Developmental Thyroid Hormone Insufficiency Research Articles

8490 The mRNA for the Nonsyndromic Deafness Gene Atp2b2 Is Reduced Following Developmental Hypothyroidism

Abstract Disclosure: E.A. Gregersen: None. R.P. Peeters: None. D. Forrest: None. D.S. Sharlin: None. Thyroid hormone (TH) is essential for auditory system development. In rodent models, low levels of TH during the early postnatal period delay cochlear tissue remodeling and alter ion channel function required for normal auditory function. TH receptors are ligand-dependent transcription factors that regulate gene expression. Considering this, auditory deficits observed in hypothyroidism are thought to be largely the result of altered expression of specific TH target genes. However, few TH-regulated genes in the developing cochlea have been reported. TH is reported to impact calcium signaling in several tissues, but whether regulators of calcium signaling are altered following developmental hypothyroidism in the cochlea is largely unexplored. In this report, we investigated the spatial and temporal expression of the ATPase, Ca++ transporting, plasma membrane 2 (Atp2b2) gene in a mouse model of developmental hypothyroidism. Timed-pregnant dams were treated with thyroid gland inhibitors to induce developmental TH insufficiency from embryonic day 12.5 (E12.5) through post-natal day 15 (P15). Untreated timed-pregnant dams served as euthyroid controls. Control and experimental cochleae were collected over a developmental period ranging from embryonic day 18.5 (E18.5) through P15. Cochleae were then processed to detect Atp2b2 mRNA by in situ hybridization or quantitative real-time PCR. In situ hybridization showed that Atp2b2 localizes to inner and outer hair cells as expected, but Atp2b2 was also observed at lower levels in the greater epithelial ridge and spiral ganglion. Quantification of Atp2b2 mRNA revealed an effect of postnatal day and treatment on Atp2b2. In euthyroid animals, Atp2b2 expression gradually increased until P10, then decreased slightly by P15. In hypothyroid animals, Atp2b2 mRNA was consistently decreased (2-2.5 fold). Hypothyroidism similarly impacted expression regardless of sex. The reduction in Atp2b2 mRNA expression in the hypothyroid animals suggests that Atp2b2 is developmentally regulated in part by thyroid hormone signaling. This study characterizes the relationship between TH and Atp2b2 expression during early cochlear development while providing insight into abnormal cochlear development due to hypothyroidism. Presentation: 6/3/2024

Effects of treadmill exercise and sex hormones on learning, memory and hippocampal brain-derived neurotrophic factor levels in transient congenital hypothyroid rats

Transient thyroid function abnormalities at birth exhibit intellectual developmental and cognitive disorders in adulthood. Given the well-known effects of physical activity and sex hormones on cognitive functions and brain-derived neurotrophic factor (BDNF), the present study examined the effects of treadmill exercise, sex hormones, and the combined treatment on learning and memory and hippocampal BDNF levels in transient congenital hypothyroid rats. To induce hypothyroidism, 6-propyl-2-thiouracil was added to the drinking water from the 6th day of gestation to the 21st postnatal day (PND). From PNDs 28 to 47, female and male pup rats received 17β-estradiol and testosterone, respectively, and about 30 min later, they were forced to run on the treadmill for 30 min once a day. On PNDs 48-55, spatial learning and memory of all rats tested in the water maze, which followed by measurement of BDNF in the hippocampus. Results showed that developmental hypothyroidism induced significant deficits in spatial learning and memory and hippocampal BDNF in both male and female rats. In both male and female hypothyroid rats, exercise and exercise plus sex hormones, but not sex hormones alone alleviated learning and memory deficits and all treatments (exercise, sex hormones, and the combined treatment) increased hippocampal BDNF. These disconnects in the effects of exercise, sex hormones and the combined treatment on behavioral and neurochemical outcomes suggest that a neurochemical mechanism other than hippocampal BDNF might contribute in the ameliorating effects of exercise on learning and memory deficits induced by developmental thyroid hormone insufficiency.

