Citation: Transl Psychiatry (2012) 2, e98, doi:10.1038/tp.2012.

24
& 2012 Macmillan Publishers Limited All rights reserved 2158-3188/12

www.nature.com/tp

Effects of maternal immune activation on gene

expression patterns in the fetal brain
KA Garbett1,5, EY Hsiao2,5, S Kalman1,3, PH Patterson2 and K Mirnics1,4

We are exploring the mechanisms underlying how maternal infection increases the risk for schizophrenia and autism in the
offspring. Several mouse models of maternal immune activation (MIA) were used to examine the immediate effects of MIA
induced by influenza virus, poly(I:C) and interleukin IL-6 on the fetal brain transcriptome. Our results indicate that all three MIA
treatments lead to strong and common gene expression changes in the embryonic brain. Most notably, there is an acute and
transient upregulation of the a, b and c crystallin gene family. Furthermore, levels of crystallin gene expression are correlated
with the severity of MIA as assessed by placental weight. The overall gene expression changes suggest that the response to MIA
is a neuroprotective attempt by the developing brain to counteract environmental stress, but at a cost of disrupting typical
neuronal differentiation and axonal growth. We propose that this cascade of events might parallel the mechanisms by which
environmental insults contribute to the risk of neurodevelopmental disorders such as schizophrenia and autism.
Translational Psychiatry (2012) 2, e98; doi:10.1038/tp.2012.24; published online 3 April 2012

Introduction
Maternal infection is a risk factor for schizophrenia and
autism. In the case of schizophrenia, a wide variety of
infections during pregnancy (viral, bacterial, parasitic) are
associated with increased risk for this disorder in the offspring.
Summing these risks, Brown and Derkits1 estimate that
430% of schizophrenia cases would be prevented if infection
could be averted in pregnant women. The fact that such a
diverse set of pathogens is associated with risk suggests that
it is the mothers response to the infection that is critical for
altering fetal brain development. In fact, the maternal response
during gestation (elevated cytokines and anti-pathogen
antibodies) is associated with the increase in schizophrenic
outcome in the offspring. Similarly for autism, a study of
410 000 cases in the Danish Medical Registry revealed an
association between viral or bacterial infection in the mother
and increased risk for the offspring.2 Also similar to schizophrenia are findings of an increased risk for autism in the
offspring if particular cytokines or chemokines are elevated in
maternal serum or amniotic fluid.3,4 The observation of a
significantly higher concordance in dizygotic twins than in nontwin siblings also suggests the importance of the maternal
fetal environment in autism.5,6
To mimic the environmental risk for development of
schizophrenia and autism, several animal models for maternal
immune activation (MIA) have been used successfully.7 First,
intranasal instillation of human influenza virus in pregnant
mice or non-human primates induces a moderate but sublethal infection, and the offspring display a series of histological
and molecular abnormalities in the hippocampus and cortex.8,9

Young and adult mouse MIA offspring10 also exhibit a

cerebellar neuropathology that is commonly found in autism.11,12 Furthermore, adult mice born to infected mothers
display behavioral abnormalities that are relevant to both
schizophrenia and autism, including deficits in social interaction, prepulse inhibition, open field and novel object exploration,10 as well as heightened responsivity to a hallucinogen.13
Second, MIA can also be induced in the absence of pathogens
by injecting pregnant rats, mice or monkeys with the synthetic
dsRNA, poly(I:C) to mimic a viral infection or with lipopolysaccharide to mimic a bacterial infection. Overall, the
behavioral results from the maternal infection, poly(I:C) and
lipopolysaccharide models is consistent, with many results
being reproduced in both rats and mice.7,14,15 Molecular and
cellular studies of adult offspring of poly(I:C)-treated rodents
reveal abnormalities that are clearly relevant to schizophrenia,
such as increased levels of GABAA receptor a2 immunoreactivity, dopamine hyperfunction, delay in hippocampal
myelination, reduced NMDA receptor expression in the
hippocampus, reduced numbers of reelin- and parvalbuminpositive cells in the prefrontal cortex, reduced dopamine D1
and D2 receptors in the prefrontal cortex and enhanced
tyrosine hydroxlyase in striatal structures.16 Third, MIA can be
induced more directly by a single injection of the cytokine
interleukin IL-6 in pregnant mice.17 This approach is based on
findings that this injection yields offspring displaying many
of the same behavioral abnormalities found in the offspring
of influenza-infected or poly(I:C)-treated dams. Moreover,
co-injection of an anti-IL-6 antibody blocks the effects of
poly(I:C), yielding offspring with normal behavior. The critical

