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Volume 5 • 2012 10.1093/biohorizons/hzs003

Novel histone deacetylase (HDAC) inhibitors with improved selectivity for HDAC2 and 3 protect against neural cell deathBenjamin Durham*

Faculty of Biological Sciences, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK.

*Corresponding author: Email: bs08bsd@leeds.ac.uk

Neurodegenerative disorders, such as motor neurone disease, Alzheimer’s disease and responses to brain traumas such as stroke, involve the unwanted death of neural cells. Although the exact underlying mechanisms leading to neural cell death are not well defined, one contributory event in many situations is the over-excitation of cells caused by too much of the neu-rotransmitter glutamate. Drugs that inhibit enzymes called histone deacetylases (HDACs) can protect neural cells from gluta-mate excitotoxicity. However, current inhibitors lack specificity and although they function in vitro, they have a substantial potential for adverse side effects in vivo. HDAC2 and 3 have been implicated in neurotoxicity and here we investigated the neuroprotective potential of three novel HDAC inhibitors that show selectivity for these. The ability of these HDAC inhibitors to protect against glutamate excitotoxicity was tested using cultured organotypic cerebral slices from 7-day-old (P7) Wistar rats. Glutamate excitotoxicity was induced by 200 µM of the glutamate transporter blocker, DL-threo-β-benzyloxyaspartate (DL-TBOA). This was applied alone and alongside 1 µM of the novel HDAC2 and 3 selective inhibitors AH51, AH61 and AH62. Neural cell viability in slices was quantified from assays using the fluorescent stains, 4′,6-diamidino-2-phenylindole and ethid-ium homodimer-1. The induction of glutamate excitotoxicity by DL-TBOA resulted in 41.3 ± 6.1% (n = 7, P < 0.01) loss in cell viability as judged by ethidium homodimer-1 staining. All three novel HDAC inhibitors significantly prevented neural cell death in response to DL-TBOA (P < 0.01), with cell viabilities of 107.5 ± 6.01% (n = 4), 97.1 ± 16.5% (n = 3) and 106.7 ± 6.45% (n = 4) for AH51, AH61 and AH62, respectively. This study has shown that inhibitors selective for HDAC2 and 3 can protect neural cells from death and thus have potential as therapeutic agents against neurotoxicity.

Key words: histone deacetylase inhibitors, HDAC2, HDAC3, neuroprotection, neurodegeneration, glutamate excitotoxicity

Submitted November 2011; accepted February 2012

IntroductionThe mechanisms that induce cell death in the central nervous system (CNS) are diverse, but many neurodegenerative diseases share some common features that can be exploited by thera-peutics. New drugs should aim to prevent cell death by acting on specific targets, and this also reduces the risk of significant side effects. A major toxic insult implicated in the pathophysi-ology of neurodegenerative diseases, including motor neurone disease, Alzheimer’s disease and stroke, is glutamate excitotox-icity (reviewed by Dong, Wang and Qin, 2009) and targeting this process is one potential therapeutic option.

Glutamate is the major excitatory neurotransmitter in the CNS; it binds to and activates ionotropic and metabotropic glutamate receptors, expressed by neurones, astrocytes, oligodendrocytes, reactive microglia and their precursors (Matute et al., 2002; Lee et al., 2010). Ionotropic receptors are cation (Na+, Ca2+ and K+) channels and include the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), kainic acid and N-methyl-d-aspartic acid (NMDA) receptors (Matute et al., 2002). Metabotropic receptors (mGluRs) are coupled with G-proteins, intracellular cascades and include mGluR1 and mGluR5 linked to the inositol tri-sphosphate (IP3)/Ca2+ signalling pathway involving calcium

