Autophagy maintains stemness by preventing senescence

Journal name:
Nature
Volume:
529,
Pages:
37–42
Date published:
DOI:
doi:10.1038/nature16187
Received
Accepted
Published online

Abstract

During ageing, muscle stem-cell regenerative function declines. At advanced geriatric age, this decline is maximal owing to transition from a normal quiescence into an irreversible senescence state. How satellite cells maintain quiescence and avoid senescence until advanced age remains unknown. Here we report that basal autophagy is essential to maintain the stem-cell quiescent state in mice. Failure of autophagy in physiologically aged satellite cells or genetic impairment of autophagy in young cells causes entry into senescence by loss of proteostasis, increased mitochondrial dysfunction and oxidative stress, resulting in a decline in the function and number of satellite cells. Re-establishment of autophagy reverses senescence and restores regenerative functions in geriatric satellite cells. As autophagy also declines in human geriatric satellite cells, our findings reveal autophagy to be a decisive stem-cell-fate regulator, with implications for fostering muscle regeneration in sarcopenia.

At a glance

Figures

  1. Altered basal autophagy in ageing muscle stem cells.
    Figure 1: Altered basal autophagy in ageing muscle stem cells.

    a, Number and area of punctate GFP–LC3 in quiescent satellite cells. Arrowheads, autophagic vesicles. b, Autophagy flux in cells from a. Cells were treated with vehicle or bafilomycin (+Baf) for 4 h before analysis. The change in mean fluorescence intensity (MFI) of GFP–LC3 in cells treated with bafilomycin is shown. c, Autophagy flux in quiescent satellite cells from old GFP–LC3 mice treated for two weeks with rapamycin or vehicle control. Satellite cells ± bafilomycin treatment as in b. d, p62 and ubiquitin (Ub) MFI from cells of the GFP–LC3 mice treated as in c. AU, arbitrary units. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. Sample numbers were n = 51 (young), n = 106 (old) cells analysed from 3 animals for a; n = 60,000 cells from 3 animals for b; n = 60,000 cells from 3 animals for c; n = 36 (control), 39 (rapamycin treated) cells from 3 animals for d. The z projections of representative images are shown. Scale bars are all 1.5 μm, apart from 5 μm for c.

  2. Defective autophagy causes numerical and functional satellite cell decline in ageing.
    Figure 2: Defective autophagy causes numerical and functional satellite cell decline in ageing.

    a, The mRFP–GFP–LC3 plasmid was transfected into young (3 months), old (24 months) and geriatric (28 months) satellite cells to enable the detection of autophagosomes (yellow) and their maturation into autolysosomes (red) in the presence or absence of bafilomycin treatment (as in Fig. 1b). The graph indicates the percentage of double-positive puncta (RFP+/GFP+) (autophagosomes) out of total puncta (RFP+/GFP+, RFP+, autophagosomes and autolysosomes). b, Quantification of p62 and ubiquitin aggregates in quiescent satellite cells from a. Co-localization staining area with respect to total cellular area. Pearson’s coefficient (r) indicates the correlation of intensity values of red and green pixels in dual-channel images. Arrowheads indicate co-localization. c, An equal number of LV-GFP-infected satellite cells from young or geriatric mice, treated for 48 h with rapamycin or LV-Atg7 infected (or controls), were transplanted into an injured mouse muscle, and analysed 4 or 28 days later (for analysis on day 28, see Extended Data Fig. 2i, j). Analysis on day 4 of GFP and Pax7 immunostaining. Quantification of GFP+ cells per muscle field. Values relative to transplanted young cells (100%). Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. The sample numbers were n = 21 (young), n = 19 (young, +bafilomycin), n = 30 (old), n = 15 (old, +bafilomycin), n = 21 (geriatric) and n = 15 (geriatric, +bafilomycin) cells analysed from 3 animals for a; n = 35 (young), n = 66 (old) and n = 104 (geriatric) cells from 3 animals for b; n = 5 engraftments per group for c. Representative images are shown. The z projections of representative images are shown. Scale bars are all 5 μm, apart from 50 μm for c.

  3. Genetic impairment of autophagy disrupts satellite cell homeostasis.
    Figure 3: Genetic impairment of autophagy disrupts satellite cell homeostasis.

    a, Satellite cell quantification by analysis of Pax7 immunostaining of muscles in three-month-old Atg7WT and Atg7ΔPax7 mice. b, Schematic of the mouse tamoxifen (Tmx) treatment and satellite cell analysis. Satellite cell quantification in Atg7WT/Atg7ΔPax7ER mice on day 30 after tamoxifen treatment as in a. c, RT–qPCR of senescence markers (7 days after tamoxifen treatment) in cells from b. d, The percentage of co-localizing γH2AX+ cells out of the total Pax7+ cells in a. e, f, Quantification of BrdU+ (e) and SA-β-gal+ (f) cells isolated from mice in a. The arrowhead indicates positive staining. g, Western blotting of cells from a; for a full scan of the gel see Supplementary Fig. 1. pS6, phosphorylated S6. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. Sample numbers were: n = 5 animals per group for a; n = 3 animals per group for bg. Representative images are shown. The z projections of representative images are shown. Scale bars, 250 μm.

  4. Autophagy loss results in mitochondrial dysfunction and accumulation of organelles, proteins and ROS.
    Figure 4: Autophagy loss results in mitochondrial dysfunction and accumulation of organelles, proteins and ROS.

    a, Lysosomal (Lamp1 and LysoTracker) and mitochondrial (MitoTracker) quantification in satellite cells from Atg7WT and Atg7ΔPax7ER mice, one month after tamoxifen treatment. Membrane potential monitored as the mean fluorescent intensity ratio of TMRM to MitoTracker Green. b, Similar quantification as in a for young and old satellite cells. Membrane potential analysis as in a. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. The sample numbers were n = 60,000 cells analysed from 3 animals for a and b. Representative images are shown. The z projections of representative images are shown. Scale bars, 5 μm.