1
Department of Psychiatry, Vanderbilt University, Nashville, TN, USA; 2Division of Biology, California Institute of Technology, Pasadena, CA, USA; 3Department of
Psychiatry, University of Szeged, Szeged, Hungary and 4Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN, USA
Correspondence: Dr K Mirnics, Department of Psychiatry, Vanderbilt University, 8130A MRB III, 465 21st Avenue South, Nashville, TN 37203, USA.
E-mail: karoly.mirnics@vanderbilt.edu
5
These two authors contributed equally to this work.
Keywords: autism; brain; crystallin; gene expression; maternal immune activation; schizophrenia

Received 09 November 2011; revised 27 February 2012; accepted 03 March 2012

Embryonic MIA transcriptome profile

KA Garbett et al

importance of IL-6 in MIA is further shown by the finding that

poly(I:C) injection in IL-6 knockout mice also yields offspring
with normal behavior.17 Flavonoids that block JAK/STAT
signaling downstream of the IL-6 receptor also block the
induction of abnormal behaviors by maternal IL-6 injection.18
Maternal IL-6 is also critical for the endocrine changes in the
placenta induced by MIA.19,20
However, the immediate effects of MIA on the fetal brain are
not well understood. It is critical to examine the very early
effects because the rise in IL-6 caused by maternal IL-6
injection is transient, as is the effect of injecting the anti-IL-6
blocking antibody in the pregnant dam. These acute effects
nonetheless lead to permanent changes in the behavior of the
offspring. To gain a better understanding of the molecular
events that take place in the MIA-exposed developing brain,
we examined the transcriptome changes associated with
three different MIA models, and identify the critical early
mediators that may contribute to the emergence of behavioral
symptoms in MIA offspring.
Materials and methods
Animals and MIA treatments. All procedures involving
animals were approved by the Caltech Animal Care and Use
Committee. Female C57BL/6J mice (The Jackson Laboratory,
Bar Harbor, ME, USA) were obtained from our in-house
breeding facility and were housed in ventilated cages under
standard laboratory conditions. Mice were mated overnight,
and the presence of a vaginal plug marked that day as
embryonic day 0 (E0). Pregnant females were not disturbed,
except for weekly cage cleaning, until E9.5 when they were
weighed and pseudo-randomly assigned to one of four groups.
Each group contained four to five pregnant females.
Treatments with human influenza virus, poly(I:C) and
recombinant IL-6 (rIL-6) were used to induce MIA at conditions that previously resulted in offspring altered behavior.10,17 To allow for time to develop a flu infection, pregnant
mice on E9.5 were anesthetized intraperitoneally with
10 mg kg1 xylazine and 100 mg kg1 ketamine and inoculated intranasally with 6000 plaque-forming units of human
influenza virus in 90 ml phosphate-buffered saline (PBS).
Pregnant mice on E12.5 were injected with saline, poly(I:C) or
rIL-6. For poly(I:C) injections, poly(I:C) potassium salt (Sigma
Aldrich, St Louis, MO, USA) was freshly dissolved in saline
and administered intraperitoneally at 20 mg kg1 based on the
weight of the poly(I:C) itself, not including the total weight of
the potassium salts. Control mice were injected with saline
alone at 5 ml g1 body weight. For rIL-6 injections, 5 mg carrierfree, mouse rIL-6 (eBioscience, San Diego, CA, USA) was
freshly dissolved in 150 ml saline and injected intraperitoneally. Pregnant mice were killed on E12.5 (3 h after poly(I:C) or
rIL-6 injection and 3 days after influenza inoculation), and the
embryonic brains were removed quickly and frozen in 1.5 ml
tubes.
Transcriptome analysis. RNA was isolated from individual
embryonic brains using PureLink RNA mini kit (Ambion,
Chicago, IL, USA). RNA quality was assessed using the
Agilent (Palo Alto, CA, USA) Bioanalyzer. To reduce gene
Translational Psychiatry

