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LPS AND HYPOXIA AFFECT TRANSCRIPTION AND 5’ HYDROXYMETHYLATION OF SLC7A5 IN THE DEVELOPING MOUSE BRAIN

Presented at the Neonatal Society Spring Meeting 2019

Authors: Eamon Fitzgerald1, James P Boardman2 and Amanda J Drake1

Institution(s)
1 British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, The Queen’s Medical Research Institute, 47, Little France Crescent; 2 MRC Centre for Reproductive Health, University of Edinburgh, The Queen’s Medical Research Institute, 47, Little France Crescent

Introduction
Preterm birth (PTB) is associated with alterations in DNA methylation at sites that influence neurodevelopment including SLC7A5, an amino acid transporter linked to autism spectrum disorder (ASD) 1, but the environmental exposures that drive epigenetic modification are uncertain. Preterm infants may be exposed to infection and hypoxia, both of which can modify the epigenome 2. We tested the hypothesis that exposure to LPS and/or hypoxia affects SLC7A5 expression in the developing mouse brain mediated by altered cytosine modifications

Methods
C57/Bl6 mice were culled at P0-P1 and 300µm forebrain slices cultured, with 4 experimental groups: (1) control (no intervention); (2) LPS (100ng/ml for 5 hours), (3) hypoxia (1% oxygen for 24 hours); and (4) LPS + hypoxia (5 hours LPS then 24 hours hypoxia). RNA/DNA was extracted, then qPCR, and 5-hydroxymethylated DNA (5hmC) immunoprecipitation-qPCR performed. N=6-12 mice (1 slice/condition) for all experiments. Animals were kept, and procedures carried out in accordance with the University of Edinburgh’s policy and Home Office regulations. Funding provided by Medical Research Scotland and Aquila Biomedical.

Results
Exposure to LPS + hypoxia (group 4), but not LPS (group 2) or hypoxia (group 3), resulted in increased expression of SLC7A5 (p=0.01) and in increased expression of the DNA demethylases Tet1 and Tet2 which convert 5-methylcytosine (classically associated with gene silencing) to 5hmC (associated with active transcription) (both p<0.05). LPS (group 2) and LPS + hypoxia (group 4) associated with increased 5hmC within a well-conserved region of SLC7A5 (p=0.04), with no effect of hypoxia (group 3), p=0.531.

Conclusions
LPS + hypoxia associates with altered expression of a candidate gene for ASD, SLC7A5. LPS and LPS + hypoxia is associated with increased 5hmC at SLC7A5. As such we propose, the effect of LPS to sensitise to subsequent hypoxia, as seen in animal models of perinatal brain injury, is at least partly mediated by 5hmC.

References
1.Sparrow, S. et al.. Transl. Psychiatry 6, e716 (2016). 2.Brigati, C. et al. Mediators Inflamm. 2010, 263914
We thank Dr Veronique Miron and Graeme Ireland for initial development and characterisation of the model.

Corresponding author e-mail address: amanda.drake@ed.ac.uk

Senior author supporting presentation on day of meeting: Prof James Boardman

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