Grant W. Anderson

Thyroid hormones and the brain

Significant progress has been made over the past 2 decades toward understanding the molecular basis of thyroid hormone action. It is now widely accepted that thyroid hormones play predominantly a nuclear role and function by regulating the transcription of specific target genes. Understanding thyroid hormone action at the tissue and organismic level requires assessment of the thyroid hormone response apparatus and identification of specific target genes. Progress toward uncovering the molecular basis of thyroid hormone action during mammalian brain development is advancing rapidly. This commentary provides a brief overview of the molecular basis of thyroid hormone action followed by three sections detailing thyroid hormone regulation of brain development at the functional, cellular, and molecular levels. Each section is followed by a discussion of unresolved issues and an analysis of our current level of understanding of each topic.

Control of Thyroid Hormone Action in the Developing Rat Brain

Thyroid hormones play important roles in brain development. The physiologic function of thyroid hormones in the developing brain is to provide a timing signal that leads to the induction of differentiation and maturation programs during precise stages of development. Inappropriate initiation of these timing events leads to asynchrony in developmental processes and a deleterious outcome. The developing brain is protected from premature thyroid hormone signaling through a variety of measures. Firstly, local brain levels of both thyroxine and triiodothyronine are controlled by ontogenically regulated patterns of production and metabolism. Secondly, developmentally regulated expression of nuclear proteins involved with the nuclear TH response apparatus control the temporal response of brain genes to thyroid hormone. Finally, developmental regulation of TH action modulating transcription factor expression also controls TH action in the developing brain. Together these molecular mechanisms cooperatively act to temporally control TH action during brain development. A description of these controlling mechanisms is the subject of this review.

American Thyroid Association Guide to investigating thyroid hormone economy and action in rodent and cell models.

BACKGROUND An in-depth understanding of the fundamental principles that regulate thyroid hormone homeostasis is critical for the development of new diagnostic and treatment approaches for patients with thyroid disease. SUMMARY Important clinical practices in use today for the treatment of patients with hypothyroidism, hyperthyroidism, or thyroid cancer are the result of laboratory discoveries made by scientists investigating the most basic aspects of thyroid structure and molecular biology. In this document, a panel of experts commissioned by the American Thyroid Association makes a series of recommendations related to the study of thyroid hormone economy and action. These recommendations are intended to promote standardization of study design, which should in turn increase the comparability and reproducibility of experimental findings. CONCLUSIONS It is expected that adherence to these recommendations by investigators in the field will facilitate progress towards a better understanding of the thyroid gland and thyroid hormone dependent processes.

Triiodothyronine is a survival factor for developing oligodendrocytes

Thyroid hormone plays an important role in oligodendrocyte development. The studies presented here suggest that thyroid hormone is required for oligodendrocyte survival during development. Oligodendrocyte precursor cells, astrocytes and microglia were cultured in a defined media. Oligodendrocyte precursor cell differentiation was induced by growth factor removal. Time course studies revealed that oligodendrocytes cultured in the presence or absence of triiodothyronine (T3) develop similarly during the first 3 days of development. Oligodendrocytes cultured in the absence of T3, however, die after developmental day 3. TdT-Mediated dUDP Nick End Labeling assay and Hoechst staining indicate that T3 rescues developing oligodendrocytes from death by apoptosis. Apoptosis is likely induced by the presence of the cytokines TNFalpha and IL-1beta. However, expression of these cytokines is not altered by thyroid hormone administration. Thus, thyroid hormone has been demonstrated to effect proliferation, myelin gene expression and now the survival of developing oligodendrocytes.

Thyroid hormone and cerebellar development

Thyroid hormone (TH) plays a key role in mammalian brain development. The developing brain is sensitive to both TH deficiency and excess. Brain development in the absence of TH results in motor skill deficiencies and reduced intellectual development. These functional abnormalities can be attributed to maldevelopment of specific cell types and regions of the brain including the cerebellum. TH functions at the molecular level by regulating gene transcription. Therefore, understanding how TH regulates cerebellar development requires identification of TH-regulated gene targets and the cells expressing these genes. Additionally, the process of TH-dependent regulation of gene expression is tightly controlled by mechanisms including regulation of TH transport, TH metabolism, toxicologic inhibition of TH signaling, and control of the nuclear TH response apparatus. This review will describe the functional, cellular, and molecular effects of TH deficit inthe developing cerebellum and emphasize the most recent findings regarding TH action in this important brain region.

