Influence of thyroid hormone and thyroid hormone receptors in the generation of cerebellar gamma-aminobutyric acid-ergic interneurons from precursor cells.

Thyroid hormones have important actions in the developing central nervous system. We describe here a novel action of thyroid hormone and its nuclear receptors on maturation of cerebellar gamma-aminobutyric acid (GABA)-ergic interneurons from their precursor cells. In rats, the density of GABAergic terminals in the cerebellum was decreased by hypothyroidism, as shown by immunohistochemistry for the GABA transporter GAT-1. This was due, at least partially, to a decreased number of GABAergic cells, because the number of Golgi II cells in the internal granular layer was decreased. GABAergic interneurons in the cerebellum differentiate from precursors expressing the Pax-2 transcription factor, generated in the subventricular zone of the embryonic fourth ventricle from where they migrate to the cerebellum. Hypothyroidism caused both decreased proliferation and delayed differentiation of precursors, with the net effect being an accumulation of immature cells during the neonatal period. The contribution of thyroid hormone receptors was studied by treating hypothyroid rats with T(3) or with the thyroid hormone receptor (TR) beta-selective agonist GC-1. Whereas treatment with T(3) reduced the number of precursors to control levels, GC-1 had only a partial effect, indicating that both TRalpha1 and TRbeta mediate the actions of T(3). Deletion of TRalpha1 in mice decreased cerebellar GAT-1 expression and Pax-2 precursor cell proliferation. It is concluded that thyroid hormone, acting through the nuclear receptors, has a major role in the proliferation and further differentiation of the Pax-2 precursors of cerebellar GABAergic cells.

Influence of thyroid hormones on maturation of rat cerebellar astrocytes.

Thyroid hormone influences brain maturation through interaction with nuclear receptors and regulation of gene expression. Their role on astrocyte maturation remains unclear. We have analyzed the role of thyroid hormone in rat cerebellar astrocyte maturation by comparing the sequential patterns of intermediate filament expression in normal and hypothyroid animals. During normal development astroglial cells sequentially express nestin, vimentin, and glial fibrillary acidic protein. Differentiated astrocytes appeared in the superior medullary vellum by postnatal day 2 and reached the white mater and internal granular layer by postnatal day 4. Intermediate filament marker expression was transiently lost from postnatal days 6 to 8 in anterior lobes, without an increased apoptosis. Vimentin expression was replaced by glial fibrillary acidic protein between postnatal days 10 and 32. The differentiated astrocytes were evenly distributed throughout the cerebellar slices, including the internal granular layer. Differences between normal and hypothyroid rats were observed starting from postnatal day 4, with lack of differentiated astrocytes in the internal granular layer. The transient decrease of astrocyte markers immunoreactivity in the anterior lobe did not take place in hypothyroid rats. The vimentin-glial fibrillary acidic protein transition was delayed and most differentiated astrocytes remained confined to the white matter. The results indicate that thyroid hormone deficiency induces a delay and a partial arrest of astrocyte differentiation. Astrocytes express thyroid hormone receptor alpha and beta subtypes suggesting that astrocytes are direct target cells of thyroid hormones.

Anxiety, memory impairment, and locomotor dysfunction caused by a mutant thyroid hormone receptor alpha1 can be ameliorated by T3 treatment.

The transcriptional properties of unliganded thyroid hormone receptors are thought to cause the misdevelopment during hypothyroidism of several functions essential for adult life. To specifically determine the role of unliganded thyroid hormone receptor alpha1 (TRalpha1) in neuronal tissues, we introduced a mutation into the mouse TRalpha1 gene that lowers affinity to thyroid hormone (TH) 10-fold. The resulting heterozygous mice exhibit several distinct neurological abnormalities: extreme anxiety, reduced recognition memory, and locomotor dysfunction. The anxiety and memory deficiencies were relieved by treatment with high levels of TH in adulthood, an effect that correlated with a normalization of GABAergic inhibitory interneurons in the hippocampal CA1 region. In contrast, a post-natal TH treatment was necessary and sufficient for ameliorating the adult locomotor dysfunction. Here, the hormone treatment normalized the otherwise delayed cerebellar development. The data thus identify two novel and distinct functions of an unliganded TRalpha1 during development and adulthood, respectively.

