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Epigenetics and Memory 1
Running head: EPIGENETICS AND MEMORY
Neuroepigenetics of Learning and Memory
A Thesis Submitted to the Faculty of
Baylor University
In Partial Fulfillment of the Requirements for the
Honors Program
By
Travis Chapman
Waco, Texas
May 2011
Epigenetics and Memory 2
Abstract
Long-term memory formation requires changes in gene expression. One mechanism for
altering gene expression involves chemical modifications of DNA or its associated
histone molecules. These “epigenetic” tags have long been studied by developmental
biologists for their role in cell differentiation, but recent evidence suggests they also
coordinate behavior in terminally differentiated neurons. Epigenetic chemical
modifications include DNA methylation as well as histone methylation, acetylation,
phosphorylation, ubiquitylation, and sumoylation. DNA methylation and histone
modifications—in particular acetylation, methylation, and phosphorylation—play a key
role in regulating memory-related behavior. Moreover, neuroscientists investigating
epigenetics have identified potential targets for therapeutic intervention in diseases like
Alzheimer’s, especially with regard to histone deacetylases (HDACs).
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Table of Contents
Introduction ………………………………………………………………………...…….4
What is epigenetics? ………………………………………………………………...........5
Histone Acetylation ……………………………………………………..……….....…...12
Histone Phosphorylation and Methylation ………………………………………...……26
DNA Methylation .............................................................................................................31
Challenges and Future Research ......................................................................................38
References ........................................................................................................................41
Epigenetics and Memory 4
Epigenetics is a contentious topic in contemporary biology. On one hand, it is the
subject of increasing media attention and excitement among scientists: both PBS and the
BBC recently had specials highlighting the revolutionary new field. Many scientists are
optimistic that understanding epigenetics will lead to a holistic picture of how the
environment and our genes interact. On the other hand, there is no clear consensus among
scientists about what epigenetics is, much less about its implications for molecular
biology or medicine. Yet the hype surrounding the epigenome continues, even as Wren
(2009) shows that about 37% of genes in the human transcriptome have no publications
characterizing their functions—a potential problem for understanding epigenetic
regulation of these genes. Though further research will tell if the mainstream media’s
popularization of epigenetics is justified by data, this new approach is already changing
how many scientific disciplines look at complex problems. One such discipline,
neuroscience, is turning to epigenetics seeking a molecular explanation for memory
formation, storage, and retrieval as well as understanding disorders of memory like
Alzheimer’s. As this paper will outline, the epigenetic approach to memory has made
significant progress in recent years—even as a young sub-discipline. The main questions
to address in order to understand this progress are as follows:
1. What is epigenetics and how have memory and dementia researchers
approached it?
2. How does epigenetics affect memory and dementia?
3. What are some challenges in this research and what are future directions for it?
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CHAPTER ONE
What is epigenetics?
Definitions for epigenetics are varied and ambiguous. As early as 1957, Conrad
Hal Waddington proposed a definition for epigenetics, though he obviously was not as
familiar with its molecular mechanisms as scientists are now. In his book The Strategy of
the Genes, he related epigenetics to epigenesis, or how genotypes give rise to phenotypes
throughout development. This definition does not incorporate contemporary molecular
biology research, so it has become largely irrelevant. Bird (2007) cites Arthur Riggs and
colleagues for proposing a more contemporary definition in 1996. Epigenetics, they
claim, is “the study of mitotically and/or meiotically heritable changes in gene function
that cannot be explained by changes in the DNA sequence” (p. 396). However, this
definition is problematic because it leaves mechanisms for these “changes” open to
interpretation and even makes heritability a necessity. Simply put, this is not consistent
with contemporary usage of the word “epigenetics.” Bird (2007) subsequently proposes a
very broad definition: “the structural adaptation of chromosomal regions so as to register,
signal, or perpetuate altered activity states” (p. 398). Levenson and Sweatt (2005), on the
other hand, argue that epigenetics “is the mechanism for the stable maintenance of gene
expression that involves physically ‘marking’ DNA or its associated proteins” (p. 109).
Broadly speaking, then, epigenetics involves relatively stable chemical modifications to
DNA or histones that in turn affect gene expression. Moreover, epigenetics does not
involve changes in the DNA sequence itself. It is also important to note that the above
definition is somewhat controversial, as many insist epigenetics should include only
heritable supra-genetic changes in the cell—as opposed to metastable or transient
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chemical markings (Bonasio, Tu, & Reinberg, 2010). This standard would exclude most
neuroscience research from epigenetics proper since neurons are terminally
differentiated, postmitotic cells. Epigenetic signals can be either cis or trans as well. Cis
signals are inherited by chromosome segregation and physically associated with DNA or
chromatin. Trans signals, on the other hand, are maintained in the cytosol by feedback
loops independent of the inherited chromatin structure (Bonasio et al., 2010). With this
definition of epigenetics as a heuristic, it is now necessary to discuss the main chemical
modifications studied by neuroscientists.
DNA methylation plays a key role in epigenetic regulation of gene expression;
and the relatively well-characterized histone modifications involved in gene regulation
are histone methylation, acetylation, and phosphorylation. This discussion of epigenetics
will be limited to the aforementioned chemical marks, though various small RNAs
(sRNAs) and even transcription factors are sometimes considered epigenetic gene
regulators.
DNA Methylation
DNA methylation occurs when a methyl (--CH3) group attaches to the 5-position
of a cytosine residue (Santi, Garrett, & Barr, 1983; Chen et al., 1991). There are four
known enzymes—called DNA methyltransferases (Dnmts)—that catalyze this reaction in
mammals: Dnmt1 (Bestor, Laudano, Mattaliano, & Ingram, 1988), Dnmt2 (Yoder &
Bestor, 1998), Dnmt3A, and Dnmt3B (Okano, Xie, & Li, 1998). Interestingly, a recently
reported enzyme called Gadd45b has demethylase activity in neurogenesis. Given
Gadd45b’s close relationship to memory research, it will be discussed extensively later in
this review. The roles of these Dnmts are varied, overlapping, and depend on the cellular
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context at any given time. That said, their functions will be discussed later. It is notable,
though, that sequence analysis has shown identical cells from the same lineage
expressing disparate DNA methylation patterns, suggesting DNA methylation patterns
are not replicated as judiciously as the nucleotides themselves (Silva, Ward, & White,
1993). This could give rise to significant variations in phenotype for individual cells or
groups of cells. Moreover, DNA methylation generally occurs at cytosine residues
followed by guanine—known as CpG dinucleotides. This is distinct from the term for
CpG-rich areas called CpG islands. CpG islands are proximal to (or in) promoters for
many “housekeeping” genes like actin (Gardiner-Garden & Frommer, 1987). It logically
follows that DNA methylation is associated with transcriptional silencing. For instance,
the protein methyl CpG-binding protein 2 (MECP2) binds to methylated DNA
(independent of the underlying DNA sequence) and has been implicated in transcriptional
silencing by recruiting histone remodeling proteins (Jones et al., 1998). Some
transcription factors—erythroblastosis 1 (ETS1), for instance—bind to unmethylated
DNA and eschew the methylated variety (Maier, Colbert, Fitzsimmons, Clark, &
Hagman, 2003). About 70% of CpG dinucleotides are methylated (Cooper & Krawczak,
1989). Finally, methylation may involve either de novo DNA methyltransferases or
maintenance DNA methyltransferases. The former initiate the transfer of a methyl group
from S-adenosyl methionine to non-methylated cytosine nucleosides while the latter add
a methyl group to the complimentary strand of hemi-methylated DNA (i.e. DNA with no
methyl group on one strand) (Day & Sweatt, 2010).
