Beatriz Morte

Perspectives in the Study of Thyroid Hormone Action on Brain Development and Function

The purpose of this review is to provide an up-to-date report on the molecular and physiologic processes involved in the role of thyroid hormone as an epigenetic factor in brain maturation. We summarize the available data on the control of brain gene expression by thyroid hormone, the correlation between gene expression and physiologic effects, and the likely mechanisms of action of thyroid hormone on brain gene expression. In addition we propose a role for unliganded thyroid hormone receptors in the pathogenesis of hypothyroidism. Finally, we review recent data indicating that thyroid hormone receptors have an impact on behavior.

Deletion of the thyroid hormone receptor α1 prevents the structural alterations of the cerebellum induced by hypothyroidism

Thyroid hormone (T3) controls critical aspects of cerebellar development, such as migration of postmitotic granule cells and terminal differentiation of Purkinje cells. T3 acts through nuclear receptors (TR) of two types, TRα1 and TRβ, that either repress or activate gene expression. We have analyzed the cerebellar structure of developing mice lacking the TRα1 isoform, which normally accounts for about 80% of T3 receptors in the cerebellum. Contrary to what was expected, granule cell migration and Purkinje cell differentiation were normal in the mutant mice. Even more striking was the fact that when neonatal hypothyroidism was induced, no alterations in cerebellar structure were observed in the mutant mice, whereas the wild-type mice showed delayed granule cell migration and arrested Purkinje cell growth. The results support the idea that repression by the TRα1 aporeceptor, and not the lack of thyroid hormone, is responsible for the hypothyroid phenotype. This conclusion was supported by experiments with the TRβ-selective compound GC-1. Treatment of hypothyroid animals with T3, which binds to TRα1 and TRβ, prevents any defect in cerebellar structure. In contrast, treatment with GC-1, which binds to TRβ but not TRα1, partially corrects Purkinje cell differentiation but has no effect on granule cell migration. Our data indicate that thyroid hormone has a permissive effect on cerebellar granule cell migration through derepression by the TRα1 isoform.

Importance of Monocarboxylate Transporter 8 for the Blood-Brain Barrier-Dependent Availability of 3,5,3′-Triiodo-l-Thyronine

Mutations of the gene expressing plasma membrane transporter for thyroid hormones MCT8 (SLC16A2) in humans lead to altered thyroid hormone levels and a severe neurodevelopmental disorder. Genetically engineered defect of the Mct8 gene in mice leads to similar thyroid hormone abnormalities but no obvious impairment of brain development or function. In this work we studied the relative role of the blood-brain barrier and the neuronal plasma cell membrane in the restricted access of T(3) to the target neurons. To this end we compared the effects of low doses of T(4) and T(3) on cerebellar structure and gene expression in wild-type (Wt) and Mct8 null male mice [Mct8-/y, knockout (KO)] made hypothyroid during the neonatal period. We found that compared with Wt animals, T(4) was considerably more potent than T(3) in the Mct8KO mice, indicating a restricted access of T(3), but not T(4), to neurons after systemic administration in vivo. In contrast, T(3) action in cultured cerebellar neurons was similar in Wt cells as in Mct8KO cells. The results suggest that the main restriction for T(3) entry into the neural target cells of the mouse deficient in Mct8 is at the blood-brain barrier.

Thyroid Hormone-Regulated Mouse Cerebral Cortex Genes Are Differentially Dependent on the Source of the Hormone: A Study in Monocarboxylate Transporter-8- and Deiodinase-2-Deficient Mice

Thyroid hormones influence brain development through the control of gene expression. The concentration of the active hormone T(3) in the brain depends on T(3) transport through the blood-brain barrier, mediated in part by the monocarboxylate transporter 8 (Mct8/MCT8) and the activity of type 2 deiodinase (D2) generating T(3) from T(4). The relative roles of each of these pathways in the regulation of brain gene expression is not known. To shed light on this question, we analyzed thyroid hormone-dependent gene expression in the cerebral cortex of mice with inactivated Mct8 (Slc16a2) and Dio2 genes, alone or in combination. We used 34 target genes identified to be controlled by thyroid hormone in microarray comparisons of cerebral cortex from wild-type control and hypothyroid mice on postnatal d 21. Inactivation of the Mct8 gene (Mct8KO) was without effect on the expression of 31 of these genes. Normal gene expression in the absence of the transporter was mostly due to D2 activity because the combined disruption of Mct8 and Dio2 led to similar effects as hypothyroidism on the expression of 24 genes. Dio2 disruption alone did not affect the expression of positively regulated genes, but, as in hypothyroidism, it increased that of negatively regulated genes. We conclude that gene expression in the Mct8KO cerebral cortex is compensated in part by D2-dependent mechanisms. Intriguingly, positive or negative regulation of genes by thyroid hormone is sensitive to the source of T(3) because Dio2 inactivation selectively affects the expression of negatively regulated genes.

