Prenatal maternal immune activation causes epigenetic differences in adolescent mouse brain

Abstract

Epigenetic processes such as DNA methylation have been implicated in the pathophysiology of neurodevelopmental disorders including schizophrenia and autism. Epigenetic changes can be induced by environmental exposures such as inflammation. Here we tested the hypothesis that prenatal inflammation, a recognized risk factor for schizophrenia and related neurodevelopmental conditions, alters DNA methylation in key brain regions linked to schizophrenia, namely the dopamine rich striatum and endocrine regulatory centre, the hypothalamus. DNA methylation across highly repetitive elements (long interspersed element 1 (LINE1) and intracisternal A-particles (IAPs)) were used to proxy global DNA methylation. We also investigated the Mecp2 gene because it regulates transcription of LINE1 and has a known association with neurodevelopmental disorders. Brain tissue was harvested from 6 week old offspring of mice exposed to the viral analog PolyI:C or saline on gestation day 9. We used Sequenom EpiTYPER assay to quantitatively analyze differences in DNA methylation at IAPs, LINE1 elements and the promoter region of Mecp2. In the hypothalamus, prenatal exposure to PolyI:C caused significant global DNA hypomethylation (t=2.44, P=0.019, PolyI:C mean 69.67%, saline mean 70.19%), especially in females, and significant hypomethylation of the promoter region of Mecp2, (t=3.32, P=0.002; PolyI:C mean 26.57%, saline mean 34.63%). IAP methylation was unaltered. DNA methylation in the striatum was not significantly altered. This study provides the first experimental evidence that exposure to inflammation during prenatal life is associated with epigenetic changes, including Mecp2 promoter hypomethylation. This suggests that environmental and genetic risk factors associated with neurodevelopmental disorders may act upon similar pathways. This is important because epigenetic changes are potentially modifiable and their investigation may open new avenues for treatment.

Citation: Transl Psychiatry (2014) 4, e434; doi:10.1038/tp.2014.80 2014 Macmillan Publishers Limited All rights reserved 2158-3188/14

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ORIGINAL ARTICLE

Prenatal maternal immune activation causes epigenetic differences in adolescent mouse brain

P Basil1, Q Li1, EL Dempster2, J Mill2,3, P-C Sham1,4, CCY Wong3 and GM McAlonan1,5

Epigenetic processes such as DNA methylation have been implicated in the pathophysiology of neurodevelopmental disorders including schizophrenia and autism. Epigenetic changes can be induced by environmental exposures such as inammation. Here we tested the hypothesis that prenatal inammation, a recognized risk factor for schizophrenia and related neurodevelopmental conditions, alters DNA methylation in key brain regions linked to schizophrenia, namely the dopamine rich striatum and endocrine regulatory centre, the hypothalamus. DNA methylation across highly repetitive elements (long interspersed element 1 (LINE1) and intracisternal A-particles (IAPs)) were used to proxy global DNA methylation. We also investigated the Mecp2 gene because it regulates transcription of LINE1 and has a known association with neurodevelopmental disorders. Brain tissue was harvested from 6 week old offspring of mice exposed to the viral analog PolyI:C or saline on gestation day 9. We used Sequenom EpiTYPER assay to quantitatively analyze differences in DNA methylation at IAPs, LINE1 elements and the promoter region of Mecp2. In the hypothalamus, prenatal exposure to PolyI:C caused signicant global DNA hypomethylation (t = 2.44, P = 0.019, PolyI:C mean69.67%, saline mean 70.19%), especially in females, and signicant hypomethylation of the promoter region of Mecp2, (t = 3.32, P = 0.002; PolyI:C mean 26.57%, saline mean 34.63%). IAP methylation was unaltered. DNA methylation in the striatum was not signicantly altered. This study provides the rst experimental evidence that exposure to inammation during prenatal life is associated with epigenetic changes, including Mecp2 promoter hypomethylation. This suggests that environmental and genetic risk factors associated with neurodevelopmental disorders may act upon similar pathways. This is important because epigenetic

changes are potentially modiable and their investigation may open new avenues for treatment.

