Research Article

NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice

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Science  17 Jun 2016:
Vol. 352, Issue 6292, pp. 1436-1443
DOI: 10.1126/science.aaf2693
  • Fig. 1 Mitochondrial dysfunction in MuSCs during aging.

    (A) GSEA demonstrates up- and down-regulated signaling pathways in MuSCs from 2-year-old mice, as compared with 3-month-old mice. This panel shows the results of our analysis of microarray data from the publicly available GEO data set (accession number GSE47177), using Kyoto encyclopedia of genes and genomes (KEGG) enrichment. Signaling pathways were ranked on the basis of normalized enrichment scores (NESs); positive and negative NESs indicate down- or up-regulation, respectively, in aged MuSCs. Specific pathways related to MuSC function are highlighted in red and blue. TGFβ, transforming growth factor–β. (B) Area-proportional Venn diagram representing 113 common genes between the significantly down-regulated genes (P < 0.05) in MuSC transcriptomes originating from aged mice [GSE47177 and GSE47401 (12)] and genes from the human mitochondrial transcriptome (26). (C to G) Young (1 month old) and aged (22 to 24 months old) C57BL/6J mice received a dietary supplement with NR for 6 weeks. (C) Quantitative real-time fluorescence polymerase chain reaction validation of transcriptional changes in mitochondrial genes of freshly sorted MuSCs. (D) Oxygen consumption rate (OCR) in freshly isolated MuSCs after 16 hours of recovery at 37°C. Pmole, picomoles. (E and F) Mitochondrial membrane potential, as measured by a tetramethylrhodamine, methyl ester (TMRM) assay (E) and cellular ATP levels (F) in freshly isolated MuSCs. (G) Relative gene expression for UPRmt genes and cell senescence markers in freshly sorted MuSCs. Data are normalized to 36b4 mRNA transcript levels. All statistical significance was calculated by Student’s t test. All data are shown as mean ± SEM (error bars). *P < 0.05; **P < 0.01; ***P < 0.001. In (C), (D), (F), and (G), n = 6 mice per group; in (E), n = 3 mice per group.

  • Fig. 2 Improved MuSC numbers and muscle function in NR-treated aged mice.

    Young (1 month old) and aged (22 to 24 months old) C57BL/6J mice received a chow diet (CD) or a CD supplemented with NR for 6 weeks. All results are compared with those of age-matched mice given a control diet. (A) NAD+ concentrations in freshly isolated MuSCs. (B and C) Percentage of fluorescence-activated cell sorting (FACS)–quantified CD34+/integrin α7+/Lin/Sca-1 MuSCs relative to the total Lin/Sca-1 cell population (B) or to muscle weight (C). (D) Representative images of PAX7- and laminin-immunostained tibialis anterior (TA) muscle. Arrows point to PAX7-positive SCs. Insets (20 μm by 20 μm) show a single MuSC. Scale bar, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (E to G) Comparison of maximal running duration (E), running distance (F), and grip strength (G) in NR-treated aged mice. (H) Hematoxylin and eosin (H&E)–stained TA tissue sections from NR-treated aged mice 7 and 14 days after CTX-induced muscle damage. Scale bar, 100 μm. (I) Images of PAX7- and laminin-immunostained TA muscle cross sections taken from NR-treated aged mice 7 days after CTX-induced muscle damage. Arrows point to PAX7-positive MuSCs. Insets (20 μm by 20 μm) show a single MuSC. Scale bar, 50 μm. (J) Quantification of MYOD1 and PAX7–double positive to PAX7-positive myofibers, performed on sections isolated 7 days after muscle damage in aged mice. (K) Newly regenerated muscle fibers stained by embryonic myosin heavy chain (eMyHC) 7 days after muscle damage in aged mice. Scale bar, 50 μm. (L) Dystrophin immunostaining of TA muscle sections in aged (16 months old) mdx mice 4 weeks after receiving transplantations of MuSCs isolated from control or NR-treated aged C56BL/6J donors. Scale bar, 100 μm. All statistical significance was calculated by Student’s t test or two-way ANOVA. All data are shown as mean ± SEM (error bars). *P < 0.05; **P < 0.01; ***P < 0.001. Main effects for treatment or age are denoted with † or ‡ symbols, respectively. † or ‡, P < 0.05; ††† or ‡‡‡, P < 0.001. In (A), n = 6 mice per group; in (B) to (D) and (H) to (K), n = 3 to 6 mice per group; in (E) to (G), n = 10 mice fed the control diet and 7 NR-treated mice; in (L), n = 12 donor mice and 3 recipient mice for each treatment. For (E) to (J), corresponding young control data can be found in fig. S2, J to O, respectively.

  • Fig. 3 NR treatment prevents MuSC senescence and improves mitochondrial function.