  5. ROS inhibition prevents senescence in aged satellite cells.
    Figure 5: ROS inhibition prevents senescence in aged satellite cells.

    a, ROS quantification in young and old satellite cells by CellROX flow cytometry. b, Mitochondria (MitoTracker) and ROS (CellROX) in satellite cells, ± 24 h bafilomycin treatment. Results represent increased MFI in the presence of bafilomycin. Representative images of young satellite cells. c, RT–qPCR of senescence markers ± Trolox. d, Quantification of BrdU+ and SA-β-gal+ cells from c, pretreated with Trolox (or controls that were not treated with Trolox), and cultured for 96 h. e, Western blotting in cells ± Trolox; for full-scan of gel see Supplementary Fig. 1. f, Geriatric cells ± Trolox 48 h pre-treatment, were transplanted and analysed as in Fig. 2c. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-tests. Sample numbers were n = 60,000 cells analysed from 3 animals for a and b; n = 3 animals per group for ce; n = 4 engraftments per group for f. Representative images are shown. The z projections of representative images are shown. Scale bars are all 5 μm apart from 50 μm for f.

  6. Epigenetic control of p16INK4a expression by ROS in autophagy-impaired satellite cells.
    Figure 6: Epigenetic control of p16INK4a expression by ROS in autophagy-impaired satellite cells.

    a, Chromatin immunoprecipitation (ChIP) for K119 ubiquitination of H2A (H2Aub) in geriatric satellite cells, in the presence or absence of 48 h of Trolox treatment. RD, regulatory domain. b, H2Aub-ChIP for Atg7WT and Atg7ΔPax7 satellite cells treated as in a. c, Western blotting for cells in b; for full gel scan see Supplementary Fig. 1. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values indicated. Sample numbers were n = 3 animals per group for ac.

  7. The reduced autophagy flux in quiescent satellite cells can be increased by pharmacological treatment in vivo.
    Extended Data Fig. 1: The reduced autophagy flux in quiescent satellite cells can be increased by pharmacological treatment in vivo.

    a, Venn diagrams of overlapping genes between a proteostasis gene set (See Supplementary Table 1) and genes significantly upregulated in quiescent satellite cells from the indicated publications or from our gene expression microarray data comparing freshly FACS isolated satellite cells from resting muscle, or muscles obtained 72 h after cardiotoxin (CTX) injury, from young, wild-type mice. b, K-means clustering analysis (performed with Gene-E, Broad Institute) of the gene expression of the autophagy-related genes during ageing. Clusters are shown with heat maps of the normalized raw data. Each column represents a different sample and each row a different gene probe. Red, increased expression; white, neutral expression; blue, decreased expression. c, Representative example of the FACS strategy and gating scheme for isolating satellite cells from mice in resting conditions. d, Pax7 and GFP immunostaining of freshly isolated satellite cells from resting muscles of young and old GFP–LC3 mice. Scale bar, 5 μm. e, Electron microscopy images of young and old satellite cells on sections of resting tibialis anterior (TA) muscle of wild-type (WT) mice. Arrowheads indicate autophagic vesicles. Scale bars, 1 μm and 0.5 μm (right and left, respectively). f, Pax7 and GFP immunostaining on tissue sections from resting tibialis anterior muscles of young and old GFP–LC3 mice. Arrowheads indicate autophagic vesicles. Scale bar, 5 μm. g, p62 and ubiquitin (Ub) MFI. Arrowheads, co-localization of p62 and ubiquitin aggregates. h, LC3 western blot of freshly isolated satellite cells from young and old, wild-type mice, treated with bafilomycin or vehicle for 4 h before collection. Graph shows LC3II quantification, after normalization with tubulin levels; for full scan see Supplementary Fig. 2. i, Quiescent satellite cells were freshly isolated from old, wild-type mice subjected to two weeks of rapamycin, spermidine or vehicle (control) treatment. Cells were treated (or not treated) with bafilomycin 4 h prior to analysis by immunostaining of LC3 marker. Z projections of representative fluorescence microscopy images are shown. Scale bars, 5 μm. j, Representative fluorescent microscopy images from Fig. 1d. Scale bar, 5 μm. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. Number of samples were n = 3 animals per group for a and b; n = 35 (young) and 66 (old) cells analysed from 3 animals for g; n = 3 animals per group for h.

  8. Reinduction of autophagy rescues proliferation and reduces senescence in geriatric satellite, thus restoring regenerative capacity.
    Extended Data Fig. 2: Reinduction of autophagy rescues proliferation and reduces senescence in geriatric satellite, thus restoring regenerative capacity.

    a, Transplanted muscles from Fig. 2c were immunostained for GFP and for Ki67, Pax7, MyoD or Mgn (to determine the distinct possible myogenic states of satellite cells in the regenerating muscle). Scale bars, 50 μm. b, Autophagy flux analysed by flow cytometry in freshly isolated satellite cells from resting muscle of GFP–LC3 mice, treated for 48 h with rapamycin or vehicle (control). Satellite cells were treated with bafilomycin or vehicle for 4 h before analysis. Results are expressed as the change in GFP–LC3 MFI in bafilomycin (−) compared to bafilomycin (+) conditions. c, Western blot analysis of pS6 protein levels in geriatric satellite cells from wild-type mice, treated for 48 h with rapamycin or vehicle (control). Graph shows pS6 quantification, normalized to tubulin; for full scan see Supplementary Information Fig. 2. d, As in Fig. 2c, percentage γH2AX+ or p16INK4a+cells from total GFP+ cells were quantified. Scale bars, 10 μm. e, Quantification BrdU+ and senescence-associated β-gal+ satellite cells, pre-treated as in Fig. 2c and analysed after 96 h. f, Quantification of senescent (senescence-associated β-gal+) satellite cells, isolated from young and geriatric wild-type mice, pre-treated for 48 h with spermidine or vehicle (control) and cultured for 96 h. g, Quantitative real-time PCR (RT–qPCR) analysis of Atg7 expression on satellite cells infected with LV-Atg7 or LV-control (LV-Co), and cultured for 96 h. h, GFP–LC3 satellite cells were infected with LV-Atg7 or LV-Co and treated with bafilomycin or vehicle for 4 h before analysis. Autophagy flux was analysed by flow cytometry and represented as in b. Representative images are shown. Scale bar, 10 μm. i, Muscle regeneration experiment by satellite cell transplantation. An equal number of satellite cells from young and geriatric mice infected with a lentivirus overexpressing the Atg7 gene (LV-Atg7) or a lentivirus control (LV-Co), which also expressed GFP, were transplanted into injured muscle of young immunodeficient mice, and collected 28 days later. GFP expression in muscles was analysed by immunostaining. Quantification of GFP+ cells (fibres) per muscle field versus transplanted control-treated satellite cells. Representative images are shown. Scale bar, 75 μm. j, EDL geriatric muscles, infected with LV-Atg7 or LV-Co, and grafted on recipient mouse muscle. Regeneration was analysed 8 days later. Frequency distribution of regenerating fibres by size. Scale bar, 25 μm. Data are mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. Number of samples were n = 60,000 cells analysed from 3 animals for b; n = 3 animals per group for c; n = 5 engraftments per group for d; n = 3 animals per group for eg; n = 60,000 cells analysed from 3 animals for h; n = 3 engraftments per group for i; n = 4 engraftments per group for j.

