PROJECT SUMMARY The overall goal of this proposal is to determine the sex chromosome genes that regulate ischemic stroke sensitivity in the aged brain, and to explore the mechanisms underlying their regulatory role. It has been increasingly recognized that stroke is a sexually dimorphic disease, however, the mechanisms underlying these sex differences are not known. The elderly constitute the majority of stroke victims, and aged women have a higher incidence, higher morbidity and higher mortality compared to age-matched men, and these differences cannot be explained solely by exposure to gonadal hormones. Previous work has shown the sex chromosome complement contributes to stroke sensitivity selectively in aged animals, when gonadal hormones are equivalent between the sexes. We have found that there is an effect of the X chromosome dosage (one X or XX) on microglial activation and immune responses. A prominent feature of the aged X chromosome is that genetic silencing of genes on the second X chromosome becomes incomplete, allowing for genes to escape from X-chromosome inactivation (XCI). This results in higher expression of these X escapee genes in XX vs. XY cells in many tissues. Kdm6a and Kdm5c are two X escapees that can regulate expression of interferon regulatory factors (IRFs) that are responsible for microglial activation through epigenetic modification. Recent work has found Kdm6a and Kdm5c are more highly expressed in microglia derived from aged female vs. male ischemic brain. Our CENTRAL HYPOTHESIS is that X chromosome complement contributes to stroke sensitivity in the aged brain, AND that the X escapee genes Kdm6a and Kdm5c epigenetically modify IRF1/3/4/5/8 in aged microglia leading to sex-specific inflammatory responses. In Aim 1 we will use the XY* mouse model to determine if the X chromosome contributes to stroke sensitivity in aged animals. Aim 2 will use an inducible conditional knock out (ICKO) animal model to test the hypothesis that Kdm6a and Kdm5c sex specifically impact on stroke outcomes through a mechanism of epigenetic modification, i.e. demethylation of H3K27me3 and H3K4me3 marks respectively. Aim 3 will test the hypothesis that X chromosome and Kdm6a/Kdm5c regulate microglial activation and immune responses through mediation of IRF1/3/4/5/8 expression. These proposed studies will investigate the Kdm6a/5c- H3k27me3/H3K4me3-IRFs signaling axes, a very innovative and novel area. We hypothesize that this pathway plays a critical role in inducing sex differences in stroke in the aged.