expression variability that was not related to MIA exposure,

RNA from each embryonic brain within a litter was pooled
into a single sample, and used for the generation of biotinlabeled sample for microarray hybridization (Supplemental
Material 1). This allowed us to identify MIA-induced gene
expression changes that were commonly found in the
embryonic brains of each litter, resulting in 45 replicates
for each of the four conditions. Thus, from a total of 138
embryos, 17 pooled RNA samples (each originating from all
the embryonic brains within an individual pregnant dam) were
generated and hybridized on a GeneChip HT MG-430 PM
24-Array Plate. On the basis of the variability in our previous
mouse experiments,21 we estimated that we had an 80%
probability to identify significant gene expression changes
that were 430%. The data were log 2 transformed using the
RMA algorithm, and differential expression was established.
A gene was considered differentially expressed if (1) the
average log 2 ratio (ALR) between the experimental treatment and control exceeded 30% (|ALR| 40.3785) and (2) the
statistical significance of differences was Po0.05 using the
Students t-test. The GenePattern software (Broad Institute,
Cambridge, CA, USA) was used for hierarchical clustering of
the gene expression intensities.
Quantitative PCR. RNA isolated from embryos residing in
the left or the right uterine horn was pooled proportionately,
so there were two samples per each pregnant dam. Individual embryonic brain RNA or pooled RNA samples were
used to generate cDNA with high-capacity cDNA reverse
transcription kit (Applied Biosystems, San Mateo, CA, USA).
SYBR Green intercalation quantitative PCR (qPCR) was run
in a 7300 real-time PCR system (Applied Biosystems) with
the following primers: crystallin gA (cryaa) (F: 50 -TCTT
CTTGGACGTGAAGCAC-30 , R: 50 -GAAATGTAGCCATGGT
CATCC-30 ), crystallin aB (cryab) (F: 50 -TGTGAATCTGGA
CGTGAAGC-30 , R: 50 -TGACAGGGATGAAGTGATGG-30 ),
crystallin bA1 (cryba1) (F: 50 -CCTGGAAAGAGGAGAATA
CCC-30 , R: 50 -TTATGATTAGCGGAACAGATGG-30 ), crystallin
bA2 (cryba2) (F: 50 -ACCAGCAAAGATGTGGGTTC-30 , R:
50 -GCCTAATGCTGGACTCTTCG-30 ), crystallin bA4 (cryba4)
(F: 50 -CACCACTCAGGTGACTACAAGC-30 , R: 50 -CCAGA
GGACACAAGGGTAGC-30 ), crystallin bB1 (crybb1) (F:
50 -TCCCAGGAACATAAGATCTGC-30 , R: 50 -ACGGTCACA
GAAGCCATAAAC-30 ), crystallin bB3 (crybb3) (F: 50 -CAG
CCGACGTAGTGACATTC-30 , R: 50 -TCATCTACGATCTCC
ATCTTGC-30 ), crystallin gB/A/C (crygb/a/c) (F: 50 -AGCG
AGATGGGAAAGATCAC-30 , R: 50 -AGTACTGGTGGCCCT
GGTAG-30 ) and crystallin gC/A (crygc/a) (F: 50 -TGCGGCT
GTATGAGAAAGAA-30 , R: 50 -CCTCGGTAGTTAGGCATC
TCA-30 ).
All of these custom-designed primers reported amplification
efficiency 490%. qPCR data were analyzed using the ddCt
method with the housekeeping gene phosphoglycerate
kinase 1 (PGK1) as a normalizer.
In addition, qPCR for the crystallin genes cryaa, cryba1 and
crybb1 was performed with 34 RNA samples pooled from the
embryos identified as residing in the left or right uterine horns.
Furthermore, the expression of cryaa, cryba1 and crybb1 was
also examined by qPCR in all 36 individual embryonic brains
originating from the influenza-treated dams.

Embryonic MIA transcriptome profile

KA Garbett et al

Correlation of placental weight with gene expression

measurements. At the time of the dissection, the weight of
the individual placentas was measured for each embryo.
Placental weight was assessed across the treatments with
a groupwise, two-tailed t-test. In addition, qPCR-reported
crystallin expression was correlated with average placental
weight across the treatments and control groups using
Pearson correlation.

