Expression of ZAKI-4 Messenger Ribonucleic Acid ... - Semantic Scholar

0013-7227/01/$03.00/0 Endocrinology Copyright © 2001 by The Endocrine Society

Vol. 142, No. 5 Printed in U.S.A.

Expression of ZAKI-4 Messenger Ribonucleic Acid in the Brain during Rat Development and the Effect of Hypothyroidism* AYESHA SIDDIQ, TAKASHI MIYAZAKI, YOSHIKO TAKAGISHI, YASUHIKO KANOU, SHIZU HAYASAKA, MINORU INOUYE, HISAO SEO, AND YOSHIHARU MURATA Department of Teratology and Genetics (A.S., Y.T., Y.K., S.H., M.I., Y.M.) and Department of Endocrinology and Metabolism (T.M., H.S.), Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan ABSTRACT We identified ZAKI-4 (also designated as DSCR1L1) as a thyroid hormone responsive gene in cultured human skin fibroblasts. Recently it has been reported that ZAKI-4 belongs to an evolutionary conserved family of proteins that function as calcineurin inhibitor. In human, ZAKI-4 and calcineurin are highly expressed in brain, where thyroid hormones play essential roles in the development during fetal and neonatal periods. In the present study, we examined the temporal and spatial expression patterns of ZAKI-4 messenger RNA (mRNA) in control and hypothyroid rat brains. Northern blot analysis revealed that ZAKI-4 mRNA was detected in both cerebral cortex and cerebellum as early as embryonic day (E)18. In the cerebral cortex, the expression level gradually increased with age, reaching a plateau at postnatal day (P)7 and remained constant thereafter until P30. A similar pattern of increase with age was also observed in hypothyroid rats; however, the magnitude of the increase was significantly reduced. In control rats, the fold increase in ZAKI-4 mRNA level from

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E CLONED ZAKI-4 (also designated as DSCR1L1, assigned by Human Nomenclature Committee) as a thyroid hormone-responsive gene from cultured human skin fibroblasts using a differential display of messenger RNAs (mRNAs) (1). The complementary DNA (cDNA) sequence analysis revealed that ZAKI-4 is transcribed as two mRNA species (3.4 and 1.4 kb), by the use of two polyadenylation signals. Both mRNAs encode a single peptide with a putative molecular mass of 21 kDa consisting of 192 amino acids. In addition to fibroblasts, ZAKI-4 mRNA is also expressed in the target tissues of thyroid hormone, such as the brain, heart, liver, and skeletal muscle in human (1). Thus, modulation of its expression by thyroid hormone status in these tissues might have an important functional consequence, which has not been studied yet, especially in brain, where ZAKI-4 mRNA is abundantly expressed. ZAKI-4 belongs to a family of evolutionary conserved proteins that harbor a conserved ISSP sequence motif (2). Received September 26, 2000. Address all correspondence and requests for reprints to: Yoshiharu Murata, Department of Teratology and Genetics, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan. E-mail: ymurata@riem. nagoya-u.ac.jp. * This study was supported, in part, by Grants-in-Aid for Scientific Research No. 11470225 (to Y.M.) and No. 12836003 (to Y.K.) from the Ministry of Education, Science, Sports and Culture, Japan.

E18 to P17 was 10.8; whereas in hypothyroid rats, it was 7.4. In cerebellum the expression level did not change with age or by thyroid status. In situ hybridization revealed that ZAKI-4 mRNA is widely expressed in neurons throughout the brain. It is noteworthy that the expression in the neurons of layer VI of the cerebral cortex was more evident in control rats than that in hypothyroid rats from P17 to P30. Though not influenced by hypothyroidism, there were several regions of the brain in which ZAKI-4 mRNA was strongly expressed. These regions were the mitral cell layer of the olfactory bulb, the substantia nigra, and the hippocampus, where calcineurin is also abundantly expressed. Therefore, it may be hypothesized that ZAKI-4 plays an important role in the development and function of the brain by modulating calcineurin function; and decrease in ZAKI-4 mRNA expression in the specific brain areas may explain, in some parts, the mechanism of abnormal brain development by hypothyroidism. (Endocrinology 142: 1752–1759, 2001)