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release from the endoplasmic reticulum (Pin and Duvoisin, 1995). During glutamate overload and excitotoxicity, over-activation of these glutamate receptors results in prolonged and massive depolarizations, excessive calcium influx into neural cells and calcium release from internal stores. Ultimately, this causes the inappropriate activation of cal-cium-dependent processes such as proteases, caspases, lipases, endonucleases, pro-apoptotic factors and the produc-tion of free radicals from the mitochondria (Arundine and Tymianski, 2003). Mitochondria act as calcium sinks, but when these organelles are overloaded with large amounts of calcium, they produce high levels of free radicals (Carriedo et al., 1998), which oxidize and damage DNA, proteins and lipids. One consequence of DNA damage is the accumulation of p53 in the nucleus and this promotes the expression of pro-apoptotic proteins that cause the mitochondrial release of cytochrome c, a major apoptotic factor that promotes the formation of the apoptosome and the activation of caspases (Jiang and Wang, 2004; Gogvadze and Orrenius, 2006). All these events lead to DNA, lipid and protein damage and breakdown, degradation of cell integrity, organelles and ulti-mately triggering necrotic or apoptotic neural cell death (Arundine and Tymianski, 2003).

Under normal conditions, over-activation of glutamate receptors does not occur due to the regulation and termina-tion of glutamatergic neurotransmission. Glutamate is removed from the extracellular space by Na+-dependent glu-tamate transporters also known as excitatory amino-acid transporters (EAATs). These are located in the plasma mem-brane of neurones and glial cells (reviewed by Matute et al., 2002). Glutamate excitotoxicity can be induced experimen-tally in neural cells by the application of an EAAT inhibitor such as DL-threo-β-benzyloxyaspartate (DL-TBOA, Shigeri, Seal and Shimamoto, 2004). Such application is a commonly used model of excitotoxicity and allows for the study of ther-apeutics on neural cell death and survival.

Inhibition of histone deacetylases can provide neuroprotectionHistone acetyltransferases (HATs) add acetyl groups and histone deacetylases (HDACs) remove acetyl groups from lysine residues in proteins. These enzymes play pivotal roles in epigenetic regulation of gene transcription by remodelling chromatin structure. The fundamental unit of chromatin is the nucleosome, composed of ~147 bp of DNA wrapped around an octamer structure of histone proteins; each histone protein has a protruding N-terminus with exposed lysine residues that are subjected to acetylation by HATs and deacetylation by HDACs (reviewed by Kornberg and Lorch, 1999). Histone acetylation causes a weaker association between DNA and histones, promoting a more open, more accessible chromatin structure, whereas deacetylation causes a tighter association between the DNA and histones, leading to a more compact, less accessible chromatin conformation for transcriptional machinery to initiate transcription (reviewed by Kornberg and Lorch, 1999). HDACs are widely

distributed in the rat brain and are expressed by both neurones and glial cells (Broide et al., 2007). There are four classes of HDACs: class I HDACs (1, 2, 3 and 8) are found within the cell nucleus where they can deacetylate histones; class II HDACs (4–7, 9 and 10) shuttle between the nucleus and the cytoplasm and as well as histones, they also deacety-late cytoplasmic proteins such as the microtubules; class III HDACs, also known as the sirtuins, couple deacetylation to NAD+ hydrolysis and the single member of class IV HDACs, HDAC11, has features in common with both class I and II HDACs (reviewed by Gregoretti, Lee and Goodson, 2004).

Class I and II HDAC inhibitors, including valproic acid, sodium phenylbutyrate and suberoylanilide hydroxamic acid (SAHA), are neuroprotective against glutamate excitotoxic-ity. Using rat neurones in vitro, both valproic acid and sodium phenylbutyrate caused the up-regulation of pro-survival and anti-apoptotic genes, including heat shock protein-70 (HSP70, Marinova et al., 2009) and Bcl-2 (Leng et al., 2010). SAHA has also been shown to be neuroprotective in a white matter ischaemic stroke model using mouse optic nerves; SAHA preserved white matter structure and function, axonal survival and oligodendrocyte survival (Baltan et al., 2011).

Although they show beneficial properties, both valproic acid and SAHA lack specificity and are associated with significant in vivo side effects, including cognitive dysfunction, headaches, sedation, nausea and vomiting, thrombosis and a reduction in both blood cell count and blood electrolytes (Armon et al., 1996; MedlinePlus NIH, 2011; Merck, 2011). An attempt to reduce these side effects but still take advantage of the promising neuroprotective ability of HDAC inhibitors would involve targeting and inhibiting specific HDACs.

