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Thyroid Research Unit, Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
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Abstract |
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 Top Abstract I. IntroductionII. Developmental SchedulesIII. Thyroid Hormone ActionIV. Molecular Actions of...V. Theories and SpeculationsVI. ConclusionsReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Developmental SchedulesIII. Thyroid Hormone ActionIV. Molecular Actions of...V. Theories and SpeculationsVI. ConclusionsReferences |
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II. Developmental Schedules |
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TopAbstractI. IntroductionII. Developmental SchedulesIII. Thyroid Hormone ActionIV. Molecular Actions of...V. Theories and SpeculationsVI. ConclusionsReferences |
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B. Developmental studies in the rat
The first systematic studies of the effects of
hypothyroidism on brain development in rats were reported by Eayrs and
his colleagues (18, 19, 20, 21). The distinguishing feature of the cerebral
cortex in hypothyroid rats is the retarded development of the neuropil.
In the hypothyroid rat, peripheral and central neuronal cell bodies are
smaller and more tightly packed. Disorders of process growth and
synaptogenesis are manifested by diminished axonal and dendritic
outgrowth, elongation and branching, and alterations in the number and
distribution of dendritic spines. Other groups have advanced
biochemical evidence of deficient development of the central nervous
system in the absence of thyroid hormone, including reduced myelination
(22, 23, 24) and delayed expression of specific enzymes (25, 26, 27). These
deficiencies are observed in neurons of the cerebral cortex, the visual
and auditory cortex, hippocampus, and cerebellum. The affected areas
can be related to the various deficits in learning and motor skills
consistently observed in hypothyroid animals (12). In the cerebellum,
thyroid hormone deprivation results in delayed proliferation and
migration of granule cells from the external to the internal granular
layer (28, 29), stunting of the dendritic arborization of the Purkinje
cells (30, 31), and diminished axonal myelination (24, 32). Consistent
with the finding of delayed cellular migration and neuronal
differentiation, Faivre et al. (33) reported a dramatic
decrease in microtubule numbers in Purkinje cells. Aniello and
colleagues (34) also demonstrated a delay in the transition from
immature to mature forms of the microtubule protein, tau, and in the
developmental expression patterns of various tubulin isotype mRNAs
(35). The developmental retardation of the rat brain resulting from
thyroid hormone deficiency can be reversed if administration of thyroid
hormone is begun before the end of the second week after birth (9, 36).
The greater the delay in hormone replacement beyond this time the less
is the chance of recovery of normal development. These age-dependent
responses to thyroid hormone deprivation differ sharply from the
reversible metabolic effects of thyroid hormone that occur in other
tissues of the rat, such as stimulation of oxygen consumption (37) or
hepatic lipogenic enzyme activities (38). These are reversible
regardless of the length of the preceding hypothyroid state or age of
the animal. Of additional interest is the finding that both adult and
neonatal rat brains fail to respond to thyroid hormone with an increase
in oxygen consumption (39).
One of the reasons for the popularity of the rat model is the relative ease with which hypothyroidism can be established in the dam, the intrauterine fetuses, and the neonates. Administration of either methimazole or propylthiouracil in drinking water or food will block the oxidation of iodide to iodine and consequently block the formation of T4 and T3 in the maternal thyroid. Since these drugs readily pass the placental barrier and are also transmitted to the suckling pups in the mother’s milk, the fetus and neonate also become profoundly hypothyroid.
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III. Thyroid Hormone Action |
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TopAbstractI. IntroductionII. Developmental SchedulesIII. Thyroid Hormone ActionIV. Molecular Actions of...V. Theories and SpeculationsVI. ConclusionsReferences |
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The interrelation of T4 and T3 in mediating the effects of thyroid hormone has assumed particular importance in the case of brain development. The brain is especially rich in type II iodothyronine 5'-deiodinase (41). The demonstration that augmented type II 5'-deiodinase activity in brains of hypothyroid animals serves to preserve the level of intracellular T3 strongly suggests that the interaction of T3 with specific nuclear receptors is a critical step in mediating thyroid hormone action in brain. However, when the level of plasma T3 is raised sufficiently, the nuclear receptors can be readily saturated (42), a finding that demonstrates that T4 is not an obligatory precursor in the generation of brain nuclear T3. In the adult rat, van Doorn et al. (43) estimated that as much as 65% of T3 in the cerebral cortex and 50% in cerebellum may be generated by conversion of T4 to T3. However, Crantz et al. (44) had earlier reported that even higher proportions of nuclear T3 (cerebrum, >80%; cerebellum, 67%) were generated as a result of local production. Ruiz de Ona and colleagues (45) demonstrated in the rat that whereas fetal brain T4 levels rose in parallel with plasma T4 during the latter days of gestation, T3 in fetal brain rose 18-fold, six times more than the 2-fold change in plasma T3. This is also a period of increasing type II 5'-deiodinase activity in brain (45). It is likely, therefore, that also in fetal brain the bulk of T3 is the product of local monodeiodination of T4.