Proteins belonging to this family have been identified in divergent species ranging from fungus to human, and they function as a calcineurin inhibitor (2). Calcineurin is a calcium- and calmodulin-activated protein phosphatase that regulates a variety of developmental and cellular processes. Calcineurin is composed of A and B subunits and dephosphorylates the transcription factor NF-AT. Then, the dephosphorylated NF-AT translocates to the nucleus and regulates gene transcription (3, 4). In brain, calcineurin plays a pivotal role in the control of memory (5, 6), synaptic plasticity, and apoptosis of the neural cells (7, 8). It has been recently shown that ZAKI-4 interacts with A subunit of calcineurin and inhibits NF-AT-mediated gene transcription (9, 10). Thyroid hormone plays an essential role for normal mammalian brain maturation and function. During the critical period of brain development in the neonatal period, lack of adequate levels of thyroid hormone, either caused by iodine deficiency or caused by developmental abnormalities of thyroid gland, results in cretinism that features severe and irreversible mental retardation (11–15). Compared with sporadic cretinism, the neurological deficits in endemic cretinism seem to be very severe, suggesting that thyroid hormone supplied from the mother is also important for fetal brain development (16). Hypothyroidism causes severe morphological abnormalities in the entire developing brain. For example, the size and number of neurons, especially the

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ZAKI-4 mRNA EXPRESSION IN RAT BRAIN DEVELOPMENT

pyramidal neurons in the neocortex and hippocampus, neurons in the olfactory bulb, and Purkinje cells in the cerebellum are reduced (11–13). The growth and arborizations of the neuronal dendrites and axonal density are also impaired (11–13), and neuronal cell migration is disturbed (11–13). In view of these profound effects of thyroid hormone deficiency on the central nervous system (CNS), neuronal genes regulated by thyroid hormone could potentially be involved in the development of the CNS. However, the exact mechanism by which hypothyroidism affects brain development remains to be elucidated. The function of ZAKI-4 as a calcineurin inhibitor and its regulation by thyroid hormone promoted us to examine the spatial and temporal expression of ZAKI-4 mRNA in the cerebral cortex and cerebellum, both in normal and hypothyroid neonates. In the present study, it was demonstrated that the increase in ZAKI-4 mRNA in the cerebral cortex, with age, was significantly attenuated in the hypothyroid pups. The reduced expression was especially evident in layer VI of the cortex, from postnatal day P17 to P30. Though not affected by hypothyroidism, the strong expression of ZAKI-4 mRNA was observed in specific brain areas, such as mitral cells of olfactory bulb, granular and pyramidal cells of hippocampus, substantia nigra where calcineurin is also highly expressed (17–21). These results suggest that ZAKI-4 is involved in the development and function of the brain by modulating calcineurin activity in vivo, and the reduction in ZAKI-4 mRNA in the specific area caused by hypothyroidism may account for one of the mechanisms of abnormal brain development in hypothyroidism. Materials and Methods Preparation of animals and samples collection All the rats were treated in accord with the principles and procedures outlined by the Committee for Animal Experiment of Nagoya University School of Medicine and Research Institute of Environmental Medicine. Pregnant Wistar rats were housed individually at 23 ⫾ 1 C and maintained on a 12-h light, 12-h dark cycle. The experimental groups consisted of control euthyroid and hypothyroid animals. Control animals received water and food ad libitum. To induce fetal and neonatal hypothyroidism, we used a combination of chemical and surgical thyroidectomy (22). Methyl mercaptoimidazol (at a concentration of 0.025%; MMI; Sigma, St. Louis, MO) was administered in drinking water, to the pregnant rats, from gestational day 10. Surgical thyroidectomy was performed on the pups on P10, and the mothers continued drinking MMI-containing water throughout the experiment. To confirm the brain hypothyroidism, cerebellar sections stained by hematoxylin and eosin were studied on both control and hypothyroid rats at the age of P0, 10, 17, 24, and 30. For the study of tissue distribution of ZAKI-4 mRNA, 10-week-old control rats were killed by decapitation; and brain, heart, liver, lung, kidney, spleen, and skeletal muscle were obtained. The brain was further dissected into three parts: cerebellum, cerebral cortex, and the rest of the brain. To study the ontogenic expression of ZAKI-4 mRNA in the brain, by Northern blotting, the cerebral cortices and cerebella were obtained on embryonic day (E)18 and P0, 10, 17, 24, and 30. To obtain brain samples from E18, the pregnant mothers were anesthetized with diethyl ether, and the fetuses were immediately removed; then, the cerebral cortices and cerebella were collected from all fetuses and frozen quickly on dry ice. Both male and female pups, from P0-P30, were killed by decapitation, and the cerebral cortices and cerebella were dissected and frozen quickly on dry ice. All the samples were stored at ⫺80 C until the analysis. To examine the spatial expression of ZAKI-4 mRNA in brain, in situ hybridization (ISH) was performed on rat brain sections, according to

