Thomas W. Bastian

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.

Fetal and Neonatal Iron Deficiency Reduces Thyroid Hormone-Responsive Gene mRNA Levels in the Neonatal Rat Hippocampus and Cerebral Cortex

Copper (Cu), iron (Fe), and thyroid hormone (TH) deficiencies produce similar defects in late brain development including hypomyelination of axons and impaired synapse formation and function, suggesting that these micronutrient deficiencies share a common mechanism contributing to these derangements. We previously demonstrated that fetal/neonatal Cu and Fe deficiencies lower circulating TH concentrations in neonatal rats. Fe deficiency also reduces whole-brain T(3) content, suggesting impaired TH action in the developing Fe-deficient brain. We hypothesized that fetal/neonatal Cu and Fe deficiencies will produce mild or moderate TH deficiencies and will impair TH-responsive gene expression in the neonatal cerebral cortex and hippocampus. To test this hypothesis, we rendered pregnant Sprague Dawley rats Cu-, Fe-, or TH-deficient from early gestation through postnatal d 10 (P10). Mild and moderate TH deficiencies were induced by 1 and 3 ppm propylthiouracil treatment, respectively. Cu deficiency did not significantly alter serum or tissue TH concentrations or TH-responsive brain mRNA expression. Fe deficiency significantly lowered P10 serum total T(3) (45%), serum total T(4) (52%), whole brain T(3) (14%), and hippocampal T(3) (18%) concentrations, producing a mild TH deficiency similar to 1 ppm propylthiouracil treatment. Fe deficiency lowered Pvalb, Enpp6, and Mbp mRNA levels in the P10 hippocampus. Fe deficiency also altered Hairless, Dbm, and Dio2 mRNA levels in the P10 cerebral cortex. These results suggest that some of the brain defects associated with Fe deficiency may be mediated through altered thyroidal status and the concomitant alterations in TH-responsive gene transcription.

Fetal and Neonatal Iron Deficiency Exacerbates Mild Thyroid Hormone Insufficiency Effects on Male Thyroid Hormone Levels and Brain Thyroid Hormone-Responsive Gene Expression

Fetal/neonatal iron (Fe) and iodine/TH deficiencies lead to similar brain developmental abnormalities and often coexist in developing countries. We recently demonstrated that fetal/neonatal Fe deficiency results in a mild neonatal thyroidal impairment, suggesting that TH insufficiency contributes to the neurodevelopmental abnormalities associated with Fe deficiency. We hypothesized that combining Fe deficiency with an additional mild thyroidal perturbation (6-propyl-2-thiouracil [PTU]) during development would more severely impair neonatal thyroidal status and brain TH-responsive gene expression than either deficiency alone. Early gestation pregnant rats were assigned to 7 different treatment groups: control, Fe deficient (FeD), mild TH deficient (1 ppm PTU), moderate TH deficient (3 ppm PTU), severe TH deficient (10 ppm PTU), FeD/1 ppm PTU, or FeD/3 ppm PTU. FeD or 1 ppm PTU treatment alone reduced postnatal day 15 serum total T4 concentrations by 64% and 74%, respectively, without significantly altering serum total T3 concentrations. Neither treatment alone significantly altered postnatal day 16 cortical or hippocampal T3 concentrations. FeD combined with 1 ppm PTU treatment produced a more severe effect, reducing serum total T4 by 95%, and lowering hippocampal and cortical T3 concentrations by 24% and 31%, respectively. Combined FeD/PTU had a more severe effect on brain TH-responsive gene expression than either treatment alone, significantly altering Pvalb, Dio2, Mbp, and Hairless hippocampal and/or cortical mRNA levels. FeD/PTU treatment more severely impacted cortical and hippocampal parvalbumin protein expression compared with either individual treatment. These data suggest that combining 2 mild thyroidal insults during development significantly disrupts thyroid function and impairs TH-regulated brain gene expression.

Iron Deficiency Impairs Developing Hippocampal Neuron Gene Expression, Energy Metabolism, and Dendrite Complexity.

