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Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline

Nature (2018) | Download Citation

Abstract

Cellular senescence, which is characterized by an irreversible cell-cycle arrest1 accompanied by a distinctive secretory phenotype2, can be induced through various intracellular and extracellular factors. Senescent cells that express the cell cycle inhibitory protein p16INK4A have been found to actively drive naturally occurring age-related tissue deterioration3,4 and contribute to several diseases associated with ageing, including atherosclerosis5 and osteoarthritis6. Various markers of senescence have been observed in patients with neurodegenerative diseases7,8,9; however, a role for senescent cells in the aetiology of these pathologies is unknown. Here we show a causal link between the accumulation of senescent cells and cognition-associated neuronal loss. We found that the MAPTP301SPS19 mouse model of tau-dependent neurodegenerative disease10 accumulates p16INK4A-positive senescent astrocytes and microglia. Clearance of these cells as they arise using INK-ATTAC transgenic mice prevents gliosis, hyperphosphorylation of both soluble and insoluble tau leading to neurofibrillary tangle deposition, and degeneration of cortical and hippocampal neurons, thus preserving cognitive function. Pharmacological intervention with a first-generation senolytic modulates tau aggregation. Collectively, these results show that senescent cells have a role in the initiation and progression of tau-mediated disease, and suggest that targeting senescent cells may provide a therapeutic avenue for the treatment of these pathologies.

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Acknowledgements

The authors thank C. H. Cho for numerous contributions to the experiments; the laboratory of C. Howe and specifically M. Standiford for help with the microglia and astrocyte cultures; M. Poeschla for assistance with the phospho-tau immunohistochemistry; G. Nelson for genotyping and animal support; B. Childs for input and assistance in Gal-TEM; the Mayo Clinic Microscopy and Cell Analysis Core and staff for assistance with flow cytometry and transmission electron microscopy; the Mayo Clinic Medical Genome Facility Gene Expression Core for RT–qPCR instrumentation; and R. Petersen and C. Howe for feedback on the manuscript. This work was supported by the Ellison Medical Foundation, the Glenn Foundation for Medical Research, the National Institutes of Health (R01AG053229), the Mayo Clinic Children’s Research Center and the Alzheimer’s Disease Research Center of Mayo Clinic (all to D.J.B.).

Reviewer information

Nature thanks M. Serrano and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Tyler J. Bussian, Asef Aziz

Affiliations

  1. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA

    • Tyler J. Bussian
    • , Jan M. van Deursen
    •  & Darren J. Baker
  2. Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN, USA

    • Asef Aziz
    • , Charlton F. Meyer
    • , Barbara L. Swenson
    • , Jan M. van Deursen
    •  & Darren J. Baker

Authors

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  2. Search for Asef Aziz in:

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Contributions

T.J.B. and A.A. performed most of the experiments. C.F.M. assisted with the senescent cell identification by Gal-TEM and FACS. B.L.S. performed immunohistochemistry assessments. J.M.v.D. assisted with experimental design and data interpretation. The manuscript was written by T.J.B. and D.J.B. All authors discussed results, made figures and edited the manuscript. D.J.B. conceived, directed and supervised all aspects of the study.

Competing interests

D.J.B. and J.M.v.D. are co-inventors on patent applications licensed to or filed by Unity Biotechnology, a company developing senolytic medicines, including small molecules that selectively eliminate senescent cells. J.M.v.D. is a co-founder of Unity Biotechnology. Research in the Baker laboratory has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with Mayo Clinic Conflict of Interest policies.

Corresponding author

Correspondence to Darren J. Baker.

Extended data figures and tables

  1. Extended Data Fig. 1 Senescent cells accumulate in PS19 mice.

    RT–qPCR analysis of senescence-associated genes in hippocampi (left) and cortices (right) of three- and ten-month-old male mice. Number of mice is as indicated, two independent experiments; normalized to the three-month wild-type group. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). Exact P values can be found in the accompanying Source Data. Source Data

  2. Extended Data Fig. 2 AP-mediated clearance selectively removes senescent cells that accumulate in the brains of PS19;ATTAC mice.

    RT–qPCR analysis of the expression of senescence markers in the hippocampus (left) and cortex (right) of six-month-old female mice, treated with either vehicle (–AP) or AP20187 (+AP). Number of mice is as indicated; normalized to the ATTAC – AP group. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). Exact P values can be found in the accompanying Source Data. Source Data

  3. Extended Data Fig. 3 Neurons do not exhibit X-Gal crystals upon Gal-TEM imaging.

    Representative electron microscopy image of neurons after SA-β-Gal staining from a six-month-old, vehicle-treated PS19;ATTAC male mouse (n = 3 male mice, 2 independent experiments). The image has been artificially coloured to show individual cell bodies. Scale bar, 10 µm.