    Aged (22 to 24 months old) C57BL/6J mice or 8-month-old SIRT1MuSC−/− mice received a dietary supplement with NR for 6 weeks. All isolated MuSCs were freshly FACS sorted for assay. Most comparative data from young mice (1 month old) are presented in fig. S3. (A and B) Immunostaining (A) and quantification (B) of γH2AX staining in freshly sorted MuSCs from aged mice. Insets (20 μm by 20 μm) show single MuSCs. (C) Single-cell gel electrophoresis (comet) assay of MuSCs from aged mice. C, chow diet; N, NR treated; NDD, nondamaged DNA; MDD, moderately damaged DNA; HDD, heavily damaged DNA. (D) Proteins levels in C2C12 myoblasts after NR treatment for 1, 3, or 6 hours. (E) Colony-formation ability assay in isolated MuSCs. (F and G) Quantification of transcript expression for cell cycle and inflammatory secretome genes (F) or OXPHOS and TCA cycle genes (G) in MuSCs. (H) Abundance of proteins from MuSCs of young (Y) and aged (A) mice fed with a chow (C) or NR (N) diet. Protein abundances were calculated using the intensities of peptides detected in the SWATH-MS maps. Roman numerals indicate corresponding OXPHOS complexes. T, TCA cycle. (I) Protein levels in MuSCs. (J and K) OCR (J) and extracellular acidification rate (ECAR) (K), in MuSCs after 16 hours of recovery at 37°C. (L) Mitochondrial membrane potential, as measured by a TMRM assay in MuSCs. (M) Cellular ATP concentration in MuSCs. (N) H&E stained TA muscle from wild-type (WT) or SIRT1MuSC−/− mice 7 days after CTX-induced muscle damage. Scale bar, 100 μm. (O to Q) Representative images (O) and quantification of PAX7-positive MuSCs in random fields of view (160 μm by 160 μm) (P) and the percentage of SIRT1-positive MuSCs (Q) in immunostained TA 7 days after CTX-induced muscle damage. Arrows in (O) point to PAX7-positive MuSCs. Insets (20 μm by 20 μm) show a single MuSC. Scale bar, 50 μm. (R) Quantification of γH2AX-positive MuSCs in immunostained TA 7 days after CTX-induced muscle damage. All statistical significance was calculated by Student’s t test or two-way ANOVA. All data are represented as mean ± SEM (error bars). *P < 0.05; **P < 0.01; ***P < 0.001. Main effects for treatment or age are denoted as † or ‡, respectively, with interactions denoted as ε. † or ‡, P < 0.05; †† or ‡‡, P < 0.01; ††† or ‡‡‡, P < 0.001. In (A) to (C) and (N) to (R), n = 3 to 6 mice per group; in (E), n = 24 repeats per group; in (F) and (G) and (J) to (M), n = 6 mice per group; in (H), protein was extracted and pooled from 6 mice in each group. For (A), (B), (E), (F), (G), and (L), corresponding young control data can be found in fig. S3, A, B, D, E, F, and G, respectively.

  • Fig. 4 Effects of NR on prohibitins, UPRmt, and MuSC senescence.

    (A) Expression of HSP60, CLPP, and prohibitins in C2C12 myoblasts upon NR treatment at the indicated time points. (B) Quantification of transcript expression for UPRmt and prohibitin genes in MuSCs from aged (22 to 24 months old) C57BL/6J mice after 6 weeks of chow or NR diets. (C) Expression of prohibitins and cell cycle proteins in C2C12 myoblasts with the combined overexpression of Phb and Phb2. (D) Expression of prohibitins and cell cycle genes with a 6-hour NR treatment in C2C12 myoblasts after a combined Phb and Phb2 short hairpin RNA knockdown. (E) H&E staining of TA muscle in NR-treated or intramuscular shPhb lentivirus–injected C57BL/6J mice 7 days after CTX-induced muscle damage. Scale bar, 100 μm. (F to H) Representative images (F) and quantification of PAX7-positive MuSCs in randomly chosen fields of view (160 μm by 160 μm) (G) and the percentage of PHB-positive MuSCs (H) in immunostained TA muscle 7 days after CTX-induced muscle damage. Arrows in (F) point to PAX7-positive MuSCs. Insets (20 μm by 20 μm) show a single MuSC. Scale bar, 50 μm. (I) Quantification of γH2AX-positive MuSCs in immunostained TA muscle cross sections taken from control and NR-treated mice 7 days after CTX-induced muscle damage. All statistical significance was calculated by Student’s t test or two-way ANOVA. All data are represented as mean ± SEM (error bars). *P < 0.05; **P < 0.01; ***P < 0.001. Main effects for treatment or Phb knockdown are denoted as † or ‡, respectively. † or ‡, P < 0.05; ††† or ‡‡‡, P < 0.001. In (B), n = 6 mice per group; in (E) to (I), n = 3 mice per group. For (B), corresponding young control data can be found in fig. S4B.

