP50 strain. Worms were scored every day (Urolithin A experiments) or every other day (NR or NMN treatments) and scored as dead when they stopped pharyngeal pumping and were unresponsive to touch. Lifespan experiments were performed with 100–150 worms/group. Kaplan–Meier survival curves of pooled populations were generated and the log-rank test was used for statistics. Swimming and pharyngeal pumping were used for healthspan evaluation following established methods21, with 10–30 worms/group (three biological repeats). Pharyngeal pumping was evaluated at adult day 4 and adult 6. Drug or vehicle treatment began at L4 stage, unless noted otherwise. Drugs used were the NAD+ precursors NR and NMN (both at 1 mM), the mitophagy inducer Urolithin A (100 μM), a SIRT1 activator SRT1720 (10 μM), and a PARP inhibitor Olaparib (500 nM). Two to seven biological repeats were performed for all experiments. OCR of C. elegans was measured using Seahorse XFe96 instrument75,82. wrn-1 and N2 C. elegans strains were synchronized by standard egg lay and L4 stage nematodes were transferred to plates +/− NR (1 mM). At days 2 and 10, worms were washed three times with M9 buffer and allowed to digest gut bacteria for 30 min prior to the start of respiration. Worms were plated in XFe96 seahorse plates in M9 buffer (15–30 worms per well). Instrument was started with six measurements of each respiratory state. First, baseline respiration was measured followed by injection of FCCP (10 μM, final concentration) to elicit maximal respiration. Then, sodium azide (40 mM, final concentration) was injected to account for non-mitochondrial respiration. Number of nematodes per well were counted for normalization.

Quantification of mitotic cells in C. elegans

For quantification of mitotic cells, immunostaining was performed83. Briefly, worms were treated with NR (1 mM) at L4 stage and germlines were isolated on adult day 1 and adult day 6 worms. Germlines were isolated on poly-l-lysine-coated slides in egg buffer (containing 25 mM HEPES, pH 7.4, 118 mM NaCl, 48 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 ) supplemented with 0.1% Tween-20 and 0.2 mM levamisol. The slides were mounted with 7 μl mounting solution containing ProLong Gold (Thermo Fisher) and 0.5 μg/ml DAPI (Sigma), followed by imaging in a confocal microscope. Number of mitotic cells was measured using ZEN 2.3 software. The distal edge of the transition zone border was defined as the first cell diameter in which two or more nuclei displayed the characteristic crescent shape.

C. elegans imaging

We performed imaging for mitochondrial network, mitochondrial content, mitophagy levels, and fat staining. To evaluate mitochondrial network in worm muscle cells, a myo-3::gfp reporter strain was imaged with Zeiss confocal microscopy, with 5 images/worm and 15 worms/group/experiment. Mitochondrial network was scored in a double-blinded manner on an arbitrary scale from 1 to 5. A score of 5 denotes a perfectly organized mitochondrial network with healthy mitochondria running parallel with the myofilament lattice. For highly fragmented and disorganized mitochondrial network morphology, we gave a score of 19. For mitochondrial content analysis, wrn-1 and N2 C. elegans strains were synchronized by standard egg lay and L4 stage nematodes were transferred to plates containing NR (1 mM), NMN (1 mM), or vehicle control. For MitoTracker staining, worms were cultivated in presence of 125 nM MitoTracker Green FM (#M7514; Life Technologies) at 20 °C for 24 h. Stained and washed worms were immobilized with levamisole before mounting on 2% agarose pads for microscopic examination with a Zeiss AxioImager Z2 epifluorescence microscope. TMRE staining (tetramethylrhodamine, ethyl ester, perchlorate; a dye that accumulates in intact, respiring mitochondria) was conducted as previously described to measure mitochondrial membrane potential10. Briefly, animals were grown at 20 °C in the presence of 150 nM TMRE for 24 h. Stained and washed worms were immobilized with levamisole before mounting on 2% agarose pads for microscopic examination with a Zeiss AxioImager Z2 epifluorescence microscope. All images were acquired under the same exposure. Average pixel intensity values were calculated by sampling images of different animals. The mean pixel intensity for each animal were calculated in these images using the ImageJ software (http://rsb.info.nih.gov/ij/). Mean values were compared using Two-way (ANOVA) variance analysis followed by the post-hoc Bonferroni’s multiple comparison test. For each experiment, at least 20–30 animals were examined for each experimental condition. Each assay was repeated at least three times. We used the Prism software package (GraphPad Software) for statistical analyses. Similarly, a MitoTracker Red dye was used for organismal mitochondrial ROS quantification10. The mitophagy reporter strain N2;Ex(pmyo-3::dsred::lgg-1;pdct-1::dct-1::gfp) (a gift from Dr.Tavernarakis10) was crossed with the wrn-1(gk99), followed by evaluation of mitophagy as detailed previously21. For imaging of mitophagy signals, we randomly took over 5 images/worm with 15 worms/group/ experiment with at least two biological repeats.