B. The role of maternal thyroid hormone in fetal brain development:
direct or indirect?
An important related issue is the role of maternal hormone in
fetal brain development. Complicating this issue is the variable extent
of placental permeability to thyroid hormones among the species
studied. Such differences result from structural features of the
placentas as well as variability in concentrations of placental
iodothyronine deiodinase activities (10, 46, 47). Early studies
suggested that maternal hormone could not cross the placenta. However,
more recent evidence (48, 49) has clearly demonstrated transplacental
transport of thyroid hormones in rat. T4 and
T3, all derived from the mother by transplacental passage,
have been detected in rat embryos before the start of secretion of the
fetal thyroid on embryonic day 17 (48, 50, 51). In contrast, there is
only very limited transfer of maternal hormone to the fetus in sheep
(10). A recent study by Vulsma et al. (52) suggested that in
human fetuses with congenital hypothyroidism, maternal-fetal transfer
of T4 may result in fetal plasma T4 levels
25–50% of those in normal infants. Since type II 5'-deiodinase
activity in brain increases in response to lowered concentrations of
T4 (53, 54), these levels of T4 might suffice
as substrate to maintain normal or near normal T3
concentrations in brain but not in other tissues (55). This would
account for the finding that most of these children will have normal
intellectual development if treated promptly at birth.
In a recent review of the literature Porterfield and Hendrich (56) pointed out that brain development in the rat pup at birth is equivalent to that observed in the human fetus at 5 to 6 months gestation, and the rat at postnatal day 10 is comparable to the human baby at birth. Care should, therefore, be exercised in applying findings with respect to the role of maternal hormone in fetal brain development in one species to any other.
In the rat, Dussault and Coulombe (57) initially estimated that placental transfer of maternal T4 is less than 1% of the fetal T4 production rate. The more recent studies of Morreale de Escobar and her colleagues (48, 58), however, argue that physiologically significant amounts of T4 and T3 are in fact transferred to the fetus from the earliest stages of gestation. As much as 20% of fetal T4 derives from transplacental transfer even after the fetal thyroid has begun to secrete hormone on fetal day 17. By sensitive RIA, Obregon et al. (48) found measurable levels of T4 and T3 in rat embryotrophoblasts as early as days 9 to 12, whereas the fetal thyroid first secretes T4 about day 17. Hormone concentrations in the fetus up to day 17 were below detectable levels if the mother had been thyroidectomized.
What remains uncertain is the developmental role of the maternal transmission of thyroid hormone. Studies by Morreale de Escobar and her colleagues (45, 59) showed that despite low levels of plasma hormone in thyroidectomized dams, both T4 and T3 levels were normal from day 17 to day 22 of gestation in the brains and plasma of fetuses with normally functioning thyroids. Although one might expect that failure of maternal to fetal hormone transfer would elicit an increase in fetal pituitary TSH secretion (59a), TSH levels near term were also unaffected by maternal hypothyroidism (49, 59). However, others have reported evidence of increased fetal TSH (60, 61). An increase in TSH would suggest that the fetal thyroid is stimulated to increase hormonal secretion to compensate for loss of the maternal contribution. Consistent with the lack of effect of maternal hypothyroidism on T3 and T4 levels in the fetal brain was the absence of detectable change in type II 5'-deiodinase activity, which is already sensitive to T4 levels at this time (45).
These results suggest that loss of the maternal contribution of hormone may not affect brain maturation during this stage of development as long as the fetal thyroid is functioning normally. Nevertheless, these fetuses had lower body and brain weights and reduced brain protein and DNA concentrations (50). Bonet and Herrera (61), however, have shown that retardation of fetal body and brain growth was only apparent if maternal hypothyroidism is present during the first half of pregnancy when the dam is undergoing marked metabolic changes associated with the onset of pregnancy. No such effect was noted if hypothyroidism was induced during the second half of pregnancy. In a series of papers, Porterfield and Hendrich (62, 63, 64) examined the effect of maternal thyroidectomy on various biochemical parameters in the fetal brain. They found that administration of GH to the thyroidectomized dams during the last days of gestation generally mitigated any of the observed deficiencies in brain growth, galactolipid accumulation, or carbohydrate utilization. They also found that GH treatment of the hypothyroid dams prevented the behavioral deficits otherwise observed in their progeny (65) and concluded "... the major factor responsible for the CNS developmental abnormalities (in progeny of hypothyroid dams) is the altered flow of nutrients or substrate to the fetus." These findings thus raise the basic question as to whether the adverse effects of maternal hypothyroidism result from a reduction in maternally transmitted T4 to the fetus or whether the effects of maternal hypothyroidism result indirectly from the untoward effects of hypothyroidism on one or more gestational processes.