  9. Genetic impairment of autophagy in young quiescent satellite cells leads to premature senescence and impaired muscle regeneration.
    Extended Data Fig. 3: Genetic impairment of autophagy in young quiescent satellite cells leads to premature senescence and impaired muscle regeneration.

    a, RT–qPCR analysis of Atg7 expression and western blot analysis of LC3, p62 and tubulin of satellite cells isolated from Atg7WT and Atg7ΔPax7 mice. Graph shows the quantification of p62 normalized to tubulin; for full scan see Supplementary Fig. 2. b, Quiescent satellite cells were freshly isolated from Atg7WT and Atg7ΔPax7 mice which had been subjected to two weeks of rapamycin or vehicle (control) treatment in vivo. Cells were treated (or controls were untreated) with bafilomycin 4 h before analysis by fluorescence microscopy. Z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. c, Quantification of satellite cells in resting muscle of three-month-old Atg7WT and Atg7ΔPax7 mice by flow cytometry analysis (α7 integrin+CD34+ cells per gram of muscle tissue). d, Representative fluorescent microscopy images from Fig. 3d. Scale bar, 10 μm. e, RT–qPCR analysis of MyoD, Mgn and Ki67 expression in freshly isolated quiescent satellite cells from resting muscle of Atg7WT and Atg7ΔPax7ER mice, 7 days after tamoxifen treatment. f, Percentage of activated satellite cells (Pax7+/MyoD+) from the total Pax7+ cells (FACS-isolated 14-h post-injury from (a)). Scale bar, 50 μm. g, pS6 and Lamp1 immunostaining of cells from a. Scale bar, 10 μm. h, γH2AX protein levels per nucleus in Pax7+ satellite cells in tibialis anterior muscles of Atg7WT and Atg7ΔPax7ER mice, 15 days post-injury. Representative images are shown. Scale bar, 25 μm. i, Pax7+ satellite cells were quantified following immunostaining on regenerating muscles of Atg7WT and Atg7ΔPax7ER mice 7 days and 15 days after cardiotoxin injury. j, Representative images of haematoxylin and eosin staining of muscles at 7 days post-injury on muscles of Atg7WT and Atg7ΔPax7ER mice. Fibre size of central-nucleated myofibres at 7 days and 28 days post-injury is quantified. Scale bar, 50 μm. k, Tibialis anterior muscles of Atg7WT and Atg7ΔPax7 mice were injured by cardiotoxin injection and 21 days later these muscles were reinjured and then subsequently analysed 21 days later (21 + 21 days post-injury). The size of central-nucleated myofibres was quantified. Representative images are shown. Scale bar, 50 μm. l, Pax7+ and Ki67+ double-positive satellite cells were quantified following immunostaining on regenerating muscles of Atg7WT and Atg7ΔPax7ER mice 7 days after cardiotoxin injury. m, An equal number of quiescent satellite cells from Atg7WT:GFP–LC3 and Atg7ΔPax7:GFP–LC3 mice (two weeks ± rapamycin pre-treatment), transplanted as in Fig. 2c, and immunostained with the indicated antibodies 4 days later. Quantification of GFP+ cells per muscle field. Values relative to transplanted young cells (100%). Representative images are shown. Scale bar, 75 μm. n, Percentage of GFP+ cells that are also Ki67+ cells in muscles from m. o, Quantification of proliferating (BrdU+) and senescent (SA-β-gal+) satellite cells, isolated from Atg7WT and Atg7ΔPax7, pre-treated for 48 h with spermidine or rapamycin (or control vehicle) and cultured for 96 h. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. The number of samples were n = 3 animals per group (a); n = 7 animals per group (c); n = 3 animals per group (el); n = 4 engraftments per group (m, n); n = 3 animals per group (o).

  10. Autophagy loss in satellite cells causes dysfunctional mitophagy and mitochondria accumulation, leading to increased ROS and senescence.
    Extended Data Fig. 4: Autophagy loss in satellite cells causes dysfunctional mitophagy and mitochondria accumulation, leading to increased ROS and senescence.

    a, p62 and ubiquitin immunostaining on freshly isolated satellite cells from resting muscle of three-month-old Atg7WT and Atg7ΔPax7ER mice, one month after tamoxifen treatment. Arrowheads indicate co-localization of p62 and Ub aggregates. Representative images are shown. Scale bar, 5 μm. b, TOM20 and Lamp1 immunostaining of quiescent satellite cells isolated from young and geriatric WT mice. Mice were subjected to two weeks of rapamycin, spermidine or Trolox (or vehicle) treatment before analysis. Co-localization was calculated as the area occupied by the immunofluorescence co-localizing staining on images with respect to the total cellular area. The Pearson’s coefficient (r) was used to analyse the correlation of the intensity values of green and red pixels in the dual-channel images. The z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. c, Mitochondria quantification by MitoTracker in quiescent satellite cells of old mice, treated with rapamycin or vehicle for two weeks. d, Mitochondria (MitoTracker labelling) in young or geriatric cells. Satellite cells, were pre-treated with CCCP for 1 h (see Methods) and ± rapamycin for 24 h. Percentage of MitoTracker MFI reduction ± rapamycin. e, For the mitochondrial membrane potential analysis, satellite cells were freshly isolated from young wild-type mice and treated for 1 h with CCCP or DMSO (control). Membrane potential (TMRM MFI/MitoTracker Green MFI ratio) of cells was calculated by flow cytometry analysis at 1 h and 24 h after CCCP treatment (being 100% the membrane potential value of control satellite cells). f, Mitochondria content was quantified by MitoTracker staining of satellite cells from young and geriatric wild-type mice treated with rapamycin or vehicle (control) for 48 h. The z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. g, Mitochondria and ROS detection by MitoTracker and CellROX staining, respectively. Co-localization was calculated as in b. The z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. h, Representative images of freshly isolated satellite cells from resting muscle of three-month-old Atg7WT and Atg7ΔPax7 mice stained with CellROX fluorescent dye and p16INK4a antibody. Scale bar, 5 μm. Data are mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. Number of samples were n = 36 (Atg7WT) and n = 38 (Atg7ΔPax7ER) cells analysed from 3 animals (a); n = 23 (young), n = 24 (control), n = 42 (rapamycin); n = 28 (spermidine) and n = 21 (Trolox) cells analysed from 3 animals (b); n = 60,000 cells analysed from 3 animals (c); n = 40,000 cells analysed from 4 animals (d); n = 30,000 cells analysed from 3 animals (e, f); n = 18 (young), 21 (control), 15 (rapamycin) and 13 (Trolox) cells analysed from 3 animals (g).

