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Endocrinology Vol. 147, No. 5 2567-2574
Copyright © 2006 by The Endocrine Society

Dynamic Nongenomic Actions of Thyroid Hormone in the Developing Rat Brain

Alan P. Farwell, Susan A. Dubord-Tomasetti, Andrzej Z. Pietrzykowski and Jack L. Leonard

University of Massachusetts Medical School, Worcester, Massachusetts 01655

Address all correspondence and requests for reprints to: Alan P. Farwell, M.D., Division of Endocrinology and Metabolism, Department of Medicine, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655. E-mail alan.farwell{at}umassmed.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two well-characterized nongenomic actions of thyroid hormone in cultured brain tissues are: 1) regulation of type 2 iodothyronine 5'deiodinase (D2) activity and 2) regulation of actin polymerization. In particular, the latter is likely to have profound effects on neuronal migration in the developing brain. In this study, we determined whether these nongenomic actions also occurred in vivo during brain development. Neonatal hypothyroidism was induced by propylthiouracil given to pregnant dams beginning on d17 of gestation and continued throughout the neonatal period. On postnatal d 14, rats were injected with either cold or [125I]-labeled iodothyronines and killed sequentially after injection. In contrast to reports in the adult rat, all three iodothyronines readily and equally entered developing brain tissues. As expected, cerebrocortical D2 activity was markedly elevated in the hypothyroid brain and both reverse T3 (rT3) and T4 rapidly decreased D2 to euthyroid levels within 3 h. Furthermore, cerebellar G-actin content in the hypothyroid rat was approximately 5-fold higher than in the euthyroid rat. Again, both rT3 and T4 rapidly decreased the G-actin content by approximately 50%, with a reciprocal increase in F-actin content to euthyroid levels without altering total actin. Neither T3 nor vehicle had any effect on D2 activity in the cortex or G- or F-actin content in the cerebellum. The thyroid hormone-dependent regulation of actin polymerization in the rat brain provides a mechanism by which this morphogenic hormone can influence neuronal migration independent of the need for altered gene transcription. Furthermore, these data suggest a prominent role for rT3 during brain development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE has long been recognized as essential for regulation of the brain developmental program (1, 2, 3, 4, 5, 6, 7). The absence of thyroid hormone during the first 3 months of life in humans results in severe mental retardation and, in a rat model, irreversible morphological abnormalities in the brain (1, 2, 3, 4, 5, 6, 7). Adequate thyroid hormone replacement in the infant with congenital hypothyroidism instituted soon after birth before these abnormalities develop successfully prevents these morphological abnormalities and allows brain development program to progress normally (8, 9). Although the consequences of hypothyroidism on brain development are well recognized, the biochemical and molecular basis of the effects of this morphogenic hormone on neuronal integration remain elusive.

The major thyroid hormone produced by the thyroid gland is T4, which is deiodinated in peripheral tissues to the transcriptionally active iodothyronine, T3 (10, 11). T4 is also deiodinated in peripheral tissues to the transcriptionally inert 3,3',5'-triiodothyronine [reverse T3 (rT3)], which is the predominant iodothyronine produced during fetal life (12). The traditional model for thyroid hormone action has T3 interacting with specific chromatin-bound ligand-activated transcription factors [thyroid hormone receptors (TRs)] (13, 14). However, the identification of developmentally important, T3-responsive genes in the brain has been difficult at best and few, if any, obvious abnormalities in brain development have been observed in mutant mice lacking all known TRs (15, 16), suggesting that T3-induced transcriptional regulation is not the sole contributor to the brain developmental program.

Nongenomic actions of the members of the steroid hormone super family are becoming increasingly important as mechanisms of hormone action (7, 17, 18). In contrast to T3-dependent transcriptional actions that occur over hours to days, nongenomic actions of thyroid hormone typically occur in seconds to minutes and often involve T4 as the ligand. Thus, for a nongenomic action to be relevant, the hormones must have rapid access to the tissue target of action. Nongenomic actions of thyroid hormone that have been described in brain cells include regulation of microfilament organization (19, 20) and regulation of type II iodothyronine 5'-deiodinase (D2) activity (21, 22, 23, 24, 25, 26). In particular, thyroid hormone-dependent regulation of microfilament organization would likely have profound effects on the developing brain because the microfilaments are essential for the pathfinding and guidance of the migrating neurite (27, 28, 29, 30, 31, 32).

