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甲状腺激素缺乏症是全球范围内的一个主要公共卫生问题，导致智力低下和不可逆转的脑损伤。该计划的长期目标是了解甲状腺激素（T3）调节哺乳动物脑发育的机制。 T3通过精确激活多个脑基因来调节大脑晚期的发育。然而，在激活之前，尽管存在T3和甲状腺激素受体，但这些基因对甲状腺激素具有抗性。因此，抑制作用损失允许T3在适当的时间激活这些基因。为了定义这种新型抑制剂效应的机制，我们将研究两种不同的脑基因，即Purkinje细胞蛋白-2（PCP-2）和髓磷脂碱性蛋白（MBP），分别以两种不同的细胞类型表达，分别以小脑Purkinje细胞和少产生细胞表示。我们提出了三个特定目标：（1）定义负责T3依赖性激活的PCP-2基因内的序列； （2）确定孤儿受体蛋白政府TF在抑制PCP-2对T3的响应中的作用； （3）确定在发育中的少突胶质细胞培养物中T3诱导MBP基因的抑制机制。第一个目标将利用凝胶移位分析和DNase I足迹来将阻遏序列定位在PCP-2基因的已确定的阻遏区域内。使用PCP-2启动子构建体构建体以复制PCP-2基因的体内发育响应，将突变并测试转基因小鼠中阻遏物作用的丧失。初步结果表明，孤儿核受体政变可能参与阻遏活性。我们将通过测量缺乏该蛋白质的小鼠中的PCP-2来测试政变的潜在作用。原发性浦肯野细胞培养物将用于测试政变在抑制对T3反应中的潜在作用。我们将使用反义技术在培养物和瞬时转染测定中敲除蛋白质过表达的蛋白质。第三个目标将通过检查其他TRE的T3响应来测试MBP TRE是否参与MBP T3诱导的抑制。我们将确定coup-tf是否是与MBP TRE结合的阻遏蛋白，如果没有，我们将克隆抑制剂蛋白。这些研究将提供有关甲状腺激素在允许哺乳动物大脑同步发育中的作用的关键信息。