  11. ROS inhibition in autophagy-impaired aged and Atg7 null satellite cells significantly restores cell proteostasis.
    Extended Data Fig. 5: ROS inhibition in autophagy-impaired aged and Atg7 null satellite cells significantly restores cell proteostasis.

    a, ROS-level quantification in quiescent satellite cells from three-month-old Atg7WT and Atg7ΔPax7 mice by CellROX flow cytometry. Representative images are shown. Scale bar, 5 μm. b, Western blot analysis of 53BP1 and parkin in satellite cells isolated from three-month-old Atg7WT and Atg7ΔPax7 mice. Tubulin control is the same tubulin control for Fig. 3g. Graph shows quantification of 53BP1 and parkin protein normalized to tubulin; for full scan see Supplementary Information Fig. 1. c, Quantification of ROS levels for satellite cells isolated from young and geriatric WT mice by flow cytometry using CellROX fluorescent dye. Satellite cells were treated with Trolox or vehicle (control) for 48 h before analysis. Results are represented as variation of MFI between young and geriatric satellite cells. d, Quantification of p62 and ubiquitin protein levels on immunostained freshly isolated satellite cells from resting muscle of old wild-type mice, in vivo treated for 2 weeks with Trolox or vehicle (control). Representative images are shown. Scale bar, 5 μm. e, Western blot analysis of LC3 and tubulin in satellite cells isolated from geriatric WT mice and treated for 48 h with Trolox or vehicle (control), in the absence or presence of bafilomycin for 4 h before analysis. Graph shows quantification of LC3II protein normalized to tubulin; for full gel scan see Supplementary Information Fig. 2. f, Autophagy flux and mitochondria in satellite cells from GFP–LC3 mice (two weeks with or without Trolox treatment). Satellite cells treated for 4 h ± bafilomycin treatment. Representative images are shown. Scale bar, 5 μm. g, The mRFP–GFP–LC3 plasmid was transfected into young or geriatric satellite cells, with 48 h treatment ± Trolox and then 4 h treatment ± bafilomycin, prior to fixation. The percentage of autophagosomes was quantified as in Fig. 2a. h, Muscle regeneration using geriatric satellite cell transplantation. An equal number of freshly isolated geriatric satellite cells, infected with GFP lentivirus and treated for 48 h with Trolox or vehicle, were transplanted into injured muscle of young immunodeficient mice. Four days later, muscles were collected and immunostained for GFP, MyoD and Mgn (to determine the possible myogenic states of satellite cells in the regenerating muscle). Representative images are shown. Scale bar, 50 μm. i, ChIP analysis for H2AK119ub (H2Aub) in satellite cells isolated from young and geriatric wild-type mice. j, Quantification of proliferating (BrdU+) and senescent (SA-β-gal+) satellite cells isolated from Atg7WT and Atg7ΔPax7 mice treated 48 h with Trolox or vehicle (control) and cultured for 96 h. k, Quantification of proliferating (BrdU+) and senescent (senescence-associated β-gal+) satellite cells isolated from Atg7WT and Atg7ΔPax7 mice and infected with LV-sh p16INK4a or LV-sh scramble, and cultured for 96 h. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-tests. P values are indicated. Number of samples were n = 60,000 cells analysed from 3 animals (a); n = 3 animals per group (b); n = 60,000 cells analysed from 3 animals (c); n = 36 (control) and n = 35 (Trolox) cells analysed from 3 animals (d); n = 3 animals per group (e); n = 60,000 cells analysed from 3 animals (f); n = 21 (young), n = 20 (young, Trolox), n = 19 (young, + bafilomycin), n = 18 (young, Trolox + bafilomycin), n = 21 (geriatric), n = 19 (geriatric, Trolox), n = 15 (geriatric, + bafilomycin) and n = 37 (geriatric, Trolox + bafilomycin) cells analysed from 3 animals (g); n = 3 animals per group (ik).

  12. Effects of p16INK4a silencing in autophagy-impaired young murine satellite cells.
    Extended Data Fig. 6: Effects of p16INK4a silencing in autophagy-impaired young murine satellite cells.

    a, Western blotting quantification of Atg7ΔPax7 satellite cells, infected with lentiviral LV-sh-p16INK4a or LV-sh-scramble and analysed 96 h later; for full gel scan see Supplementary Fig. 2. b, Atg7WT and Atg7ΔPax7 EDL, infected with LV-sh-p16INK4a or LV-sh-scramble, and grafted as Extended Data Fig. 2j. Representative eMHC-immunostaining. Scale bar, 25 μm. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. The number of samples were n = 3 animals per group (a) and n = 4 engraftments per group (b).

  13. Impaired autophagic flux in human geriatric satellite cells.
    Extended Data Fig. 7: Impaired autophagic flux in human geriatric satellite cells.

    a, Representative images of haematoxylin and eosin staining of human muscle biopsies from young (25 years old) and geriatric (95 years old) donors in resting conditions. Arrowheads indicate atrophic myofibres. Scale bar, 50 μm. b, CD56 and p16INK4a immunostaining on human muscle sections of samples described in a. Scale bar, 10 μm. c, Western blotting analysis of p62 protein in human satellite cells from young (about 25 years old) and geriatric (over 75 years old) donors, treated for 48 h ± rapamycin; for full gel scan see Supplementary Fig. 2. d, ROS and mitochondrial content analysis in human cells from treated for 48 h ± rapamycin. Graphs show MFI variation. Scale bar, 5 μm. e, Representative images from CellROX staining from d. Scale bar, 5 μm. f, Quantification of SA-β-gal+ human cells treated for 48 h ± rapamycin. Quantification was carried out 96 h after treatment. Scale bar, 200 μm. g, Quantification of proliferating (BrdU+) young and geriatric human satellite cells in culture. Representative pictures are shown. Scale bar, 25 μm. h, Western blot analysis of pS6, total S6 and tubulin in young and geriatric human satellite cells treated for 48 h with rapamycin or vehicle (control). Graphs show p62 quantification normalized to tubulin; for full scan see Supplementary Information Fig. 2. i, Immunostaining of pS6 in young and geriatric human satellite cells treated as in h. Scale bar, 75 μm. j, Scheme showing the proposed model of how age-impaired autophagy leads to muscle stem-cell senescence and regenerative decline. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-tests. P values are indicated. The number of samples were n = 3 human donors per group (ac); n = 60,000 cells analysed from 3 human donors (d), n = 3 human donors per group (fh).