Translational Psychiatry (2014) 4, e434; doi:10.1038/tp.2014.80; published online 2 September 2014

INTRODUCTIONEpigenetic processes are crucial for the regulation of genomic function and development.1 For example, DNA methylation is implicated in X-inactivation, genomic imprinting control and remote silencing of genomic regions.2 Tissue-specic epigenetic modications through CpG methylation are thought to modulate brain development, circadian rhythm and disease status.35

Consistent with this, epigenetic anomalies have been linked to complex neurodevelopmental disorders including schizophrenia and autism spectrum disorders.6,7

In early brain development, environmental exposures such as inammation,8 diet,9,10 toxins and contaminants11 disrupt developmental trajectories. For example, prenatal exposure to inammation has been implicated in the etiology of schizophrenia12

autism13 and bipolar disorder.14 Direct evidence supporting a role for maternal immune activation (MIA) during prenatal life in neurodevelopmental conditions has come from rodent studies from our group and others.1517 The consensus is that the MIA model mimics many features relevant to schizophrenia and autism in humans.18,19

Although not a genetic model, gene and protein expression differences have consistently been reported in MIA rodent models, including in the adult frontal cortex20 and the fetal whole brain.17,21,22 In an MIA model elicited by exposing pregnant

rodents to the viral analog Polyinosinic:polycytidylic acid (PolyI:C),

interleukin 6 (IL6)-induced inammation has been shown to exert an epigenetic inuence by regulating the methyltransferase gene.23 Consistent with this, IL6 is been shown to alter global DNA methylation in diseases such as oral cancer;24 however, it is unknown whether this occurs in the brain of offspring exposed to MIA.

One function of DNA methylation is to repress the transcription of repetitive retroviral elements in the genome including long interspersed element 1 (LINE1) or intracisternal A-particles (IAPs). IAPs are endogenous retroviral sequences, an important class of transposable elements that jump within the genome inducing genomic mutations and cell transformation. LINE1 elements are retrotransposons - a subclass of transposons that also modulate gene expression, especially in the developing brain.25 However, whether prenatal exposure to maternal inammation alters methylation state of these repetitive elements has not been directly examined.

An important target for epigenetic modication in models of neurodevelopment is the Methyl CpG-binding protein2 (Mecp2).26

MECP2 is involved in the timely activation and repression of gene expression. It has an important role in control of methylation27

and has a regulatory role in neuronal transcription of LINE1.25 It is

strongly associated with neurodevelopmental disorders such as Rett syndrome,28 autism,29 schizophrenia and neural tube

1Department of Psychiatry, The University of Hong Kong, Pokfulam, Hong Kong SAR, China; 2University of Exeter Medical School, Exeter University, Exeter, UK; 3MRC Social, Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, King's College London, London, UK; 4Centre for Genomic Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China and 5Department of Forensic and Neurodevelopmental Sciences, Institute of Psychiatry, Kings College London, London, UK. Correspondence: Dr GM McAlonan, Department of Forensic and Neurodevelopmental Sciences, Institute of Psychiatry, Kings College London, De Crespigny Park, Denmark Hill, London SE5 8AF, UK. E-mail: mailto:[email protected]

Web End [email protected] Received 26 February 2014; revised 2 July 2014; accepted 14 July 2014

Postnatal epigenetic effects of prenatal MIA P Basil et al

2 defects.30,31 To date however, whether Mecp2 is epigenetically

altered in the MIA model has not been investigated.

In this study we tested the hypothesis that prenatal exposure to MIA in the mouse results in global methylation differences in the brain and specically alters DNA methylation in the promoter of Mecp2. We elected to study the striatum because of its established association with schizophrenia32 and autism,33 and the hypothalamus that has an important inuence on the limbic system and is thought to have a critical role in the onset of prodromal features of schizophrenia during the pubertal/postpubertal period.34,35

MATERIALS AND METHODSAnimal model of neurodevelopmental disorders

Female and male C57BL/6N mice were bred and mated in the Laboratory Animal Unit, The University of Hong Kong. The holding space had a 12:12 h normal lightdark cycle (lights off at 1900 hours), and temperature and humidity-controlled (21 1 C, 55 5%) animal vivarium. Pregnant females were not disturbed, except for weekly cage-cleaning. Animals were maintained under ad libitum food and water supplied by the Laboratory Animal Unit. All experiments were performed in accordance with the relevant institutional and national guidelines and regulations approved by the Committee on the Use of Live Animals in Teaching and Research at The University of Hong Kong, and every effort was made to minimize the number of animals used and their suffering.

