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

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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