A report by Narayanan and Narayanan (66) indicating that maternal hypothyroidism retards the appearance of the mesencephalic nucleus of the fifth cranial nerve on gestational days 9 to 11 has yet to be confirmed. If these studies were confirmed, one would have at hand a potentially interesting model with which to further investigate the possible direct role of thyroid hormone action in early brain development. Such studies should be accompanied by efforts to define at a molecular level specific genes that are directly regulated by thyroid hormone during early stages of neural development. This would allow a distinction to be made between direct effects of deprivation of maternally derived thyroid hormone and indirect consequences resulting from the metabolic and nutritional deficiencies associated with maternal hypothyroidism.
Consistent with evidence of very limited placental transfer of thyroid hormone in the sheep (10), Potter et al. (67) found that at 50 days of gestation, before onset of fetal thyroid gland function, fetuses carried by thyroidectomized ewes exhibited normal body and brain weights. Yet, despite normal fetal plasma hormone levels throughout the remainder of gestation, there was a transient reduction in the rate of brain growth in midgestation and a lower rate of body weight gain throughout the latter half of gestation. Histological examination of the brain at late gestational stages, however, found no developmental or structural defects associated with the reduced brain weight. The findings in sheep, therefore, support the thesis that maternal hypothyroidism may indirectly result in delayed growth of the fetal carcass and brain, this despite normal fetal thyroid hormone levels. Normal differentiation of the brain will, nevertheless, be achieved so long as normal fetal thyroid function is present.
More recently, several additional reports have pointed to potential
effects of fetal hypothyroidism on aspects of brain development. Das
and Paul (68) found the ß-adrenergic receptor concentration to be
decreased in cerebral astrocytes. The level of the short form of the
G
s protein was reduced (69), although most other G proteins were
unaffected. Vega-Nunez et al. (70) observed a decrease in
the mitochondrial 16S rRNA in the fetal brain but not of other
mitochondrial RNAs. Since the mitochondrial genome is transcribed as a
single transcript, this raises a question as to the possible role of
thyroid hormone in processing of the separate RNAs. However, since in
each of these studies, maternal as well as fetal hypothyroidism was
induced by treatment with goitrogens, some uncertainty exists as to
whether these effects are due directly to fetal hormone deficiency or
to maternal hypothyroidism.
C. Intracerebral transport
There has been considerable interest in the mechanisms involved in
intracerebral transport of T4. The plasma
T4-binding protein transthyretin also is the major binding
protein in cerebrospinal fluid (71). Synthesized in the choroid plexus,
transthyretin has been postulated to facilitate the transport of
T4 across the blood-brain barrier to the brain (72, 73).
Mendel et al. (74), however, have challenged this thesis by
showing normal brain distribution of T4 in rats injected
with EMD 21388, a drug that blocks the binding of T4 to
transthyretin. More recently, Palha and colleagues (75) examined the
effect of deletion of the transthyretin gene in mice and found,
consistent with the free hormone hypothesis (76), that plasma free
T4 and T3 were unchanged. Moreover, the brain
type II 5'-deiodinase activity was unaltered, a finding also pointing
to the euthyroid status of the brain. Thus, transthyretin does not
appear to be essential for transport of thyroid hormone into the brain
or other tissues.
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IV. Molecular Actions of Thyroid Hormone in the Developing Brain |
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TopAbstractI. IntroductionII. Developmental SchedulesIII. Thyroid Hormone ActionIV. Molecular Actions of...V. Theories and SpeculationsVI. ConclusionsReferences |
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Specific nuclear receptors for T3 in adult rat brain were first identified by in vivo saturation analysis after the intravenous injection of 125I-labeled T3 together with increasing concentrations of unlabeled T3 (80). Subsequently, in vitro techniques were developed to detect and quantitate receptors by analyzing T3 binding in isolated whole nuclei or in nuclear extracts (81, 82, 83). These made it possible to detect receptors in the brains of fetal rats (39), sheep (84), and humans (85). The recognition that these sites exhibited high affinity and specificity for biologically active thyroid hormone analogs (86) strongly suggested that these structures represented the site of initiation of thyroid hormone action.