Activation and/or over-expression of specific HDACs has been associated with neurodegenerative disease and neural cell toxicity. Levels of HDAC2 in the motor cortex and the spinal cord are higher in patients with amyotrophic lateral sclerosis compared with controls (Janssen et al., 2010). Also, activation of HDAC3 resulting from phosphorylation by glycogen syn-thase kinase 3 β (GSK3β), a kinase that is widely implicated in neurodegenerative disease, promotes neural cell death (Bhat, Budd Haeberlein and Avila, 2004; Bardai and D’Mello, 2011). Furthermore, both HDAC2 and 3 negatively regulate memory (Guan et al., 2009; McQuown et al., 2011) and general class I HDAC inhibition was shown to restore some loss of memory function in an animal model of Alzheimer’s disease (Kilgore et al., 2010). Unlike the other class I HDACs, HDAC1 activity is actually neuroprotective in stroke and Huntington’s disease and inactivation or inhibition of HDAC1 leads to neurodegen-eration (Bates et al., 2006; Kim et al., 2008). Therefore, a com-pound that selectively inhibits HDAC2 and 3 but not HDAC1 should be therapeutically better for treating disorders of the brain than the currently available non-selective general class I and II HDAC inhibitors.

The studies discussed reveal that there could be therapeutic and neuroprotective potential from selectively inhibiting HDAC2 and 3. The University of Leeds developed MI192, a

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HDAC2 and 3 selective inhibitor (MI192 has low nM potency against HDAC2 and 3. Fold selectivity for HDAC1, 2 and 3 vs. HDAC3 is >250, 1.9 and 1, respectively, Cancer Research Technology, 2011; Gillespie et al., 2011) and from this pro-duced a further three HDAC2 and 3 selective inhibitors AH51, AH61 and AH62. In this study, we have assessed the neuropro-tective potential of these daughter compounds, using an organ-otypic rat cerebral slice model of glutamate excitotoxicity.

Methods

Organotypic cerebral slice culture preparationOrganotypic cerebral slices were produced as described previ-ously (Stoppini, Buchs and Muller, 1991) with modifications. Seven-day-old (P7) Wistar rats underwent cervical dislocation followed by decapitation. The brain was rapidly removed under sterile conditions and placed in ice-cold, 0.22 µm filter-sterilized (Millipore) dissection medium composed of Minimum Essential Medium Eagle with Earle’s salts and NaHCO3 (MEM, Sigma) supplemented with 1% penicillin/streptomycin (Invitrogen). The olfactory bulb and the cerebel-lum were dissected and discarded; the remaining brain (cere-brum) was washed several times with ice-cold dissection medium before being embedded in 4% molten high-strength agar (Melford) dissolved in sterile water. Coronal cerebral slices (250 µm thick) were cut using a Leica VT1000S vibra-tome, collected and the agar around the slices removed. The slices were cultured on cell culture inserts (1 µm pore size, Falcon), up to a maximum of two slices per insert. The inserts were placed in 6-well culture plates (Falcon) containing 1 ml of filter sterilized, culture medium [MEM, 25% Hanks bal-anced salt solution (HBSS, Sigma), 25% normal horse serum (Invitrogen), 11 mM NaHCO3 (Fisher Scientific), 4.6 mM L-glutamine (Sigma), 21 mM D-glucose (Fisher Scientific) and 4.2 µM L-ascorbic acid (Sigma)] pre-warmed to 37°C. Slice cultures were incubated at 37°C in a humid atmosphere at 5% CO2. After 3 days of incubation, the culture medium was replaced with 1 ml of a filter-sterilized serum-free culture medium [MEM, 25% HBSS, 11 mM NaHCO3, 4.6 mM L-glutamine, 21 mM D-glucose and 4.2 µM L-ascorbic acid and 0.3% B27 supplement (Invitrogen)] pre-warmed to 37°C.

