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

Mentions: The analogies of neuron death and dendritic atrophy in the cerebella of ATM- and HDAC4-deficient mice9,18,21,22 prompted us to examine HDAC4 in human A-T cerebella. Normally, HDAC4 immunoreactivity is found in Purkinje cell cytoplasm (Fig.1a). In A-T samples by contrast Purkinje cell nuclei had strong HDAC4 staining (Fig. 1a, b). The nuclear accumulation of HDAC4 was specific; despite structural and functional similarities23,24, HDAC5 and HDAC9 showed little nuclear accumulation (Supplementary Fig.1a). These observations were replicated in mice. HDAC4, but not HDAC5 or HDAC9 (Supplementary Fig.1b), showed significant nuclear accumulation in Atm−/− but not in wild-type mouse Purkinje cells (Fig.1a, c). Similar shifts in HDAC4 localization were found in other mouse brain regions including neocortex and brain stem (Supplementary Fig.1c). The effect was direct; nuclear accumulation of GFP-HDAC4 was found in Atm−/− neurons (Fig. 1d). Further, after exposure of cultured cortical neurons to siRNA against Atm, HDAC9 remained in the cytoplasm while endogenous HDAC4 shifted to the nucleus (Supplementary Fig. 1d).


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)

Nuclear accumulation of HDAC4 in ATM-deficient neurons leads to suppression of MEF2- and CREB-related transcriptional activitiesa) 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.
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Figure 1: Nuclear accumulation of HDAC4 in ATM-deficient neurons leads to suppression of MEF2- and CREB-related transcriptional activitiesa) 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.
Mentions: The analogies of neuron death and dendritic atrophy in the cerebella of ATM- and HDAC4-deficient mice9,18,21,22 prompted us to examine HDAC4 in human A-T cerebella. Normally, HDAC4 immunoreactivity is found in Purkinje cell cytoplasm (Fig.1a). In A-T samples by contrast Purkinje cell nuclei had strong HDAC4 staining (Fig. 1a, b). The nuclear accumulation of HDAC4 was specific; despite structural and functional similarities23,24, HDAC5 and HDAC9 showed little nuclear accumulation (Supplementary Fig.1a). These observations were replicated in mice. HDAC4, but not HDAC5 or HDAC9 (Supplementary Fig.1b), showed significant nuclear accumulation in Atm−/− but not in wild-type mouse Purkinje cells (Fig.1a, c). Similar shifts in HDAC4 localization were found in other mouse brain regions including neocortex and brain stem (Supplementary Fig.1c). The effect was direct; nuclear accumulation of GFP-HDAC4 was found in Atm−/− neurons (Fig. 1d). Further, after exposure of cultured cortical neurons to siRNA against Atm, HDAC9 remained in the cytoplasm while endogenous HDAC4 shifted to the nucleus (Supplementary Fig. 1d).

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