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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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The PP2A-A subunit, PR65, is a novel ATM target and mediates nuclear accumulation of HDAC4 in ATM-deficient neuronsa) Protein extracts from Atm+/+ and Atm−/− mouse cerebellum were immunoprecipitated with the PR65, PP2A-C or HDAC4 and blotted with phospho-[S/T]Q antibody.b) In vitro ATM kinase assays of His-tagged HDAC4, GST-tagged PR65 or PR65S401A were performed with N2a cell extract.c) Co-immunoprecipitation assays of PP2A-A and HDAC4 in lysates prepared from N2a cells with overexpression of GFP-PP2A-A (WT, S401A or S401D) and Flag-HDAC4. Lysates were immunoprecipitated with anti-Flag or anti-GFP antibodies and blotted with phospho-[S/T]Q antibody.d) Representative images of PP2A distribution in Atm+/+ and Atm−/− cultured neurons with co-expression of GFP-PP2A-A, wild-type, S401A or S401Dand mCherry-PP2A-C. Scale bar, 20 μm.e) Immunofluorescent images of endogenous or exogenous HDAC4 (green) and PP2A (red) at DIV14 in wild-type and Atm−/− primary neurons. Scale bar, 25 μm.f) Effect of inhibition of ATM activity by caffeine on the localization of GFP-HDAC4 in wild-type E16.5 cortical neurons. The five small panels to the right are isolated images of the cell body. The numeral in each of the small panels represents the time elapsed (hours) since the addition of ATM inhibitor. Scale bar, 20 μm.g) Effect of knocking down PP2A on ATM-deficient GFP-HDAC4 nuclear accumulation in neurons. Scale bar, 20 μm. Small panels as in (f).h) Immunofluorescent images of HDAC4 (green) at DIV7 in either shGapdh- or shPp2a-infected wild-type and Atm−/− primary neurons with one hour pretreatment with the PP2A-specific inhibitor, Endothall (5 μM). Scale bar, 25 μm.
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Figure 5: The PP2A-A subunit, PR65, is a novel ATM target and mediates nuclear accumulation of HDAC4 in ATM-deficient neuronsa) Protein extracts from Atm+/+ and Atm−/− mouse cerebellum were immunoprecipitated with the PR65, PP2A-C or HDAC4 and blotted with phospho-[S/T]Q antibody.b) In vitro ATM kinase assays of His-tagged HDAC4, GST-tagged PR65 or PR65S401A were performed with N2a cell extract.c) Co-immunoprecipitation assays of PP2A-A and HDAC4 in lysates prepared from N2a cells with overexpression of GFP-PP2A-A (WT, S401A or S401D) and Flag-HDAC4. Lysates were immunoprecipitated with anti-Flag or anti-GFP antibodies and blotted with phospho-[S/T]Q antibody.d) Representative images of PP2A distribution in Atm+/+ and Atm−/− cultured neurons with co-expression of GFP-PP2A-A, wild-type, S401A or S401Dand mCherry-PP2A-C. Scale bar, 20 μm.e) Immunofluorescent images of endogenous or exogenous HDAC4 (green) and PP2A (red) at DIV14 in wild-type and Atm−/− primary neurons. Scale bar, 25 μm.f) Effect of inhibition of ATM activity by caffeine on the localization of GFP-HDAC4 in wild-type E16.5 cortical neurons. The five small panels to the right are isolated images of the cell body. The numeral in each of the small panels represents the time elapsed (hours) since the addition of ATM inhibitor. Scale bar, 20 μm.g) Effect of knocking down PP2A on ATM-deficient GFP-HDAC4 nuclear accumulation in neurons. Scale bar, 20 μm. Small panels as in (f).h) Immunofluorescent images of HDAC4 (green) at DIV7 in either shGapdh- or shPp2a-infected wild-type and Atm−/− primary neurons with one hour pretreatment with the PP2A-specific inhibitor, Endothall (5 μM). Scale bar, 25 μm.

Mentions: Serine 401 of PP2A-A is a highly probable site of ATM phosphorylation32. We therefore performed immunoprecipitation with PP2A-A, PP2A-C and HDAC4 antibodies and probed the resulting Western blots with an antibody against phosphorylated serine or threonine preceding a glutamine residue (P-[S/T]Q) – the canonical ATM/ATR target site. A strong P-[S/T]Q signal was found on the PP2A-A band from wild-type but not from Atm−/− mice (Fig. 5a). No P-[S/T]Q signal was found in either genotype with PP2A-C or HDAC4 immunoprecipitates (Fig. 5a). As further proof that HDAC4 itself is not an ATM substrate, no P-[S/T]Q signal was also found in N2a cells with overexpressing Flag-HDAC4 (Supplementary Fig. 6a). We verified that S401 is the predominant ATM phosphorylation site on PP2A by in vitro kinase assays (Fig. 5b); an S401A PP2A-A mutant could not be phosphorylated by ATM. We overexpressed Flag-tagged HDAC4 with GFP-tagged isoforms of PP2A-A, and analyzed the Flag-HDAC4 immunoprecipitates for PP2A-A. We found a strong HDAC4-PP2A association with the non-phosphorylatable (S401A) PP2A-A isoform (Fig. 5c), but not with wild-type or the phosphomimetic (S401D) isoform (Fig. 5c). This was confirmed by probing PP2A-A immunoprecipitates for Flag-HDAC4 (Fig. 5c).


