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Perspectives in the Study of Thyroid Hormone Action on Brain
Development and Function
Posted 12/12/2003
Juan Bernal; Ana Guadaño-Ferraz; Beatriz Morte
Abstract and Introduction
Abstract

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.
Introduction

Brain development proceeds through precisely coordinated events in
time and space. Induction of neural tissue, differentiation and
migration of specific cell types, regional specialization or synapse
formation are major milestones in a myriad of intricately associated
events. Most of these events are largely determined by genetic
factors.[1] However, epigenetic factors, such as the thyroid hormones,
are also important because they act to control the timing and
coordination of mechanistically unrelated processes. For many years
thyroid hormone has been considered essential for brain maturation in
humans and other mammals. Deficiency of thyroid hormones during
critical periods of development leads to profound and potentially
irreversible defects of brain maturation,[2,3] and clinical syndromes
arising from thyroid hormone deficiency during fetal and postnatal
periods have been extensively reviewed elsewhere.[3,4]

The Brain as a Target of Thyroid Hormone

Most of the available evidence supports the view that thyroid hormone
does not influence the major early developmental processes such as
neural induction, neurulation, and establishment of polarity and
segmentation. Thyroid hormone is involved in regulation of later
events, such as cell migration and the formation of cortical layers,
and in neuronal and glial cell differentiation. Thus, as a consequence
of thyroid hormone deficiency during specific fetal stages, altered
cell migration in the neocortex results in less defined cortical
layering and altered distribution of callosal connections.[5]
Deficiencies of cell migration are also observed in the hippocampus,
resulting in lower number of granule cells in the dentate gyrus. A
defining feature of neonatal hypothyroidism is the delayed migration
of granule cells in the cerebellum, which results in abnormal
persistence of the external germinal layer.

Thyroid hormone also controls differentiation of not only neurons and
oligodendrocytes,[6] but also astrocytes, and microglia.[7,8] Specific
neuronal cell types are characteristically affected by hypothyroidism,
such as pyramidal cells of the neocortex and Purkinje cells of the
cerebellum. The latter show a strong dependence of thyroid hormone,
because in its absence they do not fully develop their characteristic
highly elaborated dendritic tree. On the other hand, lack of proper
differentiation of oligodendrocytes results in myelination deficits.
Deiodinases and the Control of Triiodothyronine Concentrations in the
Brain

The main active thyroid hormone is triiodothyronine (T3), which in the
brain derives in large part from 5´ deiodination of thyroxine (T4).
This pathway is closely regulated by developmental and physiologic
factors.[9] Concentrations of T3 in the brain are the result of
transfer to the brain through the blood-brain barrier, by a poorly
defined mechanism, and the local activities of deiodinases 2 (D2) and
3 (D3). D2 generates T3 from T4 by phenolic ring deiodination, whereas
D3 inactivates T4 and T3 by tyrosil ring deiodination with generation
of the inactive products reverse triiodothyronine (rT3) and 3,3´
triiodothyronine (T2), respectively. Expression of D2 and D3 in the
brain is developmentally regulated. In the rat, D2 activity is low
during the fetal period, and increases postnatally, whereas D3 follows
the opposite pattern.[10] D3 activity is high in placenta and in fetal
tissues and high activity has been detected in the uterine
implantation site.[11] This pattern of expression suggests a
protective role during development. In agreement with this, recent
data have shown that inactivation of the D3 gene leads to partial
lethality at birth and severe growth impairment.[12] It may also be
that D3 expression and the regulation of T3 generation and its
subsequent availability to target cells is a way to control the timing
of thyroid hormone signaling in a fashion similar to the control of
amphibian metamorphosis.[13] In adult animals D3 is diffusely
expressed in neurons throughout the brain.[14] In contrast, during the
late fetal and early postnatal period, D3 mRNA is highly concentrated
in discrete nuclei such as the bed nucleus of stria terminalis,
central amygdala, and the preoptic area.[15] These nuclei are involved
in ***ual differentiation of the brain during the early postnatal
period, but the functional significance for the restricted expression
of D3 in these nuclei is not known.