Results
Transcriptome changes in flu-, poly(I:C)- and rIL-6treated embryonic brains. Five pregnant dams were
treated with a single, nasal instillation of flu virus at E9.5.
The embryos were dissected on E12.5, the time of peak
maternal IL-6 expression, and brains were collected. A single
treatment of poly(I:C), rIL-6 or saline was administered to
four pregnant dams per group at E12.5. The embryonic
brains from these treatments were collected 3 h after
injection. RNA was isolated from the entire brain of
individual embryos. We obtained 31 to 36 embryos per
treatment, which totaled 138 embryonic brain samples. For
microarray analysis, embryonic brains from each litter were
pooled into a single sample. Using a 30% expression difference from saline-injected controls and a statistical significance of Po0.05, we identified 256 genes differentially
expressed in the flu model, 294 in the poly(I:C) model and
195 in the IL-6 model. Examining all of the differentially
expressed genes in a two-way (samples and genes)
unsupervised hierarchical clustering of the gene expression
intensities, the four experimental groups separate into
distinct clusters (Figure 1a) that correspond to the different
MIA treatments, with the main clusters separating the saline
treatment from all three MIA treatments. Interestingly, the
majority of the genes altered in the poly(I:C) and IL-6
treatments were upregulated, although there were as many
downregulated genes in the flu treatment, which might reflect
secondary expression changes that occurred during the 3day period following the influenza virus exposure.
Next, we tested if the transcriptome changes showed a
common expression pattern across the three different MIA
treatments. We created three groups of the most differentially
expressed genes: 256 genes in the flu model, 294 in the
poly(I:C) model and 195 in the IL-6 model. The mRNA
signature of the 256 differentially expressed transcripts in the
flu model was assessed in the poly(I:C) and rIL-6 transcriptomes. We found that, as a group, the flu model transcripts are
also significantly changed in the poly(I:C) and IL-6 models
for MIA (r 0.88, P 1.8E-84 and r 0.85, P 5.95E-72,
respectively) (Figures 1b and c). Likewise, the 195 differentially expressed genes in the IL-6-MIA group show a
significant expression difference in the flu-model and
poly(I:C)-model brains (r 0.85, P 1.42E-55 and r 0.85,
P 1.06E-54, respectively) (Figures 1d and e), and the 294
transcript changes in the IL-6 group are also significantly
present in the flu and poly(I:C) treatments (r 0.59,
P 8.78E-29 and r 0.59, P 3.9E-29, respectively)
(Figures 1f and g). This is a strong indication that all three
treatments affect similar molecular processes, although their