SAT-448 Limited Adaptive Response by Deiodinase Enzymes in the Perinatal Hypothyroid Cochlea

Thyroid hormone (TH) is essential for cochlear development and normal auditory function. Considering the importance of TH in mediating cochlear development, understanding the degree to which developing tissues can adapt to perturbations in thyroid hormone signaling is extremely important. The deiodinases (Dio2 and Dio3) are enzymes that tightly control TH availability at the tissue level and have been proposed to function as adaptive mechanisms that maintain tissue TH homeostasis. Dio2 converts thyroxine (T4) to a biologically active ligand triiodothyronine (T3); locally amplifying a T3 signal. Conversely, Dio3 inactivates T3 and T4 by converting these iodothyronines to the inactive metabolites diiodothyronine (T2) and reverse T3 (rT3), respectively. During cochlear development, Dio3 expression is high prenatally while Dio2 expression is low. During the first postnatal week, the expression levels of these enzymes invert, resulting in high expression of Dio2 and low expression of Dio3. Together, the deiodinases control the timing of postnatal cochlear remodeling; suggesting a genetic developmental clock controls TH-mediated cochlear development. Considering the role deiodinases as adaptive mechanisms, it is important to understand whether such a developmental clock can be negated during times of developmental thyroid hormone insufficiency. We hypothesize that the perinatal change in deiodinase expression is controlled by a developmental clock rather than environmental clues and therefore have limited capacity function as compensatory mechanisms in response to low TH during development. To test this, timed-pregnant mice were treated with thyroid gland inhibitors to induced hypothyroidism from gestational day 12.5 until pup sacrifice. A parallel set of untreated timed-pregnant mice served as controls. Cochlea were harvest from control and hypothyroid mice at postnatal (P) ages P1, P5, P10 and P15 for qRT-PCR, and P7 for in situ hybridization. In situ hybridization for Dio2 mRNA demonstrated a striking reduction in Dio2 mRNA in hypothyroid animals compared to euthyroid animals. Additionally, we observed a significant effect of age (p<0.001) and an interaction between age and treatment (p=0.0035) on Dio2 mRNA levels analyzed by qRT-PCR. Specifically, Dio2 mRNA levels were similar or reduced in hypothyroid animals compared to controls at P1, P5, and P10. This finding indicates that Dio2 mRNA at these ages did not respond in manner consistent with compensation. However, at P15, Dio2 mRNA levels were increased in hypothyroid animals compared to controls; a finding that is consistent with a compensatory mechanism. Taken together, our results suggest that developmental programs in tissues may be dominant over potential compensatory mechanisms and that developing tissues may be more susceptible to perturbations in tissue TH levels due to a reduced capacity to compensate.

Effect of Maternal Hypothyroidism on BDNF Expression in Developing Rat Brain

Thyroid hormone is essential for proper development of brain and modest degrees of developmental thyroid hormone insufficiency can result severe neurological deficits. We used an animal model to study the role of thyroid hormones (THs) during the gestation days and early postnatal days on developing rat brain. Dose-dependent thyroid hormone insufficiency in dams was induced by methimazole (MMI). In the first study-group thyroid hormone deficiency was induced by delivery of methimazole (MMI) to dams via drinking water at the doses of 50, 75 and 100 ppm from early gestation (GD 3) until weaning of the pups (PN20). In the second study-group hypothyroid rats were treated with 200 μg of thyroxine hormone. Pups were sacrificed on postnatal days (PN) 20. Maternal blood collected for thyroid hormone analysis. Cerebellum, medulla, hippocampus, cortex, olfactory bulbs were separated from dissected brain and relative expression of BDNF gene transcripts known to play critical roles in developing rat brain, were determined by RT-PCR. Daily body weight in dams and pups, the number of pups at birth, the eye –closure opening day and BDNF expression in brain extract was determined in the preweaning rats as a function of MMI exposure. The results indicate that genes driving important developmental processes during early brain development are sensitive to perturbations of the thyroid axis function.

Developmental Thyroid Hormone Insufficiency Induces a Cortical Brain Malformation and Learning Impairments: A Cross-Fostering Study.