D2 activity increases during the postnatal period and is sensitive to
thyroid hormone concentrations. In hypothyroidism, its increased
activity tends to normalize T3 concentrations even with greatly
reduced T4 concentrations. In contrast with D3, which is expressed in
neurons, D2 is expressed in two types of glial cells.[16,17] The
highest expression is found in the tanycytes, a specialized type of
glial cells that line the lower third of the walls of the third
ventricle. These cells send processes to the adjacent hypothalamus
where they frequently end in blood vessels, and to the median
eminence, ending in portal vessels. Thus, D2 in these cells could be
involved in providing T3 to the cerebrospinal fluid (CSF) from which
it would reach nearby structures by diffusion, and/or the portal
blood, influencing pituitary function. Stimulation of D2 in the
tanycytes by cytokines with subsequent local production of T3 and
inhibition of thyrotropin (TSH) secretion has been recently suggested
to play a role in non-thyroidal illness.[18] D2 is also expressed
throughout the brain in astrocytes and in some interneurons.[19]
Astrocytes appear therefore to have an active role not only in the
uptake of T4 from the blood through the blood-brain barrier, but also
in generating T3 and delivering it to nearby neurons. Molecular Basis
of Thyroid Hormone Action on Brain Development

Many of the effects of thyroid hormone on developmental processes in
the brain can be correlated with the controlled expression of specific
molecules. The most obvious is myelination, which is secondary to the
effects of thyroid hormone on oligodendrocyte differentiation and the
expression of oligodendrocyte specific genes.[20] Lack of thyroid
hormone during the postnatal period in rats, at the time of onset of
myelination, strongly delays the expression of many oligodendrocyte
genes, specifically the genes encoding proteins of myelin such as
myelin basic protein (MBP), proteolipid protein (PLP), and
myelin-associated glycoprotein (MAG).[21] As a result, the number of
myelinated axons in hypothyroid rats is strongly reduced.[22]

How thyroid hormone influences differentiation of neural cells is
poorly understood. It has been suggested that T3 is an instructive
factor in the early steps of oligodendrocyte generation from stem
cells, and that it controls the timing of oligodendrocyte precursor
cell differentiation.[23,24] It has also recently been shown that T3
promotes neuronal differentiation of embryonic stem cells in
culture.[25] The intimate molecular mechanisms by which thyroid
hormone promotes differentiation is unknown. Molecular suspects
include proteins regulating the cell cycle, such as E2F-1 (see Billon
et al.[24]), p53, cyclins and cyclin-dependent kinase inhibitors.[26]
Thyroid hormone control of neurotrophin expression, especially NGF,
BDN, and NT-3, may provide also an explanation for its effects on
differentiation of particular cells, such as cholinergic neurons and
Purkinje cells.[27] In many cases the effects of thyroid hormone on
differentiation are subtle but with potentially important functional
consequences. For example, in hypothyroidism there is a decreased
number of dendritic spines in pyramidal cells of the neocortex and
hippocampus, which is reversible and also observed after adult-onset
hypothyroidism.[28] Although the gross morphology of the neurons is
not appreciably modified, changes in spine density have strong
consequences on synaptic plasticity. The mechanisms by which dendritic
spines are affected by thyroid hormone are unknown.
RC3/neurogranin,[29] a dendritic spine protein is under thyroid
hormone control, both in developing and in adult animals,[30] but it
is not known whether expression of this protein is related to spine
formation.

Proper migration of neurons depends on the interaction of cell surface
receptors with proteins present in the extracellular matrix. One of
these proteins, reelin,[31] is regulated by thyroid hormone during the
late prenatal and early post-natal life in the rat.[32] Reelin
expression is essential for the orderly migration of neurons to their
specific destinations in the cerebral and cerebellar cortices, and
determines the normal pattern of cortical layers. This protein is
produced by Cajal-Retzius cells, which express another thyroid
hormone-regulated gene, prostaglandin D2 synthase.[33] Other molecules
reported to be involved in cell migration, such as laminin, tenascin
C, and L1, are also under thyroid hormone control.[34,35]

Most of the actions of thyroid hormone are exerted via nuclear
receptors, which are ligand-modulated transcription factors. The
physiologic ligand is T3, and therefore it is considered to be the
active form of thyroid hormone. Because T4 has low affinity for the
nuclear receptors, it might be considered as a prohormone, whose role
would be to deliver T3 intracellularly through 5´ deodination. There
are, however, data suggesting that T4 may have actions of its own. It
regulates D2 activity in astrocytes and has a direct action on F-actin
polymerization.[36,37] Also in astrocytes, T4 has been shown to
influence integrin-laminin interactions,[38] an important process in
cell migration.

