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. 2009 Mar 4;29(9):2824-32.
doi: 10.1523/JNEUROSCI.6186-08.2009.

Histone deacetylase inhibitors prevent p53-dependent and p53-independent Bax-mediated neuronal apoptosis through two distinct mechanisms

Takuma Uo  1 , Timothy D Veenstra, Richard S Morrison
Affiliations

Histone deacetylase inhibitors prevent p53-dependent and p53-independent Bax-mediated neuronal apoptosis through two distinct mechanisms

Takuma Uo et al. J Neurosci. .

Abstract

Pharmacological manipulation of protein acetylation levels by histone deacetylase (HDAC) inhibitors represents a novel therapeutic strategy to treat neurodegeneration as well as cancer. However, the molecular mechanisms that determine how HDAC inhibition exerts a protective effect in neurons as opposed to a cytotoxic action in tumor cells has not been elucidated. We addressed this issue in cultured postnatal mouse cortical neurons whose p53-dependent and p53-independent intrinsic apoptotic programs require the proapoptotic multidomain protein, Bax. Despite promoting nuclear p53 accumulation, Class I/II HDAC inhibitors (HDACIs) protected neurons from p53-dependent cell death induced by camptothecin, etoposide, heterologous p53 expression or the MDM2 inhibitor, nutlin-3a. HDACIs suppressed p53-dependent PUMA expression, a critical signaling intermediate linking p53 to Bax activation, thus preventing postmitochondrial events including cleavage of caspase-9 and caspase-3. In human SH-SY5Y neuroblastoma cells, however, HDACIs were not able to prevent p53-dependent cell death. Moreover, HDACIs also prevented caspase-3 cleavage in postnatal cortical neurons treated with staurosporine, 3-nitropropionic acid and a Bcl-2 inhibitor, all of which require the presence of Bax but not p53 to promote apoptosis. Although these three toxic agents displayed a requirement for Bax, they did not promote PUMA induction. These results demonstrate that HDACIs block Bax-dependent cell death by two distinct mechanisms to prevent neuronal apoptosis, thus identifying for the first time a defined molecular target for their neuroprotective actions.

