PROJECT SUMMARY In women, aging is characterized by a marked decrease in circulating estrogen (E) levels, which negatively impacts many physiological systems including sleep-wake cycles and cognitive performance. Consequently, many postmenopausal women chose to undergo E hormone replacement therapy (HRT), with the hope of reversing or alleviating these negative symptoms. Unfortunately, the underlying neuroendocrine mechanisms associated with HRT are poorly understood and the long-term efficacy of these hormonal manipulations on the central nervous system are unclear, especially in women who are overweight. Using the rhesus macaque as a translational animal model, we previously demonstrated an age-associated increase in perturbed sleep-wake cycles, and found that individuals with the most perturbed cycles showed inferior cognitive performance in a spatial memory task, as well as compromised immune responses. We also demonstrated cognitive benefits resulting from long-term administration of E to old ovariectomized females; importantly, however, the beneficial effects of E on various physiological functions were not sustained in animals maintained on a high-fat, high- sugar Western-style diet (WSD). The goal of this R21 exploratory study is to test the hypothesis that age- related molecular changes within the suprachiasmatic nucleus (SCN) contribute to disrupted sleep-wake cycles and that these are exacerbated by the marked postmenopausal attenuation of circulating E concentrations, especially when concomitantly exposed to a WSD. Therefore, our study will examine DNA methylation developments within the SCN (Aim 1), and use RNA-seq to profile gene expression changes (Aim 2), using archived brain tissue from the following pair-groups of previously-characterized rhesus macaques: 1. Young adult ovary-intact females (on a standard diet) 2. Old ovary-intact females (on a standard diet) 3. Old ovariectomized females (on a standard diet) 4. Old ovariectomized females (on a standard diet + treated with HRT for ~3 years) 5. Old ovariectomized females (on a WSD for ~3 years) 6. Old ovariectomized females (on a WSD for ~3 years + treated with HRT for ~3 years) Using a previously-validated MethylSeq approach to provide single-base resolution DNA methylation data, we will examine differential methylation CpG (DMC) and region (DMR) in the SCN – i.e., the well-established site of the master circadian clock that plays a key role in sleep maintenance and synchronizing daily physiological functions. We predict that aging, maintenance on a WSD, and insufficient E in the circulation produce functional epigenetic modifications within the core clock mechanism of the SCN that result in perturbed circadian activity-rest pattern, and in turn predispose individuals to development of Alzheimer’s disease.