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Birren et al. (23), with minor modifications. In brief, after deep anesthesia with ether, rats were perfused transcardially with 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4. Brains were dissected out and postfixed in the same solution for 20 h at 4 C. The tissues were cryoprotected in 30% sucrose in 0.1 m PBS for 2–3 days at 4 C. Then, brains were frozen in O.C.T embedding medium (10.24% w/w polyvinylalcohol, 4.26% w/w polyethylene glycol, 85.50% w/w nonreactive ingredients) in a quick-freeze CO2 chamber and kept at ⫺80 C until use. Both sagittal and frontal sections of 10-␮m thickness were prepared by cryostat at ⫺24 C. The sections were placed on ribonuclease-free 2% aminopropyltriethoxysilane (Sigma) in acetone-coated glass slides and kept at ⫺80 C until use. Rats were killed at the following postnatal ages: P3, 10, 17, 24, and 30. In all experiments, at least six animals from each experimental groups were analyzed.

Extraction of total RNA and Northern blot analysis Total RNA was extracted by the method of Chomczynski and Sachhi (24), and Northern blot analysis was performed as described previously (25). Aliquots of 15 ␮g total RNA were subjected to Northern blotting. Rat ZAKI-4 cDNA, spanning the entire open reading frame (26), was labeled with [32P␣] deoxycycidine triphosphate (specific activity, 111 TeBq/mmol; NEN Life Science Products, Boston, MA) by a Random Primed DNA Labeling Kit (Roche Molecular Biochemicals, Mannheim, Germany) and used as a probe. The membranes were rehybridized with cDNA for 18S ribosomal RNA (rRNA). Radioactivites of the bands corresponding to ZAKI-4 mRNA and rRNA were determined by Molecular Imager System (GS-363; BioRad Laboratories, Inc., Hercules, CA). ZAKI-4 mRNA levels were normalized by those of rRNA.

In situ hybridization (ISH) Both digoxigenin and [35S␣]-uridine 5⬘-triphosphate-labeled rat ZAKI-4 cRNA probes were synthesized by in vitro transcription of a pGEM-T plasmid (Promega Corp. Biotech, Madison, WI) containing the entire coding sequence of rat ZAKI-4 cDNA. The antisense probe used for ISH was transcribed by using T7 RNA polymerase after linearizing the plasmid with a restriction enzyme, SalI. The corresponding sense strand probe was transcribed by SP6 RNA polymerase after linealization of the same plasmid with NcoI. To permeabilize the tissue sections, the following series of treatments were carried out. The fixed sections were first thawed at 20 C and washed with PBS pretreated with diethylpyrocarbonate. Sections were permeabilized with 0.02% Triton X-100 in PBS, postfixed with 4% paraformaldehyde for 5 min, and washed in PBS. Then, they were acetylated with 0.25% acetic anhydride and 0.1 m triethanolamine in saline for 10 min and washed in graded ethanol. Prehybridization was performed for 3 h

FIG. 1. Tissue distribution of ZAKI-4 mRNA in adult rat. Fifteen micrograms of total RNA from each tissue was subjected to Northern blot analysis using full-length rat ZAKI-4 cDNA as a probe. The membrane was reprobed with the cDNA for rRNA. The autoradiography was performed for 7 days and for 1 h for ZAKI-4 mRNA and rRNA, respectively.