We have described the nongenomic, thyroid hormone-dependent regulation of the organization of the actin cytoskeleton both in cultured rat astrocytes (19, 33, 34) and in cultured rat granular neurons (20). In astrocytes, approximately 90% of the cellular actin is polymerized and assembled into bundles of F-actin under euthyroid conditions. In the absence of thyroid hormone, the F-actin content falls by approximately 50% and the bundled microfilaments disappear; total cellular actin remains unchanged. T4 and its metabolite, rT3, are equipotent in rapidly (~10 min) promoting actin polymerization in astrocytes, returning the F-actin content to normal and restoring the stress fibers in the absence of transcription or translation. T3 has no effect on actin polymerization at equimolar concentrations. Similarly, the F-actin content in T3-treated and hormone-free granular neurons is decreased by up to 35% compared with the T4- or rT3-treated cells, with no change in total cellular actin (20). Immunocytochemical analysis suggests that it is the F-actin that is found in the neuronal processes that is regulated by T4 and rT3 (20). These in vitro studies correlate with a report that the F-actin content in the cerebellum of 14-d-old hypothyroid rats is significantly decreased and that T4 replacement for 2 d restores the F-actin content to normal (35).

In this study, we determined the time course of action of thyroid hormone on actin polymerization and D2 activity in vivo and correlated the alterations in actin polymerization and D2 activity with entry of iodothyronines into the developing rat brain. We show that T4, T3, and rT3 all rapidly enter brain tissues after ip injection. Furthermore, we show that T4 and rT3, but not T3, dynamically regulate actin polymerization and D2 activity in the developing brain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents
Pregnant (16–17 d gestation) Sprague Dawley rats were obtained from Charles-River Labs (Kingston, NY). The study was approved by the Animal Research Committee and complies with the institutional assurance certificate of the University of Massachusetts Medical School. Neonatal hypothyroidism was induced by adding propylthiouracil (PTU) (2 mg/liter) to the drinking water of pregnant dams beginning at d 17 of gestation and continuing throughout the neonatal period.

Iodothyronines were obtained from Henning GmBH (Berlin, Germany), iopanoic acid (IOP) was obtained from Sterling Winthrop Research Institute and BSA was purchased from Sigma (St. Louis, MO). Antirabbit IgG-horseradish peroxidase conjugate was purchased from Promega (Madison, WI), rabbit polyclonal anti-actin IgG was purchased from Biomedical Technologies (Stoughton, MA), and Hybond ECL nitrocellulose was obtained from Amersham (Arlington Heights, IL). The Lumiglo chemiluminescent was obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). All other reagents were of the highest grade available.

Tissue analysis of iodothyronines
[125I]-labeled iodothyronines were synthesized by iodination of precursors with [125I]Na obtained from NEN Life Science Products (Boston, MA) following established methods (36). The entry of iodothyronines into the brain was determined in euthyroid rat pups on postnatal d 14 by ip injection of [125I]-labeled iodothyronine (20 x 106 cpm). Where indicated, animals were also injected ip with IOP (50 µg/10 g body weight), a potent inhibitor of 5'-deiodinase activity (11), to block T4 to T3 conversion. Animals were exsanguinated via the abdominal aorta and perfused with 10–20 ml iced 8 mM sodium phosphate (pH 7.4), 2.7 mM KCl, 137 mM NaCl, 1 mM IOP (PBS/IOP) at 0.5, 1, 3, and 6 h after injection. Blood samples obtained when the animals were killed were counted to determine the amount of [125I]-labeled iodothyronine absorbed after injection. Cerebral cortex and cerebellum were harvested, washed in PBS/IOP, and frozen in liquid nitrogen and weighed. Iodothyronines were extracted and analyzed by HPLC according to established procedures (37, 38). In brief, tissues were homogenized in 4 vol methanol/1 mM IOP and then extracted with 8 vol chloroform. The upper phase was collected and the bottom phase was extracted again with chloroform:methanol (2:1). The two upper phases were combined and extracted with 0.05% CaCl2. The aqueous phase was frozen, lyophilized, and resuspended in methanol. Samples were clarified by centrifugation, and the methanol was blown off under a nitrogen stream. Samples were then analyzed by HPLC and counted. The iodothyronine fractions were determined by parallel HPLC analysis of [125I]-labeled iodothyronine standards subjected to the same extraction procedures.

