Maternal immune activation induces methylation changes in schizophrenia genes

Susceptibility to schizophrenia is mediated by genetic and environmental risk factors. Infection driven maternal immune activation (MIA) during pregnancy is a key environmental risk factor. However, little is known about how MIA during pregnancy could contribute to adult-onset schizophrenia. In this study, we investigated if maternal immune activation induces changes in methylation of genes linked to schizophrenia. We found that differentially expressed genes in schizophrenia brain were significantly enriched among MIA induced differentially methylated genes in the foetal brain in a cell-type-specific manner. Upregulated genes in layer V pyramidal neurons were enriched among hypomethylated genes at gestational day 9 (fold change = 1.57 , FDR = 0.049) and gestational day 17 (fold change = 1.97 , FDR = 0.0006). We also found that downregulated genes in GABAergic Rosehip interneurons were enriched among hypermethylated genes at gestational day 17 (fold change = 1.62, FDR= 0.03). Collectively, our results highlight a connection between MIA driven methylation changes during gestation and schizophrenia gene expression signatures in the adult brain. These findings carry important implications for early preventative strategies in schizophrenia.

Got Milk? Maternal immune activation during the mid-lactational period affects nutritional milk quality and adolescent offspring sensory processing in male and female rats

The neonatal environment requires a high level of maternal demand in terms of both breastfeeding and other forms of maternal care. Previous studies have underscored the importance of these maternal factors on offspring development and behavior. However, their contribution as dynamic variables in animal models of early life stress are often overlooked. In the present study, we show that lipopolysaccharide (LPS)-induced maternal immune activation (MIA) on postnatal day (P)10 immediately elevated milk corticosterone concentrations, which recovered by P11. In contrast, both milk triglyceride and percent creamatocrit values demonstrated a prolonged decrease following inflammatory challenge. Sustained inflammatory-induced changes to the nutritional quality of milk were also evidenced by its composition of microbial communities associated with inefficient energy and lipid metabolism. Nutritional deficits in early development have been associated with metabolic dysfunction later in life. Indeed, MIA-associated changes in the nutritional profile of milk were reflected by increased adolescent offspring bodyweights. While MIA did not decrease maternal care quality, there was a significant compensatory increase in maternal licking and grooming the day that followed the inflammatory challenge. However, this did not protect against disrupted neonatal huddling or later-life alterations in sensorimotor gating and mechanical allodynia in MIA offspring. Animal models of early life stress can impact both parents and their offspring. One mechanism that can mediate the effects of such stressors is changes to maternal lactation quality which our data show can confer multifaceted and compounding effects on offspring physiology and behavior.

Repeated intermittent administration of (R)-ketamine during juvenile and adolescent stages prevents schizophrenia-relevant phenotypes in adult offspring after maternal immune activation: a role of TrkB signaling

Maternal immune activation (MIA) plays a role in the etiology of schizophrenia. MIA by prenatal exposure of polyinosinic:polycytidylic acid [poly(I:C)] in rodents caused behavioral and neurobiological changes relevant to schizophrenia in adult offspring. We investigated whether the novel antidepressant (R)-ketamine could prevent the development of psychosis-like phenotypes in adult offspring after MIA. We examined the effects of (R)-ketamine (10 mg/kg/day, twice weekly for 4 weeks) during juvenile and adolescent stages (P28–P56) on the development of cognitive deficits, loss of parvalbumin (PV)-immunoreactivity in the medial prefrontal cortex (mPFC), and decreased dendritic spine density in the mPFC and hippocampus from adult offspring after prenatal poly(I:C) exposure. Furthermore, we examined the role of TrkB in the prophylactic effects of (R)-ketamine. Repeated intermittent administration of (R)-ketamine during juvenile and adolescent stages significantly blocked the development of cognitive deficits, reduced PV-immunoreactivity in the prelimbic (PrL) of mPFC, and decreased dendritic spine density in the PrL of mPFC, CA3 and dentate gyrus of the hippocampus from adult offspring after prenatal poly(I:C) exposure. Furthermore, pretreatment with ANA-12 (TrkB antagonist: twice weekly for 4 weeks) significantly blocked the beneficial effects of (R)-ketamine on cognitive deficits of adult offspring after prenatal poly(I:C) exposure. These data suggest that repeated intermittent administration of (R)-ketamine during juvenile and adolescent stages could prevent the development of psychosis in adult offspring after MIA. Therefore, (R)-ketamine would be a potential prophylactic drug for young subjects with high-risk for psychosis.

