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Invited review
Epigenetic modifications in the nervous system and their impact upon
cognitive impairments
Andrii Rudenko
a
,
b
, Li-Huei Tsai
a
,
b
,
*
a
Picower Institute for Learning and Memory, Howard Hughes Medical Institute, USA
b
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
article info
Article history:
Received 5 November 2013
Received in revised form
23 January 2014
Accepted 24 January 2014
Available online 1 February 2014
Keywords:
Epigenetics
Central nervous system
Learning and memory
Cognitive disorders
Histone acetylation
DNA methylation and hydroxymethylation
abstract
Epigenetic regulation has been long considered to be a critical mechanism in the control of key aspects of
cellular functions such as cell division, growth, and cell fate determination. Exciting recent developments
have demonstrated that epigenetic mechanisms can also play necessary roles in the nervous system by
regulating, for example, neuronal gene expression, DNA damage, and genome stability. Despite the fact
that postmitotic neurons are developmentally less active then dividing cells, epigenetic regulation ap-
pears to provide means of both long-lasting and very dynamic regulation of neuronal function. Growing
evidence indicates that epigenetic mechanisms in the central nervous system (CNS) are important for
regulating not only specific aspects of individual neuronal metabolism but also for maintaining function
of neuronal circuits and regulating their behavioral outputs. Multiple reports demonstrated that higher-
level cognitive behaviors, such as learning and memory, are subject to a sophisticated epigenetic control,
which includes interplay between multiple mechanisms of neuronal chromatin modification. Experi-
ments with animal models have demonstrated that various epigenetic manipulations can affect cogni-
tion in different ways, from severe dysfunction to substantial improvement. In humans, epigenetic
dysregulation has been known to underlie a number of disorders that are accompanied by mental
impairment. Here, we review some of the epigenetic mechanisms that regulate cognition and how their
disruption may contribute to cognitive dysfunctions. Due to the fact that histone acetylation and DNA
methylation are some of the best-studied and critically important epigenomic modifications our research
team has particularly strong expertise in, in this review, we are going to concentrate on histone acety-
lation, as well as DNA methylation/hydroxymethylation, in the mammalian CNS. Additional epigenetic
modifications, not surveyed here, are being discussed in depth in the other review articles in this issue of
Neuropharmacology.
This article is part of the Special Issue entitled ‘Neuroepigenetic Disorders’.
Published by Elsevier Ltd.
1. Introduction
Eric Kandel recently recalled that when he and Alden Spencer
wrote a perspective on learning and memory for Physiological
Reviews in 1968, they pointed out that “there was no frame of
reference for studying memory”at that point (Kandel, 2012). Dur-
ing the next 45 years, we witnessed a tremendous progress in
learning and memory research, leading to the development of so-
phisticated conceptual frameworks to explain memory mecha-
nisms (Sweatt, 2009). Even more impressive is the fact that, despite
such progress in deciphering memory regulation, completely novel
and unexpected levels of investigations keep appearing. In this
review, we will survey some of the most recent and exciting de-
velopments in the study of learning and memory, which occur in
the field of epigenetic regulation of cognitive processes. Although
“neuroepigenetics”is a relatively young research area, a consider-
able amount of data has already been accumulated convincingly
demonstrating the critical role of epigenetic mechanisms in
memory regulation (Levenson and Sweatt, 2005; Roth and Sweatt,
2009; Lester et al., 2011; Day and Sweatt, 2011). These include
mechanisms of chromatin regulation via covalent modifications of
DNA and histone proteins, such as DNA methylation and hydrox-
ymethylation, and histone modifications (acetylation, phosphory-
lation, methylation, ubiquitylation, sumoylation, ADP ribosylation,
deamination, proline isomerization). We should also note that the
overall epigenetic control of the genome is comprised of highly
sophisticated mechanisms involving extensive crosstalk and
*Corresponding author. Picower Institute for Learning and Memory, Howard
Hughes Medical Institute, Department of Brain and Cognitive Sciences, Massachu-
setts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
E-mail address: lhtsai@mit.edu (L.-H. Tsai).
Contents lists available at ScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
0028-3908/$ esee front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.neuropharm.2014.01.043
Neuropharmacology 80 (2014) 70e82
multiple intrinsic and extrinsic feedback circuits involving
numerous different epigenetic modifications. Given the complexity
of the topic, in the current review, we will concentrate specifically
on histone acetylation, DNA methylation, and hydroxymethylation,
the former being one of the most-, while the latter one of the least-
understood epigenetic marks involved in the regulation of
cognition.
2. Histone acetylation as a key mechanism of memory
regulation
2.1. Mouse models as powerful tools to study the role of histone
acetylation in cognition
The first study examining correlation between learning and
histone acetylation in rat brain was published by Schmitt and
Matthies in (1979). A renewed interest in histone modification in
relation to cognitive processing resulted in a publication by Swank
and Sweatt (2001) showing that the exposure of mice to a novel
taste can induce long-lasting lysine acetylation and increased his-
tone acetyltransferase activity in the insular cortex. The nonspecific
histone deacetylase (HDAC) inhibitor trichostatin A (TSA) caused a
similar increase in lysine acetylation. The authors further showed
that such acetylation can be regulated by the ERK/MAP pathway,
thereby connecting novelty learning and ERK/MAP activation with
histone acetyltransferase activity and downstream histone acety-
lation (Swank and Sweatt, 2001).
Following this work, a plethora of studies tested the effects of
various non-specific histone deacetylase inhibitors including tri-
chostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), sodium
butyrate (NaBut), phenylbutyrate (PBA), and valproic acid (VPA) on
learning and memory in mice (Bourtchouladze et al., 2003; Alarcon
et al., 2004; Korzus et al., 2004; Levenson et al., 2004; Wood et al.,
2006a,b; Oliveira et al., 2007; Fischer et al., 2007; Bredy et al., 2007;
Bredy and Barad, 2008; Stefanko et al., 2009; Peleg et al., 2010;
McQuown et al., 2011; Bahari-Javan et al., 2012) and rats (Dash
et al., 2009,2010;Hawk et al., 2011). The general conclusion that
could be made from multiple experiments utilizing TSA, NaBut, and
VPA is that these treatments could ameliorate cognitive deficits and
improve various aspects of learning and memory in different ro-
dent models. Amongst the multiple examples of such improve-
ments are enhancements of spatial learning in mice that either
carry a mutant CREB-binding protein (CBP) (Korzus et al., 2004)or
experience neurodegeneration due to p25 overexpression (Fischer
et al., 2007), as well as in rats following traumatic brain injury
(Dash et al., 2009, 2010).
Chwang et al. (2006) demonstrated that Pavlovian fear condi-
tioning induces a rapid increase in the phosphorylation of histone
H3 in hippocampal CA1 that is regulated by the ERK/MAPK
pathway. A further examination of the role of ERK/MAPK cascade in
cognition showed that mice null for mitogen- and stress-activated
protein kinase 1 (MSK1), a downstream kinase of the ERK/MAPK
pathway, demonstrated deficits in fear memory as well as
decreased histone H3 acetylation and phosphorylation (Chwang
et al., 2007). These two studies demonstrate connections between
fear memory, histone acetylation, and phosphorylation in the
hippocampus, and show that this pathway of epigenetic regulation
is mediated by the ERK/MAPK cascade.
The connection between histone H3 phosphorylation/acetyla-
tion and fear memory regulation was further strengthened by the
findings of Lubin and Sweatt (2007), who showed that the inhibi-
tion of a component of the NF
k
B complex, IKK
a
, led to an impair-
ment of fear memory reconsolidation. They showed that a subset of
genes, whose expression is induced in hippocampal area CA1 by
fear conditioning, have NF
k
B regulatory elements in their promoter
regions. An analysis of the well-studied memory-related gene
Zif268 indicated that fear memory recall induced increases in his-
tone H3 phosphoacetylation and acetylation and that administra-
tion of the NF
k
B inhibitor, diethyldithiocarbamate (DDTC),
significantly attenuated both phosphoacetylation and acetylation
of histone H3 (Lubin and Sweatt, 2007).
Another interesting example of chromatin modification as a
mechanism of cognitive regulation came from findings by Genoux
et al. (2002) and Koshibu et al. (2009).Genoux et al. (2002) showed
that the Ser/Thr Protein Phosphatase type 1 (PP1), an enzyme
involved in the dephosphorylation of a wide variety of cellular
targets, serves as a molecular suppressor of learning and memory.
