BioscienceHorizons

Volume 5 2012 

10.1093/biohorizons/hzs003

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

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

Neurodegenerative disorders, such as motor neurone disease, Alzheimers 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 neurotransmitter glutamate. Drugs that inhibit enzymes called histone deacetylases (HDACs) can protect neural cells from glutamate 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 200M of the glutamate transporter blocker, DL-threo--benzyloxyaspartate
(DL-TBOA). This was applied alone and alongside 1M 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 ethidium homodimer-1. The induction of glutamate excitotoxicity by DL-TBOA resulted in 41.36.1% (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 107.56.01% (n=4), 97.116.5% (n=3) and 106.76.45%
(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

Introduction
The mechanisms that induce cell death in the central nervous
system (CNS) are diverse, but many neurodegenerative d
iseases
share some common features that can be exploited by therapeutics. 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 pathophysiology of neurodegenerative diseases, including motor neurone
disease, Alzheimers disease and stroke, is glutamate excitotoxicity (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
-amino-3-hydroxy-5-methylisoxazole-4-propionate
(AMPA), kainic acid and N-methyl-d-aspartic acid (NMDA)
receptors (Matute etal., 2002). Metabotropic receptors
(mGluRs) are coupled with G-proteins, intracellular cascades
and include mGluR1 andmGluR5 linked to the inositol trisphosphate (IP3)/Ca2+ signalling pathway involving calcium

The Author 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons
Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution,
and reproduction in any medium, provided the original work is properly cited.

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

Research article

Under normal conditions, over-activation of glutamate

receptors does not occur due to the regulation and termination of glutamatergic neurotransmission. Glutamate is
removed from the extracellular space by Na+-dependent glutamate transporters also known as excitatory amino-acid
transporters (EAATs). These are located in the plasma membrane of neurones and glial cells (reviewed by Matute etal.,
2002). Glutamate excitotoxicity can be induced experimentally in neural cells by the application of an EAAT inhibitor
such as DL-threo--benzyloxyaspartate (DL-TBOA, Shigeri,
Seal and Shimamoto, 2004). Such application is a commonly
used model of excitotoxicity and allows for the study of therapeutics on neural cell death and survival.

Inhibition of histone deacetylases can

provide neuroprotection
Histone 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 ~147bp 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 etal., 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 (47, 9 and 10) shuttle between the nucleus
and the cytoplasm and as well as histones, they also deacetylate 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 excitotoxicity. 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 etal., 2009) and Bcl-2 (Leng etal., 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 etal., 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 etal., 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 etal., 2010). Also, activation
of HDAC3 resulting from phosphorylation by glycogen synthase kinase 3 (GSK3), a kinase that is widely implicated in
neurodegenerative disease, promotes neural cell death (Bhat,
Budd Haeberlein and Avila, 2004; Bardai and DMello, 2011).
Furthermore, both HDAC2 and 3 negatively regulate memory
(Guan etal., 2009; McQuown etal., 2011) and general class I
HDAC inhibition was shown to restore some loss of memory
function in an animal model of Alzheimers disease (Kilgore
etal., 2010). Unlike the other class I HDACs, HDAC1 activity
is actually neuroprotective in stroke and Huntingtons disease
and inactivation or inhibition of HDAC1 leads to neurodegeneration (Bates etal., 2006; Kim etal., 2008). Therefore, a compound 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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release from the e ndoplasmic reticulum (Pin and Duvoisin,

1995). During glutamate overload and excitotoxicity, overactivation 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 calcium-dependent processes such as proteases, caspases,
lipases, endonucleases, pro-apoptotic factors and the production 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
etal., 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 ultimately triggering necrotic or apoptotic neural cell death
(Arundine and Tymianski, 2003).

Bioscience Horizons Volume 5 2012

Bioscience Horizons Volume 5 2012

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 etal., 2011) and from this produced a further three HDAC2 and 3 selective inhibitors AH51,
AH61 and AH62. In this study, we have assessed the neuroprotective potential of these daughter compounds, using an organotypic rat cerebral slice model of glutamate excitotoxicity.

Methods
Organotypic cerebral slice culture
preparation

Induction of glutamate excitotoxicity and

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

Assessment of cell viability

To assess cell viability in the cerebral slices, 500l of 0.6M
ethidium homodimer-1 (Invitrogen) dissolved in sterile
phosphate-buffered saline (PBS, Oxoid) was applied to the
slices. These were incubated for 30min at 37C 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
100l of 4% paraformaldehyde (PFA, Sigma) in PBS for
15min, in the dark at room temperature. Excess PFA was
removed and the slices were washed three times for 5min each
with PBS. Excess PBS was removed and the slices were tissuedried. Slides were mounted using Vectashield with
4,6-
diamidino-2-phenylindole (DAPI, Vector Laboratories)
and stored at 4C in the dark until analysis by confocal microscopy. 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 immersion objective lens. Images were taken at 405 and 555nm
wavelengths to visualize DAPI (blue) and ethidium homodimer-1 (red) fluorescence, respectively. Four randomly selected
non-overlapping images were taken from each slice and each
image was saved at 10241024 pixel resolution.
Non-biased quantification of the images was performed
independently by a blinded-observer using a 44 grid
(3000m2 per grid-square) in ImageJ (NIH). The number of
DAPI-stained nuclei and ethidium homodimer-1-stained
nuclei were counted within the 44 grid for each image. Data
are expressed as meanSEM percent cell viability taken as a
percentage of control viability, n indicates the number of independent 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.

