Presented at the Neonatal Society 2016 Spring Meeting.
Cartier J1, Allen CM1, Boardman JP2,3, Drake AJ1
1 Centre for Cardiovascular Science, University of Edinburgh, UK
2 MRC Centre for Reproductive Health, University of Edinburgh, UK
3 Centre for Clinical Brain Sciences, University of Edinburgh, UK
Background: Preterm babies are at increased risk of neurodevelopmental disorders. Astrocytes may be particularly vulnerable to damage in preterm infants and we have shown altered DNA methylation (DNAm) at astrocyte-specific genes in preterm babies, which may predict altered gene expression (1). We explored developmental changes in gene expression and DNAm at astrocyte-specific genes slc1a2 and Lrg1 in vivo and in cultured astrocytes.
Methods: Wistar rat forebrains were collected at embryonic day (E)20.5 (late gestation) and postnatal days 1 (P1) and P10. Astrocytes were isolated using positive selection of GLAST+ cells by magnetic activated cell sorting. ‘Directly isolated’ astrocytes were used for analysis of gene expression (qPCR), and DNAm (pyrosequencing). Gene expression and DNAm were also analysed in E20.5 and P1 astrocytes following 10 days in in vitro culture. The project was funded by TENOVUS Scotland.
Results: In directly isolated astrocytes, slc1a2 expression of increased with maturation between E20.5 and P10 (p < 0.01) whereas Lrg1 expression decreased with maturation (p < 0.05). DNAm at the slc1a2 promoter was extremely low and unlikely to influence gene expression. In contrast, DNAm at intragenic regions of Lrg1 decreased with maturation. In culture, isolated cells divided and developed multiple processes contacting adjacent cells; qPCR analysis for GLAST expression confirmed these cells were astrocytes. In culture, slc1a2 expression was lost but Lrg1 expression was maintained. Cell culture did not result in major changes in DNAm compared to the directly isolated astrocytes, which retained DNAm patterns similar to those at the time of isolation except for at a single CpG in the Lrg1 intragenic region at which DNAm was increased.
Conclusion: Our results confirm developmental changes in slc1a2 and report novel changes in Lrg1. DNAm at these genes may not associate with gene expression, but may reflect particular developmental stages.
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1. Sparrow, S. et al., 2016. Epigenomic profiling of preterm infants reveals DNA methylation differences at sites associated with neural function. Translational Psychiatry 6:e716–8.