Fat staining in C. elegans

Oil Red O staining was carried out as previously described with some modifications84,85. Day 7 old worms (100–200 adults) were77 washed three times with PBS then fixed in 200 μl of 1x PBS, 10% PFA and 2XMRWB (KCl 160 mM, NaCl 40 mM, NaEGTA 14 mM, 30 mM PIPES pH 7.4, 0.4 Spermine 1 mM, 1 mM Spermidine, 0.2% beta-mercaptoethanol) buffer for 1 h in an end over end mixing at room temperature. Fixed worms were washed three times with 100 mM Tris-HCl (pH 7.4). After the washes, the worms were incubated with 250 μl 40 mM DTT for 30 min at RT, followed by three washes in 1x PBS and a 15 min incubation with 70% isopropanol. Isopropanol was removed, and 1 ml of 60% Oil-Red-O dye (Sigma-Aldrich Cat. No. O9755) was added. Oil-Red-O solution was prepared by dissolving the dye in isopropanol at 5 mg/ml and equilibrating for several days. The solution was then freshly diluted with 40% water to obtain a 60% stock, allowed to sit 10 min at room temperature and filtered before using to remove insoluble material. Stained animals were incubated overnight with rocking at room temperature. The dye was then removed and 200 μl of 1x PBS with 0.01% TritonX-100 was added. Oil-Red-O absorbs light at 510 nm85. Using ImageJ we measured the average pixel intensity for a 40 pixel radius in an area behind the pharynx of each animal. A minimum of 40 animals were measured for each strain.

Mass spectrometric analysis of PAR levels

For quantitation of PAR levels in C. elegans, ~3000 worms were collected at an adult age of 7 days and snap frozen in liquid nitrogen. Thereafter, worm pellets were resuspended in 1 ml 10% TCA and subjected to five freeze-thaw cycles using liquid nitrogen and a 37 °C water bath. Then, samples were centrifuged at 3000 × g for 10 min at 4 °C. The TCA pellets were washed twice in 500 μl ice-cold 70% EtOH, air-dried, and stored at −20 °C until further processing. Afterwards PAR was purified and analyzed by mass spectrometry86,87.

Microarray using C. elegans samples

N2 and wrn-1(gk99) worms were treated with vehicle (H 2 O) or NR (1 mM final concentration) from L4 stage, followed by collection of the worm tissues on adult day 1 and adult day 7 (new drug plates were replaced with on adult day 4). The worms were then washed with M9 buffer three times and flash frozen. The samples were subjected to microarray as detailed previously21. Three independent biological samples of the worms were collected for microarray.

Electron microscopy

EM was used to examine ultrastructure of mitochondrial morphology which was performed by a US Certified Electron Microscopist Dr. J. Bernbaum. 25–30 images were randomly taken for each sample. Quantification of mitochondrial parameters in different cell types was performed using ImageJ plugin ObjectJ (length, diameter, and area). Percentage of damaged mitochondria as well as mitophagic-like events were calculated. All quantifications were performed in a double-bind manner with Two-way ANOVA used for the comparison between multiple groups.