Induction of glutamate excitotoxicity and histone deacetylase inhibitor applicationAfter a total of 5 days of slice culture, the culture medium was replaced with 1 ml of fresh, serum-free culture medium containing the experimental conditions, pre-warmed to 37°C. Cerebral slices were treated with either control (culture medium alone), 2 µM NaOH (Sigma) alone and 2 µM NaOH with 0.1% DMSO (Sigma) the drug vehicles, or 200 µM DL-TBOA (Tocris Bioscience, dissolved in 1 M NaOH) with or without 1 µM AH51, AH61 and AH62 (University of Leeds, dissolved in DMSO). Slices were then cultured at 37°C in a humid atmosphere at 5% CO2 for 3 days before analysis.

Assessment of cell viabilityTo assess cell viability in the cerebral slices, 500 µl of 0.6 µM ethidium homodimer-1 (Invitrogen) dissolved in sterile phosphate-buffered saline (PBS, Oxoid) was applied to the slices. These were incubated for 30 min at 37°C in a humid atmosphere at 5% CO2. The cerebral slices were cut from the inserts, placed on glass slides and the tissue was then fixed with 100 µl of 4% paraformaldehyde (PFA, Sigma) in PBS for 15 min, in the dark at room temperature. Excess PFA was removed and the slices were washed three times for 5 min each with PBS. Excess PBS was removed and the slices were tissue-dried. Slides were mounted using Vectashield with 4′,6- diamidino-2-phenylindole (DAPI, Vector Laboratories) and stored at 4°C in the dark until analysis by confocal micros-copy. Imaging of ethidium homodimer-1 and DAPI-stained cerebral slices was performed using an upright- configured Zeiss Observer Z1 confocal microscope with a 40× oil immer-sion objective lens. Images were taken at 405 and 555 nm wavelengths to visualize DAPI (blue) and ethidium homodi-mer-1 (red) fluorescence, respectively. Four randomly selected non-overlapping images were taken from each slice and each image was saved at 1024 × 1024 pixel resolution.

Non-biased quantification of the images was performed independently by a blinded-observer using a 4 × 4 grid (3000 µm2 per grid-square) in ImageJ (NIH). The number of DAPI-stained nuclei and ethidium homodimer-1-stained nuclei were counted within the 4 × 4 grid for each image. Data are expressed as mean ± SEM percent cell viability taken as a percentage of control viability, n indicates the number of inde-pendent cerebral slices per experimental condition. Statistical analysis was performed with SPSS (IBM) using a one-way analysis of variance (ANOVA) followed by either the Dunnett or Bonferroni post hoc tests at the 1% significance level.

ResultsSelective HDAC2 and 3 inhibitors have potential in provid-ing neuroprotection against neurodegeneration. Here, we used an organotypic rat cerebral slice model of glutamate excitotoxicity, a common pathophysiology involved in neu-rodegeneration and one that is often used to model this. Glutamate excitotoxicity and the neuroprotective ability of our novel HDAC inhibitors against this, was assessed using cultured cerebral slices obtained from 7-day-old (P7) rats by simultaneous incubation of the slices with the glutamate transporter blocker, DL-TBOA and the HDAC inhibitors. After 3 days post-exposure, slices were analysed for ethid-ium-1 homodimer and DAPI staining (Fig. 1). Ethidium homodimer-1 is a membrane-impermeable nucleic acid fluo-rescent stain, which only stains nuclei in cells when the mem-brane integrity is impaired, particularly when the cell is dead or dying, the fluorescent stain DAPI, however, is cell membrane permeable and labels all nuclei and nucleic acid of live, dead or dying cells. The amount of cell staining for both stains, for all images per condition, was quantified. The

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Figure 1. Novel HDAC2 and 3 selective inhibitors protect against DL-TBOA-induced death in cultured rat cerebral slices. Confocal images from one representative cerebral slice treated with (A) control (culture medium alone), (B) 200 µM DL-TBOA, (C) 200 µM DL-TBOA and 1 µM AH51, (D) 200 µM DL-TBOA and 1 µM AH61 or (E) 200 µM DL-TBOA and 1 µM AH62. Left panels show DAPI (blue), middle panels ethidium homodimer-1 (red) and right panels show a merged image. Arrows indicate condensed and fragmenting chromatin. Scale bar 10 µm.