Studies by Schwartz and Oppenheimer (39) identified similar sites in the brains of late gestational rats. They noted a transient surge in concentration as plasma T3 levels rise shortly after birth. Perez-Castillo et al. (87) observed that T3 receptors were present in rat brain as early as day 14 of gestation, several days before the onset of fetal thyroid function. Bradley et al. (88) and Mellstrom et al. (89), using the more sensitive technique of in situ hybridization, were able to demonstrate the presence of early but very localized expression of TRß in the fetal brain. Both TRß1 and ß2 were detected as early as embryonic day 12.5 in the portion of the otic vesicle that gives rise to the cochlea. It is of interest, however, that reports from several groups (90, 91, 92) indicated that the critical period for thyroid hormone action in cochlear development, both anatomic and functional, is limited to the perinatal period from gestational day 18 to postnatal day 5. Thus, these observations further strengthen the inference that the simple presence of TR at any given time in gestation cannot be assumed to connote a concurrent function.
In the sheep, nuclear receptors are evident by 50 days of gestation and are, as in the rat, constant until birth (84, 93) (term, 150 days). Very low levels of nuclear T3 receptors were measured in human fetal brains during the 10th week of gestation but by the 16th week concentrations increase by about 10-fold (85). Thus, in both the human and sheep fetus, nuclear receptors are already present before the onset of fetal thyroidal secretion of hormone.
B. Thyroid hormone receptor isoforms and their tissue distribution
The affinity of T3, T4, and their analogs
for the nuclear receptors in all tissues examined parallels their
biological potency when due account is taken of differential metabolism
(86) and the intensity of the biological reaction elicited is limited
by the occupation of the receptors (94). It is clear that
T3 receptors present in fetal, neonatal, and adult rat
brain do not differ with regard to their relative affinity for thyroid
hormone analogs from the receptors identified in other tissues (87, 95). These observations were initially greeted with surprise since
adult brain does not demonstrate responses to the administration of
thyroid hormone typically observed in other tissues (37, 39, 96). The
nature and mechanisms of the biochemical response of adult brain to
altered levels of thyroid hormone are still poorly understood.
An important development in our understanding of thyroid hormone action
was the cloning of the two genes coding for structurally related but
nonetheless distinctive receptor proteins, designated T3
receptor- (T3R) and T3 receptor-ß
(T3Rß), situated in the human genome on chromosomes 17
and 3, respectively (Table 1
) (reviewed
in Refs. 77, 97, and 98). Alternate splicing of the initial transcript
of the -gene generates TR1, which binds T3, and two
closely-related isoforms, collectively designated as TR2, which do
not bind T3 and the function of which remain obscure. Some
investigators have suggested that these receptor variants may compete
with the bona-fide receptors and thus attenuate the physiological
effects of T3 (99, 100). Two isoforms also arise from the
T3Rß gene, T3Rß1, the mRNA of which is
widely distributed in rat tissues, and T3Rß2, the mRNA of
which is highly concentrated in the pituitary. The T3
nuclear receptors belong to a class of closely related nuclear
transacting factors that includes the receptors for the steroid
hormones, vitamin D, retinoic acid, and cis-retinoic acid.
In common with these, the T3Rs are characterized by a
DNA-binding domain containing two zinc fingers, a carboxyl terminal
segment that contains the ligand-binding domain and transactivation
domains, and a distinctive amino-terminus whose function remains poorly
defined.
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. Recently, low levels of T3Rß2 mRNA have also
been identified in brain (101, 102). The level of functional receptor
protein can be estimated from the difference in T3 binding
capacity in nuclear extracts before and after immunoprecipitation of
the individual T3R isoforms with specific IgG (103). In the
case of TRß2, the level of its mRNA was virtually unmeasurable, yet
approximately 10% of the total nuclear binding capacity in brain is
accounted for by this isoform. This was also found to be the case in
liver, heart, and kidney (102). The T3Rß2 protein was
easily observed to concentrate in nuclei of all these tissues by
immunohistochemical analysis (102, 104). It appears possible that the
half-time of disappearance of the mRNA is substantially shorter than
the corresponding receptor protein. This would result in a situation
where a majority of cells could display the receptor protein but the
mRNA would be present at any time in only a minority of these cells
(105).