Genoux et al. (2002) showed that PP1 inhibition following learning
was able to prolong object memory. A follow-up study by Koshibu
et al. (2009) demonstrated that nuclear PP1 inhibition in the hip-
pocampus and cortex resulted in an increased object recognition
memory and led to multiple alterations in histone modifications at
the promoter of the major memory gene CREB (cAMP response
element-binding protein). These modifications included increases
in H3 serine (S)10 phosphorylation, H3 lysine (K)36 trimethylation,
as well as H3K15 acetylation (Koshibu et al., 2009). These studies
show that a protein phosphatase can regulate learning and memory
via mechanisms involving not only locus-specific (de)phosphory-
lation, but also histone acetylation, suggesting a complex rela-
tionship between different modes of epigenetic control.
When considering the growing evidence that administration of
various HDAC inhibitors can lead to improved learning and mem-
ory, it is hardly surprising that a number of recent studies have
concentrated on the role of specific HDACs in cognition. Bahari-
Javan et al. (2012) implicated a founding member of the class I
HDACs, HDAC1, in specific aspects of cognitive regulation. Over-
expression of HDAC1 in the mouse hippocampus led to an increase
of fear memory extinction, while the pharmacological inhibition of
HDAC1 impaired fear extinction (Bahari-Javan et al., 2012). Mech-
anistically, the authors showed that memory extinction training
leads to recruitment of HDAC1 to the promoter of the neuronal
activity-regulated gene, c-Fos, as well as histone H3K9 deacetyla-
tion (Bahari-Javan et al., 2012).
McQuown et al. (2011) examined a potential role for another
Class I HDAC, HDAC3, in learning and memory. Ablation of HDAC3
in the dorsal hippocampus led to an enhancement of long-term
object memory accompanied by the increased acetylation of his-
tone H4K8 and upregulation of the immediate early genes c-Fos and
Nr4a2. Surprisingly, while the memory for the location of the object
was augmented, there was no increase in memory for the object
itself. Such specificity could potentially be explained by the finding
that the expression of another class II HDAC, HDAC4, was signifi-
cantly reduced in the area of HDAC3 deletion. This discovery may
suggest that these two HDACs share a role in the regulation of
object memory, perhaps as components of the NCoR co-repressor
complex (Fischle et al., 2002). Another piece of evidence support-
ing HDAC3’s role in cognition came from a recent study by Malvaez
et al. (2013). The authors showed that administration of the
HDAC3-specific inhibitor RGF966 led to an enhancement of long-
term object memory as well as the facilitated extinction of
cocaine-seeking behavior. Mechanistically, RGF966 causes
increased acetylation of histones H4K8 in the infralimbic cortex as
well as H3K14 in infralimbic cortex, hippocampus, and nucleus
accumbens one hour following the first extinction session; how-
ever, acetylation of H2BK12 was unchanged, demonstrating the
specificity of the HDAC3 effect of histone acetylation during
learning (Malvaez et al., 2013).
In 2012, Kim et al., revealed an essential role for HDAC4 in
synaptic plasticity and memory formation. They showed that the
selective loss of HDAC4 in forebrain neurons leads to impairments
A. Rudenko, L.-H. Tsai / Neuropharmacology 80 (2014) 70e82 71
in contextual fear conditioning and the Morris water maze (MWM).
In addition, the loss of HDAC4 impairs short- and long-term syn-
aptic plasticity, as evidenced by impaired paired-pulse facilitation
and LTP, but does not affect basal synaptic transmission (Kim et al.,
2012). In addition to HDAC4, the authors generated a conditional
knock-out of another Class II HDAC, HDAC5; however, this mouse
did not exhibit any obvious behavioral impairment. Another recent,
and very comprehensive, study reported that HDAC4 is actually an
essential component of fear, as well as spatial learning, memory
(Sando et al., 2012). These investigators showed that HDAC4, which
shuttles between the nucleus and cytoplasm in response to gluta-
matergic input, regulates transcriptional programs essential for the
development of excitatory synapses, excitatory transmission, and
synaptic plasticity. Moreover, mice carrying the brain-specific
expression of a truncated HDAC4, which acts as a gain-of-
function transcriptional repressor, exhibited significant impair-
ments in spatial learning in the Barnes maze task (Sando et al.,
2012).
In 2010, some of the first data implicating Class III deacetylases
in cognition came from the report by Gao et al. (2010) which
implicated the Class III NAD-dependent deacetylase, SIRT1, in the
regulation of neuronal plasticity and memory. Mice carrying
catalytically-deficient truncated form of SIRT1 demonstrated
impaired synaptic plasticity and fear and spatial learning. The
authors went further to show that SIRT1 can restrict the expres-
sion of the micro-RNA (miRNA) miR134, which functions as a
translational repressor of critical plasticity and memory-related
genes such as Bdnf and CREB. The authors concluded that
learning and memory impairments in SIRT1 knock-out mice were
related to the upregulation of miR134 and the concomitant
decrease in BDNF and CREB protein levels (Gao et al., 2010). A
remarkable aspect of this study is that it uncovered a connection
between a known epigenetic regulator, SIRT1, and another level of
epigenetic regulation, namely, translation control via miRNAs,
suggesting an intricate cross-talk between different aspects of
epigenetic control in the nervous system. It also appears that
SIRT1 plays multiple roles regulated neurodegeneration in the
brain. The study by Donmez et al. (2010) reported that SIRT1 could
suppress beta-amyloid production by activating alpha-secretase
gene ADAM10 (Donmez et al., 2010).
2.2. Histone acetylation and HDACs in aging and cognitive decline
A number of studies have demonstrated a connection between
epigenetic abnormalities, aging, and cognitive decline. An exciting
work by Fischer et al. (2007) utilized the CK-p25 mouse line,
characterized by neuronal loss that mimics AD-like neuro-
degeneration. The authors showed that environmental enrichment
(EE) was able to reinstated learning and memory deficits in CK-p25
mice caused by neurodegeneration. Moreover, EE induced retrieval
of consolidated long-term memories, thus allowing the re-
establishment of memories that were supposedly “lost”due to
the loss of neurons. The authors discovered that EE causes an
upregulation of histone acetylation (H3K9 and K14, H4K5, K8, K12
in hippocampus as well as H3K9 and H4K5 in the cortex) and that
inhibition of HDACs by chronic sodium butyrate (NaBut) adminis-
tration mimicked the EE-induced enhancement of learning and
memory. Interestingly, despite a comparable level of neuronal loss,
NaBut-treated mice showed an increased expression of synaptic
proteins compared to saline-treated animals (Fischer et al., 2007).
This study not only demonstrated a connection between learning
and memory, EE, and epigenetic regulation, but also strongly sug-
gested that inhibition of HDAC function can serve as a powerful tool
to counteract the effects of neurodegeneration-related cognitive
decline.
A growing list of studies demonstrate the beneficial effects of
various HDAC inhibitors for the cognitive performance of mouse
lines representing models of age-related disease of cognitive
decline, such as AD. These include work by Fontán-Lozano et al.
(2008), who demonstrated that treatment with NaBut and the
HDAC inhibitor Trichostatin A (TSA) alleviate cognitive deficiencies
in a mouse model of kainic acid (KA) einduced neurodegeneration.
Moreover, they showed that the same HDAC inhibitors were able to
rescue both short and long-term memory deficits in a SAMP-8
mutant mouse strain that displays a generalized accelerated ag-
ing (Fontán-Lozano et al., 2008). Ricobaraza et al. (2012) discovered
that systemic administration of the HDAC inhibitor 4-
phenylbutyrate (PBA) reinstated defective fear learning in the
Tg2567 AD mouse model. Interestingly, learning improvement was
observed not only in 6-month-old, but also in 12 to 16-month-old,
Tg2567 animals. These authors showed that the PBA treatment and
improved cognition was associated with clearance of intraneuronal
beta-amyloid (A
b
), increased dendritic spine density, and the
upregulated expression of neuronal plasticity-related genes such as
NR2B (Ricobaraza et al., 2012).