Videos

  1. 3D reconstruction of a young satellite cell from GFP-LC3 mice.
    Video 1: 3D reconstruction of a young satellite cell from GFP-LC3 mice.
    Quiescent young satellite cells were isolated by FACS from 3-months old GFP-LC3 mice and fixed on glass slides for fluorescence microscopy analysis. 30 z stacks approximately were taken for video reconstruction as explained in the Methods section.
  2. 3D reconstruction of an old satellite cell from GFP-LC3 mice.
    Video 2: 3D reconstruction of an old satellite cell from GFP-LC3 mice.
    Quiescent old satellite cells were isolated by FACS from 24-months old GFP-LC3 mice and fixed on glass slides for fluorescence microscopy analysis. 30 z stacks approximately were taken for video reconstruction as explained in the Methods section.
  3. 3D reconstruction of an old satellite cell from GFP-LC3 mice treated with vehicle (control) and Bafilomycin 4 hours prior fixation.
    Video 3: 3D reconstruction of an old satellite cell from GFP-LC3 mice treated with vehicle (control) and Bafilomycin 4 hours prior fixation.
    Quiescent old satellite cells were treated for 4 hours with Bafilomycin and were isolated by FACS from 24-months old GFP-LC3 mice treated with vehicle (control) (i.e., the control of the Ramaycin treatment in Video 4) for two weeks in vivo. After sorting, satellite cells were fixed on glass slides for fluorescence microscopy analysis. 30 z stacks approximately were taken for video reconstruction as explained in the Methods section.
  4. 3D reconstruction of an old satellite cell from GFP-LC3 mice treated with Rapamycin and Bafilomycin 4 hours prior fixation.
    Video 4: 3D reconstruction of an old satellite cell from GFP-LC3 mice treated with Rapamycin and Bafilomycin 4 hours prior fixation.
    Quiescent old satellite cells were treated for 4 hours with Bafilomycin and were isolated by FACS from 24-months old GFP-LC3 mice treated with Rapamycin for two weeks in vivo. After sorting, satellite cells were fixed on glass slides for fluorescence microscopy analysis. 30 z stacks approximately were taken for video reconstruction as explained in the Methods section.

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References

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Author information

  1. These authors contributed equally to this work.

    • Marta Martínez-Vicente &
    • Marco Sandri

Affiliations

  1. Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative diseases (CIBERNED), E-08003 Barcelona, Spain

    • Laura García-Prat,
    • Eusebio Perdiguero,
    • Laura Ortet,
    • Vanessa Ruiz-Bonilla,
    • Susana Gutarra,
    • Antonio L. Serrano &
    • Pura Muñoz-Cánoves
  2. Neurodegenerative Diseases Research Group, Vall d’Hebron Research Institute-CIBERNED, E-08035 Barcelona, Spain

    • Marta Martínez-Vicente
  3. Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, E-08907 Barcelona, Spain

    • Javier Rodríguez-Ubreva &
    • Esteban Ballestar
  4. Advanced Fluorescence Microscopy Unit, Molecular Biology Institute of Barcelona (IBMB-CSIC), E-08028 Barcelona, Spain

    • Elena Rebollo
  5. Department of Biomedical Science, University of Padova, 35100 Padova, Italy

    • Marco Sandri
  6. Telethon Institute of Genetics and Medicine (TIGEM), 80131 Napoli, Italy

    • Marco Sandri
  7. ICREA, E-08908 Barcelona, Spain

    • Pura Muñoz-Cánoves

Contributions

L.G.-P. designed and performed most experiments, analysed data, interpreted results and wrote the manuscript. A.L.S. and E.P. designed and performed experiments, and helped in interpreting results and editing the manuscript. M.M.-V. and M.S. helped in designing and interpreting some experiments and results and editing the manuscript. L.O., V.R.-B. and S.G. performed some experiments and provided technical support. E.R. provided technical support in microscopy. J.R.-U. and E.B. performed ChIP experiments and helped in interpreting results. P.M.-C. conceived the project, designed experiments, interpreted results and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Microarray data have been deposited into the NCBI Gene Expression Omnibus under accession number GSE70376.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: The reduced autophagy flux in quiescent satellite cells can be increased by pharmacological treatment in vivo. (758 KB)

    a, Venn diagrams of overlapping genes between a proteostasis gene set (See Supplementary Table 1) and genes significantly upregulated in quiescent satellite cells from the indicated publications or from our gene expression microarray data comparing freshly FACS isolated satellite cells from resting muscle, or muscles obtained 72 h after cardiotoxin (CTX) injury, from young, wild-type mice. b, K-means clustering analysis (performed with Gene-E, Broad Institute) of the gene expression of the autophagy-related genes during ageing. Clusters are shown with heat maps of the normalized raw data. Each column represents a different sample and each row a different gene probe. Red, increased expression; white, neutral expression; blue, decreased expression. c, Representative example of the FACS strategy and gating scheme for isolating satellite cells from mice in resting conditions. d, Pax7 and GFP immunostaining of freshly isolated satellite cells from resting muscles of young and old GFP–LC3 mice. Scale bar, 5 μm. e, Electron microscopy images of young and old satellite cells on sections of resting tibialis anterior (TA) muscle of wild-type (WT) mice. Arrowheads indicate autophagic vesicles. Scale bars, 1 μm and 0.5 μm (right and left, respectively). f, Pax7 and GFP immunostaining on tissue sections from resting tibialis anterior muscles of young and old GFP–LC3 mice. Arrowheads indicate autophagic vesicles. Scale bar, 5 μm. g, p62 and ubiquitin (Ub) MFI. Arrowheads, co-localization of p62 and ubiquitin aggregates. h, LC3 western blot of freshly isolated satellite cells from young and old, wild-type mice, treated with bafilomycin or vehicle for 4 h before collection. Graph shows LC3II quantification, after normalization with tubulin levels; for full scan see Supplementary Fig. 2. i, Quiescent satellite cells were freshly isolated from old, wild-type mice subjected to two weeks of rapamycin, spermidine or vehicle (control) treatment. Cells were treated (or not treated) with bafilomycin 4 h prior to analysis by immunostaining of LC3 marker. Z projections of representative fluorescence microscopy images are shown. Scale bars, 5 μm. j, Representative fluorescent microscopy images from Fig. 1d. Scale bar, 5 μm. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. Number of samples were n = 3 animals per group for a and b; n = 35 (young) and 66 (old) cells analysed from 3 animals for g; n = 3 animals per group for h.