C. Interactions of ligand, receptor, and DNA
The past decade has witnessed an extraordinary explosion of
insight into the interactions of T3 and its receptor with
other nuclear proteins and specific sequences of DNA. The nature of
these interactions are generally believed to constitute the molecular
mechanism by which the hormone regulates gene expression. Extensive
reviews of this process have recently appeared (77, 106). Briefly, the
thyroid hormone receptor binds to DNA sequences generically designated
as thyroid hormone response elements (TREs), in the regulatory region
of target genes. The TRE characteristically consists of two DNA
hexamers, termed half-sites, with the consensus sequence AGGTCA. Often
the half-sites form a direct repeat separated by four bases although
the orientation of the half-sites to each other and their spacing may
vary considerably (106). Although T3R may occupy the two
half-sites as homodimers, available evidence suggests that the
T3R may preferentially form heterodimers on the TRE with
receptors for cis-retinoic acid or other proteins. The
potential for a given DNA sequence to act as a TRE can be verified by
demonstrating that it confers responsivity to T3 regulation
upon a reporter gene in transient transfections. More recent
investigations have shown that nuclear proteins may influence the
expression of target genes by binding to the receptor without
themselves binding directly to the DNA (107, 108, 109). Since such proteins
may function in a positive or negative fashion, they have been
designated as "co-activators" and "co-repressors."
D. Ontogeny of thyroid hormone receptor isoforms in brain
The identification of the various receptor isoforms prompted
efforts to define the developmental pattern in the appearance of each
in brain. Of interest was the finding that Northern analysis failed to
show T3Rß gene expression in fetal brain (110).
Immunoprecipitation studies also failed to show evidence of
T3Rß1 or T3Rß2 binding activity and
indicated that T3R1 accounted for the total binding
capacity in fetal brain (103, 111). As indicated earlier, the work of
Bradley et al. (88, 112) and Mellstrom and colleagues (89)
makes it likely that some TRß is present early in restricted segments
of the brain. A dramatic 40-fold rise in T3Rß1 mRNA
concentration begins at the time of birth (Fig. 1) and reaches maximal levels by
postnatal day 10 (110, 113). In contrast, the levels of
T3R1 and T3R2 mRNA, already high in the
prenatal state, increase only transiently by a factor of 2 in the first
days after birth. The T3R1 and T3R2 mRNA
levels subsequently fall to adult levels by postnatal day 15. More
recent studies showed that approximately 60% of the total
T3 nuclear binding capacity in the adult rat brain is due
to T3R1, 30% to T3Rß1, and the remaining
10% to T3Rß2 (Table 1) (102).
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The nearly simultaneous rise in T3Rß1 and serum
T3 raised the possibility that the increase in T3per se was responsible for the induction of
T3Rß1. Studies by Brown and colleagues earlier noted a
similarly coordinated surge in T3Rß and serum
T3 levels in tadpoles during normal metamorphosis and
precocious administration of T3 to the tadpoles induced a
rise in the T3Rß mRNA (115). Competence, the ability of
the young tadpole to prematurely metamorphose in response to
T3, correlates with induction of the T3Rß
mRNA (116). Ranjan et al. (117) have more recently
demonstrated the presence of a TRE in the promoter of the amphibian
T3RßA gene. Presumably T3 induces the
expression of this gene through an interaction with
T3R1. However, in the neonatal rat, a surge in TRß1
mRNA identical to that in normal pups was demonstrated in pups rendered
completely hypothyroid (110). Presumably, in the rat the rise in
T3Rß mRNA is governed by other developmental factors.
E. Search for T3-responsive genes in the neonatal rat
brain
Despite the abundant data indicating the importance of thyroid
hormone in brain development and the presence of all
T3-receptor isoforms in brain, there was and continues to
be a surprising lack of information of the specific brain genes
regulated by T3. Muñoz and colleagues (118) have
tested the effect of hypothyroidism on a series of mRNAs in the
developing brain. One of these, RC3 or neurogranin, has been
extensively investigated. This mRNA is of neuronal origin and begins to
accumulate between the 5th to 7th postnatal days and reaches maximum
levels by day 12. In hypothyroid rat pups, the rate of accumulation was
blunted and even at 30 days of age, the mRNA concentration was about
half that in normal controls (119). The mechanism of T3
regulation of this mRNA is unclear since to date a functional hormone
response element has not been identified in the promoter region of the
gene (120). Two genes, glial fibrillary acidic protein and glutamine
synthetase, expressed in astrocytes were unaffected by the change in
thyroid state, as was a series of unidentified brain-specific mRNAs. In
contrast, levels of mRNA for myelin-associated genes specifically
expressed in oligodendroglia, including myelin basic protein (MBP),
myelin-associated glycoprotein (MAG), and proteolipid protein (PLP),
were all reduced by at least 50% in the hypothyroid brains. Although
the concentrations of microtubule-associated proteins (MAP), MAP-1,
MAP-2, and tau, are affected by altered thyroid state, the mRNA levels
do not change perceptibly (34, 121, 122, 123). However, Aniello et
al. (34) have demonstrated that thyroid hormone can, directly or
indirectly, regulate the timing of the splicing mechanism responsible
for replacement of juvenile tau mRNA variants with the adult varieties.
