Estrogen modulation of hypothalamic arcuate neurons

描述(申请人提供)：下丘脑弓状核中的一群神经元表达前阿片黑素皮质素(POMC)，这是一种多肽前体，当被切割时产生α-黑素细胞刺激素(α-MSH)和β-内啡肽(β-END)。这些产品参与了许多体内平衡功能的调节，如生殖、液体平衡、食欲、压力和动力。虽然雌激素对POMC基因表达的影响已为人们所熟知，但其分子机制尚未阐明。本研究旨在探讨雌激素作用于弓状POMC神经元导致α-MSH和β-END释放增加的分子机制。比较下丘脑基因表达变化的初步微阵列结果显示，PI3激酶p55伽马上调，这是PI3-激酶信号通路的一部分。利用单细胞逆转录聚合酶链式反应和原位杂交技术，确定PI3Kp55Gamma是否定位于POMC弓状神经元。其次，我将通过核糖核酸酶保护实验和实时定量聚合酶链式反应来验证基因芯片发现的PI3Kp55Gamma受雌激素的调控。最后，我将利用全细胞记录来确定PI3K通路在POMC弓状神经元雌激素信号转导中的作用。

DESCRIPTION (provided by applicant): A population of neurons in the arcuate nucleus of the hypothalamus expresses proopiomelanocortin (POMC), a polypeptide precursor that when cleaved yields alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin (beta-end). These products are involved in the regulation of many homeostatic functions such as reproduction, fluid balance, appetite, stress and motivation. Although, it is well established that estrogen affects POMC gene expression, the molecular mechanisms have not been elucidated. This proposal aims to investigate the molecular mechanisms of estrogen action on the arcuate POMC neurons leading to increased release of alpha-MSH and beta-end. Preliminary microarray results comparing gene expression changes in the hypothalamus showed the upregulation of PI3 kinase p55gamma, which is part of the PI3-kinase signaling pathway. Using single-cell reverse-transcription PCR and in situ hybridization, I will determine if PI3Kp55gamma is localized in POMC arcuate neurons. Second, I will validate the microarray finding that PI3Kp55gamma is regulated by estrogen using RNase protection assays and quantitative real-time PCR. Finally, I will determine the function of the PI3K pathway in estrogen signaling in POMC arcuate neurons using whole-cell recordings.