Whether these nongenomic actions of T4 actually contribute in vivo to
the effects of thyroid hormone on neural cell migration is however
unknown. Work by Davis et al.[39] also suggests that T4 has biologic
activity per se. In this case, the action of T4 is mediated via the
microtubule-associated protein (MAP) kinase pathway and
phosphorylation of the T3 receptor and other targets.[39] So far it is
not known how this activity of T4 may contribute to the overall
physiologic activities of the thyroid hormones. With respect to the
nongenomic actions, mice deficient in all forms of nuclear T3
receptors have no signs of hyperthyroidism in the face of highly
elevated circulating T4 and T3, suggesting that most effects of
thyroid hormone are mediated through the nuclear receptors.[40]
Therefore, the contribution of nongenomic actions to the overall
effects of thyroid hormone is not yet clear. Davis and Davis[41] have
suggested that at least for the heart, nongenomic actions of thyroid
hormone determine the basal activity of transporters and ion channels.
If this concept is extrapolated to the brain, the nongenomic actions
of thyroid hormone could be important in neurotransmission.

Another long-standing controversial topic is the role of the
mitochondria in the overall effects of thyroid hormone in brain and
other tissues.[4,42] It is clear that thyroid hormone influences
mitochondrial function indirectly through the control of
nuclear-encoded mitochondrial genes. In addition, the expression of
mitochondrial-encoded genes are also influenced by the thyroidal
status, and T3 has direct actions on isolated mitochondria in
vitro.[43] Therefore, direct effects of T3 on the mitochondria in vivo
appear likely. Truncated forms of TR?1 and RXR? have been described in
the mitochondria,[44] and it is proposed that T3 may affect
mitochondrial transcription in a way similar to the action on the
nucleus. These topics have been recently reviewed in detail.[45]
Patterns and Mechanisms of Gene Regulation by Thyroid Hormone in the
Brain

During the past 10 years, we and others have identified a number of
genes under thyroid hormone control in the brain (for a review, see
Bernal[4]). This list of genes is likely to be greatly enlarged when
global ****ysis of gene expression using cDNA arrays technologies are
used to identify thyroid hormone target genes. Most of the genes
identified so far are expressed and regulated by thyroid hormone
during the postnatal period (Fig. 1), and the role of thyroid hormone
is to accelerate the normal physiologic process of upregulation or
downregulation that these genes experience after birth. A good example
is provided by the myelin genes, which are induced a few days after
birth, in parallel with the timing of oligodendrocyte differentiation
and the myelination wave. In the absence of thyroid hormone,
ac***ulation of myelin gene products, mRNA, and protein proceeds at a
slower rate, and final normal concentrations are attained but later in
development than in normal animals. Other genes show a region-specific
dependence of T3. One example is RC3. This gene is expressed in
subsets of neurons of the cerebrum, and thyroid hormone is needed to
achieve normal expression in discrete regions such as layer VI of the
neocortex and retrosplenial region, caudate nucleus, and dentate
gyrus. The gene is expressed in other regions such as the upper layers
of neocortex, and pyramidal cells of hippocampus, but it is not
sensitive to thyroid hormone in these locations despite the presence
of T3 receptors. Because RC3 is regulated by T3 directly at the
transcriptional level,[46,47] the most likely explanation for such a
region-specific control is that regulation of this gene is based on a
combinatorial distribution of transcription factors, including T3
receptors. Therefore, in sensitive regions, T3 receptors might
complement the pool of transcription factors needed for target gene
expression.