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FIG. 2. Ontogenic expression of ZAKI-4 mRNA in the cerebral cortex and effect of hypothyroidism on the expression profile. Total RNA (15 ␮g) from cerebral cortex of both control and hypothyroid animals, on E18 and on P0, 10, 17, 24, and 30, was subjected to Northern blot analysis for ZAKI-4. The membrane was reprobed with the cDNA for rRNA. A, Representative autoradiograph showing ZAKI-4 mRNA after 7 days of exposure. An autoradiograph of rRNA, obtained by 1 h of exposure, is also shown. B, Densitometric analysis. ZAKI-4 mRNA levels normalized by those of rRNA are presented as mean ⫾ SE (n ⫽ 5). *, P ⬍ 0.0001 vs. E18 in control group; #, P ⬍ 0.0001 vs. E18 in hypothyroid group; ⫹, P ⬍ 0.01; ⫹⫹, P ⬍ 0.001 between control and hypothyroid; NS, not significant. at 55 C in the prehybridization solution [50% formamide (Nacalai Tesque Inc., Kyoto, Japan), 10% dextran sulfate (Sigma), 5 ⫻ Denhardt’s solution (1 ⫻ Denhardt’s solution is 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, 0.02% BSA), 0.1 m Tris-HCl (pH 7.4), 0.4 m NaCl, 5 mm EDTA, 0.1% SDS, 250 ␮g/ml yeast transfer RNA, 250 ␮g/ml salmon sperm DNA, and 100 mm dithiothreitol]. Hybridization was performed in the prehybridization solution containing either 1 ␮g/ml of the digoxigenin-labeled cRNA or 106 cpm/ml of the 35 S-labeled cRNA at 55 C for 16 h. The sections were washed twice in 50% formamide and 2 ⫻ SSC at 55 C for 15 min and then incubated with 0.2 ␮g/ml RNAseA (Roche Molecular Biochemicals) in a solution [0.5 m NaCl, 0.05 m EDTA, and 0.05 m Tris-HCl (pH 7.5)] for 30 min at 37 C. After sequential washing at 20 C with 100 mm dithiothreitol in 2, 1, 0.5, and 0.1 ⫻ SSC each for 5 min, the sections were further rinsed twice with 0.1 ⫻ SSC and dehydrated with ethanol before autoradiography. The slides were exposed to x-ray films (Eastman Kodak Co., Rochester, NY) for 7–10 days and dipped into NTB2 emulsion (Eastman Kodak Co.) for 2 weeks at 4 C. After the exposure, slides were developed with Eastman Kodak Co. D19, fixed, and counterstained with cresyl violet. Samples that were hybridized with the sense cRNA were used as control.

Statistical analysis Statistical analysis was carried out using one-way ANOVA followed by Fisher’s protected least-significant-difference analysis. P values less than 0.05 were considered as significant differences.

Results Confirmation of brain hypothyroidism

Histological examination was performed on the cerebellum of rats treated with MMI and thyroidectomy (data not shown). The external granule cells remained in the hypothyroid rat on P24, when they had already disappeared in the control rat. This retardation of granular cell migration in the cerebellum is a typical finding of brain hypothyroidism (27). In addition, other features of neonatal hypothyroidism, such as severe growth retardation and delayed eye opening (28), were also noted.


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FIG. 3. Ontogenic expression of ZAKI-4 mRNA in the cerebellum and effect of hypothyroidism on the expression profile. Total RNA (15 ␮g) from cerebellum of both control and hypothyroid animals, on E18, P0, 10, 17, 24, and 30, was subjected to Northern blot analysis for ZAKI-4. The membrane was reprobed with the cDNA for rRNA. A, Representative autoradiograph of ZAKI-4 mRNA obtained by 7 days of exposure. An autoradiograph for rRNA, obtained by 1 h of exposure, is presented in the lower panel. B, Densitometric analysis. ZAKI-4 mRNA levels normalized by those of rRNA are presented as mean ⫾ SE (n ⫽ 5).

