Nuclear accumulation of HDAC4 in ATM deficiency promotes neurodegeneration in ataxia telangiectasia

Journal name:
Nature Medicine
Volume:
18,
Pages:
783–790
Year published:
DOI:
doi:10.1038/nm.2709
Received
Accepted
Published online

Abstract

Ataxia telangiectasia is a neurodegenerative disease caused by mutation of the Atm gene. Here we report that ataxia telangiectasia mutated (ATM) deficiency causes nuclear accumulation of histone deacetylase 4 (HDAC4) in neurons and promotes neurodegeneration. Nuclear HDAC4 binds to chromatin, as well as to myocyte enhancer factor 2A (MEF2A) and cAMP-responsive element binding protein (CREB), leading to histone deacetylation and altered neuronal gene expression. Blocking either HDAC4 activity or its nuclear accumulation blunts these neurodegenerative changes and rescues several behavioral abnormalities of ATM-deficient mice. Full rescue of the neurodegeneration, however, also requires the presence of HDAC4 in the cytoplasm, suggesting that the ataxia telangiectasia phenotype results both from a loss of cytoplasmic HDAC4 as well as its nuclear accumulation. To remain cytoplasmic, HDAC4 must be phosphorylated. The activity of the HDAC4 phosphatase, protein phosphatase 2A (PP2A), is downregulated by ATM-mediated phosphorylation. In ATM deficiency, enhanced PP2A activity leads to HDAC4 dephosphorylation and the nuclear accumulation of HDAC4. Our results define a crucial role of the cellular localization of HDAC4 in the events leading to ataxia telangiectasia neurodegeneration.

At a glance

Figures

  1. Nuclear accumulation of HDAC4 in ATM-deficient neurons leads to the suppression of MEF2- and CREB-related transcriptional activities.
    Figure 1: Nuclear accumulation of HDAC4 in ATM-deficient neurons leads to the suppression of MEF2- and CREB-related transcriptional activities.

    (a) Paraffin sections of human cerebellar cortex from controls and patients with ataxia telangiectasia (A-T) and cryostat sections of Atm+/+ and Atm−/− mouse cerebella immunostained with antibody to HDAC4 using either horseradish peroxidase immunocytochemistry (brown) or immunofluorescence (green). Aldolase C (red) is a cytoplasmic marker of Purkinje cells. White boxes indicate areas magnified in the two single-channel inserts. (b,c) The percentage of Purkinje cells with nuclear accumulation of HDAC4 are shown in human ataxia telangiectasia samples (b) and Atm−/− mice (c). Values represent the percentage of the total Purkinje cell population (aldolase C counts). Each bar represents the average of three independent experiments. Error bars, s.e.m. *P < 0.05 by Student's t test. (d) Images of endogenous HDAC4 (endo-HDAC4) and exogenous HDAC4 (GFP-HDAC4) traffic in cultured neocortical neurons from both Atm+/+ and Atm−/− embryos. (e) Protein extracts from Atm+/+ and Atm−/− mouse cerebella immunoprecipitated (IP) with HDAC4 and blotted with antibodies to MEF2A. IgG, immunoglobulin G. (f) Protein extracts from Atm+/+ and Atm−/− mouse cerebella immunoprecipitated with HDAC4 and blotted with antibodies to CREB. (g) After ChIP with antibodies to MEF2A from Atm+/+ and Atm−/− cerebela, a quantitative real-time PCR analysis was performed for the presence of specific MEF2A target genes. (h) After ChIP with antibodies to CREB from Atm+/+ and Atm−/− cerebella, a quantitative real-time PCR analysis was performed for the presence of specific CREB target genes. *P < 0.01 by Student's t test (f,h). Gapdh was used as a control. Error bars, s.e.m. All the qPCR primers used are listed in Supplementary Table 2. (i,j) Validation of the effect of nuclear HDAC4 on the MEF2A-DNA and CREB-DNA interactions in Atm−/− neurons. Nuclear extracts (NE) from Atm+/+ and Atm−/− neurons with lentiviral Hdac9 shRNA (shHdac9) and Hdac4 shRNA (shHdac4) infection were incubated with biotin-labeled probes, as indicated.

  2. Nuclear accumulation of HDAC4 leads to global effects on histone acetylation and neuronal gene expression.
    Figure 2: Nuclear accumulation of HDAC4 leads to global effects on histone acetylation and neuronal gene expression.

    (a) Fluorescent images of sections from Atm+/+ and Atm−/− mouse cerebella immunostained for two histones (H3 and H4) and the acetylated histones (AcH3 and AcH4), as indicated. (b) Protein extracts of cortices and cerebella from wild-type and Atm−/− mice probed with antibodies to various histones, with the histones labeled to the left of the gels. Cont, control. (c,d) Quantification of three repetitions of the experiment illustrated in panel b. Error bars, s.d. *P < 0.05 by Student's t test. (eg) Fragmented chromatin immunoprecipitated with antibodies to H3, AcH3 or AcH4, as indicated, and quantified using real-time PCR. The primers used for the qPCR are listed in Supplementary Table 2. Error bars, s.e.m. (h) An illustration of the HDAC4 ChIP-seq alignment and peaks. A 2.7-Mb sample region of chromosome 1 (Chr1) shows the density of coverage of 35-nt sequencing tags from input DNA or ChIP from wild-type (blue) or Atm−/− (red) mouse brain. WT-HDAC4, wild-type HDAC4; Atm−/−-HDAC4, HDAC4 in Atm−/− (knockout) mice.

  3. Inhibition of HDAC4 and blocking the nuclear accumulation of HDAC4 partially reverses the ataxia telangiectasia phenotype.
    Figure 3: Inhibition of HDAC4 and blocking the nuclear accumulation of HDAC4 partially reverses the ataxia telangiectasia phenotype.

    (a) TSA injection reverses neuronal degeneration markers in the Atm−/− cerebellum. Fluorescent images of Atm−/− brain sections immunostained for cleaved caspase-3, as well as for PCNA and cyclin D1. The white arrows indicate labeled Purkinje cells. (b) Quantification of the degeneration markers for the experiment shown in a. Each bar represents the average of three independent experiments. Error bars, s.e.m. (c) Immunoblot assays of neuronal and cell-cycle proteins in cerebellar lysates prepared from DMSO- or TSA-injected wild-type and Atm−/− mice. (d) Quantification of the western blot bands shown in c. Error bars, s.d. (e) Effects of TSA on the motor function of Atm−/− and wild-type mice. Motor performance was measured as the average latency before falling from a rotarod. Each treatment group consisted of 4–6 mice. *P < 0.05 by analysis of variance (ANOVA). (f,g) Effects of TSA on the spontaneous locomotor activities (f) and the exploratory activities (g) in Atm−/− mice, as observed by open-field test. Data are means ± s.e.m.

  4. HDAC4 cytoplasmic localization requires phosphorylation of HDAC4 and is independent of DNA damage.
    Figure 4: HDAC4 cytoplasmic localization requires phosphorylation of HDAC4 and is independent of DNA damage.

    (a) The effect of Hdac4 shRNA on caspase-3 activation in Atm−/− neurons. Activation of caspase-3 (red) was used as an index of impending neurodegeneration; microtubule-associated protein 2 (Map2) (green) was used as a neuronal marker. Eto, etoposide; NT, no treatment; shGapdh, Gapdh shRNA. (b) Cell death quantified by counting the number of activated caspase-3–immunostained cells and expressing these numbers as a percentage of the total Map2-stained neurons. Data are mean ± s.e.m. (c) Mice treated with or without 5 Gy whole-body irradiation (IR). Cryostat sections of Atm+/+ and Atm−/− cerebella immunostained for HDAC4 (green) and H2A histone family member X (γ−H2AX) or phospho-Ser15 of p53 (both in red). At least three pairs of age-matched mice were used for each experiment. White boxes indicate areas magnified in the two single-channel insets. (d) Immunoblot assays of HDAC4 and phospho-S632 of HDAC4 in nuclear (Nuc) or cytoplasmic (Cyt) extracts prepared from Atm+/+ and Atm−/− mouse cerebella. Heat shock protein 90 (Hsp90) and HDAC1 were used as cytoplasmic and nuclear markers, respectively. (e) A quantification of the bands shown in d. *P < 0.05 by Student's t test. (f) Immunoblot assays of HDAC4 and phospho-HDAC4 in protein extracts prepared from frozen cerebellar samples from four human controls without ataxia telangiectasia and four individuals with ataxia telangiectasia. (g,h) Coimmunoprecipitations showing the interaction between HDAC4 and 14-3-3 protein in lysates of cerebellar tissue from human control and ataxia telangiectasia brains. WB, western blot. (i) Coimmunoprecipitations showing the association of HDAC4 with the PP2A subunits in lysates of cerebellar tissue from human control and ataxia telangiectasia brain. IB, immunoblot.