  4. Extended Data Fig. 4 Increased expression of senescence-associated genes is observed in astrocytes and microglia isolated from PS19 mice.

    a–e, Gating strategy (a) for FACS isolation of living astrocytes (b), microglia (c), oligodendrocytes (d) and neuron-enriched CD56+ cells (e) from cortices of six-month-old wild-type and PS19 mice. b, Astrocyte (CD11bCD45O1GLAST+CD56) fraction (left) and RT–qPCR analysis (right). c, Microglia (CD11b+CD45+O1GLAST+CD56) fraction (left) and RT–qPCR analysis (right). d, Oligodendrocyte (CD11bCD45O1+GLASTCD56) fraction (left) and RT–qPCR analysis (right). e, Neuron-enriched CD56+ (CD11bCD45O1GLASTCD56+) fraction (left) and RT–qPCR analysis (right). Individual numbers of independent mouse cell population isolations are indicated in the parentheses above p16Ink4a columns (2 independent experiments). Data are mean ± s.e.m. *P < 0.05; **P < 0.01 (unpaired two-sided t-tests with Welch’s correction). Exact P values can be found in the accompanying Source Data. Source Data

  5. Extended Data Fig. 5 Verification of the identity of cell populations isolated by FACS.

    a–d, RT–qPCR analysis of cell-identity markers from cell populations isolated from six-month-old wild-type and PS19 mice: Aqp4 expression enriched in astrocytes (a), Cx3cr1 expression enriched in microglia (b), Olig2 expression enriched in oligodendrocytes (c) and Nefl expression enriched in neurons (d). Expression is normalized to intact cortices of six-month-old wild-type mice (n = 4 biologically independent cell isolations for each group, 2 independent experiments). Data are mean ± s.e.m. ***P <  < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). Exact P values can be found in the accompanying Source Data. Source Data

  6. Extended Data Fig. 6 AP administration does not erroneously eliminate non-senescent glial cells isolated from ATTAC mice.

    a, CD11b staining of primary microglia treated with IFNγ (200 ng ml−1), LPS (100 ng ml−1) or a combination of both (n = 3 biologically independent samples). b, Quantification of TUNEL-positive bodies in basal or activated microglia (n = 4 wild-type and 8 ATTAC cultures for each treatment group, 2 independent experiments). c, GFAP staining of primary astrocytes treated with IFNγ, LPS or a combination of both as described in a (n = 3 biologically independent samples). d, Quantification of the change in confluency over 24 h in basal or activated astrocytes (n = 4 biologically independent cultures of each genotype and treatment). Scale bars, 100 μm (a, c). Data are mean ± s.e.m. *P < 0.05; ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test (b, d)). Exact P values can be found in the accompanying Source Data. Source Data

  7. Extended Data Fig. 7 Administration of AP does not broadly eliminate cells or increase proliferation of microglia.

    a, Quantification of TUNEL-positive bodies (as a percentage of all cells) at the transition between the CA2 and CA3 within the hippocampus after a short-term AP administration in six-month-old mice (n = 3 mice per genotype and treatment group). b, Quantification of IBA1/EdU double-positive cells in the hippocampus and cortex of six-month-old mice that were administered AP beginning at weaning age (n = 4 mice per genotype and treatment group). Data are mean ± s.e.m. We note that no comparison is statistically significant (one-way ANOVA with Tukey’s multiple comparisons test). Exact P values can be found in the accompanying Source Data. Source Data

  8. Extended Data Fig. 8 Senescent cells promote gliosis.

    a, RT–qPCR analysis of Gfap, S100b and Cd11b in the hippocampi of six-month-old male mice (n = 5 mice per group; normalized to the ATTAC – AP group). b, RT–qPCR analysis as in a in hippocampi of six-month-old female mice (number of mice as indicated; normalized to the ATTAC – AP group). c, Representative GFAP immunohistochemistry staining in the hippocampus of six-month-old vehicle and AP-treated ATTAC and PS19;ATTAC female mice (n = 4 mice per group, 2 independent experiments). d, Representative IBA1 staining in the hippocampus of six-month-old vehicle and AP-treated ATTAC and PS19;ATTAC female mice (n = 4 mice per group, 2 independent experiments). Scale bar, 100 µm (c) and 50 µm (d). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). Exact P values can be found in the accompanying Source Data. Source Data

  9. Extended Data Fig. 9 AP treatment attenuates tau phosphorylation.

    a, Ponceau-S loading controls for the western-blot lysates of the whole brain of six-month-old mice (shown in Fig. 3a) for total tau (left) and phosphorylated tau (S202/T205; right). b, Quantification of the western-blot analysis of the whole brain of six-month-old mice for soluble tau (left), soluble phosphorylated tau (S202/T205; middle), and insoluble phosphorylated tau (S202/T205; right). Numbers of biologically independent mice are indicated in parentheses, data are from ≥3 independent experiments. c, Immunostaining of the cortex of six-month-old mice for tau protein phosphorylated at T231 (top) and S396 (bottom; n = 4 mice per group, 2 independent experiments). Scale bar, 100 µm. Data are mean ± s.e.m. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). Exact P values can be found in the accompanying Source Data. Source Data

  10. Extended Data Fig. 10 Vision-based novel-object discrimination remains intact in AP-treated PS19;ATTAC mice.

    Objects used for novel-object recognition during the training and testing phases for visual discrimination (left) and the average ratio for the number of investigations (right, n = 8 female mice per group). Data are mean ± s.e.m. **P < 0.01; ***P < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test). Exact P values can be found in the accompanying Source Data. Source Data

Supplementary information

  1. Supplementary Figure 1

    This file contains the uncropped scans with size marker indications.

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DOI

https://doi.org/10.1038/s41586-018-0543-y

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