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Figures

Figure 1.
Figure 1.
HDACIs prevent p53-mediated neuronal apoptosis through inhibition of the p53–Bax pathway. A, HDACIs protect against camptothecin-induced neuronal cell death. p53+/+ neurons were treated with CPT (2.5 μm) alone or in combination with SB (2 mm) or TSA (200 nm) with DMSO used as a vehicle control. Representative phase-contrast images at 24 h after treatment are shown. B, Neuronal survival was quantitatively assessed by morphology (Xiang et al., 1996), by counting healthy cells in the same field at time 0 and 24 h after treatment for each condition as shown in supplemental Figure 3A, available at www.jneurosci.org as supplemental material. Mean ± SD of two fields each from three cultures (n = 6; results confirmed in 3 independent experiments). Asterisk indicates significantly different from all other conditions (p < 0.0001, one-way ANOVA using Tukey's post hoc test). Other comparisons between the conditions show no significant difference (p > 0.05). C, Effect of TSA on p53 expression. p53+/+ neurons were treated with CPT and/or TSA for 12 h, fixed and stained for p53 and a neuronal marker, MAP2, with Hoechst 33258 staining. Representative images are shown from three independent experiments. D, Effect of TSA on p53 and cleaved caspase-3 protein levels. Protein samples were prepared from Bax+/+ and Bax−/− neurons treated with CPT and/or TSA for 12 h and subjected to Western blotting. β-Actin was used as an internal loading control for all blots unless otherwise stated. Representative data are shown from two independent experiments. E, HDACI treatment reduces caspase cleavage activity. p53+/+ neurons were harvested at 12 h after treatment with CPT and/or TSA (DMSO as a vehicle control). Cytosolic extracts were prepared and evaluated for zDEVD-AFC cleavage activity. The data represent the mean ± SD of relative fluorescence units (RFU)/mg protein (n = 3 cultures per condition; results confirmed in 3 independent experiments). Asterisk indicates significantly different from all other conditions (p < 0.0001, one-way ANOVA using Tukey's post hoc test). DMSO versus CPT plus TSA, p = 0.63. Other comparisons between the conditions showed no significant difference (p > 0.05). F, TSA blocks etoposide (ETO)-induced caspase-3 cleavage. p53+/+ neurons were treated with ETO (5 μm) and/or TSA (200 nm) for 12 h and analyzed by Western blotting as described in D. Representative data are shown from two independent experiments. G, TSA blocks caspase-3 cleavage induced by Nutlin-3a. p53+/+ neurons were treated with nutlin-3a (10 μm) and/or TSA (200 nm). Protein samples were prepared 12 h after treatment and analyzed for p53 and cleaved caspase-3. Representative data are shown from two independent experiments. H, HDACI blocks caspase-3 activation induced by heterologous expression of human p53. p53−/− neurons were infected with adenovirus expressing human p53 (Ad-p53) or β-galactosidase (Ad-LacZ) at 50 MOI for 24 h and then, after washing with virus-free media, treated with CPT (2.5 μm) in the presence or absence of SB (2 mm). DMSO was used as a vehicle control. Cellular exacts were prepared 12 h after treatment and analyzed for total p53 protein and cleaved caspase-3. Representative data are shown from two independent experiments. I, TSA blocks caspase-9 cleavage induced by CPT treatment. p53+/+ neurons were treated with CPT and/or TSA. Protein samples were prepared 12 h after treatment and analyzed for caspase-9. The arrow and arrowhead indicate full-length and cleaved caspase-9, respectively. Representative data are shown from two independent experiments. J, TSA blocks Bax activation induced by CPT treatment. p53+/+ neurons were treated with CPT and/or TSA (DMSO as vehicle control) for 12 h, then lysed and subjected to immunoprecipitation with anti-Bax (6A7) antibody for detecting activated Bax protein. Total extracts (input) and immune complexes (IP: 6A7) were analyzed by Western blotting using anti-Bax (N-20) antibody. The intensity of each band corresponding to the Bax protein was quantitated using ImageJ 1.41o software (National Institutes of Health, Bethesda, MD). The data are presented as the ratio of the immunoprecipitated Bax band relative to the respective Bax input signal. Representative data are shown from three independent experiments.
Figure 2.
Figure 2.
HDACIs reversibly and selectively block p53-dependent transcriptional activation of PUMA. A, B, p53+/+ neurons were cotreated with CPT and SAHA (10 μm) for 13 h (“Pre-” treatment) and then immediately harvested (0 h in “post-treatment”) or provided fresh media with CPT, CPT plus SAHA, CPT plus actinomycin D (ActD; 2 μm), or CPT plus cycloheximide (CHX; 10 μg/ml), and then harvested at the indicated times after switching the media. DMSO was used as a vehicle control. Protein samples were analyzed for cleaved caspase-3 (A) and PUMA and p21WAF1/CIP1 (p21) (B) by Western blotting. Representative data are shown from two independent experiments. C, Expression levels of PUMA and PHLDA3 were analyzed by semiquantitative RT-PCR (see Materials and Methods) from total RNA extracted from cells collected at the indicated times after switching the initial media. Ribosomal protein S12 (RPS12) was analyzed as an internal control for RNA loading for each sample reaction. Representative data are shown from two independent experiments.
Figure 3.
Figure 3.
HDACI does not prevent p53-dependent caspase-3 activation and cell death in SH-SY5Y neuroblastoma. A, SH-SY5Y cells were treated with CPT (0.5 μm) and/or TSA (200 nm). DMSO served as a vehicle control. Cells were lysed for assessment of zDEVD-AFC cleavage activity 12 h after treatment (mean ± SD of relative fluorescence units/mg protein, n = 3 cultures per condition; results confirmed in three independent experiments). DMSO versus TSA, p = 0.35; all other combinations are significantly different (p < 0.001–0.01). One-way ANOVA using Tukey's post hoc test. B, SH-SY5Y cells were treated as described in A for 24 h, and cell death was assessed by staining with EthD-1. Representative data are shown from three independent experiments. C, HDACI blocks PUMA induction but does not prevent NOXA induction in camptothecin-treated SH-SY5Y cells. Protein samples were prepared from SH-SY5Y cells treated with CPT and/or TSA for 12 h and subjected to Western blotting. NOXA was detected on a different gel from that used for detection of PUMA, p53 and β-actin. The same set of protein extracts were applied to both gels. The nonspecific bands (N.S.) migrating at ∼12 kDa on the NOXA blot provide an indication of protein loading. Representative data are shown from two independent experiments.
Figure 4.
Figure 4.
HDACIs prevent p53-independent activation of caspase-3, which occurs independently of PUMA induction. A, p53+/+ and p53−/− neurons were treated with staurosporine (STS; 0.5 μm) in the presence or absence of class I and II HDAC inhibitors (SB, 2 mm; SAHA, 10 μm; TSA, 200 nm) or sirtuin inhibitors [nicotinamide (NAM), 10 mm; sirtinol, 50 μm]. DMSO was used as a vehicle control. Protein extracts were prepared 8 h later and analyzed for cleaved caspase-3. Representative data are shown from two independent experiments. B, p53−/− neurons treated with STS and/or TSA were fixed 7 h after treatment and immunostained for cleaved caspase-3 and β-tubulin III, a neuronal marker (TuJ1 antigen). Representative data are shown from two independent experiments. C, The data represent the mean ± SD of the total number of cleaved caspase-3-positive cells in four random fields within a reticule (824 × 660 μm) from three cultures (n = 3). TSA versus TSA plus STS, p = 0.84; all other combinations are significantly different (p < 0.002–0.05). One-way ANOVA using Tukey's post hoc test. D, TSA blocks cleavage of caspase-9 induced by STS treatment. p53−/− neurons were treated with STS and/or TSA. Protein samples were prepared 7 h after treatment and analyzed for caspase-9. An arrow and an arrowhead indicate full-length and cleaved caspase-9, respectively. Representative data are shown from two independent experiments. E, p53+/+ and p53−/− neurons were treated for 24 h with 3-NP at the indicated concentrations in the presence or absence of TSA (200 nm). p53+/+ neurons were also treated for 12 h with CPT to induce p53-dependent caspase-3 activation. Protein extracts were analyzed for expression of p53 and cleaved caspase-3. The nonspecific bands (N.S.) migrating at ∼35 kDa on the p53 blot provide an indication of protein loading. Representative data are shown from three independent experiments. F, Bax+/+ and Bax−/− neurons were treated for 12 h with the Bcl-2 inhibitor (BCL2-I; 8 μm) in the presence or absence of TSA. Bax+/+ neurons were also treated for 12 h with CPT to induce p53-dependent caspase-3 activation. Protein extracts were analyzed for expression of p53 and cleaved caspase-3. Representative data are shown from two independent experiments. G, Protein extracts were prepared from p53−/− neurons treated with STS (0.5 μm, 8 h), BCL2-I (8 μm, 12 h), and 3-NP (1 mm, 24 h) and analyzed for expression of PUMA. Representative data are shown from two independent experiments.
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