  2. Extended Data Figure 2: Reinduction of autophagy rescues proliferation and reduces senescence in geriatric satellite, thus restoring regenerative capacity. (558 KB)

    a, Transplanted muscles from Fig. 2c were immunostained for GFP and for Ki67, Pax7, MyoD or Mgn (to determine the distinct possible myogenic states of satellite cells in the regenerating muscle). Scale bars, 50 μm. b, Autophagy flux analysed by flow cytometry in freshly isolated satellite cells from resting muscle of GFP–LC3 mice, treated for 48 h with rapamycin or vehicle (control). Satellite cells were treated with bafilomycin or vehicle for 4 h before analysis. Results are expressed as the change in GFP–LC3 MFI in bafilomycin (−) compared to bafilomycin (+) conditions. c, Western blot analysis of pS6 protein levels in geriatric satellite cells from wild-type mice, treated for 48 h with rapamycin or vehicle (control). Graph shows pS6 quantification, normalized to tubulin; for full scan see Supplementary Information Fig. 2. d, As in Fig. 2c, percentage γH2AX+ or p16INK4a+cells from total GFP+ cells were quantified. Scale bars, 10 μm. e, Quantification BrdU+ and senescence-associated β-gal+ satellite cells, pre-treated as in Fig. 2c and analysed after 96 h. f, Quantification of senescent (senescence-associated β-gal+) satellite cells, isolated from young and geriatric wild-type mice, pre-treated for 48 h with spermidine or vehicle (control) and cultured for 96 h. g, Quantitative real-time PCR (RT–qPCR) analysis of Atg7 expression on satellite cells infected with LV-Atg7 or LV-control (LV-Co), and cultured for 96 h. h, GFP–LC3 satellite cells were infected with LV-Atg7 or LV-Co and treated with bafilomycin or vehicle for 4 h before analysis. Autophagy flux was analysed by flow cytometry and represented as in b. Representative images are shown. Scale bar, 10 μm. i, Muscle regeneration experiment by satellite cell transplantation. An equal number of satellite cells from young and geriatric mice infected with a lentivirus overexpressing the Atg7 gene (LV-Atg7) or a lentivirus control (LV-Co), which also expressed GFP, were transplanted into injured muscle of young immunodeficient mice, and collected 28 days later. GFP expression in muscles was analysed by immunostaining. Quantification of GFP+ cells (fibres) per muscle field versus transplanted control-treated satellite cells. Representative images are shown. Scale bar, 75 μm. j, EDL geriatric muscles, infected with LV-Atg7 or LV-Co, and grafted on recipient mouse muscle. Regeneration was analysed 8 days later. Frequency distribution of regenerating fibres by size. Scale bar, 25 μm. Data are mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. Number of samples were n = 60,000 cells analysed from 3 animals for b; n = 3 animals per group for c; n = 5 engraftments per group for d; n = 3 animals per group for eg; n = 60,000 cells analysed from 3 animals for h; n = 3 engraftments per group for i; n = 4 engraftments per group for j.

  3. Extended Data Figure 3: Genetic impairment of autophagy in young quiescent satellite cells leads to premature senescence and impaired muscle regeneration. (781 KB)

    a, RT–qPCR analysis of Atg7 expression and western blot analysis of LC3, p62 and tubulin of satellite cells isolated from Atg7WT and Atg7ΔPax7 mice. Graph shows the quantification of p62 normalized to tubulin; for full scan see Supplementary Fig. 2. b, Quiescent satellite cells were freshly isolated from Atg7WT and Atg7ΔPax7 mice which had been subjected to two weeks of rapamycin or vehicle (control) treatment in vivo. Cells were treated (or controls were untreated) with bafilomycin 4 h before analysis by fluorescence microscopy. Z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. c, Quantification of satellite cells in resting muscle of three-month-old Atg7WT and Atg7ΔPax7 mice by flow cytometry analysis (α7 integrin+CD34+ cells per gram of muscle tissue). d, Representative fluorescent microscopy images from Fig. 3d. Scale bar, 10 μm. e, RT–qPCR analysis of MyoD, Mgn and Ki67 expression in freshly isolated quiescent satellite cells from resting muscle of Atg7WT and Atg7ΔPax7ER mice, 7 days after tamoxifen treatment. f, Percentage of activated satellite cells (Pax7+/MyoD+) from the total Pax7+ cells (FACS-isolated 14-h post-injury from (a)). Scale bar, 50 μm. g, pS6 and Lamp1 immunostaining of cells from a. Scale bar, 10 μm. h, γH2AX protein levels per nucleus in Pax7+ satellite cells in tibialis anterior muscles of Atg7WT and Atg7ΔPax7ER mice, 15 days post-injury. Representative images are shown. Scale bar, 25 μm. i, Pax7+ satellite cells were quantified following immunostaining on regenerating muscles of Atg7WT and Atg7ΔPax7ER mice 7 days and 15 days after cardiotoxin injury. j, Representative images of haematoxylin and eosin staining of muscles at 7 days post-injury on muscles of Atg7WT and Atg7ΔPax7ER mice. Fibre size of central-nucleated myofibres at 7 days and 28 days post-injury is quantified. Scale bar, 50 μm. k, Tibialis anterior muscles of Atg7WT and Atg7ΔPax7 mice were injured by cardiotoxin injection and 21 days later these muscles were reinjured and then subsequently analysed 21 days later (21 + 21 days post-injury). The size of central-nucleated myofibres was quantified. Representative images are shown. Scale bar, 50 μm. l, Pax7+ and Ki67+ double-positive satellite cells were quantified following immunostaining on regenerating muscles of Atg7WT and Atg7ΔPax7ER mice 7 days after cardiotoxin injury. m, An equal number of quiescent satellite cells from Atg7WT:GFP–LC3 and Atg7ΔPax7:GFP–LC3 mice (two weeks ± rapamycin pre-treatment), transplanted as in Fig. 2c, and immunostained with the indicated antibodies 4 days later. Quantification of GFP+ cells per muscle field. Values relative to transplanted young cells (100%). Representative images are shown. Scale bar, 75 μm. n, Percentage of GFP+ cells that are also Ki67+ cells in muscles from m. o, Quantification of proliferating (BrdU+) and senescent (SA-β-gal+) satellite cells, isolated from Atg7WT and Atg7ΔPax7, pre-treated for 48 h with spermidine or rapamycin (or control vehicle) and cultured for 96 h. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. The number of samples were n = 3 animals per group (a); n = 7 animals per group (c); n = 3 animals per group (el); n = 4 engraftments per group (m, n); n = 3 animals per group (o).