Transcriptional induction of RC3/neurogranin by thyroid hormone: differential neuronal sensitivity is not correlated with thyroid hormone receptor distribution in the brain

RC3/neurogranin is a calmodulin-binding protein kinase C substrate, located in dendritic spines of forebrain neurons. It has been implicated in post-synaptic signal transduction events involving Ca2+ and calmodulin leading to many forms of synaptic plasticity. RC3 gene expression is under developmental and physiological regulation. The main physiological regulator appears to be thyroid gland activity. Hypothyroidism decreased RC3 mRNA concentration in the brain of post-natal day 22 rats. The affected areas included layer 6 of cerebral cortex, layers 2-3 of retrosplenial cortex, dentate gyrus and the caudate whereas others were not affected by hypothyroidism, such as upper layers of cerebral cortex, the pyramidal layer of the hippocampus and the amygdala. A single administration of triiodothyronine (T3) induced a significant transcriptional increase of RC3 mRNA in hypothyroid rats, 24 h after administration. Differential sensitivity to thyroid hormone was not related to differential expression of T3 receptor isoforms or the T3 receptor inhibitory variant alpha2. Therefore, it is likely that cell sensitivity to thyroid hormone in the brain depends on T3 receptor-associated factors.

THE HUMAN RC3 GENE HOMOLOG, NRGN CONTAINS A THYROID HORMONE-RESPONSIVE ELEMENT LOCATED IN THE FIRST INTRON

NRGN is the human homolog of the neuron-specific rat RC3/neurogranin gene. This gene encodes a postsynaptic 78-amino acid protein kinase substrate that binds calmodulin in the absence of calcium, and that has been implicated in dendritic spine formation and synaptic plasticity. In the rat brain RC3 is under thyroid hormone control in specific neuronal subsets in both developing and adult animals. To evaluate whether the human gene is also a target of thyroid hormone we have searched for T3-responsive elements in NRGN cloned genomic fragments spanning the whole gene. Labeled DNA fragments were incubated with T3 receptors (T3R) and 9-cis-retinoic acid receptors and immunoprecipitated using an anti T3R antibody. A receptor-binding site was localized in the first intron, 3000 bp downstream from the origin of transcription. Footprinting analysis revealed the sequence GGATTAAATGAGGTAA, closely related to the consensus T3-responsive element of the direct repeat (DR4) type. This sequence binds the T3R-9-cis-retin...NRGN is the human homolog of the neuron-specific rat RC3/neurogranin gene. This gene encodes a postsynaptic 78-amino acid protein kinase substrate that binds calmodulin in the absence of calcium, and that has been implicated in dendritic spine formation and synaptic plasticity. In the rat brain RC3 is under thyroid hormone control in specific neuronal subsets in both developing and adult animals. To evaluate whether the human gene is also a target of thyroid hormone we have searched for T3-responsive elements in NRGN cloned genomic fragments spanning the whole gene. Labeled DNA fragments were incubated with T3 receptors (T3R) and 9-cis-retinoic acid receptors and immunoprecipitated using an anti T3R antibody. A receptor-binding site was localized in the first intron, 3000 bp downstream from the origin of transcription. Footprinting analysis revealed the sequence GGATTAAATGAGGTAA, closely related to the consensus T3-responsive element of the direct repeat (DR4) type. This sequence binds the T3R-9-cis-retinoic acid receptors heterodimers, but not T3R monomers or homodimers, and is able to confer regulation by T3R and T3 when fused upstream of the NRGN or thymidine kinase promoters. The data reported in this work suggest that NRGN is a direct target of thyroid hormone in human brain, and that control of expression of this gene could underlay many of the consequences ofhypothyroidism on mental states during development as well as in adult subjects.

Thyroid hormone transporters—functions and clinical implications

Thyroid hormones regulate many metabolic and developmental processes, including key functions in the brain, and mutations in a transporter specific for thyroid hormone lead to severe neurological impairment. In this Review, Bernal and colleagues discuss the physiological importance and clinical implications of thyroid hormone transport, with a particular focus on brain development.

Thyroid hormone regulation of rhes, a novel Ras homolog gene expressed in the striatum

Thyroid hormone action on brain development is essentially exerted through regulation of the expression rate of a number of genes some of which have been identified in the past 10 years. In the present work we describe the thyroid hormone regulation of a novel Ras homolog which we have named Rhes (Ras homolog enriched in striatum). The rhes cDNA was previously isolated in subtractive hybridization experiments aimed at identifying cDNA clones corresponding to genes expressed preferentially in the rat striatum. The sequence was found to encode a small GTP-binding protein of the Ras family with highest homology to the dexamethasone-inducible Dexras1. Here we show that rhes mRNA and protein in the striatum are strongly dependent on the thyroidal status. Developmentally, Rhes was regulated such that in normal rats there was an increased rhes mRNA content in the striatum after postnatal day 5 (P5). Rhes concentration in hypothyroid rats was similar to that of normal rats at P5, but the subsequent age-dependent increase was blunted. The administration of a single T3 dose to hypothyroid rats normalized rhes mRNA concentration in 8 h, whereas it took 24 h, or more, to normalize the expression of rc3, another T3-dependent brain gene, involved in PKC signaling. Double in situ hybridization using rhes and rc3 riboprobes showed that the bulk of rhes signal was located in cells expressing rc3. Given the relevance of small GTPases in signal transduction it is very likely that control of rhes, in addition to rc3, is of relevance to explain the actions of thyroid hormone in the striatum, a region of the brain especially vulnerable in neurological cretinism.

Thyroid Hormone Action in the Adult Brain: Gene Expression Profiling of the Effects of Single and Multiple Doses of Triiodo-L-Thyronine in the Rat Striatum

Thyroid hormones have profound effects on mood and behavior, but the molecular basis of thyroid hormone action in the adult brain is relatively unknown. In particular, few thyroid hormone-dependent genes have been identified in the adult brain despite extensive work carried out on the developing brain. In this work we performed global analysis of gene expression in the adult rat striatum in search for genomic changes taking place after administration of T(3) to hypothyroid rats. The hormone was administered in two different schedules: 1) a single, large dose of 25 microg per 100 g body weight (SD) or 2) 1.5 microg per 100 g body weight once daily for 5 d (RD). Twenty-four hours after the single or last of multiple doses, gene expression in the striatum was analyzed using Codelink microarrays. SD caused up-regulation of 149 genes and down-regulation of 88 genes. RD caused up-regulation of 18 genes and down-regulation of one gene. The results were confirmed by hybridization to Affymetrix microarrays and by TaqMan PCR. Among the genes identified are genes involved in circadian regulation and the regulation of signaling pathways in the striatum. These results suggest that thyroid hormone is involved in regulation of striatal physiology at multiple control points. In addition, they may explain the beneficial effects of large doses of thyroid hormone in bipolar disorders.

Characterization of the promoter region and flanking sequences of the neuron-specific gene RC3 (neurogranin)

RC3 encodes a thyroid hormone-dependent, calmodulin-binding, protein kinase C substrate (neurogranin, p17) present in the dendritic spines of discrete neuronal populations in the forebrain. Its physiological role could be related to synaptic plasticity, memory, and other processes. In the present work we have isolated and sequenced 2.4 kbp of genomic DNA upstream from the origin of transcription and determined its nucleotide sequence. The major features of the RC3 promoter are the absence of TATA and CAAT boxes and the presence of an Initiator sequence surrounding the cap site. By sequence analysis we identified several cis-acting regulatory elements, among them response elements for retinoic acid and steroid (glucocorticoids/progesterone) hormone receptors. An oligonucleotide containing the retinoic acid responsive element bound to retinoic acid receptors specifically in vitro and conferred retinoic acid regulation to a heterologous promoter after transfection in COS-7 cells. Retinoic acid and dexamethasone, respectively, increased activity of the RC3 promoter in neuroblastoma cells when a deletion construct containing the retinoic acid and the glucocorticoid responsive elements was cotransfected with retinoic acid receptor or glucocorticoid receptor expression vectors. When added together all-trans retinoic acid and dexamethasone had additive effects. Despite the fact that RC3 expression in vivo is thyroid hormone-dependent, no evidence for the presence of a thyroid hormone responsive element was found within the 2.4 kbp flanking region analyzed and thyroid hormone did not increase reporter activity after cotransfection of suitable constructs with thyroid hormone receptor expression vectors. Our results suggest that the expression of RC3 in vivo could be subject to complex physiological signals, including retinoids and steroid hormones in addition to thyroid hormones.