Actions of Estrogen & Environmental Estrogens on Neurons

描述(申请人提供)：拟议研究的广泛长期总体目标是了解雌激素(E2)和雌激素受体(ER)在神经元发育中的生理作用，并确定环境雌激素(EES)在多大程度上可以在这些发育中的神经元中模仿或抑制E2的影响。E2介导的快速信号转导机制尚不清楚。这些操作因细胞类型的不同而有很大差异，甚至在同一细胞中也可能有所不同。除了生殖组织和乳腺癌，神经系统细胞是临床上非常重要的E2靶点。环境雌激素，也被称为内分泌干扰物，是一组不同的化合物，可以模仿或拮抗E2的正常作用。EES对发育中的神经系统的影响程度尚不清楚。拟议的实验使用雌激素敏感的新生大鼠小脑神经元的原代培养和发育中的小脑作为非性别相关的神经元模型，将解决以下特定目标-特定目标1是确定结构多样化的EES是否调节发育中的小脑神经元中的ERK1/2信号。假设在E2反应的神经元中，EES以类E2的方式快速调节ERK1/2信号和神经元生理，单个EES的化学结构的差异决定了它们激活ERK信号和/或拮抗E2的快速作用的能力。具体目的2是确定E2介导的ERK信号快速激活背后的信号机制的性质。推测质膜上定位的ERP是E2/EE诱导ERK信号转导的中介体。从原代颗粒细胞和海马神经细胞培养的膜E2结合蛋白将被亲和纯化，并通过LC-串联质谱仪进行鉴定。免疫细胞化学和功能丧失实验被用来证明识别的候选膜内质网的功能。ERA、ERP和孤儿G蛋白偶联受体GPR30作为膜ER的能力将直接被解决。

DESCRIPTION (provided by applicant): The broad long-term overall goal of the proposed research is to understand the physiological role of estrogen (E2) and estrogen receptors (ERs) during neuronal development and to determine to what extent environmental estrogens (EEs) can mimic or inhibit the effects of E2 in these developing neurons. Mechanisms of rapid E2-mediated signal transduction are not well understood. These actions vary greatly from cell type to cell type, and may even vary in the same cell. In addition to reproductive tissues & breast cancers, cells of the nervous system are a clinically very important E2 targets. Environmental estrogens, also known as endocrine disrupting chemicals, are a diverse group of compounds that can mimic or antagonize the normal actions of E2. The extent to which EEs impact the developing nervous system is unclear. Proposed experiments using primary cultures of estrogen-sensitive neonatal rat cerebellar neurons and the developing cerebellum as non-sexually related neuronal models, will address the following Specific Aims - Specific Aim 1 is to determine whether or not structurally diverse EEs modulate ERK1/2 signaling in developing cerebellar neurons. It is hypothesized that in E2-responsive neurons, EEs rapidly modulate ERK1/2 signaling and neuronal physiology in an E2-like fashion, and that differences in the chemical structure of individual EEs determine their ability to activate ERK-signaling &/or antagonize the rapid actions of E2. Specific Aim 2 is to determine the nature of the signaling mechanism underlying E2-mediated rapid activation of ERK-signaling. It is hypothesized that a plasma membrane, localized version of ERp is acting as the mediator of rapid E2/EE-induced ERKsignaling. Membrane E2-binding proteins from primary granule cell and hippocampal neuronal cultures will be affinity purified and identified through LC-tandem mass spectrometry. Immunocytochemical and loss-of-function experiments are proposed to demonstrate the function of identified candidate membrane ERs. The ability of ERa, ERp, and the orphan G-protein coupled receptor GPR30 to act as the membrane ER will be directly addressed.