Manuscript Click here to view linked References Richetto et al. 2016 Genome-Wide DNA Methylation Changes in a Mouse Model of Infection-Mediated Neurodevelopmental Disorders Juliet Richetto1§, Renaud Massart2,3,§, Ulrike Weber-Stadlbauer1, Moshe Szyf3, Marco A. Riva4,5, Urs Meyer1* 1Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland. 2UMR7216 Epigenetics and Cell Fate, University Paris Diderot, Paris, France 3Department of Pharmacology and Therapeutics, McGill University, Montreal, QC, Canada 4Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy. 5Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy. §These authors contributed equally to the present study. *Corresponding author. Correspondence: Urs Meyer, Ph.D. Institute of Pharmacology and Toxicology University of Zurich-Vetsuisse Winterthurerstrasse 260, 8057 Zurich, Switzerland E-mail: [email protected] Tel.: +41 44 635 88 44; Fax.: +41 44 635 89 10 Running title: DNA Methylation Changes After Prenatal Infection. Key words: Autism; epigenetics; DNA methylation; maternal immune activation; poly(I:C); schizophrenia. Number of words in Abstract / Main Text: 249 / 4,722 Number of Tables / Figures: 1 / 7 Number of Supplemental Information: 5 1 Richetto et al. 2016 ABSTRACT Background: Prenatal exposure to infectious or inflammatory insults increases the risk of neurodevelopmental disorders. Using a well-established mouse model of prenatal viral- like immune activation, we examined whether this pathological association involves genome-wide DNA methylation differences at single nucleotide resolution. Methods: Prenatal immune activation was induced by maternal treatment with the viral mimetic poly(I:C) in middle (gestation day 9) or late (gestation day 17) gestation. Following behavioral and cognitive characterization of the adult offspring, unbiased capture array bisulfite sequencing was combined with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantitative real-time PCR analyses to quantify DNA methylation changes and transcriptional abnormalities in the medial prefrontal cortex of immune-challenged and control offspring. GO term enrichment analysis was used to explore shared functional pathways of genes with differential DNA methylation. Results: Adult offspring of immune-challenged mothers displayed hyper- and hypomethylated CpGs at numerous loci and at distinct genomic regions, including genes relevant for GABAergic differentiation and signaling (e.g., DLX1, LHX5, LHX8), Wnt signaling (WNT3, WNT8A, WNT7B), and neural development (e.g., EFNB3, MID1, NLGN1, NRX2). Altered DNA methylation was associated with transcriptional changes of the corresponding genes. The epigenetic and transcriptional effects were dependent on the offspring’s age and were markedly influenced by the precise timing of prenatal immune activation. Conclusions: Prenatal viral-like immune activation is capable of inducing stable DNA methylation changes in the medial prefrontal cortex. These long-term epigenetic modifications are a plausible mechanism underlying the disruption of prefrontal gene transcription and behavioral functions in subjects with prenatal infectious histories. 2 Richetto et al. 2016 INTRODUCTION Prenatal exposure to infectious or inflammatory insults is an environmental risk factor for brain disorders with neurodevelopmental components, including schizophrenia (1,2), autism (3,4), and bipolar disorder (5,6). This epidemiological association is further supported by translational work in animal models demonstrating abnormal brain development and behavioral dysfunctions following prenatal administration of infectious pathogens or immune activating agents (7-10). Cytokine-associated inflammatory events, together with downstream pathophysiological effects such as oxidative stress and (temporary) macronutrient and micronutrient deficiency, seem to be critical in mediating the adverse effects of maternal infection on the fetal system (2,11,12). These pathological processes can affect somatic cell development and change the offspring’s neurodevelopmental trajectories, which in turn can lead to the emergence of behavioral and cognitive disturbances in later life (13-16). Epigenetic modifications may be critical for mediating alterations in brain development and behavioral functions in response to in-utero immune challenges. Epigenetic mechanisms stably program genome activity and gene expression without altering the DNA sequence (17-19). Rodent models of prenatal immune activation now begin to reveal epigenetic alterations in the offspring’s brain and have already provided evidence for altered DNA methylation (20,21), histone modifications (22,23), and