Expression of ZAKI-4 mRNA in different tissues of rat

We first examined the tissue distribution of ZAKI-4 mRNA by Northern blot analysis, in 10-week-old rat tissue. As shown in Fig. 1, ZAKI-4 mRNA was depicted as two bands at 3.4 and 1.4 kb. As observed in humans (1), these two mRNA species are likely to be generated by alternative polyadenylation. ZAKI-4 mRNA was expressed abundantly in the brain and heart. The expression was observed in three regions of the brain (cerebellum, cerebral cortex, and the rest of the brain), indicating the wide distribution of ZAKI-4 mRNA in the brain. The expression was also detected in kidney, liver, and skeletal muscles; but their amount was much less than those in brain and heart. ZAKI-4 mRNA was not detected in lung or spleen in the present experimental condition. Developmental expression of ZAKI-4 mRNA in normal and hypothyroid rats

We next compared the ontogenic expression of ZAKI-4 mRNA in cerebral cortex and the cerebellum in control and hypothyroid rats. As shown in Fig. 2, in the cerebral cortex,

ZAKI-4 mRNA was detected as early as E18. Then, the expression increased gradually and significantly with age, reaching a plateau at P17. A similar pattern of ZAKI-4 mRNA expression was observed in hypothyroid animals. However, after P10, the levels were significantly lower than those in control animals. There was a 10.8-fold increase in the mRNA expression, from E18 to P17, in the control; whereas the increase was 7.4-fold in hypothyroid animals. Figure 3 shows the ontogenic profile of ZAKI-4 mRNA expression in cerebellum. The expression was also detected as early as E18 in cerebellum. However, in contrast to cerebral cortex, the level of expression did not change significantly until P30. Moreover, there was no significant difference in ZAKI-4 mRNA level between control and hypothyroid animals. Spatial expression of ZAKI-4 mRNA during brain development

The spatial expression of ZAKI-4 mRNA in the brain of control and hypothyroid rats was studied by ISH during

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FIG. 4. Comparison of the distribution of ZAKI-4 mRNA in rat brain between control and hypothyroid rats. Negative film images of ISH with 35 S-labeled rat ZAKI-4 cRNA antisense probe in control (A–C) and hypothyroid (D–F) rats on P10 (A and D), P17 (B and E), and P30 (C and F). G, Hybridization with the cRNA sense probe. The hybridization signals are depicted as white dots. Ob, Olfactory bulb; Cx, cerebral cortex; VI, sixth layer of the cerebral cortex; Hp, hippocampus; Thn, thalamic nucleus; Sn, substantia nigra; Ce, cerebellum; Pn, pontine nucleus; scale bar, 5 mm.

their development. In both control and hypothyroid rats, the expression of ZAKI-4 mRNA was widely distributed, specifically in the neurons of the entire brain, from P3 to the end of the observation (P30) (Fig. 4, A–F). The expression was also observed as early as E18 (data not shown). In some specific areas, the intense signal was detected; these were: olfactory bulb, cerebral cortex, hippocampus, part of thalamic nuclei, substantia nigra, cerebellum, and pontine nucleus. No signal

was detected when sense probes were used for the hybridization (Fig. 4G). In the olfactory bulb, the mitral cell layer showed strong expression throughout the development in both control and hypothyroid rats (Fig. 4, A–F). All mitral cells were intensely labeled with digoxigenin-labeled ZAKI-4 antisense probe (Fig. 5A). The moderately labeled cells were also present in the glomerular layer and internal granular layers. In the


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FIG. 5. Cell type-specific distribution of ZAKI-4 mRNA, in a control rat brain, by ISH with digoxigenin-labeled cRNA probes. A, Olfactory bulb. Strong expression of the ZAKI-4 mRNA is present in mitral cells (arrowheads in inset) of the mitral cell layer (MI). The glomerular layer (Gl), external plexiform layer (EPl), and internal granular layer (Igr) also express the ZAKI-4mRNA moderately. Scale bar, 100 ␮m. B, Hippocampus. Granule cells in dentate gyrus (DG) and pyramidal cells in CA1–3 are intensely labeled. Scale bar, 200 ␮m; C, cerebral cortex. The expression of ZAKI-4 mRNA is shown in neurons of layers I–VI. The inset shows pyramidal cells in layer V. Scale bar, 100 ␮m.

