Targeting cellular senescence prevents age-related bone loss in mice

Journal name:
Nature Medicine
Volume:
23,
Pages:
1072–1079
Year published:
DOI:
doi:10.1038/nm.4385
Received
Accepted
Published online

Aging is associated with increased cellular senescence, which is hypothesized to drive the eventual development of multiple comorbidities1. Here we investigate a role for senescent cells in age-related bone loss through multiple approaches. In particular, we used either genetic (i.e., the INK-ATTAC 'suicide' transgene encoding an inducible caspase 8 expressed specifically in senescent cells2, 3, 4) or pharmacological (i.e., 'senolytic' compounds5, 6) means to eliminate senescent cells. We also inhibited the production of the proinflammatory secretome of senescent cells using a JAK inhibitor (JAKi)3, 7. In aged (20- to 22-month-old) mice with established bone loss, activation of the INK-ATTAC caspase 8 in senescent cells or treatment with senolytics or the JAKi for 2–4 months resulted in higher bone mass and strength and better bone microarchitecture than in vehicle-treated mice. The beneficial effects of targeting senescent cells were due to lower bone resorption with either maintained (trabecular) or higher (cortical) bone formation as compared to vehicle-treated mice. In vitro studies demonstrated that senescent-cell conditioned medium impaired osteoblast mineralization and enhanced osteoclast-progenitor survival, leading to increased osteoclastogenesis. Collectively, these data establish a causal role for senescent cells in bone loss with aging, and demonstrate that targeting these cells has both anti-resorptive and anabolic effects on bone. Given that eliminating senescent cells and/or inhibiting their proinflammatory secretome also improves cardiovascular function4, enhances insulin sensitivity3, and reduces frailty7, targeting this fundamental mechanism to prevent age-related bone loss suggests a novel treatment strategy not only for osteoporosis, but also for multiple age-related comorbidities.

At a glance

Figures

  1. Clearance of p16Ink4a+ senescent cells prevents age-related bone loss.
    Figure 1: Clearance of p16Ink4a+ senescent cells prevents age-related bone loss.

    (a) Experimental design for testing the effect of senescent cell clearance using a transgenic approach on age-related bone loss: 20-month-old female INK-ATTAC mice were randomized to either vehicle (n = 13) or AP20187 (n = 16) treatments (intraperitoneally (i.p) twice weekly) for 4 months. (b,c) RT–qPCR analysis of p16Ink4a (b) and EGFP (encoded by the INK-ATTAC transgene) (c) mRNA expression levels in osteocyte-enriched cells derived from the bones of mice. (d,e) Representative images (n > 30 images per animal, 13 vehicle-treated and 16 AP20187-treated) of a senescent (SEN) osteocyte (magnification, 100×) (d) versus a nonsenescent (non-SEN) osteocyte (magnification, 100×) (e) according to the senescence-associated distention of satellites (SADS, see arrows in d) assay in cortical bone diaphysis (scale bars, 2 μm). (f) Quantification of the percentage of senescent osteocytes in mice treated with either vehicle or AP20187 according to the SADS assay. (g,h) RT–qPCR analysis of p16Ink4a (g) and EGFP (h) mRNA expression levels in perigonadal adipose tissue. (i) Representative microcomputed tomography (μCT) images (n = 13 vehicle-treated and 16 AP20187-treated mice) of bone microarchitecture at the lumbar spine of vehicle-treated versus AP20187-treated mice. (jn) Quantification of μCT-derived bone volume fraction (BV/TV; %) (j), trabecular number (Tb.N; 1/mm) (k), trabecular thickness (Tb.Th; mm) (l), trabecular separation (Tb.Sp; mm) (m), and structure model index (SMI, a measure of plate/rod morphology, with lower numbers being better) (n) at the lumbar spine. (o) Representative μCT images (n = 13 vehicle-treated and 16 AP20187-treated mice) of bone microarchitecture at the femur. (p,q) Quantification of μCT-derived cortical thickness (Ct.Th, mm) (p) and micro-finite-element analysis (μFEA)-derived failure load (N, Newton (i.e., a measure of bone strength)) (q). (ru) Histomorphometric quantification at the femoral endocortical surface of osteoclast numbers per bone perimeter (N.Oc/B.Pm; /mm) (r), osteoblast numbers per bone perimeter (N.Ob/B.Pm; /mm) (s), endocortical mineral apposition rate (MAR; mcm/d) (t), and endocortical bone formation rate per bone surface (BFR/BS; mcm3/mcm2/d) (u); n = 8/group. (v,w) Mineralization of osteoblastic MC3T3 cells exposed to control (CON) or senescent (SEN) conditioned medium (CM) (n = 3/group) (v), with quantification of eluted alizarin red dye (w). Data represent mean ± s.e.m. (error bars). *P < 0.05; **P < 0.01; ***P < 0.001 (independent samples t-test or Wilcoxon rank–sum test, as appropriate).