Metabolomics

Three independent methods were applied for 3 types of purposes as summarized and detailed below. Except human blood samples (one biological sample per person), samples of three biological repeats from human cell culture or the cell culture media from the human cells were used for data collection. The descriptions of all metabolomics methods and data complies were in line with the community requirements.

For single NAD + detection in the human primary fibroblasts (Fig. 1, three biological repeats of samples) or blood samples (one biological sample per person), a commercial kit (NAD/NADH Assay Kit, Colorimetric, #ab65348) was used for NAD + detection per manufacturer’s instructions. Briefly, cells were freshly collected (5 million cells/group), quickly washed with 1xPBS (cold), followed by NAD + detection per manufacture’s protocol. All the procedures were performed on ice or at 4 °C to minimize NAD + metabolism.

For NAD + and its-related metabolites within cells, samples of three biological repeats from primary human cell culture were used for the experiments (Fig. 2 and Supplementary Fig. 3). Experiments were performed using liquid chromatography-mass spectrometry (LC-MS) 33,88 at Dr. Charles Brenner´s lab (Detailed below).

For extracellular metabolites, cell culture media were collected 24 h after primary human cell culture (three biological repeats, with data in Supplementary Fig. 4). Samples were then subjected LC-MS (including Label-free QqQ metabolomics and post-processing and bioinformatic analysis) at Dr. Costas A. Lyssiotis´s lab (Detailed below).

NAD+ and its related metabolites within cells

Liquid chromatography-mass spectrometry (LC-MS)33,88 was used. To detect intracellular NAD+ metabolites (including NMN, NAD+, NAAD, ADPR, MeNAM, NA, Inosine, IMP, NADP, cytidine, AMP, ADP, NAM etc.), cells were treated with NR for 24 h, followed by collection of cells for LC-MS as detailed elsewhere88. Internal standards: Stable isotope analogs of nucleotides and nucleoside were grown in a yeast broth with universally labeled 13C glucose, resulting in all ribose rings being fully labeled. NAAD is the only analyte of interest not having a labeled analog, 13C 10 NAD was used as its internal standard. For the second analysis a mix of 18O-NR, 18O-Nam, d 4 -NA, d 3 -MeNam, and d 3 - methyl-4-pyridone-3-carboxamide was used as internal standard. For methodology: Cell pellets were received on dry ice and stored at −80 °C. To extract metabolites both sets of internal standards were added along with 400 µL of hot ethanol/HEPES buffer as described previously, working four samples at a time. Extracted samples were stored on ice until all were complete. Samples were then heated at 55 °C with shaking for 3 min. Samples were centrifuged at 16.1 rcf, 4 °C. The supernatants were transferred to fresh microcentrifuge tubes. Calibrators and QC samples were prepared by adding both internal standards and both sets of calibrators to microcentrifuge tubes, 400 µL of hot ethanol/HEPES buffer was added and solvent for all samples, calibrators, and controls was removed in a vacuum centrifuge. For LCMS, the number of pmol of analytes in the samples were determined in two LCMS analyses, which were performed back to back. Separation and quantitation of analytes were performed with a Waters Acquity LC interfaced with a TQD mass spectrometer (Waters) operated in positive ion multiple reaction monitoring mode. NAM related analytes were analyzed using an acid separation, whereas nucleotides and most nucleosides were analyzed using an alkaline separation, each using a dedicated 2.1 × 100 mm Hypercarb column (Thermo Fisher Scientific) held at 60 °C89. The autosampler was held at 8 °C. The source temperature of the mass spectrometer was 150 °C and the desolvation temperature was 350 °C. Cone voltages and Collision energies were optimized for each MRM. The LC conditions for the NAM analysis were: Flow 0.20 mL/min, mobile phase “C” 10 mM ammonium acetate + 0.1% formic acid; mobile phase “D” acetonitrile + 0.1% formic acid. For the NAD run the flow rate was 0.353 mL/min, mobile phase “C” 7.5 mM ammonium acetate + 0.05% ammonium hydroxide; mobile phase “D” acetonitrile + 0.05% ammonium hydroxide. The gradients were:

NAM NAD Time (min) % C % D Time (min) % C % D 0 95 5 0 97 3 1.8- 95 5 1.8- 97 3 11 60 40 14 50 50 11.3- 60 40 14.1 10 90 14.3- 10 90 16.2- 10 90 14.4 95 5 17.1 97 3 20 95 5 22 97 3

Extracellular metabolites

To detect extracellular metabolites, cells were treated with NR for 24 h followed by collection of cell culture media. Cell culture media from three biological repeats/group was used to run LC-MS (including Label-free QqQ metabolomics and post-processing and bioinformatic analysis). Plain MEM media (incubated that had been in cell culture incubator for 24 h) was used as internal background control. Agilent 1290 UHPLC and 6490 Triple Quadrupole (QqQ) Mass Spectrometer (LC-MS) were used in this study. Agilent MassHunter Optimizer and Workstation Software LC/MS Data Acquisition for 6400 Series Triple Quadrupole B.08.00 was used for standard optimization and data acquisition. Agilent MassHunter Workstation Software Quantitative Analysis Version B.0700 for QqQ was used for data analysis. For reversed-phase chromatography (RPC), a Waters Acquity UPLC BEH TSS C18 column (2.1 × 100 mm, 1.7 μm) was used with mobile phase (A) consisting of 0.5 mM NH4F and 0.1% formic acid in water; mobile phase (B) consisting of 0.1% formic acid in acetonitrile. Gradient program: mobile phase (B) was held at 1% for 1.5 min, increased to 80% in 15 min, then to 99% in 17 min and held for 2 min before going to initial condition and held for 10 min. For hydrophilic interaction chromatography (HILIC), a Waters Acquity UPLC BEH amide column (2.1 × 100 mm, 1.7 μm) was used with mobile phase (A) consisting of 20 mM ammonium acetate, pH 9.6 in water; mobile phase (B) consisting of acetonitrile. Gradient program: mobile phase (B) was held at 85% for 1 min, decreased to 65% in 12 min, then to 40% in 15 min and held for 5 min before going to initial condition and held for 10 min. Both columns were at 40 °C and 3 μl of each sample was injected into the LC-MS with a flow rate of 0.2 ml/min. Calibration of TOF MS was achieved through Agilent ESI-Low Concentration Tuning Mix. Optimization was performed on the 6490 QqQ in the positive or negative mode for the RPC or HILIC respectively for each of 220 standard compounds to get the best fragment ion and other MS parameters for each standard. Retention time for each of 220 standards was measured from a pure standard solution or a mix standard solution. The LC-MS/MS method was created with dynamic MRMs with RTs, RT windows and MRMs of all 220 standard compounds. Key parameters of AJS ESI in both the positive and the negative acquisition modes are: Gas temp 275 ̊C, Gas Flow 14 l/min, Nebulizer at 20 psi, SheathGasHeater 250 ̊C, SheathGasFlow 11 l/min, and Capillary 3000 V. For MS: Delta EMV 200 V or 350 V for the positive or negative acquisition mode respectively and Cycle Time 500 ms and Cell Acc 4 V for both modes. The QqQ data was pre-processed with Agilent MassHunter Workstation Software Quantitative Analysis and post-processed for further quality control in the programming language R. We calculated coefficient of variation (CV) across replicate samples for each metabolite given a cut-off value of peak areas in both the positive and the negative modes. We then compared distributions of CVs for the whole dataset for a set of peak area cut-off values of 0, 1000, 5000, 10,000, 15,000, 20,000, 25,000, and 30,000 in each mode. A noise cut-off value of peak areas in each mode was chosen by manual inspection of the CV distributions: 5000 for the positive mode and 5000 for the negative mode. Each sample is then normalized by the total intensity of all metabolites to reflect the same protein content as a normalization factor. We then retained only those metabolites with at least 2 replicate 6 measurements. The remaining missing value in each condition for each metabolite was filled with the mean value of the other replicate measurements. Finally, the abundance of each metabolite in each sample was divided by the median of all abundance levels across all samples for proper comparisons, statistical analyses, and visualizations among metabolites. The statistical significance test was done by a two-tailed t-test with a significance threshold level of 0.1. The p-values were not adjusted in favor of subsequent manual inspection and more flexible biological interpretation. Those metabolites with p-value < 0.1 and CV < 1 were defined to be differential metabolites. Pathway analysis of differential metabolites was done using the webtool of MetaboAnalyst with default settings (metaboanalyst.ca). All other bioinformatics analyses including graphs and plots were also done using R/Bioconductor.