Histone Modifications
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Before discussing histone modifications, it is important to review the structure of
histone molecules. Core histones are organized in an octamer of four subunits, with each
nucleosome containing two copies of every subunit: H2A, H2B, H3, and H4. In addition,
each subunit has N-terminus “tails” that project beyond the rest of the protein. These tails
are important in transcriptional regulation and memory, which will be addressed later.
The two “linker” histones, H1 and H5, hold the histone octamer and DNA together as a
“nucleosome,” but these will be discussed less than the others in this essay. The DNA
wraps around each octamer with about 150 bp and may be either tightly or loosely
wrapped depending on the chemical environment. This packaging results in two different
kinds of chromatin: heterochromatin and euchromatin. Constitutive heterochromatin is
generally tightly packed, found at centromeres and telomeres, and transcriptionally silent.
Euchromatin, on the other hand, is the loosely packed form of chromatin associated with
transcriptional activation. Another kind, facultative heterochromatin, silences gene
expression in some cell types and activates it in others.
Major histone modifications include methylation, acetylation, phosphorylation,
sumoylation, and ubiquitylation. SUMO and ubiquitin are small proteins that attach to
histone and other molecules as post-translational modifiers. Their roles in memory
regulation via histone modification remain mysterious, although there are numerous
examples of post-translational changes on memory-related molecules. For example,
ubiquitin-mediated proteolysis degrades the regulatory subunit of PKA, causing deficits
in long term memory (Chain, Schwartz, & Hegde, 1999). Methylation, acetylation, and
phosphorylation involve the addition of a methyl (CH3) group, acetyl (COCH3) group, or
phosphate (PO4) group, respectively, to certain amino acids of histone tails. For instance,
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molecules called histone acetyltransferases (HATs) transfer an acetyl group from acetyl-
CoA to lysine residues on histones. Complementarily, histone deacetylases (HDACs)
catalyze the removal of acetyl groups on lysine residues. There are also histone
methyltransferases (HMTs) and demethylases for transfer of a methyl group to or from
histones, respectively. Kinases and phosphatases add and remove phosphate groups,
respectively. Drugs called HDAC inhibitors pharmacologically inhibit the activity of
HDACs—which has therapeutic potential since increasing histone acetylation (and thus
gene expression) may help ameliorate memory loss. Table 1 and Figure 1 summarize
known HDAC isoforms, HDAC inhibitors associated with them, and the sites of DNA
and histone modifications (image credit: Roth & Sweatt, 2009). These basic molecular
changes will be discussed throughout this essay; below is a specific example of
epigenetic change in neural development that shows the enormous impact of epigenetics
in the nervous system.
Table 1.
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Figure 1.
Neural cell differentiation is an example of the potent effects of chromatin
remodeling. Clearly neurons have striking differences from other cells in the body.
Besides maintaining an ionic gradient conducive to action potential firing, they have a
distinctive molecular toolkit involved in excitability and synapse function. So neurons
must develop this functional distinctiveness from other cell types. Promoter regions for
neuronal genes contain a neuron-restrictive silencer element (NRSE) (Maue, Kraner,
Goodman, & Mandel, 1990; Mori, Schoenherr, Vandenbergh, & Anderson, 1992). Other
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cell types ubiquitously express a transcription factor, RE-1-silencing transcription factor
(also known as REST or NRSF) that silences expression of neuronal genes (Chong et al.,
1995). Transcriptional repression with REST involves changes in chromatin structure.
Two proteins, SIN3A and CoREST, bind to REST and affect HDAC1 and HDAC2,
respectively (Huang, Myers, & Dingledine, 1999). Although expression of SIN3A
mirrors expression levels of REST, CoREST is more restricted—perhaps because SIN3A
mediates most NRSE-associated gene silencing whereas CoREST serves a particular role
in subtypes of neural cells (Grimes et al., 2000). Both SIN3A and CoREST increase
histone deacetylase activity by their interaction with the aforementioned HDACs. The
REST/CoREST complex is also associated with increased DNA methylation, and it
targets not only genes with the NRSE but also genes proximal to an NRSE (Lunyak et al.,
2002). So silencing of neural genes in non-neural cells occurs by the activity of a
chromatin remodeling complex that decreases histone acetylation and increases DNA
methylation. Epigenetic markings thus have an enormous impact on neural phenotype.
The specific neuroepigenetic correlates of learning and memory will be discussed
extensively throughout this review. Particular attention will be given to the better-
characterized correlates of learning and memory, including DNA methylation, histone
acetylation, histone phosphorylation, and ubiquitylation.
Epigenetics and Memory 12
CHAPTER TWO
How does epigenetics affect memory and dementia?
Histone Acetylation
The best-characterized epigenetic modifications in memory research are histone
acetylation, phosphorylation, and methylation as well as DNA methylation. Thus, these
will be addressed in detail throughout this review.
Two influential studies by Alarcón et al. (2004) and Korzus et al. (2004)
addressed the importance of altered histone acetylation patterns in memory formation.
Scientists used a model of Rubinstein-Taybi syndrome (RTS) to investigate the
epigenetic cause of the severe mental retardation seen in RTS. This model originally
piqued the interest of researchers in learning and memory because RTS is caused by a
mutation in the CREB binding protein (CBP) gene, and CREB has been reported as a
major protein involved in the late phase of long-term potentiation (LTP). First,
researchers investigated the phenotype of a heterozygous (cbp+/-) mutant mouse, which
shows symptoms similar to RTS in humans. Despite no change in baseline motor activity,
anxiety, or short-term memory, the mice showed significant deficits in long-term memory
as measured by contextual fear conditioning, cued fear conditioning, and novel object
recognition paradigms (Alarcon et al., 2004). However, they showed normal performance
in the Morris water maze task—the authors suggest this is due to practice in this task (in
contrast, fear conditioning depended on one noxious event). Moreover, there were
electrophysiological signs of late phase LTP deficits in the cbp+/- mice at Schaffer
collateral synapses. Mutant mice expressing a constitutively active form of CREB did not
show normal memory capacity, suggesting there is another mechanism involved. Alarcón
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and colleagues suggest histone acetylation as a possible mechanism. They tested their
hypothesis pharmacologically, giving cbp +/- mice the histone deacetylase (HDAC)
inhibitor SAHA. In addition to causing increased H2B acetylation, SAHA restored
contextual memory and electrophysiological response to wild-type levels. Korzus et al.
