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. 2020 Oct;40(10):2081-2097.
doi: 10.1177/0271678X19884742. Epub 2019 Nov 7.

HDAC4 and HDAC5 form a complex with DREAM that epigenetically down-regulates NCX3 gene and its pharmacological inhibition reduces neuronal stroke damage

Luigi Formisano  1 , Giusy Laudati  1 , Natascia Guida  2 , Luigi Mascolo  1 , Angelo Serani  1 , Ornella Cuomo  1 , Maria Cantile  1 , Francesca Boscia  1 , Pasquale Molinaro  1 , Serenella Anzilotti  2 , Vincenzo Pizzorusso  1 , Gianfranco Di Renzo  1 , Giuseppe Pignataro  1 , Lucio Annunziato  2
Affiliations

HDAC4 and HDAC5 form a complex with DREAM that epigenetically down-regulates NCX3 gene and its pharmacological inhibition reduces neuronal stroke damage

Luigi Formisano et al. J Cereb Blood Flow Metab. 2020 Oct.

Abstract

The histone deacetylases (HDACs)-dependent mechanisms regulating gene transcription of the Na+/Ca+ exchanger isoform 3 (ncx3) after stroke are still unknown. Overexpression or knocking-down of HDAC4/HDAC5 down-regulates or increases, respectively, NCX3 mRNA and protein. Likewise, MC1568 (class IIa HDACs inhibitor), but not MS-275 (class I HDACs inhibitor) increased NCX3 promoter activity, gene and protein expression. Furthermore, HDAC4 and HDAC5 physically interacted with the transcription factor downstream regulatory element antagonist modulator (DREAM). As MC1568, DREAM knocking-down prevented HDAC4 and HDAC5 recruitment to the ncx3 promoter. Importantly, DREAM, HDAC4, and HDAC5 recruitment to the ncx3 gene was increased in the temporoparietal cortex of rats subjected to transient middle cerebral artery occlusion (tMCAO), with a consequent histone-deacetylation of ncx3 promoter. Conversely, the tMCAO-induced NCX3 reduction was prevented by intracerebroventricular injection of siDREAM, siHDAC4, and siHDAC5. Notably, MC1568 prevented oxygen glucose deprivation plus reoxygenation and tMCAO-induced neuronal damage, whereas its neuroprotective effect was abolished by ncx3 knockdown. Collectively, we found that: (1) DREAM/HDAC4/HDAC5 complex epigenetically down-regulates ncx3 gene transcription after stroke, and (2) pharmacological inhibition of class IIa HDACs reduces stroke-induced neurodetrimental effects.

Keywords: HDAC class II; MC1568; NCX3; neuroprotection.

