Hsa_circ_0000527 Downregulation Suppresses the Development of Retinoblastoma by Modulating the miR-27a-3p/HDAC9 Pathway.

BACKGROUND: Accumulating evidence indicates that the progression of retinoblastoma (RB) may involve circRNA dysfunction. We aimed to disclose the role of hsa_circ_0000527 and its potential functional mechanism in RB. METHODS: The expression of hsa_circ_0000527, miR-27a-3p and histone deacetylase 9 (HDAC9) mRNA was monitored using quantitative real-time polymerase chain reaction (qPCR). Functional assays, including cell proliferation and apoptosis, were investigated using cell counting kit-8 (CCK-8) assay, colony formation assay and flow cytometry assay. The expression of apoptosis-associated proteins and HDAC9 protein was detected by western blot. The targeting relationship between miR-27a-3p and hsa_circ_0000527 or HDAC9 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Besides, Xenograft models were constructed to confirm the effect of hsa_circ_0000527 in vivo. RESULTS: Hsa_circ_0000527 and HDAC9 were upregulated, while miR-27a-3p was downregulated in RB tissues and cells. Hsa_circ_0000527 downregulation repressed RB cell proliferation and induced RB cell apoptosis. MiR-27a-3p was a target of hsa_circ_0000527, and hsa_circ_0000527 suppressed the expression of miR-27a-3p. MiR-27a-3p inhibition reversed the role of hsa_circ_0000527 downregulation. In addition, HDAC9 was a target of miR-27a-3p, and hsa_circ_0000527 indirectly regulated HDAC9 expression by targeting miR-27a-3p. MiR-27a-3p restoration inhibited RB cell proliferation and promoted apoptosis, which was reversed by HDAC9 overexpression. Hsa_circ_0000527 downregulation could inactivate the PI3K/AKT pathway. Moreover, hsa_circ_0000527 downregulation blocked tumor growth rate in vivo. CONCLUSION: hsa_circ_0000527 downregulation blocked the progression of RB by regulating the miR-27a-3p/HDAC9 pathway, which might be associated with the inactivation of the PI3K/AKT pathway.

Long Noncoding RNA TTC39A-AS1 Promotes Breast Cancer Tumorigenicity by Sponging MicroRNA-483-3p and Thereby Upregulating MTA2.

INTRODUCTION: In recent years, the regulatory activities of long noncoding RNAs have received increasing attention as an important research focus. This study aimed to characterize the expression and detailed roles of TTC39A antisense RNA 1 (TTC39A-AS1) in breast cancer (BC), in addition to concentrating on its downstream mechanisms. METHODS: Quantitative RT-PCR was performed to determine the expression levels of TTC39A-AS1, microRNA-483-3p (miR-483-3p), and metastasis-associated gene 2 (MTA2). Further, the detailed functions of TTC39A-AS1 in BC cells were confirmed using the Cell Counting Kit 8 assay, flow cytometric analysis, and Transwell cell migration and invasion assays. The targeting relationship between TTC39A-AS1, miR-483-3p, and MTA2 in BC was predicted via bioinformatics analysis and further confirmed by performing the luciferase reporter assay and RNA immunoprecipitation. RESULTS: TTC39A-AS1 was present in high levels in BC; this result was confirmed in our sample cohort and The Cancer Genome Atlas database. Patients with BC with a high level of TTC39A-AS1 had a shorter overall survival than those with a low level of TTC39A-AS1. Functionally, the absence of TTC39A-AS1 accelerated cell apo-ptosis but retained cell proliferation, migration, and invasion. Mechanistically, TTC39A-AS1 functioned as a competing endogenous RNA in BC by sponging miR-483-3p and thereby indirectly increasing MTA2 expression. Finally, rescue experiments revealed that the tumor-inhibiting actions of TTC39A-AS1 knockdown on the malignant characteristics of BC cells could be reversed by inhibiting miR-483-3p or upregulating MTA2. CONCLUSION: The newly identified TTC39A-AS1/miR-483-3p/MTA2 pathway was revealed to be a critical regulator in the tumorigenicity of BC, possibly offering a novel therapeutic direction for the anticancer treatment of BC.

