PROJECT SUMMARY Sex differences impact the prevalence and degree of frailty. Women have a higher prevalence of more severe frailty compared to age-matched men. A key difference between the sexes is their exposure to distinct steroid hormone profiles across the life course. In studying how E2 (17β-estradiol) regulates metabolism across female reproductive stages we recently identified a small population of ERα (estrogen receptor alpha) neurons in the hypothalamus that drive spontaneous physical activity in mice in response to E2. These neurons coordinate energy expenditure to support changes in behavior and physiology across the female reproductive cycle. We predict that disengagement of these neurons after menopause contributes to metabolic and functional declines that predispose women to frailty. However, using CRISPRa (CRISPR-mediated gene activation) we can bypass ERα-dependent regulation of these neurons and dramatically increase physical activity levels in female and male mice. As sedentary behavior is a risk factor for chronic diseases, including frailty, and interventions that promote a more active lifestyle improve health and longevity, the goal of this R21 is to determine if we can leverage CRISPRa gene engineering in these nominally estrogen-sensitive neurons to restore and maintain physical activity levels and associated health benefits as females age. In aim 1 we will determine if stimulation of these activity-promoting neurons in E2-depleted mice is sufficient to ameliorate sex differences in frailty phenotype and hypothalamic neuroinflammation. Longitudinal assessments of frailty phenotype along with terminal examination of histological markers of neuroinflammatory stress in these hypothalamic neurons will be performed in CRISPRa stimulated female and male mice as well as sedentary controls. In response to exercise the liver produces several secreted factors that act systemically to improve neuronal and metabolic health. The liver also expresses ERα and exhibits distinct gene expression programs in males and females. In aim 2 we will determine if there are sex differences in the hepatic response to physical activity that ultimately contribute to female bias in frailty. To that end we will use proximity labeling and mass spectrometry to specifically examine physical activity-dependent changes in the liver secretome of female and male mice. Collectively, we anticipate that this integrative approach will rationalize sex differences in frailty and may identify new translational opportunities.