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Figures

Figure 1.
Figure 1.
Effect of class IIa HDAC inhibitor MC1568 on ncx3 promoter activity, gene, and protein expression in cortical neurons. (a) Effect of 24 h of TSA (100 nM), MS-275 (5 µM), and MC1568 (5 µM) exposure on Acetylated-Histone H4 protein expression. Cells were treated with TSA, MS-275, and MC1568 for a 2 h pulse (n = 3). *p ≤ 0.05 versus vehicle, one-way ANOVA, followed by Tukey’s multiple comparison test. (b) Effect of TSA (100 nM), MS-275 (5 µM), and MC1568 (5 µM) exposure on ncx3 promoter activity (n = 5). *p ≤ 0.05 versus pGL3-basic; **p ≤ 0.05 versus pGL3-basic and pGL3-ncx3 + vehicle, one-way ANOVA, followed by Tukey’s multiple comparison test. (c) Effect of MC1568 (5 µM) on ncx3 promoter activity measured after 6, 12, 24 and 48 h treatment (n = 5). *p ≤ 0.05 versus pGL3-basic; **p ≤ 0.05 versus pGL3-basic and pGL3-ncx3 + vehicle, one-way ANOVA, followed by Tukey’s multiple comparison test. (d) Effect of 12, 24, and 48 h of MC1568 (5μM) treatment on ncx3 gene expression (n = 5). *p ≤ 0.05 versus vehicle, one-way ANOVA, followed by Tukey’s multiple comparison test. (e, f) Representative WB with quantification of NCX3 (e) and NCX1 (f) after 24 h treatment with MC1568 (5 µM) in cortical neurons (n = 3). *p < 0.05 versus vehicle, Student t test.
Figure 2.
Figure 2.
HDAC4 and HDAC5 down-regulated ncx3 gene and protein expression. (a, b) ncx3 gene expression and representative WB with quantification in cortical neurons transfected with the following constructs: (1) Empty Vector, (2) HDAC4 Vector, (3) HDAC5 Vector, (4) HDAC7 Vector, and (5) HDAC9 Vector. (c, d) ncx3 gene expression and representative WB with quantification in cortical neurons transfected with the following siRNAs: (1) siCTL, (2) siHDAC4, (3) siHDAC5, (4) siHDAC7, and (5) siHDAC9. The cells were processed after 24 h of transfection for qRT-PCR and immunoblotting (n = 5). *p ≤ 0.05 versus empty vector or siCTL, one-way ANOVA, followed by Tukey’s multiple comparison test.
Figure 3.
Figure 3.
HDAC4 and HDAC5 bound to ncx3 brain promoter via downstream regulatory element antagonist modulator (DREAM). (a–c) ChIP analysis of the ncx3 brain promoter performed with: (a) anti acetylated-histone H4, (b) anti-HDAC4, and (c) anti-HDAC5 in cortical neurons. Acetylation status of histone H4 (n = 3) and binding activity of HDAC4 (n = 5) and HDAC5 (n = 7) are graphically represented as the percentage of vehicle. *p ≤ 0.05 versus vehicle, Student t test. (d, e) Representative Western blot showing immunoprecipitation between DREAM and HDAC4 and between DREAM and HDAC5 in cortical neurons. IgG was used as a negative control. (f) Representative WB with quantification in cortical neurons after 24 h of transfection with siDREAM (n = 3). *p < 0.05 versus siCTL, Student t test. (g–i) ChIP analysis of the ncx3 brain promoter performed with: (g) anti-acetylated-histone H4, (h) anti-HDAC4, and (j) anti-HDAC5 in cortical neurons. Acetylation status of histone H4 (n = 5) and binding activity of HDAC4 (n = 5) and HDAC5 (n = 4/5) are graphically represented as the percentage of siCTL. *p ≤0.05 versus siCTL, Student t test. (j) Luciferase activity of ncx3 gene promoter wild type (pGL3-ncx3) or mutated in DRE-site (pGL3-ncx3-DREmut), with or without MC1568 (5 μM), in cortical neurons (n = 3). *p ≤ 0.05 versus pGL3basic; **p ≤ 0.05 versus pGL3basic and pGL3-ncx3 + vehicle, one-way ANOVA, followed by Tukey’s multiple comparison test.
Figure 4.
Figure 4.