Increased Susceptibility and Intrinsic Apoptotic Signaling in Neurons by Induced HDAC3 Expression.

Purpose: Inhibition or targeted deletion of histone deacetylase 3 (HDAC3) is neuroprotective in a variety neurodegenerative conditions, including retinal ganglion cells (RGCs) after acute optic nerve damage. Consistent with this, induced HDAC3 expression in cultured cells shows selective toxicity to neurons. Despite an established role for HDAC3 in neuronal pathology, little is known regarding the mechanism of this pathology. Methods: Induced expression of an HDAC3-mCherry fusion protein in mouse RGCs was accomplished by transduction with AAV2/2-Pgk-HDAC3-mCherry. Increased susceptibility to optic nerve damage in HDAC3-mCherry expressing RGCs was evaluated in transduced mice that received acute optic nerve crush surgery. Expression of HDAC3-FLAG or HDAC3-mCherry was induced by nucleofection or transfection of plasmids into differentiated or undifferentiated 661W tissue culture cells. Immunostaining for cleaved caspase 3, localization of a GFP-BAX fusion protein, and quantitative RT-PCR was used to evaluate HDAC3-induced damage. Results: Induced expression of exogenous HDAC3 in RGCs by viral-mediated gene transfer resulted in modest levels of cell death but significantly increased the sensitivity of these neurons to axonal damage. Undifferentiated 661W retinal precursor cells were resilient to induced HDAC3 expression, but after differentiation, HDAC3 induced GFP-BAX recruitment to the mitochondria and BAX/BAK dependent activation of caspase 3. This was accompanied by an increase in accumulation of transcripts for the JNK2/3 kinases and the p53-regulated BH3-only gene Bbc3/Puma. Cell cycle arrest of undifferentiated 661W cells did not increase their sensitivity to HDAC3 expression. Conclusions: Collectively, these results indicate that HDAC3-induced toxicity to neurons is mediated by the intrinsic apoptotic pathway.

Lysine Deacetylase Substrate Selectivity: A Dynamic Ionic Interaction Specific to KDAC8.

Lysine acetylation and deacetylation are critical for regulation of many cellular proteins. Despite the importance of this cycle, it is unclear how lysine deacetylase (KDAC) family members discriminate between acetylated proteins to react with a discrete set of substrates. Potential short-range interactions between KDAC8 and a known biologically relevant peptide substrate were identified using molecular dynamics (MD) simulations. Activity assays with a panel of peptides derived from this substrate supported a putative ionic interaction between arginine at the -1 substrate position and KDAC8 D101. Additional assays and MD simulations confirmed this novel interaction, which promotes deacetylation of substrates. Verification that a negatively charged residue at the 101 position is necessary for the ionic interaction and observed reactivity with the substrates was performed using KDAC8 derivatives. Notably, this interaction is specific to KDAC8, as KDAC1 and KDAC6 do not form this interaction and each KDAC has a different specificity profile with the peptide substrates, even though all KDACs could potentially form ionic interactions. When reacted with a panel of putative human KDAC substrates, KDAC8 preferentially deacetylated substrates containing an arginine at the -1 position. KDAC8 D101-R(-1) is a specific enzyme-substrate interaction that begins to explain how KDACs discriminate between potential substrates and how different KDAC family members can react with different subsets of acetylated proteins in cells. This multi-pronged approach will be extended to identify other critical interactions for KDAC8 substrate binding and determine critical interactions for other KDACs.

Sulforaphane attenuates cisplatin-induced hearing loss by inhibiting histone deacetylase expression.