The MIA model was prepared following the procedures previously reported in our laboratory and others.18,36 Potassium salt of PolyI:C was obtained from Sigma-Aldrich (Gillingham, UK) and dissolved in saline. A dose of 5 mg kg1 in an injection volume of 5 ml kg1, prepared on the day of injection, was administered to pregnant dams on gestation day 9 via the tail vein under gentle physical restraint. The resulting offsprings were weaned and sexed at postnatal day 21. The pups were weighed and littermates of the same sex were caged three to four per cage.

At 6 weeks, mice were killed by cervical dislocation, and their brains were removed quickly. The hypothalamus and striatum were collected in1.5-ml tubes using careful dissection methods referring the Allen Mouse Brain Atlas37 and frozen in liquid nitrogen for storage. Ten to fty milligrams of brain tissue were homogenized on ice in a new reaction tube with freshly made and autoclaved lysis buffer (10 mM Tris-HCl, 0.1 M EDTA,0.5% sodium dodecyl sulfate) using a DNase-free, sterile plastic pestle. Samples were lysed by incubation with 300 ml of lysis buffer and 3.3 l Proteinase K (18.5 mg ml1) for 16 h in a water bath at 50 C. Samples were mixed thoroughly to break up any unlysed tissue and 1.65 l Proteinase K(18.5 mg ml1) was added to the sample followed by 1-h incubation at 50 C. Proteinase K was inactivated by incubating samples at 65 C for 30 min. Lysates were transferred to 2 ml eppendorf tubes and extracted with

300 ml of phenolchloroformisoamyl alcohol (25:24:1) buffered to pH 7.5 by inverting 20 times and centrifuging at 13 000 r.p.m. for 15 min. The aqueous layer was extracted twice with 300 ml of chloroformisoamyl alcohol (24:1) in a new reaction tube, by inverting 20 times and centrifuging at 13 000 r.p.m. for 15 min. The aqueous layer was incubated with 75 l of 10 M ammonium acetate (NH4Ac) and 600 ml ice-cold 100%

ethanol in a new reaction tube at 20 C for 1 h. The supernatant was removed after centrifugation at 13 000 r.p.m. for 15 min and the pellet was washed with 1 ml of 70% ethanol. DNA was pelleted by centrifuging at 13 000 r.p.m. for 10 min. The pellets were left to air dry for 1 h and resuspended in 100 l of TE buffer. A NanoDrop ND-1000 was used to quantify the DNA samples, and the quality and size of samples were checked using a 1% agarose gel using gel electrophoresis.

Sodium bisulte conversionIn all, 500 ng of genomic DNA from each sample was treated with sodium bisulte in duplicate, using the EZ 96-DNA methylation kit (Zymo Research, Orange, CA, USA), following the manufacturer's standard protocol. The kit exploits the three-step reaction that takes place between cytosine and sodium bisulte where unmethylated cytosine is converted to uracil. The DNA was resuspended in 30 l of distilled water and stored at 20 C until further use.

LINE1 and IAP assayWe measured highly repetitive element methylation, blinded to epidemiological exposure condition. Previously reported sequences of forward

and reverse primers of LINE1 and IAP38,39 Sigma-Aldrich were used for analysis with the Sequenom EpiTYPER (San Diego, CA, USA), using universally methylated DNA as a methylated reference (EMD Millipore Corporation, Darmstadt, Germany), and an unmethylated DNA as negative control. The LINE1 and IAP element assays were designed to cover more than 600 000 genomic locations, which share a consensus sequence.40

Mecp2 promoter DNA methylation assayMecp2 promoter DNA methylation was quantied using the Sequenom

EpiTYPER platform, and primers for the target region were designed using EpiDesigner (http://www.epidesigner.com

Web End =www.epidesigner.com, Sequenom). Six CpG sites were quantied in an amplicon spanning 282 bp in the promoter and the rst exon (Please see Supplementary Table 1 for primer details). All EpiTYPER assays were tested using a standard curve with methylated and unmethylated DNA. Genomic DNA was isolated and 1 g DNA was bisulte-treated as described above using the EZ DNA methylation kit (Zymo Research).

