Effects of maternal immune activation on gene expression patterns in the fetal brain

Effects of maternal immune activation on gene

expression patterns in the fetal brain

KA Garbett

1,5

, EY Hsiao

2,5

,SKa

lma

1,3

, PH Patterson

and K Mirnics

1,4

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

and

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)

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 Derkits

estimate that

30% 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 mother’s 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

10 000 cases in the Danish Medical Registry revealed an

association between viral or bacterial infection in the mother

and increased risk for the offspring.

Also similar to schizo-

phrenia 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 non-

twin 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.

First,

intranasal instillation of human influenza virus in pregnant

mice or non-human primates induces a moderate but sub-

lethal infection, and the offspring display a series of histological

and molecular abnormalities in the hippocampus and cortex.

8,9

Young and adult mouse MIA offspring

also exhibit a

cerebellar neuropathology that is commonly found in aut-

ism.

11,12

Furthermore, adult mice born to infected mothers

display behavioral abnormalities that are relevant to both

schizophrenia and autism, including deficits in social interac-

tion, prepulse inhibition, open field and novel object explora-

tion,

as well as heightened responsivity to a hallucinogen.

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 lipopoly-

saccharide 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 GABA

receptor

2 immunor-

eactivity, dopamine hyperfunction, delay in hippocampal

myelination, reduced NMDA receptor expression in the

hippocampus, reduced numbers of reelin- and parvalbumin-

positive cells in the prefrontal cortex, reduced dopamine D1

and D2 receptors in the prefrontal cortex and enhanced

tyrosine hydroxlyase in striatal structures.

Third, MIA can be

induced more directly by a single injection of the cytokine

interleukin IL-6 in pregnant mice.

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

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

Department of Psychiatry, Vanderbilt University, Nashville, TN, USA;

Division of Biology, California Institute of Technology, Pasadena, CA, USA;

Department of

Psychiatry, University of Szeged, Szeged, Hungary and

Vanderbilt 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

These two authors contributed equally to this work.

Keywords:

autism; brain; crystallin; gene expression; maternal immune activation; schizophrenia

Citation: Transl Psychiatry (2012) 2, e98,

doi:10.1038/tp.2012.24

&

www.nature.com/tp

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.

Flavonoids that block JAK/STAT

signaling downstream of the IL-6 receptor also block the

induction of abnormal behaviors by maternal IL-6 injection.

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 Ja

ckson 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 con-

ditions that previously resulted in offspring altered beha-

vior.

10,17

To allow for time to develop a flu infection, pregnant

mice on E9.5 were anesthetized intraperitoneally with

10 mg kg

xylazine and 100 mg kg

ketamine and inocu-

lated intranasally with 6000 plaque-forming units of human

influenza virus in 90

l 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 kg

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

body weight. For rIL-6 injections, 5

g carrier-

free, mouse rIL-6 (eBioscience, San Diego, CA, USA) was

freshly dissolved in 150

l saline and injected intraperitone-

ally. 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

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 biotin-

labeled 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 4–5 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,

we estimated that we had an 80%

probability to identify significant gene expression changes

that were

30%. 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 treat-

ment and control exceeded 30% (|ALR|

0.3785) and (2) the

statistical significance of differences was

o

0.05 using the

Student’s

-test. The G

enePattern 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. Indi-

vidual 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

cryaa

) (F: 5

-TCTT

CTTGGACGTGAAGCAC-3

,R:5

-GAAATGTAGCCATGGT

CATCC-3

), crystallin

cryab

) (F: 5

-TGTGAATCTGGA

CGTGAAGC-3

,R:5

-TGACAGGGATGAAGTGATGG-3

crystallin

A1 (

cryba1

) (F: 5

-CCTGGAAAGAGGAGAATA

CCC-3

,R:5

-TTATGATTAGCGGAACAGATGG-3

), crystallin

A2 (

cryba2

) (F: 5

-ACCAGCAAAGATGTGGGTTC-3

,R:

-GCCTAATGCTGGACTCTTCG-3

), crystallin

A4 (

cryba4

)

(F: 5

-CACCACTCAGGTGACTACAAGC-3

,R:5

-CCAGA

GGACACAAGGGTAGC-3

), crystallin

B1 (

crybb1

) (F:

-TCCCAGGAACATAAGATCTGC-3

,R:5

-ACGGTCACA

GAAGCCATAAAC-3

), crystallin

B3 (

crybb3

) (F: 5

-CAG

CCGACGTAGTGACATTC-3

,R:5

-TCATCTACGATCTCC

ATCTTGC-3

), crystallin

B/A/C (

crygb/a/c

) (F: 5

-AGCG

AGATGGGAAAGATCAC-3

,R:5

-AGTACTGGTGGCCCT

GGTAG-3

) and crystallin

C/A (

crygc/a

) (F: 5

-TGCGGCT

GTATGAGAAAGAA-3

,R:5

-CCTCGGTAGTTAGGCATC

TCA-3

All of these custom-designed primers reported amplification

efficiency

90%. 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

etal

Translational Psychiatry

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

-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-6-

treated 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 dif-

ference from saline-injected controls and a statistical signi-

ficance of

o

0.05, 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 3-

day 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 transcrip-

tomes. 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 (

0.88,

1.8E-84 and

0.85,

5.95E-72,

respectively) (Figures 1b and c). Likewise, the 195 differen-

tially expressed genes in the IL-6-MIA group show a

significant expression difference in the flu-model and

poly(I:C)-model brains (

0.85,

1.42E-55 and

0.85,

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 (

0.59,

8.78E-29 and

0.59,

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 (

30%

expression change and

o

0.05 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

cryab

cryba2

cryba4

and

crygc/a

. The qPCR data are

highly correlated with the microarray data (flu:

0.82,

0.007; poly(I:C):

0.84,

o

0.005; rIL-6:

0.89,

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 flu-

exposed 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,

0.001;

cryba1

: ddCt

1.39,

0.01;

crybb1

: ddCt

2.00,

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

the frontal cortex and hippocampus of 15-week-old pups

(

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 (

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

Embryonic MIA transcriptome profile

KA Garbett

etal

Translational Psychiatry

saline-treated dams (

15) at 12 months of age. Thus, we

conclude that the upregulation of crystallin genes observed

across the three MIA models is transient.

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:

0.00016; poly(I:C):

0.03439; IL-6:

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

0.85,

0.072;

cryba1

0.72,

0.138;

crybb1

0.92,

0.041, respectively). Thus,

the response of the crystallin genes corresponds closely with

the degree of MIA.

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.

Figure 1

The transcriptome of the embryonic brain is altered in three maternal immune activation (MIA) models. (

) All differentially expressed transcripts in the MIA

treatments were subjected to a two-way (horizontal: genes; vertical: samples) unsupervised clustering using GenePattern. Gene expression differ

ences are color-coded (red—

increased expression; blue—decreased 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. (

–

)

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 t

he 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 coho

rts. Thus, the

pattern of differentially expressed genes in the flu treatment was tested in the poly(I:C) and rIL-6 cohorts (

and

, respectively), the pattern of differentially expressed genes in

the rIL-6 treatment was tested in the flu and poly(I:C) cohorts (

and

, respectively) and the pattern of differentially expressed genes in the poly(I:C) treatment was assessed in

the flu and rIL-6 cohorts (

and

, 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.

Embryonic MIA transcriptome profile

KA Garbett

etal

Translational Psychiatry

Discussion

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

and

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

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

cryaa

), crystallin

B/C/A (

crygb/c/a

), crystallin

C/A (

crygc/a

), crystallin

B1 (

crybb1

), crystallin

A2 (

cryba2

), crystallin

(

crybb3

), crystallin

A1 (

cryba1

), crystallin

A4 (

cryba4

), crystallin

cryab

)) on

pools of RNA derived from the three MIA treatments. The

axis denotes average

log 2 ratios reported by microarrays, whereas the ddCt qPCR values are plotted on

the

axis. Note that the qPCR and microarray-reported expression changes are

highly correlated across the three MIA models.