INTRODUCTION: Cruciferous vegetables are a rich source of sulforaphane (SFN), which acts as a natural HDAC inhibitor (HDACi). Our previous study found that HDACi could restore histone acetyltransferase/histone deacetylase (HAT/HDAC) balance in the cochlea and attenuate gentamicin-induced hearing loss in guinea pigs. Here, we investigated the protective effect of SFN on cisplatin-induced hearing loss (CIHL). METHODS: Thirty rats were randomly divided into 3 equal groups: the control group, cisplatin group, and SFN+cisplatin group. Rats were injected with SFN (30 mg/kg once a day) and cisplatin (7 mg/kg twice a day) for 7 days to investigate the protective role of SFN on CIHL. We observed auditory brainstem response (ABR) threshold shifts and immunostained cochlear basilar membranes of rats. For in vitro experiments, we treated HEI-OC1 cells and rat cochlear organotypic cultures with SFN (5, 10, and 15 µM) and cisplatin (10 µM). Immunofluorescence, cell viability, and protein analysis were performed to further analyze the protective mechanism of SFN on CIHL. RESULTS: SFN (30 mg/kg once a day) decreased cisplatin (7 mg/kg twice a day)-induced ABR threshold shifts and outer hair cell loss. CCK-8 assay showed that cisplatin (10 µM) reduced the viability of HEI-OC1 cells to 42%, and SFN had a dose-dependent protective effect. In cochlear organotypic cultures, we found that SFN (10 and 15 µM) increased cisplatin (10 µM)-induced myosin 7a+ cell count and restored ciliary morphology. SFN (5, 10, and 15 µM) reversed the cisplatin (10 µM)-induced increase in HDAC2, -4, and -5 and SFN (15 µM) reversed the cisplatin (10 µM)-induced decrease in H3-Ack9 [acetyl-histone H3 (Lys9)] protein expression in HEI-OC1 cells. Neither cisplatin nor cisplatin combined with SFN affected the expression of HDAC7, or HDAC9. CONCLUSION: SFN prevented disruption of the HAT/HDAC balance, protecting against CIHL in rats.

Apocynin Prevents Anxiety-Like Behavior and Histone Deacetylases Overexpression Induced by Sub-Chronic Stress in Mice.

Anxiety disorders are common mental health diseases affecting up to 7% of people around the world. Stress is considered one of the major environmental risk factors to promote anxiety disorders through mechanisms involving epigenetic changes. Moreover, alteration in redox balance and increased reactive oxygen species (ROS) production have been detected in anxiety patients and in stressed-animal models of anxiety. Here we tested if the administration of apocynin, a natural origin antioxidant, may prevent the anxiety-like phenotype and reduction of histone acetylation induced by a subchronic forced swimming stress (FSS) paradigm. We found that apocynin prevented the enhanced latency time in the novelty-suppressed feeding test, and the production of malondialdehyde induced by FSS. Moreover, apocynin was able to block the upregulation of p47phox, a key subunit of the NADPH oxidase complex. Finally, apocynin prevented the rise of hippocampal Hdac1, Hdac4 and Hdac5, and the reduction of histone-3 acetylation levels promoted by FSS exposure. In conclusion, our results provide evidence that apocynin reduces the deleterious effect of stress and suggests that oxidative stress may regulate epigenetic mechanisms.

Expression and prognostic analyses of HDACs in human gastric cancer based on bioinformatic analysis.

ABSTRACT: Gastric cancer (GC) is a common cancerous tumor, and is the third leading cause of cancer mortality worldwide. Although comprehensive therapies of GC have been widely used in clinical set ups, advanced gastric cancer carries is characterized by poor prognosis, probably due to lack of effective prognostic biomarkers. Mammalian histone deacetylase family, histone deacetylases (HDACs), play significant roles in initiation and progression of tumors. Aberrant expression of HDACs is reported in many cancer types including gastric cancer, and may serve as candidate biomarkers or therapeutic targets for GC patients.Gene Expression Profiling Interactive Analysis was used to explore mRNA levels of HDACs in GC. Kaplan-Meier plotter was used to determine the prognostic value of HDACs mRNA expression in GC. Genomic profiles including mutations of HDACs were retrieved from cBioPortal webserver. A protein-protein interaction network was constructed using STRING database. GeneMANIA was used to retrieve additional genes or proteins related to HDACs. R software was used for functional enrichment analyses.Analysis of mRNA levels of HDAC1/2/4/8/9 showed that they were upregulated in GC tissues, whereas HDAC6/10 was downregulated in GC tissues. Aberrant expression of HDAC1/3/4/5/6/7/8/10/11 was all correlated with prognosis in GC. In addition, expression levels of HDACs were correlated with different Lauren classifications, and clinical stages, lymph node status, treatment, and human epidermal growth factor receptor 2 status in GC.The findings of this study showed that HDAC members are potential biomarkers for diagnosis or prognosis of gastric cancer. However, further studies should be conducted to validate these findings.