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control (culture medium alone) slice cell viability was 81.7 ± 3.9% (n = 6) and this was expressed as 100%. For all other experimental conditions, viability was quantified as mean ± SEM percent of cell viability, expressed as a percent-age of the control viability.

For slices treated with the vehicle conditions, cell viability was not significantly different to the control slices, incuba-tion with both 2 µM NaOH and 0.1% DMSO had a viability of 102 ± 12.4% (n = 3) and 2 µM NaOH alone the viability was 108 ± 3.9% (n = 3). Slices treated with 200 µM DL-TBOA showed a significant amount of cell death and the number of viable cells was reduced by 41.3 ± 6.1% (n = 7, compare Figs 1A with B and 2). The dead cells showed indi-cations of DNA breakdown, fragmentation and condensing of the chromatin that occurs during apoptosis (Aoyama et al., 2005); this was demonstrated by the punctate ethidium homodimer-1 staining. These data show that our model of glutamate excitotoxicity induces a significant amount of neu-ral cell death. We then tested our novel HDAC inhibitors to see if this cell death could be prevented.

To test the neuroprotective efficacy of the novel HDAC2 and 3 inhibitors, they were co-applied with 200 µM DL-TBOA and cell viability was assessed. AH51, AH61 and AH62 (1 µM) significantly reduced cell death brought upon by the application of DL-TBOA (P < 0.01) and resulted in a level of cell viability not significantly different from control (culture medium alone) slice cultures or from each other; 107.5 ± 6.01% (n = 4), 97.1 ± 16.5% (n = 3) and

106.7 ± 6.45% (n = 4) for AH51, AH61 and AH62, respec-tively (Figs 1C–E and 2). Our novel HDAC inhibitors pre-vented neural cell in our model of glutamate excitotoxicity, and these initial results show future promise, selective HDAC2 and 3 inhibitors still retain the beneficial neuropro-tective effects associated with the general non-selective HDAC inhibitors and may be useful as therapeutic agents against neural cell toxicity.

DiscussionHere, we have shown that three HDAC2 and 3 selective inhibitors, AH51, AH61 and AH62, are neuroprotective against glutamate excitotoxicity; a pathological process involved in many different neurodegenerative disorders. AH51, AH61 and AH62 were effective neuroprotective agents and because of their selectivity for HDAC2 and 3, these compounds are predicted to have better therapeutic potential, due to their lack of efficacy to inhibit HDAC1 and other HDACs, which can contribute to the production of side effects associated with currently available general non-selec-tive class I and II HDAC inhibitors.

This study shows the possibility of replacing non-specific HDAC inhibitors such as valproic acid and SAHA with more selective compounds as potential therapies for some neural disorders. As discussed earlier and reviewed by Kazantsev and Thompson (2008) and Chuang et al. (2009), general non-selective HDAC inhibitors have been well reported as efficacious neuroprotective agents against glutamate excito-toxicity. Our novel selective HDAC2 and 3 inhibitors fully protected against neural cell death in our disease model; therefore, our study shows the potential of moving away from general inhibitors towards more selective ones, such as those against HDAC2 and 3 and there is no compromise in neuroprotective efficacy. The exact molecular mechanisms of neuroprotection by inhibiting HDAC2 and 3 are not yet known. However, it is likely that the mechanisms responsible involve the increased transcription of pro-survival and anti-apoptotic genes, but may also involve the prevention of neu-rotoxicity associated with increased HDAC2 and 3 activities.