The continuing search for HDAC inhibitors with improved
specificity and efficacy has produced novel compounds with
potentially interesting biological properties. For example, Song
et al. (2013) reported the development of two HDAC inhibitors
with longer half-lives and better bloodebrain barrier penetration
then most known HDAC inhibitors, namely, a mercaptoacetamide-
based Class II HDAC inhibitor (coded as W2) and a hydroxamide-
based Class I and II inhibitor (coded as I2). An examination of W2
and I2 in vitro showed that W2 decreased both A
b
40 and A
b
42
amyloid peptide species, while I2 decreased only A
b
40. I2 also
decreased expression of
b
- and
g
-secretase proteins, while W2
reduced only
g
-secretase expression. Interestingly, they also found
that administration of W2 decreased A
b
levels in addition to
decreasing tau phosphorylation at Thr181 while rescuing cognitive
impairment in aged hAPP 3xTg AD mice. A recent study by Fass
et al. (2013) reported the discovery of Crebinostat, a novel cogni-
tive enhancer. This study is remarkable as it employed a chemical
biology approach to search for novel HDAC inhibitors. The authors
performed a systematic screening of a library of small molecules
structurally related to known HDAC inhibitors using activation of
CREB-mediated transcription as readout. Following the identifica-
tion of Crebinostat, the authors showed that its systematic
administration for a period of ten days resulted in an enhancement
of fear learning in wild type mice. They also showed that treatment
with Crebinostat increased the dendritic density of Synapsin 1
immunoreactive puncta as well as the expression of Bdnf in
cultured neurons.
Gräff et al. (2012) established a major role for HDAC2 in the
epigenetic blockade apparent in the neurodegenerating brain. One
of the findings by this group was an intriguing connection between
neurotoxic insults, such as H
2
O
2
or A
b
oligomers, and increased
HDAC2 expression. They showed that such regulation occurs
through the activation of glucocorticoid-response elements found
in the HDAC2 promoter. This mode of regulation may explain at
least some of AD-related HDAC2-dependent neuronal pathologies.
Moreover, it can tie together such phenomena as stress, increased
HDAC2 expression, and potential cognitive and psychiatric abnor-
malities observed in neurodegenerative as well as neuropsychiatric
disease. An interesting example of a connection between chronic
stress and HDAC2 function was reported by Uchida et al. (2011),
who showed that chronic stress could trigger elevation of HDAC2
expression and an increase of its binding to the promoter of glial
cell-derived neurotrophic factor (Gdnf) and the resulting down-
regulation of Gdnf expression in the nucleus accumbens (NAcc).
Overexpression of a dominant-negative form of HDAC2 led to an
A. Rudenko, L.-H. Tsai / Neuropharmacology 80 (2014) 70e8272
increase in Gdnf expression and reduced depressive-like behaviors
in stressed mice (Uchida et al., 2011).
A recent study by Gräff et al. (2013) reported a connection be-
tween neurodegeneration, cognitive decline, and caloric restriction
(CR). The authors analyzed CK-p25 mice and showed that a dietary
regimen of CR significantly delayed the onset of synaptic loss,
neurodegeneration, and cognitive dysfunction in these mice. They
also found that CR caused activation of SIRT1 as well as significant
reductions in acetylation of the target histone H3K56. Surprisingly,
administration of small molecule activator of SIRT1, SRT3657, was
able to recapitulate the neuroprotective effects of CR by reducing
synapse loss and improving learning and memory. This finding
suggests that SIRT1 may be a critical component of CR-based
amelioration of neurodegeneration, and points toward the thera-
peutic potential of SIRT1-related treatments.
Two recent studies implicate two Class II HDACs, HDAC5 and
HDAC6, in AD-related memory dysfunction. Agis-Balboa et al.
(2013) bred an HDAC5 loss-of-function mutation into a back-
ground of A
b
PP/PS1-21 mice, which exhibit an aggressing A
b
pla-
que deposition and age-dependent memory impairment. They
tested wild type, HDAC5
/
,A
b
PP/PS1-21, and the compound A
b
PP/
PS1-21; HDAC5
/
10-month old mice in various cognitive para-
digms and discovered that, surprisingly, the ablation of HDAC5
further impaired, rather than rescued, learning in the A
b
PP/PS1-21
HDAC5
/
animals. HDAC5 itself was found to be important for the
consolidation of context and tone-dependent fear memory. Anal-
ysis of the plaque loads and synaptic markers in A
b
PP/PS1-21 and
A
b
PP/PS1-21 HDAC5
/
brains revealed no significant differences
between the groups.
Unlike HDAC5, HDAC6 downregulation appears to ameliorate
AD progression. Govindarajan et al. (2013) generated A
b
PP/PS1-21;
HDAC6
/
mice and performed analyses very similar to Agis-Balboa
et al. (2013). They found that impairments in associative fear
learning and spatial learning, observed in A
b
PP/PS1-21 mice, were
restored in A
b
PP/PS1-21; HDAC6
/
animals. Molecular analyses of
HDAC6 targets indicated that the loss of HDAC6 significantly
increased
a
-tubulin K40 acetylation and restored impaired mito-
chondria trafficking in the hippocampus of A
b
PP/PS1-21 mice.
An interesting study by Peleg et al. (2010) carried out a
comprehensive epigenomic analysis of age-related memory
impairment in mice using 3-, 8- and 16-month old C57BL/6 ani-
mals. They found that 16-month old mice exhibited significant
declines in spatial and fear memory. An analysisof multiple histone
acetylation marks demonstrated that cognitive decline in aged
mice correlates specifically with decreases in the acetylation of
histone H4K12. Notably, treatment of aged mice with SAHA both
improved cognitive performance and restored H4K12 levels, indi-
cating that regulation of H4K12 acetylation may be indeed a critical
step in aging-related epigenetic regulation (Peleg et al., 2010). This
remarkable finding demonstrates that not only individual HDAC
enzymes, but also individual histone modifications, may have
distinct and critically important roles in the epigenetic regulation of
cognition.
2.3. HDAC2 as a critical molecular component in cognitive
regulation
Studies using various pharmacological HDAC inhibitors to
investigate neural roles of histone acetylation have contributed
significantly to our understanding of the importance of histone
acetylation as a mechanism of cognitive control. However, accu-
mulating evidence, some already cited, clearly points to specific
roles for individual HDAC enzymes in different aspects of cognitive
regulation (Wood et al., 2006a,b; Akhtar et al., 2009; Gao et al.,
2010; McQuown et al., 2011; Bahari-Javan et al., 2012; Kim et al.,
2012; Sando et al., 2012; Malvaez et al., 2013). An exciting study
by Guan et al. (2009) demonstrated that a Class I HDAC, HDAC2, acts
as a critical negative regulator of learning and memory. The authors
discovered that overexpression of HDAC2, but not the closely
related HDAC1, causes impairments in synaptic plasticity and
memory formation. Interestingly, they showed that the commonly
used HDAC inhibitor, SAHA, did not produce further memory
improvement in HDAC2-null mice, suggesting that HDAC2 serves as
a primary target for the well-documented cognitive improvements
ascribed to SAHA. At the cellular level, Guan et al. (2009) observed
that HDAC2 knock-out leads to increases, while its overexpression
leads to decreases, in synapse number. Moreover, they found that
HDAC2, but not HDAC1, is typically strongly associated with the
promoter regions of multiple genes involved in the regulation of
synapse development, neuronal plasticity, and learning and
memory, such as Egr1,Homer1,Arc,Nrx1/3,Shank3,PSD95,CREB,
CBP,GLUR1/2, and NR2A/2B (Guan et al., 2009). Altogether, these
findings strongly suggest that HDAC2 acts a critical negative
constraint on learning and memory by suppressing a variety of
genes important for synaptic plasticity and cognition. This
remarkable discovery showcased a specific role for an individual
HDAC enzyme in regulating multiple genes critical for cognition.
The surprising aspects of this report are the findings that HDAC2,
but not its frequent heterodimer partner HDAC1, is critical for
controlling multiple memory-related genes and that many of these
genes are regulated by neuronal activity. Interestingly, a study by
Nott et al. (2008) demonstrated that activity-regulated BDNF can
induce nitrosylation of the chromatin-bound HDAC2 at Cys 262 and
Cys 274, which promotes HDAC2 release from the chromatin. This
leads to an increase in the acetylation of histones associated with
neurotrophin-dependent gene promoters in rat cortical neurons.
These researchers also showed that siRNA-mediated silencing of
HDAC2 causes an increase in the average dendritic length, total
dendritic length, and the number of dendritic branches per neuron
(Nott et al., 2008). This study not only implicates HDAC2 in
neurotrophin-depended neuronal signaling, but also suggests a
mechanism for the activity-dependent regulation of HDAC2 func-
tion in neurons.