Whole worm body proteomics

For the whole worm body proteomics, samples of four biological repeats were collected followed by data generation and quantification using methods reported previously21. Both control and drug treated worms were cultured in the presence of 5-Fluoro-deoxyuridine (5-FdUrd, 50 µM) to prevent development of progeny in order to produce a synchronized population. L4 stage N2 and wrn-1 worms were treated with NR (1 mM) for up to 7 days. Subsequently, protein extractions (with extraction buffer 40 mM Tris-HCl pH7.4, 150 mM NaCl, 0.01%, NP-40, 2 mM EDTA; plus protease inhibitors) were performed from four independent experiments. Protein concentration is perfomred with Xpose according to manufactures instructions. Protein extracts (25 μg) were precipitated with 400 μl of ice-cold acetone at −20 °C. After overnight precipitation, samples were centrifuged at 16,000 g for 30 min at 4 °C and the supernatant was discarded. Proteins were re-dissolved in 50 μL of 6 M urea in 50 mM ammonium bicarbonate, pH 7.8. Subsequently, 2.5 μL of 200 mM DTT (Sigma-Aldrich, Norway) was added and the samples were incubated for 30 min at 30 °C to reduce the disulfide bridges. Thiols were then alkylated with 7.5 μL of 200 mM iodoacetamide (IAA Sigma-Aldrich, Oslo, Norway) (1 h, 30 °C, dark), following which the excess of IAA was quenched with 10 μL of 200 mM DTT (30 min, 30 °C). After reducing the urea concentration down to 1 M, proteins were digested with sequencing grade modified trypsin (Promega, Madison, WI, USA) for 1 h at 37 °C, followed by 15 h incubation at 30 °C. The digestion was terminated by adding 5 μL of 50% formic acid and the generated peptides were purified using a Strata C18-E SPE column (Phenomenex, Værløse, Denmark), and dried using a Speed Vac concentrator (Eppendorf, Hamburg, Germany). Tryptic digests from four replicates of each sample were analyzed using an Ultimate 3000 nano-UHPLC system (Dionex, Sunnyvale, CA, USA) connected to a Q Exactive mass spectrometer (ThermoElectron, Bremen, Germany) equipped with a nano electrospray ion source. For liquid chromatography separation, an Acclaim PepMap 100 column (C18, 3 μm beads, 100 Å pore size, 75 μm inner diameter, 50 cm length) was used. A flow rate of 300 nL/min was employed with a solvent gradient of 4–35% B in 207 min, to 50% B in 20 min and then to 80% B in 2 min. Solvent A was aqueous 0.1% formic acid and solvent B contained 0.1% formic acid in 90% acetonitrile. The mass spectrometer was operated in the datadependent mode to automatically switch between MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 300 to 2000) were acquired in the Orbitrap with resolution R = 70,000 at m/z 200, after accumulation to a target of 1,000,000 ions in the C-trap. The maximum allowed ion accumulation times were 100 ms. The method used allowed sequential isolation of up to the ten most intense ions, depending on signal intensity (intensity threshold 1.7e4), for fragmentation using higher collision induced dissociation (HCD) at a target value of 1e5 charges and a resolution R = 17,500. Target ions already selected for MS/MS were dynamically excluded for 60 s. The isolation window was 2 m/z units without offset. The maximum allowed ion accumulation for the MS/MS spectrum was 60 ms. For accurate mass measurements, the lock mass option was enabled in MS mode and the polydimethylcyclosiloxane ions generated in the electrospray process from ambient air were used for internal recalibration during the analysis (with protocol from Fang et al.21).

