Elucidation of Molecular mechanism of thyroid hormone action on brain development and function

甲状腺激素在大脑发育中起重要作用已被广泛认识，但其确切机制尚未阐明。1996年，我们发现ZAKI-4是一个甲状腺激素应答基因。该基因至少有两个同工型，即ZAKI-4 α和β，可通过转录起始和剪接产生。我们最近证明了这两种异构体都能抑制钙调磷酸酶的活性。钙调磷酸酶是一种依赖钙调蛋白的磷酸酶，在神经组织中大量表达。由于已知钙调神经磷酸酶在大脑发育和功能中起着关键作用，我们假设甲状腺激素通过调节钙调神经磷酸酶的作用对大脑发育起作用，因此设计了本研究项目。在本研究中，我们旨在阐明三点；甲状腺激素对成年小鼠组织中ZAKI-4 α和β mrna表达的影响、ZAKI-4在蛋白水平上的空间和细胞内分布，以及通过产生ZAKI-4基因敲除小鼠在体内的作用。至于甲状腺激素对ZAKI-4异构体mRNA表达的影响，我们发现甲状腺功能减退小鼠的大脑皮层中ZAKI-4 α mRNA的表达降低，而甲状腺功能亢进诱导心脏中ZAKI-4 β mRNA的表达。其他组织中ZAKI-4 α和β mRNA的表达不受甲状腺状态的影响。由此可见，甲状腺激素在体内也能调控ZAKI-4亚型的表达，且这种调控具有亚型特异性和组织特异性。为了研究ZAKI-4蛋白在大脑中的空间分布和亚细胞分布，我们制备了抗ZAKI-4抗体，证实了ZAKI-4蛋白在大脑中广泛分布。其中嗅球、海马、大脑皮层和小脑的免疫反应最强烈。在亚细胞定位方面，ZAKI-4免疫反应仅限于神经元体，未在胶质细胞中检测到。ZAKI-4蛋白与钙调磷酸酶的双重免疫染色显示，它们在神经元的胞体和树突外周共定位。从这些结果，我们可以证明ZAEI-4蛋白定位在它们能够抑制钙调磷酸酶活性的区域。敲除ZAKI-4的新一代小鼠仍在进行中。我们确定了小鼠ZAKI-4基因的基因组结构，并构建了靶向载体。通过同源重组，我们获得了携带靶向构建体的小鼠胚胎干细胞。将胚胎干细胞注入囊胚，获得嵌合小鼠。现在我们正在研究靶向结构是否通过生殖细胞传播。少

It has been well recognized that thyroid hormone plays an important role in the brain development, however, its precise mechanism has not been elucidated yet. In 1996,we identified ZAKI-4 as a thyroid hormone-responsive gene. From this gene at least two isoforms, ZAKI-4 α and β, are generated by alternative transcriptional initiation and splicing. We have recently demonstrated that both isoforms inhibit calcineurin activity. Calcineurin is a calcium-calmodulin dependent phosphatase and abundantly expressed in neural tissues. Since calcineurin is known to play a pivotal role in brain development and function, we have hypothesized that thyroid hormone exerts its action on brain development by regulating the calcineurin action and, thus, have designed the present research project. In this study, we aimed to elucidate three points ; the effect of thyroid hormone on the expression of ZAKI-4 α and β mRNAs in adult mouse tissues, spatial and intracellular distribution of ZAKI-4 at its protein … More level, and the role in vivo by generating a knock-out mouse of the ZAKI-4 gene. As for the effect of thyroid hormone on the expression of ZAKI-4 isoform mRNAs, we found that expression of ZAKI-4 α mRNA was decreased in the cerebral cortex of hypothyroid mice whereas hyperthyroidism induced the expression of ZAKI-4 β mRNA in the heart. Expression of neither ZAKI-4 α nor β mRNA was influenced by thyroid status in other tissues. It is thus indicated that thyroid hormone regulate the expression of ZAKI-4 isoforms also in vivo and the regulation is isoform-and tissue-specific. In order to study the spatial and subcellular distribution of ZAKI-4 proteins in the brain, we prepared anti-ZAKI-4 antibody and demonstrated that ZAKI-4 proteins are distributed widely in the brain. Especially in olfactory bulb, hippocampus, cerebral cortex and cerebellum, the most intense immunoreactivity was found. As for the subcellular localization, ZAKI-4 immunoreactivity was confined to neuronal somata and was not detected in glia. The double immunostaining of ZAKI-4 proteins and calcineurin revealed that they were co-localized in the periphery of the soma and dendrites of neurons. From these result, we could demonstrate that ZAEI-4 proteins are localized in the area where they are able to inhibit calcineurin activity. Generation of ZAKI-4 knockout mice is still on the way. We determined the genomic structure of mouse ZAKI-4 gene and constructed the targeting vector. By homologous recombination, we obtained mouse ES cells which carry the targeting construct. We injected the ES cells into the blastocyst and obtained the chimeric mice. Now we are examining whether the targeting construct is transmitted by the germ cells. Less