Aberrant maturation of astrocytes in thyroid hormone receptor alpha 1 knockout mice reveals an interplay between thyroid hormone receptor isoforms.

Although the effects of thyroid hormones on the development of neurons and oligodendrocytes are well documented, less is known about the hormonal effects on astrocytes. Our analyses of cerebellar slices from 2-month-old T(3) receptor protein (TR)alpha1-deficient mice show that mature astrocytes, Golgi epithelial cells, and their Bergmann processes had strongly reduced glial fibrillary acidic protein (GFAP) and nestin immunoreactivity, in contrast to wild-type mice. Furthermore, the Bergmann processes exhibited an irregular GFAP staining. A similar expression of nestin and GFAP was observed in 11-d-old (P11) mutant pups. Surprisingly, however, hypothyroidism normalized the appearance of these markers in the P11 mutants, suggesting that liganded TR beta is detrimental to astroglial cell differentiation in the absence of TR alpha 1. To test this hypothesis, hypothyroid mice were treated from birth until P11 with the TR beta-selective ligand GC-1. This treatment was devastating in the TR alpha 1(-/-) mice, causing little if any nestin or GFAP immunoreactivity, whereas the wild-type mice were normal. The results thus indicate an important interplay between thyroid hormone receptor isoforms in astroglial cell maturation.

Differential effects of triiodothyronine and the thyroid hormone receptor beta-specific agonist GC-1 on thyroid hormone target genes in the b ain.

The availability of synthetic thyroid hormone receptor agonists provides a valuable tool to analyze whether specific receptor isoforms mediate specific physiological responses to thyroid hormone. GC-1 is a thyroid hormone analog displaying selectivity for thyroid hormone receptor beta. We have analyzed the effect of GC-1 on expression of thyroid hormone target genes in the cerebrum and cerebellum. Congenitally hypothyroid rats were treated with single daily doses of either T3 or GC-1. Both compounds similarly induced Purkinje cell protein-2 (PCP-2) in the cerebellum. Expression of RC3 and Rhes in the caudate, and hairless, neurotrophin-3, Reelin, and Rev-ErbAalpha in the cerebellum, was analyzed by in situ hybridization on postnatal d 16. Hypothyroidism strongly decreased expression of RC3 and Rhes in the caudate, and hairless, Rev-ErbAalpha, and neurotrophin-3 in the cerebellum, and increased Reelin. T3 treatment normalized the expression of all genes. However, GC-1 effectively normalized expression of Rhes and Reelin only. The lack of a GC-1 effect on most cerebellar genes can be explained by the known distribution of thyroid hormone receptor alpha and beta isoforms. However, in the caudate, RC3 and Rhes are expressed in the same cells, and therefore, they may represent specific gene responses linked to specific thyroid hormone receptor isoforms.

Deletion of the thyroid hormone receptor alpha 1 prevents the structural alterations of the cerebellum induced by hypothyroidism.

Thyroid hormone (T3) controls critical aspects of cerebellar development, such as migration of postmitotic granule cells and terminal differentiation of Purkinje cells. T3 acts through nuclear receptors (TR) of two types, TRalpha1 and TRbeta, that either repress or activate gene expression. We have analyzed the cerebellar structure of developing mice lacking the TRalpha1 isoform, which normally accounts for about 80% of T3 receptors in the cerebellum. Contrary to what was expected, granule cell migration and Purkinje cell differentiation were normal in the mutant mice. Even more striking was the fact that when neonatal hypothyroidism was induced, no alterations in cerebellar structure were observed in the mutant mice, whereas the wild-type mice showed delayed granule cell migration and arrested Purkinje cell growth. The results support the idea that repression by the TRalpha1 aporeceptor, and not the lack of thyroid hormone, is responsible for the hypothyroid phenotype. This conclusion was supported by experiments with the TRbeta-selective compound GC-1. Treatment of hypothyroid animals with T3, which binds to TRalpha1 and TRbeta, prevents any defect in cerebellar structure. In contrast, treatment with GC-1, which binds to TRbeta but not TRalpha1, partially corrects Purkinje cell differentiation but has no effect on granule cell migration. Our data indicate that thyroid hormone has a permissive effect on cerebellar granule cell migration through derepression by the TRalpha1 isoform.