Tissue harvest and hormone assays
On postnatal d 14 (P14), PTU-treated rat pups were injected ip with T4, rT3, T3, or vehicle (1 M NaCl, 0.1% BSA, 100 µl volume) and animals were killed at 1, 3, and 6 h after injection. Where indicated, animals were also injected ip with IOP (50 µg/10 g body weight) to block T4 to T3 conversion. Control animals included postnatal d 14 euthyroid and PTU-treated animals and were killed at 0 and 6 h. In all experiments, animals were weighed, killed by decapitation and their blood collected for hormone assays. The cerebral cortex and cerebellum were rapidly isolated, washed in iced 8 mM sodium phosphate (pH 7.4), 2.7 mM KCl, 137 mM NaCl (PBS), frozen in liquid nitrogen, and kept at –70 C until use.

Serum TSH was measured in duplicate by RIA using materials obtained from the National Pituitary Agency, National Institutes of Health (Bethesda, MD). Serum T4, T3, and rT3 were determined in duplicate by specific RIAs.

Actin analysis
Cerebellar samples were thawed and tissues were homogenized in 5 vol actin buffer [50 mM Tris (pH 6.8), 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride] and diluted to 1 mg protein/ml. Samples were solubilized by the addition of an equal volume of actin buffer containing 1% Triton X-100 (final Triton concentration 0.5%). The insoluble cytoskeletal pellet, containing F-actin, was separated from the soluble fraction, containing G-actin, by centrifugation in a microfuge at room temperature (39, 40, 41). The cytoskeletal pellet was solubilized in depolymerization buffer [1.5 M guanidine HCl, 1 M sodium acetate, 1 mM CaCl2, 1 mM ATP, 20 mM Tris HCl (pH 7.4)]. Both fractions were analyzed by immunoblot using a polyclonal rabbit antiactin IgG followed by an antirabbit IgG-horseradish peroxidase conjugate. Slot blots were developed with the Lumiglo chemiluminescent system and analyzed by scanning densitometry.

D2 analysis
Cerebral cortex samples were thawed and homogenized in 40 vol of 250 mM sucrose, 10 mM dithiothreitol (DTT), 1 mM EDTA, 20 mM HEPES (pH 7.0). D2 activity was determined in homogenates by the iodide release method at 2 nM rT3 and 20 mM DTT in the presence of 1 mM PTU (22). Units are expressed as fmol I– released/h.

Statistical methods
Results were analyzed by single-factor ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of the entry of iodothyronines into brain tissues
Before iodothyronines are able to enter the central nervous system (CNS), they must pass across the blood-brain barrier or through the choroid plexus-cerebrospinal fluid barrier (42, 43, 44). Although many studies have shown that iodothyronines readily enter the brain (24, 38, 45, 46, 47, 48, 49), there is still some debate as to possible differential entry of iodothyronines into the CNS. To study rapid actions of iodothyronines in the brain, we examined the time course of entry of iodothyronines into the cerebral cortex and the cerebellum after ip injection of [125I]-labeled iodothyronines into postnatal d 14 euthyroid rat pups.