Figure 4

Crystallin expression is variable in individual brains from flu-exposed

embryos. Expression of crystallin

cryaa

), crystallin

A1 (

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.

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

axis denotes the magnitude of expression change

measured in ddCT (1Ct

2-fold change) and the

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.

Embryonic MIA transcriptome profile

KA Garbett

etal

Translational Psychiatry

changing. Nonetheless, the common gene expression pat-

terns 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 dysregula-

tion 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

B-

crystallin protein levels are detected in the frontal cortex of

autistic brains.

In addition, anti-

B crystallin antibodies are

reported to be elevated in the sera of autistic subjects

compared with controls.

Moreover, increased expression

B-crystallin is detected in Rett syndrome brains,

and

B-crystallin is found in inclusions in brains from patients with

Fragile X-associated tremor/ataxia syndrome.

In schizo-

phrenia, altered expression of both

-crystallin and

-crystallin

are reported in the anterior cingulate cortex

and prefrontal

cortex.

27,28

Furthermore, dysregulated brain crystallin levels

are implicated in the manifestation of a variety of neurod-

egenerative disorders, including Parkinson’s disease, Alzhei-

mer’s disease, multiple sclerosis, Creutzfeldt–Jakob disease

and Alexander disease.

29–35

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

B-crystallin in Alzheimer’s disease or experimental

autoimmune encephalitis mice causes worsened symp-

toms.

36,37

These effects in experimental autoimmune

encephalitis mice are in turn ameliorated by systemic delivery

of recombinant

B-crystallin,

as are the effects of experimental

stroke in mice.

Similarly, transgenic overexpression of

B-crystallin ameliorates symptoms in a mouse model of

Alexander disease, a fatal developmental neurodegenerative

disorder originating in astrocytes.

In addition,

A-crystallin

exhibits anti-apoptotic protective effects in experimentally

induced inflammation of the uvea,

and pretreatment with

-crystallin blocks systemically induced inflammation in the

brain.

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,

A-crystallin-mediated

inhibition of caspase-3 rescues Pax6-deficient dopaminergic

neurons from apoptosis.

Likewise,

B-crystallin protects

against Fas/APO-1-induced cell death

in vitro.

Crystallins can

also confer neuroprotection by modulating cytoplasmic

calcium levels. Retinal

B2-crystallin, for example, coloca-

lizes with synaptotagmin 1, a primary calcium sensor, as well

as calmodulin, a major calcium-binding protein,

and several

crystallins harbor Greek key motifs that sequester calcium

ions.

44–46

These properties in

- and

B2-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,

B-crystallin modulates

nuclear factor-

B activity based on its phosphorylation

status,

and pretreatment with

B-crystallin prevents nuclear

factor-

B-induced neurotoxic effects.

Moreover,

B-crystal-

lin administration

in vitro

and

in vivo

downregulates tumor

necrosis factor-

and nitric oxide synthase in activated

microglia.

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 crystal-

lin 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

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 environmen-

tally induced oxidative damage,

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.

Simi-

larly, 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.

In addition, atonal homolog 7 (

atoh7

)is

involved in the development of the optic nerve.

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 expres-

sion 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

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

etal

Translational Psychiatry

regions? Finally, what is the critical time window for modulat-

ing 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 dif-

ferentiation and axonal growth,

42,43

overexpression of crystal-

lins (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

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 cortex

and

communication between the various brain regions is altered,

leading to permanent changes in connectivity and neuro-

chemistry.

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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Embryonic MIA transcriptome profile

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etal

Translational Psychiatry