Upregulation of histone acetylation reverses organic anion transporter 2 repression and enhances 5-fluorouracil sensitivity in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The failure of chemotherapy in HCC patients is partly due to inadequate intracellular drug accumulation caused by abnormally expressed drug transporters. Human organic anion transporter 2 (hOAT2), a transporter mainly expressed in liver and kidney, is responsible for uptake of various antineoplastic drugs such as 5-fluorouracil (5-FU). Among 32 pairs of human HCC samples, we preliminarily found that OAT2 was suppressed in HCC tissues compared with matched tumor-adjacent tissues at both mRNA and protein levels, which resulted in 5-FU resistance in HCC. However, the epigenetic regulatory mechanisms of OAT2 downregulation have not been investigated. In this study, we first proved it was histone hypoacetylation rather than DNA hypermethylation that participated in transcriptional repression of OAT2 in two HCC cell lines (BEL-7402 and SMMC-7721). In general, there were two pathways confirmed using tissues and cells: 1) Increased histone deacetylase sirtuin 7 (SIRT7) mediated loss of histone 3 lysine 18 acetylation (H3K18ac) at the promoter of OAT2 and inhibited its transcription. 2) More histone deacetylase 7 (HDAC7) instead of lysine acetyltransferase 8 (KAT8) enrichment at the promoter of OAT2 led to low levels of histone 4 lysine 16 acetylation (H4K16ac). Further, we found that histone deacetylases inhibitor vorinostat (SAHA) could reverse histone hypoacetylation state to activate OAT2 transcription and enhance uptake of classic OAT2 substrate zidovudine. Therefore, we evaluated the effect of combining SAHA and 5-FU and the results demonstrated that SAHA could sensitize HCC cells to 5-FU. Collectively, we proposed such a combination treatment to overcome 5-FU resistance in HCC from the perspective of epigenetically restoring OAT2.

HDAC3 Downregulation Improves Cerebral Ischemic Injury via Regulation of the SDC1-Dependent JAK1/STAT3 Signaling Pathway Through miR-19a Upregulation.

Histone deacetylase (HDAC) inhibitors can protect the brain from ischemic injury. This study aimed to identify the regulation of HDAC3 in cerebral ischemic injury. Middle cerebral artery occlusion (MCAO) was performed to establish a mouse model with cerebral ischemic injury, in which expression of HDAC3 and miR-19a was evaluated using RT-qPCR. In MCAO mice with silencing of HDAC3, infarct volume was determined using 2,3,5-triphenyl tetrazolium chloride (TTC) staining, and serum levels of TNF-α, IL-6, and IL-8 were measured using ELISA. An in vitro model was constructed in human umbilical vein endothelial cells (HUVECs) with oxygen-glucose deprivation/reoxygenation (OGD/R), followed by gain- and loss-of-function experiments. Relationships among miR-19a, HDAC3, and syndecan-1 (SDC1) were explored using RIP, ChIP, and dual-luciferase reporter assays. The expression of HDAC3, SDC1, JAK1, and STAT3 along with the extent of JAK1 and STAT3 phosphorylation was measured by Western blot analysis. HUVEC viability, apoptosis, and angiogenesis were assessed by CCK-8, flow cytometry, and angiogenesis assays in vitro separately. We found elevated HDAC3 and downregulated miR-19a expression in the MCAO mice. Decreased TNF-α, IL-6, and IL-8 serum levels were observed in response to silencing of HDAC3. HDAC3 inhibited the expression of miR-19a, which in turn targeted SDC1, leading to JAK1/STAT3 signaling pathway activation. HDAC3 overexpression or miR-19a inhibition repressed HUVEC viability and angiogenesis but enhanced HUVEC apoptosis. Our data unraveled the mechanism whereby HDAC3 inhibition ameliorated cerebral ischemic injury by activating the JAK1/STAT3 signaling pathway through miR-19a-mediated SDC1 inhibition.