All three iodothyronines were readily absorbed in to the bloodstream after ip injection (5–10% injected dose/ml at 1 h) and remained detectable throughout the experimental period (2–5% injected dose/ml at 6 h). In the cerebral cortex (Fig. 1Go, top), total [125I] reached a peak at 1 h after the injection of T4 and rT3, whereas a peak was not observed until 3 h after the injection of T3. At all time points, T3 was 2- to 3-fold more abundant in the cortex than either T4 or rT3 (Fig. 1Go, bottom). The tissue:plasma ratio at 3 h was similar with all three iodothyronines, indicating equal access to the cerebral cortex (Table 1Go). The steady decline of T4 from 1–6 h in the cortex was blocked by the concurrent administration of IOP, a potent inhibitor of both type I and type II 5'-deiodinases (50). Levels of rT3 remained relatively stable over the 6-h period. In the cerebellum, peak tissue levels of total [125I] were achieved within 1 h after injection of rT3 and within 3 h after the injection of T4 and T3 (Fig. 2Go, top). Peak tissue levels of rT3 were achieved in the cerebellum by 1 h and then fell to a plateau level from 3–6 h (Fig. 2Go, bottom). T3 steadily increased to peak tissue levels in the cerebellum by 3 h then plateaued, whereas T4 reached peak tissue levels by 3 h, followed by a slow decrease by 6 h. As observed in the cortex, the concurrent administration of IOP blocked the decrease in T4 tissue levels from 3–6 h; instead T4 levels continued to steadily rise the cerebellum for up to 6 h after IOP administration. Similarly, the tissue:plama ratio at 3 h in the cerebellum was the same for all three iodothyronines and approximately 2-fold higher than in the cortex (Table 1Go). These studies show that all iodothyronines rapidly and equally enter developing brain tissues and remain in the CNS for at least 6 h after a bolus injection.


Figure 1
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  		FIG. 1. Entry of iodothyronines into the developing rat cerebral cortex. Postnatal d 14 euthyroid rat pups were injected ip with [125I]-labeled iodothyronines (20 x 106 cpm) and tissues were harvested at the indicated times. Tissues were extracted with chloroform:MeOH and analyzed by HPLC as discussed in Materials and Methods. Top, Total tissue [125I]. Results are presented as the percent injected dose of each individual iodothyronine normalized to the amount of tissue and represent the mean of results obtained in two separate experiments with six animals/experiment (n = 12 total). Bottom, Tissue iodothyronine content. Results are presented as the ratio of [125I]-labeled iodothyronine to the total tissue [125I] normalized to the amount of tissue and represent the mean of results obtained in two separate experiments with six animals/experiment (n = 12 total). T4/IOP, [125I]-labeled T4 (20 x 106 cpm) and IOP (50 µg/10 g body weight) were injected ip at the same time.

 

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  		TABLE 1. Plasma and tissue concentrations of iodothyronines 3 h after ip injection

 

Figure 2
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  		FIG. 2. Entry of iodothyronines into the developing rat cerebellum. Postnatal d 14 euthyroid rat pups were injected ip with [125I]-labeled iodothyronines (20 x 106 cpm) and tissues were harvested at the indicated times. Tissues were extracted with chloroform:MeOH and analyzed by HPLC as discussed in Materials and Methods. Top, Total tissue [125I]. Results are presented as the percent injected dose of each individual iodothyronine normalized to the amount of tissue and represent the mean of results obtained in two separate experiments with six animals/experiment (n = 12 total). Bottom, Tissue iodothyronine content. Results are presented as the ratio of [125I]-labeled iodothyronine to the total tissue [125I] normalized to the amount of tissue and represent the mean of results obtained in two separate experiments with six animals/experiment (n = 12 total). T4/IOP, [125I]-labeled T4 (20 x 106 cpm) and IOP (50 µg/10 g body weight) were injected ip at the same time.