Some differences are evident among studies with respect to thyroid
hormone regulation of nerve growth factor. Whereas all investigators
observe changes in the NGF protein levels (124, 125), thyroid hormone
is not consistently seen to affect the mRNA concentrations (118, 125, 126).
Thyroid hormone deprivation in neonatal rats was long known to result in a diminished rate of myelin production and the concentration of each of its component proteins, an impairment that can be reversed by thyroid hormone administration (reviewed in Refs. 8 and 9). Studies by Farsetti and colleagues (127, 128) have demonstrated that the gene for myelin basic protein is directly regulated by thyroid hormone. These investigators identified a sequence in the promoter of the mouse MBP gene at position -186 to -169 that was effective in the hormonal transactivation of a reporter gene in NIH3T3 cells. Thyroid hormone also regulates the mRNA levels of other myelin proteins, PLP, MAG, and 2',3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) (129, 130). However, whereas Tosic et al. (129) reported that the hormone increased transcriptional activity of the MAG gene, Rodriguez-Pena and colleagues (130) found no difference in transcriptional rate in normal, hypo-, or hyperthyroid rats and attributed the hormonal effect to stabilization of the newly transcribed mRNA. Bogazzi et al. (131) have also described a sequence in the promoter region of the PLP gene that can be activated by thyroid hormone in transient transfection assays.
In an effort to identify potential T3 target genes, our
laboratory examined, by Northern analysis, total RNA from whole brain
genes expressed prominently in cerebellar Purkinje cells. Studies by
Nordquist et al. (132) had already identified three such
genes that were also developmentally regulated. The differentiation of
cerebellar Purkinje cells is known to be thyroid hormone-dependent and
to occur in the early neonatal period (Fig. 2) (30, 31). The genes examined included
calbindin, the myo-inositol-triphosphate (IP-3) receptor, and Purkinje
cell protein-2 (Pcp-2). The latter codes for a protein of unknown
function that is expressed exclusively in Purkinje cells in cerebellum
and in the bipolar cells of the retina (132).
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, show a similar
pattern for each of the four genes studied. Hypothyroidism resulted in
a substantial delay in the rise of each of the four mRNAs. However, by
days 30–40 the same level of gene expression was attained as in the
hypothyroid pups supplemented with T3 despite maintenance
of the hypothyroid state. These observations underscore the importance
of timing in normal brain development since the profound deficit noted
in Purkinje cell maturation (30, 31) in hypothyroid pups occurs despite
ultimate normalization of the expression of these genes.
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suggest that gene expression in response
to T3 is characterized by three phases: Phase 1, a
refractory state that exists throughout the prenatal period in the rat;
Phase 2, the T3 responsive period from postnatal day 3 to
postnatal day 20; and Phase 3, starting after day 20 during which gene
expression is independent of T3 regulation. This may not,
however, be typical of all genes regulated by T3.
Other experiments have attempted to define the mechanisms by which
thyroid hormone regulates the expression of these genes. Studies in our
laboratory have examined the gene coding for Pcp-2. Since expression of
this gene in brain is confined to Purkinje cells, Pcp-2 appeared to be
suitable as a model of neuronal as contrasted to glial gene expression.
Initial transfection assays using constructs with the native Pcp-2
promoter showed only weak T3-induced augmentation of gene
expression (133). Subsequent studies, however, demonstrated that a
potential TRE sequence consisting of three half-sites ligated to a
heterologous promoter conferred T3 responsivity on the
reporter gene (134). Further, a sequence of 68 bp immediately 3' of the
TRE appeared to play an important role in silencing the response of the
gene to T3 (Fig. 4).
Excision of the 68-bp sequence from the native Pcp-2 promoter resulted
in robust T3-mediated regulation of gene expression (135).
More recent studies suggest this sequence may bind an inhibitory
nucleoprotein that serves to restrain premature gene regulation by
T3 in the late fetal stages (135). Since the sequence did
not result in attenuation of basal transcription, we have designated
the 68-bp sequence as a response silencer region.
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1 mRNA are already present in the undifferentiated
O-2A cell. The capacity of the cultured cell to respond to
T3 with enhanced MBP gene expression coincides with the
appearance of T3Rß1 mRNA on day 2 of culture.