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)

The PP2A-A subunit, PR65, is a novel ATM target and mediates nuclear accumulation of HDAC4 in ATM-deficient neuronsa) Protein extracts from Atm+/+ and Atm−/− mouse cerebellum were immunoprecipitated with the PR65, PP2A-C or HDAC4 and blotted with phospho-[S/T]Q antibody.b) In vitro ATM kinase assays of His-tagged HDAC4, GST-tagged PR65 or PR65S401A were performed with N2a cell extract.c) Co-immunoprecipitation assays of PP2A-A and HDAC4 in lysates prepared from N2a cells with overexpression of GFP-PP2A-A (WT, S401A or S401D) and Flag-HDAC4. Lysates were immunoprecipitated with anti-Flag or anti-GFP antibodies and blotted with phospho-[S/T]Q antibody.d) Representative images of PP2A distribution in Atm+/+ and Atm−/− cultured neurons with co-expression of GFP-PP2A-A, wild-type, S401A or S401Dand mCherry-PP2A-C. Scale bar, 20 μm.e) Immunofluorescent images of endogenous or exogenous HDAC4 (green) and PP2A (red) at DIV14 in wild-type and Atm−/− primary neurons. Scale bar, 25 μm.f) Effect of inhibition of ATM activity by caffeine on the localization of GFP-HDAC4 in wild-type E16.5 cortical neurons. The five small panels to the right are isolated images of the cell body. The numeral in each of the small panels represents the time elapsed (hours) since the addition of ATM inhibitor. Scale bar, 20 μm.g) Effect of knocking down PP2A on ATM-deficient GFP-HDAC4 nuclear accumulation in neurons. Scale bar, 20 μm. Small panels as in (f).h) Immunofluorescent images of HDAC4 (green) at DIV7 in either shGapdh- or shPp2a-infected wild-type and Atm−/− primary neurons with one hour pretreatment with the PP2A-specific inhibitor, Endothall (5 μM). Scale bar, 25 μm.
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Figure 5: The PP2A-A subunit, PR65, is a novel ATM target and mediates nuclear accumulation of HDAC4 in ATM-deficient neuronsa) Protein extracts from Atm+/+ and Atm−/− mouse cerebellum were immunoprecipitated with the PR65, PP2A-C or HDAC4 and blotted with phospho-[S/T]Q antibody.b) In vitro ATM kinase assays of His-tagged HDAC4, GST-tagged PR65 or PR65S401A were performed with N2a cell extract.c) Co-immunoprecipitation assays of PP2A-A and HDAC4 in lysates prepared from N2a cells with overexpression of GFP-PP2A-A (WT, S401A or S401D) and Flag-HDAC4. Lysates were immunoprecipitated with anti-Flag or anti-GFP antibodies and blotted with phospho-[S/T]Q antibody.d) Representative images of PP2A distribution in Atm+/+ and Atm−/− cultured neurons with co-expression of GFP-PP2A-A, wild-type, S401A or S401Dand mCherry-PP2A-C. Scale bar, 20 μm.e) Immunofluorescent images of endogenous or exogenous HDAC4 (green) and PP2A (red) at DIV14 in wild-type and Atm−/− primary neurons. Scale bar, 25 μm.f) Effect of inhibition of ATM activity by caffeine on the localization of GFP-HDAC4 in wild-type E16.5 cortical neurons. The five small panels to the right are isolated images of the cell body. The numeral in each of the small panels represents the time elapsed (hours) since the addition of ATM inhibitor. Scale bar, 20 μm.g) Effect of knocking down PP2A on ATM-deficient GFP-HDAC4 nuclear accumulation in neurons. Scale bar, 20 μm. Small panels as in (f).h) Immunofluorescent images of HDAC4 (green) at DIV7 in either shGapdh- or shPp2a-infected wild-type and Atm−/− primary neurons with one hour pretreatment with the PP2A-specific inhibitor, Endothall (5 μM). Scale bar, 25 μm.
Mentions: Serine 401 of PP2A-A is a highly probable site of ATM phosphorylation32. We therefore performed immunoprecipitation with PP2A-A, PP2A-C and HDAC4 antibodies and probed the resulting Western blots with an antibody against phosphorylated serine or threonine preceding a glutamine residue (P-[S/T]Q) – the canonical ATM/ATR target site. A strong P-[S/T]Q signal was found on the PP2A-A band from wild-type but not from Atm−/− mice (Fig. 5a). No P-[S/T]Q signal was found in either genotype with PP2A-C or HDAC4 immunoprecipitates (Fig. 5a). As further proof that HDAC4 itself is not an ATM substrate, no P-[S/T]Q signal was also found in N2a cells with overexpressing Flag-HDAC4 (Supplementary Fig. 6a). We verified that S401 is the predominant ATM phosphorylation site on PP2A by in vitro kinase assays (Fig. 5b); an S401A PP2A-A mutant could not be phosphorylated by ATM. We overexpressed Flag-tagged HDAC4 with GFP-tagged isoforms of PP2A-A, and analyzed the Flag-HDAC4 immunoprecipitates for PP2A-A. We found a strong HDAC4-PP2A association with the non-phosphorylatable (S401A) PP2A-A isoform (Fig. 5c), but not with wild-type or the phosphomimetic (S401D) isoform (Fig. 5c). This was confirmed by probing PP2A-A immunoprecipitates for Flag-HDAC4 (Fig. 5c).

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