  5. The PP2A-A subunit, PR65, is a ATM target and mediates nuclear accumulation of HDAC4 in ATM-deficient neurons.
    Figure 5: The PP2A-A subunit, PR65, is a ATM target and mediates nuclear accumulation of HDAC4 in ATM-deficient neurons.

    (a) Protein extracts from Atm+/+ and Atm−/− mouse cerebella immunoprecipitated with PP2A-A, PP2A-C or HDAC4 and blotted with antibodies to phospho-Ser/Thr-Gln. MW, molecular weight. (b) In vitro ATM kinase assays of His-tagged HDAC4 (His-HDAC4), GST-tagged PP2A-A (GST–PP2A-A) or PP2A-A with the S401A mutation performed with N2a cell extract. (c) Coimmunoprecipitation assays of PP2A-A and HDAC4 in lysates prepared from N2a cells with overexpression of various forms of GFP–PP2A-A (WT, S401A or S401D) and Flag-HDAC4. Lysates were immunoprecipitated with antibodies to Flag or GFP and were blotted with antibodies to phospho-Ser/ThrGln. (d) Immunofluorescent images of endogenous or exogenous HDAC4 (green) and PP2A-A (red) in wild-type and Atm−/− primary neurons that were in culture for 14 d. White boxes indicate areas magnified in the two single-channel insets. (e) Representative images of PP2A distribution in Atm+/+ and Atm−/− cultured neurons with coexpression of GFP–PP2A-A and mCherry–PP2A-C with wild-type, S401A or S401D PP2A-A. Scale bar, 20 μm. (f) Effect of the inhibition of ATM activity by caffeine on the localization of GFP-HDAC4 in wild-type embryonic day 16.5 cortical neurons. The five small panels to the right are isolated images of the cell body. The number in each of the small panels represents the time elapsed (h) since the addition of the ATM inhibitor. (g) Effect of knocking down PP2A on ATM-deficient GFP-HDAC4 nuclear accumulation in neurons using caffeine plus Pp2a shRNA (shPp2a). The small panels on the right are as described in f. (h) Immunofluorescent images of HDAC4 (green) in wild-type and Atm−/− primary neurons infected with shGapdh or shPp2a lentivirus after 3 d in culture and treated with the PP2A-specific inhibitor endothall after 7 d in culture.

  6. Blocking nuclear HDAC4 activity rescues the Atm-/- phenotype in vivo and in vitro.
    Figure 6: Blocking nuclear HDAC4 activity rescues the Atm−/− phenotype in vivo and in vitro.

    (a,b) Representative images of PCNA-stained and cleaved-caspase-3–stained (red) Purkinje cells showing the effects of the lentiviral delivery of various HDAC4 mutants (green) on degenerative progression in Atm−/− mouse cerebella. 4A, NLS (nuclear localization signal) mutant HDAC4 (cytoplasmic); L1062A, nuclear export mutant HDAC4 (nuclear); 3SA, non-phosphorylatable HDAC4 (nuclear). White boxes indicate areas magnified in the two single-channel insets. (c) Rotarod tests showing the average latency to fall for wild-type and Atm−/− mice after injection of different HDAC4 and S401D lentiviral particles. *P < 0.001 by ANOVA. (d,e) Open-field tests showing the effects of different lentiviral particles of HDAC4 and PP2A-A S401D on the spontaneous locomotor activity (d) and exploratory activity (e) of Atm−/− mice. Each treatment group consisted of 4–6 mice. Data are means ± s.e.m.