Attenuated transcriptional response to pro-inflammatory cytokines in schizophrenia hiPSC-derived neural progenitor cells

Maternal immune activation (MIA) during prenatal development is an environmental risk factor for psychiatric disorders including schizophrenia (SZ). This link is particularly well established in the case of schizophrenia. Converging lines of evidence from human and animal model studies have suggested that elevated cytokine levels in the maternal and fetal compartments are an important indication of the mechanisms driving this association. However, there is variability in susceptibility to the psychiatric risk conferred by MIA, likely influenced by genetic factors. How MIA interacts with a genetic profile susceptible to SZ is challenging to test in animal models. To address this gap, we examined whether differential gene expression responses occur in forebrain-lineage neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSC) generated from three individuals with a diagnosis of schizophrenia and three healthy controls. Following acute (24 hour) treatment with either interferon-gamma (IFNγ; 25 ng/μl) or interleukin (IL)-1β (10 ng/μl), we identified, by RNA sequencing, 3380 differentially expressed genes (DEGs) in the IFNγ-treated control lines (compared to untreated controls), and 1980 DEGs in IFNγ-treated SZ lines (compared to untreated SZ lines). Of these, 359 genes responded differently to IFNγ treatment in SZ relative to control lines, which were enriched for genes differentially expressed in adult SZ cases and in pathways related to the regulation of antigen processing, neuronal post-synaptic density, and the presynapse. There were no differentially expressed genes in the IL-1β-treatment conditions after Benjamini-Hochberg correction. Gene set enrichment analysis however showed that IL-1β impacts immune function and neuronal differentiation. Collectively, our data suggest that a) SZ NPCs show an attenuated transcriptional response to IFNγ treatment compared to control NPCs; b) IL-1β may be less relevant to NPC cultures than IFNγ and c) the genes differentially regulated in schizophrenia lines are primarily mitochondrial, loss-of-function, pre- and post-synaptic genes. Our findings particularly highlight early synaptic development as a key target for future research into the mechanisms of association between maternal immune activation and schizophrenia risk.

Interferon-ɣ Exposure of Human iPSC-derived Neurons Alters Major Histocompatibility Complex I and Synapsin I Protein Expression

Human epidemiological data links maternal immune activation during gestation with increased risk for neurodevelopmental disorders including schizophrenia. Animal models of maternal immune activation (MIA) provide causal evidence for this association and strongly suggest that inflammatory cytokines act is a critical link between maternal infection and aberrant offspring brain and behavior development. This includes evidence for reduced synapse formation, consistent with post-mortem and in vivo evidence of reduced synaptic density in schizophrenia. However, to what extent specific cytokines are necessary and sufficient for these effects remains unclear. Using a human cellular model, we recently demonstrated that acute exposure to interferon-ɣ (IFNɣ) recapitulates molecular and cellular phenotypes associated with neurodevelopmental disorders. Here, we extend this work to test whether IFNɣ affects synapse formation in an induced neuron model that generates forebrain glutamatergic neurons. Using immunocytochemistry and quantitative PCR, we demonstrate that acute IFNɣ exposure results in significantly increased MHCI expression at the mRNA and protein level. Furthermore, acute IFNɣ exposure decreases synapsin I protein in neurons but does not affect synaptic gene mRNA levels. Interestingly, complement component 4A (C4A) mRNA is also significantly increased following acute IFNɣ exposure. This study builds on our previous work by showing that IFNɣ-mediated disruption of relevant synaptic proteins can occur at early stages of synapse formation, potentially contributing to neurodevelopmental disorder phenotypes such as schizophrenia.

Adolescent THC Treatment Does Not Potentiate the Behavioral Effects in Adulthood of Maternal Immune Activation

Both in utero exposure to maternal immune activation and cannabis use during adolescence have been associated with increased risk for the development of schizophrenia; however, whether these exposures exert synergistic effects on brain function is not known. In the present study, mild maternal immune activation (MIA) was elicited in mice with prenatal exposure to polyinosinic-polycytidylic acid (poly(I:C)), and ∆9-tetrahydrocannabinol (THC) was provided throughout adolescence in cereal (3 mg/kg/day for 5 days). Neither THC nor MIA pretreatments altered activity in assays used to characterize hyperdopaminergic states in adulthood: amphetamine hyperlocomotion and prepulse inhibition of the acoustic startle reflex. Adolescent THC treatment elicited deficits in spatial memory and enhanced spatial reversal learning in adult female mice in the Morris water maze, while exposure to MIA elicited female-specific deficits in fear extinction learning in adulthood. There were no effects in these assays in adult males, nor were there interactions between THC and MIA in adult females. While doses of poly(I:C) and THC were sufficient to elicit behavioral effects, particularly relating to cognitive performance in females, there was no evidence that adolescent THC exposure synergized with the risk imposed by MIA to worsen behavioral outcomes in adult mice of either sex.

