REGULATION OF BRAIN DEVELOPMENT BY THYROID HORMONE

甲状腺激素对大脑发育的调节

• 批准号: 6517472
• 负责人: CARY N MARIASH
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517472
• 关键词: DNA footprinting; antisense nucleic acid; brain; cerebellar Purkinje cell; gel mobility shift assay; gene expression; gene mutation; genetically modified animals; hormone regulation /control mechanism; laboratory mouse; laboratory rat; molecular cloning; myelin basic proteins; nerve /myelin protein; neuroendocrine system; neurogenesis; nuclear receptors; nucleic acid sequence; oligodendroglia; thyroid hormones

Thyroid hormone deficiency during fetal and early childhood development is a major world-wide public health problem causing mental retardation and irreversible brain damage. The long term goal of this program is to understand the mechanism by which thyroid hormone (T3) regulates mammalian brain development. T3 regulates late brain development by activating several brain genes at a precise time. However, prior to this activation these genes are resistant to thyroid hormone despite the presence of T3 and thyroid hormone receptors. Thus, the loss of repression allows T3 to activate these genes at the appropriate time. To define the mechanism of this novel repressor effect we will study two different brain genes, Purkinje cell protein-2 (Pcp-2) and myelin basic protein (MBP), each expressed in two different cell types, cerebellar Purkinje cells and oligodenrocytes, respectively. We propose three specific aims: (1) to define the sequences within the Pcp-2 gene responsible for the repression of T3-dependent activation; (2) establish the role of the orphan receptor protein COUP-TF in repressing the response of Pcp-2 to T3; (3) determine the mechanism of repression for T3 induction of MBP gene in developing oligodendrocyte cultures. The first aim will utilize gel shift assays and DNAse I footprinting to localize the repressor sequence within an identified repressor region of the Pcp-2 gene. The identified sequence will be mutated and tested for loss of repressor action in transgenic mice using a Pcp-2 promoter construct shown to duplicate the in vivo developmental response of the Pcp-2 gene. Preliminary results suggest that the orphan nuclear receptor COUP-TF may be involved in the repressor activity. We will test the potential role of COUP- TFI by measuring Pcp-2 in mice deficient in this protein. Primary Purkinje cell cultures will be used to test the potential role of COUP- TFII in repressing the response to T3. We will use anti-sense technology to knockout the protein in culture and transient transfection assays to overexpress the protein. The third aim will be accomplished by examining the T3 response of other TREs to test if the MBP TRE is involved in repression of the T3 induction of MBP. We will determine if COUP-TF is the repressor protein that binds to the MBP TRE, and if not we will clone the repressor protein. These studies will provide critical information on the role of thyroid hormone in allowing synchronous development of the mammalian brain.

胎儿和幼儿发育期间的甲状腺激素缺乏是一个重大的世界性公共卫生问题，导致智力低下和不可逆转的脑损伤。该项目的长期目标是了解甲状腺激素 (T3) 调节哺乳动物大脑发育的机制。 T3 通过在精确的时间激活多个大脑基因来调节晚期大脑发育。然而，在激活之前，尽管存在 T3 和甲状腺激素受体，这些基因仍对甲状腺激素具有抗性。因此，抑制的丧失使得T3能够在适当的时间激活这些基因。为了确定这种新型阻遏效应的机制，我们将研究两种不同的大脑基因，即浦肯野细胞蛋白-2 (Pcp-2) 和髓磷脂碱性蛋白 (MBP)，它们分别在两种不同的细胞类型（小脑浦肯野细胞和少突胶质细胞）中表达。我们提出了三个具体目标：（1）定义 Pcp-2 基因内负责抑制 T3 依赖性激活的序列； (2) 建立孤儿受体蛋白COUP-TF在抑制Pcp-2对T3反应中的作用； (3)确定在少突胶质细胞培养物中抑制MBP基因T3诱导的机制。第一个目标是利用凝胶位移测定和 DNAse I 足迹法将阻遏物序列定位在 Pcp-2 基因的已识别阻遏物区域内。所鉴定的序列将被突变，并使用显示复制 Pcp-2 基因体内发育反应的 Pcp-2 启动子构建体测试转基因小鼠中阻抑作用的丧失。初步结果表明孤儿核受体COUP-TF可能参与阻遏活性。我们将通过测量缺乏这种蛋白质的小鼠中的 Pcp-2 来测试 COUP-TFI 的潜在作用。原代浦肯野细胞培养物将用于测试 COUP-TFII 在抑制 T3 反应中的潜在作用。我们将使用反义技术敲除培养物中的蛋白质，并使用瞬时转染测定来过度表达蛋白质。第三个目标将通过检查其他 TRE 的 T3 反应来实现，以测试 MBP TRE 是否参与抑制 MBP 的 T3 诱导。我们将确定 COUP-TF 是否是与 MBP TRE 结合的阻遏蛋白，如果不是，我们将克隆阻遏蛋白。这些研究将提供有关甲状腺激素在哺乳动物大脑同步发育中的作用的关键信息。