HDAC3 Activity within the Nucleus Accumbens Regulates Cocaine-Induced Plasticity and Behavior in a Cell-Type-Specific Manner.

Epigenetic mechanisms regulate processes of neuroplasticity critical to cocaine-induced behaviors. This includes the Class I histone deacetylase (HDAC) HDAC3, known to act as a negative regulator of cocaine-associated memory formation within the nucleus accumbens (NAc). Despite this, it remains unknown how cocaine alters HDAC3-dependent mechanisms. Here, we profiled HDAC3 expression and activity in total NAc mouse tissue following cocaine exposure. Although chronic cocaine did not affect expression of Hdac3 within the NAc, chronic cocaine did affect promoter-specific changes in HDAC3 and H4K8Ac occupancy. These changes in promoter occupancy correlated with cocaine-induced changes in expression of plasticity-related genes. To causally determine whether cocaine-induced plasticity is mediated by HDAC3's deacetylase activity, we overexpressed a deacetylase-dead HDAC3 point mutant (HDAC3-Y298H-v5) within the NAc of adult male mice. We found that disrupting HDAC3's enzymatic activity altered selective changes in gene expression and synaptic plasticity following cocaine exposure, despite having no effects on cocaine-induced behaviors. In further assessing HDAC3's role within the NAc, we observed that chronic cocaine increases Hdac3 expression in Drd1 but not Drd2-cells of the NAc. Moreover, we discovered that HDAC3 acts selectively within D1R cell-types to regulate cocaine-associated memory formation and cocaine-seeking. Overall, these results suggest that cocaine induces cell-type-specific changes in epigenetic mechanisms to promote plasticity important for driving cocaine-related behaviors.SIGNIFICANCE STATEMENT Drugs of abuse alter molecular mechanisms throughout the reward circuitry that can lead to persistent drug-associated behaviors. Epigenetic regulators are critical drivers of drug-induced changes in gene expression. Here, we demonstrate that the activity of an epigenetic enzyme promotes neuroplasticity within the nucleus accumbens (NAc) critical to cocaine action. In addition, we demonstrate that these changes in epigenetic activity drive cocaine-seeking behaviors in a cell-type-specific manner. These findings are key in understanding and targeting cocaine's impact of neural circuitry and behavior.

CYP2C9 inhibits the invasion and migration of esophageal squamous cell carcinoma via downregulation of HDAC.

Cytochrome P450 2C9 (CYP2C9) is involved in the metabolism of cancer drugs and exogenous carcinogens. In our study, CYP2C9 was downregulated in multiple cohorts of human esophageal squamous cell carcinoma (ESCC). Until now, its role and epigenetic regulation of CYP2C9 repression in ESCC remain poorly understood. CYP2C9 repression in collected ESCC patient tumor tissues was demonstrated by RT-qPCR and Western blot. The histone acetylation level was carried out by the treatment of histone deacetylase inhibitor TSA and RNA interference. Epigenetic analysis revealed that the increased expression of CYP2C9 in KYSE-150 and TE1 cells was characterized by inhibition of HDAC8 and HDAC1, respectively. TSA decreased the levels of HDAC occupancy around CYP2C9 promoter region greatly. Overexpression of CYP2C9 reduced the invasion and migration of ESCC cells.

NADPH levels affect cellular epigenetic state by inhibiting HDAC3-Ncor complex.