 
Analysis of the rapid actions of thyroid hormone in the developing cerebral cortex and cerebellum
The above data clearly show that all iodothyronines rapidly enter the developing brain; thus, we were able to proceed and examine and compare the dynamic effects of iodothyronines on brain processes. Neonatal hypothyroidism was induced by adding PTU (2 mg/liter) to the drinking water of pregnant dams beginning at d 17 and continuing throughout the neonatal period. On postnatal d 14, rats were injected ip with T4 (200 ng/10 g body weight), T3 (40 ng/10 g body weight), rT3 (200 ng/10 g body weight). Animals were killed and blood and tissues harvested 1–6 h after injection. Shown in Fig. 3Go are the serum hormone concentrations in the experimental animals. PTU treatment resulted in undetectable T4 and rT3 concentrations and TSH concentrations 5- to 6-fold greater than that observed in the euthyroid animals. As observed previously (24, 51, 52), serum T3 levels remained detectable in the PTU-treated animals at approximately 25% the level seen in the euthyroid animals. After the injection of T4, serum T4 concentrations rose steadily to a maximum level at 3 h that was approximately 2- to 3-fold higher than in the euthyroid animals. Importantly, the only animals with detectable circulating T4 were the euthyroid and T4-treated animals. Also after T4 injection, serum T3 concentrations steadily increased to euthyroid levels and rT3 concentrations increased to approximately 8-fold greater than that observed in euthyroid animals over the 3- to 6-h time period, whereas TSH concentrations steadily fell to euthyroid levels by 6 h. After T3 injection, serum T3 concentrations increased to slightly above euthyroid levels (and higher than observed after T4 injection) by 3 h then declined to euthyroid levels over the following 3 h. TSH concentrations also decreased by approximately 50% by 6 h after injection of T3, with no change in serum T4 or rT3 concentrations in the T3-treated animals. Similarly, rT3 injection altered only serum rT3 concentrations, increasing the levels to approximately 8-fold above that observed in euthyroid animals over the 3- to 6-h time period. As expected, there were no changes in serum hormone concentrations from the PTU-treated baseline in the animals injected with vehicle.


Figure 3
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  		FIG. 3. Serum levels of iodothyronines after ip injection. On postnatal d 14, PTU-treated rat pups were injected ip with T4 [200 ng/10 g body weight (BW)], T3 (40 ng/10 g BW), rT3 (200 ng/10 g BW), or vehicle and animals were killed at 1, 3, and 6 h after injection. Control postnatal d 14 euthyroid and PTU-treated animals were killed at 0 and 6 h. Blood was collected for hormone assays as discussed in Materials and Methods. Results are presented as the mean of values obtained in triplicate animals in three experiments (n = 9).

 
Dynamic regulation of D2 activity in the cerebral cortex
Regulation of D2 activity in the brain is a well-characterized nongenomic action of thyroid hormone and is a sensitive indicator of the cellular levels of thyroid hormone in the cerebral cortex (7, 22, 24, 26, 34, 40, 53). D2 catalyzes T4 to T3 conversion within the brain and enzyme activity is inversely proportional to serum T4 levels. In rat astrocytes, T4 dynamically regulates D2 activity by activating actin-based endocytosis leading to the internalization of the enzyme and directing it to recycling pathways within the cell (26, 34, 40, 54, 55). Using D2 as a marker of nongenomic thyroid hormone action, we examined the effects of individual iodothyronines on D2 activity in the developing rat cerebral cortex.

As shown previously (21, 22, 56), D2 activity in the hypothyroid cerebral cortex is approximately 10-fold greater than that found in the euthyroid rat brain (Fig. 4Go). The injection of vehicle alone or of T3 at sufficient doses to produce euthyroid serum levels (Fig. 3Go) had no effect on D2 activity in the cerebral cortex over the 6 h period. In contrast, injection of both T4 and rT3 decreased D2 activity in the cerebral cortex by approximately 50% and 75%, respectively, within 30 min of injection (Fig. 4Go). D2 activity decreased to levels observed in the euthyroid cortex within 3 h after either T4 or rT3 injection and remained stable from 3–6 h after injection. At all time points, D2 activity was significantly lower in either the T4- or rT3-treated rats compared with either the T3 or vehicle-treated rats (P < 0.05). The effects of T4 and rT3 on D2 activity in the cerebral cortex are dose dependent, whereas T3 continued to have no effect on D2 activity at equimolar concentrations (Table 1Go).


Figure 4
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  		FIG. 4. Effect of iodothyronines on D2 activity in the developing rat cerebral cortex. On postnatal d 14, PTU-treated rat pups were injected ip with T4 [200 ng/10 g body weight (BW)], T3 (40 ng/10 g BW), rT3 (200 ng/10 g BW), or vehicle, and animals were killed at 1, 3, and 6 h after injection. Where indicated, animals were also injected ip with IOP (50 µg/10 g BW). Control postnatal d 14 euthyroid and PTU-treated animals were killed at 0 and 6 h. Tissues were harvested and frozen in liquid nitrogen before use as discussed in Materials and Methods. D2 activity was determined in tissue homogenates at 2 nM rT3 and 20 mM DTT in the presence of 1 mM PTU. Results are presented as the mean of values obtained in triplicate animals in three experiments (n = 9). Units = fmol I– released/h.