hippocampus, granule cells in the dentate gyrus and pyramidal cells in the CA1–3 were intensely labeled (Fig. 5B); and the expression pattern was similar during the development (Fig. 4, A–F). Substantia nigra was one of the most intensely labeled areas in the brain (Fig. 6, A and B). The strong expression of ZAKI-4 mRNA was found in the pars compacta from P3, whereas the expression was moderate in pars reticulata (Fig. 6, C and D). In the cerebellum, the external and internal granular layers expressed ZAKI-4 mRNA in the early age of development; however, the expression was gradually confined to the internal granular layer, with migration of cells from the external to internal granular layer. In addition, ZAKI-4 was also expressed in part of thalamic nuclei and pontine nucleus in all ages during development

(Fig. 4, A–F). In the areas described above, the expression pattern of ZAKI-4 was not affected by the thyroid status. In contrast, hypothyroidism affected the pattern of ZAKI-4 mRNA expression in cerebral cortex. Neurons including pyramidal cells expressed the ZAKI-4 mRNA in all layers (I–VI) (Fig. 5C). Among these layers, the most intense labeling was found in layer VI, from P17 to P30, in the normal rats (Fig. 4, B and C). However, in hypothyroid animals, the prominent labeling in layer VI was not observed in this period (Fig. 4, E and F). Discussion

The present study revealed that tissue distribution of ZAKI-4 mRNA expression is similar between human and rat.

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FIG. 6. Expression of ZAKI-4 mRNA in substantia nigra. A and C, Control; B and D, hypothyroid. A and B, negative film images of frontal sections; scale bar, 0.5 mm. C and D, darkfield photomicrographs; scale bar, 200 ␮m. Note the higher expression of ZAKI-4 mRNA in pars compacta (Snpc) than in pars retuculata (Snpr).

In both species, ZAKI-4 mRNA was highly expressed in brain and heart. In these tissues, it has been demonstrated that calcineurin is also abundantly expressed and plays an important role (5, 6, 17, 29, 30). Because recent studies have revealed that a family of ZAKI-4 proteins, such as DSCR-1, yeast Rcn 1, inhibits calcineurin function in vitro when they are overexpressed (2), our present results on the tissue distribution suggest a possible role of ZAKI-4 as a natural calcineurin regulator in vivo. Several genes have been identified in brain as thyroid hormone-responsive (15). These genes could be grouped according to the cell lineage. A group of genes, such as myelin basic protein (31) and proteolipid protein (32), are expressed in oligodendrocytes and play a role in myelination (15). The second group includes Purkinje cell protein-2 (33) and neurogranin/RC3 (28), which are expressed in neurons, but the functions of these proteins are not well understood yet. ZAKI-4 could also be categorized in the second group, because ZAKI-4 mRNA is mainly expressed in neuronal cells (Fig. 5). The present study demonstrated that ZAKI-4 mRNA was expressed from the rat fetal period to adulthood and widely distributed throughout the brain. However, the effect of hypothyroidism on the expression of ZAKI-4 mRNA in the brain was limited in a specific area after P10. Northern blot analysis revealed that the expression of ZAKI-4 mRNA was

