Abstract WP344: Abdullah Al mamun

Abstract

Introduction: Ischemic stroke is becoming increasingly recognized as a sexually dimorphic disease. However, the mechanisms underlying the sex dichotomy are complicated and remain elusive. We have reported that the sex chromosome complement contributes to the stroke sensitivity in aged animals. Kdm6a and Kdm5c, two genes escaping from X chromosome inactivation (XCI), are demethylases of H3K27me3 and H3K4me3 respectively. The two genes have higher expression in XX vs. XY cardiac tissue and are implicated in mediating expression of inflammatory mediators. We hypothesize that Kdm6a and Kdm5c are also differently expressed in brains of mice with two vs. one copy of X chromosome, and lead to sex differences in post-stroke inflammation and stroke outcomes in aged mice. Methods: Aged (18-22 months) WT C57BL/6 and FCG mice (XXF, XYF, XXM, and XYM) were subjected to a 60-minute middle cerebral artery occlusion (MCAO). Stroke outcomes including infarct size and behavior deficits were evaluated at 3d of MCAO. Initial screening of X-escapee genes was performed by RNAseq. Microglial activation and peripheral immune cell infiltration were determined by flow cytometry (FC). Expression of Kdm6a and kdm5c was evaluated by RT-PCR and IHC. Epigenetic markers (H3K27me1/3 and H3K4me1/3) downstream to Kdm6a and kdm5c were examined by Western blot. Results: RNAseq data revealed a significantly higher mRNA levels of Kdm6a and Kdm5c in WT female vs. male microglia. WT female brains had dramatic increase of both kdm6a and kdm5c expression after MCAO compared to male brains, corresponding to higher levels of H3K27me1 and H3K4me1 in females. Brain mRNA levels of Kdm6a and Kdm5c were also significantly increased in XXF/XXM compared to XYF/XYM FCG mice. Importantly, microglia from XXF/XXM mice express more Kdm6a and kdm5c than XYF/XYM microglia, accompanied by higher expression of H3K27me1 and H3K4me1 in XXF and XXM mice brains. XXF/XXM microglia had higher levels of intracellular pro-inflammatory cytokines (TNFα, IL1β, iNOS, IL6) after stroke. Conclusion: Mice with two X chromosomes have robust expression of kdm6a and kdm5c in microglia that leads to high epigenetic modification of inflammatory gene expression. XCI escaping may be critical in mediating stroke sensitivity in aged mice.