Actions of Estrogen & Environmental Estrogens on Neurons

描述（由申请人提供）：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）在神经元发育过程中的生理作用，并确定环境雌激素（EE）在多大程度上可以模拟或抑制E2在这些发育神经元中的作用。快速E2介导的信号转导机制还不清楚。这些作用在不同的细胞类型之间有很大的不同，甚至在同一个细胞中也可能不同。除了生殖组织和乳腺癌，神经系统细胞也是临床上非常重要的E2靶点。环境雌激素，也称为内分泌干扰化学品，是一组不同的化合物，可以模拟或拮抗E2的正常作用。EE对发育中的神经系统的影响程度尚不清楚。使用雌激素敏感的新生大鼠小脑神经元和发育中的小脑作为非性相关神经元模型的原代培养物的拟议实验将解决以下具体目标-具体目标1是确定结构上不同的EE是否调节发育中的小脑神经元中的ERK 1/2信号传导。据推测，在E2反应神经元，EE快速调节ERK 1/2信号和神经元生理学在E2样的方式，并在个别EE的化学结构的差异决定了他们的能力，激活ERK信号和/或拮抗E2的快速行动。具体目标2是确定E2介导的ERK信号快速激活的信号传导机制的性质。据推测，质膜，ERp的本地化版本是作为快速E2/EE诱导的ERK信号转导的介质。将亲和纯化来自原代颗粒细胞和海马神经元培养物的膜E2结合蛋白，并通过LC-串联质谱法鉴定。免疫细胞化学和功能丧失的实验，提出了证明的候选人膜ER的功能。ER α、ER β和孤儿G蛋白偶联受体GPR 30作为膜ER的能力将被直接解决。

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.