Results
Selective HDAC2 and 3 inhibitors have potential in providing neuroprotection against neurodegeneration. Here, we
used an organotypic rat cerebral slice model of glutamate
excitotoxicity, a common pathophysiology involved in neurodegeneration 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 ethidium-1 homodimer and DAPI staining (Fig. 1). Ethidium
homodimer-1 is a membrane-impermeable nucleic acid fluorescent stain, which only stains nuclei in cells when the membrane 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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Organotypic cerebral slices were produced as described previously (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, 0.22m filtersterilized (Millipore) dissection medium composed of
Minimum Essential Medium Eagle with Earles salts and
NaHCO3 (MEM, Sigma) supplemented with 1% penicillin/
streptomycin (Invitrogen). The olfactory bulb and the cerebellum were dissected and discarded; the remaining brain (cerebrum) 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 (250m thick) were cut using a Leica VT1000S vibratome, collected and the agar around the slices removed. The
slices were cultured on cell culture inserts (1m pore size,
Falcon), up to a maximum of two slices per insert. The inserts
were placed in 6-well culture plates (Falcon) containing 1ml
of filter sterilized, culture medium [MEM, 25% Hanks balanced salt solution (HBSS, Sigma), 25% normal horse serum
(Invitrogen), 11mM NaHCO3 (Fisher Scientific), 4.6mM
L-glutamine (Sigma), 21mM D-glucose (Fisher Scientific) and
4.2M L-ascorbic acid (Sigma)] pre-warmed to 37C. Slice
cultures were incubated at 37C in a humid atmosphere at
5% CO2. After 3 days of incubation, the culture medium was
replaced with 1ml of a filter-sterilized serum-free culture
medium [MEM, 25% HBSS, 11mM NaHCO3, 4.6mM
L-glutamine, 21mM D-glucose and 4.2M L-ascorbic acid
and 0.3% B27 supplement (Invitrogen)] pre-warmed to 37C.

Research article

Research article

Bioscience Horizons Volume 5 2012

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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) 200M DL-TBOA, (C) 200M DL-TBOA and 1M AH51,
(D) 200M DL-TBOA and 1M AH61 or (E) 200M DL-TBOA and 1M 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 10m.

Bioscience Horizons Volume 5 2012

control (culture medium alone) slice cell viability was

81.73.9% (n=6) and this was expressed as 100%. For all
other experimental conditions, viability was quantified as
meanSEM percent of cell viability, expressed as a percentage of the control viability.

To test the neuroprotective efficacy of the novel HDAC2

M
and 3 inhibitors, they were co-applied with 200
DL-TBOA and cell viability was assessed. AH51, AH61 and
AH62 (1M) significantly reduced cell death brought upon
by the application of DL-TBOA (P<0.01) and resulted in
alevel of cell viability not significantly different from
control(culture medium alone) slice cultures or from each
other; 107.56.01% (n=4), 97.116.5% (n=3) and

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

106.76.45% (n=4) for AH51, AH61 and AH62, respectively (Figs 1CE and 2). Our novel HDAC inhibitors prevented neural cell in our model of glutamate excitotoxicity,
and these initial results show future promise, selective
HDAC2 and 3 inhibitors still retain the beneficial neuroprotective effects associated with the general non-selective
HDAC inhibitors and may be useful as therapeutic agents
against neural cell toxicity.

Discussion
Here, 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-selective 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 etal. (2009), general
non-selective HDAC inhibitors have been well reported as
efficacious neuroprotective agents against glutamate excitotoxicity. 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 antiapoptotic genes, but may also involve the prevention of neurotoxicity associated with increased HDAC2 and 3 activities.
Cell culture models have been used to test HDAC inhibitors as neuroprotective agents against glutamate excitotoxicity (Marinova etal., 2009; Leng etal., 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 interplay 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 proportion 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 prevented DL-TBOA-induced cell death, suggesting that HDAC
inhibitors protect all cell types susceptible to DL-TBOA-

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For slices treated with the vehicle conditions, cell viability

was not significantly different to the control slices, incubation with both 2M NaOH and 0.1% DMSO had a viability
of 10212.4% (n=3) and 2M NaOH alone the viability
was 108
3.9% (n=
3). Slices treated with 200
M
DL-TBOA showed a significant amount of cell death and the
number of viable cells was reduced by 41.36.1% (n=7,
compare Figs 1A with B and 2). The dead cells showed indications of DNA breakdown, fragmentation and condensing
of the chromatin that occurs during apoptosis (Aoyama
etal., 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 neural cell death. We then tested our novel HDAC inhibitors to
see if this cell death could be prevented.

Research article

Research article

mediated death. Using cell-specific immunohistochemical

labelling and specific brain regions alongside the viability
assay, one could directly correlate neural cell death with specific 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 predominantly affected in different neurodegenerative diseases.

Conclusion
Selective 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.

Acknowledgements
I 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.

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

Author biography
B.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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Dong, X. X., Wang, Y. and Qin, Z. H. (2009) Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases, Acta Pharmacologica Sinica, 30 (4), 379387.

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