  4. Extended Data Figure 4: Autophagy loss in satellite cells causes dysfunctional mitophagy and mitochondria accumulation, leading to increased ROS and senescence. (495 KB)

    a, p62 and ubiquitin immunostaining on freshly isolated satellite cells from resting muscle of three-month-old Atg7WT and Atg7ΔPax7ER mice, one month after tamoxifen treatment. Arrowheads indicate co-localization of p62 and Ub aggregates. Representative images are shown. Scale bar, 5 μm. b, TOM20 and Lamp1 immunostaining of quiescent satellite cells isolated from young and geriatric WT mice. Mice were subjected to two weeks of rapamycin, spermidine or Trolox (or vehicle) treatment before analysis. Co-localization was calculated as the area occupied by the immunofluorescence co-localizing staining on images with respect to the total cellular area. The Pearson’s coefficient (r) was used to analyse the correlation of the intensity values of green and red pixels in the dual-channel images. The z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. c, Mitochondria quantification by MitoTracker in quiescent satellite cells of old mice, treated with rapamycin or vehicle for two weeks. d, Mitochondria (MitoTracker labelling) in young or geriatric cells. Satellite cells, were pre-treated with CCCP for 1 h (see Methods) and ± rapamycin for 24 h. Percentage of MitoTracker MFI reduction ± rapamycin. e, For the mitochondrial membrane potential analysis, satellite cells were freshly isolated from young wild-type mice and treated for 1 h with CCCP or DMSO (control). Membrane potential (TMRM MFI/MitoTracker Green MFI ratio) of cells was calculated by flow cytometry analysis at 1 h and 24 h after CCCP treatment (being 100% the membrane potential value of control satellite cells). f, Mitochondria content was quantified by MitoTracker staining of satellite cells from young and geriatric wild-type mice treated with rapamycin or vehicle (control) for 48 h. The z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. g, Mitochondria and ROS detection by MitoTracker and CellROX staining, respectively. Co-localization was calculated as in b. The z projections of representative fluorescence microscopy images are shown. Scale bar, 5 μm. h, Representative images of freshly isolated satellite cells from resting muscle of three-month-old Atg7WT and Atg7ΔPax7 mice stained with CellROX fluorescent dye and p16INK4a antibody. Scale bar, 5 μm. Data are mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. Number of samples were n = 36 (Atg7WT) and n = 38 (Atg7ΔPax7ER) cells analysed from 3 animals (a); n = 23 (young), n = 24 (control), n = 42 (rapamycin); n = 28 (spermidine) and n = 21 (Trolox) cells analysed from 3 animals (b); n = 60,000 cells analysed from 3 animals (c); n = 40,000 cells analysed from 4 animals (d); n = 30,000 cells analysed from 3 animals (e, f); n = 18 (young), 21 (control), 15 (rapamycin) and 13 (Trolox) cells analysed from 3 animals (g).

  5. Extended Data Figure 5: ROS inhibition in autophagy-impaired aged and Atg7 null satellite cells significantly restores cell proteostasis. (552 KB)

    a, ROS-level quantification in quiescent satellite cells from three-month-old Atg7WT and Atg7ΔPax7 mice by CellROX flow cytometry. Representative images are shown. Scale bar, 5 μm. b, Western blot analysis of 53BP1 and parkin in satellite cells isolated from three-month-old Atg7WT and Atg7ΔPax7 mice. Tubulin control is the same tubulin control for Fig. 3g. Graph shows quantification of 53BP1 and parkin protein normalized to tubulin; for full scan see Supplementary Information Fig. 1. c, Quantification of ROS levels for satellite cells isolated from young and geriatric WT mice by flow cytometry using CellROX fluorescent dye. Satellite cells were treated with Trolox or vehicle (control) for 48 h before analysis. Results are represented as variation of MFI between young and geriatric satellite cells. d, Quantification of p62 and ubiquitin protein levels on immunostained freshly isolated satellite cells from resting muscle of old wild-type mice, in vivo treated for 2 weeks with Trolox or vehicle (control). Representative images are shown. Scale bar, 5 μm. e, Western blot analysis of LC3 and tubulin in satellite cells isolated from geriatric WT mice and treated for 48 h with Trolox or vehicle (control), in the absence or presence of bafilomycin for 4 h before analysis. Graph shows quantification of LC3II protein normalized to tubulin; for full gel scan see Supplementary Information Fig. 2. f, Autophagy flux and mitochondria in satellite cells from GFP–LC3 mice (two weeks with or without Trolox treatment). Satellite cells treated for 4 h ± bafilomycin treatment. Representative images are shown. Scale bar, 5 μm. g, The mRFP–GFP–LC3 plasmid was transfected into young or geriatric satellite cells, with 48 h treatment ± Trolox and then 4 h treatment ± bafilomycin, prior to fixation. The percentage of autophagosomes was quantified as in Fig. 2a. h, Muscle regeneration using geriatric satellite cell transplantation. An equal number of freshly isolated geriatric satellite cells, infected with GFP lentivirus and treated for 48 h with Trolox or vehicle, were transplanted into injured muscle of young immunodeficient mice. Four days later, muscles were collected and immunostained for GFP, MyoD and Mgn (to determine the possible myogenic states of satellite cells in the regenerating muscle). Representative images are shown. Scale bar, 50 μm. i, ChIP analysis for H2AK119ub (H2Aub) in satellite cells isolated from young and geriatric wild-type mice. j, Quantification of proliferating (BrdU+) and senescent (SA-β-gal+) satellite cells isolated from Atg7WT and Atg7ΔPax7 mice treated 48 h with Trolox or vehicle (control) and cultured for 96 h. k, Quantification of proliferating (BrdU+) and senescent (senescence-associated β-gal+) satellite cells isolated from Atg7WT and Atg7ΔPax7 mice and infected with LV-sh p16INK4a or LV-sh scramble, and cultured for 96 h. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-tests. P values are indicated. Number of samples were n = 60,000 cells analysed from 3 animals (a); n = 3 animals per group (b); n = 60,000 cells analysed from 3 animals (c); n = 36 (control) and n = 35 (Trolox) cells analysed from 3 animals (d); n = 3 animals per group (e); n = 60,000 cells analysed from 3 animals (f); n = 21 (young), n = 20 (young, Trolox), n = 19 (young, + bafilomycin), n = 18 (young, Trolox + bafilomycin), n = 21 (geriatric), n = 19 (geriatric, Trolox), n = 15 (geriatric, + bafilomycin) and n = 37 (geriatric, Trolox + bafilomycin) cells analysed from 3 animals (g); n = 3 animals per group (ik).