Statistical analysisGlobal DNA methylation was quantied in the two brain regions of interest (the hypothalamus and striatum) from 22 PolyI:C-exposed and 17 saline-exposed 6-week-old mice using the Sequenom EpiTYPER platform. The sample characteristics are shown in detail in Table 1. The samples were handled together to reduce experimental/handling bias at all stages of experiments. In all group comparisons each animal was treated as an individual subject. Both LINE1 and IAP assays measure a proxy of global DNA methylation, and Pearson correlation testing was used to identify any correlation between assays. A general linear model (GLM) was used to identify any main effect of group or sex or any interaction with unpaired t-tests to explore the data post hoc. Grubbs test using extreme studentized deviate method was used to detect outliers with an alpha cutoff 40.01.

The criterion for statistical signicance was set as Po0.05 and analyses were conducted using R (http://www.R-project.org/

Web End =http://www.R-project.org/) and SPSS. The effect sizes reported are the absolute change (delta) in DNA methylation.

RESULTSAltered global methylation in the adolescent brain following prenatal immune challenge

Quantitative DNA methylation was measured across LINE1 elements to give a proxy of global DNA methylation across ~ 600 000 repeats in the mouse genome.41,42

LINE1 methylation in the hypothalamusThere was a signicant main effect of treatment (F (1, 36) = 5.72, = 0.516, P = 0.022), with no effect of sex (F (1, 36) = 1.27, = 0.248, P = 0.266) on the mean LINE1 methylation, and 12.18% of variance was explained with the GLM used. This resulted from signicant hypomethylation in the hypothalamus of the PolyI:C group (69.66%, s.d. = 0.62) compared with the saline group(70.19%, s.d. = 0.72; t = 2.44, P = 0.019, delta methylation = 0.53; see Table 2), especially in females (t = 3.04, P = 0.005, PolyI:C group mean 69.45, s.d. = 0.59, saline group mean 70.26, s.d. = 0.71, delta methylation = 0.81).

Table 1. Animal numbers

Group PolyI:C Saline

Litter 6 weeks M1 M2 M3 M4 M5

Sex (male:female) 3:3 3:6 2:5 4:5 3:5

Total 22 17

PolyI:C group (litter M1, M2 and M3) and saline group (M4 and M5) with the number of males and females in each litter. Animals used in the experiment were, as far as possible, kept free from any interventions that could further affect their epigenetic status.

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Table 2. PolyI:C and saline group comparison results of different assays

LINE1 IAP Mecp2

Mean s.d. P-value Mean s.d. P-value Mean s.d. P-value

Hypothalamus0.696 0.006 0.265 0.067 PolyI:C F 0.694 0.005 0.935 0.014 F 0.290 0.0370.019* 0.002** M 0.700 0.005 M 0.148 0.042

F 0.005** 0.47 F 0.000***0.701 0.007 0.346 0.059M 0.885 M 0.202

Saline F 0.702 0.007 0.931 0.012 F 0.362 0.046 M 0.700 0.007 M 0.266 0.068

Striatum0.707 0.011 0.930 0.050 0.295 0.179PolyI:C 0.29 0.85 0.37 Saline 0.704 0.010 0.936 0.066 0.345 0.121

Abbreviations: F, Female; IAP, intracisternal A particle; LINE1, long interspersed elements; M, Male; Mecp2, Methyl CpG Binding Protein2. Mean methylation of all the markers for each assay. *Po0.05, **Po0.005 and ***Po0.0005.

The rst CpG in the LINE1 element in the hypothalamus was differentially methylated in adolescent stage after PolyI:C administration in prenatal life, main effect of Group (F (1, 36) = 5.77, = 1.503, P = 0.021). In all, 9.08% of the variance was explained by the GLM used. There was no signicant main effect of sex (F (1,36) = 0.08, = 0.183, P = 0.775); however, there was a signicant Group x Sex interaction (F (1, 35) = 5.54, = 2.846, P = 0.024) and the GLM explained 19.27% of variance. This was explained by signicant hypomethylation in the PolyI:C group (t = 2.42, delta methylation = 1.49, P = 0.02; PolyI:C group mean = 27.68%, saline group = 29.17%, s.d. = 2.03). Again, differences were most pronounced in females (t = 4.11, delta methylation = 2.62, Po0.001).