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References

  1. Barzilai, A., Biton, S. & Shiloh, Y. The role of the DNA damage response in neuronal development, organization and maintenance. DNA Repair (Amst.) 7, 10101027 (2008).
  2. Bundey, S. Clinical and genetic features of ataxia-telangiectasia. Int. J. Radiat. Biol. 66, S23S29 (1994).
  3. Lavin, M.F. & Shiloh, Y. The genetic defect in ataxia-telangiectasia. Annu. Rev. Immunol. 15, 177202 (1997).
  4. Savitsky, K. et al. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 268, 17491753 (1995).
  5. Sedgwick, R.P. & Boder, E. Progressive ataxia in childhood with particular reference to ataxia-telangiectasia. Neurology 10, 705715 (1960).
  6. D'Mello, S.R. Histone deacetylases as targets for the treatment of human neurodegenerative diseases. Drug News Perspect. 22, 513524 (2009).
  7. Grozinger, C.M. & Schreiber, S.L. Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14–3-3–dependent cellular localization. Proc. Natl. Acad. Sci. USA 97, 78357840 (2000).
  8. Haberland, M., Montgomery, R.L. & Olson, E.N. The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat. Rev. Genet. 10, 3242 (2009).
  9. Majdzadeh, N., Morrison, B.E. & D'Mello, S.R. Class IIA HDACs in the regulation of neurodegeneration. Front. Biosci. 13, 10721082 (2008).
  10. Yang, X.J. & Seto, E. The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat. Rev. Mol. Cell Biol. 9, 206218 (2008).
  11. Darcy, M.J., Calvin, K., Cavnar, K. & Ouimet, C.C. Regional and subcellular distribution of HDAC4 in mouse brain. J. Comp. Neurol. 518, 722740 (2010).
  12. Grozinger, C.M., Hassig, C.A. & Schreiber, S.L. Three proteins define a class of human histone deacetylases related to yeast Hda1p. Proc. Natl. Acad. Sci. USA 96, 48684873 (1999).
  13. Wang, A.H. et al. HDAC4, a human histone deacetylase related to yeast HDA1, is a transcriptional corepressor. Mol. Cell. Biol. 19, 78167827 (1999).
  14. Majdzadeh, N. et al. HDAC4 inhibits cell-cycle progression and protects neurons from cell death. Dev. Neurobiol. 68, 10761092 (2008).
  15. McKinsey, T.A., Zhang, C.L., Lu, J. & Olson, E.N. Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation. Nature 408, 106111 (2000).
  16. Wang, A.H. et al. Regulation of histone deacetylase 4 by binding of 14–3-3 proteins. Mol. Cell. Biol. 20, 69046912 (2000).
  17. Zhao, X. et al. The modular nature of histone deacetylase HDAC4 confers phosphorylation-dependent intracellular trafficking. J. Biol. Chem. 276, 3504235048 (2001).
  18. Bolger, T.A. & Yao, T.P. Intracellular trafficking of histone deacetylase 4 regulates neuronal cell death. J. Neurosci. 25, 95449553 (2005).
  19. Chen, B. & Cepko, C.L. HDAC4 regulates neuronal survival in normal and diseased retinas. Science 323, 256259 (2009).
  20. Youn, H.D., Grozinger, C.M. & Liu, J.O. Calcium regulates transcriptional repression of myocyte enhancer factor 2 by histone deacetylase 4. J. Biol. Chem. 275, 2256322567 (2000).
  21. Kuljis, R.O., Xu, Y., Aguila, M.C. & Baltimore, D. Degeneration of neurons, synapses, and neuropil and glial activation in a murine Atm knockout model of ataxia-telangiectasia. Proc. Natl. Acad. Sci. USA 94, 1268812693 (1997).
  22. Xu, Y. et al. Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects, and thymic lymphoma. Genes Dev. 10, 24112422 (1996).
  23. Backs, J., Backs, T., Bezprozvannaya, S., McKinsey, T.A. & Olson, E.N. Histone deacetylase 5 acquires calcium/calmodulin-dependent kinase II responsiveness by oligomerization with histone deacetylase 4. Mol. Cell. Biol. 28, 34373445 (2008).