(2004) investigated this phenomenon further with a different mouse model of RTS. Given
that the aforementioned mutant mice are susceptible to developmental abnormalities that
could confound results, Korzus and colleagues (2004) developed a mouse with spatial
and temporal restriction of CBP deficits. In particular, CA1 and dentate gyrus
hippocampal neurons express a form of CBP without histone acetyltransferase (HAT)
activity only after treatment with tetracycline. These (CBP{HAT+/-}) mice are
substantially impaired in their ability to perform a visual-paired comparison task as well
as the Morris water maze task (for spatial memory). However, additional practice
restored performance on the Morris task, lending credence to the hypothesis Alarcon et
al. (2004) gave about their RTS model’s Morris task abilities. Termination of the
tetracycline diet as well as treatment with the HDAC inhibitor trichostatin A (TSA)
ameliorated the memory-related symptoms of the RTS model mice, indicating that the
HAT activity of CBP is critical to memory formation (Korzus, Rosenfeld, & Mayford,
2004). These results have been further confirmed and elaborated on. Although TSA was
assumed to work by nonspecifically increasing gene expression, it has actually been
shown to regulate expression of specific genes in a CREB:CBP-dependent mechanism
(Vecsey et al., 2007).
Researchers at Baylor College of Medicine validated a critical role for histone
acetylation in memory later that year. Levenson et al. (2004) first trained mice in a
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contextual fear conditioning paradigm and collected tissue samples from the
hippocampus either 1 hour or 24 hours afterwards. Compared to control subjects, fear-
conditioned mice showed a two-fold increase in acetylation of histone H3 (Lys-14) after
1 hour. However, the increase was transient; they observed decreased acetylation (normal
levels) after 24 hours. Mice unable to form an association between the unconditioned
stimulus and the context in which it was received also showed normal acetylation levels
(a well-characterized phenomenon called latent inhibition). This indicates that memory
formation requires increased acetylation in certain areas of the chromosomes—a
transformation of heterochromatin. Their findings are also persuasive because they
treated other mice with NMDA or MEK antagonists, relating canonical LTP pathways to
the putative mechanism of chromatin remodeling. As predicted by other studies,
acetylation levels were normal and mice failed to consolidate significant fear
conditioning when treated with NMDA or MEK antagonists. In addition, treatment of
mice with HDAC inhibitors (TSA or sodium butyrate) enhanced induction of LTP,
demonstrated as an increase in freezing in the fear conditioning paradigm. This
enhancement of LTP was due, at least in part, to a three to five-fold
increase in acetylation of histone H4. They also found electrophysiological hallmarks
similar to Alarcón et al. (2004) at Schaffer collateral synapses (Levenson et al., 2004).
The dependence of latent inhibition on H4 changes and fear conditioning on H3 changes
suggests a “histone code” for specific types of memories (Levenson & Sweatt, 2005).
Understanding HDAC inhibition as an area for therapeutic intervention in
learning and memory processes will likely require basic knowledge of the roles of
HDACs themselves. As mentioned before, there are 11 known HDAC isoforms, so
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identifying discrete roles for each of them is itself a daunting task. HDAC1 research will
be addressed later, as it ties in with a neurodegeneration study by Fischer and colleagues
(2007). HDAC2 and HDAC3, however, have been characterized as negative regulators of
learning and memory.
Guan et al. (2009) implicates HDAC2, and excludes HDAC1, in negative
regulation of memory formation and synaptic plasticity. First, they created mutant mouse
lines overexpressing HDAC1 and HDAC2 (called HDAC1OE mice and HDAC2OE,
respectively) in areas important for memory, such as pyramidal neurons in the
hippocampus. The HDAC1 and HDAC2 coding sequences were placed in frame with
endogenous Tau, with Tau mutants showing no memory deficits compared to wild type
mice. HDAC2OE mice had about 70% less histone 4 lysine 12 (H4K12) acetylation
compared to wild type mice as well as significantly less H4K5 acetylation but no change
in Ac-H4K14. HDAC1OE mice also had significantly less overall acetylated lysine, but
there was no effect on H4K12 and H4K5 as in HDAC2OE mice. HDAC1OE mice had no
deficits in associative conditioning (freezing in tone-dependent and contextual fear
conditioning). On the other hand, HDAC2OE mice showed ~20% less freezing in tone-
dependent conditioning as well as ~45% less freezing in contextual fear conditioning
experiments. HDAC1OE mice also had similar escape latency and quadrant affiliation as
wild type mice in the Morris water maze task. HDAC2OE mice had much higher escape
latency than HDAC1OE and wild type mice in addition to spending much less time in the
quadrant containing the platform. Correspondingly, HDAC2 knockout mice had more
histone acetylation as well as increased freezing behavior. Gross anatomical analysis of
HDAC KO and OE mice indicated no developmental abnormalities as well. With regard
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to spine density in the CA1 region of the hippocampus, HDAC2OE mice had
significantly less than wild type mice while HDAC2 KO mice had much more. An
immunoreactive synaptophysin assay also showed less synapse formation in HDAC2OE
mice and more synapse formation in HDAC2 KO mice. HDAC inhibition (HDACi) with
SAHA restored memory functioning in HDAC2OE mice. Finally, HDAC2 KO mice had
increased electrophysiological signs of LTP (fEPSPs) over 40 minutes of testing while
HDAC2OE mice showed decreased LTP. In addition, a variety a memory-related genes
interact with HDAC2 at their promoter regions, and HDAC2 preferentially associates
with CoREST. This could pave the way for a potential mechanism by which HDAC2
functions. Given the important role CoREST plays in neuronal gene repression (outlined
above), it is plausible that HDAC2 mediates learning and memory by interacting with
basic neuronal regulatory mechanisms. Taken together, this indicates a critical role for
HDAC2 in memory formation, particularly as a negative regulator of molecular,
behavioral, and electrophysiological signs of memory in mice (Guan et al., 2009).
HDAC3 performs a similar role as HDAC2, according to McQuown et al. (2011).
Researchers probed the role of HDAC3 using a genetic and pharmacological approach.
Using a combination of mutant mice and viral infection, they created homozygous
deletions of HDAC3 in area CA1 in the dorsal hippocampus. Moreover, they used the
selective inhibitor RGFP136 to pharmacologically block normal HDAC3 functioning in
the hippocampus. Assayed by immunohistochemistry, both conditions increased histone
acetylation in CA1. As expected, genetic and pharmacological HDAC3 manipulation
enhanced performance in long-term memory tests. There was also increased hippocampal
expression of the memory-related genes nuclear receptor subfamily 4 group A, member 2
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(Nr4a2) and c-fos. However, hippocampus-specific delivery of small interfering RNA
(siRNA) targeting Nr4a2 blocked the memory enhancements associated with HDAC3
deletion (but not pharmacological inhibition), suggesting a potential mechanism for
HDAC3’s negative regulation of long-term memory (McQuown et al., 2011).
Besides a role in associative and spatial memory formation, histone acetylation
changes are also implicated in animal models of Alzheimer’s and neurodegeneration. It
has been previously demonstrated that recovery of learning and memory in mice with
significant brain atrophy and loss of memory is associated with environmental
enrichment (EE). Findings by Fischer, Sananbenesi, Wang, Dobbin, and Tsai (2007)
show that histone proteins are involved in synaptic plasticity and mediate the memory-
influencing effects of environmental enrichment for mice.