Chicken Ovalbumin Upstream Promoter-Transcription Factor (COUP-TF) Modulates Expression of the Purkinje Cell Protein-2 Gene A POTENTIAL ROLE FOR COUP-TF IN REPRESSING PREMATURE THYROID HORMONE ACTION IN THE DEVELOPING BRAIN

The cerebellar Purkinje cell-specific PCP-2 gene is transcriptionally activated by thyroid hormone during the 2nd and 3rd weeks of postnatal life in the rat. In contrast, thyroid hormone has no detectable effects on PCP-2 expression in the fetal rat. We now present data that suggest that the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor (COUP-TF) represses triiodothyronine (T3)-dependent transcriptional activation of PCP-2 in the immature Purkinje cell. Gel shift assays show that the PCP-2 A1TRE and adjoining sequences (−295/−199 region) bind to rat and mouse brain nucleoproteins in a developmentally regulated fashion and that one of these nucleoproteins could be the orphan nucleoprotein COUP-TF. In support of this hypothesis, in vitro translated COUP-TF binds to the −295/−199 region and COUP-TF represses T3-dependent activation of the PCP-2 promoter in transient transfection analyses. Finally, immunohistochemical studies reveal that COUP-TF is specifically expressed in the immature fetal and early neonatal Purkinje cell and that this expression diminishes coincident with thyroid hormone induction of PCP-2 expression. Our findings are consistent with the hypothesis that the presence or absence of inhibitory proteins bound to the thyroid hormone response element of T3-responsive genes governs the responsivity of these genes to thyroid hormone during brain development.

Perinatal Iron and Copper Deficiencies Alter Neonatal Rat Circulating and Brain Thyroid Hormone Concentrations

Copper (Cu), iron (Fe), and iodine/thyroid hormone (TH) deficiencies lead to similar defects in late brain development, suggesting that these micronutrient deficiencies share a common mechanism contributing to the observed derangements. Previous studies in rodents (postweanling and adult) and humans (adolescent and adult) indicate that Cu and Fe deficiencies affect the hypothalamic-pituitary-thyroid axis, leading to altered TH status. Importantly, however, relationships between Fe and Cu deficiencies and thyroidal status have not been assessed in the most vulnerable population, the developing fetus/neonate. We hypothesized that Cu and Fe deficiencies reduce circulating and brain TH levels during development, contributing to the defects in brain development associated with these deficiencies. To test this hypothesis, pregnant rat dams were rendered Cu deficient (CuD), FeD, or TH deficient from early gestation through weaning. Serum thyroxine (T(4)) and triiodothyronine (T(3)), and brain T(3) levels, were subsequently measured in postnatal d 12 (P12) pups. Cu deficiency reduced serum total T(3) by 48%, serum total T(4) by 21%, and whole-brain T(3) by 10% at P12. Fe deficiency reduced serum total T(3) by 43%, serum total T(4) by 67%, and whole-brain T(3) by 25% at P12. Brain mRNA analysis revealed that expression of several TH-responsive genes were altered in CuD or FeD neonates, suggesting that reduced TH concentrations were sensed by the FeD and CuD neonatal brain. These results indicate that at least some of the brain defects associated with neonatal Fe and Cu deficiencies are mediated through reductions in circulating and brain TH levels.