 
Dynamic regulation of actin polymerization in the cerebellum
We next examined the effect of iodothyronines on the polymerization state of the microfilaments in the cerebellum. Previous studies showed that the cerebellum of hypothyroid postnatal d 14 rats had a lower amount of F-actin than the euthyroid rat cerebellum, with no change in total actin content (35). We confirmed those findings and show that the F-actin content in the hypothyroid rat cerebellum is 30–40% lower than in the euthyroid rat cerebellum at postnatal d 14 (Fig. 5Go; Table 2Go, P < 0.05 vs. euthyroid animal). In addition, the G-actin content in the cerebellum in the hypothyroid rat pups is greater than 2-fold higher than that found in the euthyroid rats (P < 0.001 vs. euthyroid animal). As shown previously (35), total cerebellar actin content was unchanged in all conditions examined in this study (data not shown). Acute administration of either T3 or vehicle did not affect either the G-actin or F-actin content in the hypothyroid rat cerebellum of the 6-h period. In contrast, T4 and rT3 both rapidly decreased G-actin content (P < 0.001 vs. PTU-treated animal) and increased the F-actin content in the PTU-treated rat cerebellum to that observed in the euthyroid cerebellum (P < 0.05 vs. PTU-treated animal) within 3 h (Fig. 5Go). These effects persisted for at least 6 h. The T4- and rT3-dependent changes in G- and F-actin content are concentration-dependent, whereas T3 has no effect even at high levels (Table 2Go). These data indicate that T4 and rT3, but not T3, dynamically regulate actin polymerization in the developing rat cerebellum.


Figure 5
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  		FIG. 5. Effect of iodothyronines on the G- and F-actin content in the developing rat cerebellum. On postnatal d 14, PTU-treated rat pups were injected ip with T4 [200 ng/10 g body weight (BW)], T3 (40 ng/10 g BW), rT3 (200 ng/10 g BW), or vehicle, and animals were killed at 1, 3, and 6 h after injection. Where indicated, animals were also injected ip with IOP (50 µg/10 g body weight). Control postnatal d 14 euthyroid and PTU-treated animals were killed at 0 and 6 h. Tissues were harvested and frozen in liquid nitrogen before use as discussed in Materials and Methods. Tissues were homogenized then solubilized with 0.5% Triton X-100. The soluble G-actin was separated from the insoluble F-actin fibers by centrifugation and the actin content in the soluble and insoluble fraction was analyzed by Western blot with an antiactin IgG as discussed in Materials and Methods. The G-actin content is expressed as a percent of the PTU-treated control animals, whereas the F-actin content is expressed as a percent of the euthyroid control animals. Results are presented as the mean of values obtained in triplicate animals in at least two experiments (n = at least 6).

 

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  		TABLE 2. Dose-dependent effects of iodothyronines on D2 activity and G- and F-actin content in the P14 brain

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the adult rat, local deiodination of T4 serves as the major source of brain tissue T3 and rT3 (24, 50, 57), potentially confounding the interpretation of T4- and rT3-specific actions in the brain. In particular, previous studies performed in adult rats suggested that access of rT3 to brain tissues was markedly less than that of T4 and T3 (24, 45), with local deiodination of T4 serving as the primary, if not sole, source of rT3 in the cerebral cortex (24). In this paper, we show that serum T4, rT3, and T3 all have equal and rapid access to cerebral cortical and cerebellar tissues in the developing rat. A likely explanation for this apparent discrepancy is the increased permeability to thyroid hormones of the blood:brain barrier in the developing brain (58, 59, 60). Because circulating T4, rT3, and T3 absorbed after ip injection readily enter developing brain tissues, this study allows the direct evaluation of individual iodothyronines on the nongenomic regulation of D2 activity in the cerebral cortex and actin polymerization in the cerebellum of the developing rat.

