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

Li J, Chen J, Ricupero CL, Hart RP, Schwartz MS, Kusnecov A, Herrup K - Nat. Med. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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.

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HDAC4 cytoplasmic localization requires its phosphorylation and is independent of DNA damage.a) Effect of shHdac4 on caspase3 activation in Atm−/− neurons. Activation of caspase3 (red) was used as an index of impending neurodegeneration; Map2 (green) was used as a neuronal marker. Scale bar, 50 μm.b) Cell death was quantified by counting the number of activated caspase3 immunostained cells and expressing these numbers as a percentage of the total Map2-stained neurons (* = p < 0.05).c) Mice were treated with or without 5 Gy whole-body irradiation. Cryostat sections of Atm+/+ and Atm−/− cerebellum were immunostained for HDAC4 (green) and γ–H2AX or phospho-S15 of p53 (both in red). Scale bar, 50 μm.At least three-pair of age-matched animals were used for each experiment.d) Immunoblot assays of HDAC4 and phospho-S632-HDAC4 in nuclear or cytoplasmic extracts prepared from Atm+/+ and Atm−/− mouse cerebellum. Hsp90 and HDAC1 were used as cytoplasmic and nuclear marker respectively.e) Quantification of the bands shown in (d) reveals a significant decrease in the ratio of phosphorylated to non-phosphorylated HDAC4 in Atm−/− mouse cerebellum (* = p < 0.05).f) ) Immunoblot assays of HDAC4 and phospho-HDAC4 in protein extracts prepared from frozen cerebellar samples of 4 human controls and 4 individuals with A-T.g-h) Co-immunoprecipitations show the interaction between HDAC4 and 14-3-3 in lysates of cerebellar tissue from human control and A-T brain.i) Co-immunoprecipitations show the association of HDAC4 with PP2A subunits in lysates of cerebellar tissue from human control and A-T brain.
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Figure 4: HDAC4 cytoplasmic localization requires its phosphorylation and is independent of DNA damage.a) Effect of shHdac4 on caspase3 activation in Atm−/− neurons. Activation of caspase3 (red) was used as an index of impending neurodegeneration; Map2 (green) was used as a neuronal marker. Scale bar, 50 μm.b) Cell death was quantified by counting the number of activated caspase3 immunostained cells and expressing these numbers as a percentage of the total Map2-stained neurons (* = p < 0.05).c) Mice were treated with or without 5 Gy whole-body irradiation. Cryostat sections of Atm+/+ and Atm−/− cerebellum were immunostained for HDAC4 (green) and γ–H2AX or phospho-S15 of p53 (both in red). Scale bar, 50 μm.At least three-pair of age-matched animals were used for each experiment.d) Immunoblot assays of HDAC4 and phospho-S632-HDAC4 in nuclear or cytoplasmic extracts prepared from Atm+/+ and Atm−/− mouse cerebellum. Hsp90 and HDAC1 were used as cytoplasmic and nuclear marker respectively.e) Quantification of the bands shown in (d) reveals a significant decrease in the ratio of phosphorylated to non-phosphorylated HDAC4 in Atm−/− mouse cerebellum (* = p < 0.05).f) ) Immunoblot assays of HDAC4 and phospho-HDAC4 in protein extracts prepared from frozen cerebellar samples of 4 human controls and 4 individuals with A-T.g-h) Co-immunoprecipitations show the interaction between HDAC4 and 14-3-3 in lysates of cerebellar tissue from human control and A-T brain.i) Co-immunoprecipitations show the association of HDAC4 with PP2A subunits in lysates of cerebellar tissue from human control and A-T brain.

Mentions: To determine whether HDAC4 played a role in the neuronal DNA damage response, we exposed cultures of wild-type neurons to low doses of etoposide. This enhanced the activation of caspase-3, but the effect was 10-fold greater in Atm−/− neurons (Fig. 4a, b). When shRNA against HDAC4 was used, however, the cell death induced by etoposide increased in wild-type with little effect on Atm−/− cells (Fig. 4a, b). Thus loss of cytoplasmic HDAC4 makes neurons more sensitive to DNA damage. Yet HDAC4 itself does not respond to DNA damage. We exposed mice to 5 Gy of whole body irradiation, activating ATM in wild-type, but not in Atmtm1Bal mutants (Fig. 4c). The cytoplasmic location of HDAC4 nonetheless remained unchanged in both genotypes (Fig. 4c).


