SUMMARY Nearly two-thirds of the more than 6 million Americans living with Alzheimer’s disease (AD) are women; however, the molecular mechanisms underlying this sex difference in AD vulnerability are largely unknown. Prior research has reported lower levels of estrogen in the brain of women with AD. The long-term objective of this application is to define the role of estrogen deficiency in the observed sex-specific difference in AD vulnerability. Aromatase, which converts androgens to estrogens, is mainly expressed in the brain and gonads of mice. We demonstrated that knockout of aromatase locally in the mouse brain (bArKO) or totally in whole body (tArKO) causes sex- specific memory deficits in old female mice. This phenotype is more severe in tArKO mice with estrogen deficiency throughout the body vs. bArKO mice with estrogen deficiency only in the brain. Estrogen exerts its biological action via binding to estrogen receptors (ERs). Treatment with the selective ERα agonist propylpyrazoletriol rescued memory loss in female tArKO mice. Old female tArKO, but not old male tArKO mice, exhibited fewer neurons in the hippocampus compared to wild-type littermates. RNA-seq, qPCR, and immunofluorescence analyses of the hippocampus from young or old bArKO mice of both sexes revealed astrocyte reactivity and related gene expression and morphologic changes only in old female bArKO mice. These data suggested a novel mechanism in which estrogen deficiency leads to astrocyte reactivity. We hypothesize that deficiency of estrogen/ERα signaling in the brain after menopause causes sex-specific astrocyte reactivity, resulting in AD-related neuropathology and memory loss, leading to increased AD vulnerability in females. To ascertain the mechanisms connecting estrogen deficiency in the brain to sex differences in AD vulnerability, we propose the following aims: 1. Determine whether reversal of aromatase deletion and estrogen deficiency reduces astrocyte reactivity and protects against neurodegeneration and memory loss. We will use transgenic and pharmacological approaches to restore brain aromatase expression or estrogen action in bArKO and tArKO mice at different ages. The estrogen/ERα-mediated genomic mechanisms responsible for the suppression of sex-specific hippocampal astrocyte reactivity, neurodegeneration, and memory loss will be determined using integrative genome-wide analyses employing single-nucleus (sn) RNA-seq, ERα-ChIP-seq, and snATAC-seq on freshly isolated hippocampal astrocytes. 2. Determine whether conditional knockout of ERα in hippocampal astrocytes of APPNL-G-F mice accelerates astrocyte reactivity, memory loss, and AD-related neuropathology. In parallel, we will assess estrogen levels, expression of aromatase and ERα, and sex-specific expression of reactive astrocyte-related genes in existing human datasets and postmortem hippocampal tissues and their correlation with the severity of AD pathology and memory deficits in medical records of women and age-matched men with AD. We expect that the findings from the proposed studies will identify new drug targets and lead to novel therapeutical strategies for the prevention and treatment of AD.

NARRATIVE The reasons why women are more vulnerable than men to developing Alzheimer’s disease (AD) are unknown. We created novel and clinically relevant mouse models that link age-related estrogen deficiency with sex-based differences in brain astrocyte reactivity, neurodegeneration, and memory loss. We propose to use these models to understand the mechanisms underlying the sex difference in AD vulnerability in older women and whether treatments that restore estrogen action in the brain can prevent AD development in women via rescuing astrocyte function.