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gradually increased in the cerebral cortex during development and that the increase was attenuated by hypothyroidism from P10 through the end of observation period (P30). This is the first demonstration in vivo that ZAKI-4 mRNA expression in brain is decreased by hypothyroidism. Furthermore, ISH revealed that the most prominent effect of hypothyroidism was restricted to layer VI in the cerebral cortex. As reported previously in human skin fibroblasts, T3 enhances the transcription of ZAKI-4 gene indirectly, because it was completely abolished by the pretreatment with cycloheximide (1). It is thus indicated that T3 induces synthesis of some protein(s) and this protein(s) enhances the transcription of the ZAKI-4 gene. Therefore, the effect of hypothyroidism exerted on the restricted area in the brain could be attributable to the differential expression of the protein(s). Because it was demonstrated that neonatal hypothyroidism affects ZAKI-4 mRNA expression in the cerebral cortex, especially in the layer VI, one could assume that thyroid hormone affects the calcineurin activity in the developing brain. However, no information is available, so far, explaining how calcineurin activity in the cerebral cortex is affected by neonatal hypothyroidism. Interestingly, thyroid hormone-responsive genes, reported in the brain so far, are subjected to temporal and spatial regulation by T3. Thyroid hormone-dependent induction of myelin basic protein, Purkinje cell protein-2, calbindin, and IP3 receptor mRNAs is temporal, being observed from P1 to P15, but the effect no longer exists at P45 (34). On the other hand, the effect of hypothyroidism on the expression of RC3 mRNA is similar to that on ZAKI-4 mRNA, to some extent. The expression is influenced by long-term hypothyroidism (from P10 to P15 of the rat neonatal period through adulthood), but there is an exquisite regional selectivity of thyroid hormone action on RC3 expression (28), as in the case of ZAKI-4. In cerebral cortex, for example, the influence of hypothyroidism on the RC3 expression is limited to layer VI (28). The strong expression of ZAKI-4 mRNA in specific brain areas would provide important information for understanding the role of ZAKI-4 protein in the CNS. Because ZAKI-4 has been shown to inhibit calcineurin function in vitro (9, 10), it may also function as a calcineurin inhibitor in vivo. In rat brain, it has been reported that calcineurin is expressed in neurons throughout the brain (17–20), especially in the caudatoputamen, hippocampus, and substantia nigra (17, 18). On the other hand, the expression was not detected in glial cells (17, 21). Therefore, it seems that calcineurin and ZAKI-4 mRNAs are expressed coordinately in different brain areas. Several reports have indicated that calcineurin is involved in synaptic plasticity and thus plays a role in memory and learning (5, 6). Our finding that ZAKI-4 is also strongly expressed in hippocampus would suggest that ZAKI-4 regulates the calcineurin function in this area and is involved in memory and learning. In the present study, the strong expression of ZAKI-4 mRNA was also demonstrated in mitral cells of olfactory bulb and substantia nigra pars compacta, which are parts of the dopaminergic system of the brain. Cell death in this system gives rise to the degenerative diseases of the brain, such as Parkinson’s disease. Because it has been shown that


calcineurin induces neuronal apoptosis (35, 36), uncontrolled calcineurin activity may induce neurodegenerative disorders. In fact, there are several reports that cyclosporin A, an inhibitor of calcineurin, exerts beneficial effects on dopaminergic neurons and dopamine-mediated behaviors and thus has been listed as a potential therapeutic agent for Parkinson’s disease and other movement disorders (37–39). Therefore, it could be hypothesized that ZAKI-4 plays a role in maintaining the dopaminergic system and that the failure of the expression would lead to neurodegenerative disorders. In summary, we presented that ZAKI-4 is expressed abundantly in rat brain and heart. In the brain, ZAKI-4 mRNA was widely expressed, mainly in neurons of the entire brain. In addition, a strong expression was observed in some areas, such as olfactory bulb, substantia nigra, cerebral cortex, and pontine nucleus, and a part of thalamic nulei. Hypothyroidism induced a significant decrease in the expression of ZAKI-4 mRNA in cerebral cortex during the development after P10, especially in layer VI of the cerebral cortex. Although a further study is required to elucidate how thyroid hormone regulates calcineurin activity in the developing brain, the decreased ZAKI-4 expression by neonatal hypothyroidism may lead to alteration in calcineurin activity and result in abnormal brain development. Acknowledgments We thank Dr. Juan Bernal, Instituto de Investigaciones Biomedicas, Madrid, Spain, for his valuable suggestions on our study. We also thank Dr. Satoshi Kakiya, First Department of Internal Medicine, Nagoya University, School of Medicine, for providing us technical instruction for ISH; and Dr. Yoshitaka Hayashi, Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, for his careful reviewing of the manuscript.

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