NADPH has long been recognized as a key cofactor for antioxidant defence and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We find that the reduction of cellular NADPH levels, achieved by silencing malic enzyme or glucose-6-phosphate dehydrogenase, impairs global histone acetylation and transcription in both adipocytes and tumour cells. These effects can be reversed by supplementation with exogenous NADPH or by inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator nuclear receptor corepressor 2 (Ncor2; SMRT) or Ncor1, thereby impairing HDAC3 activation. Interestingly, NADPH and the inositol tetraphosphate molecule Ins(1,4,5,6)P4 appear to bind to the same domains on HDAC3, with NADPH having a higher affinity towards HDAC3 than Ins(1,4,5,6)P4. Thus, while Ins(1,4,5,6)P4 promotes formation of the HDAC3-Ncor complex, NADPH inhibits it. Collectively, our findings uncover a previously unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3.

Targeting HDAC3 in the DBA/2J spontaneous mouse model of glaucoma.

High intraocular pressure (IOP) is the most common risk factor associated with glaucoma in humans. While lowering IOP is effective at reducing the rate of retinal ganglion cell (RGC) loss, to date, no treatment exists to directly preserve these cells affected by damage to the optic nerve. Recently, histone deacetylase-3 (HDAC3) has become a potential therapeutic target because it plays an important role in the early nuclear atrophic events that precede RGC death. Conditional knockout or inhibition of HDAC3 prevents histone deacetylation, heterochromatin formation, apoptosis, and eventual RGC loss following acute optic nerve injury. Using these approaches to repress HDAC3 activity, we tested whether targeting HDAC3 protects RGCs from ganglion cell-specific BRN3A expression loss, total somatic cell loss, and optic nerve degeneration in the DBA/2J mouse model of spontaneous glaucoma. Targeted ablation of Hdac3 activity did not protect RGCs from axonal degeneration or somatic cell death in the DBA/2J mouse model of glaucoma. However, inhibition of HDAC3 activity using RGFP966 conferred mild protection against somatic cell loss in the ganglion cell layer in aged DBA/2J mice. Further experimentation is necessary to determine whether other class I HDACs may serve as potential therapeutic targets in chronic models of glaucoma.

The microRNA miR-134-5p induces calcium deposition by inhibiting histone deacetylase 5 in vascular smooth muscle cells.

Calcium deposition in vascular smooth muscle cells (VSMCs) is a form of ectopic ossification in blood vessels. It can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that the microRNA miR-134-5p potentiates inorganic phosphate (Pi)-induced calcium deposition in VSMCs by inhibiting histone deacetylase 5 (HDAC5). Using miRNA microarray analysis of Pi-treated rat VSMCs, we first selected miR-134-5p for further evaluation. Quantitative RT-PCR confirmed that miR-134-5p was increased in Pi-treated A10 cells, a rat VSMC line. Transfection of miR-134-5p mimic potentiated the Pi-induced increase in calcium contents. miR-134-5p increased the amounts of bone runt-related transcription factor 2 (RUNX2) protein and bone morphogenic protein 2 (BMP2) mRNA in the presence of Pi but decreased the expression of osteoprotegerin (OPG). Bioinformatic analysis showed that the HDAC5 3'untranslated region (3'UTR) was one of the targets of miR-134-5p. The luciferase construct containing the 3'UTR of HDAC5 was down-regulated by miR-134-5p mimic in a dose-dependent manner in VSMCs. Overexpression of HDAC5 mitigated the calcium deposition induced by miR-134-5p. Our results suggest that a Pi-induced increase of miR-134-5p may cause vascular calcification through repression of HDAC5.

Chidamide combined with paclitaxel effectively reverses the expression of histone deacetylase in lung cancer.

The role of histone deacetylases (HDACs) in lung cancer has been extensively studied. Inhibition of HDAC activities have been used as a new cancer treatment strategy. To date, many HDAC inhibitors have been shown to induce apoptosis and inhibit tumorigenesis. Chidamide (CS055) is a new member of HDAC inhibitors. In China, Chidamide has been approved for the treatment of relapsed or refractory peripheral T-cell lymphoma. However, the efficacy of Chidamide in non-small cell lung cancer remains unclear. In this study, we used lung cancer primary cells and investigated the effects of Chidamide combined with paclitaxel on lung cancer. We found that Chidamide combined with paclitaxel effectively inhibited the expression and activity of HDAC in primary lung cancer cells, induced their apoptosis and blocked cell cycle. Chidamide combined with paclitaxel may therefore provide a new promising therapeutic treatment for lung cancer.