Click to zoom Figure 1. (click image to zoom) Thyroid
hormone-regulated genes in rat brain. The dependency of gene
expression on thyroid hormone supply is represented schematically
along a time line from conception (C) through embryonic (E) and
postnatal (P) development. Birth is indicated by P0. Most genes are
affected by the thyroidal status only during a limited period of
development, which is indicated by the horizontal bars. NGF, TrkA, and
RC3 are also thyroid hormone-dependent during adult life. A few genes,
such as those encoding NSP-A, Oct-1, and RC3 have also been shown to
be regulated during embryonic development. The inset delimited by the
discontinuous lines illustrates the regional and temporal pattern of
thyroid hormone regulation of myelin genes which follows the
myelination wave, from the caudal to rostral regions. BS, brain stem;
CB, cerebellum; MB, midbrain; Hipp, hippocampus. At the bottom of the
figure, the approximate time of first detection of TR? and of TR?, the
appearance of the thyroid gland, and the peak generation of neurons,
astrocytes and oligodendrocytes. Modified from Bernal[42] with
permission from Elsevier.

Another example of region-specific control is ZAKI-4. This gene
encodes a calcineurin inhibitor expressed throughout the brain,[48]
but sensitive to T3 only in layer VI of neocortex. Regulation of this
gene by T3 is indirect because it requires new protein synthesis. In
this case, it is likely that in layer VI, T3 regulates expression of a
protein required for ZAKI-4 expression. Recent evidence suggests that
the mechanism of ZAKI-4 induction by thyroid hormone involves
activation of phosphoinositide-3 kinase.[49]

The temporal patterns of thyroid hormone-dependent gene expression in
the brain suggest that the critical period of thyroid hormone
sensitivity is limited to the first 2-3 postnatal weeks in the rat. In
humans the sensitive period would correspondingly start after
midpregnancy. However, there may be a bias in this concept, derived
from the fact that most searches for thyroid hormone-dependent genes
in the brain have been made during the postnatal period, at the peak
of T3 receptor expression and occupancy. From the data illustrated in
Figure 1, it can be seen that the period of thyroid hormone
sensitivity for some genes extends to the adult period (RC3, TrkA, and
NGF) whereas for others it starts during the fetal period (tenascin C,
L1, reelin). Earlier ages were not ****yzed. In other studies[50,51]
it was found that maternal hormones influenced fetal brain expression
of RC3, NSP-A, and Oct-1. Maternal hormones have recently been
demonstrated to play an important role in cell migration in the fetal
neocortex.[52] The application of global ****ysis of gene expression
using suitable models of fetal hypothyroidism may help to identify
T3-regulated genes during fetal brain development.

The primary action of thyroid hormone on gene expression is mediated
through interaction of the T3 receptors with responsive elements
located in gene regulatory regions.[53] Some of the genes known to be
responsive to thyroid hormone in the brain contain triiodothyronine
response elements (TREs) and in some cases the action of T3 has been
shown to be at the transcriptional level in vitro. Genes containing
TREs in their promoter or intronic regions include those encoding
myeline basic protein,[54] the Purkinje cell-specific gene (PCP2),[55]
which encodes a G protein nucleotide exchange factor,[56] the
calmodulin binding and protein kinase C (PKC) substrate RC3,[47]
prostaglandin D2 synthetase[57,58] the transcription factor
hairless,[59] the neuronal cell adhesion molecule (NCAM),[60] and the
early response gene NGFI-A.[61] Expression of other genes are
regulated at the levels of mRNA stability (acetyl cholinester-ase),
protein translation (MAP2), or mRNA splicing (tau). Regulation of
splicing might be indirect, and subsequent to a primary action on the
transcription of splicing regulators.[62] Role of Thyroid Hormone
Receptors

In mammals, T3 receptors are the products of two genes known as TR?
and TR? that encode nine protein products that arise by alternative
splicing and differential promoter usage. The TR? gene encodes five
protein products (TR?1, TR?2, TR?3, and the truncated products DTR?1
and DTR?2) from which only TR?1 binds T3. The TR? gene encodes four T3
binding proteins, of which TR?1, TR?2, and TR?3 also bind to
responsive elements in DNA. In addition, a truncated protein, DTR?3
binds T3 but not DNA. It may be said, therefore, that there are two
types of receptors, ? and ?, and four different receptor isoforms. The
physiologic role of the nonreceptor proteins is at present still
unclear.