A study by Gräff et al. (2012) built upon previous work to
demonstrate that HDAC2 plays a critical role in cognitive impair-
ments in a mouse model of neurodegeneration as well as in human
Alzheimer’s disease (AD). In the model of induced severe neuro-
degeneration, the CK-p25 mouse (Cruz et al., 2003, 2006), the au-
thors found that these animals not only exhibit multiple cognitive
impairments but also have increased levels of HDAC2 expression in
the hippocampus and prefrontal cortex. This increase in HDAC2
leads to decreased histone acetylation and the down-regulation of
multiple memory-related genes, many of which were previously
shown to be under negative regulation by HDAC2 (Guan et al.,
2009). Importantly, the knock-down of HDAC2 in the hippocam-
pus of CK-p25 mice led to a rescue of the gene expression, synapse
abundance, and plasticity, as well as cognitive performance (Gräff
et al., 2012). The authors went further to show that brain samples
from human patients with sporadic AD also exhibited elevated
levels of HDAC2, but not HDA1 and HDAC3, immunoreactivity in
the hippocampus. Based on their findings, the authors conclude
that HDAC2 can act as a critical component of an “epigenetic
blockade”in the neurodegenerating brain, and suggest that inhi-
bition of HDAC2 may serve as a component in a more efficacious
strategy for AD treatment. Additional evidence for the role of
HDAC2 as negative regulator of cognition came from a recent study
by Morris et al. (2013). Theyshowed that the conditional ablation of
HDAC2 in postmitotic forebrain neurons significantly accelerated
the rate of extinction of fear memory and conditioned test aversion
in mice, while not affecting episodic memory. Interestingly, these
A. Rudenko, L.-H. Tsai / Neuropharmacology 80 (2014) 70e82 73
mutant mice also showed improved performance in an attention
set-shifting task. Based upon their data, the authors conclude that
HDAC2 may have specific roles in different types learning, and is
particularly important in associative learning tasks (Morris et al.,
2013).
A study by Hanson et al. (2013) reported that HDAC2 could cell-
autonomously negatively regulate excitatory synaptic function in
pyramidal neurons in CA1. Moreover, in addition to suppressing
excitatory function, HDAC2 appeared to enhance inhibitory syn-
aptic function in hippocampus. These findings led the authors to
suggest that shift in the balance of excitation/inhibition favoring
excitation could contribute to beneficial effects of suppressing
HDAC2 in models of cognitive impairment (Hanson et al., 2013).
An intriguing and potentially important connection between
HDAC2 and HIV-related neuropathology was suggested by Saiyed
et al. (2011). They showed that a treatment with the neurotoxic
HIV-1 protein Tat, which can induce neuronal apoptosis and exac-
erbate HIV-related neurodegeneration, can lead to an increased
HDAC2 expression in a neuroblastoma cell line. They also found
that Tat treatment impairs the expression of some critical memory
and neuronal plasticity-related genes, such as CREB. Importantly,
knock-down of HDAC2 appeared to reverse various Tat-mediated
effects in neuroblastoma cell lines and human primary neurons.
These findings indicate that the “epigenetic blockade”of cognitive
function in neurodegeneration shown by Gräff et al. (2012) may
also be relevant to mental impairment observed in HIV-associated
neurocognitive disorder (HAND) and suggest a possible avenue for
HAND treatment (Saiyed et al., 2011).
Recently, Kurita et al. (2012) suggested that HDAC2 might
function as an important component of chromatin-related neuro-
psychiatric regulation. First, they found that the expression of
HDAC2, but not HDAC1 or HDAC4, was elevated in the frontal cortex
of human schizophrenic patients treated with atypical antipsy-
chotics. They then showed that chronic treatment with the anti-
psychotic clozapine caused a downregulation of mGluR2 expression
that was associated with decrease in histone H3 acetylation at the
mGluR2 promoter in both human and mouse frontal cortex. Inter-
estingly, treatment with clozapine caused an increase in binding of
HDAC2 to the mGluR2 promoter. Additionally, overexpression of
HDAC2 in the frontal cortex induced schizophrenia-like behaviors
and administration of the HDAC inhibitor SAHA reversed the
clozapine-induced repression of mGluR2. This interesting study
demonstrates that HDAC2 may function as a negative constraint on
the effects of antipsychotics, and suggests that inhibitors of HDAC2
can serve as an additional therapeutic tool for treating disorders
such as schizophrenia (Kurita et al., 2012).
2.4. Rubinstein-Taybi syndrome
Rubinstein-Taybi syndrome (RTS) was the first to be discovered,
and remains the best characterized disorder in which cognitive
impairment results from epigenetic abnormalities. This genetic
disorder is characterized by anatomical abnormalities and severe
mental retardation (Rubinstein and Taybi, 1963). Despite its rare
instance (w1 in 125,000 to 1 in 720,000 births), RTS patients ac-
counts for approximately 1 in 300 cases of institutionalized pa-
tients with mental retardation (Hallam and Bourtchouladze, 2006).
Petrij et al. (1995) reported the initial discovery that RTS can result
from a mutation in the histone acetyltransferase (HAT) protein,
CREB-binding protein (CBP). They discovered that RTS can occur
due to a re-arrangement in the chromosomal locus 16p13.3 or as a
heterozygous point mutation in CBP. More recent work demon-
strates that a fraction of RTS cases may arise as a result of mutations
affecting p300, a protein closely related to CBP (Roelfsema et al.,
2005).
Tanaka et al. (1997) reported the development of the first mouse
model of RTS, in which the CBP gene was deleted. Homozygous
mutant embryos died in utero and heterozygous animals (CBP
þ/
)
were viable but exhibited various RTS-like skeletal abnormalities
and growth retardation (Tanaka et al., 1997). This mouse line served
as one of the earliest models used to assess the role of neuro-
epigenetic regulation in cognition (Alarcon et al., 2004). Behavioral
characterization of CBP
þ/
mice showed that they exhibited long-
term fear and object memory impairments (Alarcon et al., 2004)
in addition to disruptions in hippocampal L-LTP and reduced
acetylation of histone H2B. The inhibition of HDAC activity led to an
enhancement of L-LTP and a rescue of memory deficits in the CBP
þ/
animals (Alarcon et al., 2004).
Oike et al. (1999) developed another RTS mouse model via the
creating an insertion in the CBP gene that results in a C-terminally
truncated protein (residues 1e1084) containing the CREB-binding
domain (CBP
þ/C-term
), which was thought to act as a dominant-
negative inhibitor of the CBP protein. Similarly to the line by
generate by Tanaka et al. (1997), the homozygous mutant animals
died embryonically. Heterozygous CBP
þ/C-term
mice exhibited
RTS-like retardation of growth and osseous maturation, cardiac and
skeletal abnormalities (Oike et al., 1999). Similarly to CBP
þ/
ani-
mals, CBP
þ/C-term
animals exhibited to have normal short-term
but deficient long-term memory with impairments in passive
avoidance, fear conditioning, and object recognition (Oike et al.,
1999; Bourtchouladze et al., 2003). Bourtchouladze et al. (2003)
reasoned that as CBP acts as a major CREB co-factor, other drugs
that can modulate CREB function, may be effective in treating RTS-
related symptoms. Cell-cased drug screen indicated that phos-
phodiesterase 4 (PDE4) inhibitors were particularly effective in
enhancing CREB function. Therefore, they administered one of
those inhibitors, rolipram, and showed that it restored object
recognition memory in CBP
þ/C-term
mice (Bourtchouladze et al.,
2003).
Korzus et al. (2004), showed that the activity of the histone CBP,
is critical for memory consolidation (Chan and La Thangue, 2001).
In this work, mice carrying a mutation that abolishes the HAT ac-
tivity of CBP (CBP {HAT
} mice) exhibited normal acquisition of
new information and short-term memory, but impaired long-term
memory. Specifically, these mice had deficiencies in declarative
memory, as assessed by visual-paired compression (VPC) test, and
spatial learning, as assessed by the Morris water maze (MWM) test.
Interestingly, CBP {HAT
} animals did not show any impairment in
fear memory, suggesting that the mutation affected only specific
types of memory. The authors showed that disruption of CBP ac-
tivity in the mutant animals caused down-regulation of the in vivo
expression of a neuronal-activity regulated gene, c-Fos, which is
critically important for the regulation of neuronal plasticity and
memory consolidation (Bourtchuladze et al., 1994). Administration
of the HDAC inhibitor TSA increased levels of histone H3 acetylation
in the hippocampus and rescued memory impairment in the VPC
test.