Overexpression of HDAC6, but not HDAC3 and HDAC4 in the penumbra after photothrombotic stroke in the rat cerebral cortex and the neuroprotective effects of α-phenyl tropolone, HPOB, and sodium valproate.

Epigenetic processes play important roles in brain responses to ischemic injury. We studied effects of photothrombotic stroke (PTS, a model of ischemic stroke) on the intracellular level and cellular localization of histone deacetylases HDAC3, HDAC4 and HDAC6 in the rat brain cortex, and tested the potential neuroprotector ability of their inhibitors. The background level of HDAC3, HDAC4 and HDAC6 in the rat cerebral cortex was relatively low. HDAC3 localized in the nuclei of some neurons and few astrocytes. HDAC4 was found in the neuronal cytoplasm. After PTS, their levels in penumbra did not change, but HDAC4 appeared in the nuclei of some cells. Its level in the cytoplasmic, but not nuclear fraction of penumbra decreased at 24, but not 4 h after PTS. HDAC6 was upregulated in neurons and astrocytes in the PTS-induced penumbra, especially in the nuclear fraction. Unlike HDAC3 and HDAC4, HDAC6 co-localized with TUNEL-positive apoptotic cells. Inhibitory analysis confirmed the involvement of HDAC6, but not HDAC3 and HDAC4 in neurodegeneration. HDAC6 inhibitor HPOB, HDAC2/8 inhibitor α-phenyl tropolone, and non-specific histone deacetylase inhibitor sodium valproate, but not HDAC3 inhibitor BRD3308, or HDAC4 inhibitor LMK235, decreased PTS-induced infarction volume in the mouse brain, reduced apoptosis, and recovered the motor behavior. HPOB also restored PTS-impaired acetylation of α-tubulin. α-phenyl tropolone restored acetylation of histone H4 in penumbra cells. These results suggest that histone deacetylases HDAC6 and HDAC2 are the possible molecular targets for anti-ischemic therapy, and their inhibitors α-phenyl tropolone, HBOP and sodium valproate can be considered as promising neuroprotectors.

Stimulation of toll-like receptor 4 downregulates the expression of α7 nicotinic acetylcholine receptors via histone deacetylase in rodent microglia.

Microglia have both protective and degenerative roles in the central nervous system. The α7 nicotinic acetylcholine receptor (nAChR) is crucial in the regulation of the neuroprotective role in microglia. Recent studies have demonstrated decreased expression of α7 nAChR in brain in response to neuroinflammation, but the mechanism mediating the downregulation of the α7 nAChR has yet to be elaborated. Treatment of microglial cell line BV2 cells or rat primary cultured microglia with the inflammogen lipopolysaccharide (LPS) significantly decreased the expression of α7 nAChR mRNA in a time and concentration-dependent manner. The effects of LPS were prevented by pretreatment with TAK-242, a toll-like receptor 4 (TLR4) blocker. The LPS-induced downregulation of α7 nAChR was also prevented with trichostatin A, a histone deacetylase (HDAC) inhibitor, but not 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor. Further pharmacological probing revealed that HDAC2 and HDAC3 were involved in the effects of LPS. Treatment of BV2 cells with LPS significantly reduced acetylation of histone H3 at lysine 9 of the α7 nAChR promoter. The current findings demonstrate that inflammation-evoked activation of TLR4 leads to the reduction of the neuroprotective function of microglia through the downregulation of the α7 nAChR. Also, histone modification could be crucial in the regulation of the neuroprotective role of microglia during neuroinflammatory states.

Advanced glycation end products inhibit neural stem cell differentiation via upregualtion of HDAC3 expression.