Iron deficiency (ID), with and without anemia, affects an estimated 2 billion people worldwide. ID is particularly deleterious during early-life brain development, leading to long-term neurological impairments including deficits in hippocampus-mediated learning and memory. Neonatal rats with fetal/neonatal ID anemia (IDA) have shorter hippocampal CA1 apical dendrites with disorganized branching. ID-induced dendritic structural abnormalities persist into adulthood despite normalization of the iron status. However, the specific developmental effects of neuronal iron loss on hippocampal neuron dendrite growth and branching are unknown. Embryonic hippocampal neuron cultures were chronically treated with deferoxamine (DFO, an iron chelator) beginning at 3 days in vitro (DIV). Levels of mRNA for Tfr1 and Slc11a2, iron-responsive genes involved in iron uptake, were significantly elevated in DFO-treated cultures at 11DIV and 18DIV, indicating a degree of neuronal ID similar to that seen in rodent ID models. DFO treatment decreased mRNA levels for genes indexing dendritic and synaptic development (i.e. BdnfVI,Camk2a,Vamp1,Psd95,Cfl1, Pfn1,Pfn2, and Gda) and mitochondrial function (i.e. Ucp2,Pink1, and Cox6a1). At 18DIV, DFO reduced key aspects of energy metabolism including basal respiration, maximal respiration, spare respiratory capacity, ATP production, and glycolytic rate, capacity, and reserve. Sholl analysis revealed a significant decrease in distal dendritic complexity in DFO-treated neurons at both 11DIV and 18DIV. At 11DIV, the length of primary dendrites and the number and length of branches in DFO-treated neurons were reduced. By 18DIV, partial recovery of the dendritic branch number in DFO-treated neurons was counteracted by a significant reduction in the number and length of primary dendrites and the length of branches. Our findings suggest that early neuronal iron loss, at least partially driven through altered mitochondrial function and neuronal energy metabolism, is responsible for the effects of fetal/neonatal ID and IDA on hippocampal neuron dendritic and synaptic maturation. Impairments in these neurodevelopmental processes likely underlie the negative impact of early life ID and IDA on hippocampus-mediated learning and memory.

Maternal iron supplementation attenuates the impact of perinatal copper deficiency but does not eliminate hypotriiodothyroninemia nor impaired sensorimotor development

Copper, iron and iodine/thyroid hormone (TH) deficiencies disrupt brain development. Neonatal Cu deficiency causes Fe deficiency and may impact thyroidal status. One purpose of these studies was to determine the impact of improved iron status following Cu deficiency by supplementing the diet with iron. Cu deficiency was produced in pregnant Holtzman [Experiment 1 (Exp. 1)] or Sprague-Dawley [Experiment 2 (Exp. 2)] rats using two different diets. In Exp. 2, dietary Fe content was increased from 35 to 75 mg/kg according to NRC guidelines for reproduction. Cu-deficient (CuD) Postnatal Day 24 (P24) rats from both experiments demonstrated lower hemoglobin, serum Fe and serum triiodothyronine (T3) concentrations. However, brain Fe was lower only in CuD P24 rats in Exp. 1. Hemoglobin and serum Fe were higher in Cu adequate (CuA) P24 rats from Exp. 2 compared to Exp. 1. Cu- and TH-deficient rats from Exp. 2 exhibited a similar sensorimotor functional deficit following 3 months of repletion. Results suggest that Cu deficiency may impact TH status independent of its impact on iron biology. Further research is needed to clarify the individual roles for Cu, Fe and TH in brain development.

Fetal and neonatal iron deficiency but not copper deficiency increases vascular complexity in the developing rat brain.

Objectives: Anemia caused by nutritional deficiencies, such as iron and copper deficiencies, is a global health problem. Iron and copper deficiencies have their most profound effect on the developing fetus/infant, leading to brain development deficits and poor cognitive outcomes. Tissue iron depletion or chronic anemia can induce cellular hypoxic signaling. In mice, chronic hypoxia induces a compensatory increase in brain blood vessel outgrowth. We hypothesized that developmental anemia, due to iron or copper deficiencies, induces angiogenesis/vasculogenesis in the neonatal brain. Methods: To test our hypothesis, three independent experiments were performed where pregnant rats were fed iron- or copper-deficient diets from gestational day 2 through mid-lactation. Effects on the neonatal brain vasculature were determined using quantitative real-time polymerase chain reaction to assess mRNA levels of angiogenesis/vasculogenesis-associated genes and GLUT1 immunohistochemistry to assess brain blood vessel density and complexity. Results: Iron deficiency, but not copper deficiency, increased mRNA expression of brain endothelial cell- and angiogenesis/vasculogenesis-associated genes (i.e. Glut1, Vwf, Vegfa, Ang2, Cxcl12, and Flk1) in the neonatal brain, suggesting increased cerebrovascular density. Iron deficiency also increased hippocampal and cerebral cortical blood vessel branching by 62 and 78%, respectively. Discussion: This study demonstrates increased blood vessel complexity in the neonatal iron-deficient brain, which is likely due to elevated angiogenic/vasculogenic signaling. At least initially, this is probably an adaptive response to maintain metabolic substrate homeostasis in the developing iron-deficient brain. However, this may also contribute to long-term neurodevelopmental deficits.