LINE1 methylation in the striatumThere was no signicant main effect of group, or sex, or any Group x Sex interaction on the mean LINE1 methylation in the striatum (PolyI:C group mean = 70.79, s.d. = 1.15 and saline group mean =70.41, s.d. = 1.01).

IAP assayIAP promoters were not differentially methylated in the hypothalamus or striatum in the PolyI:C and saline control groups, see Table 2. LINE1 and IAP assays for the mean global methylation were not correlated (P = 0.298, r = 0.170).

Mecp2 promoter methylationThe mean methylation of Mecp2 in the polyI:C-exposed hypothalamus was lower than in saline groups (PolyI:C group = 26.57%, s.d. = 6.70; saline group = 34.63%, s.d. = 5.97). There was a signicant effect of group and sex in the mean methylation of the Mecp2 promoter region in the hypothalamus: (F (1, 26) = 24.08, =

7.937, Po0.001) and (F (1, 26) = 34.49, = 12.384, Po0.001), respectively. In total, 67.18% of variance in Mecp2 methylation was explained by the GLM used. There was no signicant Group x Sex interaction. Post hoc tests conrmed Mecp2 promoter hypomethylation in the PolyIC group (t = 3.39, delta methylation = 8.05, P = 0.003) and in females (t = 4.35, delta methylation = 7.14, Po0.001). Please see Figure 1 and Table 2.

Individual CpG sites 2 and 4 were signicantly differentially methylated in each group (F (1, 26) = 13.12, = 9.870, P = 0.001; F (1, 26) = 11.71, = 6.244, P = 0.002, respectively) and sex (F (1,26) = 10.84, = 11.294, P = 0.003; F (1, 26) = 13.08, = 8.604,

P = 0.001, respectively). The mean methylation for CpG site 2; PolyI:C group = 20.35%, s.d. = 8.71, saline group = 30.33%, s.d. = 7.24 and CpG site 4; PolyI:C group mean = 30.58%, s.d. = 6.21, saline group = 36.91%, s.d. = 5.19. There was no signicant Group x Sex interaction. A total of 15.63% of variance in CpG 2 methylation and 45.22% of variance in CpG 4 methylation were explained by the GLM used.

Mean methylation of Mecp2 in polyI:C-exposed striatum was also lower than controls but this difference did not reach statistical signicance (please see Figure 1 and Table 2).

DISCUSSIONThis is the rst study to directly examine the epigenetic consequences of prenatal exposure to MIA in the adolescent rodent brain. We found signicant DNA hypomethylation in the hypothalamus and a similar trend in the striatum of offspring exposed to MIA compared with saline-exposed controls. In addition, the Mecp2 gene promoter region was signicantly hypomethylated in the hypothalamus of MIA-exposed group but not in the striatum. These effects were most pronounced in females.

LINE1 methylationApproximately 50% of the mammalian genome comprises the retrotransposon-associated repeat sequences, with LINE1 elements and LTR elements (including IAP) accounting for 20%40

and 10%, respectively.43 LINEs are ~ 6-kb-long elements with an internal polymerase II promoter encoding two open reading frames and account for a great amount of somatic mosaicism.44

LINE1 elements transpose through RNA intermediates, assembling with their own encoded proteins through reverse transcription. LINE1 is highly active and can create indels, new splice sites which ne-tune gene expression.45 LINE1 transposons may also disrupt coding regions, and their epigenetic suppression by causing indels is vital during the early course of development.46 Inappropriate suppression caused by altered DNA methylation across these elements may lead to adverse consequences in the adult life.47

Consistent with this role in development, LINE1 has been reported to be differentially methylated and highly susceptible to retrotransposition in neurodevelopmental disorders.25 For exam

ple, hyperactive retrotransposition and higher copy number of LINE1 have been reported in schizophrenia and observed in the

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Figure 1. Hypomethylated Mecp2 promoter in the hypothalamus of PolyI:C-exposed offspring. y axis shows methylation in percentage with error bar as s.e.m., histograms represent saline- and polyI:C-treated male and female groups. (a) Mecp2 promoter methylation in the hypothalamus of polyI:C- and saline-exposed male and female groups. (**P-value is calculated from a stratied t-test between saline- and polyI:C-affected females). (b) Mecp2 promoter methylation in the striatum of polyI:C and saline-exposed male and female groups.

polyI:C MIA model.48 Our observation of LINE1 hypomethylation in the same MIA model would be expected to increase retro-transposition and may provide one possible path linking inammation with the increased risk of schizophrenia.