Fischer et al. (2007) used CK-p25 Tg mice—in which a doxycycline diet induces
expression of the neurotoxic protein p25—to induce symptoms of dementia to compare
with a control. Neuronal populations were cut in half in the cingulate cortex—assayed by
the presence of NeuN, a neuron-specific, nuclear protein. The transgenic mice were split
into two groups: those reared with EE and those reared without EE. The EE group
recovered long-term memories significantly better than the non-EE group, with about
30% savings for the water maze and fear conditioning tasks—even when p25-induced
changes in anxiety and locomotor activity were taken into account. In addition, the EE
and non-EE transgenic groups showed similar brain atrophy levels, suggesting EE leads
to the recovery of long-term memories. Finally, environmental enrichment resulted in
significant lysine acetylation increases on histones H3 and H4 in both the cortex and
hippocampus. The acetylation effect continued over 2 weeks of measurements after
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behavioral memory tests. Levels of synaptophysin assessed by an immunoreactivity assay
in the anterior cingulate cortex indicated significantly more synapses in environmentally
enriched animals as well.
To investigate whether chromatin remodeling mimics the effects of environmental
enrichment, Fischer et al. (2007) used the HDAC inhibitor sodium butyrate (SB). Some
Tg mice were given daily injections of SB to induce chromatin remodeling while others
were treated with saline solution. Similar to the EE and non-EE groups, the SB group
showed about 45% savings for fear conditioning and about 30% savings for the water
maze task compared to mice injected with saline solution. Given a period of 4 weeks for
consolidation of a fear-conditioning task, the SB-injected mice had similar improvement
over mice injected with saline solution. In many tests SB-treated mice performed as well
as or nearly as well as wild type mice. Moreover, there were more synapses and up-
regulated histone acetylation of H3 and H4 lysine residues despite no apparent increase in
the number of neurons. This supports the hypothesis that increasing histone-tail
acetylation mimics the memory-enhancing effects of environmental enrichment, induces
synaptic rewiring, and even leads to the recovery of inaccessible long-term memories in
mice (Fischer et al., 2007).
Given the importance of characterizing HDAC functioning to understand how
HDAC inhibition works, researchers also considered the effects of the CK-p25 Tg mouse
model on HDAC1. Cell cycle changes and DNA damage are becoming important areas of
interest in neurodegeneration research, and expression of p25 initiates many hallmarks of
aberrant cell cycle activity and DNA damage before atrophy occurs. Messenger RNA
levels of many cell cycle markers are up-regulated by p25 toxicity in the hippocampus,
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including cyclin B, cyclin E, cdc25a, p21, MCM3, MCM6, and MCM7. In addition,
protein levels of the DNA damage markers gH2AX and Rad51 were greatly up-regulated
in the forebrain after p25 induction. Increased comet tails on a micrograph and increased
immunoreactivity of damage markers in fluorescence imaging also indicated DNA
damage. Loss of HDAC1 by RNAi or pharmacological inhibition of HDAC1 activity
with the class I HDACi MS-275 yielded similar results as p25 toxicity (i.e. DNA damage,
aberrant cell cycle activity, and atrophy). More directly linking p25 cell damage and
HDAC1 function, overexpression of HDAC1 in p25 cells protected against cell toxicity
and DNA damage. This approach to ameliorate p25 damage was effective in cultured
neurons as well as in an in vivo ischemia model (Kim et al., 2008). It is therefore clear
HDAC1 plays a role in neurodegeneration in the p25 model of cell toxicity, but
researchers are also interested in traditional Alzheimer’s pathology.
The traditional molecular hallmarks of AD, amyloid plaques and neurofibrillary
tangles, are also important to incorporate in an epigenetic perspective on memory.
Although the data is somewhat preliminary given the relative youth of epigenetics as a
discipline, there is accumulating evidence that directly implicates epigenetics in
canonical Alzheimer’s pathways. Amyloid-β precursor protein (APP) is cleaved in the
transmembrane domain by γ-secretase. This cleavage results in amyloid β plaques on the
extracellular side as well as a more mysterious domain on the intracellular side.
Cao and Südhof (2001) investigated the APP cytoplasmic (intracellular) domain
using a two-hybrid system. The two-hybrid system allows researchers to investigate
interactions among proteins by taking advantage of the distinct functional units, or
domains, in transcription factors. Transcription factors typically have a DNA binding
Epigenetics and Memory 20
domain (DBD) as well as an activation domain (AD). To accomplish the two-hybrid
approach, researchers first attach a protein of interest to the DBD of a transcription factor
(this fusion protein is called the “bait”). Second, a protein that presumably interacts with
the domain of interest is attached to the AD of a transcription factor (this protein is called
the “prey”). If there is, in fact, an interaction between the “bait” and “prey” proteins,
transcription will be greatly up-regulated on a reporter gene (usually lacZ). This is
because the interacting proteins bring the DBD and AD in such close proximity that they
can function similarly to the normal transcription factor and thus initiate transcription. Of
course, if there is little or no interaction between two proteins there will be little or no up-
regulation of transcription of the reporter gene. The most common transcription factors
used in the two-hybrid approach are yeast Gal4 and bacterial LexA (Little, 2010).
Cao and Südhof (2001) characterized protein-protein interactions of the APP
cytoplasmic domain using cell culture models (PC12, HEK293, COS, and HeLa cells).
The DBDs of Gal4 or LexA were fused with full length, intracellular APP695. They also
cotransfected the cells with a Gal4/LexA-dependent plasmid containing luciferase (the
reporter gene) to analyze transcription changes. There was a small change in transcription
levels in the Gal4/LexA transcription factor fusion APP, but >2000-fold transcription
increases occurred in cells cotransfected with Fe65. This indicates interaction between
exogenous APP and Fe65. Additionally, the increase in transcription did not occur in
APP with a point mutation that blocks Fe65 binding; nor was there an increase in
transcription when the “prey” was Mint1/X11 (i.e. the effect is specific to Fe65). Cao and
Südhof (2001) reported similar results in all cell types they tested. Both PTB1 and PTB2
were required domains for Fe65 functioning in their assay. Additionally, the same two-
Epigenetics and Memory 21
hybrid assay was used with a different reporter in COS cells to examine interactions
between Fe65 and a HAT reported in many cancer studies, Tip60. There was a dramatic
transcriptional increase when Gal4-Tip60 was co-expressed with Fe65 and APP, but
either no increase or very little increase with mutant APP, Fe65 alone, or APP alone. This
implies that Tip60 histone acetyltransferase forms a complex with both Fe65 and the
cytoplasmic domain of cleaved APP to remodel chromatin and increase gene
expression—directly linking epigenetics and canonical Alzheimer’s pathways (Cao &
Südhof, 2001). Since epigenetic modifications happen in the nucleus (where
chromosomes are localized), Cao and Südhof (2001) determined by fluorescence
microscopy that Tip60 is in the nucleus under every condition tested while Fe65 is
nuclear only in the absence of APP (or in the presence of mutant APP). APP was
localized in the cytoplasm, but because of assay limitations this did not preclude the
possibility that some (<5%) APP might be in the nucleus.
