HDAC inhibitor protects chronic cerebral hypoperfusion and oxygen-glucose deprivation injuries via H3K14 and H4K5 acetylation-mediated BDNF expression

Abstract

Vascular dementia (VaD) is the second most common cause of dementia, but the treatment is still lacking. Although many studies have reported that histone deacetylase inhibitors (HDACis) confer protective effects against ischemic and hypoxic injuries, their role in VaD is still uncertain. Previous studies shown, one HDACi protected against cognitive decline in animals with chronic cerebral hypoperfusion (CCH). However, the underlying mechanisms remain elusive. In this study, we tested several 10,11-dihydro-5H-dibenzo[b,f]azepine hydroxamates, which act as HDACis in the CCH model (in vivo), and SH-SY5Y (neuroblastoma cells) with oxygen-glucose deprivation (OGD, in vitro). We identified a compound 13, which exhibited the best cell viability under OGD. The compound 13 could increase, in part, the protein levels of brain-derived neurotrophic factor (BDNF). It increased acetylation status on lysine 14 residue of histone 3 (H3K14) and lysine 5 of histone 4 (H4K5). We further clarified which promoters (I, II, III, IV or IX) could be affected by histone acetylation altered by compound 13. The results of chromatin immunoprecipitation and Q-PCR analysis indicate that an increase in H3K14 acetylation leads to an increase in the expression of BDNF promoter II, while an increase in H4K5 acetylation results in an increase in the activity of BDNF promoter II and III. Afterwards, these cause an increase in the expression of BDNF exon II, III and coding exon IX. In summary, the HDACi compound 13 may increase BDNF specific isoforms expression to rescue the ischemic and hypoxic injuries through changes of acetylation on histones.

Keywords: HDAC; OGD; histone acetylation; histone deacetylase inhibitor; vascular dementia.

Figure 1

SH‐SY5Y neuroblastoma cells were subjected to OGD (hypoxia) injury in the presence of different concentrations of DMSO (control) and/or compounds 3‐13. A‐K, Relative cell viability was assessed through MTT assays. Data are presented as the mean ± standard error of the mean (SEM) of four experiments. *P < 0.05 compared with the group treated with DMSO under OGD condition

Figure 2

Effect of compound 13 on the protein levels of BDNF in the SH‐SY5Y cells under OGD condition (A‐B), cortex (right cortex, CR; left cortex, CL) (C) or hippocampus (H) (D) of CCH mice. The protein levels of BDNF were measured after BCCAO for 3 mo. β‐actin served as the loading control. The densities were normalized to β‐actin, and each bar represents the mean ± SEM of four independent experiments (n = 4, *P < 0.05, **P < 0.01 vs indicated group). The viability of cells treated with compound 13 in the presence or not of a neutralizing antibody of BDNF was noted. E, The cell viability was examined through MTT assay in cells exposed to OGD treated with compound 13 in the presence of a neutralizing antibody of BDNF (1, 3 or 10 µg/mL). Data are presented as the mean ± SEM of four experiments (n = 4, *P < 0.05 vs indicated group)

Figure 3

Effect of compound 13 on histone acetylation (H3K14 or H4K5) in the cells exposed to OGD injury. The levels of histone acetylation (Ac‐H3K14 (A, B) and Ac‐H4K5 (C, D)) were studied after OGD for 24 h with the Western blotting analysis. β‐actin served as the loading control. The densities were normalized to β‐actin, and each bar represents the mean ± SEM of four independent experiments (n = 4, *P < 0.05, **P < 0.01 vs indicated group)

Figure 4

Effect of compound 13 on the BDNF promoter (I, II, III, IV or IX) around H3K14 or H4K5 in the cells exposed to OGD injury. A‐J, The expression of BDNF promoters was studied with chromatin IP, followed by qPCR analysis after OGD for 24 h. Fold expression was normalized to input control, and each bar represents the mean ± SEM of four independent experiments (n = 4, *P < 0.05, **P < 0.01 vs indicated group)

Figure 5

Effect of compound 13 on BDNF exon (I‐IX, II‐IX, III‐IX, IV‐IX or IX) in the cells exposed to OGD injury. A‐E, The expression of BDNF exons was studied with qPCR analysis after OGD for 24 h. The fold expression was normalized to GAPDH, and each bar represents the mean ± SEM of four independent experiments (n = 4, *P < 0.05, **P < 0.01 vs indicated group)

Figure 6

A proposed mechanism by which HDACi protects from injuries via BDNF expression