Effect of insults associated with preterm birth on mouse brain development

Abstract

Preterm birth (PTB) is associated with an increased incidence of many neurodevelopmental disorders. The mechanisms underlying this association are unclear, but insults associated with PTB such as hypoxia, inflammation and stress can affect brain development. In this thesis I examine the effects of these insults on the mouse brain at times with neurodevelopmental relevance to PTB. Mice when born are roughly equivalent in terms of brain development to a human at 24 weeks of gestation and mature to term equivalence by postnatal day (P) 10. As such, this course of postnatal brain development in mice provides an opportunity to study insults associated with PTB at timepoints neurodevelopmentally relevant to PTB in humans. DNA methylation is crucial to the fine spatial and temporal control of gene expression associated with normal brain development. Altered DNA methylation (5-methylcytosine; 5mC) has been shown in the brains of individuals with autism spectrum disorders, schizophrenia and other neurodevelopmental disorders. Additionally, preterm birth is associated with altered DNA methylation at several key genes associated with neurodevelopment. However, many of these studies cannot distinguish 5mC and 5- hydroxymethylation (5hmC), which is generated from 5mC by the Ten-eleven translocation (TET) enzymes. The TET enzymes are highly susceptible to environmental perturbations and are therefore a good candidate for epigenetic dysregulation associated with PTB related insults. I hypothesised that common PTB-related insults such as inflammation and/or hypoxia would affect the expression of the TET enzymes and this would be associated with altered DNA hydroxymethylation. To test this hypothesis, I used a forebrain slice culture model and showed that hypoxia results in increased expression of the TET enzymes. Previous studies have shown that DNA hydroxymethylation accumulates at classic hypoxic response genes following hypoxia in the cancer environment. I tested the applicability of this to brain development and found significant accumulation of 5hmC in these areas following hypoxia, demonstrating the relevance of this mechanism to neonatal hypoxia. Early life stress is also thought to be important in PTB. To test effects of stress on brain development, I developed a novel paradigm of early life stress in mice (modified maternal separation or MMS). MMS consists of separating a pup from its mother for 1.5 hours/ day from P4-P6. During this separation period the pup is moved into a supine position, from where it will struggle to return to the prone position and the process is repeated. I hypothesised that MMS would affect the transcriptome and DNA methylome in the perinatal period and result in altered behaviour in adulthood. Using 3’ mRNA sequencing and DNA methylation immunoprecipitation-sequencing I found subtle alterations in gene expression and profound alterations in DNA methylation in the hypothalamus immediately following MMS. In adulthood, I demonstrated that pups exposed to MMS have a hyperactivity phenotype in the elevated plus and open field mazes but do not display hyperactivity under habitual conditions. At 4 months of age, there were no changes in candidate gene expression in the hypothalamus, but there were changes in the expression of genes associated with stress signalling in the hippocampus following MMS. Finally, there were no persistent changes in DNA methylation in candidate regions in the hypothalamus. I also showed an NFkB signature in the hypothalamic transcriptome following MMS. NFkB signalling is classically associated with inflammation, I therefore hypothesised that inflammation, induced by LPS, would potentiate subsequent MMS. Using immunohistochemistry, I found no difference in specific cell populations in the cortex or hippocampus. I then performed 3’ mRNA sequencing to assess the transcriptome wide effect of LPS and MMS in the cortex. I showed enrichment of NFkB binding sites among differentially expressed genes in MMS but there was no additional effect of prior LPS exposure. Finally, I sought to explore whether LPS and/or MMS affected synaptic pruning in the hippocampus, however there was no evidence for altered expression of genes associated with synaptic pruning. In summary, in this thesis I show that LPS, hypoxia and early life stress can affect mouse brain development. These results further the knowledge around how insults associated with PTB may affect brain development and neurodevelopmental outcome.