DREAM, HDAC4, and HDAC5 bound to ncx3 brain promoter, determining its sequence deacetylation in the periischemic temporoparietal cortex of tMCAO rats. (a–c) Representative WB with quantification of DREAM, HDAC4, and HDAC5 in cortex of rats subjected to 24 h and 48 h of tMCAO (n = 5). *p < 0.05 versus Sham, one-way ANOVA, followed by Tukey’s multiple comparison test. Confocal double labeling experiments displaying the co-expression of DREAM (d), HDAC4 (e), and HDAC5 (f) with the neuronal marker NeuN in the ipsilateral temporoparietal cortex of sham-operated animals (left panels) and in the ipsilateral periischemic temporoparietal cortex 24 h after tMCAO surgery (right panels). Dashed lines delimit the cortical ischemic core and periischemic region. Scale bars 100 µm. ChIP analysis of the ncx3 brain promoter performed with: (g) anti-DREAM, (h) anti-HDAC4, (i) anti-HDAC5, and (j) anti-Acetylated-Histone H4 in cortex of rats subjected to 24 h of tMCAO. Binding activity of DREAM, HDAC4, HDAC5 and acetylation status of Histone H4 are graphically represented as the percentage of sham-operated animals (n = 5). *p ≤ 0.05 versus Sham, Student t test .
Figure 5.
Figure 5.
HDAC4, HDAC5, and DREAM knockdown prevented ncx3 gene and protein expression reduction in the periischemic temporoparietal cortex oftMCAO rats. (a–b) Effect of 24 h and 48 h of tMCAO on: (a) ncx3 gene (n = 5) and (b) protein expression (n = 5). *p ≤ 0.05 versus Sham, one-way ANOVA, followed by Tukey’s multiple comparison test. (c–e) Representative WB with quantification in rats subjected to siRNAs intracerebroventricular (icv) injection against HDAC4, HDAC5 or DREAM and euthanized after 24 h (n = 3). *p < 0.05 versus siCTL, Student t test. Effect of 24 h of siHDAC4, siHDAC5, and siDREAM icv administration in rats subjected to 24 h of tMCAO on ncx3 (f) gene (n = 5) and (g) protein expression (n = 5). *p ≤ 0.05 versus Sham and ^p ≤ 0.05 versus Sham and tMCAO + siCTL, one-way ANOVA, followed by Tukey’s multiple comparison test.
Figure 6.
Figure 6.
MC1568 reduced neuronal death in cortical neurons exposed to OGD/Reoxy. (a, b) Effect of 24 h and 48 h of reoxygenation (Reoxy) after 3 h of OGD on: (a) ncx3 gene (n = 5) and (b) protein expression (n = 3) in cortical neurons. *p ≤ 0.05 vs CTL, one-way ANOVA, followed by Tukey’s multiple comparison test. (c, d) Effect of MC1568 (5μM) in cortical neurons subjected to OGD followed by 48 h of reoxygenation on: (c) ncx3 gene (n = 5) and (d) protein expression (n = 3). *p ≤ 0.05 versus Vehicle; #p ≤ 0.05 versus OGD/Reoxy 48 h + Vehicle, one-way ANOVA, followed by Tukey’s multiple comparison test. (e) Effects of MC1568 (5μM) exposure or siNCX3 transfection on cell death of cortical neurons subjected to OGD/Reoxy for 48 h, as evaluated by LDH assay (n = 5). *p ≤ 0.05 versus vehicle; #p ≤ 0.05 versus OGD/Reoxy 48h; ^p ≤ 0.05 versus MC1568 + siCTL + OGD/Reoxy 48h, one-way ANOVA, followed by Tukey’s multiple comparison test.
Figure 7.
Figure 7.
MC1568 ameliorated ischemic damage in rats subjected to tMCAO. (a–c) Representative WB with quantification of HDAC4 (n = 5), HDAC5 (n = 5) and Acetylated-Histone H4 protein (n = 5) expression after 24 h of tMCAO and effect of icv administered MC1568. *p ≤ 0.05 versus Sham; #p ≤ 0.05 versus tMCAO + vehicle, one-way ANOVA, followed by Tukey’s multiple comparison test. (d) % of brain infarct volume in male rats subjected to tMCAO after icv administration of: (a) siCTL + Vehicle, (b) siCTL + MC1568, (c) siNCX3 + Vehicle, and (d) siNCX3 + MC1568. Representative brain slices from each experimental group are shown at the top of each column (n = 5). Ischemic rats were euthanized 24 h after tMCAO of the % of the infarct volume compared to the ipsilateral hemisphere. *p ≤0.05 vs tMCAO + siCTL + Vehicle, two-way ANOVA, followed by Newman–Keuls test.
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