Altered DNA methylation as a potential mediator of environmental risk for neurodevelopmental disorders ts with evidence from other systems. For example, prenatal exposure to the antiepilepsy drug valproate is associated with increased risk of autism spectrum disorders in the offspring,49 and this drug has been recognized to cause widespread epigenetic reprogramming especially DNA hypomethylation.50

IAP methylationIAPs are autonomous retrotransposon elements with long terminal repeats and all the necessary transcriptional regulatory elements. These class-II endogenous retroviruses transpose through retroviral mechanisms in the cisternae of the endoplasmic reticulum.51 IAPs add a signicant amount of genetic variabilities to the genome with differential tissue-specic expression and are capable of interfering gene function in the brain.52,53 The

complexity and the size of IAP make it less common and less likely to occupy the vital regions in the genome. Thus, IAP assays target completely different elements than LINE1, with a lower representation over the genome,54 which may account for the sparsity of ndings using this assay in our study, and the lack of evidence linking IAP alterations with neurodevelopmental disorders.

Mecp2 methylationMECP2 has a well-established role in Retts syndrome but has also been implicated, by our group and others, in idiopathic neurodevelopmental disorders such as autism and schizophrenia.55,56 MECP2 is an abundant neuronal protein that

indirectly mediates gene transcription by recruiting transcription factor cAMP responsive element-binding protein 1, depending on the methylation status of the DNA.27 This action alters the expression of many genes in the hypothalamus57 and beyond, including LINE1. Aberrant promoter methylation of MECP2 itself is thought to contribute to a defective biological network and potentially to the etiology of neurodevelopmental disorders.58

Here we report a signicant association between MIA and Mecp2 promoter hypomethylation. The polyI:C used in this MIA model is known to cause an increase in IL6.59 MECP2 in turn may suppress IL6 expression;60 therefore, we speculate that hypomethylation, leading to a higher level of MECP2, may be involved in silencing genes that are activated by the immune response. Changes in Mecp2 could theoretically inuence the expression of

other developmental genes previously reported to be altered in the MIA model, for example, brain-derived neurotrophic factor61

and Reelin, Glutamate decarboxylase 6762 and cAMP response element-binding protein;63 histone deacetylases and DNA (cyto-sine-5)-methyltransferase 1.26,64 A theoretical construct showing a functional protein association network potentially inuenced by MECP2 is shown in Supplementary Information and Figure 1.65

More specically, in terms of the dopamine system, closely linked to disorders such as schizophrenia, we speculate that removing a methylation brake on Mecp2 gene expression would promote or even overdevelop dopaminergic neurons. This is because the converse - low levels of MECP2 in dopamine neurons - results in fewer dendrites, less membrane surface area and even a compromised nigrostriatal pathway.66

PolyIC caused greater LINE1 hypomethylation in the hypothalamus of female offspring; however, although the Mecp2 promoter was more hypomethylated in females in both polyI:C- and saline-exposed groups, there was no signicant Sex x Group interaction. Although neurodevelopmental disorders such as autism are generally more prevalent in males than females, this is not the case for schizophrenia,67 and Rett syndrome is predominantly diagnosed in females.68 Moreover, consistent with our results, genome-wide methylation studies of schizophrenia and other psychiatric conditions have reported global hypomethylation in females.69,70 This excess of epigenetic modication in the female sex may also be relevant to the suggestion that environmental exposures are relatively more important in females with neuro-developmental disorders.71

Regional methylation changesHypomethylation of Mecp2 and LINE1 in an adolescent offspring exposed to MIA was most pronounced in the hypothalamus. Hypothalamuspituitaryadrenal axis developmental changes, and especially hypothalamic dopamine systems, are strongly implicated in the onset of schizophrenia.35,72 Consistent with this evidence, MECP2 has been found to mediate dopaminergic precursor differentiation in the ventral midbrain especially in the developing hypothalamus via Dlk1.73 Similarly, aminergic neuro-transmitter concentration is regulated by MECP2 through rate-limiting enzymes involved in synthesis and degradation, which may contribute to phenotypes relevant to schizophrenia and autism.74