Analysis of gene expression and DNA methylation in a cognitive phenotype model of schizophrenia

<p>The underpinning hypothesis of this study is that the environmental insults implicated in schizophrenia cause epigenetic changes that trigger deleterious gene expression, resulting in deviations from normal neurodevelopment. The behavioural abnormalities in schizophrenia can be grouped into the three common classes of symptoms: positive, negative, and cognitive. Cognitive symptoms are symptoms that impair cognitive processing and have detrimental effects on individuals with schizophrenia. Maternal immune activation refers to a rat model that stimulates a maternal immune system with an infection or infectious-like stimulus resulting in adverse phenotypes. A cognitive phenotype, maternal immune activation (MIA) model of schizophrenia was employed to use epigenetic markers to discover what deleterious genes drive the cognitive deficits phenotype. Previous work has discerned many changes in gene expression that are implicated in schizophrenia. A hypothesis-driven approach was utilized to determine whether previously studied candidate genes are relevant in the cognitive symptoms of schizophrenia in this cognitive-phenotype model. It was found that prenatal treatment of lipopolysaccharide (LPS) (which is the major outer membrane component of gram-negative bacteria and mimics bacterial infection) on prenatal day 10 and 11 led to changes in mRNA levels in the prefrontal cortex of adolescent rats. Typically, an increase in the amount of transcript in the LPS condition compared to the saline condition, or a greater variability in the amount of transcript between replicates in the LPS condition than the saline condition, was observed. Statistical analysis revealed that these changes did not met statistical significance. To build towards a whole genome DNA methylation analysis, two discrete approaches were used. The first utilized bisulfite modification and investigated changes in candidate genes as a precursor to genome-wide BS-sequencing. DNA methylation was measured across CpG rich regions and an absence of DNA methylation was detected in these regions in both the LPS and saline conditions in the candidate genes. The second approach utilized a long-read sequencing platform to establish the feasibility of a bisulfite conversion-free method for whole-genome DNA methylation approach within our lab. Through the establishment of this method factors that affect the reliability, quality, and accuracy of the final sequencing product were explored. Many of which were in the downstream-from-sequencing, data analysis component of the process. Discoveries were also made regarding how much data would be needed to make direct DNA methylation detection feasible. The data presented here demonstrated that the cognitive-phenotype MIA model had altered gene expression correlating with previously measured behavioural cognitive deficits in the prefrontal cortex in genes that were known to be associated with schizophrenia. To extend this further, a whole genome approach would be needed to discover novel drivers of the phenotype. In the current study, headway was made towards the development and establishment of a whole genome DNA methylation detection method to further this continued aim.</p>

Analysis of gene expression and DNA methylation in a cognitive phenotype model of schizophrenia

<p>The underpinning hypothesis of this study is that the environmental insults implicated in schizophrenia cause epigenetic changes that trigger deleterious gene expression, resulting in deviations from normal neurodevelopment. The behavioural abnormalities in schizophrenia can be grouped into the three common classes of symptoms: positive, negative, and cognitive. Cognitive symptoms are symptoms that impair cognitive processing and have detrimental effects on individuals with schizophrenia. Maternal immune activation refers to a rat model that stimulates a maternal immune system with an infection or infectious-like stimulus resulting in adverse phenotypes. A cognitive phenotype, maternal immune activation (MIA) model of schizophrenia was employed to use epigenetic markers to discover what deleterious genes drive the cognitive deficits phenotype. Previous work has discerned many changes in gene expression that are implicated in schizophrenia. A hypothesis-driven approach was utilized to determine whether previously studied candidate genes are relevant in the cognitive symptoms of schizophrenia in this cognitive-phenotype model. It was found that prenatal treatment of lipopolysaccharide (LPS) (which is the major outer membrane component of gram-negative bacteria and mimics bacterial infection) on prenatal day 10 and 11 led to changes in mRNA levels in the prefrontal cortex of adolescent rats. Typically, an increase in the amount of transcript in the LPS condition compared to the saline condition, or a greater variability in the amount of transcript between replicates in the LPS condition than the saline condition, was observed. Statistical analysis revealed that these changes did not met statistical significance. To build towards a whole genome DNA methylation analysis, two discrete approaches were used. The first utilized bisulfite modification and investigated changes in candidate genes as a precursor to genome-wide BS-sequencing. DNA methylation was measured across CpG rich regions and an absence of DNA methylation was detected in these regions in both the LPS and saline conditions in the candidate genes. The second approach utilized a long-read sequencing platform to establish the feasibility of a bisulfite conversion-free method for whole-genome DNA methylation approach within our lab. Through the establishment of this method factors that affect the reliability, quality, and accuracy of the final sequencing product were explored. Many of which were in the downstream-from-sequencing, data analysis component of the process. Discoveries were also made regarding how much data would be needed to make direct DNA methylation detection feasible. The data presented here demonstrated that the cognitive-phenotype MIA model had altered gene expression correlating with previously measured behavioural cognitive deficits in the prefrontal cortex in genes that were known to be associated with schizophrenia. To extend this further, a whole genome approach would be needed to discover novel drivers of the phenotype. In the current study, headway was made towards the development and establishment of a whole genome DNA methylation detection method to further this continued aim.</p>