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

阐明甲状腺激素对大脑发育和功能作用的分子机制

基本信息

批准号：
14370324
负责人：
MURATA Yoshiharu
金额：
$ 7.49万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370324/
关键词：
thyroid hormone ZAKI-4 brain calcineurin knockout mouse immunohistochemistry 相同組換え

项目摘要

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

甲状腺激素在脑发育中的重要作用已被公认，但其确切机制尚未阐明。1996年，我们确定ZAKI-4为甲状腺激素反应基因。从该基因至少产生两种亚型，ZAKI-4 α和β，通过选择性转录起始和剪接。我们最近已经证明，这两种亚型抑制钙调磷酸酶活性。钙调神经磷酸酶是一种钙-钙调蛋白依赖性磷酸酶，在神经组织中大量表达。由于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作用对大脑发育发挥作用，因此设计了本研究项目。本研究旨在阐明甲状腺激素对ZAKI-4 α和β mRNA在成年小鼠组织中表达的影响，ZAKI-4蛋白的空间和细胞内分布， ...更多信息水平，并通过产生ZAKI-4基因的敲除小鼠来研究体内作用。至于甲状腺激素对ZAKI-4亚型mRNA表达的影响，我们发现甲状腺功能低下小鼠大脑皮质中ZAKI-4 α mRNA的表达减少，而甲状腺功能亢进小鼠心脏中ZAKI-4 β mRNA的表达增加。其他组织中ZAKI-4 α和β mRNA的表达均不受甲状腺状态的影响。因此表明甲状腺激素也在体内调节ZAKI-4亚型的表达，并且该调节是亚型特异性和组织特异性的。为了研究ZAKI-4蛋白在脑内的空间和亚细胞分布，我们制备了抗ZAKI-4抗体，证明ZAKI-4蛋白在脑内广泛分布。在嗅球、海马、大脑皮层和小脑中，ZAKI-4的免疫反应最强，在亚细胞定位上，ZAKI-4仅在神经元胞体中表达，而在胶质细胞中不表达。ZAKI-4蛋白和钙调神经磷酸酶的双重免疫组化染色显示，它们共同定位于神经元的索马和树突的外周。从这些结果中，我们可以证明ZAEI-4蛋白定位于它们能够抑制钙调磷酸酶活性的区域。ZAKI-4基因敲除小鼠的产生仍在进行中。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ES细胞。将胚胎干细胞注射到囊胚内，获得嵌合小鼠。现在，我们正在检查靶向结构是否由生殖细胞传播。少