Neonatal Infection of Mice With Lactate Dehydrogenase-elevating Virus Results in Suppression of Humoral Antiviral Immune Response but does not Alter the Course of Viraemia or the Polyclonal Activation of B Cells and Immune Complex Formation

Neonatal infection of FVB mice with lactate dehydrogenase-elevating virus (LDV) prevented the normal formation of anti-LDV antibodies observed in mice infected at 5 days of age or older. Even 22 weeks post-infection, the concentration of circulating anti-LDV antibodies in neonatally infected mice was insignificant. However, the time course and level of persistent viraemia were the same in neonatally infected mice lacking anti-LDV antibodies as in mice infected at 5 or 15 days of age which developed normal antiviral immune responses. The results support the view that LDV replication in mice is unaffected by antiviral immune responses and instead is primarily dependent on the rate of regeneration of LDV-permissive macrophages. This view is further supported by the following findings. Treatment of mice with cyclophosphamide or dexamethasone, which are known to increase plasma LDV levels, increased the proportion of LDV-permissive macrophages in the peritoneum. Injection of mice with interleukin-3, which is known to stimulate macrophage development, increased plasma LDV levels in persistently infected mice 10- to 100-fold. During the first month of age when mice possess a higher proportion of LDV-permissive macrophages than older mice and peritoneal macrophages exhibit self-sustained growth, the persistent plasma LDV titres were also 10- to 100-fold higher than in older mice. The polyclonal activation of B cells induced by LDV that results in a permanent elevation of IgG2a or IgG2b in the circulation, and the formation of 180K to 300K immune complexes containing IgG2a or IgG2b were also the same in neonatally infected mice and mice infected 5 or 15 days after birth. Thus, the polyclonal activation of B cells occurs in the absence of an antiviral humoral immune response and the immune complexes do not contain anti-LDV antibodies. The immune complexes probably consist of autoantibodies formed in the course of the polyclonal activation of B cells and their cellular antigens.

Competitive inhibition of organic anion transporting polypeptide 1c1-mediated thyroxine transport by the fenamate class of nonsteroidal antiinflammatory drugs.

Organic anion transporting polypeptide (Oatp) 1c1 is a high-affinity T(4) transporter with narrow substrate specificity expressed at the blood-brain barrier. A transport model using cells overexpressing Oatp1c1 was created to identify novel Oatp1c1 substrates and inhibitors. Rat Oatp1c1 was cloned and stably expressed in human embryonic kidney 293 cells. Oatp1c1-transfected human embryonic kidney 293 cells transported (125)I-labeled T(4) in a time-dependent manner that was completely abolished in the presence of excess unlabeled T(4). Next, various compounds, including inhibitors of thyroid hormone uptake, were screened for inhibitory effects on Oatp1c1-mediated T(4) uptake. Phenytoin (64%), indocyanine green (17%), fenamic acid (68%), diclofenac (51%), and meclofenamic acid (33%) all reduced T(4) uptake by Oatp1c1 when assayed at concentrations of 10 microM. Dose-response assays for the fenamic acids, iopanoic acid, indocyanine green, and phenytoin revealed IC(50) values for Oatp1c1 T(4) uptake below or near the blood plasma levels after therapeutic doses. Further kinetic assays and reciprocal plot analyses demonstrated that the fenamic acid diclofenac inhibited in a competitive manner. Finally, microvessels were isolated from adult rat brain and assessed for T(4) uptake. Ten micromolar of fenamate concentrations inhibited T(4) microvessel uptake with a similar hierarchical inhibition profile [fenamic acid (43%), diclofenac (78%), and meclofenamic acid (85%)], as observed for Oatp1c1 transfected cells. Oatp1c1 is expressed luminally and abluminally in the blood-brain barrier endothelial cell, and exhibits bidirectional transport capabilities. Together, these data suggest that Oatp1c1 transports fenamates into, and perhaps across, brain barrier cells.

Organic Anion-Transporting Polypeptides at the Blood-Brain and Blood-Cerebrospinal Fluid Barriers

Organic anion-transporting polypeptides (Oatps) are solute carrier family members that exhibit marked evolutionary conservation. Mammalian Oatps exhibit wide tissue expression with an emphasis on expression in barrier cells. In the brain, Oatps are expressed in the blood-brain barrier endothelial cells and blood-cerebrospinal fluid barrier epithelial cells. This expression profile serves to illustrate a central role for Oatps in transporting endo- and xenobiotics across brain barrier cells. This chapter will detail the expression patterns and substrate specificities of Oatps expressed in the brain, and will place special emphases on the role of Oatps in prostaglandin synthesis and in the transport of conjugated endobiotics.