effects might be somewhat different because of the timing

difference in flu exposure and potential downstream signaling
differences. A comprehensive list of the differentially
expressed genes and their expression levels is provided in
Supplemental Material 2.
Of the transcripts with most changed expression (430%
expression change and Po0.05 in all three treatments), 41
genes were altered in both IL-6 and flu treatment, 31 in
poly(I:C) and flu, 17 in poly(I:C) and IL-6 and 12 genes were
similarly changed in all three MIA models. A hierarchical
clustering of the expression intensities of these 12 genes
clearly separates the saline-treated controls from the flu,
poly(I:C) and IL-6 treatments (Figure 2).
Common gene expression changes among treatments point
to involvement of the crystallin gene family. Surprisingly,
5 of the 12 genes that are upregulated in all three MIA
treatments belong to the crystallin gene family: cryaa, crybb3,
crybb1, cryba1 and crygb/crygc. To validate this finding with
an independent method, we generated cDNA from pooled
RNA from embryos in each dam. To inquire about a possible
effect of location in the uterus, RNA was separately pooled
from embryos residing in the left and the right uterine horns, so
there were two samples for each dam. For more detailed view
on the crystallin family of genes, we also tested the expression
of cryab, cryba2, cryba4 and crygc/a. The qPCR data are
highly correlated with the microarray data (flu: r 0.82, P
0.007; poly(I:C): r 0.84, Po0.005; rIL-6: r 0.89, P 0.001)
(Figure 3). No obvious effect of position is apparent.
As the expression of these crystallin genes is highly
coordinated, we selected only cryaa, cryba1 and crybb1 for
further qPCR analysis using RNA from the individual fluexposed embryonic brains. The upregulation of crystallin
transcripts observed by microarray is confirmed by the qPCR
analysis of the 36 individual embryonic RNA samples. Flu
treatment results in significantly increased cryaa, cryba1
and crybb1 transcript levels (cryaa: ddCt 1.97, P 0.001;
cryba1: ddCt 1.39, P 0.01; crybb1: ddCt 2.00, P
0.0002) (Figure 4). However, these data also reveal a
remarkable individual variability in flu-induced crystallin
upregulation, which does not correspond to the uterine
position of the embryos (flu: s.d. 2.8; saline: s.d. 1.6).
This indicates that flu exposure does not equally affect all
exposed embryonic brains within each dam, which could
potentially contribute to eventual behavioral variability.
Crystallin family upregulation is transient. As the
crystallin family genes are significantly upregulated at both
3 h following IL-6 and poly(I:C) treatment and at 3 days
following flu treatment, we asked if these expression
changes persist throughout the lifespan of the offspring.
We examined the expression of cryaa, cryba1 and crybb1 in
the frontal cortex and hippocampus of 15-week-old pups
(n 12) from poly(I:C)- and saline-treated dams. We find no
significant difference between these groups in the expression
of these genes. Furthermore, no difference is found in the
frontal cortex between 6-month-old (n 6) offspring of
saline-, rIL-6- and poly(I:C)-treated dams. Similarly, in a
different cohort of mice, no difference in crystallin expression
is seen in the frontal cortex and hippocampus of poly(I:C)- and
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Embryonic MIA transcriptome profile

KA Garbett et al

Figure 1 The transcriptome of the embryonic brain is altered in three maternal immune activation (MIA) models. (a) All differentially expressed transcripts in the MIA
treatments were subjected to a two-way (horizontal: genes; vertical: samples) unsupervised clustering using GenePattern. Gene expression differences are color-coded (red
increased expression; bluedecreased expression), and the intensity of the color corresponds to the magnitude of gene expression change. Note that the experimental
groups separate into distinct clusters that correspond to the different MIA treatments, with the main clusters separating the saline treatment from all three MIA treatments. (bg)
Three data set-driven gene groups were created, each containing genes that were differentially expressed in one of the three MIA models (256 genes in the flu model, 294 in
the poly(I:C) model and 195 in the recombinant IL-6 (rIL-6) model). We then examined the expression of each of these gene groups in the other two MIA cohorts. Thus, the
pattern of differentially expressed genes in the flu treatment was tested in the poly(I:C) and rIL-6 cohorts (b and c, respectively), the pattern of differentially expressed genes in
the rIL-6 treatment was tested in the flu and poly(I:C) cohorts (d and e, respectively) and the pattern of differentially expressed genes in the poly(I:C) treatment was assessed in
the flu and rIL-6 cohorts (f and g, respectively). Note that the gene expression patterns uncovered in any of the three MIA treatments show a very strong, highly significant
correlation to the other two MIA treatments.

Figure 2 The most significantly changed transcripts in all three maternal

immune activation (MIA) treatments include multiple members of the crystallin gene
family. Clustering was performed as described in Figure 1a. Note that, of the 12
gene expression changes observed in all three MIA conditions, 5 are mRNA species
belonging to the crystallin gene family.

saline-treated dams (n 15) at 12 months of age. Thus, we

conclude that the upregulation of crystallin genes observed
across the three MIA models is transient.
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Crystallin expression level correlates with placental

weight. MIA has a strong effect on development, and at
least part of this effect is mediated through the influence of
maternal IL-6 on the placenta.19,20 To test whether there is a
relationship between the effects of MIA on the placenta and
on the fetal brain, we measured the placental weight of
individual embryos from the three MIA conditions (Figure 5).
The average placental weight is significantly decreased in
all three MIA treatments compared to saline treatment
(flu: P 0.00016; poly(I:C): P 0.03439; IL-6: P 0.0041).
Furthermore, brain expression levels of cryaa, cryba1 and
crybb1 determined by qPCR are highly correlated with the
decrease in the average placental weight obtained for all
conditions (cryaa: r 0.85, P 0.072; cryba1: r 0.72,
P 0.138; crybb1: r 0.92, P 0.041, respectively). Thus,
the response of the crystallin genes corresponds closely with
the degree of MIA.

