Thyroid Hormone and Retinoic Acid Regulation of Gene Expression

描述（由申请人提供）： 三碘甲腺原氨酸（T3）和视黄酸（RA）是正常神经元分化和生长所必需的。我们已经利用神经元细胞系和分化成神经元的胚胎干（ES）细胞来鉴定T3和RA基因靶标。甲状腺激素受体（TR）β是神经元中表达的主要亚型，具有与TR β不同的特征。在上一个资助期，我们确定了RA和T3在神经发育中的联系。RA刺激单羧酸转运蛋白8（Mct 8）甲状腺激素转运蛋白的表达，进而促进神经元T3摄取。据报道，患有Mct 8、Allan-Herndon-达德利综合征的基因突变的人存在严重的智力迟钝和神经功能缺损，这些个体对T3治疗无效。创伤性脑损伤（TBI）模型显示血清和脑中T3水平降低。本项目将重点关注T3和RA在促进神经生长和分化以及从损伤中恢复中的作用。我们将确定T3和RA基因调控和信号转导通路的调制机制。我们将确定甲状腺转运蛋白的功能作用，特别是MCT 8和MCT 10，以及它们对神经元生长、分化和神经元特异性基因表达的影响。我们已经开发出一种技术，小鼠ES细胞分化成锥体神经元，一个独特的模型，研究T3在大脑中的作用。我们还将研究急性和慢性TBI啮齿动物模型标本特定脑区的基因表达。我们将使用甲状腺转运抑制剂和转运蛋白mRNA敲低来确定T3转运的功能重要性。甲状腺激素类似物DITPA不需要Mct 8神经转运蛋白进入神经元，将成为探索甲状腺转运重要性的补充工具。我们将使用抑制剂和敲除途径成分，以确定Wnt/β catenin和MEK/ERK MAPK途径在调节神经元增殖和甲状腺激素转运中的作用。我们将利用遗传学方法来确定TR和TR对T3介导的基因的作用，以促进神经分化，生长和延长神经元的存活。我们将评估锥体神经元中对生长和分化重要的已知T3调节基因，并基于TR结合进行全基因组ChIP-Seq项目，以识别新的T3调节基因。我们将确定调节T3-调节神经元生长和分化的因子的作用，包括鸡卵清蛋白上游转录因子（COUP-TF 1）和钙调蛋白依赖性激酶IV（CamKIV）的作用。最后，我们将测试急性和慢性TBI模型后啮齿动物脑区中T3信号通路基因的表达。我们的假设是，RA对信号转导通路的特异性作用和T3对核基因表达的特异性作用促进了神经元的分化和生长，延长了神经元的存活，并且对损伤的反应可能重演了神经元生长和分化的发育模式。我们的目标是确定有潜力在TBI等条件下促进神经分化和生长的治疗靶点。

DESCRIPTION (provided by applicant): Triiodothyronine (T3) and retinoic acid (RA) are essential for normal neuronal differentiation and growth. We have utilized neuronal cell lines, and embryonic stem (ES) cells differentiated into neurons, to identify T3 and RA gene targets. Thyroid hormone receptor (TR) � is the predominant isoform expressed in neurons and has features distinct from those of TR�. In the previous grant period we identified a link between RA and T3 in neural development. RA stimulates expression of the Monocarboxylate Transporter 8 (Mct8) thyroid hormone transporter, which in turn promotes neuronal T3 uptake. Profound mental retardation and neurologic deficits are reported in humans with gene mutations that inactivate Mct8, Allan-Herndon-Dudley Syndrome, and these individuals are refractory to treatment with T3. Traumatic Brain Injury (TBI) models show reduced levels of T3 in the serum and brain. This project will focus on the role of T3 and RA in promoting neural growth and differentiation as well as recovery from injury. We will identify the mechanisms of T3 and RA gene regulation and modulation of signal transduction pathways. We will determine the functional role of thyroid transporters, especially MCT8 and MCT10, and their influence on neuronal growth, differentiation, and neuron-specific gene expression. We have developed a technique to differentiate mouse ES cells into pyramidal neurons, a unique model to study T3 action in the brain. We will also study gene expression in specific brain areas of specimens from rodent models of acute and chronic TBI. We will use thyroid transport inhibitors and transporter mRNA knockdowns to determine the functional importance of T3 transport. The thyroid hormone analog, DITPA, does not require the Mct8 neural transporter to enter neurons and will be a complimentary tool to probe the importance of the thyroid transport. We will use inhibitors and knockdowns of pathway components to determine the role of the Wnt/� catenin and MEK/ERK MAPK pathways in regulation of neuronal proliferation and thyroid hormone transport. We will utilize genetic approaches to determine the role of TR� and TR� on T3-mediated genes to promote neural differentiation, growth and to prolong neuronal survival. We will evaluate known T3-regulated genes in pyramidal neurons important for growth and differentiation, as well as performing a genome-wide ChIP-Seq project, based on TR� binding, to identify new T3-regulated genes. We will determine the role of factors that modulate T3-regulation of neuronal growth and differentiation, including the actions of Chicken Ovalbumin Upstream Transcription Factor (COUP-TF1) and Calmodulin-Dependent Kinase IV (CamKIV). Finally, we will test expression of T3 signaling pathway genes in rodent brain areas after an acute and chronic TBI model. Our hypothesis is that specific actions of RA on signal transduction pathways and T3 on nuclear gene expression promote neuronal differentiation and growth and prolong neuronal survival, and the response to injury may recapitulate the developmental patterns of neuronal growth and differentiation. Our goal is to identify therapeutic targets with the potential to promote neural differentiation and growth in conditions such as TBI.