There is additional evidence suggesting the APP intracellular domain (AICD)
functions in a similar manner to the Notch intracellular domain (NICD) (Sastre et al.,
2001; Kimberly, Zheng, Guenette, & Selkoe, 2001). However, an important study notes
that the AICD also has a pathway of its own and regulates its own precursor’s
transcription via the multiprotein complex described by Cao and Südhof (2001) (von
Rotz et al., 2004). Using a combination of confocal microscopy with inducible
fluorescence tagging and co-immunoprecipitation, von Rotz and colleagues (2004)
determined that Tip60 localized in the nucleus. However, fluorescent AICD localized in
the nucleus with Tip60 only in the presence of Fe65 in HEK293 cells. The AICD-Fe65-
Tip60 complex (called AFT) had structural damage in a mutant of Tip60, abolishing the
Epigenetics and Memory 22
spherical nuclear spots that indicated nuclear localization of AFT. Gamma-secretase
inhibitors also blocked formation of the AFT complex. Besides the AFT complex, Tip60
and AICD also form an association with the APP adaptor protein Jip1b. The AICD-Jip1b-
Tip60 (AJT) complex serves a similar function as AFT, with Jip1b aiding in transport of
AICD to the nucleus and docking it to Tip60. Consistent with Cao and Südhof (2001),
Mint1/X11α trapped AICD in the cytosol. Finally, there is an apparent positive feedback
mechanism involved in AICD signaling. Induced expression of AICD resulted in
increased expression of APP and Tip60, but not the Notch-effector gene Hes1 (von Rotz
et al., 2004). Although present studies of APP intracellular signaling are far from
providing a comprehensive view of how epigenetics and amyloid-β are connected, there
is already evidence that epigenetic intervention in APP signaling could lead to better
treatments of Alzheimer’s.
Additional evidence for epigenetic involvement in APP signaling comes from a
mutant mouse model, APP/PS1dE9. APPswe/PS1dE9 mice have contextual memory
deficits after 6 months of life due to overexpression of presenilin-1 (PS1) and the
presence of a Swedish mutation, which is linked to early-onset AD. Kilgore et al. (2010)
demonstrated the beneficial effects of HDACi on class I HDACs in the aforementioned
AD model mouse. Chronic (2-3 week), intraperitoneal injection (IP) of sodium valproate,
sodium butyrate, or vorinostat (SAHA) into the mutant mouse enhanced histone
acetylation and contextual memory task performance to wild type levels. Amelioration of
memory function in the mutant mice happened without affecting baseline exploratory
behavior or immediate freezing. Thus, state-dependent effects were not an issue and
HDACi of class I HDACs is a potential therapeutic pathway for Alzheimer’s researchers.
Epigenetics and Memory 23
Most strikingly, SAHA affected each class of HDAC to achieve its therapeutic effect.
Given that vorinostat is already a T-cell lymphoma drug marketed as Zolinza, clinical
trials in AD would be much easier to pursue. As expected, memory improvement
correlated with histone acetylation increases (Kilgore et al., 2010).
Besides its relationship with APP biology, Alzheimer’s is also an age-related
disease diagnosed most frequently in people over 65 years of age. Given the likelihood of
memory-related problems occurring later in life, Peleg et al. (2010) designed an
experiment to relate hippocampal histone acetylation changes to aging in mice. Levels of
histone tail acetylation in three-month-old mice were compared to sixteen-month-old
mice in a contextual fear conditioning task. Given that mice live 26-28 months on
average, 16-month-old mice could be considered elderly. Behaviorally, 16-month-old
mice performed significantly less freezing and had increased platform-locating latency in
the Morris task (besides spending significantly less time in the target quadrant). The
lysine acetylation assay was performed in control mice as well as mice 10 min., 30 min.,
60 min., and 24 hours after fear conditioning training. Residues analyzed for acetylation
changes include H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16. The histone
acetylation profile of every group was similar with two notable exceptions. H4K5
showed increased acetylation at 30 min. after fear conditioning in 16-month-old mice
while there was no such increase in 3-month-olds. Since other time points showed a
similar acetylation profile in young and aged mice at H4K5, this result is much less eye-
catching than the following one. H4K12 acetylation did not increase in 16-month-old
mice as a response to fear conditioning at any time point, whereas in 3-month-old mice
every fear-conditioned group had increased acetylation. This happened without any
Epigenetics and Memory 24
change in baseline HAT/HDAC or H4 levels in the cell. This led researchers to conclude
that deregulation of H4K12 in aged rats after fear conditioning leads to their impaired
memory performance. Furthermore, microarray analysis of hippocampal gene expression
1 hour after fear conditioning showed 2229 gene transcript changes (increase or decrease)
in 3-month-old mice, with 1539 of those genes previously linked to associative learning.
On the other hand, 16-month-old mice had only 6 gene expression changes after fear
conditioning. Some gene expression profiles deciphered by data mining were also
verified with real-time PCR. Baseline gene expression was virtually identical in 3-month
vs. 16-month control groups. Taken together, this means deregulation of H4K12
acetylation following fear conditioning in aged mice results in drastically different gene
expression profiles than young mice. Similar to other studies, SAHA treatment of 16-
month-old mice increased freezing behavior in an associative learning task. Interestingly,
H4K12 acetylation increased significantly in response to SAHA treatment whereas no
other lysine residues had a statistically significant increase, including H4K5 (which had
increased acetylation at the 30 min. time point in the old group). Finally, H4K12
acetylation increased at the promoter regions of the formin 2 and Prkca genes, and a
variety of other genes had increased H4K12 acetylation at coding regions. Increased
H4K12 acetylation at coding regions correlated with increased gene expression as well.
There was no change in the 16-month-old group’s ability to find a visible platform,
exploratory behavior, or response to foot shock; various markers of hippocampal
plasticity and integrity were similar between young and old mice. Thus, aging’s negative
effect on memory capacity in mice is related to H4K12 acetylation changes rather than
loss of structural integrity, exploratory behavior, or response to foot shock (Peleg et al.,
Epigenetics and Memory 25
2010). Epigenetics could, therefore, offer insight into the cognitive decline seen with
natural aging.
Epigenetics and Memory 26
CHAPTER THREE
How does epigenetics affect memory and dementia?
Histone Phosphorylation and Methylation
There has recently been an explosion of knowledge gathered about the
relationship between histone acetylation, DNA methylation, and memory, with
comparatively little work done on histone phosphorylation and methylation. At the very
least, however, the latter two chemical alterations appear important in early hippocampal
memory consolidation. Notably, there is currently no work published on prefrontal cortex
changes with respect to histone phosphorylation and methylation, which puts this
research significantly behind other epigenetic approaches. That is not to say work is not
currently being done on this. The Lubin lab at the University of Alabama has already
given oral presentations of forthcoming work relating histone methylation to prefrontal
cortex memory consolidation.
It has been previously reported that cellular stress and activation of immediate
early genes correlates with increases in histone phosphorylation (Thomson, Clayton, &
Mahadevan, 2001). Two early studies have also indicated the importance of histone
phosphorylation in early memory consolidation in the hippocampus. Crosio, Heitz, Allis,
Borrelli, and Corsi (2003) demonstrated that multiple signaling pathways can trigger
histone phosphorylation in the hippocampus. Researchers used a dopaminergic receptor
agonist (SKF82958), mACh receptor agonist (pilocarpine), and a kainate glutamate
receptor agonist (kainic acid), all of which induced chromatin remodeling in hippocampal
neurons. Marks of chromatin remodeling included H3 serine 10 phosphorylation and
lysine 14 acetylation. Validating previous results, activation of these signaling pathways
Epigenetics and Memory 27
also increased c-fos transcription (Crosio et al., 2003). However, these results do not
necessarily indicate that histone phosphorylation is important in learning and memory.
