Estrogen, Astrocyte Reactivity, and Sex Differences in Alzheimer's Disease

总结 在600多万患有阿尔茨海默病（AD）的美国人中，近三分之二是女性;然而， AD易感性性别差异的分子机制在很大程度上是未知的。先前的研究 曾报道女性AD患者大脑中雌激素水平较低。本申请的长期目标是 目的是确定雌激素缺乏在观察到的AD易感性性别特异性差异中的作用。芳香酶， 其将雄激素转化为雌激素，主要在小鼠的脑和性腺中表达。我们证明 敲除小鼠大脑中的局部芳香化酶（bArKO）或整个身体中的芳香化酶（tArKO）会导致性行为- 老年雌性小鼠的特定记忆缺陷。这种表型在具有雌激素的tArKO小鼠中更严重 在整个身体中缺乏雌激素的bArKO小鼠与仅在大脑中缺乏雌激素的bArKO小鼠相比。雌激素发挥其 通过结合雌激素受体（ER）发挥生物学作用。选择性ER α激动剂治疗 丙基吡唑三醇挽救了雌性tArKO小鼠的记忆丧失。年老的雌性tArKO，但不是年老的雄性tArKO小鼠， 与野生型同窝仔相比，在海马体中表现出较少的神经元。RNA-seq、qPCR和 对来自两种性别的年轻或年老bArKO小鼠的海马的免疫荧光分析显示， 星形胶质细胞反应性和相关基因表达以及仅在老年雌性bArKO小鼠中的形态学变化。这些 数据提示了一种新的机制，其中雌激素缺乏导致星形胶质细胞反应性。我们假设 绝经后大脑中雌激素/ER α信号的缺乏会导致性别特异性星形胶质细胞反应， 导致AD相关的神经病理学和记忆丧失，导致女性AD易感性增加。到 为了确定大脑中雌激素缺乏与AD易感性性别差异之间的联系机制， 提出以下目标：1.确定是否逆转芳香化酶缺失和雌激素缺乏 减少星形胶质细胞的反应性，防止神经变性和记忆丧失。我们将使用转基因 以及在bArKO和tArKO中恢复脑芳香化酶表达或雌激素作用的药理学方法 不同年龄的老鼠雌激素/ER α介导的基因组机制负责抑制 性别特异性海马星形胶质细胞反应性、神经变性和记忆丧失将使用 使用单核（sn）RNA-seq、ER α-ChIP-seq和snATAC-seq进行全基因组整合分析， 新鲜分离的海马星形胶质细胞。2.确定海马ER α的条件性敲除是否 APPNL-G-F小鼠的星形胶质细胞加速星形胶质细胞反应性、记忆丧失和AD相关的神经病理学。在 同时，我们将评估雌激素水平，芳香化酶和ER α的表达， 现有人类数据集和死后海马组织中的反应性星形胶质细胞相关基因及其 与AD病理严重程度和女性和年龄匹配的医疗记录中的记忆缺陷相关 男性AD我们希望拟议研究的结果将确定新的药物靶点， 用于预防和治疗AD的新治疗策略。

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.