  2. Senescent-cell clearance by treatment with senolytics (D + Q) prevents age-related bone loss.
    Figure 2: Senescent-cell clearance by treatment with senolytics (D + Q) prevents age-related bone loss.

    (a) Experimental design for testing the effect of senescent cell clearance through periodic treatment with D + Q on age-related bone loss: 20-month-old male C57BL/6 mice were randomized to either vehicle (n = 13) or D + Q (n = 10) treatments (once monthly by oral gavage) for 4 months. (b) RT–qPCR analysis of p16Ink4a mRNA expression levels in osteocyte-enriched cells derived from bones. (c) Quantification of the percentage of senescent osteocytes in cortical bone diaphysis using the SADS assay. (dg) Analysis of perigonadal adipose tissue p16Ink4a mRNA expression levels (d), and staining for senescence-associated β-galactosidase- (SA-β-gal) positive cells (e, vehicle; f, D + Q) (see arrows (in e); magnification, 10×; scale bars, 400 μm; 100 μm in inset in (e)), with quantification of the percentage of SA-β-gal-positive cells (g). (h) Representative μCT images (n = 13 vehicle-treated and 10 D + Q–treated mice) of bone microarchitecture at the lumbar spine of vehicle-treated versus D + Q–treated male C57BL/6 mice. (im) Quantification of μCT-derived bone volume fraction (BV/TV; %) (i), trabecular number (Tb.N; 1/mm) (j), trabecular thickness (Tb.Th; mm) (k), trabecular separation (Tb.Sp; mm) (l), and structure model index (SMI) (m) at the lumbar spine. (n) Representative μCT images (n = 13 vehicle-treated and 10 D + Q–treated mice) of bone microarchitecture at the femur. (o,p) Quantification of μCT-derived cortical thickness (Ct.Th, mm) (o) and failure load (N) (p). (qt) Histomorphometric quantification at the femoral endocortical surface of osteoclast numbers per bone perimeter (N.Oc/B.Pm; /mm) (q), osteoblast numbers per bone perimeter (N.Ob/B.Pm; /mm) (r), endocortical mineral apposition rate (MAR; mcm/d) (s), and endocortical bone formation rate per bone surface (BFR/BS; mcm3/mcm2/d) (t) (n = 8/group). Data represent mean ± s.e.m. (error bars). *P < 0.05; **P < 0.01; ***P < 0.001 (independent samples t-test or Wilcoxon rank–sum test, as appropriate).

  3. The SASP increases osteoclastogenesis in vitro by promoting the survival of monocyte osteoclast progenitors.
    Figure 3: The SASP increases osteoclastogenesis in vitro by promoting the survival of monocyte osteoclast progenitors.

    (a) Representative images (from three individual sets of cell cultures) of tartrate-resistant acid phosphatase (TRAP)-stained osteoclast cultures (magnification, 4×; scale bars, 1,000 μm) pre-treated with vehicle (negative control), M-CSF (25 ng/ml), control (CON) conditioned medium (CM), or senescent (SEN) CM (n = 3/group). (b) Osteoclast numbers per well following 4 d of osteoclast differentiation of bone marrow cells pre-treated with CON CM or SEN CM. (c) RT–qPCR analysis of osteoclast genes in bone marrow cells pre-treated with CON CM or SEN CM following 2 d of osteoclast differentiation; gene expression was denoted as fold-change relative to vehicle (negative control) pre-treated osteoclast cultures (n = 3/group). (d,e) Flow cytometric analysis of CD115+ (monocyte marker) and Rank (osteoclast progenitor marker) cells in nonadherent population after 24 h of treatment with CON CM or SEN CM. (d) Representative images (from five individual sets of cell cultures) of flow cytometric gating with the CD115+/Rank monocyte population outlined in red (5.4% in CON CM and 14.2% in SEN CM). (e) Average CD115+/Rank percentages in CON CM versus SEN CM (n = 5/group; see Supplementary Fig. 15 for flow cytometry gating strategy). (f,g) Apoptosis, as measured by cleavage of a luminogenic caspase-3/7 substrate (RLU), in whole marrow (f) and monocyte-enriched cultures (g) following 24 h of treatment with CON CM or SEN CM (n = 5/group). (h,i) Representative images (from five individual sets of cell cultures) of TRAP-stained osteoclast cultures (magnification, 4×; scale bars, 1,000 μm) (h) and osteoclast numbers per well (i) after 3 d of osteoclast differentiation of bone marrow cells pre-treated with CON CM, SEN CM, or CM from senescent cells treated with 0.6-μM JAKi (SEN/JAKi) (n = 5/group). (j) Osteoclast numbers per well after 3 d of osteoclast differentiation of bone marrow cells pre-treated with SEN CM in the presence of control IgG (20 μg/ml), anti-PAI-1 (5 μg/ml), anti-IL-6 (5 μg/ml), or anti-IL-8 (20 μg/ml) (n = 5/group); osteoclast numbers are expressed relative to SEN CM control. Data represent mean ± s.e.m. (error bars). *P < 0.05; **P < 0.01; ***P < 0.001 (independent samples t-test or Wilcoxon rank–sum test, as appropriate); P < 0.01 (one-way analysis of variance (ANOVA) versus CON CM followed by Bonferroni post hoc test); P < 0.01 one-way (ANOVA versus SEN CM followed by the Bonferroni post hoc test).