Nearly simultaneously with Korzus et al. (2004), Alarcon et al.
(2004) showed that mice haploinsufficient for the same HAT, CBP,
exhibited various cognitive impairments that included decreased
fear and object recognition memory. These learning impairments in
CBP
þ/
mice appeared to be task-specific, similar to the CBP {HAT
}
mutants. Interestingly, unlike CBP {HAT
} animals, CBP
þ/
mice
demonstrated normal spatial learning, although they showed im-
pairments in object recognition and fear memory. The specificity of
the CBP
þ/
phenotype is also emphasized by the observation that
these mice showed reduced acetylation of histone H2B, but normal
overall acetylation of H2A, H3, and H4. CBP mutant mice also
exhibited normal early-phase long-term potentiation (LTP; E-LTP)
but showed deficiencies in the induction of late-phase LTP (L-LTP),
A. Rudenko, L.-H. Tsai / Neuropharmacology 80 (2014) 70e8274
which requires new protein synthesis. The application of the HDAC
inhibitor SAHA restored histone H2B acetylation levels as well as L-
LTP induction in hippocampal slices from CBP
þ/
mice. Accordingly,
in vivo administration of SAHA in CBP
þ/
mice rescued the long-
term fear memory deficit in these animals. These findings led the
authors to conclude that chromatin acetylation is a critical
component of long-term plasticity and memory storage. They also
suggested that CBP
þ/
mice may serve as a suitable animal model of
Rubinstein-Taybi syndrome.
Other studies followed by developing additional mouse models
of RTS based on various CBP mutations (Wood et al., 2005, 2006a,b;
Barrett et al., 2011; Valor et al., 2011). Consistently with the earlier
findings, different CBP mutant lines exhibited various degrees
impairment in long-term memory. Wood et al. (2006a,b) demon-
strated that not only CBP is important for fear and object memory
but that its memory-related function critically depends on the KIX
domain responsible for interacting with several transcription fac-
tors. Mutating this domain led to impairment in long-term fear and
object recognition memory. Furthermore, a study by Vecsey et al.
(2007) demonstrated that disruption of CREB-binding KIX
domain of CBP clocks the ability of HDAC inhibition to rescue de-
fects in E-LTP caused by disrupting CREB/CBP interaction (Vecsey
et al., 2007). These findings suggest that a functional copy of CBP
is necessary to mediate positive effect of HDAC inhibitors on plas-
ticity and cognitive impairments in CBP mutant animals. A study by
Valor et al. (2011) investigated ablation of CBP in the postmitotic
forebrain neurons, which allowed them to circumvent embryonic
lethality of the conventional CBP knock-outs. They found that loss
of CBP resulted in a strong reduction in neuronal histone acetyla-
tion, with a primary targets being H2A and H2B. Interestingly, CBP
ablation had a very modest effect on the immediate-early gene
expression, did not affect neuronal viability, and showed normal
fear as well as special memory, but exhibited impairment in long
term object recognition memory (Valor et al., 2011). A relatively
mild phenotype observed by Valor et al. (2011) can be explained by
the difference in relative importance of the CBP activity in em-
bryonic and postnatal cells. Indeed, the study by Wang et al. (2010)
demonstrated that CBP binds to neuronal and glial gene promoters
and regulates global histone acetylation in the mouse embryonic
cortex, and that CBP is essential for differentiation of neuronal as
well as glial cells (Wang et al., 2010). Additionally, a recent study by
Tsui et al. (2014) reported that CBP expressed within the embryonic
medial ganglionic eminence regulates the differentiation of in-
terneurons from ventral forebrain neuronal precursors, thus
implicating CBP in global regulation of the major inhibitory
neurotransmission system of the brain (Tsui et al., 2014).
Studies by Oliveira et al. (2007) and Oliveira et al. (2011) showed
mutations in HAT p300, closely related to CBP, also cause long-term
memory deficits as well as histone acetylation abnormalities.
Inhibitory truncated form of p300 lacking both the HAT and the
transcriptional activation domain (
D
p300), caused decreased levels
of histone H3K4 and H3K14 acetylation.
D
p300 animals also
exhibited defects in long-term object recognition and contextual
fear memory, but not in spatial memory (Oliveira et al., 2007, 2011).
A study by Viosca et al. (2010) described a comprehensive exami-
nation of adult p300
þ/
mice (Yao et al., 1998;Viosca et al., 2010).
Interestingly, while these animals did not show significant alter-
ations in histone acetylation and gene expression, they exhibited
impaired growth and facial dysmorphia eanatomical features
associated with RTS in humans, as well as abnormal gait and mild
cognitive impairment. Overall defects observed in p300
þ/
animals
were substantially weaker then in the reported CBP
þ/
mutants
highlighting a major effect of CBP HAT activity on multiple neuro-
logical and phenotypes and cognitive impairment associated with
RTS (Viosca et al., 2010). Very exciting recent study reported
generation of lymphoblastoid cell lines from nine human patients
with RTS carrying different mutations in the CBP gene locus and
representing a wide clinical variability of the syndrome (Lopez-
Atalaya et al., 2012). Similar to the results obtained from CBP
mutant mouse models, the authors discovered significantly
decreased levels of H2A and H2B acetylation in lymphoblastoid RTS
lines. Importantly, treatment of the cell cultures from the mutant
lines showing the highest level of H2B acetylation deficit, with
HDAC inhibitor TSA, resulted in an increased acetylation of the
histone H2B compared with the mutant and control lines treated
with a vehicle (Lopez-Atalaya et al., 2012). This exciting result in-
dicates that similar to the mouse models it is possible to rescue
histone acetylation deficits in human cells derived from RTS pa-
tients, which a great news for developing potential treatments for
RTS.
Overall, studies using mouse RTS models as well as initial ex-
periments using human-derived cell lines, strongly suggest that
disruption of histone acetylation serves as a mechanism underlying
cognitive deficits in RTS and provided a great example of epigenetic
therapeutic approach to treat such deficits.
3. DNA methylation and hydroxymethylation as mechanisms
of neuronal regulation
3.1. DNA methylation
DNA methylation is covalent modification of DNA catalyzed by a
family of DNA methyltransferase enzymes (DNMTs). It has been
implicated in regulation of gene expression and chromatin struc-
ture affecting wide variety of biological processes including trans-
posable elements silencing, gene imprinting, chromosomal
inactivation, and embryonic stem (ES) development (Jaenisch and
Bird, 2003). Disruption of proper DNA methylation was shown to
lead to various pathological processes such as neurodevelopmental
and cancer (Portella and Esteller, 2010). Mammalian DNA methyl-
ation often occurs at both promoter areas and in the gene bodies in
context of CpG dinucleotides. However recent studies reported a
relatively high amount of non-CG methylation including CHG and
CHH (where H is A, C, or T) (Portella and Esteller, 2010). Very recent
study reported an extremely comprehensive genome-wide, base-
resolution analysis of DNA methylation in neurons and glial cells in
the human and mouse brain during different developmental stages
and discovered that DNA methylation is critical for brain develop-
ment (Lister et al., 2013). Surprisingly, they discovered that non-CG,
CH methylation strongly accumulates in neurons through early
childhood and adolescence and becomes the more prevalent form
of methylation in mature neurons. Lister et al. (2013) also discov-
ered that positions of individual mCGs and mCHs are highly
conserved between different neurons and individuals indicating
that DNA methylation is a highly controlled process, and that
neuronal and glial methylation landscapes appear to be very
different, suggesting a role of DNA methylation in sculpting the cell
identity (Lister et al., 2013).
The first indications that DNA methylation may play a role in
cognition came from a series of studies carried out in David Sweatt’
Lab. Miller and Sweatt (2007) used Pavlovian fear conditioning and
discovered that this behavioral paradigm leads to an upregulation
of the mRNA of de novo DNMTs, DNMT3A and DNMT3B, in the rat
hippocampus. The infusion of DNMT inhibitors, 5-azadeoxycytidine
(5-Aza) and zebularine (zeb), into the hippocampus following fear
conditioning caused in impairment in fear memory in animals.