Thyroid hormones (THs) are essential for brain development, but few rodent models exist that link TH inefficiency to apical neurodevelopmental endpoints. We have previously described a structural anomaly, a heterotopia, in the brains of rats treated in utero with propylthiouracil (PTU). However, how the timing of an exposure relates to this birth defect is unknown. This study seeks to understand how various temporal treatments of the mother relates to TH insufficiency and adverse neurodevelopment of the offspring. Pregnant rats were exposed to PTU (0 or 3 ppm) through the drinking water from gestational day 6 until postnatal day (PN) 14. On PN2 a subset of pups was cross-fostered to a dam of the opposite treatment, to create 4 conditions: pups exposed to PTU prenatally, postnatally, during both periods, or not at all (control). Both PTU and TH concentrations were characterized in the mother and offspring over time, to capture the dynamics of a developmental xenobiotic exposure. Brains of offspring were examined for heterotopia presence and severity, and adult littermates were assessed for memory impairments. Heterotopia were observed under conditions of prenatal exposure, and its severity increased in animals in the most prolonged exposure group. This malformation was also permanent, but not sex biased. In contrast, behavioral impairments were limited to males, and only in animals exposed to PTU during both the gestational and postnatal periods. This suggests a distinct TH-dependent etiology for both phenotypes, and illustrates how timing of hypothyroxinemia can induce abnormal brain structure and function.

Adult hippocampal neurogenesis is impaired by transient and moderate developmental thyroid hormone disruption

The hippocampus maintains a capacity for neurogenesis throughout life, a capacity that is reduced in models of adult onset hypothyroidism. The effects of developmental thyroid hormone (TH) insufficiency on neurogenesis in the adult hippocampus, however, has not been examined. Graded degrees of TH insufficiency were induced in pregnant rat dams by administration of 0, 3 or 10ppm of 6-propylthiouracil (PTU) in drinking water from gestational day (GD) 6 until weaning. Body, brain, and hippocampal weight were reduced on postnatal day (PN) 14, 21, 78 and hippocampal volume was smaller at the 10 but not 3ppm dose level. A second experiment examined adult hippocampal neurogenesis following developmental or adult onset hypothyroidism. Two male offspring from 0 and 3ppm exposed dams were either maintained on control water or exposed to 3ppm PTU to create 4 distinct treatment conditions (Control-Control; Control-PTU, PTU-Control, PTU-PTU) based on developmental and adult exposures. Beginning on the 28th day of adult exposure to 0 or 3ppm PTU, bromodeoxyuridine (BrdU, 50mg/kg, ip) was administered twice daily for 5days, and one male from each treatment was sacrificed 24h and 28days after the last BrdU dose and brains processed for immunohistochemistry. Although no volume changes were seen in the hippocampus of the neonate at 3ppm, thinning of the granule cell layer emerged in adulthood. Developmental TH insufficiency produced a reduction in newly born cells, reducing BrdU+ve cells at 1 with no further reduction at 28-days post-BrdU. Similar findings were obtained using the proliferative cell marker Ki67. Neuronal differentiations was also altered with fewer doublecortin (Dcx) expressing cells and a higher proportion of immature Dcx phenotypes seen after developmental but not adult TH insufficiency. An impaired capacity for neurogenesis may contribute to impairments in synaptic plasticity and cognitive deficits previously reported by our laboratory and others following moderate degrees of developmental TH insufficiency induced by this PTU model.

Effects of maternal hypothyroidism during pregnancy on learning, memory and hippocampal BDNF in rat pups: Beneficial effects of exercise

Hypothyroidism during early development leads to numerous morphological, biochemical and functional changes in developing brain. In this study, we investigated the effects of voluntary and treadmill exercise on learning, memory and hippocampal BDNF levels in both hypothyroid male and female rat pups. To induce hypothyroidism in the mothers, 6-propyl-2-thiouracil (PTU) was added to their drinking water (100mg/L) from their embryonic day 6 to their postnatal day (PND) 21. For 14days, from PNDs 31 to 44, the rat pups were trained with one of the two different exercise protocols, namely the mild treadmill exercise and the voluntary wheel exercise. On PNDs 45–52, a water maze was used for testing their learning and memory ability. The rats were sacrificed one day later and their BDNF levels were then measured in the hippocampus. The findings of the present study indicate that hypothyroidism during the fetal period and the early postnatal period is associated with the impairment of spatial learning and memory and reduced hippocampal BDNF levels in both male and female rat offspring. Both the short-term treadmill exercise and the voluntary wheel exercise performed during the postnatal period reverse the behavioral and neurochemical deficits induced by developmental thyroid hormone insufficiency in both male and female rat offspring. The findings of this study thus demonstrate a marked reversibility of both behavioral and neurochemical disorders induced by developmental thyroid hormone insufficiency through the performance of exercise.