For data analysis, raw data were acquired using Xcalibur v2.5.5 and raw files were processed to generate peak list in Mascot generic format (.mgf) using ProteoWizard release version 3.0.331. Database searches were performed using Mascot in-house version 2.4.0 to search the SwissProt database (Caenorhabditis elegans, 19.05.2015, 26156 proteins) assuming the digestion enzyme trypsin, at maximum one missed cleavage site, fragment ion mass tolerance of 0.02 Da, parent ion tolerance of 10 ppm and oxidation of methionine, acetylation of the protein Nterminus as variable modifications and carbamidomethylation of cysteine as fixed modification. Scaffold (version Scaffold_4.3.4, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at >99.0% probability.

Real-time PCR

Cells were treated with/without NR (1 mM) for 24 h. Total RNA was extracted with TriZol (Invitrogen, Carlsbad, CA, USA) reagent, reverse-transcribed using iScript cDNA Synthesis Kit (BIO-RAD, Hercules, CA, USA). mRNA levels were quantified by real-time PCR using a SYBR Green quantitative PCR kit (Thermo Fisher Scientific, Waltham, MA USA) on the MyiQ iCycler real-time PCR detection system (BIO-RAD, Hercules, CA, USA), and then normalized to GAPDH using the 2−ΔΔCT calculation method90. Primer sequences used in this study are as follows: NMNAT1 forward, 5′-TCTCCTTGCTTGTGGTTCATTC-3′ and reverse, 5′-TGACAACTGTGTACCTTCCTGT-3′; GAPDH forward, 5′-GAGTCAACGGATTTGGTCGT-3′ and reverse, 5′-GACAAGCTTCCCGTTCTCAG-3′91. For the real-time PCR in worms, we collected 50 worms/group and performed PCR using samples from three biological replicates. Primers used for the PCR were detailed elsewhere10,37. Values are the means of at least three independent experiments, and standard deviations are indicated as error bars.

Western blots

Western blotting was used to examine protein expression following methods detailed previously21. Briefly, primary human cells (HT01, WRN-Kd, WS01 cells) were collected and prepared using 1x RIPA buffer (Cell Signaling, #9806 S) containing protease inhibitors (Bimake, #B14002) and phosphatase inhibitors (Bimake, #B15002). Proteins were separated on 4–12% Bis-Tris gel (ThermoFisher Scientific, #NP0336BOX) and probed with antibodies. Chemiluminescence detection was performed using a ChemiDoc XRS System. Antibodies used were: β-actin (Santa Cruz, #sc-1616), WRN (Santa Cruz, # sc-5629), CD38 (Santa Cruz, # sc-374650), CD157 (R&D systems, #AF4736), CD73 (R&D systems, #AF5795), PAR (TREVIGEN, #4336-BPC-100), PARP1 (Cell signaling, #9542), AMPK (Cell signaling, #5831), pAMPK (Thr172) (Cell signaling, #2535), pULK1 (Ser555) (Cell signaling, #5869), ULK1 (Cell signaling, #6439), p62 (Cell signaling, #39749), Bcl2L13 (ThermoFisher, # PA5–15043), LC3 (Novus, #NB100–2220), PSD95 (Cell signaling, #3450). All other antibodies were obtained from Cell signaling. Gamma adjustment was used to reduce dark background when necessary. Quantification was performed using ImageJ. Unless elsewhere stated, all the 1st antibodies were with 1000× dilution, while 2nd antibodies were with 10,000× dilution. The original western blots were in the Source Data file with molecular markers labeled.

Data collection and statistical analysis

Double-blinded methods were used in the C. elegans studies (including lifespan, healthspan) and experiments requiring imaging. We used two-tailed unpaired t-test for comparison between two groups, or One-Way ANOVA or Two-way ANOVA (followed by Tukey’s test) for comparison among multiple groups. All data were presented as mean ± S.E.M. as indicated with *p value <0.05 considered statistically significant. For lifespan studies, p values were derived from log-rank calculations.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.