Surprisingly, retraining of the animals treated with DNMT in-
hibitors after the first training on the test day 1 led to significant
increase in freezing levels on a test day 2. This finding suggests that
the effects of DNMT inhibition and, supposedly, DNA methylation,
A. Rudenko, L.-H. Tsai / Neuropharmacology 80 (2014) 70e82 75
are transient and plastic. Analysis of the of methylation and
expression of a memory suppressor gene, PP1, showed contextual
fear conditioning cause an increase in DNA methylation and
decrease in its expression f PP1. Intra-CA1 infusion of 5-Aza
following fear conditioning was able to reverse methylation and
expression changes of PP1. The authors also showed that fear
conditioning caused methylation and expression changes of the
memory-promoting gene reelin and that those changes were
opposite to the ones related to PP1: de-methylation and increased
transcription. Interestingly, the they also found that methylation
and expression changes of both PP1 and reelin returned to a base-
line level 24 h after fear conditioning training, indicating that
learning-induced locus-specific DNA methylation can be highly
dynamic (Miller and Sweatt, 2007). Miller et al. (2008) confirmed
that pharmacological inhibition of DNMT immediately after fear
conditioning impairs memory consolidation. Interestingly, the also
found that CA1 administration of 5-Aza immediately after fear
conditioning led to a decrease in a training-induced levels of his-
tone H3 acetylation suggesting that DNA methylation may regulate
learning-related histone acetylation. Moreover, administration of
the HDAC inhibitor NaBut together with 5-Aza alleviated the effects
of 5-Aza on memory consolidation as well as synaptic plasticity,
indicating that increase in histone acetylation can override DNMT
inhibition in cognitive regulation. (Miller et al., 2008).
An overlap and potential interaction between DNA methylation
and histone acetylation is a very interesting an intriguing topic
(Chiurazzi et al., 1999). A long-standing view was that initially
established DNA methylation patterns dictate following formation
of local histone acetylation landscapes via activity of histone
modifying proteins, such as HDACs, found in multiprotein com-
plexes that can bind DNA methylated areas (Eden et al., 1998).
However, a study by Cervoni and Szyf (2001) demonstrated, rather
unexpectedly, that local histone acetylation states can direct DNA
demethylation activity thus establishing DNA methylation patterns
in mammalian cells. Moreover, they showed that histone acetyla-
tion eDNA demethylation sequence can be established in a cell-
cycle and replication-independent fashion (Cervoni and Szyf,
2001). The effect of HDAC inhibitor, TSA, on DNA methylation sta-
tus demonstrated by Cervoni and Szyf (2001) in a cell culture sys-
tem, was also observed in vivo by Weaver et al. (2004). This
remarkable study demonstrated that rat maternal behavior was
able to induce epigenetic programming in the offspring, inducing
changes in DNA methylation and histone acetylation at the gluco-
corticoid receptor (GR) gene promoter in the hippocampus. Addi-
tionally, Weaver et al. (2004) showed that an
intracerebroventricular infusion of TSA resulted in abolishing group
differences not only in histone acetylation but also in DNA
methylation and GR expression as well as hypothalamic-pituitary-
adrenal (HPA) axis response to stress (Weaver et al., 2004). Future
investigation of the molecular mechanisms regulating histone
acetylation and DNA methylation will undoubtedly shed more light
on potential hierarchical interactions between these two key
mechanisms of epigenetic regulation and the role of such in-
teractions in cognitive regulation.
Following their earlier work on the role of DNA methylation in
learning and memory, Miller et al. (2010) reported that contextual
fear conditioning induced hypermethylation of the memory-
related gene calcineurin (CaN) within 24 h after in prefrontal cor-
tex of the trained rats. Interestingly, such time course appears
correspond to the memory consolidation and shifting the newly
formed memory to the cortex, suggesting an importance of DNA
methylation in these processes. The authors found that fear
condition-induced methylation of CaN in the cortex persisted 30
days after training even when expression of CaN returned to a
baseline. Intra-ACC Infusions of 5-Aza, zeb, or another DNMT
inhibitor, RG108, 30 days after fear conditioning impaired long-
term memory. These findings led the authors to suggest that DNA
methylation in the prefrontal cortex is important for memory sta-
bility and that DNA methylation canserve as a marker for a memory
trace.
In addition to the earlier studies using pharmacological inhibi-
tion of DNMTs, Feng et al. (2010) generated mouse lines with
forebrain-specific deletions of two DNMTs, de novo Dnmt3a and a
maintenance Dnmt1. We should mention that in the earlier study
Fan et al. (2001) showed that conditional deletion of Dnmt1 in
postmitotic neurons leads to the global genomic hypomethylation
and death of Dnmt1
-
neurons. Interestingly, such critical role of
Dnmt1 appears to take place only in postnatal brain as its deletion
does not affect neuronal survival in prenatal brain. Feng et al. (2010)
reported that single Dnmt1 and Dnmt3a knock-outs have normal
synaptic plasticity as well as fear learning. Surprisingly, a double
knock-out of Dnmt3a and Dnmt1 exhibited a decrease of late-phase
LTP, enhancement of LTD as well as an impairment in long-term
contextual fear memory and spatial memory. These unexpected
findings suggest that despite their non-overlapping roles in the
other tissues, Dnmt3a and Dnmt1 may have somewhat redundant
function in postmitotic neurons, specifically related to regulating
learning and memory as well as neuronal plasticity.
Despite growing strong evidence that DNA methylation plays an
important role in regulating various neuronal functions (LaSalle
et al., 2013) potential connections between various brain disor-
ders and aberrant DNA methylation remain very unclear. Siegmund
et al. (2007) reported methylation analysis of the 50 selected genes
in the human cerebral cortex in relation to aging process and in
association with AD and schizophrenia. Interestingly, while they
showed that multiple genes (8 erobust increase in methylation
across the lifespan and 16 esharp rise in methylation after birth)
underwent age-related hypermethylation, there was very little
connection between AD, schizophrenia and methylation dynamics.
Whether this result is due to particular features of selected set of
analyzed genes, specific brain area or, indeed, a lack of obvious
relation between the disease in question and neuronal methylation
profiles is not currently clear.
3.2. DNA hydroxymethylation
A novel epigenetic mark, 5-hydroxymethylcytosine was first
discovered in mammalian DNA in 1972 (Penn et al., 1972). How-
ever, in order to gain serious attention, this mark had to be “re-
discovered”much later: Kriaucionis and Heintz (2009) detected
the presence of 5 hmC in the cerebellar neurons and Tahiliani et al.
(2009) reported that ten-eleven translocation 1 (Tet1) protein can
convert 5 mCe5 hmC in mammals. Further studies reported that
5 hmC is specifically abundant in the embryonic stem cells (ESC)
(w4.4% of all CpGs in mouse ESCs) as well as in the brain (w0.6%
of all Cs in Purkinje cells). Search for Tet1 erelated proteins
yielded two additional members of Tet family: Tet2 and Tet3. All
Tets were shown to function as oxyglutarate- and iron-dependent
dioxygenases. As the presence of 5 h mC depends on the preex-
isting 5 mC it suggests that Tets specifically catalyze 5 mC e5 hmC
conversion.
Most of the studies on 5 hmC and Tet proteins concentrated on
ESCs and induced pluripotent stem cells (iPSCs) and provided
multiple evidence that Tets as well as 5 hmC are involved in
regulating pluripotency, self-renewal, and differentiation potential
of these cells thus playing a critical role in early embryonic devel-
opment (Ito et al., 2010; Koh et al., 2011). Interestingly, knock-out of
aTet1 was shown not to affect pluripotency in ESCs and was
compatible with embryonic and postnatal development (Dawlaty
A. Rudenko, L.-H. Tsai / Neuropharmacology 80 (2014) 70e8276
et al., 2011). However, one should keep in mind a high likelihood of
potential functional redundancy amongst members of Tet family.
