Effects of neonatal hypothyroidism on rat brain gene expression.

To define at the molecular biological level the effects of thyroid hormone on brain development we have examined cDNA clones of brain mRNAs and identified several whose expression is altered in hypothyroid animals during the neonatal period. Clones were identified with probes prepared by subtractive or differential hybridization, and those corresponding to mRNAs altered in hypothyroidism were further studied by Northern blot analysis. Using RNA prepared from whole brains, no effect of hypothyroidism was found on the expression of the astroglial gene coding for glial fibrillary acidic protein. Among genes of neuronal expression, no significant alterations were found in the steady state levels of mRNAs coding for neuron-specific enolase, microtubule-associated protein-2, Tau, or nerve growth factor. N-CAM mRNA increased slightly in hypothyroid brains. In contrast a 2- to 3-fold decrease was found in the mRNA coding for a novel neuronal gene, RC3. This is the first neuronal gene known to be significantly altered at the mRNA level by thyroid hormone deprivation. The abundance of the mRNAs for the major myelin proteins proteolipid protein, myelin basic protein, and myelin-associated glycoprotein, expressed by oligodendrocytes, were also decreased in hypothyroid brains. Developmental studies on RC3 and myelin-associated glycoprotein expression indicated that the corresponding mRNAs accumulate in the brain of normal rats during the first 15-20 days of neonatal life. A similar accumulation occurred in hypothyroid brains, but at much reduced levels. The results demonstrate that thyroid hormone controls the steady state levels of particular mRNAs during brain development.     

Manipulating thyroid status alters endoplasmic reticulum calcium homeostasis in rat cerebellum

Thyroid-related hormones regulate the efficiency and expression of sarco-endoplasmic reticulum calcium ATPases in cardiac and skeletal muscle. However, little is known about the relationship between thyroid hormones and calcium (Ca2+) homeostasis in the brain. It is hypothesized that manipulating rat thyroid hormone levels would induce significant brain Ca2+ adaptations consistent with clinical findings. Adult male Sprague-Dawley rats were assigned to one of three treatment groups for 28 days: control, hypothyroid (6-n-propyl-2-thiouracil (PTU), an inhibitor of thyroxine (T4) synthesis), and hyperthyroid (T4). Throughout, rats were given weekly behavioral tests. Ca2+ accumulation decreased in the cerebellum in both hyper- and hypothyroid animals. This was specific to different ER pools of calcium with regional heterogeneity in the response to thyroid hormone manipulation. Behavioral tasks demonstrated sensitivity to thyroid manipulation, and corresponded to alterations in calcium homeostasis. Ca2+ accumulation heterogeneity in chronic hyper- and hypothyroid animals potentially explains clinical manifestations of altered thyroid status.     

Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain

The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA⁺RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T₃-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T₄ intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005)     

Identification of genes mediating thyroid hormone action in the developing mouse cerebellum

Despite the indispensable role thyroid hormone (TH) plays in brain development, only a small number of genes have been identified to be directly regulated by TH and its precise mechanism of action remains largely unknown, partly because most of the previous studies have been carried out at postnatal day 15 or later. In the present study, we screened for TH-responsive genes in the developing mouse cerebellum at postnatal day 4 when morphological alterations because of TH status are not apparent. Among the new candidate genes selected by comparing gene expression profiles of experimentally hypothyroid, hypothyroid with postnatal thyroxine replacement, and control animals using oligoDNA microarrays, six genes were confirmed by real-time PCR to be positively ( orc1l, galr3, sort1, nlgn3, cdk5r2 , and zfp367 ) regulated by TH. Among these, sort1 , cdk5r2, and zfp367 were up-regulated already at 1 h after a single injection of thyroxine to the hypothyroid or control animal, suggesting them to be possible primary targets of the hormone. Cell proliferation and apoptosis examined by BrdU incorporation and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed that hypothyroidism by itself did not enhance apoptosis at this stage, but rather increased cell survival, possibly through regulation of these newly identified genes.     

