J Mol Neurosci
DOI 10.1007/s12031-015-0665-8
Liver X Receptor Agonist Modifies the DNA Methylation Profile
of Synapse and Neurogenesis-Related Genes in the Triple
Transgenic Mouse Model of Alzheimer’s Disease
A. G. Sandoval-Hernández 1 & H. G. Hernández 4 & A. Restrepo 1 & J. I. Muñoz 2 &
G. F. Bayon 3 & A. F. Fernández 3 & M. F. Fraga 3 & G. P. Cardona-Gómez 2 & H. Arboleda 4 &
Gonzalo H. Arboleda 1,5
Received: 19 March 2015 / Accepted: 8 October 2015
# Springer Science+Business Media New York 2015
Abstract The liver X receptor agonist, GW3965, improves
cognition in Alzheimer’s disease (AD) mouse models. Here,
we determined if short-term GW3965 treatment induces chang-
es in the DNA methylation state of the hippocampus, which are
associated with cognitive improvement. Twenty-four-month-
old triple-transgenic AD (3xTg-AD) mice were treated with
GW3965 (50 mg/kg/day for 6 days). DNA methylation state
was examined by modified bisulfite conversion and hybridiza-
tion on Illumina Infinium Methylation BeadChip 450 k arrays.
The Morris water maze was used for behavioral analysis. Our
results show in addition to improvement in cognition methyla-
tion changes in 39 of 13,715 interrogated probes in treated
3xTg-AD mice compared with untreated 3xTg-AD mice.
A. G. Sandoval-Hernández and H. G. Hernández contributed equally to
this work.
Electronic supplementary material The online version of this article
(doi:10.1007/s12031-015-0665-8) contains supplementary material,
which is available to authorized users.
* Gonzalo H. Arboleda
gharboledab@unal.edu.co
1
Grupo de Muerte Celular, Instituto de Genética, Universidad
Nacional de Colombia, Bogotá, Colombia
2
Área de Neurobiología Celular y Molecular, Grupo de Neurociencias
de Antioquia, Universidad de Antioquia, Medellín, Colombia
3
Cancer Epigenetics Laboratory, Institute of Oncology of Asturias
(IUOPA), Hospital Universitario Central de Asturias (HUCA),
Universidad de Oviedo, Oviedo, Spain
4
Grupo de Neurociencias, Universidad Nacional, Bogotá, Colombia
5
Departamento de Patología, Facultad de Medicina, Universidad
Nacional de Colombia, Bogotá, Colombia
These changes in methylation probes include 29 gene loci.
Importantly, changes in methylation status were mainly from
synapse-related genes (SYP, SYN1, and DLG3) and
neurogenesis-associated genes (HMGB3 and RBBP7). Thus,
our results indicate that liver X receptors (LXR) agonist treat-
ment induces rapid changes in DNA methylation, particularly
in loci associated with genes involved in neurogenesis and syn-
aptic function. Our results suggest a new potential mechanism
to explain the beneficial effect of GW3965.
Keywords Alzheimer’s disease . Liver X receptors . Triple
transgenic mice . GW3965
Introduction
Alzheimer’s disease (AD) is an age-dependent neurodegenera-
tive disorder and a major public health problem. The hippocam-
pus is involved in learning and memory and is the earliest
affected site in AD (Braak et al. 1993). The dentate gyrus of
the hippocampus plays an essential role in normal hippocampal
function throughout life, and is one of the main areas for adult
neurogenesis, which is affected in AD (Ming and Song 2011).
DNA methylation is an important epigenetic modification
of the genome that occurs on cytosine residues at carbon 5 of
the pyrimidine ring of simple sequences termed CpG dinucle-
otides and subsequently controls gene expression. DNA meth-
ylation at CpG dinucleotides and 2 kb upstream and down-
stream from CpG dinucleotides is strongly associated with
stable transcriptional repression (Jones 2012). Thus, DNA
methylation controls expression of many genes and is in-
volved in important cellular processes including embryonic
development, chromatin structure, and X chromosome inacti-
vation. Recently, a growing number of reports have shown

aberrant changes in DNA methylation patterns in many hu-
man diseases (Coppieters et al. 2014). In the human AD brain,
two independent analyses of genomic DNA methylation (the
Bmethylome^) showed variations in the methylation pattern
compared with controls, which correlated with the pathologi-
cal stage of the disease (De Jager et al. 2014; Masliah et al.
2013). In addition, similar changes in the methylation pattern
were also observed in the brain of AD mouse models
(Hernandez et al. 2014; Sanchez-Mut et al. 2013; Barrachina
and Ferrer 2009).
Liver X receptors (LXR) belong to the nuclear receptor
superfamily that regulates DNA transcription (Wojcicka
et al. 2007). Transcriptional activity of LXR is regulated by
binding to its natural ligands, most of which are oxygenated
metabolites of cholesterol (i.e., oxysterols) (Mitro et al. 2007),
and by interaction with additional co-regulatory proteins.
Some of these co-regulatory proteins directly regulate epige-
netic changes, including the SWItch/Sucrose Non-Fermentable
chromatin-remodeling factors that induce changes in DNA
methylation and promote transcriptional activation (Kim et al.
2012; O’Malley et al. 2008; Rosenfeld et al. 2006; Banine et al.
2005). However, nuclear receptor function, and in particular
their control over DNA methylation during brain development,
in adulthood, and their impact on human diseases remain poor-
ly understood (Otaegui-Arrazola et al. 2010). LXR can be
pharmacologically modulated by synthetic agonists such as
GW3965, a selective agonist for LXRα (EC50 = 190 nM)
and LXRβ (EC50 = 30 nM) (Collins et al. 2002) that can
induce LXR activation in the brain because of its ability to
cross the blood–brain barrier (Xu et al. 2013; Donkin et al.
2010; Fitz et al. 2010).
Recently, LXR activation was shown to improve cognitive
alterations based on its ability to regulate gene expression and,
in particular, apolipoprotein E (ApoE) and ATP-binding cas-
sette transporter (ABCA1), in mouse models of AD (Donkin
et al. 2010; Jiang et al. 2008). Moreover, LXR knockout mice
on an amyloidogenic mouse model background develop a
severe amyloid-β (Aβ) pathology (Zelcer et al. 2007), sug-
gesting that LXR is an important mediator of Aβ metabolism
and a plausible therapy for AD.
To identify novel potential mechanisms that play a role in
cognitive improvement following LXR treatment in a murine
model of AD, we examined the effect of short-term treat-
ment of GW3965 in the triple transgenic mouse model of
AD (3xTg-AD), using a microarray platform to compare
DNA methylation changes in the hippocampus of treated
and untreated mice. We found that cognitive improvement
conferred by the LXR agonist was associated with broad
changes in the DNA methylation pattern of 39 (of 13,715)
interrogated probes in treated compared with untreated
3xTg-AD mice. Interestingly, the most important changes
were observed in synapse (SYP, SYN1, and CASK)- and
neurogenesis (HMGB3 and RBBP7)-related genes.
J Mol Neurosci
Materials and Methods
Animals Triple-transgenic AD (3xTg-AD) mice (Oddo et al.
2003) and wild-type (WT) mice from the house colony from
the species pathogen-free animal facility of Sede de
Investigación Universitaria at the University of Antioquia
(Medellín-Colombia) were used. Animals were kept in a 12-
h dark/light cycle and received food and water ad libitum. A
total of 24 3xTg-AD mice (24-month-old) and 7 WT mice
(24-month-old) were used. Female animals were randomly
distributed within the groups. Animals were treated orally
everyday with the LXR agonist GW3965 (50 mg/kg) or with
vehicle (dimethyl sulfoxide; 20 mg/mL) for six consecutive
days, in groups of five animals in regular cages. Animals
were handled following the Colombian (Law 84 of 1989
and Resolution 8430 of 1993) and international regulations
and standards on animal welfare and conformed to the An-
imal Research: Reporting In Vivo Experiments (ARRIVE)
guidelines (Kilkenny et al. 2010).
Morris Water Maze Hippocampal-dependent spatial memo-
ry was examined using the water maze paradigm (Morris et al.
1982). The apparatus consisted of a white pool (100 cm di-
ameter and 54 cm high). The tank was filled to a depth of
35 cm with opaque water with non-toxic white gouache paint,
which remained at a constant temperature (22 ± 2 °C). The
platform (10 cm diameter) was located 1.5 cm below the
water surface during spatial learning and 1 cm above the
water surface during the visible session. Extra-maze visual
cues around the room remained in fixed positions through-
out the experiment.
Initial Spatial Training The aim was for the mice to acquire a
learning task, in which they were trained to find the platform,
within two daily sessions for 5 days. Each session consisted of
four successive trials, and each trial began with the mouse
placed pseudo-randomly in one of four starting positions. Be-
fore the initial trial, the mice were trained to stay for 30 s on
the platform. Forty-eight hours after the learning phase, the
animals were tested for retention during a 90-s probe trial
without the platform. During the probe trial, the latency to
reach the exact platform location was determined. To control
for any difference between experimental groups in visual–
motor abilities or motivation, latencies to reach the platform
were determined using a visible platform (four trials) at the
end of the retention phase. Behavior was recorded with an
automated system (Viewpoint, Lyon, France).
DNA Extraction and DNA Methylation Microarrays Mice
were anesthetized with ketamine/xylazine, the brains ex-
tracted, manually dissected, and immediately frozen in liq-
uid nitrogen for DNA isolation using phenol-chloroform,
as previously described (Masliah et al. 2013). To identify
J Mol Neurosci
global DNA methylation changes, the Illumina Infinium
450 k DNA Methylation assay (Illumina, San Diego, CA,
USA) was performed in three 3xTg-AD mice that received
GW3965 and three mice that received vehicle. The 450 k
protocol was performed at the Genome Quebec Facilities
(Montreal, Canada), according to the manufacturer’s pro-
tocols and as previously described (Wong et al. 2013). In
brief, DNA conversion was performed using the EZ DNA
Methylation Kit (Zymo Research Corp., Orange, CA,
USA) and then subsequently hybridized to an Illumina
Infinium 450 k DNA Methylation BeadChip and scanned
(iScan, Illumina). Finally, the previously described adapta-
tions for data analysis of the mouse genome were used
(Wong et al. 2013). These adaptations are based on human
genome similarity and enable reliable evaluation of 13,715
probes in a mouse using the Illumina Infinium 450 k DNA
Methylation assay (Wong et al. 2013).
Histology of Aβ Plaques and Neurofibrillary Tangles Mice
were anesthetized with ketamine/xylazine and perfused
transcardially with 4 % paraformaldehyde in phosphate-
buffered saline, then the brains were removed and transferred
into 30 % sucrose. Brains were cut into 50-μm coronal sec-
tions using a vibratome (Leica VT 1000S, Leica, Nussloch,
Germany) and stored at −20 °C in anti-freezing medium until
further processing. For diaminobenzidine (DAB) staining,
sections were pretreated for 20 min at room temperature in
0.1 M phosphate buffer (PB) (pH 7.4) with methanol (PB/
methanol 1:1) and 1 % hydrogen peroxide (to block endoge-
nous peroxidase). Afterwards, sections were rinsed and incu-
bated for 1 h in 0.1 M PB with 1 % BSA and 0.3 % Triton
X-100. Sections were incubated with primary antibodies in the
same buffer overnight at 4 °C. Primary antibodies that rec-
ognized phosphorylated tau at S202/205 in mice and
humans (Biernat et al. 1992) (AT8, 1:500; Thermo Scien-
tific, Rockford, IL, USA) and 6E10 of human APP/Aβ
(SIG-39320; Signet/Covance, Dedham, MA, USA) were
used. Subsequently, sections were incubated with a bio-
tinylated mouse secondary antibody and then ABC–HRP
complex (Pierce Biotechnology; Rockford, IL, USA) for
2 h. Detection using DAB was performed. Sections were
dehydrated and covered with mounting solution and ob-
served using a zoom microscope (Axio-Zoom-V16, Zeiss,
Munich, Germany). Images were acquired using the Zeiss
Axio Cam HRc digital color camera (1388 × 1040 pixels)
with ZEN pro software (Zeiss). For each captured image,
semi-quantitative analysis of appropriate areas was per-
formed by segmentation analysis using ImageJ software
(National Institutes of Health, Bethseda, MD, USA). Areas
identified as positive for 6E10 or AT8 immunoreactivity
after thresholding were individually inspected to confirm
the presence of plaques or hyperphosphorylated tau. The
mean area value was averaged per treatment in each
genotype group for the number of animals per group, as
indicated in the figure legends. For group comparisons,
statistical analysis was performed by analysis of variance
with Bonferroni post test. Thioflavin-S (Thio-S) staining
was assessed as described previously (Ly et al. 2011). In
brief, slides were rinsed consecutively in ethanol (70 then
80 % for 1 min each). Next, slides were rinsed with dis-
tilled water twice and covered with glycerol jelly. Images
were captured by confocal fluorescence microscopy (×20
magnification) (Nikon Eclipse C1 plus; Nikon Instruments
Inc., Melville, NY, USA).
Western Blotting Following pharmacological treatment, all
animals received behavioral training and then the brains were
extracted and manually dissected. Hippocampal tissue was
homogenized in 300 μL ice-cold RIPA buffer (Pierce Biotech-
nology) containing complete protease inhibitor cocktail tab-
lets and phosphatase inhibitor cocktail tablets (Complete,
PhosSTOP; Roche Applied Science, Mannheim, Germany)
using a PRO Scientific homogenizer (Oxford, CT, USA) at
full speed for 20 s. Homogenates were sonicated at 30 %
output for 10 s, then centrifuged (12,500 g) at 4 °C for
45 min in a microcentrifuge (Thermo Scientific). The protein
concentration of supernatant fractions was determined using
the BCA protein assay kit (Thermo Scientific) and 20 μg of
protein samples run on polyacrylamide gels at 100 V. After
electrophoresis, proteins were transferred to polyvinylidene
fluoride (PVDF) membranes (Amersham Hybond™-P; GE
Healthcare, Buckinghamshire, UK) and incubated in a
blocking buffer (5 % powder milk in TBS-Tween 20) for 1 h
at room temperature until probed. PVDF membranes were
then incubated with 5 mL anti-synapsin-I antibody, (1:1000,
ab64581; Abcam, Cambridge, UK) in a blocking buffer over-
night at 4 °C, washed three times in TBS-Tween (5 min/wash),
followed by incubation with a peroxidase conjugated second-
ary antibody for 1 h (1:2000) and then washed three times
with TBS-Tween. Bound antibody was detected using the
ECL system (Thermo Scientific).
Results
GW3965 Treatment Restores Cognitive Deficits
in 3xTg-AD Mice
We initially examined the effect of LXR agonist treatment on
cognition using the Morris water maze (WMW) task to deter-
mine if short-term LXR activation contributes to improvement
of spatial learning and memory in 3xTg-AD mice. In the
learning task (Fig. 1a), WT mice performed significantly bet-
ter than untreated 3xTg-AD mice on 5-day trials (P < 0.001).
Treating 3xTg-AD mice with GW3965 resulted in no signif-
icant difference with WT. In the retention task, which was

Fig. 1 LXR agonist restores memory and cognition in 3xTg-AD mice.
Spatial learning and memory were examined using the MWM after 6 days
of treatment with the LXR agonist GW3965. a Learning task: treated
3xTg-AD mice took significantly less time to learn the location of the
hidden platform. Statistically significant differences were detected by
analyzed in a 4× proximity design (Fig. 1b), WT animals spent
significantly more time near the platform than untreated 3xTg-
AD (P < 0.001). Further, GW3965-treated 3xTg-AD mice
spent significantly more time near the platform than untreated
3xTg-AD (P < 0.01) and were not significantly different from
WT mice. These results indicate that in 3xTg-AD mice,
GW3965 improves hippocampal-dependent behavior tested
by the MWM.
GW3965 Treatment Does Not Modify the Pathological
Hallmarks of AD
To determine if LXR activation induces changes in amyloid
plaque load and phosphorylated tau, and also if these changes
are associated with the observed cognitive improvement, we
performed immunohistochemistry using DAB detection. No
significant differences in positive immunoreactivity for anti-
Aβ (6E10) or anti-PHF1 (AT8) were observed between treat-
ed and untreated 3xTg-AD mice (Fig. 2).
DNA-Methylation Data Analysis
Hippocampal DNA was analyzed using Illumina Infinium
450 k DNA Methylation microarrays adjusted for the mouse
genome. A previous report found that only a subset of 13,715
probes bind identically to genetic sequences within the mouse
genome (Wong et al. 2013). We used the minfi Bioconductor
R package (Aryee et al. 2014) to import and normalize raw
data intensities. DNA methylation density plots in mouse
samples (Fig. 3a) showed the same distribution as in hu-
man samples (Fig. 3b), as previously reported (Wong et al.
2013). To obtain reliable intensities, we excluded probes
with detection values of P > 0.001. After subset-quantile
within array normalization (SWAN) (Aryee et al. 2014),
robust Bayesian analysis was performed using the Limma
Bioconductor R package (Teschendorff et al. 2013) to
J Mol Neurosci
two-way analysis of variance (ANOVA). ***P < 0.001. b Retention tasks
by 4× analysis: time spent in the area near the platform (in sec). Signif-
icant differences were detected by ANOVA followed by Bonferroni mul-
tiple comparison test
identify differentially methylated probes. Beta mean bar
plots did not show group differences in DNA methylation
patterns for the probes evaluated (Fig. 3c).
To identify differentially methylated probes, we used the
multiple testing correction, Benjamini–Hochberg false dis-
covery rate, as well as a delta beta filter of 0.05 (Table 1).
We detected 47 differentially methylated probes
(P < 0.0003) with P values corresponding to the false discov-
ery Benjamini–Hochberg correction (P < 0.1). We observed a
decrease in DNA methylation for a specific subset of probes,
as shown in the density plot (Fig. 3d). In addition, we exclud-
ed results with beta value differences less than 10 % (delta
beta filter of 0.1). Thus, overall, we identified 39 probes, cor-
responding to 29 genes, which were differentially methylated
(Table S1). Classification and function of each differentially
methylated gene were performed by an extensive PubMed
literature search. The differentially methylated genes were
found to be particularly related to synaptic function, prolifer-
ation, and neurogenesis (Tables 1 and 2).
GW3965 Treatment Restores Expression of Specific
Synapse-Related Genes
We found that compared with untreated 3xTg-AD mice,
GW3965 treatment of 3xTg-AD mice promotes significant
hypo-methylation of synapse-related genes (Table 1). These
genes include synapsin-1, synaptophysin, and synapse-
associated protein 102 (SAP-102), expression of which has
previously been shown to be decreased in human AD brain
(Proctor et al. 2010; Qin et al. 2004). Thus, we next deter-
mined if the DNA methylation changes observed following
GW3965 treatment of 3xTg-AD mice play a regulatory role in
expression of two specific synaptic proteins: synapsin-1
(shows decreased DNA-methylation) and postsynaptic densi-
ty protein 95 (PSD95) (no change in DNA methylation).
GW3965 treatment significantly restored expression of
J Mol Neurosci
Fig. 2 GW3965 treatment does not produce detectable changes in
histopathological AD hallmarks. Amyloid plaque deposition and hyper-
phosphorylated tau in brains of GW3965-treated 3xTg-AD mice exam-
ined by immunohistochemistry. Representative micrographs showing the
hippocampus. a Aβ immunohistochemistry was examined using anti-
body anti-Aβ (6E10). b Hyper-phosphorylated tau was examined using
antibody anti-PHF1 (AT8). No significant differences were observed in
synapsin-1 protein in 3xTg-AD mice compared with untreated
3xTg-AD mice (P > 0.01, n = 4) (Fig. 4a, c) but did not change
PSD95 expression (Fig. 4a, b).
Discussion
LXR activation is a promising therapeutic target for AD, as
demonstrated by its ability to improve cognitive function in
murine models of AD. Here, we investigated the potential
mechanisms associated with the beneficial effects exerted by
LXR agonist treatment in 24-month-old 3xTg-AD mice, a
murine model of AD. We used the 3xTg-AD animals at this
particularly old age, as they are likely to suffer from a
detrimental aging process that mediates gene silencing as
a consequence of the human transgenes. We treated this
model short term to identify the first DNA methylation
anti-Aβ- or PHF1-positive areas in the hippocampus or cortex of treated
3xTg-AD mice. No significant change in c Aβ-positive area or d neuro-
fibrillary tangles, was observed in the hippocampus of treated 3xTg-AD
mice. Data are expressed as mean ± SEM. Statistical analysis was per-
formed by one-way analysis of variance followed by Tukey’s multiple
comparison test. Female mice, n = 3 per 3xTg-AD group
changes that occur and that are potentially related to the
mechanism of action of this agonist.
First, we found that short-term GW3965 treatment
(50 mg/kg/day for 6 days) improved performance on learning
and retention tasks in treated 3xTg-AD compared with un-
treated 3xTg-AD mice. Similar behavioral results have been
previously reported using the same treatment in a contextual
fear-conditioning paradigm in the double transgenic AD
mouse model (Tg2576), which is a purely amyloidogenic
model (Jiang et al. 2008). Second, functional studies have
shown that LXR agonist treatment contributes to Aβ clear-
ance after 4 months of treatment (Jiang et al. 2008). However,
we did not find significant changes in the pathological hall-
marks of AD expressed in the 3xTg-AD mice model, most
probably owing to the short-term treatment, and more time
may be required to replicate the significant amyloid burden
reduction observed in the previous study (Jiang et al. 2008).
Moreover, transgene differences in the 3xTg-AD mice
 J Mol Neurosci

Fig. 3 Quality control. The beta

density plot after probe selection
in our assay (a) is similar to that
performed on the human sample
included (b). The average
methylation ratio is similar with
or without treatment (c).
Therefore, changes in
methylation derived from the
treatment appear to be specific for
genomic loci (rather than a
general effect). The volcano plot
illustrates that the epigenetic
differences are mainly due to
decreased DNA methylation in a
specific set of probes (d, left side)

Table 1 Differentially
methylated synapses genes Gene Probe code P value Related function References
Name

Dlg3 23429746 0.00007 Regulation of NMDA signals pathway; (Han et al. 2011; Qu et al.
role in synaptic plasticity; mutations in 2009; Proctor et al.
this gene are associated with X-linked 2010)
mental retardation; is significantly de-
creased human AD patients
Syn1 00779763 0.00014 Localization at cytoplasmic surface of (Qin et al. 2004)
synaptic vesicles; role in synaptogenesis
and the modulation of neurotransmitter
release; is significantly decreased in
humans AD patients
Syp 10818284 0.00011 Integral membrane protein present in small (Proctor et al. 2010; Sze
synaptic vesicles; is significantly et al. 1997)
decreased in humans AD patients
Gpc4 07312966 0.00002 Localized in pre- and postsynaptic mem- (de Wit et al. 2013;
branes of excitatory synapses; belongs to Decktor et al. 1990;
GPC family associated with Aβ plaques van Horssen et al.
2001)
Tspan7 cg04345928 0.00008 Role in the control of neurite outgrowth; (Wakabayashi et al.
regulatory protein of γ-secretase com- 2009)
plex and activity
Cask 19638040 0.00020 Scaffold protein, is located at synapses in (Moog et al. 1993)
the brain
Il1rapl1 13077484 0.00022 Mediates excitatory synapse formation; (Ramos-Brossier et al.
mutation cause intellectual disabilities 2015; Yasumura et al.
2014)
J Mol Neurosci

Table 2 Differentially
methylated neurogenesis and Gene Probe P value Related function References
proliferation genes Name codes

Sox-3 21773962 0.00006 Involve in embryonic and primary (Rogers et al. 2013;
neurogenesis and mental retardation Laumonnier et al.
2002; Archer et al.
2011)
Hmgb3 12284142 0.00007 Regulates hematopoietic stem cell self- (Nemeth et al. 2006)
25685741 0.00009 renewal and differentiation
Zfx 06019215 0.00020 Control the self-renewal of human (Harel et al. 2012)
embryonic stem cells
pp90RSK2 17794813 0.00025 Codifies for serine/threonine kinases that (Anjum and Blenis
regulates mitogen-activated kinase 2008; Carriere et al.
(MAPK); controlling growth, 2008)
proliferation and migration
Zic3 06915321 0.00022 Pluripotency regulator of embryonic stem (Declercq et al. 2013;
13613682 0.00009 cells; induce conversion of human Kumar et al. 2012;
fibroblast to neuronal progenitor cells Lim et al. 2010)

compared with the Tg2576 mice, and also the strong pa-
thology of the very old 3xTg-AD mice used here, may
have also contributed.
Third, the hippocampus is essential for cognitive function
and recent evidence indicates that learning and long-term
memory depend on histone modifications and dynamic
changes in DNA methylation (for a review see Day and
Sweatt 2011). These changes in DNA methylation can be
transitory (for hours) or long-lasting (persist for weeks). The
first evidence for transitory DNA methylation changes was
detected on CpG dinucleotides of the Reelin gene after
contextual fear conditioning (Miller and Sweatt 2007). In-
terestingly, Reelin and ApoE exert their effects by direct
interaction with ApoE receptors (ApoER2 and VLDLR)
and LXR agonists upregulate ApoE protein expression.
Similarly, persistent epigenetic changes have been described
for cortical DNA methylation of CpG dinucleotides of cal-
cineurin, a memory-suppressing gene (Miller et al. 2010). In
the context of AD, we found changes in DNA methylation of
synapse-related genes associated with LXR activation and
specifically within DNA regions that codify for proteins lo-
calized at synapses, either at the presynaptic terminal, e.g.,
synapsin-1 and synaptophysin, or the postsynaptic terminal,
e.g., SAP-102. These genes are reduced in the postmortem
brain from AD patients (Proctor et al. 2010; Qin et al. 2004;
Sze et al. 1997) and show decreased methylation following
GW3965 treatment in the present analysis. Because changes
in gene expression are frequently reported to be necessary
for synaptic plasticity, which is one of the main mecha-
nisms associated with learning and memory (Flavell and
Greenberg 2008), it is interesting that LXR activation by
GW3965 treatment rapidly causes increased transcriptional

Fig. 4 GW3965 increases protein expression of synapsin-1 but not
PSD95. Hippocampal levels of synapsin-1 and PSD95 were examined
by Western blot analysis. a Representative Western blot images of
synapsin-1 (Syn1), PSD95, and β-actin in the hippocampus of three
different groups of animals: WT, GW3965-treated 3xTg-AD, and untreat-
ed 3xTg-AD mice. b Densitometric quantification of PSD95 Western blot
results in hippocampal lysates. c Densitometric analysis of Syn1 Western
blot results in hippocampal lysates. Samples from each mouse were nor-
malized to actin and expressed as fold change. Data are expressed as
mean ± SEM. Statistical analysis was performed by one-way analysis
of variance and Student’s t test comparison between groups, n = 4 per
group. **P < 0.01 compared with WT; &&P < 0.01 compared with un-
treated 3XTg-AD

activity of synapse-related proteins and appears to counter-
act the epigenetic modifications mediated by the patholog-
ical condition. Fourth, we also found changes in DNA
methylation of genes that regulate proliferation and are
most likely associated with regulating neuronal precursors
in the subgranular zone of the hippocampus (Levison and
Goldman 1993; Paterson et al. 1973), a primary niche for
neuronal stem cells (NSC) and the site of adult neurogenesis.
Dysfunction in hippocampal neurogenesis is associated with
AD in different transgenic animal models, namely those ex-
pressing mutant presenilin-1 and mutant amyloid precursor
protein (APP) and in the 3xTg-AD (Rodriguez et al. 2008;
Donovan et al. 2006; Chevallier et al. 2005; Haughey et al.
2002). It has been suggested that control of NSC proliferation
either by extrinsic factors (Wnt3, Wnt10b, Wnt2, BDNF, and
Reelin) or intrinsic factors (Sox2, Sox4, Sox11, Ngn2,
NeuroD1, and NeuroD2) is dynamically regulated by epige-
netic modification of DNA or histones (Covic et al. 2010). In
agreement with this, LXR promotes neurogenesis in vitro in
human embryonic stem cells, and in vivo in the ventral mid-
brain during development (Theofilopoulos et al. 2013; Ma
et al. 2009; Sacchetti et al. 2009), supporting our findings
of changes in neurogenesis-related genes. Further analysis
on the impact of LXR agonists on NSC gene regulation
described in the present model are needed.
Interestingly, we also found changes in DNA methylation
in additional genes such as FMR2P, PTCHD1, RPS6KA3, and
SLC6A8 (Table S1), which are associated with X-linked men-
tal retardation and/or autism (Chaudhry et al. 2014; Bensaid
et al. 2009; Rosenberg et al. 2004; Merienne et al. 1999).
SLC6A8 is involved in creatine transport into cells and was
recently demonstrated to be required for energy control in the
brain and associated with impaired metabolism in different
neuronal disorders including AD (Burklen et al. 2006; Gallant
et al. 2006). However, the role of most of these genes on
synaptic function and development remains unclear, as well
as their role in AD physiopathology.
Previous research has shown cognitive improvement by
LXR agonist treatment with increased ABCA1 and ApoE
protein expression (Donkin et al. 2010; Jiang et al. 2008).
Both these genes are subjected to epigenetic regulation, and
ABCA1 epigenotypes are associated with familial hypercho-
lesterolemia, aging, and coronary artery disease (Guay et al.
2014; Guay et al. 2012). Regarding ApoE, a CpG dinucleotide
was found within the 3′-coding region of the gene, which was
associated with expression levels at the APOE locus. In addi-
tion, the CpG dinucleotide is highly methylated in the human
postmortem brain (Yu et al. 2013). More recently, methylation
of an APOE CpG dinucleotide was shown and associated to
the pathological process of AD (Chibnik et al. 2015). LXR
agonists are well known to increase ABCA1 gene expression
in murine double transgenic models of AD, which promotes
increasing proteolytic pathways of Aβ and mediates cognitive
J Mol Neurosci
improvement. However, the relationship mediated by LXR
and methylation changes at the ABCA1 loci are not known.
Unfortunately, the ApoE1 and Abca1 genes are not present in
the gene subset of the Illumina 450 k Infinium DNA Methyl-
ation microarray and therefore require a specific analysis ap-
proach (Wong et al. 2013).
In conclusion, we have demonstrated changes in the DNA-
methylation pattern mediated by short-term treatment with an
LXR agonist (GW3965) in the 3xTg-AD mouse model.
Previous reports have shown that LXR agonists produce
cognitive improvements with short-term treatment (Jiang et al.
2008) and also in 21-month-old animals (Vanmierlo et al.
2011) in a mechanism that is independent of Aβ plaques.
Our results suggest an alternative mechanism of action to ex-
plain the cognitive changes associated with short-term LXR
agonist treatment in AD mice models, namely by mediating
specific DNA hypo-methylation and re-expression of genes
related to synaptic function in the hippocampus, one of the
main affected regions in AD. We also observed changes in
neurogenesis-related genes, and both pathways are widely
described to have a role in the physiopathology and pro-
gression of AD.
Acknowledgments We thank Dr. Jon Collins (GlaxoSmithKline,
Stevenage, UK) for providing GW683965A. This study was funded by
Colciencias (Contract Nos. 401-2011 and 498-2012).
Compliance with Ethical Standards Animals were handled following
the Colombian (Law 84 of 1989 and Resolution 8430 of 1993) and
international regulations and standards on animal welfare and conformed
to the Animal Research: Reporting In Vivo Experiments (ARRIVE)
guidelines (Kilkenny et al. 2010).
Conflict of Interest The authors declare that they have no competing
interests.
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