Diabetes mellitus (DM) is a highly prevalent chronic systemic disease, which may cause cognitive decline and degenerative change of the brain. Neuronal differentiation defects of neural stem cells (NSCs) played an important role in the development and progression of diabetes-associated cognitive decline (DACD), but the intrinsic pathological mechanism remains largely unclear. In the present study, we demonstrated that expression level of HDAC3 was upregulated in diabetic mice with reduced learning and memory abilities and in cultured NSCs after advanced glycation end products (AGEs) induction. In addition, AGEs interfered with normal differentiation of the cultured NSCs, and knocking down the expression of HDAC3 could partially attenuate the inhibitory effect of AGEs on NSCs differentiation. Findings in this study demonstrate that HDAC3 may serve as an experimental clue for revealing the pathogenesis of DACD.

Inhibition of histone deacetylase 3 by MiR-494 alleviates neuronal loss and improves neurological recovery in experimental stroke.

HDAC3 is an essential negative regulator of neuronal plasticity and memory formation. Although a chemical inhibitor has been invented, little is known about its endogenous modulators. We explored whether miR-494 affects HDAC3-mediated neuronal injury following acute ischemic stroke. A substantial increase in plasma miR-494 was detected in AIS patients and was positively associated with the mRS at one year after symptom onset. The miR-494 levels were transiently increased in the infarcted brain tissue of mice. In contrast, miR-494 levels were reduced in neurons but increased in the medium after OGD. Intracerebroventricular injection of miR-494 agomir reduced neuronal apoptosis and infarct volume at the acute stage of MCAO, promoted axonal plasticity and long-term outcomes at the recovery stage, suppressed neuronal ataxin-3 and HDAC3 expression and increased acetyl-H3K9 levels in the ipsilateral hemisphere. In vitro studies confirmed that miR-494 posttranslationally inhibited HDAC3 in neurons and prevented OGD-induced neuronal axonal injury. The HDAC3 inhibitor increased acetyl-H3K9 levels and reversed miR-494 antagomir-aggravated acute cerebral ischemic injury, as well as brain atrophy and long-term functional recovery. These results suggest that miR-494 may serve as a predictive biomarker of functional outcomes in AIS patients and a potential therapeutic target for the treatment of ischemic stroke.

Epigenetic suppression of liver X receptor ß in anterior cingulate cortex by HDAC5 drives CFA-induced chronic inflammatory pain.

BACKGROUND: Liver X receptors (LXRs), including LXRα and LXRß, are key regulators of transcriptional programs for both cholesterol homeostasis and inflammation in the brain. Here, the modes of action of LXRs and the epigenetic mechanisms regulating LXRß expression in anterior cingulate cortex (ACC) of chronic inflammatory pain (CIP) are investigated. METHODS: The deficit of LXR isoform and analgesic effect of LXR activation by GW3965 were evaluated using the mouse model of CIP induced by hindpaw injection of complete Freund's adjuvant (CFA). The mechanisms involved in GW-mediated analgesic effects were analyzed with immunohistochemical methods, ELISA, co-immunoprecipitation (Co-IP), Western blot, and electrophysiological recording. The epigenetic regulation of LXRß expression was investigated by chromatin immunoprecipitation, quantitative real-time PCR, and sequencing. RESULTS: We revealed that CFA insult led to LXRß reduction in ACC, which was associated with upregulated expression of histone deacetylase 5 (HDAC5), and knockdown of LXRß by shRNA led to thermal hyperalgesia. Co-IP showed that LXRß interacted with NF-κB p65 physically. LXRß activation by GW3965 exerted analgesic effects by inhibiting the nuclear translocation of NF-κB, reducing the phosphorylation of mitogen-activated protein kinases (MAPKs) in ACC, and decreasing the promoted input-output and enhanced mEPSC frequency in ACC neurons after CFA exposure. In vitro experiments confirmed that HDAC5 triggered histone deacetylation on the promoter region of Lxrß, resulting in downregulation of Lxrß transcription. CONCLUSION: These findings highlight an epigenetic mechanism underlying LXRß deficits linked to CIP, and LXRß activation may represent a potential novel target for the treatment of CIP with an alteration in inflammation responses and synaptic transmission in ACC.