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

Li J, Chen J, Ricupero CL, Hart RP, Schwartz MS, Kusnecov A, Herrup K - Nat. Med. (2012)

HDAC4 cytoplasmic localization requires its phosphorylation and is independent of DNA damage.a) Effect of shHdac4 on caspase3 activation in Atm−/− neurons. Activation of caspase3 (red) was used as an index of impending neurodegeneration; Map2 (green) was used as a neuronal marker. Scale bar, 50 μm.b) Cell death was quantified by counting the number of activated caspase3 immunostained cells and expressing these numbers as a percentage of the total Map2-stained neurons (* = p < 0.05).c) Mice were treated with or without 5 Gy whole-body irradiation. Cryostat sections of Atm+/+ and Atm−/− cerebellum were immunostained for HDAC4 (green) and γ–H2AX or phospho-S15 of p53 (both in red). Scale bar, 50 μm.At least three-pair of age-matched animals were used for each experiment.d) Immunoblot assays of HDAC4 and phospho-S632-HDAC4 in nuclear or cytoplasmic extracts prepared from Atm+/+ and Atm−/− mouse cerebellum. Hsp90 and HDAC1 were used as cytoplasmic and nuclear marker respectively.e) Quantification of the bands shown in (d) reveals a significant decrease in the ratio of phosphorylated to non-phosphorylated HDAC4 in Atm−/− mouse cerebellum (* = p < 0.05).f) ) Immunoblot assays of HDAC4 and phospho-HDAC4 in protein extracts prepared from frozen cerebellar samples of 4 human controls and 4 individuals with A-T.g-h) Co-immunoprecipitations show the interaction between HDAC4 and 14-3-3 in lysates of cerebellar tissue from human control and A-T brain.i) Co-immunoprecipitations show the association of HDAC4 with PP2A subunits in lysates of cerebellar tissue from human control and A-T brain.
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Figure 4: HDAC4 cytoplasmic localization requires its phosphorylation and is independent of DNA damage.a) Effect of shHdac4 on caspase3 activation in Atm−/− neurons. Activation of caspase3 (red) was used as an index of impending neurodegeneration; Map2 (green) was used as a neuronal marker. Scale bar, 50 μm.b) Cell death was quantified by counting the number of activated caspase3 immunostained cells and expressing these numbers as a percentage of the total Map2-stained neurons (* = p < 0.05).c) Mice were treated with or without 5 Gy whole-body irradiation. Cryostat sections of Atm+/+ and Atm−/− cerebellum were immunostained for HDAC4 (green) and γ–H2AX or phospho-S15 of p53 (both in red). Scale bar, 50 μm.At least three-pair of age-matched animals were used for each experiment.d) Immunoblot assays of HDAC4 and phospho-S632-HDAC4 in nuclear or cytoplasmic extracts prepared from Atm+/+ and Atm−/− mouse cerebellum. Hsp90 and HDAC1 were used as cytoplasmic and nuclear marker respectively.e) Quantification of the bands shown in (d) reveals a significant decrease in the ratio of phosphorylated to non-phosphorylated HDAC4 in Atm−/− mouse cerebellum (* = p < 0.05).f) ) Immunoblot assays of HDAC4 and phospho-HDAC4 in protein extracts prepared from frozen cerebellar samples of 4 human controls and 4 individuals with A-T.g-h) Co-immunoprecipitations show the interaction between HDAC4 and 14-3-3 in lysates of cerebellar tissue from human control and A-T brain.i) Co-immunoprecipitations show the association of HDAC4 with PP2A subunits in lysates of cerebellar tissue from human control and A-T brain.
Mentions: To determine whether HDAC4 played a role in the neuronal DNA damage response, we exposed cultures of wild-type neurons to low doses of etoposide. This enhanced the activation of caspase-3, but the effect was 10-fold greater in Atm−/− neurons (Fig. 4a, b). When shRNA against HDAC4 was used, however, the cell death induced by etoposide increased in wild-type with little effect on Atm−/− cells (Fig. 4a, b). Thus loss of cytoplasmic HDAC4 makes neurons more sensitive to DNA damage. Yet HDAC4 itself does not respond to DNA damage. We exposed mice to 5 Gy of whole body irradiation, activating ATM in wild-type, but not in Atmtm1Bal mutants (Fig. 4c). The cytoplasmic location of HDAC4 nonetheless remained unchanged in both genotypes (Fig. 4c).

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus