Lead Induces Similar Gene Expression Changes in Brains
of Gestationally Exposed Adult Mice and in Neurons
Differentiated from Mouse Embryonic Stem Cells
Francisco Javier Sánchez-Martín1, Yunxia Fan1, Diana M. Lindquist2, Ying Xia1, Alvaro Puga1*
1 Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati, College of Medicine, Cincinnati, Ohio, United States of
America, 2 Cincinnati Children's Hospital Medical Center, Department of Radiology, Cincinnati, Ohio, United States of America
Exposure to environmental toxicants during embryonic life causes changes in the expression of developmental genes
that may last for a lifetime and adversely affect the exposed individual. Developmental exposure to lead (Pb), an
ubiquitous environmental contaminant, causes deficits in cognitive functions and IQ, behavioral effects, and attention
deficit hyperactivity disorder (ADHD). Long-term effects observed after early life exposure to Pb include reduction of
gray matter, alteration of myelin structure, and increment of criminal behavior in adults. Despite growing research
interest, the molecular mechanisms responsible for the effects of lead in the central nervous system are still largely
unknown. To study the molecular changes due to Pb exposure during neurodevelopment, we exposed mice to Pb in
utero and examined the expression of neural markers, neurotrophins, transcription factors and glutamate-related
genes in hippocampus, cortex, and thalamus at postnatal day 60. We found that hippocampus was the area where
gene expression changes due to Pb exposure were more pronounced. To recapitulate gestational Pb exposure in
vitro, we differentiated mouse embryonic stem cells (ESC) into neurons and treated ESC-derived neurons with Pb for
the length of the differentiation process. These neurons expressed the characteristic neuronal markers Tubb3, Syp,
Gap43, Hud, Ngn1, Vglut1 (a marker of glutamatergic neurons), and all the glutamate receptor subunits, but not the
glial marker Gafp. Importantly, several of the changes observed in Pb-exposed mouse brains in vivo were also
observed in Pb-treated ESC-derived neurons, including those affecting expression of Ngn1, Bdnf exon IV, Grin1,
Grin2D, Grik5, Gria4, and Grm6. We conclude that our ESC-derived model of toxicant exposure during neural
differentiation promises to be a useful model to analyze mechanisms of neurotoxicity induced by Pb and other
Citation: Sánchez-Martín FJ, Fan Y, Lindquist DM, Xia Y, Puga A (2013) Lead Induces Similar Gene Expression Changes in Brains of Gestationally
Exposed Adult Mice and in Neurons Differentiated from Mouse Embryonic Stem Cells. PLoS ONE 8(11): e80558. doi:10.1371/journal.pone.0080558
Editor: Xianglin Shi, University of Kentucky, United States of America
Received September 15, 2013; Accepted October 15, 2013; Published November 19, 2013
Copyright: © 2013 Sánchez-Martín et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants 5 R01 ES010807 and R21 ES020048. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: Alvaro.Puga@uc.edu
Lead, a non-biodegradable metal, is one of the most
ubiquitous persistent toxicants present in the environment.
Unlike other heavy metals, like iron, copper or manganese,
lead has no known biological functions , but, because of its
physico-chemical properties of high malleability, ductility,
softness, low melting point and resistance to corrosion, it has
many industrial uses, including manufacturing of pipes, lead-
based paints, ceramic glazes, batteries, pottery, and
Occupational and accidental lead poisoning have been
described since the time of the ancient Romans [2,3]. Pb
inhibits detoxification enzymes,
cholesterol metabolism, causing
dysfunction, hypertension and encephalopathy [4–6], and
several reproductive system problems, including reduction of
libido, delay in puberty and infertility [3,7,8]. Chronic and low-
dose exposure to Pb during prenatal life and early childhood
damages the CNS. Solid epidemiological evidence has linked
Pb exposure during early childhood to deficits in cognitive
functions and IQ, behavioral effects, and attention deficit
hyperactivity disorder (ADHD) [9–11]. Importantly, early life
exposure to Pb can have remote effects later in life, producing
persistent injury in adults, including gray matter volume loss in
prefrontal cortex [12,13]. Other long-term consequences of
childhood lead exposure include changes of myelin structure in
white matter  and low level of activation in brain areas
associated with language function, such as left frontal cortex
and left middle temporal gyrus . Neurochemically, lead
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decreases the brain concentration of important metabolites,
such as N-acetyl aspartate,
phosphocreatinine, and a composite of glutamate and
glutamine . Behaviorally, blood lead levels during childhood
have been correlated to an increase of criminal behavior in
adult life [9–11,16]. Altogether, these data suggest that lead
exposure during early life may produce irreversible neuronal
dysfunction and reorganization that last into adult life.
There is a significant body of evidence in support of this
contention. Twenty-three years after exposure to Pb during
infancy, Macaca fascicularis monkeys showed elevated
expression of Alzheimer disease-related genes, including β-
amyloid precursor protein (APP) and β-site APP cleaving
enzyme 1 (BACE1), and an increase of total amyloid plaques in
the cortex . Significantly, the aging brains of these monkeys
that were exposed to lead as infants also showed changes in
the epigenetic machinery, with a reduction in total DNA
methylation, DNA methyltransferases-1 and -3A, and methyl
CpG binding protein-2 levels, and modifications of histone
marks critical for the regulation of gene expression [17,18].
Similar results have been observed in the cortical region of
rodents, in which early life-Pb exposure altered gene
expression patterns and global methylation profiles [19,20], and
in zebrafish (Danio rerio), where Pb altered neurological
development pathways and caused neurotoxicity [21,22].
Recent studies using primary neuronal cultures indicate that
lead exposure during childhood may negatively modify
important neuronal pathways implicated in the late effects
observed during adult life, especially affecting pathways
implicated in synaptic plasticity, learning, memory, and cell
survival, including modification of the N-Methyl-D-Aspartate
(NMDA) receptor architecture [23,24], changes of the activity of
Ca2+/calmodulin dependent protein kinase II (CaMKII),
phosphorylation of transcription factor CREB, and expression
and translocation of brain-derived neurotrophic factor (BDNF)
[25,26]. Pb exposure also represses the expression of
presynaptic vesicular proteins implicated in neurotransmitter
release, such as synaptobrevin (VAMP1) and synaptophysin
(SYN), while it increases p75 neurotrophin receptor (p75(NTR))
levels and alters TrkB-p75(NTR) colocalization in glutamate
In this study we have initiated a test of the hypothesis that
the Pb-induced alterations that occurs during neural
development may be responsible for the behavior and cognitive
impairment observed in adult life. We exposed mice to Pb in
utero and found that these mice presented alterations in the
expression of several genes, including neural markers,
neurotrophins, transcription factors, and glutamate-related
genes. We also induced mouse ESCs to differentiate into
neurons and treated them with Pb during the differentiation
process and observed that some of the changes induced by Pb
in the brains of mice were also found in vitro. This in vitro
model may serve as a powerful tool to study the mechanisms
of lead and other environmental neurotoxicants.
Materials and Methods
Mice and lead exposure
C57BL/6J mice were housed in the vivarium at Cincinnati
Children's Hospital Medical Center under controlled conditions
of temperature, humidity, and lighting, and provided standard
mouse chow and water ad libitum. All experimental procedures
conducted with these animals have been approved by the
University of Cincinnati and Cincinnati Children's Animal Care
and Use Committees. Female mice were exposed to 0 or 3
ppm of lead acetate in drinking water from 8 weeks prior to
mating, through gestation and until postnatal day PD10. This
treatment regimen insured steady state lead levels in the dam
at the time of gestation. From each treatment group, brain
tissues of 3 male offspring, including cortex, hippocampus, and
thalamus were collected at PD60.
Mouse embryonic stem cells culture, in vitro
differentiation and treatments
Undifferentiated C57BL/6-C2 mouse mESC  were
cultured in ES medium, consisting of Dulbecco’s modified
Eagle’s medium (DMEM, Gibco) supplemented with 15% (v/v)
ESC qualified fetal bovine serum (knockout serum replacement
FBS, Gibco), 2 mM L-glutamine (Gibco), 0.1 mM 2-β-
mercaptoethanol (βME, Gibco), 0.1 mM non-essential amino
acids (NEAA, Gibco), 1 mM sodium pyruvate (Gibco), 100 U/ml
penicillin (Gibco), 100 μg/ml streptomycin (Gibco), and 1000
U/ml leukemia inhibitory factor (LIF, Millipore). Cells were
plated in 0.1% (w/v) gelatin-coated dishes at 37°C in a
humidified atmosphere with 5% CO2, and passaged every two
days. Neural differentiation followed protocols previously
described by others . Briefly, to obtain cellular aggregates
(CA), 4 x 106 undifferentiated mESCs were plated in
bacteriological dishes for 8 days using CA medium, consisting
of DMEM supplemented with 10% fetal bovine serum (FBS,
Gibco), 2 mM L-glutamine, 550 nM βME, 0.1 mM NEAA, 100
U/ml penicillin, and 100 µg/ml streptomycin. On day 4, the CA
medium was made to contain 5 μM retinoic acid (Sigma-
Aldrich). To obtain a cellular suspension, cells were trypsinized
and plated at 2 x 105 cells/cm2 in 24-well or 6-well plates
coated with poly-DL-ornithine (Sigma-Aldrich) and laminin
(Roche), using N2 medium, consisting of a 1:1 mixture of
DMEM and DMEM nutrient mixture F-12 ham (Sigma-Aldrich)
supplemented with 2 mM L-glutamine, 50 μg/ml BSA, 100 nM
putrescine, 20 nM progesterone, 30 nM sodium selenite, 25
μg/ml insulin, 25 μg/ml transferrin, 100 U/ml penicillin, and 100
μg/ml streptomycin. After 48 hours, the N2 medium was
replaced with complete medium, consisting of DMEM
supplemented with 2 mM L-glutamine, 50 μg/ml BSA, 1X
serum-free medium supplement B27 (GIBCO), 30 nM sodium
selenite, 25 μg/ml insulin, 25 μg/ml transferrin, 100 U/ml
penicillin, and 100 μg/ml streptomycin. The cells were
maintained at 37°C in a humidified atmosphere with 5% CO2.
When needed, cells were treated with lead acetate (Sigma-
Aldrich) at different concentrations for the duration of neural
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Cell lysates and Western immunoblot analysis
Western immunoblots were performed as previously
described . Briefly, cells were lysed by sonic disruption on
ice in NETN buffer. Samples were cleared from debris by
centrifugation at 14000 rpm for 15 minutes. Protein
concentration of the supernatants was determined by the
Bradford assay. Tissue samples were homogenized prior to
lysis using an Ultra-Turrax T25 homogenizer (Janke& Kunkel,
Ika® Labortechnik). Proteins were separated in 8% SDS-
polyacrylamide gels and transferred to polyvinylidene fluoride
(PVDF) membranes by electroblotting. Immunoblots were
probed with antibodies specific for neuronal β3 tubulin (TUBB3)
(Abcam), glial fibrillary acidic protein (GFAP) (Millipore) and β-
actin (Sigma-Aldrich) for 2 hours at room temperature in TBS-T
buffer containing 5% (w/v) non-fat milk. After washing, blots
were incubated with the appropriate horseradish peroxidase-
modified secondary antibodies in TBS-T for 1 hour at room
temperature. Protein bands were visualized by incubation with
chemiluminescent PicoWest Super Signal (Pierce) and
exposure to X-Ray film. Band intensity was evaluated using
Cellular aggregates were dissociated with trypsin and the
cells were grown on coverslips at 2 x 105 cells/cm2. After 3
days in culture, cells, termed 3-DIV cells for 3-days-in-vitro,
were fixed with 4% paraformaldehyde for 15 minutes at room
temperature, washed twice with PBS and cells were
permeabilized and blocked in a blocking solution containing
0.05% (v/v) Triton X-100 and 2% (w/v) BSA in PBS. Primary
antibodies were incubated in blocking solution for 2 hours at
room temperature at 1:500 for TUBB3, 1:500 for vesicular
glutamate transporter 1 (VGLUT1) (Synaptic Systems),
and1:1000 for GFAP. Samples were washed twice in a
washing solution containing 0.05% (v/v) Triton X-100 in PBS
and incubated with the appropriate secondary antibodies in
blocking solution at room temperature for 1 hour at 1:100 for
Alexa 488 anti-mouse, and 1:100 for Alexa 488 anti-rabbit
(Molecular Probes). After the second antibody was removed,
samples were washed twice with washing solution and
incubated with 2 μg/ml Hoechst in PBS for 10 minutes.
Coverslips were mounted onto slides using 44% glycerol in
PBS. Micrographs were taken at 40X using an Axioplan Zeiss
fluorescent microscope equipped with an AxioCam ERc5s and
Zeiss’ Zen Microscopy suite application.
Reverse transcription and quantitative real-time RT-
Total RNA was isolated from undifferentiated and
differentiated cells by using TRIzol reagent (Ambion) and from
tissues by using a RNeasy Minikit (Qiagen). Reverse
transcription was performed using random hexamer primers
and SuperScript III transcriptase (Invitrogen) as previously
described . Quantitative real-time RT-PCR was used to
quantify the expression levels of different genes, using Gapdh
mRNA as the normalization standard. Primers used are shown
in Table S1. Raw data is shown as 2 to the power of - ΔΔCt,
where ΔΔCt = (CtGene - CtGapdh)Assay.
Active caspase-3 analysis by flow cytometry
trypsinized to obtain isolated cells and collected by
centrifugation at 200xg for 5 minutes. After washing twice with
PBS, active caspase-3 was assayed with a FITC Active
Caspase-3 Apoptosis Kit (BD Pharmigen) following the
manufacturer’s protocols and processed by flow cytometry.
and cellular aggregates were
All differentiation experiments were done using at least three
independent cultures. Data are shown as the mean ± SEM.
Group comparisons were made using one-way ANOVA
followed by post hoc Tukey test. A p-value < 0.05 was
considered statistically significant.
Gene expression changes induced by gestational Pb
Prenatal and early childhood lead exposure are associated
with adverse cognitive, neurobehavioral and motor outcomes,
suggesting altered brain structure and function and, by
extension, altered gene expression patterns in the CNS .
These alterations can be extended also to adolescence and
adult life as a consequence of peak blood Pb levels above 5-10
µg/dl during childhood [15,32]. To study gene expression
changes induced by Pb in the brain of mice, we exposed
female mice to 0 or 3 ppm of lead acetate in drinking water
from 8 weeks prior to mating, through gestation and until
postnatal day PD10, and collected brain tissues of 3 male
offspring from each treatment group at PD60. For gene
expression analyses we chose cortex, hippocampus, and
thalamus because of their implication in learning and memory
formation [33–35] and the known loss of gray mass volume in
cortex resulting from Pb exposure in humans . Recently, it
has been shown that 27 ppm of gestational lead exposure in
drinking water produced a concentration of Pb in blood of 10
μg/dl at PN10 in C57BL/6J mice . Therefore, we used one
tenth the amount of this metal of what has been considered the
action level and human-equivalent of Pb.
We used quantitative real-time RT-PCR (qRT-PCR) to
measure the expression of several neural markers implicated in
vesicle release (Syn1, Vamp1, and Syp), neurite and axon
growth (Tubb3, Nes, and Gap43), axon maturation and
synaptogenesis (Reln), mRNA stabilization (Hud), BDNF
vesicle transport (Htt), and transcription factors involved in
neuronal differentiation (Ngn1, NeuroD1, Sox3, and Sox4)
(Figure 1, blue bars). We also examined the expression of the
neurotrophins Bdnf, Ngf, Nt3 and Nt4 (Figure 1, orange bars),
which are produced and secreted by neural and glial cells and
are implicated in cell proliferation, maturation survival, axonal
outgrowth, and neural plasticity . We included transcription
factors with key roles in the CNS, such as Sp1 and the
members of the CREB and NF-κβ families (Figure 1, pink
bars), and glutamate-related genes (Figure 1, green bars),
including the different subunits of the glutamate receptors
Grin1, Grin2A-D, and Grin3A-B, for the glutamate N-methyl-D-
aspartate (NMDA) receptors, Grik1-5 for the kainate receptors,
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Gria1-4 for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) receptors, Grm1-8 for the metabotropic receptors,
and Vglut1-2 for the vesicular glutamate transporters. Of the
three tissues tested, hippocampus showed more Pb-induced
gene expression changes than the others. The neural markers
Hud, Syn1, Htt, Vamp1, and Ngn1, the member of the NF-κβ
family crel, and the glutamate-related genes Grin2A, Grin3B,
Grik2, Grm4, and Grm8 were up-regulated, while the neural
markers NeuroD1 and Sox3, the neurotrophins Bdnf exon IX
and Ngf, the transcription factor Creb1 and Creb2, the
members of the NF-κβ family Nfκb2, Rela, and Relb, and the
subunits for glutamate receptors Grin1, Grin2D, Gria1, Gria4,
and Grm6 were down-regulated (Figure 1A). Bdnf exon IX
contains the coding region, whereas exon IV, a non-coding
exon, is up-regulated through calcium-dependent neural
activity [39,40]. In cortex and thalamus, Pb exposure led more
often to up-regulation of gene expression than to down-
regulation. In cortex, the neural marker Reln, the member of
the CRE family Creb5, the transcription factor Sp1, and Grin3B,
Grik1, Grik2, Grik3, Gria1, Grm4 and Grm6 glutamate
receptors were up-regulated, whereas exons IX and IV of the
neurotrophin Bdnf and Nfκb2 were down-regulated (Figure 1B).
In thalamus, the neural markers Nes, Vamp1, Ngn1, and Sox3,
the neurotrophins Ngf and Nt4, the members of the NF-κβ
family Rela and Relb, the transcription factor Sp1, and
glutamate-related genes Grin2A, Grin2B, Grin2C, Grin2D,
Grin3A, Grik2, Gria1, Vglut1, and Grm6 were up-regulated
while the for glutamate receptor subunits Grik3, Grik5, Gria2,
Gria3, and Gria4 were down-regulated (Figure 1C). These
results, observed 2 months after the time of exposure, support
the conclusion that early-life exposure to Pb may produce
irreversible changes that extend to adult life, producing
alterations in the expression pattern of the genes analyzed.
Neural differentiation of mouse ESC produces
To identify molecular changes taking place during
embryogenesis and neurodevelopment as a consequence of
extended Pb exposure we used an in vitro model of neural
differentiation of mouse ESCs. Unlike other toxicological
models of primary neuron cultures or neuronal cell lines, such
as PC12 or SHSY5H cells, developmental effects of Pb can be
better studied in these cells because addition of Pb to the
culture medium during the differentiation process allows us to
determine whether changes that take place in vivo during
gestational Pb-exposure occur also in Pb-treated neurons
differentiated in vitro.
We followed protocols previously established by others to
induce differentiation of C57BL/6-C2 mouse ESC along
neuronal lineages . To verify the extent of neuronal
differentiation and determine the level of contamination with
glial cells, we used western immunoblot analysis for the neural
marker TUBB3 and the glial marker GFAP. After 3 days in vitro
(3-DIV) following cellular
immunoblot analyses showed that 3-DIV cells expressed high
levels of TUBB3 and no detectable GFAP (Figure 2A). As a
positive control, a total protein extract from mouse brain
showed high levels of both
proteins (Figure 2A).
Immunofluorescence analyses verified that most of the cells
expressed TUBB3 and that there were no detectable cells
positive for GFAP. Furthermore, immunofluorescence analyses
showed that our protocol produced glutamatergic neurons, as
demonstrated by the expression of VGLUT1 (Figure 2B), a
marker of glutamatergic neurons [41,42]. Immunofluorescence
quantification indicated that 85 % of the cells were TUBB3 and
VGLUT1 positive (Figure S1). In addition, 9% of the cells in the
culture were positive for active caspase-3, an indication of
apoptosis (see below).
To further characterize the neural differentiation process of
the ESC, we analyzed the expression of pluripotency and
neural markers by qRT-PCR. Compared to undifferentiated
ESC, the pluripotency genes, Nanog, Oct4, Gdf3, and Sox2
were significantly down-regulated (Figure 3A). Among the
neural markers analyzed there were genes involved in vesicle
release (Syn1, Vamp1, and Syp), axon growth and maturation
and synaptogenesis (Tubb3, Nes, Gap43, and Reln),
transcription factors (Ngn1, NeuroD1, Sox3, and Sox4), BDNF
vesicle transport (Htt), and mRNA stabilization (Hud). All the
neural genes tested were significantly up-regulated, although to
different extents (Figure 3B). In addition, we used qRT-PCR to
assay for the subunits of NMDA, kainate, AMPA, and
metabotropic receptors for glutamate, and for the glutamate
transporters Vglut1 and Vglut2. As for the neural markers, all
the glutamate-related genes were up-regulated relative to their
expression in ESC (Figure 3C), supporting the view that the ES
cells had differentiated into a pure culture of glutamatergic
Lead exposure diminishes cell numbers in cellular
aggregates and changes gene expression pattern
during ESC neural differentiation
Treatment of cultured rat embryonic hippocampi neurons
with micromolar Pb concentrations has been shown to
decrease the levels of presynaptic vesicular proteins implicated
in neurotransmitter released, CREB phosphorylation, and
BDNF signaling [25,26]. In order to verify these results in our
experiments and mimic gestational Pb exposure in vitro, we
treated differentiating ES cells with Pb acetate for the length of
the neuronal differentiation process and until day 3 in vitro. At 1
µM Pb the number of cells resulting from CA disaggregation
was reduced by more than 90% relative to control untreated
cells (Figure 4A). The effect of Pb treatment was clearly
concentration dependent, reducing survival by 30 and 6% of
control after treatment with 0.1 and 0.01 µM Pb, respectively
(Figure 4A). We chose 0.1 µM as the concentration for all
Pb toxicity has been related to cell death by apoptosis .
To determine whether the loss of cells observed after CA
disaggregation was caused by apoptosis, we measured active
caspase-3 expression at different time points during CA
formation. Interestingly, we found no difference in active
caspase-3 levels at any point between control and Pb-treated
cells during the whole process of CA formation, neither in the
supernatant nor in the CA fraction (Figure 4B), compared to
staurosporine as a positive control. This result indicates that
loss of cells from the cellular aggregates is unlikely to result
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from apoptosis and more likely to be due to Pb interference
with the competence of the cells to aggregate.
The neurons resulting from Pb treatment of ESC during the
differentiation process are TUBB3 and VGLUT1 positive,
morphologically indistinguishable from untreated control
neurons, as determined by immunofluorescence analysis
(Figure 5A and 5B). Expression of the pluripotency genes
Nanog, Oct4, Gdf3, and Sox2 was slightly up-regulated in
these neurons, but not significantly different from their
expression in untreated control neurons (Figure 6, green bars).
The neural marker Ngn1 was up-regulated after Pb treatment,
whereas Syp, Htt, Gap43, and Reln were marginally down-
regulated in the presence of Pb, although the difference was
not statistically significant (Figure 6, blue bars). We also
examined the differentiated neurons for expression of the
neurotrophins and the transcription factors analyzed in the
mouse brain tissues. Bdnf exon IV was down-regulated in cells
treated with 0.1 µM Pb, whereas Bdnf exon IX was up-
regulated (Figure 6, orange bars). Creb1 was slightly down-
regulated, with no significant difference to their expression in
control neurons, and Rela was up-regulated by Pb. The rest of
the genes analyzed did not change their expression (Figure 6,
pink bars). To complete the analysis, we measured the
expression of subunits for NMDA, kainate, AMPA, and
metabotropic receptors for glutamate, and for the glutamate
transporters Vglut1 and Vglut2. Pb down-regulated Grin1 and
Grin2D of the NMDA receptor subunits, Grik1 and Grik5 of the
kainate receptor subunits, Gria4 of the AMPA receptor
Figure 1. Expression patterns of genes altered in mice brain after 0 or 3 ppm of gestational Pb exposure. (A),
Hippocampus; (B), cortex; and (C), thalamus at PND60 were dissected from 3 male mice in each group. Gene expression as
determined from qRT-PCR levels was normalized to Gapdh expression in each condition and expressed relative to the
corresponding level in controls with no Pb treatment. Tissues from each mouse were processed individually and the data shown
represents the mean ± SEM of the three mice. (*) p< 0.05.
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subunits, Grm1 and Grm6 of the metabotropic receptor, and
Vglut1 of the glutamate transporters. Grin2A, Grin2B, Grin2C,
Grin3B, Gria2, and Grm4 were marginally different from their
expression in control neurons (Figure 6, yellow bars). These
results show that Pb exhibits a repressive effect on the
expression of glutamate receptors, leading to the conclusion
that Pb treatment during neural differentiation process of ESC
in vitro causes changes in gene expression patterns that affect
multiple neuronal transport processes, including calcium
A side-by-side comparison of Pb effects on gene expression
in ESC-derived 3-DIV neurons and brain tissues reveals that
more frequently repression is the consequence of Pb treatment
in both cases. Thus, Pb up-regulates Ngn1 in vitro and in
hippocampus, while it down-regulates the glutamate receptor
subunits Grin1, Grin2D, Gria4, and Grm6. Ngn1 is also up-
regulated by Pb in the thalamus. Bdnf is down-regulated in vitro
and in the cortex and the glutamate receptor subunits Grm3
and Grm4 subunits are down-regulated in vitro and in thalamus
(Figure 7). These similarities in gene expression responses
between mouse brain tissues gestationally exposed to Pb on
the one hand, and ESC-derived neurons differentiated in the
presence of Pb, on the other, support the concept that this
model of toxic exposure during neural differentiation may be
useful to analyze the mechanisms of toxicity by lead and other
In this study we have found that mice exposed to Pb in utero
show gene expression changes in adult life and that many of
these changes can also be found in Pb-treated neurons
differentiated in vitro from mouse ES cells. We followed the
Figure 2. Expression of TUBB3, VGLUT1, and GFAP proteins. (A) Western immunoblot analysis of the neural marker TUBB3
and the glial marker GFAP. Representative western blots are shown for mouse embryonic stem cells (mESC), for neurons obtained
from mESC neural differentiation 3 days after CA disaggregation (3DIV Neuron), and for mouse brain. Experiments were performed
in three individual cultures and the mean ± SEM expression quantified relative to β-actin and shown below each lane. (B)
Fluorescence detection of TUBB3, GFAP, and VGLUT1 proteins were carried out in neurons obtained from mESC neural
differentiation 3 days after CA disaggregation. The third panel shows the merge for each pair of proteins.
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expression of genes coding for neural markers, neurotrophins,
transcription factors, and glutamate-receptors at postnatal day
60 in three different brain regions, namely hippocampus,
cortex, and thalamus. Among the three brain areas analyzed,
hippocampus was the one to show more Pb-induced gene
expression changes for the gene tested (Hud, Syn1, Htt,
Vamp1, Ngn1, NeuroD1, Sox3, Bdnf exon IX, Ngf, Creb1,
Creb2, Crel, NFκβ2, Rela, Relb, Grin1, Grin2A, Grin2D,
Grin3B, Grik2, Gria1, Gria4, Grm4, Grm6, and Grm8), followed
by thalamus (Nes, Vamp1, Ngn1, Sox3, Ngf, Nt4, Rela, Relb,
Sp1, Grin2A, Grin2B, Grin2C, Grin2D, Grin3A, Grik2, Grik3,
Grik5, Gria1, Gria2, Gria3, Gria4, Vglut1, and Grm6) and cortex
(Reln, Bdnf exon IX and IV, Creb5, NFκβ2, Sp1, Grin3B, Grik1,
Grik2, Grik3, Gria1, Grm4, and Grm6). After a side-by-side
comparison of the effects of Pb on the gene expression in
ESC-derived neurons and brain tissues, we observed that
Ngn1 was up-regulated and Grin1, Grin2D and Grm6 were
down-regulated in vitro and in hippocampus, being Ngn1 also
up-regulated in thalamus. Bdnf exon IV and Grik5 were down-
regulated in vitro and in cortex and thalamus, respectively.
Finally, Gria4 was repressed in vitro, in hippocampus, and
Figure 3. Gene expression pattern of neurons obtained from mESC neural differentiation 3 days after CA
disaggregation. Total RNA was isolated from neurons obtained from mESC neural differentiation 3 days after CA disaggregation.
Gene expression was normalized to Gapdh expression. The oligonucleotides used for amplification are described in Table S1. Gene
expression was analyzed by qRT-PCR for, (A), pluripotency markers Nanog, Oct4, Sox2, Gdf3; (B), for neural markers; and (C), for
glutamate receptors subunits and vesicular glutamate transporters. The data shown are the mean ± SEM from three independent
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Recently, it has been described that reelin (RELN),
implicated in axon growth and maturation, is decreased in
zebrafish telencephalon  when exposed to Pb during
development. Although we did observe a slightly down-
regulation in the in vitro model, Reln was up-regulated in the
cortex after gestational exposure to Pb, without any alteration
in the other brain tissues
synaptophysin (SYP) phosphorylation as a result of Pb
exposure have been described recently in hippocampal primary
cultures, suggesting that this disturbance may produce
impairment in vesicular release in synapses [26,27]. The same
study found changes in the phosphorylation status of huntingtin
(HTT), which acts as a transcription factor and a regulator of
BDNF vesicle transport in physiological conditions, and when
mutated, is responsible for Huntington’s disease . None of
these genes, however, were affected by gestational Pb
exposure in our studies, being marginally down-regulated in the
3-DIV neurons treated with Pb. In contrast, mRNA levels for the
transcription factor gene neurogenin 1 (Ngn1), implicated in
neural differentiation programs and cell fate , are
significantly elevated in both our in vitro and in vivo models.
Although we do not detect any morphological alterations when
the cells are treated with Pb, our gene expression data suggest
that the differentiation process may be compromised.
The ionotropic receptors NMDA, kainate, and AMPA, and the
G-protein-coupled metabotropic receptor for glutamate are
involved in excitatory transmission with important effects on
analyzed. Decreases in
synaptic plasticity that are implicated in neuronal processes
such as learning and memory . Recently, chronic exposure
to Pb of primary hippocampal neurons has been shown to
cause modifications on NMDA receptor composition as a result
of decreases in the expression of GRIN1, GRIN2A, and
GRIN2B [23,24]. Our results suggest that Pb alters the
expression of the different glutamate receptors subunits and
that this effect may cause an imbalance of calcium
homeostasis responsible for the impairment of other signaling
pathways. In our in vivo studies, Grin2A was up-regulated in
hippocampus and thalamus of mice treated gestationally with
Pb, but we did not observe any expression changes in cortex
or in cultures of glutamatergic neurons. Additionally, Grin1 was
down-regulated in hippocampus and ESC-derived neurons
exposed to Pb. Calcium conductance increases when the
kainate receptors contain the Grik5 subunit, while when they
contain Grik1 and Grik2 they are less permeable to calcium
[48,49]. Although there were more alterations in gene
expression in other subunits of NMDA, kainate, AMPA, and
metabotropic receptors by Pb in both models, Grik5 was down-
regulated in both ESC-derived neurons treated with Pb and in
thalamus, and Grik2 was up-regulated in cortex and thalamus
of mice treated gestationally with Pb. If as a consequence of
Pb treatment the glutamate receptors are reorganized, their
functional properties may be sufficiently affected as to disrupt
calcium homeostasis and signaling.
Figure 4. Effect of Pb in cellular aggregate formation. mESC were incubated with the indicated Pb concentrations during the
complete process of CA formation. (A) After CA disaggregation, cell numbers were recorded. (B) Active caspase-3 determinations.
Cells were treated with 0 (control) or 0.1 µMPb (Pb) during the 8 days of CA formation process. CAs were disaggregated every two
days and active caspase-3 was measured in the cultures (Control, yellow bars; Pb, pink bars). The supernatant was taken every two
days and active caspase-3 was measured in the cells in suspension (Control, green bars; Pb, blue bars). Neurons obtained from
mESC neural differentiation 3 days after CA disaggregation were also analyzed (3DIV, orange bar). Staurosporine (St, red bars)
was used as a positive control of active caspase-3 induced apoptosis. The experiments were performed in three independent
cultures. (*) p< 0.05.
Lead Disrupts Neuronal Gene Expression
PLOS ONE | www.plosone.org8November 2013 | Volume 8 | Issue 11 | e80558
Calcium signaling through the glutamate receptor has been
shown to increase the level of CREB-dependent Bdnf
transcripts containing exon IV , an effect that is down-
regulated by Pb without affecting the expression of other non-
coding exons . In our experiments, Bdnf exon IX transcripts
are repressed after Pb treatment in both hippocampus and
cortex, being not affected in thalamus and increased in the
neurons obtained from neural differentiation of mESCs. These
discrepancies suggest that other non-coding exon(s) may play
a role in the maintenance of Bdnf expression. The transcripts of
the coding exon IV are down-regulated in cortex and our in
vitro model, and not affected in hippocampus and thalamus.
BDNF expression is highly controlled by different regulatory
mechanisms, including epigenetic mechanisms, and mitogen-
activated protein kinases, phosphoinositide-3 kinase and
phospholipase-γ pathways . As the neurotrophins are
implicated in cell survival, differentiation, axon growth and
guidance, synapses formation, and memory formation, the
outcomes observed after Pb treatment provide a putative
mechanism by which this metal may induce long-term
potentiation (LTP) and spatial memory impairments. The role
played by neurotrophins in such physiological events has
recently been reinforced by the findings that antibodies against
BDNF and NT4 blocked long-term recognition memory in rats
. Interestingly, we find that Bdnf exon IV and Grin1 are
down-regulated in cortex and hippocampus of mice treated
gestationally with Pb and in 3-DIV neurons exposed to Pb
during the neural differentiation process, suggesting that these
changes may extend into adulthood, beyond the time when the
organism was exposed to the agent.
Pb toxicity has been associated with apoptosis  and
shown to induce an increase of caspase-3 cleavage and a
decrease of intracellular glutathione levels in human SH-SY5Y
neuroblastoma cells . In our hands, however, 0.1 µM Pb
Figure 5. Immunofluorescence detection of TUBB3 (A) and VGLUT1 (B). Neurons obtained from mESC neural differentiation 3
days after CA disaggregation were untreated (control) or treated with 0.1 µM (Pb) during the whole differentiation process. The third
panel shows the merge for each pair of proteins. The experiment shown is representative of three independent cultures.
Lead Disrupts Neuronal Gene Expression
PLOS ONE | www.plosone.org9November 2013 | Volume 8 | Issue 11 | e80558
reduced cell numbers after CA disaggregation without inducing
concomitant cell death directly and without activating
caspase-3. These results are remarkably similar to those
described for low-concentration, long-term treatment of neural
stem cells derived from E15 rat cortex, striatum, and ventral
mesencephalon, and mouse bone marrow-mesenchymal stem
cells, which show that cell proliferation is slightly altered at low
Pb concentration and considerably
concentration [54,55]. We tested treatment of ESC with
different Pb concentrations in the range of 0.01 to 0.1 µM for 10
days and we did not observe any reduction of cell proliferation
(Figure S2). Other in vivo studies have also shown
discrepancies on casape-3 activation due to long-term
exposure, suggesting that Pb effects may be region-, time-,
and concentration-specific [56,57].
It is paramount to determine the molecular mechanisms by
which Pb produces neurotoxicity. Although the use of lead has
been reduced in the last few decades, its non-biodegradable
nature and ubiquitous presence make it an environmental
agent of general concern. Developmental or early-life exposure
to Pb may produce CNS disorders detectable later in
affected at high
adulthood. The theory of the fetal origin of adult disease states
that during development, exposure to environmental agents
such as heavy metals, or to stressful situations like poor
nutrition, may adversely contribute to adult pathogenesis
[58,59], possibly by reprogramming specific gene expression
patterns. Early-life exposure to lead may produce persistent
changes in the mechanisms that regulate gene expression,
contributing to adult neurological pathologies. Specifically,
children are more sensitive to Pb exposure because absorption
is greater in early life that in adults  and during the
gestational period, Pb crosses the placenta and blood-brain
barrier reaching the developing fetal brain . Reduction of
gray matter and alteration of the myelin structure and important
metabolites are among the remote effects that early-life
exposure to chronic and low-dose Pb produce in the CNS of
adults . Among its many other adverse effects, Pb is
implicated in hepatic, renal, vascular, reproductive, and
nervous diseases [3,4,7,8]. Additionally, lead accumulates in
bones and blood, increasing the body burden and extending
exposure well beyond the time of direct contact. Our current
studies have developed a suitable in vitro model that uses
Figure 6. Expression patterns of genes altered in ESC-derived neurons after Pb treatment. mESC were untreated or treated
with 0.1 µM of Pb during the whole neural differentiation process. Total RNA was isolated from neurons obtained from mESC neural
differentiation 3 days after CA disaggregation. Gene expression was normalized to Gapdh expression in each condition and
expressed relative to the corresponding level in untreated controls. Green bars represent pluripotency markers; blue bars, neural
markers; orange bars, neurotrophins; pink bars, transcription factors; and yellow bars, glutamate receptors subunits and vesicular
glutamate transporters. The data shown are the mean ± SEM of three independent replicates. (*) p< 0.05.
Lead Disrupts Neuronal Gene Expression
PLOS ONE | www.plosone.org10November 2013 | Volume 8 | Issue 11 | e80558
mouse ESC differentiation to recapitulate neural gestational
exposure to lead, which may serve to characterize the
molecular mechanisms of lead neurotoxicity.
Figure S1. Quantification of TUBB3 and VGLUT1 positive
cells. After fluorescence detection of TUBB3 and VGLUT1
neurons obtained from mESC, the percentage of TUBB3 and
VGLUT1 positive cells was calculated as the Number of
immunopositive cells/Total number of cells x100. At least five
micrographs from different assays were analyzed. (*) p<0.05.
Figure S2. Effects of Pb in mouse ESC cell number. Mouse
ES cells were treated with the indicated concentration of Pb
during ten days. Cell numbers were recorded every other day
at the time that the cells were passaged. Cell numbers were
normalized to the number of cells plated on day 0.
Table S1. List of primers used for real-time RT-PCR.
We thank Qin Wang, Hisaka Kurita, Vinicius Carreira, Chia-I
Ko, and Jerry Ovesen for critically reading the manuscript.
Conceived and designed the experiments: AP YX DML.
Performed the experiments: FJSM DML YF. Analyzed the data:
FJSM YX AP. Contributed reagents/materials/analysis tools:
YX. Wrote the manuscript: FJSM AP.
Figure 7. Expression patterns of genes with similar alterations in neural differentiated neurons treated with 0.1 µMPb and
in mouse brain gestationally exposed to 3 ppm Pb. Neurons were treated with 0.1 µM Pb or left untreated for the length of the
neural differentiation process. Mouse tissues were the same as in Figure 1. Gene expression was normalized to Gapdh expression
and expressed relative to the corresponding levels in untreated or unexposed controls with no Pb treatment. The data shown are
the mean ± SEM of three independent determinations.
Lead Disrupts Neuronal Gene Expression
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