  4. Suppression of the SASP by treatment with the JAK1/2 inhibitor ruxolitinib prevents age-related bone loss.
    Figure 4: Suppression of the SASP by treatment with the JAK1/2 inhibitor ruxolitinib prevents age-related bone loss.

    (a) Experimental design for testing the effect of SASP inhibition induced by ruxolitinib on age-related bone loss: 22-month-old male C57BL/6 mice were randomized to either vehicle (n = 8) or JAKi (n = 7) treatments daily for 2 months. (b) Representative μCT images of bone microarchitecture at the lumbar spine (n = 8 vehicle-treated and n = 7 JAKi-treated mice). (cg) Quantification of μCT-derived bone volume fraction (BV/TV; %) (c), trabecular number (Tb.N; 1/mm) (d), trabecular thickness (Tb.Th; mm) (e), trabecular separation (Tb.Sp; mm) (f), and SMI (g). (h) Representative μCT images (n = 8 vehicle-treated and n = 7 JAKi-treated mice) of bone microarchitecture at the femoral metaphysis. (in) Quantification of femoral metaphysis μCT-derived BV/TV (%)(i), Tb.N (1/mm) (j), Tb.Th (mm) (k), Tb.Sp (mm) (l), SMI (m), and failure load (N) (n). Femoral metaphysis trabecular quantification of osteoclast numbers per bone perimeter (N.Oc/B.Pm; 1/mm) (o) and osteoblast number per bone perimeter (N.Ob/B.Pm; 1/mm) (p). Data represent mean ± s.e.m. (error bars). *P < 0.05; **P < 0.01; ***P < 0.001 (independent samples t-test). (q,r) Comparing the effects of anti-resorptive versus senolytic therapies on bone metabolism. (q) (1) Senescent cells (SCs) accumulate in the bone microenvironment with aging, where they (2) increase bone resorption by osteoclasts (OCs) and (3) decrease bone formation by osteoblasts (OBs). (4) Anti-resorptive therapy inhibits or eliminates OCs and decreases bone resorption; owing to (5) coupling, there is a concomitant reduction in bone formation. (r) (1) Senolytic therapy reduces the burden of (2) SCs, which in turn (3) suppresses bone resorption with (4) either an increase (cortical bone) or maintenance (trabecular bone) in bone formation, leading to (5) uncoupling between OCs and OBs.

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Author information

  1. These authors contributed equally to this work.

    • Joshua N Farr,
    • Ming Xu &
    • Megan M Weivoda

Affiliations

  1. Robert and Arlene Kogod Center on Aging and Division of Endocrinology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.

    • Joshua N Farr,
    • Ming Xu,
    • Megan M Weivoda,
    • David G Monroe,
    • Daniel G Fraser,
    • Jennifer L Onken,
    • Brittany A Negley,
    • Jad G Sfeir,
    • Mikolaj B Ogrodnik,
    • Christine M Hachfeld,
    • Nathan K LeBrasseur,
    • Matthew T Drake,
    • Robert J Pignolo,
    • Tamar Pirtskhalava,
    • Tamara Tchkonia,
    • Merry Jo Oursler,
    • James L Kirkland &
    • Sundeep Khosla

Contributions

J.N.F. performed most of the experiments and analyses on INK-ATTAC and D + Q–treated mice. M.X. generated conditioned medium and defined the SASP mechanism. M.M.W. performed osteoclast cell culture experiments. M.X. and M.M.W. performed most of the experiments and analyses on JAKi-treated mice. D.G.M. provided technical guidance. D.G.F., J.L.O., B.A.N., J.G.S., M.B.O., C.M.H., T.P., T.T., N.K.L., M.T.D., R.J.P., and M.J.O. assisted with various aspects of the experiments and analyses. J.N.F., M.X., M.M.W., T.T., J.L.K., and S.K. contributed to the design of experiments. J.N.F., M.W., and S.K. wrote the manuscript with input from all co-authors. S.K. directed and supervised all aspects of the study in collaboration with J.L.K. All authors reviewed the manuscript.

Competing financial interests

J.L.K., T.T., and T.P. have a financial interest related to this research. A patent on senolytic drugs (WO2015116735A1) is held by Mayo Clinic. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and was conducted in compliance with Mayo Clinic Conflict of Interest policies.

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