As noted previously, regulation of D2 activity in the adult cerebral cortex is a well-characterized nongenomic action of thyroid hormone (21, 22, 53, 56). This study shows that regulation of D2 by T4 and rT3 also occurs in the developing brain. As reported in the adult brain (21, 22, 53, 56), D2 activity in the developing cerebral cortex is markedly increased in the absence of thyroid hormone. Acute administration of either T4 or rT3 caused cerebrocortical D2 levels in the hypothyroid rat pups to fall to euthyroid levels within 3 h, whereas injection of T3 at concentrations sufficient to raise serum T3 levels above the normal range failed to have any effect over that seen with vehicle alone (Fig. 4Go). Importantly, injection of the hypothyroid rat pups with rT3 increased serum levels of only rT3 (Fig. 3Go). Thus, because serum T4 levels remained undetectable and serum T3 levels remained low and unchanged, the fall in D2 activity in the hypothyroid rat cortex after injection of rT3 is a direct effect of this inactive iodothyronine.

Underlying the regulation of D2 activity is the nongenomic regulation of actin polymerization by thyroid hormone in brain cells (19). The expression of actin is developmentally regulated in the rat brain (35, 61, 62). At birth, the F-actin content in the rat brain is low and, beginning at approximately postnatal d 5, steadily increases to adult levels over the next 2 wk (35, 61, 62). Peak actin content is observed around postnatal d 8, after which levels fall to adult levels by the third week of life. Thyroid hormone has been reported to have no effect on the total actin content in the developing brain (35), despite the observation that the peak actin mRNA content in the cerebellum is delayed by a few days in the hypothyroid rat (63). However, the F-actin content in the developing hypothyroid rat cerebellum was reported to remain low until postnatal d 15–16, before steadily increasing to adult levels (35). Treatment of the hypothyroid rat with T4 normalized the F-actin content in the cerebellum within 4 d (35).

In this study, we confirm the observation (35) that the F-actin content in the hypothyroid rat cerebellum at postnatal d 14 is significantly lower than in the euthyroid animal (Fig. 5Go). Importantly, we show that the F-actin content in the hypothyroid rat cerebellum is rapidly (within 3 h) restored to normal by the administration of either T4 or rT3. The increase in the F-actin content is associated with a reciprocal fall in the G-actin content, indicating changes in the polymerization state of the cellular actin as opposed to a change in the total cellular actin content. In contrast, T3 had no effect on the F-actin content in the hypothyroid rat cerebellum (Fig. 5Go). Because T3 readily enters cerebellar tissues (Fig. 2Go), the inability for T3 to modulate F-actin content in the cerebellum is not due to restricted access to the brain intracellular compartment. Similar to the actions of rT3 on D2 activity, the increase in cerebellar F-actin content after injection of rT3 is clearly a direct action of this iodothyronine. These in vivo data correlate well with previous in vitro observations on the regulation of actin polymerization by T4 and rT3 in cultured rat astrocytes (19, 40, 41) and rat neurons (20). Similar to our observation in the cerebellum (Table 2Go), T4 and rT3 are greater than 100-fold more potent than T3 in promoting actin polymerization in cultured rat astrocytes and neurons.

Thyroid hormone-dependent regulation of actin polymerization is likely to play a major role in the brain developmental program. The microfilaments in both the neurons and the astrocytes play a key role during neuronal migration in the developing brain. The ability to regulate actin polymerization in the migrating neurite is essential to interpret extracellular guidance cues used by the migrating cell to follow specific pathways to their destination (27, 28, 29, 30, 32, 64). Chemical disruption of actin polymerization in cultured neurons markedly impairs neuronal growth cone motility and pathfinding ability in vitro (32, 65, 66, 67). Furthermore, the T4- and rT3-dependent regulation of actin polymerization in neurons markedly blunts neuronal migration and neuronal process outgrowth in vitro (20).