Interacting role of thyroxine and growth hormone in the hepatic synthesis of alpha 2u-globulin and its messenger RNA

Hypophysectomy completely abolishes and thyroidectomy results in a 90% reduction in the hepatic content of alpha 2u-globulin and its mRNA in the male rat. Thyroid hormone is also known to be required for the synthesis and secretion of pituitary growth hormone. In the hypothyroid rat either thyroxine or growth hormone was found to increase the activity and number of sequences of the mRNA for alpha 2u-globulin (measured by translational assay and hybridizational analysis with a cloned cDNA probe) to the euthyroid level. Treatment of hypophysectomized rats with a hormone combination containing growth hormone but not thyroxine increased the hepatic level of the mRNA for alpha 2u-globulin to that of normal animals. From these results we conclude that thyroxine indirectly influences the hepatic concentration of the mRNA for alpha 2u-globulin through its effect on pituitary growth hormone. Although administration of growth hormone to hypothyroid animals raised the hepatic concentration of alpha 2u-globulin mRNA to the euthyroid level, synthesis of alpha 2u-globulin remained low (50% of the normal). Complete recovery of alpha 2u-globulin synthesis required thyroxine. Therefore, in addition to an indirect effect on the hepatic level of alpha 2u-globulin mRNA, thyroxine also directly influences the synthesis of this protein. This direct effect of thyroxine on alpha 2u-globulin synthesis seems to be exerted at a step distal to the formation of mature mRNA.     

Thyroid hormones modulate ornithine decarboxylase in the immature rat cerebellum

In this study, we measured ornithine decarboxylase (ODC) activity as a potential parameter to evaluate the response of the developing rat brain to thyroid hormones. In cerebellum, neonatal hyperthyroidism (40 micrograms thyroxine/100 g body weight daily from birth) increased ODC activity at 2 and 5 days of age and then accelerated its developmental decline. Conversely, ODC activity was decreased in 2- and 5-day-old hypothyroid rats (propylthiouracil to the mother), but it was not significantly different from normal thereafter. No significant differences were observed in the forebrain following either treatment. In hypothyroid rat cerebellum, a single injection of triiodothyronine (T3, 100 micrograms/100 g 18 h before sacrifice) increased significantly ODC activity at all ages. A dose-response study showed that 0.5 micrograms T3/100 g is sufficient to obtain maximal stimulation. Finally, administration of antiserum against rat growth hormone had no significant effect on ODC response to T3. These results show that ODC is a useful marker of thyroid state and tissue response in the neonatal rat cerebellum.     

Ontogenic pattern of dopamine, acetylcholine, and acetylcholinesterase in the brains of normal and hypothyroid rats.

The influence of neonatal thyroidectomy (Tx) on developmental changes in dopamine (DA), acetylcholine (ACh), and acetylcholinesterase (AChE) was studied in the whole brain of rats. In control animals, brain levels of ACh gradually increased and attained adult values at the 70th day. In contrast, AChE activity showed a rapid increase between the 7th and 30th days. Levels of DA were low during the early postnatal life but markedly increased to reach adult values of 1.47 mug/g at the 30th day, after which no further enhancement was noted. Neonatal Tx interfered with the normal growth of the animals, decreased brain weights, and markedly influenced the developmental pattern of both DA and ACh in the brain. The concentration of DA in 30-day-old hypothyroid rats was 46% of the control values. In contrast, brain ACh levels in Tx rats were consistently above those seen in controls, being significantly higher, by 49 and 64%, at 15 and 30 days, respectively. Activity of AChE in brains of hypothyroid animals was not significantly different from that in controls. Treatment of Tx rats with thyroid hormone virtually restored the levels of DA and ACh to values in control animals.     

Rapid increase of mitochondrial uncoupling protein and its mRNA in stimulated brown adipose tissue. Use of a cDNA probe

The administration of thyroxine to neonatal rats stimulates RNA synthesis by neuronal nuclei isolated from the developing rat brain cortex. Glial nuclei are relatively resistant to thyroxine treatment. The activity of neuronal RNA polymerase II is particularly stimulated by the hormone. Thyroxine also affects neuronal chromatin structure as shown by changes in the relative proportion of different subnuclear fractions obtained by gentle micrococcal nuclease digestion of nuclei from hormone-treated rats.     