They only show that hippocampal neurons use it in response to stimulation.
More directly implicating histone phosphorylation in learning processes, Chwang,
O’Riordan, Levenson, and Sweatt (2006) found phosphorylation and acetylation
increases (on H3S10 and H3K14) as a result of ERK/MAPK activation in vitro. The
process was NMDA receptor-dependent since the NDMA-R anatagonist MK801 blocked
the effect. More importantly, though, contextual fear conditioning increased area CA1
histone phosphorylation transiently (1-2 hours after training). MEK works just upstream
of ERK in the ERK signaling cascade, and administration of the MEK inhibitor SL327
blocked H3S10 phosphorylation changes after fear conditioning. This result was verified
with a phospho-ERK assay, which made sure phosphorylation (activation) of ERK was,
in fact, inhibited by SL327 (Chwang et al., 2006). Although phenomenological results are
important, it is clinically beneficial to identify target kinases and phosphatases involved
in epigenetic modification for therapeutic intervention. ERK/MEK signaling has long
been considered an important pathway in modulation of gene expression, but an
important study by Lubin and Sweatt (2007) elucidates the role of a well-studied immune
response kinase in memory reconsolidation.
Memory maintenance requires reconsolidation after recall. In rats, contextual fear
conditioning normally involves shocking the animal in a novel context (the conditioning
chamber) and testing freezing behavior after the initial training. Reestablishment of
contextual conditioned fear (CCF) memory, accomplished by re-exposing the animal to
the novel context, can be blocked with inhibition of protein synthesis—ERK/MAPK
Epigenetics and Memory 28
inhibition, for instance (Lubin & Sweatt, 2007). Lubin and Sweatt (2007) specifically
looked at signaling with the transcription factor nuclear factor kappa B (NF-κB) to see
whether there was an effect on histone phosphorylation during reconsolidation.
It is first necessary to review information on NF-κB signaling, which has been
primarily implicated in response to inflammation. NF-κB is inhibited by inhibitor kappa
B (IκB); thus modification of IκB is required in order to activate NF-κB. IκB proteins are
marked for degradation when they are phosphorylated by the IκB kinase (IKK) complex.
The IKK complex has α, β, and regulatory γ subunits. Once IKK frees IκB from NF-κB,
NF-κB translocates to the nucleus and binds genes with the κB consensus sequence in
their promoter. However, components of the NF-κB signaling pathway have been shown
to add phosphate and acyl groups to histones in nonneuronal cells, so the NF-κB DNA
binding complex is likely not the whole story; this was the impetus for investigating
chromatin remodeling by NF-κB in hippocampus cells.
First, the NF-κB inhibitor diethyldithiocarbamate (DDTC) was administered (via
IP injection) to a group of rats after a memory test. Although vehicle and DDTC rats
exhibited similar freezing behavior during the first memory test, freezing decreased in
DDTC-treated rats during tests 2 days and 7 days after training, suggesting NF-κB
inhibition interferes with CCF memory. Phosphorylated IKKα serine 180—and not IKKβ
serine 181—levels also increased in area CA1 1 hour after training. DDTC treatment
blocked the increase. Increased IKKα phosphorylation only occurred in rats re-exposed
to the context after initial context + shock training, as there was no such increase in
animals only exposed to the context (over 1 or 2 days) or only given contextual fear
conditioning. DNA binding activity of NF-κB assessed by an electrophoretic mobility
Epigenetics and Memory 29
shift assay (EMSA) also rose following conditioning and attenuated in DDTC groups.
DDCT injection ultimately resulted in decreased H3 phosphorylation after CCF training.
Surprisingly, pharmacological inhibition of IKK with sulfasalazine (SSZ) reversed the
behavioral and molecular effects of context re-exposure. Another pharmacological tool,
SN50, blocks NF-κB’s normal interaction with DNA binding sites. SN50 administration
decreased freezing behavior in the CCF paradigm, but there was no effect on histone
phosphorylation. On the other hand, IKKα inhibition decreased phosphorylation at the
promoters of the immediate early gene Zif268 as well as IκBα. Taken together, the
results impart a critical role for IκB kinase (IKK) in regulation of chromatin structure
after CCF memory training in area CA1 of the hippocampus. Regulation of chromatin
structure by IKKα happens upstream of regulation by NF-κB’s transcription complex to
affect learning and memory (Lubin & Sweatt, 2007). The kinase activity of IKKα thus
represents a promising target for therapeutic intervention in diseases of memory.
Histone methylation’s involvement in memory-related processes remains more
mysterious than other epigenetic factors. Gupta et al. (2010) was the first to investigate
hippocampus-specific histone methylation patterns after fear conditioning. Unlike
previous epigenetic marks discussed in this review, researchers demonstrated
transcriptional activation and silencing with histone methylation. This is due to the well-
characterized capacity of lysine residues to be monomethylated, dimethylated, or
trimethylated. Each of these result in a different transcriptional hallmark depending on
the lysine residue of interest. Tissue collection 1 hour after fear conditioning from area
CA1 of the hippocampus showed an increase in both H3 trimethylation at lysine 4
(H3K4me3) as well as H3K9me2. H3K9 dimethylation, however, decreased below
Epigenetics and Memory 30
control levels 24 hours after fear conditioning while H3K4 trimethylation returned to the
baseline. Latent inhibition (i.e. exposure to the context before context+shock pairing)
decreased freezing behavior and blocked the transient increase in H3K4me3, so proper
timing is crucial for transient trimethylation. Deficits in a methyltransferase, Mll, with
specific activity on H3K4 resulted in decreased freezing behavior. Finally, promoters
regions for Zif268 and BDNF also had an increase in H3K4me3 after fear conditioning.
HDAC inhibition with sodium butyrate also helped increase trimethylation and decrease
dimethylation, confirming pervious studies. The trimethylation increase correlated with
altered DNA methylation at Zif268 promoters, increased mRNA levels, and altered
MeCP2 binding (Gupta et al., 2010). Histone methylation therefore functions to alter
memory consolidation in area CA1 of the hippocampus in concert with histone
acetylation and phosphorylation. However, there is currently no evidence of histone
methylation as a mark of memory maintenance. This concludes the discussion of histone
modifications in learning and memory, but DNA methylation also impacts learning-
related behavior.
Epigenetics and Memory 31
CHAPTER FOUR
How does epigenetics affect memory and dementia?
DNA Methylation
As outlined previously, DNA methylation involves the enzyme-catalyzed addition
of a methyl (--CH3) group at the 5’ position of cytosine pyrimidine rings. There is
increasing evidence that transcriptional regulation by DNA methylation aids in both
memory formation in the hippocampus as well as memory maintenance in the cortex.
Miller and Sweatt (2007) reported an important role for DNA methylation by
DNMTs in contextual fear conditioning experiments. Context + shock animals showed
dramatically up-regulated mRNA levels of DNMT3A, DNMT3B, and c-fos 30 minutes
following fear conditioning (compared to context only animals) in area CA1 of the
hippocampus. Infusion of global DNMT inhibitors 5-azadeoxycytidine (5-aza) or
zebularine (zeb) into area CA1 of the hippocampus immediately after training in a
contextual fear conditioning task blocked freezing behavior. This effect was not seen in
animals injected with a DNMT inhibitor 6 hours after training. Notably, the decrease in
freezing was transient—DNMT inhibited animals in later tests had similar freezing times
as control animals in the test 1 day after training. A CpG island analyzed in the memory
suppressor gene protein phosphatase 1 (PP1) was dramatically more methylated than
context-only controls 1 hour after learning, and DNMT inhibition occluded this effect
while increasing mRNA of PP1 in area CA1. Reelin (Reln) methylation also decreases
after fear conditioning; the effect is larger with DNMT inhibition. These results suggest a
critical role for covalent modification of DNA in memory formation in CA1 of the
hippocampus (Miller & Sweatt, 2007).
Epigenetics and Memory 32
Similar to studies on the native function of HDACs in the chapter on histone
acetylation, some DNMTs have been analyzed for specific functional properties. Feng et
al. (2010) implicates Dnmt1 and Dnmt3a in synaptic function, learning, and memory.
Although mice with mutations in Dnmt1, Dnmt3a, or Dnmt3b are not viable, Feng et al.
(2010) developed a conditional knockout model for adult forebrain neurons that eschews
viability issues—more specifically they used a CaMKIIa-Cre93 transgene to induce
deletion exclusively in postmitotic neurons postnatally. Researchers knocked out Dnmt1,
Dnmt3a, or both; no significant gene knockout occurred by postnatal day 3 (P3), but by
P14 and into 4 months of life knockout was accomplished. Stereological analysis of
double knockout (DKO) mice revealed slightly decreased hippocampus and dentate gyrus
volume while single knockout mice had no obvious anatomical differences from control
mice. Regardless, optical fractionator analysis indicated that the decrease in total volume
was not a result of atrophy but rather a decrease in volume of individual neurons. First,
DKO mice had disrupted LTP at Schaffer collateral-CA1 synapses and enhanced LTD in
a stimulation protocol that normally fails to induce LTD. Behaviorally, DKO mice took
more time to find the platform in the Morris water maze and spent less time in the target
quadrant than control mice with no change in baseline swimming speeds. This indicates
deficits in spatial learning and memory in DKO mice. Knockout animals also exhibited
less freezing behavior 24 hours after contextual fear conditioning, another indicator of
learning and memory deficiency. Microarray and real time PCR analysis revealed
upregulated immune genes important in learning and memory, including MHC-I and
Stat1. Interestingly, Reln and PP1 levels did not change in DKO mice. These genes will
be discussed in more detail later. Bisulfite sequencing also showed significantly less
Epigenetics and Memory 33
DNA methylation at the Stat1 promoter from -895 to -1,010 bp in DKO mice. This did
not occur in single knockout mice, suggesting Dnmt1 and Dnmt3a compensate for one
another. In addition, demethylation occurred only in cells that tested positive for NeuN in
fluorescence-activated cell sorting (FACS)—an indicator of neuronal populations.
Finally, demethylation was verified with a variant of mass spectrometry, and bisulfite
sequencing found demethylation on the promoters for Dhh, Kcne1, and two regions of
Pten. Overall, these results strongly suggest Dnmt1 and Dnmt3a work together to
maintain methylation patterns in adult forebrain neurons. Their regulation of DNA
methylation ultimately perpetuates proper synaptic function for learning and memory
(Feng et al., 2010).
One of the most important results with regard to DNA methylation comes from
Miller et al (2010). While previous studies investigated transient epigenetic marks after
learning in animals, Miller et al. (2010) found long-lasting DNA methylation patterns
maintaining memory. Since behavioral memories persist multiple molecular lifetimes,
maintaining these memories would likely involve such a self-perpetuating mechanism.
Consequently, DNA methylation is a good candidate for maintenance of transcriptional
repression. However, one of the most compelling aspects of Miller et al. (2010) is its
description of DNA methylation as a potential mechanism for remote memory
maintenance in a prefrontal cortical area called the anterior cingulate cortex (ACC). It has
been demonstrated previously that the ACC maintains remote contextual fear memory. In
particular, activity-dependent expression of c-fos and Zif268 (genes implicated in
memory) increases during remote memory maintenance in the ACC. Moreover, a null α-
CaMKII mutation and pharmacological inactivation of the ACC with lidocaine block
Epigenetics and Memory 34
remote memory (Frankland, Bontempi, Talton, Kaczmarek, & Silva, 2004). However, a
mechanism for long-term memory storage remains elusive. The prefrontal cortex is a
popular candidate for involvement in epigenetic memory maintenance—hippocampal
epigenetic marks return to baseline levels after a maximum of about one day after
learning. There are now results implicating DNA methylation in the consolidation
process. Miller et al. (2010) first used immunoprecipitation to examine dorsomedial
prefrontal cortex (dmPFC) DNA methylation levels on learning-related genes that have
large GC-rich CpG islands in rodents. The immediate early gene Erg1 was demethylated
at all time points. On the other hand, reelin (Reln) was hypermethylated 1 hour after
training, with levels of hypermethylation decreasing over time. This result is somewhat
surprising since reelin is considered a positive regulator of memory and hypermethylation
is associated with transcriptional silencing. Finally, methylation of the phosphatase and
memory suppressor gene calcineurin (CaN, Ppp3ca) did not change shortly after training.
Rather, hypermethylation measured by bisulfite sequencing occurred within 1 day of
training and persisted in tests both 7 days and 30 days after training. This is more
intuitive than results for Reln since CaN is a memory suppressor gene. Thus, CaN was
used as a proxy for global dmPFC DNA methylation changes occurring after contextual
fear conditioning. To further confirm that the observed DNA methylation changes
represent associative learning persisting in the cortex, a group of rats tested 7 days post-
training were injected with the NMDA antagonist MK-801. Besides blocking acquisition
of the fear memory, MK-801 administration interfered with dmPFC hypermethylation of
CaN and Reln (with no effect on Erg1), suggesting that specific environmental signals
induce hypermethylation of CaN and Reln. Notably, hypermethylation of CaN was
Epigenetics and Memory 35
accompanied by considerable decreases in CaN mRNA and protein levels 30 days after
training. But none of this determines whether DNA methylation is a necessary factor in
maintaining remote memory. Pharmacological inhibitors of DNMTs (5-
azadeoxycytidine, zebularine, RG108) administered in the ACC (a subregion of the
dmPFC) 30 days after training decreased freezing behavior by 45-60% in the memory
test, in addition to reducing CaN DNA methylation levels and increasing CaN mRNA
levels. Intra-ACC injections of DNMT inhibitors had no effect on fear memory 2 days
after training; nor was there an effect in these rats 30 days after training, so
administration of DNMT inhibitors to disrupt remote memory in the ACC must occur
after necessary cortical DNA methylation events. Moreover, there was no indication of
major damage to the ACC itself (Miller et al., 2010).
DNA methylation also appears to be crucial in BDNF’s ability to spur dendritic
growth and positively regulate learning and memory. Lubin, Roth, and Sweatt (2008)
first found a large increase in BDNF transcription in area CA1 of the hippocampus 2
hours after fear conditioning. The increase was specific to exon IV of BDNF, but others
increased slightly in context-only controls. Fear conditioning was accompanied by
methylation decreases at exons I and IV, and methylation increases at exon VI of BDNF.
