Thyroid Hormone and Retinoic Acid Regulation of Gene Expression

三碘甲腺原氨酸(T3)和维甲酸(RA)对于正常的神经元分化和 成长。我们利用了神经细胞系，并将胚胎干细胞分化为 神经元，识别T3和RA基因靶点。甲状腺激素受体(TR)是主要的 异构体在神经元中表达，具有不同于tr？的特征。在上一次拨款中 在此期间，我们发现了RA和T3在神经发育中的联系。RA刺激血管紧张素转换酶的表达 单羧酸转运体8(Mct8)甲状腺激素转运体，进而促进 神经元对T3的摄取。据报道，患有严重精神发育迟滞和神经缺陷的人 使Mct8失活的基因突变，Allan-Herndon-Dudley综合征，这些人是 对T3治疗无效。创伤性脑损伤(TBI)模型显示脑组织中T3水平降低 血清和大脑。本项目将重点研究T3和RA在促进神经生长和 分化以及损伤后的恢复。我们将确定T3和RA基因的作用机制 信号转导通路的调节和调制。我们将确定功能角色 甲状腺转运蛋白，特别是MCT8和MCT10及其对神经元生长的影响 分化和神经元特异性基因表达。我们已经开发了一种技术来区分 小鼠ES细胞转化为锥体神经元，是研究T3在大脑中作用的独特模型。我们还将 急、慢性啮齿动物模型标本特定脑区基因表达的研究 TBI。我们将使用甲状腺转运蛋白抑制剂和转运蛋白基因敲除来确定 T3转运的功能重要性。甲状腺激素类似物DITPA不需要Mct8 神经转运体进入神经元，并将是一个补充工具来探索的重要性 甲状腺运输。我们将使用抑制剂和敲除途径组件来确定 Wnt/β连环蛋白和MEK/ERK MAPK信号通路在神经元增殖调控中的作用 甲状腺激素的运输。我们将利用遗传方法来确定tr？和 转录因子T_3介导的基因可促进神经分化、生长和延长神经元存活。 我们将评估锥体神经元中已知的T3调节基因，这些基因对生长和 分化，以及执行全基因组芯片-序列项目，基于tr？结合，以 识别新的T3调节基因。我们将确定调节T_3调节的因素的作用。 神经元的生长和分化，包括鸡卵清蛋白上游的作用 转录因子(COUP-TF1)和钙调蛋白依赖的激酶IV(CamKIV)。最后，我们会 急、慢性颅脑损伤后大鼠脑区T3信号通路基因的表达 模特。我们的假设是RA在信号转导通路上的特定作用，而T_3在信号转导通路上的作用 核基因表达促进神经元分化和生长，延长神经元存活时间， 对损伤的反应可能概括了神经元生长和发育的模式 差异化。我们的目标是确定具有促进神经功能的潜在治疗靶点。 在TBI等条件下分化生长。

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.