Transactivation studies in the cultured oligodendrocytes confirmed that
the T3 effect is mediated by the TRE first characterized by
Farsetti et al. (128). This model thus replicates, in a
striking fashion, the pattern of response of MBP observed in the brain
in vivo. During phase 1 in culture, the O-2A cell does not
synthesize MBP nor is the MBP gene responsive to T3. During
differentiation (phase 2), T3 accelerates the rate of
expression of the MBP gene. In phase 3, the mature oligodendrocyte no
longer responds to T3 but MBP synthesis occurs at maximal
rates independent of the presence or absence of the hormone. Perhaps,
as in the case of other brain genes, the role of T3 in the
maturing oligodendrocyte in culture is to augment the expression of
target genes at a predetermined time point in the developmental
schedule. Based on preliminary data (see below), we postulate that the
appropriate time point at which T3 acts is determined by
the disappearance of suppressor factors that in the fetal state inhibit
either basal gene expression or T3-regulation (Fig. 5).
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raises the
possibility that the regulatory mechanism is identical for each.
However, an examination in our laboratory of 1100 bp of the calbindin
upstream region has failed to provide evidence for a functional TRE as
determined by failure to demonstrate T3 regulation in
transient transfection experiments (D. J. Carlson, K. A. Strait, H. L.
Schwaltz, and J. H. Oppenheimer, unpublished data). Although there is
precedent for the existence of a TRE this far removed from the start
site of transcription (137), these findings raise the possibility that
the expression of this gene is mediated by an intermediate agent. The
lag time between individual components of the reaction mechanism may be
too short to allow recognition of separate pathways. Lindholm et
al. (138) have presented evidence that T3 acts only
indirectly on Purkinje cell differentiation and calbindin synthesis by
causing increased secretion of neurotrophin-3 from the cerebellar
granule cells. However, contrary to these findings, Alvarez-Dolado
et al. (125) failed to observe an effect of hypothyroidism
on neurotrophin-3 mRNA concentrations in cerebellum. The study of the expression of the MBP and Pcp-2 genes suggests that the basic interaction of T3 with target genes in brain is similar to the action of thyroid hormone in other tissues. However, the identity and function of the direct target genes of thyroid hormone in brain remain largely unknown.
G. Extranuclear actions of T4?
There continues to be interest in the possibility that
thyroid hormone exerts extranuclear effects. In general, evidence
supporting this hypothesis appears sparse (77). In the case of the
brain, however, there is one action of thyroid hormone that clearly
appears to be independent of nuclear mechanisms, regulation of the type
II iodothyronine 5'-deiodinase activity (41). This enzyme plays an
important role in defending the brain against the effects of
iodine deficiency and hypothyroidism. Studies by Leonard and his
colleagues of the regulation of this enzyme by T4 both
in vivo (53, 54) and in cultured astrocytes (78)
demonstrated that the action of the hormone is quite rapid and is not
blocked by cycloheximide or actinomycin D. T4 regulation
was disrupted by cytochalasin B (139), suggesting that down-regulation
of enzyme activity by T4 required an intact actin
cytoskeleton. Their studies also showed that T4 acted to
maintain the polymerization state of the actin. They propose that in
the presence of T4, type II 5'-deiodinase binds to F actin,
after which the enzyme is internalized by way of the microfilamentous
cytoskeleton and targeted to an endosomal pool (79, 140). Since
cellular migration, neurite outgrowth, and dendritic spine formation
are dependent on interactions of the actin cytoskeleton with other
cellular proteins (141, 142), Leonard and colleagues suggest that the
action of T4 on the actin cytoskeleton may also influence
these aspects of neuronal differentiation and, consequently, cell-cell
interactions in brain. Actin polymerization in the cerebellum is
reduced in the hypothyroid rat and restored by T4
treatment; however, there are contradictory reports (143, 144) as to
whether the kinetics of response to hormone in vivo are
consistent with those observed in cell culture. Further efforts are
required to establish that similar mechanisms may be operating in
vivo and in vitro.
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The role of TR1 in the prenatal rat central nervous system remains
unclear. It is possible that TR1 may have a ligand-independent
function in early brain development (145). The dramatic increase in
circulating T3 in the early neonate results from augmented
pituitary TSH production, increased thyroidal T4 secretion,
and increased peripheral T4 monodeiodination. The almost
simultaneous rise in serum T3 and the level of brain TRß1
in contrast to the near constancy of TR1 originally suggested to us
that the T3Rß1 isoform mediated the developmental effects
of T3. In recent collaborative studies with Forrest
et al., who developed a T3Rß null strain of
mice (146), we observed no significant differences between mutant and
wild type mice with respect to the developmental expression of the
Pcp-2 and MBP genes (147). Forrest et al. (146) had earlier
demonstrated that the absence of the T3Rß did not result
in deficiencies in most behavioral or neuroanatomical parameters.