  • Fig. 5 Increased SC number and function in NR-treated mdx mice.

    mdx mice received a dietary supplement with NR for 10 weeks. All results were compared with those of mdx mice given a control diet. (A) β-Gal staining of MuSCs isolated from C57BL/10SnJ or mdx mice and cultured in vitro for three generations. Scale bar, 10 μm. (B to D) FACS contour plots of Sca-1/Lin cells isolated from muscle. Percentages of the CD34+/integrin α7+/Lin/Sca-1 MuSC populations are noted in red in the contour plots (B) and are quantified relative to the total Lin/Sca-1 cell population (C) or muscle weight (D). (E) Immunostaining of eMyHC+ fibers in tissue sections of NR-treated mdx mice 7 days after CTX-induced muscle damage. Scale bar, 100 μm. (F to H) FACS contour plots (F), quantification (G), and distribution (H) of MuSC autofluorescence as a measure of the relative NAD(P)H concentration (where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate) upon ultraviolet light excitation. Autofluorescence emission was detected with a wavelength of 450 nm. The arrowhead in (H) points to the highly autofluorescent SC population. (I) Quantification of β-Gal staining of FACS-sorted MuSCs from C57BL/6J (B6 with CD), untreated (mdx with CD), or NR-treated mdx (mdx with NR) mice challenged with phosphate-buffered saline or NR for 6 hours in vitro. (J) Immunostaining showing γH2AX and cleaved caspase-3 in MuSCs cultured in vitro for three generations. The arrow points to a γH2AX-positive nucleus. Scale bar, 10 μm. (K) H&E staining of tissue sections from NR-treated aged mdx mice (16 months old) with 7 days of recovery after CTX-induced muscle damage. Scale bar, 100 μm. All statistical significance was calculated by Student’s t test or one-way ANOVA. All data are represented as mean ± SEM (error bars). *P < 0.05; **P < 0.01; ***P < 0.001. In (A) to (H), (J), and (K), n = 3 to 5 mice per treated group; in (I), n = 3 mice and n = 6 in vitro treatments. More than 500 cells were quantified for each condition.

  • Fig. 6 NR improves NSC and McSC function and increases the life span of aged C57BL/6J mice.

    Aged (22 to 24 months old) C57BL/6J mice received a dietary supplement with NR for 6 weeks. (A and B) Representative images (A) and quantification (B) of EdU-positive NSCs in the SVZ from young and aged mice after NR treatment. Scale bar, 50 μm. (C and D) Representative images (C) and quantification (D) of Ki67- and DCX-positive NSCs in the SVZ, harvested from young and aged mice treated with or without NR. Arrows in (C) point to Ki67-positive NSCs. Scale bar, 50 μm. (E and F) Representative images (E) and quantification (F) of c-KIT and TRP2 double-positive McSCs in the bulge of hair follicles from dorsal skin, harvested from young and aged mice treated with or without NR. Arrows in (E) point to double-positive McSCs. Scale bar, 50 μm. (G) Kaplan-Meier survival curves of control and NR-treated aged mice, with the NR treatment beginning at 2 years (700 days) of age. (H) Hazard rate decreased under NR treatment. Individual life spans were collected and used to estimate the hazard function of each population, using numerical differentiation of the Kaplan-Meier survival estimator (solid lines). The shaded areas represent the 95% confidence bands of the true hazard. P values were calculated with the use of a Cox proportional hazards model. All statistical significance was calculated by Student’s t test or two-way ANOVA, except in (G) and (H). All data are represented as mean ± SEM (error bars). *P < 0.05; **P < 0.01; ***P < 0.001. Main effects for treatment or age are denoted as † or ‡, respectively. †† or ‡‡, P < 0.01; ††† or ‡‡‡, P < 0.001. In (A) to (F), n = 6 mice per group; in (H), n = 30 mice per treated group.

Supplementary Materials

  • NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice

    Hongbo Zhang, Dongryeol Ryu, Yibo Wu, Karim Gariani, Xu Wang, Peiling Luan, Davide D'Amico, Eduardo R. Ropelle, Matthias P. Lutolf, Ruedi Aebersold, Kristina Schoonjans, Keir J. Menzies,Johan Auwerx

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S6
    • Captions for Tables S1 to S6
    • Full Reference List
    Table S1
    GSE47177_KEGG analysis of genes increase and decrease expression in aged muscle stem cell.
    Table S2
    GSE47104_KEGG analysis of genes increase and decrease expression in aged muscle stem cell.
    Table S3
    GSE47401_KEGG analysis of genes increase and decrease expression in aged muscle stem cell.
    Table S4
    Lists of down- and upregulated genes in MuSC aging.
    Table S5
    List of Primers for qRP-PCR
    Table S6
    SWATH-MS quantification of proteins from freshly isolated MuSCs

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