Since the “re-discovery”of 5 hmC considerable effort has been
made to uncover its exact functional significance. The leading hy-
pothesis has been that 5 hmC serves as an intermediate product in
an oxidative DNA demethylation pathway. This is a very exciting
hypothesis as unlike DNA methylation, the mechanisms of DNA
demethylation remain very poorly understood. Initial discovery
that 5 hmC and Tet protein are specifically abundant in rodent brain
(Kriaucionis and Heintz, 2009; Szulwach et al., 2011) suggested that
Tets as well as 5 hmC may have important roles in postmitotic
neurons of the central nervous system (CNS). Ma et al. (2009) re-
ported that neuronal activity cause strong induction of the member
of Gadd45 protein family, Gadd45. They also demonstrated
downregulation of Gadd45b expression in hippocampus led a
decrease in neuronal activity-induced adult neurogenesis. More-
over blocking Gadd45b expression resulted in significant decrease
in activity-induced demethylation of Bdnf IX and Fgf1B promoters
as well as expression of the corresponding genes. Additionally,
overexpression of Gadd45b appeared to promote in vivo demethy-
lation. These very interesting results indicated that there is a locus-
specific active DNA demethylation occurring in adult mouse brain
that Gadd45b is important for that process (Ma et al., 2009). Guo
et al. (2011a) built upon this study and reported an exciting dis-
covery showing that activity-dependent demethylation of Bdnf IX
and Fgf1B promoters in dentate gyrus neurons was completely
abolished by knocking down endogenous Tet1. Moreover, they also
showed that AID/APOBEC deaminases may work in concert with
Tet1 to promote activity-induced DNA demethylation in vivo.
Complementary approached using human cells demonstrated that
Tet1 can mediate DNA demethylation via 5 hmC generation and
that 5 hmC-demethylaiton requires a base excision repair (BER)
pathway (Guo et al., 2011a,b). Thus, this study provided a direct
evidence of 5 hmC demethylase activity in mammalian cells and
implicated Tet1 in active DNA demethylation in CNS in vivo.
The hypothesis that 5 hmC acts as an intermediate in active DNA
demethylation and abundant evidence that DNA methylation often
serves as a means to downregulate gene expression suggested that
there should be a positive correlation between 5 hmC content and
transcriptional activity of genomic regions. Recent findings from
the genomic studies in mammalian CNS appear to support this
conclusion (Szulwach et al., 2011; Mellén et al., 2012; Lister et al.,
2013). First attempts to map genomic 5 hmC distribution in the
mouse and human brain discovered stable and dynamically
modified loci during neuronal development and ageing and sug-
gested that 5 hmC may play an important role in these processes
(Szulwach et al., 2011). The wholeegenome analysis of 5 hmC dy-
namics during mammalian brain development discovered that
5 hmC is enriched within active genomic regions in both fetal and
adult mouse brain and, importantly, that despite lower absolute
levels of hmCC in the fetal brain, the adult patterns of hmCG are
already forming in utero in both neurons and astrocytes. Impor-
tantly, developmentally demethylated CG regions appear to be
enriched for hmCG bases, which suggests that hmCG may be poised
at dormant genomic regions in order to facilitate DNA demethyla-
tion later in development. Whole-genome bisulfate sequencing
analysis of Tet2/mouse brain uncovered an increase in hyper-
methylated regions suggesting that Tet2 enzyme may be involved
in mCG demethylation during development (Lister et al., 2013).
In a very recent study, Rudenko et al. (2013) performed a
comprehensive analysis of Tet1/mice (Tet1KO; Dawlaty et al.,
2011) and discovered downregulation of multiple neuronal
activity-regulated genes, including Npas4 and c-Fos, in the cortex
and hippocampus of these animals. Tet1KO mice also exhibited
abnormal hippocampal long-term depression and impaired
memory extinction. Analysis of Npas4, indicated that its promoter
region is hypermethylated in both behaviorally naive Tet1KO mice
and following memory extinction training, which may account for
the downregulation of Npas4 as well as its downstream targets
(Ramamoorthi et al., 2011). Based on their findings, Rudenko et al.
(2013) propose that Tet1 is important for maintaining locus-specific
DNA hypomethylation, particularly at the promoters of at least
some of the neuronal activity-regulated genes, which may play a
critical role in keeping these genes poised for rapid transcriptional
activation (Fig. 1). Such regulation may allow an increased tran-
scriptional flexibility and provide a basis for genomic regulation of
various aspects of cognition including memory extinction
(Rudenko et al., 2013).
Another study on the role of Tet1 in the brain by Kaas et al.
(2013) reported that Tet1 itself can be regulated by neuronal ac-
tivity and that its overexpression leads to global changes in the
levels of modified cytosine. They also showed that Tet1 regulates
expression of neuronal-activity regulated genes important for
learning and memory and that overexpression of either Tet1 or
catalytically inactive Tet1 mutant in the dorsal hippocampus
resulted in impairment in long-term fear memory formation (Kaas
et al., 2013).
Identities of the protein readers of 5 hmC mark only very
recently have started to get discovered. Spruijt et al. (2013) used a
DNA pull-down followed by quantitative mass-spectrometry-based
proteomics to identify readers of 5 hmC and 5 mC in mouse ESCs,
neuronal progenitor cells (NPCs), and adult brain tissue. They
discovered that each of these marks recruits a distinct set of pro-
teins, including previously uncharacterized Wdr76 and Thy28 that
preferentially bind 5 hmC in the brain and NPCs, and brain-specific
Thap11/Ronin. Only three proteins showed interaction with both
5 mC and 5 hmC: MeCP2, Uhrf1, and Thy28 (Spruijt et al., 2013).
Interestingly, while MeCP2 protein, which has long been implicated
in chromatin and transcriptional regulation through 5 mC binding,
has been recently demonstrated to bind 5 mC and 5 hmC-con-
taining DNA with similar affinity in the mouse cerebellum (Mellén
et al., 2012). The discovery that MeCP2 acts as one of the major
readers of 5 hmC is very intriguing and potentially highly signifi-
cant. Finding that Rett-syndrome causing MeCP2 R133C mutation
preferentially inhibits 5 hmC binding can add not only to our un-
derstanding of the role of this protein in chromatin regulation but
also provide a significant insight into the mechanisms of Rett
syndrome (Mellén et al., 2012).
3.3. Rett syndrome
A most well-known and best studied human epigenetic disorder
related to DNA methylation is Rett syndrome (RTT). RTT is an X-
linked dominant neurodevelopmental disorder with a cognitive
impairment component. As male carriers normally die due X-
hemizygocity, RTT manifestations occur postnatally only in females
at a rate about 1 in 10,000 live births (Rett, 1966; Wan et al., 1999;
Chahrour and Zoghbi, 2007). RTT is a progressive disorder with
patients developing normally for the first 6e18 month of age, but
stagnating later, developing microcephaly by the second year of life,
muscle hypotonia, weight loss, and overall growth retardation.
Affected individuals develop various features of autistic behavior,
including a loss of linguistic skills, stereotypic hand movements,
loss of eye-to-eye contact, and social withdrawal. Moreover, RTT
causes progressive cognitive impairment (Chahrour and Zoghbi,
2007). Brains of the RTT patients were shown to have signifi-
cantly reduced both gray and white matter as well as dendritic
spines atrophy (Naidu et al., 2001; Johnston et al., 2001; Chapleau
et al., 2009). Using information from a very rare familiar case of
RTT, Huda Y. Zoghbi and colleagues mapped Xq28 candidate region
A. Rudenko, L.-H. Tsai / Neuropharmacology 80 (2014) 70e82 77
being responsible for the disorder (Amir et al., 1999). Screening of
candidate genes uncovered mutation in methyl-CpG binding pro-
tein 2 (MeCP2) (Lewis et al., 1992; Meehan et al., 1992) as a reason
for more then 95% of classic RTT cases. Most of the mutations arise
in de novo paternal germline and, interestingly often involve a C to T
transition at CpG dinucleotides (Chahrour and Zoghbi, 2007). Initial
studies indicated that MeCP2 acts as transcriptional co-repressor,
which can specifically bind CpG dinucleotides, recruit other co-
repressor factors including Sin3a, HDAC1 and 2, and inhibit tran-
scription from the methylated promoters (Nan et al., 1997; Jones
et al., 1998; Nikitina et al., 2007). Interestingly, later studies re-
ported that although MeCP2 indeed can act as a transcriptional
repressor, it also may play role as a context-dependent transcrip-
tional activator by associating with CREB at specific promoters
(Chahrour et al., 2008).