Thyroid hormone-, carbohydrate, and age-dependent regulation of a methylation site in the hepatic S14 gene

The rat hepatic S14 gene has served as a model of thyroid hormone regulation of gene expression. Earlier studies of the S14-containing chromatin region demonstrated that a cytosine residue at position 625 (C-625) in the 3' untranslated exon was hypermethylated in hepatic DNA derived from hypothyroid animals. This observation was consistent with the markedly reduced level of expression of the S14 gene in these rats. The current studies have extended these observations to groups of rats in various thyroidal states. By using the restriction enzyme Hhal, the percent demethylation of this site was quantitated (hypothyroid, 9.3%; euthyroid, 19.2%; hyperthyroid, 66.6%). Moreover, the level of methylation was shown to be reversible as the thyroidal state was altered. Our data also indicate that these changes are probably independent of de novo DNA synthesis. Kinetic studies of the demethylation of this cytosine residue after T3 administration showed no change for at least 1 day and maximal change after about 4 days. This contrasts with the significant rise in S14 mRNA evident within 30 min and suggests that demethylation plays no role in the acute induction of this gene by T3. Carbohydrate feeding, another stimulus of S14 expression, similarly caused the demethylation of this cytosine residue. Earlier studies had demonstrated that mRNA S14 expression was not detectable in rat pups before about 20 days of age and continued to rise through the first year of life. Consistent with those findings, S-14 C-625 was fully methylated up to 15 days of age. Progressive demethylation then occurred up to 12 months of age. These results indicate that increased demethylation of a specific site in the 3' untranslated region of the S14 gene, possibly resulting from augmented excision repair processes, is correlated with increased expression of the gene.     

The C-terminal TPR domain of Tom70 defines a family of mitochondrial protein import receptors found only in animals and fungi

In fungi and animals the translocase in the outer mitochondrial membrane (TOM complex) consists of multiple components including the receptor subunit Tom70. Genome sequence analyses suggest no Tom70 receptor subunit exists in plants or protozoans, raising questions about its ancestry, function and the importance of its activity. Here we characterise the relationships within the Tom70 family of proteins. We find that in both fungi and animals, a conserved domain structure exists within the Tom70 family, with a transmembrane segment followed by 11 tetratricopeptide repeat motifs organised in three distinct domains. The C-terminal domain of Tom70 is highly conserved, and crucial for the import of hydrophobic substrate proteins, including those with and those without N-terminal presequences. Tom70 likely arose after fungi and animals diverged from other eukaryote lineages including plants, and subsequent gene duplication gave rise to a paralogue specific to the Saccharomyces group of yeasts. In animals and in fungi, Tom70 plays a fundamental role in the import of precursor proteins, by assisting relatively hydrophobic regions of substrate proteins into the translocation channel in the outer mitochondrial membrane. Proteins that function equivalently to Tom70 may have arisen independently in plants and protists.     

Role of Thyroid Hormone and Nerve Growth Factor in the Development of Choline Acetyltransferase and Other Cell-Specific Marker Enzymes in the Basal Forebrain of the Rat

The effects of treatment with L-thyroxine (subcutaneously 0.3 microgram/g body weight daily from birth, i.e., day 1) and 2.5S nerve growth factor (NGF; intraventricularly 2 micrograms on 1, 3, 5, 7, and 9 postnatal days), separately and together, were studied on the biochemical development of different cell types in the basal forebrain of 10-day-old rats. The development of cholinergic, gamma-aminobutyric acid-ergic (GABAergic), and glutamatergic neurons was monitored respectively in terms of choline acetyltransferase (ChAT), glutamate decarboxylase (GAD), and glutaminase activities, whereas glutamine synthetase (GS) and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase) activities were used to judge the maturation of astroglial and oligodendroglial cells. Treatment with either thyroid hormone or NGF from birth significantly increased the expression of ChAT activity in the basal forebrain of neonatal rats. When both agents were administered to the same animal, in agreement with our earlier in vitro findings, the stimulation in ChAT activity was much greater than the sum of the individual effects. In hypothyroid rats, significant effects of NGF at the low doses used were not detectable, although the increase of ChAT activity induced by thyroxine was potentiated by NGF in these animals. Under the present experimental conditions neither thyroxine nor NGF treatment had an appreciable effect on the activities of glutaminase, GS, and lactate dehydrogenase. However, the administration of thyroxine markedly increased CNPase activity in normal rats, whereas in hypothyroid rats the effect on both CNPase and GAD was also significant. Similar elevations in CNPase and GAD activities were not observed after NGF treatment, suggesting that the effect of NGF was specific to the cholinergic cells.(ABSTRACT TRUNCATED AT 250 WORDS)