Restoration of HDAC1 Enzymatic Activity after Stroke Protects Neurons from Ischemia/Reperfusion Damage and Attenuates Behavioral Deficits in Rats

Abstract

A therapeutic approach for promoting neuroprotection and brain functional regeneration after strokes is still lacking. Histone deacetylase 1 (HDAC1), which belongs to the histone deacetylase family, is involved in the transcriptional repression of cell-cycle-modulated genes and DNA damage repair during neurodegeneration. Our previous data showed that the protein level and enzymatic activity of HDAC1 are deregulated in stroke pathogenesis. A novel compound named 5104434 exhibits efficacy to selectively activate HDAC1 enzymatic function in neurodegeneration, but its potential in stroke therapy is still unknown. In this study, we adopted an induced rat model with cerebral ischemia using the vessel dilator endothelin-1 to evaluate the potential of compound 5104434. Our results indicated compound 5104434 selectively restored HDAC1 enzymatic activity after oxygen and glucose deprivation, preserved neurite morphology, and protected neurons from ischemic damage in vitro. In addition, compound 5104434 attenuated the infarct volume, neuronal loss, apoptosis, DNA damage, and DNA breaks in cerebral ischemia rats. It further ameliorated the behavioral outcomes of neuromuscular response, balance, forepaw strength, and functional recovery. Collectively, our data support the efficacy of compound 5104434 in stroke therapy and contend that it can be considered for clinical trial evaluation.

Keywords: DNA damage; HDAC1; apoptosis; cylinder test; mNSS; stroke.

Figure 1

HA treatment restored HDAC1 enzymatic activity and protected neuronal damage after OGD in vitro. (A) HDAC1 activity assay was conducted 24 h after OGD. HDAC1 was isolated from protein lysates extracted from primary neuron by immunoprecipitation, and then HDAC1 activity was detected by the assay kit. (B) Cell viability was evaluated by MTT assay 24 h after OGD. (C) Protein lysates extracted from primary neuron were subjected to ROS assay 24 h after OGD. (D) Neuronal damage was evaluated by LDH assay 24 h after OGD. n = 6. * Denotes p < 0.05, ** denotes p < 0.01.

Figure 2

HA treatment preserved neurite outgrowth pattern in terms of total outgrowth, process length, process number, and branch number in vitro. (A) Representative figures of immunocytochemistry staining for NeuN and MAP2 in primary neuron 24 h after OGD. (B–E) High-throughput-acquired neuron images were analyzed in terms of total outgrowth, process length, process number, and branch number. n = 6. * Denotes p < 0.05, ** denotes p < 0.01.

Figure 3

HA treatment attenuated HDAC1 enzymatic dysfunction and ischemia volume of the brain in rats. (A) Flow chart of experimental design in vivo. (B) HDAC1 enzymatic activity assay for evaluation of the dose–effect of HA. n = 6 (C) Representative figures of TTC staining that were conducted at post-stroke day 3 in rats with endothelin-1-induced brain ischemia. Quantified data of ischemia volume from rats with brain ischemia. n = 8. * Denotes p < 0.05; *** denotes p < 0.001.

Figure 4

HA treatment alleviated neuronal apoptosis in the brain of rats after ischemic stroke. (A) Representative figures of immunofluorescent staining for NeuN and cleaved caspase-3 were conducted in rats with brain ischemia 3 days after stroke. (B) Quantified data of mean cell number with NeuN immunoreactivity in each view. (C) Quantified data of mean cell number with cleaved caspase-3 immunoreactivity in each view. (D) Quantified data of mean cell number with double-positive immunoreactivity of NeuN and cleaved-caspase-3 in each view. Data quantification accumulated twenty image views across six brain sections per brain from cortex penumbra. n = 8. * Denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.

Figure 5

HA treatment attenuated neuronal DNA damage in the brain of rats after ischemic stroke. (A) Representative figures of immunofluorescent staining for NeuN and γH2AX were conducted in rats with brain ischemia 3 days after stroke. (B) Quantified data of mean cell number with γH2AX immunoreactivity in each view. (C) Quantified data of mean cell number with double-positive immunoreactivity of γH2AX and NeuN in each view. Data quantification accumulated twenty image views across six brain sections per brain from cortex penumbra. n = 8. ** Denotes p < 0.01, *** denotes p < 0.001.

Figure 6

HA treatment ameliorated DNA breaks in the brain of rats after ischemic stroke. (A) Representative figures of TUNEL staining were conducted in rats with brain ischemia 3 days after stroke; immunofluorescent co-staining for NeuN was conducted to label neurons. (B) Quantified data of mean cell number with TUNEL signal in each view. (C) Quantified data of mean cell number with TUNEL signal and NeuN immunoreactivity in each view. Data quantification accumulated twenty image views across six brain sections per brain from cortex penumbra. n = 8. ** Denotes p < 0.01, *** denotes p < 0.001.

Figure 7

HA treatment improved the behavioral outcomes of rats after ischemic stroke. (A) Data of mNSS were acquired from rats with ischemic stroke at post-stroke day (PSD) 1, 3, 7. (B) Data of balance beam test were acquired from rats with ischemic stroke at PSD 1, 3, 7. (C) Data of hanging test were acquired from rats with ischemic stroke at PSD 1, 3, 7. (D) Data of cylinder test were acquired from rats with ischemic stroke at PSD 1, 3, 7. n = 8. * Denotes p < 0.05, *** denotes p < 0.001.

Figure 8

HA treatment improved synaptic density in the rats with cerebral ischemia. (A) Representative figures of immunofluorescent staining for PSD95. White boxes noted region with amplification at the lower panel. Data were acquired in the cortex penumbra. (B) Quantification of PSD95 immunoreactive density. n = 6 per group. ** Denotes p < 0.01, *** denotes p < 0.001.