Concerning whether different receptor isoforms subserve different
physiologic functions by selectively regulating specific genes, the
current view is that the receptor isoforms are mostly equivalent in
their biologic activity in vivo, including binding affinity for
T3,[63] and that the different physiologic roles of each receptor
depends on their particular patterns of expression. Thus, TR? is
involved in regulation of pituitary, liver, and cochlear function,
whereas TR? regulates cardiac function, body temperature, gut
maturation, and lymphocyte development. These specific effects of each
receptor type are mainly due to their tissue distribution. In the
cerebellum, TR? is expressed in the granular cells, whereas TR? is
expressed in the Purkinje cells. Therefore, it is not surprising that
the effects of T3 on migration if granular cells is mediated by TR?,
whereas those on differentiation of Purkinje cells are mediated by
TR?.[64] Another recent example (see below) is the finding of
quantitative differences in TR?1 and TR?1 expression among subsets of
GABAergic interneurons of the cerebral cortex and hippocampus, which
is correlated with effects on behavior.[65] A different case has been
made on the control of thermogenesis in brown adipose tissue, where
the use of the TR?-selective ligand GC-1 has allowed to define
specific functions for TR?1 and TR?1.[66]

A prominent role of TR?1 in brain development and function may be
deduced from its relative expression in cerebrum and cerebellum,
accounting for approximately 70%- 80% of total T3 receptor
binding.[67] Given the widespread distribution of T3 receptors in the
brain it is noteworthy that mutant mice lacking TR?1, TR?, or both, do
not display obvious signs of developmental abnormalities. One possible
explanation for this paradox is that in the absence of ligand,
transcriptional repression or abnormal regulation of transcription by
the unliganded receptor is responsible for the effects of profound
hypothyroidism. This is supported by two lines of evidence. One was
provided by Samarut et al.,[68] which showed that congenitally
hypothyroid, Pax 8-deficient mice, which die during the first weeks of
life, can be rescued by TR?1 gene deletion. Other evidence was
provided by our group in studies on cerebellar development in
TR?1-deficient mice.[64] In these studies we showed that contrary to
what happens in wild-type mice, the mutant mice do not show delayed
granular cell migration and arrested Purkinje cell differentiation
after induction of neonatal hypothyroidism. It appears, therefore,
that these cerebellar alterations which constitute one of the
hallmarks of neonatal hypothyroidism in rodents are the result of
negative influences of the unliganded TR?1 cerebellar expression of a
mutated form of TR?1 with dominant negative activity strongly arrested
Purkinje cell differentiation.[69] Role of Thyroid Hormone Receptors
in Behavior

As pointed out above, absence of thyroid hormone receptors is not
associated with obvious developmental alterations. The possibility
exists, however, that absence of receptor during development may lead
to subtle alterations derived from disturbances in correct maturation
of specific neurons. A role of TR?1 in early neuronal differentiation
has been suggested from studies using stem cells in culture.[25,70] It
is likely that receptor deficiency is therefore associated with
alterations in differentiation or migration of selected neuronal
groups leading to subtle anomalies of brain function, not easily
related to receptor function. Behavioral ****ysis of
receptor-deficient mice may disclose alterations not previously
expected on the basis of the known classical effects of thyroid
hormone. TR?-deficient mice did not present any alterations in several
tests including the open field test, the Morris water maze test, or
contextual fear conditioning tests.[71] However, female mice with
deletions of either TR?1 or TR?1 show opposite response to estrogens
on *** behavior:[72] absence of TR?1 led to an increase ***ual
behavior, whereas absence of TR?1 reduced ***ual behavior. In other
terms, ligand-activated TR?1 would be an inhibitor of ***ual response,
whereas liganded TR?1 would have the opposite function. The data for
TR?1 fit with results from Pfaff and coworkers[73] that showed that
thyroid hormone may interfere with the actions of estrogens on female
*** responses. At the molecular level it was shown that TR may
interfere with estrogen induction of responsive genes by interactions
at the level of the estrogen responsive elements of target genes.[73]
The fact that TR?1 behaved differently than TR?1 in these behavioral
paradigms may be because of additional roles of TR?1 in other forms of
behavior.