The direction of Mecp2 methylation differences in the striatum of MIA exposed mice relative to controls was similar to those observed in the hypothalamus; however, group differences did not reach statistical signicance. We also observed a main effect of sex - females were generally undermethylated relative to males in

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both PolyI:C and control groups in both regions of interest. Thus, whereas low power may have limited any group differences in methylation in the striatum, it is also possible that lower methylation in females of both groups may have obscured group differences in methylation in the striatum.

Advantages and limitations of the studyAn organisms phenotype is produced by its genotypes interaction with the environment and thus epigenetic changes inuence gene expression, DNA repair and recombination. Under the inuence of these epigenetic mechanisms, genetically identical cells, tissues and organs show variation and become unique during the course of development.75 However, investigating epigenetic etiological mechanisms in the brain, and understanding the effect of epigenetic modications resulting from the prenatal assaults in humans, is extremely challenging. This is partly because studies are indirect, relying upon peripheral blood, buccal swabs and other tissues.

Therefore, animal models can help to better control the environmental exposures and homogeneity of the sample. Moreover, the animal model allows DNA isolation from specic regions of the brain. This is important because patterns of DNA methylation are region- and tissue-specic,76,77 and peripheral

tissue samples collected in humans are unlikely to reect what is happening in the brain.78 However, although animal models allow us to sample brain, there are still technical constraints and it must be acknowledged that assays such as LINE145 and IAP provide only a proxy of global methylation.6

We also accept that the current study targeted restricted regions of the brain; therefore, we cannot say whether our ndings are truly region-specic or more generalized. Even though carefully dissected out, DNA was isolated from tissue comprising a mix of cells with possibly different epigenetic proles. Assays of single cell or minimal numbers of isolated cells could have improved cell type accuracy but also have restricted generalization. Furthermore, we used LINE1 and IAP assays only to obtain a proxy of global DNA methylation. These assays cannot reveal the individual genes affected, nor tell us whether the differences observed have functional implications. Future work will need to be conducted to ascertain the functionality of these methylation changes (especially the signicant, but very small, group difference in LINE1 DNA methylation in the hypothalamus).

Finally, we acknowledge that alternative design would be to consider each litter as a single experimental unit with the assumption that the exposure of fetuses within one litter is identical. However, this assumption may not always hold as each fetus in the mouse uterus has been shown to have a distinct blood perfusion pattern;79 therefore, exposures may not necessarily be identical. On balance, we wished to optimize genetic similarity so that the focus would be on environmental exposure. We therefore elected to treat each an offspring as a subject, and enter data from each littermate separately into the analysis.

SummaryThis study examined epigenetic differences in the hypothalamus and striatum of the adolescent brain following exposure to MIA in prenatal life, a risk factor for schizophrenia and related neurodevelopmental conditions. We observed differences in both global methylation and Mecp2 promoter methylation that may have relevance to neurodevelopmental disorders. In particular, hypomethylation of the Mecp2 promoter may have widespread repercussions on gene expression and deserves further exploration as a possible biomarker for environmental modications linked to prenatal assault. Further exploration of epigenetic mechanisms operating in neurodevelopmental conditions is warranted because these gene modications are potentially

modiable and this approach may open new avenues for treatment.

CONFLICT OF INTEREST

The authors declare no conict of interest.

ACKNOWLEDGMENTS

We thank members of the Psychiatric Epigenetic group for Infrastructural support for aspects of the study at Social, Genetic and Developmental Psychiatry, Department of Forensic and Neurodevelopmental Sciences, Kings College London and Statistical Genetics group, Department of Psychiatry, The University of Hong Kong (HKU). PB is supported by a Post Graduate Scholarship, HKU and the Graduate Research Exchange Scheme, Faculty of Medicine, HKU. The experimental work was supported by Hong Kong Universities General Research Fund award GRF_HKU 774710M. We also acknowledge ZEE Foundation, Hong Kong for their nancial support. GM is a member of the EU-AIMS consortium.

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