Cell culture models have been used to test HDAC inhibi-tors as neuroprotective agents against glutamate excitotoxic-ity (Marinova et al., 2009; Leng et al., 2010), but our model provides a more appropriate environment of the CNS with a mixture of cell types, which homogeneous cell cultures do not. Neurodegeneration in the CNS involves a complex inter-play between neurones and glial cells, so using intact cerebral slices maintains such a mix of different cell types and the interactions between them. We did not determine whether one specific cell type was more affected than others, what propor-tion of cell death was derived from neurones or glial cells and what possible changes in the interactions between glial cells and neurones took place. Nevertheless, HDAC inhibition pre-vented DL-TBOA-induced cell death, suggesting that HDAC inhibitors protect all cell types susceptible to DL-TBOA-

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Figure 2. Comparison of cell viability in cerebral slices between DL-TBOA and the novel HDAC2 and 3 selective inhibitors. Cerebral slices were treated with control (culture medium alone, n = 6), DL-TBOA (200 µM) alone (n = 7) or with 1 µM AH51 (n = 4), AH61 (n = 3) or AH62 (n = 4). Data shown are mean ± SEM percent cell viability expressed as a percentage of control, #P < 0.01 compared with control, *P < 0.01 compared with DL-TBOA.

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mediated death. Using cell-specific immunohistochemical labelling and specific brain regions alongside the viability assay, one could directly correlate neural cell death with spe-cific cell type and also assess any changes in glial and neurone interactions. By selecting specific brain regions such as the cortex, hippocampus or the substantia nigra, our selective HDAC2 and 3 inhibitors can also be assessed to see whether they better protect specific CNS regions and those predomi-nantly affected in different neurodegenerative diseases.

General class I and II HDAC inhibitors are not only neu-roprotective against glutamate excitotoxicity, but have been shown to be protective against other neurotoxic insults. Ryu et al. (2005) used organotypic mouse spinal cord slices from mice with mutant superoxide dismutase-1 (SOD1) amyo-trophic lateral sclerosis. By administering the general class I and II HDAC inhibitor sodium phenylbutyrate, they saw a concentration-dependent increase in motor neurone survival. Coinciding with this finding was increased histone acetyla-tion and prevention of apoptosis through an increase in the expression of Bcl-2 and a reduction in the release of cyto-chrome c from the mitochondria. Also, in a standard model of Parkinson’s disease, 1-methyl-4-phenylpyridinium (MPP+) induced, valproic acid prevented dopaminergic neurone loss in cell culture (Kidd and Schneider, 2010) but also partially prevented the degeneration of the substantia nigra and striatum in in vivo rat models of the disease (Kidd and Schneider, 2011). Chen et al. (2006) showed that valproic acid increases the expression of neurotrophins in dopaminer-gic neurones and glial cell cultures; including brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. These neurotrophins play prominent roles in neural cell development, neural cell survival and synaptic plasticity and therefore are ideal candidates to be increasingly expressed to promote neuroprotection and neural cell function. The novel HDAC2 and 3 selective inhibitors AH51, AH61 and AH62, used in our study, fully protected neural cells from glutamate excitotoxicity like that observed by others with the general class I and II HDAC inhibitors, and like the more general inhibitors, our selective ones may provide more wide-spread protection and be effective in treating various neuro-degenerative disorders.

ConclusionSelective HDAC inhibitors are a promising prospect for the future treatment of neural cell death induced by glutamate excitotoxicity. This study has shown that novel HDAC2 and 3 selective inhibitors can solely protect against this form of toxicity in an organotypic cerebral slice model. In addition, these compounds could have further promise as therapeutic agents in other forms of neurodegeneration.

AcknowledgementsI would like to thank Dr Ian C. Wood for his insightful contribution and providing his laboratory resources for this

study. Furthermore, I thank Katy Burnage, Domenic Manfredi and Laura Anne Willis for their contributions with obtaining the results. Finally, I thank Mohamed Al-Griw, for obtaining the rat brains and providing the organotypic slice culture and confocal microscopy training.

FundingThis study was funded by the Faculty of Biological Sciences, University of Leeds, as part of the undergraduate final year project scheme.

Author biographyB.D. graduated in July 2011 with a First Class BSc (Hons) in Medical Sciences from the Faculty of Biological Sciences, University of Leeds. In September 2011, he was shortlisted for the Best European Biology Student of the Year at the Science, Engineering and Technology (SET) Awards. He has a keen interest in uncovering the molecular mechanisms that control gene expression in human disease with a desire to develop and test therapies that reverse or compensate for this. B.D. started a PhD with Dr Ian C. Wood in October 2011.

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