Adult onset-hypothyroidism: alterations in hippocampal field potentials in the dentate gyrus are largely associated with anaesthesia-induced hypothermia.

Thyroid hormone (TH) is essential for a number of physiological processes and is particularly critical during nervous system development. The hippocampus is strongly implicated in cognition and is sensitive to developmental hypothyroidism. The impact of TH insufficiency in the foetus and neonate on hippocampal synaptic function has been fairly well characterised. Although adult onset hypothyroidism has also been associated with impairments in cognitive function, studies of hippocampal synaptic function with late onset hypothyroidism have yielded inconsistent results. In the present study, we report hypothyroidism induced by the synthesis inhibitor propylthiouracil (10 p.p.m., 0.001%, minimum of 4 weeks), resulted in marginal alterations in excitatory postsynaptic potential (EPSP) and population spike (PS) amplitude in the dentate gyrus measured in vivo. No effects were seen in tests of short-term plasticity, and a minor enhancement of long-term potentiation of the EPSP slope was observed. The most robust synaptic alteration evident in hypothyroid animals was an increase in synaptic response latency, which was paralleled by a failure to maintain normal body temperature under anaesthesia, despite warming on a heating pad. Latency shifts could be reversed in hypothyroid animals by increasing the external heat source and, conversely, synaptic delays could be induced in control animals by removing the heat source, with a consequent drop in body and brain temperature. Thermoregulation is TH- dependent, and anaesthesia necessary for surgical procedures posed a thermoregulatory challenge that was differentially met in control and hypothyroid animals. Minor increases in field potential EPSP slope, decreases in PS amplitudes and increased latencies are consistent with previous reports of hypothermia in naive control rats. We conclude that failures in thyroid-dependent temperature regulation rather than direct action of TH in synaptic physiology are responsible for the observed effects. These findings stand in contrast to the synaptic impairments observed in adult offspring following developmental TH insufficiency, and emphasise the need to control for the potential unintended consequences of hypothermia in the interpretation of hypothyroid-induced changes in physiological systems, most notably synaptic transmission.

Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates

Brain-derived neurotrophic factor (BDNF) is a neurotrophin critical for many developmental and physiological aspects of CNS function. Severe hypothyroidism in the early neonatal period results in developmental and cognitive impairments and reductions in mRNA and protein expression of BDNF in a number of brain regions. The present study examined the impact of modest levels of developmental thyroid hormone insufficiency on BDNF protein expression in the hippocampus, cortex and cerebellum in the neonatal and adult offspring of rat dams treated throughout pregnancy and lactation. Graded levels of hormone insufficiency were induced by adding propylthiouracil (PTU, 0, 1, 2, 3 and 10ppm) to the drinking water of pregnant dams from early gestation (gestational day 6) until weaning of the pups. Pups were sacrificed on postnatal days (PN) 14 and 21, and ~PN100, and trunk blood collected for thyroid hormone analysis. Hippocampus, cortex, and cerebellum were separated from dissected brains and assessed for BDNF protein. Dose-dependent reductions in serum hormones in dams and pups were produced by PTU. Consistent with previous findings, age and regional differences in BDNF concentrations were observed. However, no differences in BDNF expression were detected in the preweanling animals as a function of PTU exposure; yet dose-dependent alterations emerged in adulthood despite the return of thyroid hormone levels to control values. Males were more affected by PTU than females, BDNF levels in hippocampus and cortex were altered but not those in cerebellum, and biphasic dose–response functions were detected in both sexes. These findings indicate that BDNF may mediate some of the adverse effects accompanying developmental thyroid hormone insufficiency, and reflect the potential for delayed impact of modest reductions in thyroid hormones during critical periods of brain development on a protein important for normal synaptic function.

Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency

Thyroid hormones are critical for the development and maturation of the central nervous system. Although somatic and neurological effects are well documented following severe thyroid hormone deprivation, much less is known of the functional consequences of moderate levels of hormone insufficiency. We have previously demonstrated that severe thyroid hormone reductions in the postnatal period are associated with impairments in synaptic transmission in the dentate gyrus. The present study was performed to examine the dose–response relationships of moderate levels of hormone disruption on synaptic function in the dentate gyrus in an in vivo preparation and to determine the effects on spatial learning. Pre- and postnatal thyroid hormone insufficiency was induced by administration of 3 or 10 ppm propylthiouracil (PTU) to pregnant and lactating dams via the drinking water from gestation day (GD) 6 until postnatal day (PN) 30. This regimen produced a 47% and 65% reduction in serum T4, in the dams of the low and high-dose groups, respectively. At the time of testing of adult offspring, hormone status had returned to control levels. In littermates, field potentials evoked in the dentate gyrus in response to stimulation of the perforant path were assessed under urethane anesthesia. The data reveal dose-dependent reductions in synaptic transmission and impairments in long-term potentiation (LTP) of the EPSP component of the compound field potential. In contrast, LTP of the population spike measure was paradoxically enhanced. Spatial learning in the Morris water maze was profoundly impaired in high-dose animals. Although the majority of subjects in the low-dose group eventually acquired the task, their acquisition rate lagged behind control values. Reversal learning was assessed in all animals reaching criterion performance and found to be impaired in PTU-exposed animals relative to controls. These data support previous findings in area CA1 in vitro, extend observations associated with dentate gyrus synaptic function to a lower dose range, and provide correlative evidence of behavioral disruption in a hippocampal-dependent learning task following developmental thyroid hormone insufficiency.

Impairment in Short-Term but Enhanced Long-Term Synaptic Potentiation and ERK Activation in Adult Hippocampal Area CA1 Following Developmental Thyroid Hormone Insufficiency*

Thyroid hormones are critical for the development and maturation of the central nervous system. Insufficiency of thyroid hormones during development impairs performance on tasks of learning and memory that rely upon the hippocampus and impairs synaptic function in young hypothyroid animals. The present study was designed to determine if perturbations in synaptic function persist in adult euthyroid animals exposed developmentally to insufficient levels of hormone. Pre- and postnatal thyroid hormone insufficiency was induced by administration of 3 or 10 ppm propylthiouracil (PTU) to pregnant and lactating dams via the drinking water from gestation day (GD) 6 until postnatal day (PN) 30. This regimen produced a graded level of hormonal insufficiency in the dam and the offspring. Population spike and population excitatory postsynaptic potentials (EPSP) were recorded at the pyramidal cell layer and the stratum radiatum, respectively, in area CA1 of hippocampal slices from adult male offspring. PTU exposure increased baseline synaptic transmission, reduced paired-pulse facilitation, and increased the magnitude of the population spike long-term potentiation (LTP). Phosphorylation of the extracellular signal-regulated kinases (ERK1 and ERK2) was increased as a function of LTP stimulation in slices from PTU-exposed adult animals. On the other hand, no differences in the basal levels of synaptic proteins implicated in synaptic plasticity (total ERK, synapsin, growth-associated protein-43, and neurogranin) were detected. These results reinforce previous findings of persistent changes in synaptic function and, importantly extend these observations to moderate levels of thyroid hormone insufficiency that do not induce significant toxicity to the dams or the offspring. Such alterations in hippocampal synaptic function may contribute to persistent behavioral deficits associated with developmental hypothyroidism.

Mode of Action: Developmental Thyroid Hormone Insufficiency—Neurological Abnormalities Resulting From Exposure to Propylthiouracil

Because thyroid hormone is essential for normal brain development before and after birth, environmental chemicals that interfere with thyroid hormone signaling can adversely affect brain development. Adverse consequences of thyroid hormone insufficiency depend both on severity and developmental timing, indicating that environmental antithyroid factors may produce different effects at different developmental windows of exposure. Mechanistic studies can provide important insight into the potential impact of chemicals on human thyroid function, but relevance to humans must be systematically evaluated. This kind of analysis depends on data sets that include information about animals and humans. The drug 6-n-propyl-2-thiouracil (PTU) is used in animals to experimentally manipulate serum thyroid hormone levels, and in humans to treat patients, including pregnant women, with Graves' disease. A systematic analysis of the mode of action (MOA) of PTU in rats and in humans discloses similar modes of action. While the analysis predicts that PTU doses that produce thyroid hormone insufficiency in humans would adversely affect the developing brain, careful monitoring of PTU administration in pregnant and lactating humans keeps infant serum thyroid hormone levels within the normal range.