Clearly, in the mouse the T3Rß1 does not appear to be
essential in mediating the postnatal stimulation of the two target
genes examined, Pcp2 and MBP, or of brain morphogenesis. These
conclusions are consistent with the clinical observations that patients
with the thyroid hormone resistance syndrome due to homozygous deletion
of the T3Rß gene demonstrate normal mental development
(148). However, the coordination of the rise in T3 and
T3Rß1 could represent an effort to increase the number of
available receptors for the optimal development of specialized systems
such as the cochlear-vestibular apparatus. The studies by Forrest
et al. (149) demonstrated that although the absence of
T3Rß did not affect cochlear morphogenesis, these animals
do suffer a major deficiency in auditory function. This is also
consistent with the presence of deaf-mutism found in patients with
thyroid hormone resistance due to deletion of the T3Rß
gene (150).
Although it is clear that the postnatal surge in thyroid hormone concentration in the rat is essential for normal brain development, it was not known whether premature increase in T3 would result in early expression of responsive genes or other evidence of precocious development. We recently observed that injection of large doses of T4 into pregnant rats on gestational day 15, sufficient to raise the fetal brain T3 to adult levels for the remainder of pregnancy failed to elicit premature expression of the Pcp-2, calmodulin kinase IV, and myelin basic protein genes (151). Nor were there detectable changes at gestational day 21 in morphological development of the cerebellum in the fetuses with increased brain T3 levels or in fetuses made hypothyroid by methimazole treatment from day 14 of gestation. These findings suggest that yet-to-be defined cellular processes render the cells responsive to thyroid hormone action in the postnatal period. Moreover, in rat fetuses treated with excess T3 or with methimazole only in the last 6 days of gestation, there was no effect on brain weight, DNA, RNA, or protein content (151). However, it is important to emphasize again the importance of the differences in the developmental patterns of rat, sheep, and human. As pointed out previously, the T3-sensitive postnatal period of brain development in the rat occurs during intrauterine gestation in humans and sheep. Thus, alterations in the thyroid status of the human fetus during the latter half of pregnancy may have important developmental ramifications. In the sheep, thyroidectomy in utero leads to major deficiencies in morphogenesis (16, 17).
Barres et al. (152) have pointed out that proliferating oligodendrocyte precursors, O-2A cells, require antecedent development before they are susceptible to the differentiating effects of T3. In the case of the O-2A precursors, sensitivity to T3 is established only after the cells undergo at least several cycles of division. The changes we have observed in the developing brain may well fit an analogous pattern.
The nature of the developmental factors that allow target genes to become sensitive to the action of T3 remains unclear. One could postulate the developmental expression of a cofactor required for thyroid hormone effectiveness. Alternatively, suppressor mechanisms could operate in the fetal brain to prevent thyroid hormone-induced effects. Preliminary studies have identified one of these suppressor proteins as the orphan receptor, COUF-TF. Gel shift studies in our laboratory (158) have revealed nuclear proteins that bind to the upstream promoter region of the T3 target genes, Pcp-2 and MBP. These proteins rapidly disappear at about the same time that these genes become sensitive to the action of thyroid hormone.
The mechanism responsible for the later ligand-independent expression of genes that are responsive to T3 early in the postnatal period remains unclear. Studies of the MBP promoter transfected into cultured oligodendrocytes showed that the ligand-independent effect is abolished when the TRE is mutated (136). This raises the possibility that the late-phase MBP gene may be induced by the unliganded receptor or another transacting factor capable of binding to the TRE.
In this context, it is of interest to contrast these findings with the role of thyroid hormone in amphibian metamorphosis (reviewed in 153 . In the rat, differentiation, however defective, continues despite delayed growth in the absence of T3. Moreover, there is no evidence for a premature response of brain target genes to the early administration of T3 (151). To the contrary, Gudernatsch (154, 155) first showed that premature administration of thyroid hormone to tadpoles caused them to undergo early metamorphosis. Although thyroidectomy blocks metamorphosis, tadpoles continue to grow to an extremely large size (156, 157). It, therefore, appears that there is a fundamental difference in the mechanisms by which T3 affects amphibian metamorphosis and those regulated by T3 in mammalian brain.
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Footnotes |
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1 Supported by Grant AM 19–812 from the NIH. 
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Pcp-2-CAT)
responded to T3 with a several fold greater transcription
rate. Fold induction is defined as the transcription rate in the
presence of T3 relative to that in the absence of
T3.