Embryonic MIA transcriptome profile

KA Garbett et al

Figure 3 Quantitative PCR (qPCR) validates crystallin gene expression

changes across the maternal immune activation (MIA) models. The expression of
nine genes was tested (crystallin aA (cryaa), crystallin gB/C/A (crygb/c/a), crystallin
gC/A (crygc/a), crystallin bB1 (crybb1), crystallin bA2 (cryba2), crystallin bB3
(crybb3), crystallin bA1 (cryba1), crystallin bA4 (cryba4), crystallin aB (cryab)) on
pools of RNA derived from the three MIA treatments. The X axis denotes average
log 2 ratios reported by microarrays, whereas the ddCt qPCR values are plotted on
the Y axis. Note that the qPCR and microarray-reported expression changes are
highly correlated across the three MIA models.

Figure 5 Crystallin expression is correlated with placental weight. quantitative

PCR (qPCR) expression levels of cryaa (red), cryba1 (green) and crybb1 (blue) are
correlated with mean placental weights across the maternal immune activation
(MIA) groups and controls. The X axis denotes the magnitude of expression change
measured in ddCT (1Ct 2-fold change) and the Y axis depicts average placental
weight. Squares denote saline treatment, diamonds denote poly(I:C) treatment,
triangles denote IL-6 treatment and circles denote flu treatment. Note that the
average placental weight is significantly different between the controls and all three
MIA treatments, and that the magnitude of all the crystallin expression changes is
highly correlated with placental weight.

Discussion

Figure 4 Crystallin expression is variable in individual brains from flu-exposed

embryos. Expression of crystallin aA (cryaa), crystallin bA1 (cryba1) and crybb1
was assessed by quantitative PCR (qPCR) across the individual brains of 36 flu and
36 saline embryos. Each symbol represents a phosphoglycerate kinase 1 (PGK1)normalized gene expression level in a single embryonic brain. The dashed gray line
denotes the reliable transcript detection threshold by qPCR. Note the upregulation
of the three crystallin genes in the flu group compared with controls, and the
considerable variability of crystallin expression levels between the flu-exposed
embryonic brains.

This study of the acute effects of MIA on the embryonic brain

transcriptome reveals that: (1) all three MIA treatments
(flu, poly(I:C) and IL-6) evoke strong gene expression
changes in the embryonic brain; (2) the three MIA treatments
yield diverse as well as overlapping transcriptome signatures;
(3) all MIA treatments strongly upregulate the crystallin gene
family, represented by a, b and g members, in the embryonic
brain; (4) the upregulation of crystallin genes is an acute
reaction that does not persist into adulthood; and (5) the level
of crystallin gene expression is correlated with the degree of
MIA as measured by placental weight. In addition, our data
also reveal a remarkable variability in crystallin induction
among the individual brains in the flu MIA model, indicating
that this form of MIA does not affect all of the embryos equally
within each dam.
Although we find that the three MIA models induce a
number of genes in common, there are also significant
differences among the groups. This can be attributed in part
to differences in the molecular action of the three perturbation
agents, and also to the differences in the time point of the flu,
poly(I:C) and IL-6 exposure. The efficiency of flu exposure
and disease time course is challenging to control, and the
flu-triggered cytokine response is gradual and takes days to
develop. The time point chosen for IL-6 and poly(I:C) injection
(E12.5) approximately mimics the peak of IL-6 induction
following flu infection (E9.5).10,17 However, cytokine induction
is longer lasting following infection than it is for the other two
treatments. Likewise, the molecular effects of poly(I:C) versus
IL-6 at 3 h after exposure may not be fully equivalent, resulting
in both common and divergent gene expression changes at
the same time point. Thus, we believe that the overall data
argue that the transcriptome differences we observe are
part of the same process, providing molecular snapshots
of canonical, transient expression changes that are rapidly
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KA Garbett et al

