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, 18 (5), 783-90

Nuclear Accumulation of HDAC4 in ATM Deficiency Promotes Neurodegeneration in Ataxia Telangiectasia

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Nuclear Accumulation of HDAC4 in ATM Deficiency Promotes Neurodegeneration in Ataxia Telangiectasia

Jiali Li et al. Nat Med.

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.

Figures

Figure 1
Figure 1
Nuclear accumulation of HDAC4 in ATM-deficient neurons leads to suppression of MEF2- and CREB-related transcriptional activities a) Paraffin sections of human cerebellar cortex from controls and A-T patients and cryostat sections of Atm+/+ and Atm−/− mouse cerebellum were immunostained with HDAC4 antibody using either HRP immunocytochemistry (brown) or immunofluorescence (green). Aldolase C (red) immunostaining was used as a cytoplasmic marker of Purkinje cells. Scale bar, 50μm. b-c) The percentage of Purkinje cells with nuclear accumulation of HDAC4 were shown from A-T 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 denote SEM. (* = p < 0.05). d) Images of endogenous and exogenous HDAC4 traffic in cultured neocortical neurons from both Atm+/+ and Atm−/− embryos. Scale bar, 20μm. e, g) Protein extracts from Atm+/+ and Atm−/− mouse cerebella were immunoprecipitated with HDAC4 and blotted with MEF2A (e) or CREB (g) antibodies. f - h) Following ChIP with MEF2A or CREB antibody from Atm+/+ and Atm−/− cerebellum, quantitative real-time PCR analysis was performed for the presence of specific MEF2A (f) or CREB (h) target genes (* = p < 0.01). Gapdh was used as a control. All q-PCR primers are listed in Table S1. i, j) Validation of the effect of nuclear HDAC4 on MEF2A-DNA and CREB-DNA interaction in Atm−/− neurons. Nuclear extracts (NE) from Atm+/+and Atm−/− neurons with lentiviral shHdac9 and shHdac4 infection were incubated with biotin-labeled probes as indicated.
Figure 2
Figure 2
Nuclear accumulation HDAC4 leads to global effects on histone acetylation and neuronal gene expression a) Fluorescent images of Atm+/+ and Atm−/− cerebellum sections immunostained for different histones and acetylated histones as indicated. b) Protein extracts of cortex and cerebellum from wild-type and Atm−/− mice were probed with various histone antibodies as labeled to the left of the gels. c-d) Quantification of 3 repetitions of the experiment illustrated in panel I. Error bars denotes standard deviations. * = p<0.05 (by Student’s T-test). e-g) Fragmented chromatin was immunoprecipitated with the antibodies indicated and quantified with real-time PCR. The primers used for q-PCR are listed in Table S1. Statistical analysis was carried out using Student’s t test. Error bars represent SEM h) An illustration of the HDAC4 ChIP-seq alignment and peaks. A 2.7 Mb sample region of chromosome 1 shows the density of coverage of 35 nt sequencing tags from input DNA or ChIP from wild type (blue) or Atm−/− (red) mouse brain.
Figure 3
Figure 3
Inhibition of HDAC4 and blocking its nuclear accumulation partially reverses the A-T 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 PCNA and cyclin D1. White arrows indicate the labeled Purkinje cells. Scale bar, 25μm. b) Quantification of the degeneration markers for the experiment illustrated in (a). Each bar represents the average of three independent experiments; error bars denote SEM. c) Immunoblot assays of neuronal and cell cycle genes in mice cerebella lysates prepared from DMSO- or TSA-injected wild-type and Atm−/− mice) d) Quantification of western blot bands illustrated in panel (c). Error bars denotes standard deviations. e) Effects of TSA on the motor function of Atm−/− and wild-type animals. Motor performance measured as the average latency before falling from a rota-rod. Each treatment group consisted of 4-6 animals. f-g) Effects of TSA on the spontaneous locomotor activities (f) and the exploratory activities (g) in Atm−/−mice were observed by open-field test. Data are presented as mean values ± SEM.
Figure 4
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.
Figure 5
Figure 5
The PP2A-A subunit, PR65, is a novel ATM target and mediates nuclear accumulation of HDAC4 in ATM-deficient neurons a) 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.
Figure 6
Figure 6
a-b) Representative images of PCNA- and cleaved caspase3-stained Purkinje cells show the effects of lentiviral delivery of different HDAC4 mutants on degenerative progression in Atm−/− mouse cerebellum.. NLS-HDAC4 (cytoplasmic) = 4A; nuclear export mutant HDAC4 (nuclear) = L1062A; Non-phosphorylatable HDAC4 (nuclear) = 3SA. Scale bar, 50 μm. c) Rota-rod tests show average latency to fall for wild type (+/+) and Atm−/− animals after injection of different HDAC4 as well as S401D lentiviral particles. d-e) Open–field tests show effects of different lentiviral HDAC4 as well as S401D on the spontaneous locomotor activity (d) and exploratory activity (e) in Atm−/−mice. Each treatment group consisted of 4-6 animals. Data are presented as mean values ± SEM.

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