Regulation of actin polymerization in the supporting astrocytes is also essential for the organization of neuronal guidance molecules such as laminin on the astrocyte surface during brain development (68, 69, 70, 71, 72, 73, 74). Disruption of the microfilaments in cultured astrocytes also disrupts the formation of laminin arrays on the cell surface (75). We have previously shown that the nongenomic thyroid hormone-dependent regulation of F-actin content in astrocytes modulates integrin:laminin interactions in astrocytes that, in turn, modulates the ability of astrocytes to fix and position laminin on the cell surface after secretion (34, 75). This in vitro modulation of laminin patterning on the astrocyte surface also occurs in vivo because the appearance of laminin in the molecular layer in the developing hypothyroid rat cerebellum is both delayed and diminished compared with the euthyroid cerebellum (76), without any change in laminin mRNA expression. Because the present study shows that thyroid hormone dynamically regulates actin polymerization in the developing brain, these data suggest that the thyroid hormone-dependent modulation of F-actin:integrin:laminin interactions occurs in vivo and is the likely etiology of the altered laminin appearance in the hypothyroid cerebellum.

This study also shows that rT3, an iodothyronine that has previously been considered an inactive metabolite of T4, indeed has direct regulatory actions on in vivo processes in the developing brain. Previous studies reported that a single injection of rT3 decreased the pyknotic index in the hypothyroid neonatal cerebellum to euthyroid levels (77). The ability of rT3 to increase the F-actin content in the developing cerebellum provides a mechanism for this observation because pathfinding and migration would be restored to the migrating neurites in the presence of rT3. This would lead to more cells reaching their target destination and surviving rather than dying and becoming pyknotic. The regulation of F-actin content and D2 activity by rT3 in the developing brain is the first demonstration of in vivo processes directly modulated by this iodothyronine.

Our data suggest that the nongenomic regulation of actin polymerization plays a major role in neuronal migration during brain development. Indeed, although transcriptional gene regulation is the major mechanism of thyroid hormone action, alternative mechanism(s) of action need to be considered since the demonstration that mutant mice lacking all known TRs exhibit apparently normal brain development (15, 16). Others have suggested a role for the unliganded receptor in thyroid hormone-mediated brain development because TRs bind to DNA in the unliganded state and in vitro studies have shown that the unliganded receptor can exert a negative effect on transcription (78). Furthermore, inactivation of TR{alpha} prevented a hypothyroid phenotype in congenitally hypothyroid Pax8(–/–) mice (79), although subsequent studies have suggested a more complex explanation for these observations (80). However, when considering unliganded TRs as mediators of the abnormities observed in the hypothyroid brain, it is important to remember that the obligatorily unliganded TR{alpha}2 is the most abundant TR isoform in the brain throughout development (81). Furthermore, like the unliganded T3-binding TRs, TR{alpha}2 has been reported to function as a transcriptional inhibitor in vitro (82, 83). Importantly, there has been no direct in vivo demonstration of regulated gene repression by either the unliganded T3-binding TRs or by TR{alpha}2; instead, all in vivo data have been inferential (i.e. hypothyroid mice exhibiting a more benign phenotype in the absence of expression, as noted above). Thus, it seems likely that non-TR-mediated processes are likely to contribute to the action of thyroid hormone on the developmental program of the brain.

In summary, we have shown that thyroid hormone dynamically regulates D2 activity and actin polymerization in the developing rat brain and that it likely does so via a nongenomic mechanism of action. The thyroid hormone-dependent regulation of actin polymerization in the rat brain provides mechanism by which this morphogenic hormone can influence neuronal migration and development independent of the need for altered gene transcription. Furthermore, because rT3 is equipotent to T4 in the nongenomic regulation D2 activity and microfilament organization in both astrocytes and neurons (7, 19, 20, 33, 34), and rT3 is the most abundant iodothyronine during fetal life (12), these data suggest a prominent role for this iodothyronine during brain development.


   Footnotes
 
This work was supported by National Institutes of Health Grant DK 49998 (to A.P.F). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

Authors A.P.F., S.A.D.-T., A.Z.P., and J.L.L. have nothing to disclose.

First Published Online February 9, 2006

Abbreviations: CNS, Central nervous system; D2, type II iodothyronine 5'-deiodinase; DTT, dithiothreitol; F-actin, filamentous actin; IOP, iopanoic acid; PTU, propylthiouracil; TR, thyroid hormone receptor.

Received October 6, 2005.

Accepted for publication January 31, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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