The appropriate increase or decrease in BDNF exon mRNA was confirmed with real time
PCR. Similar to previous studies, DNMT inhibition (with zeb and RG108) reversed these
behavioral and molecular hallmarks and changed BDNF expression. Researchers also
found alterations in chromatin remodeling as measured by histone acetylation as a result
of contextual fear conditioning and DNMT inhibition; and the process was NMDA
receptor-dependent (Lubin et al., 2008). Similar results were reported by Martinowich et
Epigenetics and Memory 36
al. (2003) for cultured neurons after depolarization. CpG methylation of the exon IV
promoter decreased after depolarization while BDNF synthesis increased. This increase
in transcription involved changes in a chromatin remodeling complex. The MeCP2-
HDAC1-mSin3A repression complex dissociated from the BDNF promoter, allowing the
increase in transcription (Martinowich et al., 2003). Taken together, Martinowich et al.
(2003) and Lubin, Roth, and Sweatt (2008) indicate that BDNF’s important role in
dendrite survival and learning depends on DNA methylation.
As mentioned in the introduction, molecules with DNA demethylase activity have
been elusive in mammals. Even the existence of demethylases was the subject of
controversy. After all, a large amount of energy is required to break the covalent carbon
bonds of methylated DNA, and this could be prohibitive on a realistic biological
timescale. There have previously been reports of such proteins in C. elegans, but Ma et
al. (2009) was the first to characterize a mammalian protein with specific demethylase
activity and a role in adult neurogenesis, Gadd45b. Electroconvulsive treatment (ECT) of
adult mice causes upregulation of hippocampal neurogenesis, and Ma et al. (2009) found
a large induction of Gadd45b mRNA (growth arrest and DNA-damage-inducible protein
45 beta) 1-3 hours after ECT. What’s more, in situ hybridization also confirmed the
increase in Gadd45b after ECT. Even exploration of a novel environment significantly
increased Gadd45b expression in the dentate gyrus (measured 1 hour after exploration by
immunostaining). Gadd45a had already been demonstrated as a demethylase in human
cultured cells, so Gadd45b was a good candidate to investigate for demethylase activity
controlling neurogenesis induction. Intriguingly, the dramatic mRNA increase was not
seen for Gadd45a and Gadd45g in the hippocampus, implicating Gadd45b specifically in
Epigenetics and Memory 37
the ECT/exploratory response process. The Gadd45b induction process depends on the
NMDA receptor, as a NMDAR antagonist (CPP) administered in vivo before ECT
specifically blocked Gadd45b up-regulation. A cell proliferation assay with BrdU in the
dentate gyrus also showed that shRNA-induced knockdown of Gadd45b abolished
normal ECT-dependent proliferation. Finally, the frequency of methylation at individual
CpG sites of BDNF IX and FGF-1B increased to control levels in Gadd45b KO mice
after ECT. The methylation increase was accompanies by decreased dendritic length of
samples in the dentate gyrus (Ma et al., 2009). Since Gadd45b modulates neurogenesis
and learning-related gene expression with demethylase activity, it will be the subject of
more extensive research in the coming years. It is an exciting result because of its
potential to give neural DNA methylation research another avenue for therapeutic
intervention (similar to HDAC inhibition). This could be an important advance in
treatment of Alzheimer’s disease, as age-specific DNA methylation pattern alterations
have already been reported in late-onset AD patients (Wang, Oelze, & Schumacher,
2008). Normal aging even correlates with loss of genomic 5-methyldeoxycytidine
(Wilson, Smith, Ma, & Cutler, 1987).
Epigenetics and Memory 38
CHAPTER FIVE
Challenges and Future Directions
Given that epigenetics is a relatively new discipline, the literature is currently
somewhat vague. For instance, most epigenetics of memory studies incorporate HDACs
like sodium butyrate, which modulates expression of a large number of genes in a
shotgun attempt to increase those that actually help memory. However, scientists do not
fully understand what all of these genes do—or in other words why increased expression
of a set of genes results in enhanced performance for mice in a fear conditioning task.
And getting to that point will take time. It will require advanced knowledge of very
specific HDACs, HDAC inhibitors, HATs, DNMTs, etc.—knowledge that could require
decades of research and substantial funding. Targeting specific histone residues to
improve memory is a daunting task in itself, not to mention figuring out which ones
improve memory without deleterious side-effects. That is not to say it is a far-fetched
idea, though. EnVivo Pharmaceuticals recently announced that a drug similar in structure
to SAHA reached phase 2 clinical trials for treatment of Alzheimer’s, according to
private correspondence with one of their consultants, David Sweatt. Given the current
intrigue with results like Fischer et al. (2007), in which the memory-related symptoms of
neurodegeneration were greatly decreased despite the atrophy, funding may soon be
easier to acquire for this research. After all, clinical implications this compelling have
certainly received attention from governments seeking decreased medical care costs for
the elderly and start-up companies looking for a panacea. While some clinical trials with
HDACs have already failed in phase 3, it may only be a matter of time before the right
molecules are tested if current mouse models are truly revelatory; the appeal of HDACs
Epigenetics and Memory 39
or HATs for the treatment of diseases may not just be hype. Sananbenesi and Fischer
(2009) argue that many inherited and sporadic epigenetic deregulations in brain diseases
seem causally involved in disease progression unlike any other potential treatment
options. This could be why HDAC inhibitors have both neuroprotective and
neurodegenerative potential—epigenetics might be the key bottleneck to understanding
these diseases. Though the “histone code” is not nearly as well understood as DNA’s
traditional “code,” deciphering it may be challenging and exciting for neuroscience.
Indeed, there is even debate about whether the epigenetic marks outlined above integrate
to form a histone code at all. Beyond this problem, there are few studies indicating that
lifelong memories could be stored by such a histone code. The first studies suggesting
this possibility relate long term epigenetic changes to early life stress and nurturing
(Roth, Lubin, Funk, & Sweatt, 2009; Weaver et al., 2004). Still, it will take more research
to fully answer the question of how lifelong memories are stored. Epigenetic states will
even differ between brain regions to integrate behavior, and understanding this could take
years of research as well (Day & Sweatt, 2011). Epigenetic modifications could very well
act independently to affect gene expression.
Another interesting implication of neuroepigenetics is its challenge to
physiological psychology’s doctrine of Hebbian learning—“cells that fire together wire
together.” While it may be true that interactions at thousands of synapses for each cell
create neural circuits, epigenetics would require some rethinking of the initial hypothesis.
Chemical changes in the nucleus of one cell could completely silence genes involved in
plasticity, and thus shut down the neural circuit. So there could be at least two
Epigenetics and Memory 40
mechanisms at play in the process of behavioral plasticity—epigenetic and Hebbian
(Roth & Sweatt, 2009).
Regardless of these challenges, one thing remains clear: epigenetic manipulation
is one of the most promising avenues for therapeutic intervention in Alzheimer’s right
now. An epigenetic perspective could bridge the gap between known genetic
predispositions for Alzheimer’s and the necessary cellular events that trigger it over time.
Epigenetics and Memory 41
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