Eltrombopag, a thrombopoietin mimetic, crosses the blood-brain-barrier and impairs iron-dependent hippocampal neuron dendrite development.

Essentials Potential neurodevelopmental side effects of thrombopoietin mimetics need to be considered. The effects of eltrombopag (ELT) on neuronal iron status and dendrite development were assessed. ELT crosses the blood–brain barrier and causes iron deficiency in developing neurons. ELT blunts dendrite maturation, indicating a need for more safety studies before neonatal use.

Thyroid hormone-dependent formation of a subcortical band heterotopia (SBH) in the neonatal brain is not exacerbated under conditions of low dietary iron (FeD)

Thyroid hormones (TH) are critical for brain development and insufficiencies can lead to structural abnormalities in specific brain regions. Administration of the goitrogen propylthiouracil (PTU) reduces TH production by inhibiting thyroperoxidase (TPO), an enzyme that oxidizes iodide for the synthesis of TH. TPO activity is iron (Fe)-dependent and dietary iron deficiency (FeD) also reduces circulating levels of TH. We have previously shown that modest degrees of TH insufficiency induced in pregnant rat dams alters the expression of TH-responsive genes in the cortex and hippocampus of the neonate, and results in the formation of a subcortical band heterotopia (SBH) in the corpus callosum (Royland et al., 2008, Bastian et al., 2014, Gilbert et al., 2014). The present experiment investigated if FeD alone was sufficient to induce a SBH or if FeD would augment SBH formation at lower doses of PTU. One set of pregnant rats was administered 0, 1, 3, or 10ppm of PTU via drinking water starting on gestational day (GD) 6. FeD was induced in a 2nd set of dams beginning on GD2. A third set of dams received the FeD diet from GD2 paired with either 1ppm or 3ppm PTU beginning on GD6. All treatments continued until the time of sacrifice. On PN18, one female pup from each litter was sacrificed and the brain examined for SBH. We observed lower maternal, PN2 and PN18 pup serum T4 in response to PTU. FeD reduced serum T4 in pups on PN16, but did not affect serum T4 in dams or PN2 pups. Neither did FeD in combination with PTU alter T4 levels in dams on PN18 or pups on PN2 compared to PTU treatment alone. By PN16, however more severe T4 reductions were observed in pups when FeD was combined with PTU. SBH increased with increasing dosage of PTU, but counter to our hypothesis, no SBH was detected in the offspring of FeD dams. As such, T4 levels in dams and newborn pups rather than older neonates appear to be a better predictor SBH associated with TH insufficiency. These data indirectly support previous work indicating prenatal TH insufficiency but not postnatal TH insufficiency in offspring is required for SBH formation.

Thyroid Hormone Regulation of Mammalian Reproductive Development and the Potential Impact of Endocrine-Disrupting Chemicals

Thyroid hormones are essential for growth and development of all vertebrate organisms. A growing literature reveals that thyroid hormones are centrally important in the development of reproductive tissues. In males, the predominant target is the developing testis with aberrant Sertoli and Leydig cell development and accompanying testicular dysfunction. Hypothyroidism has also been associated with precocious puberty in a distinct subpopulation of adolescent girls. The hypothalamic–pituitary–thyroid axis is the target of numerous endocrine-disrupting chemicals. Intriguingly, many of these chemicals also perturb the development and function of mammalian reproductive tissues. We now review the key literature detailing the role of thyroid hormones in mammalian reproductive development and provide an analysis of the evidence linking thyroidal disruption by polychlorinated biphenyl, polybrominated diphenyl ether, phthalate, triclosan, and bisphenol A exposure, with maldevelopment of mammalian reproductive systems.