micro-RNA expression (24) that persist into adolescence and/or adulthood. Some of the epigenetic modifications induced by prenatal immune activation have been found to correlate with altered transcription of the corresponding genes and with behavioral deficits (21). These studies, however, were based on targeted approaches and did not assess global DNA methylation profiles across the entire genome. Hence, it remains unknown whether prenatal immune activation may cause altered DNA methylation in selected genomic regions or loci, or alternatively, whether such changes extend to the entire genome. The present study is the first to examine genome-wide DNA methylation differences at single nucleotide resolution by capture array bisulfite sequencing of promoters and 3 Richetto et al. 2016 enhancers using a well-established mouse model of prenatal viral-like immune activation. The model is based on maternal administration of the viral mimetic poly(I:C) (= polyriboinosinic-polyribocytidilic acid), which induces a cytokine-associated viral-like acute phase response in maternal and fetal compartments, including the fetal brain (10). Prenatal poly(I:C) treatment in rodents leads to multiple behavioral and cognitive disturbances in offspring, many of which are implicated in developmental psychiatric disorders such as schizophrenia and autism (7-12). The prenatal poly(I:C) exposure model thus offers a unique opportunity to explore genome-wide DNA methylation changes following prenatal exposure to an etiologically relevant risk factor of developmental psychiatric disorders. Since the precise timing of prenatal immune activation can critically influence the nature and/or severity of the long-term brain abnormalities (25-27), we compared alterations in DNA methylation in response to prenatal immune activation in middle and late gestation. We focused on DNA methylation profiles in the prefrontal cortex (PFC) in view of its role in developmental psychiatric disorders such as schizophrenia and autism (28-30). MATERIALS AND METHODS Animals and Prenatal Immune Activation C57Bl6/N mice were used throughout the study. Female mice were subjected to a timed mating procedure as established previously (31,32). As described in the Supplementary Information, pregnant dams on gestation day (GD) 9 or 17 were randomly assigned to receiving either a single injection of poly(I:C) (potassium salt; Sigma–Aldrich, Buchs, St. Gallen, Switzerland) or vehicle. Poly(I:C) (5 mg/kg) was dissolved in sterile pyrogen-free 0.9% NaCl (vehicle) solution to yield a final concentration of 1 mg/ml and was administered intravenously (i.v.) into the tail vein under mild physical constraint. The two gestational windows (i.e., GD 9 and 17) were selected based on our previous studies assessing the influence of the precise timing of prenatal viral-like immune activation (25,26). GD 9 in the mouse roughly corresponds to human gestational weeks 4 to 5 in 4 Richetto et al. 2016 terms of limbic neurogenesis, whereas GD 17 corresponds to human gestational weeks 28 to 29 in terms of cortical neurogenesis (http://translatingtime.net/translate). The allocation and housing of neonatal and adult offspring of dams injected with poly(I:C) on GD 9 (POL-GD9 offspring) or GD 17 (POL-GD17 offspring) and vehicle- exposed control offspring (CON offspring) are described in the Supplementary Information. All procedures described in the present study had been previously approved by the Cantonal Veterinarian’s Office of Zurich, and all efforts were made to minimize the number of animals used and their suffering. Behavioral Testing Behavioral testing in adult offspring started on postnatal day (PND) 100. The tests included paradigms assessing spatial recognition memory, social interaction, and prepulse inhibition (PPI) of the acoustic startle reflex. These tests were selected based on their relevance to neurodevelopmental disorders with infectious and inflammatory components, including schizophrenia and autism (33,34). A detailed description of the test apparatuses and procedures is provided in the Supplementary Information. Each animal underwent each behavioral test in the following order: (i) spatial recognition memory test, (ii) social interaction test, and (iii) PPI test. A test-free resting period of 2 days was imposed between individual tests. Collection of Brain Samples All adult offspring were first subjected to behavioral testing prior to the molecular analyses (Supplementary Information). They were killed by decapitation two weeks after completion of behavioral testing. The brains were rapidly extracted from the skull (within < 20 s) and placed on an ice-chilled