PROJECT SUMMARY/ABSTRACT Several pharmacological compounds have shown promise as interventional strategies for extending longevity. One recently studied compound shown to improve healthspan and longevity is 17α-estradiol (17α-E2). 17α-E2 is a diastereomer of 17β-estradiol (17β-E2) with considerably less binding affinity for classical estrogen receptors than 17β-E2. The NIA Interventions Testing Program has shown that 17α-E2 extends lifespan in male, but not female, mice at two doses. The mechanisms that promote sexually divergent responses to 17α-E2 are poorly understood and are aligned with the increasing recognition that aging and the incidence of specific diseases often differ between the sexes. This project will explore potential mechanisms by which 17α-E2 modulates aging and metabolism in a sex-specific manner. Successful completion of this project will determine: 1) if estrogen receptor α (ERα) is required for 17α-E2 to elicit benefits on healthspan, disease pathology, and lifespan, 2) if 17α-E2 modulates metabolic parameters predominantly through hypothalamic ERα-mediated signaling, 3) if the beneficial effects of 17α-E2 on metabolic parameters can be mimicked by activating hypothalamic ERα through chemogenetic approaches, and 4) if the elimination of endogenous estrogen production render female mice responsive to 17α-E2-mediated effects on metabolism, and if this is ERα dependent. We hypothesize that 17α- E2 signals through ERα in male mice to enhance healthspan and longevity and that these effects are at least partially mediated through the hypothalamus. We also hypothesize that female mice lacking endogenous estrogens will beneficially respond to 17α-E2 in an ERα-dependent manner. Aim 1: Determine if 17α-E2 improves healthspan and lifespan in an ERα-dependent manner. We will evaluate the effects of chronic 17α-E2 treatment on indices of healthspan and lifespan in wild-type and global ERαKO mice. Aim 2: Determine if 17α- E2 elicits metabolic benefits through ERα in the hypothalamus. We will evaluate the effects of 17α-E2 treatment on metabolic and healthspan parameters in mice with a hypothalamus-specific deletion of ERα using stereotaxic delivery of adeno-associated virus (AAV) that drives expression of Cre in ERα-flox mice. Aim 3: Determine if chemogenetic activation of hypothalamic ERα-expressing cells can mimic the health benefits observed with 17α- E2 treatment. We will evaluate the effects of stimulating hypothalamic ERα-expressing cells on metabolic and healthspan outcomes via site-specific expression of AAV Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in ERα-flox mice. Aim 4: Determine if the elimination of endogenous estrogen production renders female mice responsive to 17α-E2 treatment. We will evaluate the effects of 17α-E2 treatment on metabolic and healthspan parameters in wild-type and global ERαKO female mice undergoing ovariectomy or 4-vinylcyclohexene diepoxide-induced ovarian insuffici...