We have ****yzed whether deletion of TR?1 would lead to behavioral
alterations in adult mice using several behavioral tests,[65] the
first of which was the open-field test to assess locomotor behavior in
a new environment. In this test the animals were more prone to display
"freezing," (i.e., to stay motionless) (Fig. 2). This indicated that
the mice had high emotionality. In a second battery of tests, the
animals learn to associate an innocuous stimulus (the conditional
stimulus), which might be either the context (as in contextual fear
conditioning) or a cue, for example a sound (as is cued fear
conditioning), with an aversive stimulus (unconditional stimulus) that
is usually an electric shock delivered through metal rods in the cage
floor. As shown in Figure 2, there were no differences in behavior at
24 hours of training. However, the extinction of the response was
surprisingly delayed in the context fear conditioning test. In other
words, TR?1-deficient mice have a reduced capacity to forget negative
memories. The results of these experiments, combined with data on the
microstructure of the hippocampus and cerebral cortex, as well as the
cellular distribution of thyroid hormone receptors have led us to
hypothesize that TR?1 is involved in the specification of hippocampal
neuronal circuits involved in behavior.[65]

Click to zoom Figure 2. (click image to zoom) Behavior of adult
TR?1-deficient mice (solid boxes) in the open field (A) and the
contextual (B) and cued (C) fear conditioning tests. The behavior of
control wild-type mice is represented in open boxes. In the open field
test the animals were placed in the center of a circular open field of
140 cm diameter and 32 cm height, and the behavior and locomotor
activity were videotaped. A significant number of TR?1-deficient mice
stayed motionless (freezing), displayed less rearing, and spent less
time in the center area of the field than the wild-type mice. The fear
conditioning tests took place in Pavlovian conditioning cages. In this
context, fear conditioning, which depends on the integrity of both
amygdala and hippocampus, the mice learn to associate the context with
an aversive stimulus (i.e., an electric shock). Extinction of the
response took much more time for the TR?1 knockout mice. In cued-fear
conditioning, a test that depends on the integrity of the amygdala,
the mice learn to associate the aversive stimulus with a cue, usually
a sound. In this test, both learning (24-hour test) and the extinction
of the response (1 week) were similar in knockout and wild-type mice.
The results suggested a lesion at the level of the hippocampus, and
were correlated with a decreased number of GABAergic terminals in the
CA1 field. Reproduced from Guadaño-Ferraz et al.[65] with permission.

Conclusions

From the data ac***ulated during the past 10 years, we are starting to
understand the general features of thyroid hormone action in the brain
in molecular terms. Knowledge in this field has, however, had a very
strong limitation because of the complexity of the target organ under
study, which requires the convergence of several disciplines. In
addition to a more precise understanding of the classic effects of
thyroid hormone deficiency or excess, the ****ysis of mice with
genetically altered thyroid hormone receptors are providing surprising
and unexpected results. In particular, the notion that some features
of the hypothyroid phenotype may be the result of altered
transcriptional regulation by the unliganded receptors more than to
the lack of hormone per se. In addition, the indication that thyroid
hormone receptors may have a role in behavior opens new fields of
inquiry and raises the intriguing possibility that receptor mutations
may underlie psychiatric forms of behavior in humans.

Funding Information

Supported by grants BF12002-00489 of Ministerio de Ciencia y
Tecnología, and 08.5/0044.1/2000 from Comunidad de Madrid. Reprint
Address

Dr. Juan Bernal, Instito de Investigaciones Biomedicas, Arturo
Duperier 4, 28029 Madrid, Spain. E-mail: jbernal@iib.uam.es


Juan Bernal, Ana Guadaño-Ferraz, and Beatriz Morte, Instituto de
Investigaciones Biomédicas Alberto Sols, Consejo Superior de
Investigaciones Científicas y Universidad Autónoma de Madrid, Madrid,
Spain
Thyroid 13(11):1005-1012, 2003. © 2003 Mary Ann Liebert, Inc.

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