changing. Nonetheless, the common gene expression patterns across the three MIA models likely give rise to the
behavioral deficits that are common in the offspring of all three
treatments such as prepulse inhibition, social interaction and
open-field exploration.10,17 This possibility provides strong
motivation to further pursue the potential effects of dysregulation of these particular genes on brain development, with a
primary focus on the role of the crystallin gene family.
That elevated brain crystallin expression is common among
three MIA mouse models aligns well with studies implicating
crystallins in human autism and schizophrenia. Altered aBcrystallin protein levels are detected in the frontal cortex of
autistic brains.22 In addition, anti-aB crystallin antibodies are
reported to be elevated in the sera of autistic subjects
compared with controls.23 Moreover, increased expression
of aB-crystallin is detected in Rett syndrome brains,24 and
aB-crystallin is found in inclusions in brains from patients with
Fragile X-associated tremor/ataxia syndrome.25 In schizophrenia, altered expression of both a-crystallin and m-crystallin
are reported in the anterior cingulate cortex26 and prefrontal
cortex.27,28 Furthermore, dysregulated brain crystallin levels
are implicated in the manifestation of a variety of neurodegenerative disorders, including Parkinsons disease, Alzheimers disease, multiple sclerosis, CreutzfeldtJakob disease
and Alexander disease.2935
Although the exact role of crystallins in these disorders is
unclear, studies in animal models suggest that upregulated
crystallin levels serve a neuroprotective function. Knocking
out aB-crystallin in Alzheimers disease or experimental
autoimmune encephalitis mice causes worsened symptoms.36,37 These effects in experimental autoimmune
encephalitis mice are in turn ameliorated by systemic delivery
of recombinant aB-crystallin,37 as are the effects of experimental
stroke in mice.38 Similarly, transgenic overexpression of
aB-crystallin ameliorates symptoms in a mouse model of
Alexander disease, a fatal developmental neurodegenerative
disorder originating in astrocytes.39 In addition, aA-crystallin
exhibits anti-apoptotic protective effects in experimentally
induced inflammation of the uvea,40 and pretreatment with
a-crystallin blocks systemically induced inflammation in the
brain.41
Recent studies have shown that, in addition to their
canonical roles as molecular chaperones, crystallins serve a
wide variety of other functions that may underlie their
protective roles in vivo. For example, aA-crystallin-mediated
inhibition of caspase-3 rescues Pax6-deficient dopaminergic
neurons from apoptosis.42 Likewise, aB-crystallin protects
against Fas/APO-1-induced cell death in vitro. Crystallins can
also confer neuroprotection by modulating cytoplasmic
calcium levels. Retinal bB2-crystallin, for example, colocalizes with synaptotagmin 1, a primary calcium sensor, as well
as calmodulin, a major calcium-binding protein,43 and several
crystallins harbor Greek key motifs that sequester calcium
ions.4446 These properties in a- and bB2-crystallins are
thought to confer their ability to promote neurite outgrowth
during retinal repair.43,47 Another pathway of crystallin activity
is through the regulation of oxidative stress and acute phase
immune responses. For instance, aB-crystallin modulates
nuclear factor-kB activity based on its phosphorylation
status,48 and pretreatment with aB-crystallin prevents nuclear
Translational Psychiatry