A number of RTT mouse models were generated by either
ablating or mutagenizing MeCP2. Rudolf Jaenisch and colleagues
generated two mouse RTT models by knocking out MeCP2 in the
germline and conditionally ablating it using Nestin-Cre (Chen et al.,
2001). Both mutant lines appeared to recapitulate a number of
human RTT features: they were healthy and fertile for the first few
weeks of age but progressively developed abnormal behaviors
including nervousness, body, trembling and occasionally hard
respiration at five weeks. At later stages, the mutants displayed
hypoactivity, trembling, and weight loss. Most mutants died at
about 10 weeks of age. Autopsies of the mutant mice revealed a
decrease in the overall brain size as well as neuronal size in some
brain areas coincident with increased neuronal density, which is
consistent with findings in human RTT patients (Chen et al., 2001;
Bauman et al., 1995). MeCP2 ablation cause less severe phenotypes
in mice compared to human RTT: hemizygous males developed
obvious abnormalities and died as young adults and heterozygous
females remained healthy for a long time. Huda Y. Zoghbi’group
generated a mouse mutant line carrying MeCP2 truncation with a
stop codon after codon 308 (MeCP2
308/y
). This mutant form elimi-
nates the C-terminal third of the coding MeCP2 sequence and
supposed to display similarity to a number of human RTT muta-
tions that have truncations in a similar region (Shahbazian et al.,
2002). Interestingly, MeCP2
308/y
male mice did not display any
obvious abnormalities in the brain and CNS neuronal morphology
and showed normal fear and spatial learning. However, additional
behavioral characterization demonstrated that the mutant animals
exhibited impairment in their motor function and abnormal social
interaction. The authors also observed an increase in a histone H3,
but not histone H4, acetylation in the cortex and cerebellum of the
mutant mice, which was interpreted as a result of inability of
truncated MeCP2 to recruit HDAC complexes to a chromatin.
Another mutant line was generated by Gemelli et al. (2006) by
conditional ablation of MeCP2 in forebrain neurons using CaMKII-
Cre93. The mutant mice, in which MeCP2 is ablated postnatally,
exhibit normal locomotor activity but abnormal coordination.
Additionally, they show an increased anxiety, impaired social in-
teractions and deficits in fear memory (Gemelli et al., 2006). Very
interesting model reported by Yasui et al. (2014) represents a point
mutation in the translational start codon of MeCP2 exon 1 resulting
in an ablation of MeCP2-e1 isoform. The mutant mice that express
only MeCP2 e2-isoform, were shown to develop forelimb stereo-
typy, hindlimb clasping, excessive grooming, hypoactivity,
abnormal anxiety, and sociability, thus recapitulating a number of
RTT-related deficits (Yasui et al., 2014).
Interestingly, transcriptional profiling of the forebrain, cerebral
cortex and hippocampal tissues from Nestin-Cre;MeCP2
2lox/y
and
CaMKII-Cre93;MeCP2
2lox/y
mutant mice of different ages did not
revel any dramatic changes in gene expression (Tudor et al., 2002).
Additional analyses of potential transcriptional changes in MeCP2
mutant mice also revealed very subtle alterations (Horike et al.,
2005; Nuber et al., 2005). These findings look rather intriguing
considering a strong impact MeCp2 mutations have on various as-
pects of neurological phenotype. Potential answer to this puzzle
was provided by Chen et al. (2003) who demonstrated that MeCP2
control expression of an activity-dependent gene, Bdnf, which in
turn was shown to regulate wide array of neuronal functions. They
reported that in the absence of neuronal activity, MeCP2 binds to
the Bdnf promoter III negatively regulating its expression. In
response to activity, MeCP2 gets phosphorylated and is released
from the Bdnf promoter, thus enabling expression (Chen et al.,
2003;Chang et al., 2006). Further studies by Michael Greenberg’
s group have demonstrated that MeCP2 can bind to the neuronal
genome in a histone-like fashion, and that phosphorylation of
MeCP2 at S421 regulates excitatory-inhibitory balance and den-
dritic development in neurons, and controls behavioral responses
to novelty (Cohen et al., 2011). Moreover, Ebert et al. (2013) has also
showed that activity-dependent phosphorylation of MeCP2 at T308
blocks its interaction with nuclear co-repressor (NCoR) leading to
disruption of MeCP2 function as transcriptional repressor. They
demonstrated that MeCP2 T308A (T to A conversion) knock-in mice
Fig. 1. Rudenko et al. (2013) proposed that Tet1 is important for maintaining locus-specific DNA hypomethylation, particularly at the promoters of at least some of the neuronal
activity-regulated genes, such as Npas4,Fos, and Arc. Such hypomethylation may be critical for keeping these genes poised for rapid transcriptional activation upon activity-based
stimulation. Lack of Tet1 as well as promoter hypomethylation may promote binding of repressor complexes inhibiting transcription.
A. Rudenko, L.-H. Tsai / Neuropharmacology 80 (2014) 70e8278
showed attenuation of behavioral stimulus-dependent activation of
Bdnf and Npas4. They authors speculate that such loss of activity-
dependent MeCP2 function may contribute to a human RTT.
Very recently, Baker et al. (2013) reported two new mouse
models of RTT. In these lines, MeCP2 was mutated at either amino
acid 270 (MeCP2-R270X) or 273 (MeCP2-G273X), following the
data from humane male RTT patients, showing that symptom
severity can be differently influences by these two loci. Interest-
ingly, despite the fact that transcriptional dysregulation is similar
between MeCP2-R270X and MeCP2-G273X, R270X mice had sub-
stantially more severe phenotype then G273X. The authors
discovered that a conserved AT-hook domain is disrupted n MeCP2-
R270X but not in MeCP2-G273X mice leading to
a
-thalassemia/
mental retardation syndrome X liked protein (ATRX) mislocaliza-
tion in G273X animals. These findings suggest the mechanism by
which AT-hook domain of MeCP2 may determine the clinical
course of RTT.
4. Conclusions and future directions
This review summarized a series of evidence clearly implicating
epigenetic mechanisms as an important level of regulation of the
nervous system. We decided to focus on analyzing just a set of
epigenetic modifications in neurons: the histone acetylation and
DNA methylation/hydroxymethylation, although it is clear that
many more epigenetic tools are used, often in combination, to
mediate multiple aspects of neuronal function efrom a regulation
of specific gene expression to a coordinated response to compli-
cated behavioral experiences.
One of the general conclusions we would like to make is that
there is enough evidence to consider epigenetic regulation a critical
mechanism of neuronal function. Second, we would like to state
that multiple epigenetic regulatory mechanisms can be very dy-
namic in postmitotic neurons. For example, DNA methylation and
de-methylation that have long been considered very robust and
long-lasting ways to create epigenomic annotations, now appear to
also serve as means of very fast responses to behavioral stimuli
(Levenson et al., 2006; Miller and Sweatt, 2007). Epigenetic control
of activity-regulated gene expression can also provide a very
powerful tool to convey neuronal responses (Haggarty and Tsai,
2011; Kosik et al., 2012; Ebert et al., 2013). Third, it is getting
more and more apparent that epigenetic control is a critical
mechanism of normal cognitive regulation (Day and Sweatt, 2011).
Studies in the area of epigenetics of mental processes, can signifi-
cantly improve our basic understanding of such critical aspects of
human function as memory formation and consciousness. More-
over, cognitive neuroepigenetics can potentially lead to very
exciting discoveries that could revolutionize treatments of various
mental impairments.
Some of the future directions and challenges in the field of
neuroepigenetics are related to a multiplicity and complexity of the
regulatory mechanisms. For example, it would be important to
better understand the role of the “histone code”in chromatin
regulation and consequently, to gain a deeper insights into which
enzymes may be responsible for different aspects of creating and
“de-ciphering”this code. Related to that, is an issue of specificity of
different regulatory mechanisms preferentially modulating
different neuronal processes. De-construction of these mechanisms
would not only increase our understanding of the basic processes
but also would allow us to highlight particular routs of potential
therapeutic intervention. For example, development of more se-
lective and potent HDAC inhibitors can in a not too distant future
dramatically improve efficiency of available treatments of cognitive
disorders. An opposite aspect of specificity is a multitude of
epigenetic mechanisms that can act in concert to regulate a single
behavioral outcome. Future studies including, development of
more technically advanced ways to perform genome-wide analysis
of different epigenetic marks, should bring us a better under-
standing of epigenetic hierarchy and networks regulating various
cognitive processes, which in turn should also help to create better
therapeutic solutions for mental disorders.
Conflict of interest
The authors declare no conflict of interests.
Acknowledgments
The authors apologize to the colleagues whose work was not
cited due to space limitations. We thank Dr. Alison Mungenast for
critical reading of the manuscript and all the members of Tsai lab
for helpful discussions. A.R is a recipient of the NARSAD Young
Investigator Award, L.-H.T. is an Investigator of the Howard Hughes
Medical Institute.
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