  6. Extended Data Figure 6: Effects of p16INK4a silencing in autophagy-impaired young murine satellite cells. (263 KB)

    a, Western blotting quantification of Atg7ΔPax7 satellite cells, infected with lentiviral LV-sh-p16INK4a or LV-sh-scramble and analysed 96 h later; for full gel scan see Supplementary Fig. 2. b, Atg7WT and Atg7ΔPax7 EDL, infected with LV-sh-p16INK4a or LV-sh-scramble, and grafted as Extended Data Fig. 2j. Representative eMHC-immunostaining. Scale bar, 25 μm. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-test. P values are indicated. The number of samples were n = 3 animals per group (a) and n = 4 engraftments per group (b).

  7. Extended Data Figure 7: Impaired autophagic flux in human geriatric satellite cells. (453 KB)

    a, Representative images of haematoxylin and eosin staining of human muscle biopsies from young (25 years old) and geriatric (95 years old) donors in resting conditions. Arrowheads indicate atrophic myofibres. Scale bar, 50 μm. b, CD56 and p16INK4a immunostaining on human muscle sections of samples described in a. Scale bar, 10 μm. c, Western blotting analysis of p62 protein in human satellite cells from young (about 25 years old) and geriatric (over 75 years old) donors, treated for 48 h ± rapamycin; for full gel scan see Supplementary Fig. 2. d, ROS and mitochondrial content analysis in human cells from treated for 48 h ± rapamycin. Graphs show MFI variation. Scale bar, 5 μm. e, Representative images from CellROX staining from d. Scale bar, 5 μm. f, Quantification of SA-β-gal+ human cells treated for 48 h ± rapamycin. Quantification was carried out 96 h after treatment. Scale bar, 200 μm. g, Quantification of proliferating (BrdU+) young and geriatric human satellite cells in culture. Representative pictures are shown. Scale bar, 25 μm. h, Western blot analysis of pS6, total S6 and tubulin in young and geriatric human satellite cells treated for 48 h with rapamycin or vehicle (control). Graphs show p62 quantification normalized to tubulin; for full scan see Supplementary Information Fig. 2. i, Immunostaining of pS6 in young and geriatric human satellite cells treated as in h. Scale bar, 75 μm. j, Scheme showing the proposed model of how age-impaired autophagy leads to muscle stem-cell senescence and regenerative decline. Data show mean ± s.e.m. Comparisons by two-sided Mann–Whitney U-tests. P values are indicated. The number of samples were n = 3 human donors per group (ac); n = 60,000 cells analysed from 3 human donors (d), n = 3 human donors per group (fh).

Supplementary information

Video

  1. Video 1: 3D reconstruction of a young satellite cell from GFP-LC3 mice. (6.84 MB, Download)
    Quiescent young satellite cells were isolated by FACS from 3-months old GFP-LC3 mice and fixed on glass slides for fluorescence microscopy analysis. 30 z stacks approximately were taken for video reconstruction as explained in the Methods section.
  2. Video 2: 3D reconstruction of an old satellite cell from GFP-LC3 mice. (6.95 MB, Download)
    Quiescent old satellite cells were isolated by FACS from 24-months old GFP-LC3 mice and fixed on glass slides for fluorescence microscopy analysis. 30 z stacks approximately were taken for video reconstruction as explained in the Methods section.
  3. Video 3: 3D reconstruction of an old satellite cell from GFP-LC3 mice treated with vehicle (control) and Bafilomycin 4 hours prior fixation. (10.77 MB, Download)
    Quiescent old satellite cells were treated for 4 hours with Bafilomycin and were isolated by FACS from 24-months old GFP-LC3 mice treated with vehicle (control) (i.e., the control of the Ramaycin treatment in Video 4) for two weeks in vivo. After sorting, satellite cells were fixed on glass slides for fluorescence microscopy analysis. 30 z stacks approximately were taken for video reconstruction as explained in the Methods section.
  4. Video 4: 3D reconstruction of an old satellite cell from GFP-LC3 mice treated with Rapamycin and Bafilomycin 4 hours prior fixation. (7.2 MB, Download)
    Quiescent old satellite cells were treated for 4 hours with Bafilomycin and were isolated by FACS from 24-months old GFP-LC3 mice treated with Rapamycin for two weeks in vivo. After sorting, satellite cells were fixed on glass slides for fluorescence microscopy analysis. 30 z stacks approximately were taken for video reconstruction as explained in the Methods section.

PDF files

  1. Supplementary Figures (1.4 MB)

    This file contains Supplementary Figures 1-2. Supplementary Figure 1 contains uncropped Western blot scans with size indications corresponding to Figures 3g, 5e, 6c and Extended Data Figure 5b. Supplementary Figure 2 contains uncropped Western blot scans with size indications corresponding to Extended Data Figures 6a, 7c, 1h, 2c, 3a, 5e and Figure 7i.

Excel files

  1. Supplementary Table (181 KB)

    This file contains Supplementary Table 1, a gene list of proteostasis stem cells; Gene list of quiescence genes belonging to each proteostasis pathway, including autophagy pathways and genes; Dataset of upregulated genes in quiescent muscle.

Additional data