factor-kB-induced neurotoxic effects.49 Moreover, aB-crystallin administration in vitro and in vivo downregulates tumor
necrosis factor-a and nitric oxide synthase in activated
microglia.50
Given this literature, the induction of crystallins in response
to MIA can be seen as an attempt of the developing brain to
counteract the immune system challenge. Importantly, the
crystallin upregulation we observe in embryos in response to
MIA cannot be detected in the hippocampus and the frontal
cortex of MIA-treated adolescent mice, indicating that crystallin induction is transient and characteristic of the acute phase
of the MIA exposure. Furthermore, comparing our data to
previous transcriptome studies of MIA offspring suggests that
the fetal and adolescent/adult brain transcriptome changes do
not share significant commonalities.17,51,52
In addition to crystallin upregulation, several other genes
are altered in all three models of MIA tested here. For some of
them (mip, si, tnnc2), there is insufficient knowledge about the
function of the proteins they encode, which makes it difficult
to envision their particular roles in the brain, while other
transcripts are likely to play an important role in brain
development. Aldh1a1 is an important player in environmentally induced oxidative damage,53 and this might explain its
strong upregulation in these MIA models. As an enzyme,
ALDH1A1 exerts a cellular protective role by metabolizing
highly reactive aldehydes, including ethanol metabolite
acetaldehyde and major products of lipid peroxidation.
ALDH1A1 is best known for converting retinaldehyde to
retinoic acid, and altered retinoic acid levels in the brain might
disrupt normal anteroposterior neural development.54 Similarly, sterol-C4-methyl oxidase-like protein (SC4MOL) is
localized to the endoplasmic reticulum membrane and is
believed to be involved in cholesterol biosynthesis, which
when dysregulated has clinical manifestations in neurological
development.55 In addition, atonal homolog 7 (atoh7) is
involved in the development of the optic nerve.56 Therefore,
the upregulation of these three genes, aldh1a1, sc4mol and
atoh7, may represent an important change in the molecular
environment of the embryonic brain that could result in
aberrant neural development. Finally, we find that the expression levels of many genes with known roles in cell cycle
regulation, neuronal development and differentiation (for
example, cyclin-dependent kinase 12, cyclin-dependent
kinase 17, insulin-like growth factor binding protein 3, neural
cell adhesion molecule 1 and 2, transforming growth factor b2,
transducer of ERBB2, 2, and so on) are highly correlated with
the expression of the crystallins (Supplemental Material 2).
The common gene expression pattern across the various
MIA treatments, in conjunction with previous findings, argues
that IL-6 is a critical mediator of MIA, and that crystallins are
important molecular mediators of this process. This raises
several important questions. First, is the IL-6/crystallin
cascade a promising target for molecular intervention in the
context of schizophrenia and autism treatment or prevention?
Second, are the currently used anti-inflammatory therapies
(which show promise as adjuvant therapy in schizophrenia)
modulating the IL-6/crystallin pathway, and acting through this
mechanism? Third, how is crystallin overexpression in the
embryonic brain disrupting normal development, and does it
alter the typical connectivity between the developing brain

Embryonic MIA transcriptome profile

KA Garbett et al

regions? Finally, what is the critical time window for modulating the IL-6/crystallin pathway during which we can reverse
the detrimental behavioral effects of MIA? The answers to
these critical questions remain mostly unknown to date, and
will have to be answered by comprehensive follow-up
experiments.
Overall, based on our findings and the known molecular
effects of crystallins, we propose that the induction of the
crystallin genes in response to MIA is a neuroprotective
attempt of the developing brain to counteract environmental
stress. However, this response is likely to have detrimental
consequences. Owing to their additional roles in neuronal differentiation and axonal growth,42,43 overexpression of crystallins (and other genes such as aldh1a1, sc4mol and atoh7)
might tip the delicate balance between neurogenesis and
differentiation in the embryonic brain. This view is strongly
supported by the observation that poly(I:C) MIA leads to
decreased hippocampal and cortical neurogenesis.57,58 We
also propose that once the immune activation disappears, the
normal brain developmental program resumes, but with a
time lag. As a result, layer formation in the cortex59 and
communication between the various brain regions is altered,
leading to permanent changes in connectivity and neurochemistry.60 This ultimately results in the various behavioral
abnormalities that are found in the offspring of all three MIA
models. Finally, we also argue that this cascade of events
might parallel the mechanisms by which environmental insults
contribute to the risk of neurodevelopmental disorders such
as schizophrenia and autism.
Conflict of interest
The authors declare no conflict of interest.

Acknowledgements. We are thankful for the microarray work performed

by the Genome Sciences Resource at Vanderbilt University and for valuable
comments of the manuscript provided by Martin J Schmidt. The experiments were
supported by NIH R01 MH067234 (KM), MH079299 (KM), Cure Autism Now (PHP),
McKnight Foundation Neuroscience of Brain Disorder Award (PHP), NIH RO1
MH067978 (PHP), Autism Speaks Dennis Weatherstone Pre-Doctoral Fellowship
(EYH) and NRSA 5 T32GM07737 (EYH).

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