  24. Méjat, A. et al. Histone deacetylase 9 couples neuronal activity to muscle chromatin acetylation and gene expression. Nat. Neurosci. 8, 313321 (2005).
  25. Korzus, E., Rosenfeld, M.G. & Mayford, M. CBP histone acetyltransferase activity is a critical component of memory consolidation. Neuron 42, 961972 (2004).
  26. Nervi, C. et al. Inhibition of histone deacetylase activity by trichostatin A modulates gene expression during mouse embryogenesis without apparent toxicity. Cancer Res. 61, 12471249 (2001).
  27. Yoshida, M., Kijima, M., Akita, M. & Beppu, T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J. Biol. Chem. 265, 1717417179 (1990).
  28. Barlow, C. et al. Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell 86, 159171 (1996).
  29. Liu, Y., Randall, W.R. & Schneider, M.F. Activity-dependent and -independent nuclear fluxes of HDAC4 mediated by different kinases in adult skeletal muscle. J. Cell Biol. 168, 887897 (2005).
  30. Paroni, G. et al. PP2A regulates HDAC4 nuclear import. Mol. Biol. Cell 19, 655667 (2008).
  31. Shi, Y. Serine/threonine phosphatases: mechanism through structure. Cell 139, 468484 (2009).
  32. Matsuoka, S. et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316, 11601166 (2007).
  33. Chan, J.K., Sun, L., Yang, X.J., Zhu, G. & Wu, Z. Functional characterization of an amino-terminal region of HDAC4 that possesses MEF2 binding and transcriptional repressive activity. J. Biol. Chem. 278, 2351523521 (2003).
  34. Miska, E.A. et al. HDAC4 deacetylase associates with and represses the MEF2 transcription factor. EMBO J. 18, 50995107 (1999).
  35. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
  36. Li, H. et al. The Sequence Alignment/Map (SAM) format and SAMtools. Bioinformatics. 25, 20782079 (2009).
  37. Fejes, A.P. et al. FindPeaks 3.1: a tool for identifying areas of enrichment from massively parallel short-read sequencing technology. Bioinformatics 24, 17291730 (2008).
  38. Kaemmerer, W.F. et al. In vivo transduction of cerebellar Purkinje cells using adeno-associated virus vectors. Mol. Ther. 2, 446457 (2000).
  39. Cortés, M.L., Oehmig, A., Perry, K.F., Sanford, J.D. & Breakefield, X.O. Expression of human ATM cDNA in Atm-deficient mouse brain mediated by HSV-1 amplicon vector. Neuroscience 141, 12471256 (2006).
  40. Cooper, J.F. & Kusnecov, A.W. Methylmercuric chloride induces activation of neuronal stress circuitry and alters exploratory behavior in the mouse. Neuroscience 148, 10481064 (2007).

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Affiliations

  1. Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, Rutgers University, Piscataway, New Jersey, USA.

    • Jiali Li,
    • Jianmin Chen,
    • Christopher L Ricupero,
    • Ronald P Hart &
    • Karl Herrup
  2. Department of Psychology, Rutgers University, Piscataway, New Jersey, USA.

    • Melanie S Schwartz &
    • Alexander Kusnecov

Contributions

J.L. and K.H. designed the experiments, analyzed data and wrote the manuscript. C.L.R. and R.P.H. developed, carried out and analyzed data for the ChIP-seq analyses. J.L. and J.C. carried out the immunocytochemistry experiments. J.C. performed all of the qPCR experiments. J.L. and A.K. carried out the mouse cerebellar lentiviral injections. M.S.S., J.L. and A.K. carried out behavioral tests.

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The authors declare no competing financial interests.

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    Supplementary Figures 1–8, Supplementary Tables 1–3 and Supplementary Methods

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