Thyroid hormone (T3) plays a critical role in the development of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically regulated by T3 during brain development are largely unknown. By using a subtractive hybridization technique we have isolated a number of cDNAs that represented mitochondrial genes (12S and 16S rRNAs and cytochrome c oxidase subunit I). The steady state level of all three RNAs was reduced in hypothyroid animals during the postnatal period and T3 administration restored control levels. During fetal life the level of 16S rRNA was decreased in the brain of hypothyroid animals, suggesting a prenatal effect of thyroid hormone on brain development. Since T3 does not affect the amount of mitochondrial DNA, the results suggest that the effect of T3 is at transcriptional and/or postranscriptional level. In addition, the transcript levels for two nuclear-encoded mitochondrial cytochrome c oxidase subunits: subunits IV and VIc were also decreased in the brains of hypothyroid animals. Hypothyroidism-induced changes in mitochondrial RNAs were followed by a concomitant 40% decrease in cytochrome c oxidase activity. This study shows that T3 is an important regulator of mitochondrial function in the neonatal brain and, more importantly, provides a molecular basis for the specific action of this hormone in the developing brain. (J. Clin. Invest. 1995. 96:893-899.)

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We recently demonstrated that thyroid hormone is an important regulator of mitochondrial gene expression during brain development. To gain further insights into the consequences of this regulation, we have performed functional and structural analysis of brain mitochondria from control and hypothyroid neonatal rats. Flow cytometric analysis showed a significant decrease in the mitochondrial transmembrane potential in hypothyroid animals compared with controls, which was reversed after 48 h, but not after 2 h, of thyroid hormone administration, suggesting that the functional alterations observed are the consequence of changes in mitochondrial gene expression. In addition, band shift studies showed a protein bound to the rat mitochondrial promoter differentially regulated by thyroid state. Electron microscopic analysis of cerebral cortex, striatum, and hippocampus revealed marked differences in the morphology of neuronal mitochondria from control and hypothyroid neonates. Hypothyroid mitochondria presented a decrease in the area of the inner membrane plus cristae in all areas studied, except for the hippocampal CA1 neurons and nonneuronal cell types. The observations reported here provide a basis for the known biochemical action of thyroid hormone on brain development.

CCAAT/enhancer binding proteins (C/EBP) are a family of transcription factors with a highly conserved basic/leucine zipper (bZIP) domain that has been implicated in the transcriptional control of genes involved in cell growth and differentiation. We have previously demonstrated that the expression of C/EBPalpha and C/EBPbeta genes is regulated by thyroid hormone in rat liver during development. The aim of the present study was to explore the molecular mechanisms underlying the control of C/EBPalpha gene expression by thyroid hormone. To achieve this goal, we isolated and characterized a genomic clone containing 1171 bp of the 5'-flanking region of the rat C/EBPalpha gene. This fragment was an active promoter in MB492 cells, an immortalized brown adipocyte cell line that expresses the endogenous C/EBPalpha gene in a T3-dependent manner. Sequence analysis suggested the presence of three thyroid hormone response elements, TRE-1 (-602/-589), TRE2 (-411/-396), and TRE3 (-376/-350). The results of deletion, mutagenesis, and gel mobility shift analysis disclosed that only TRE-1, an ER2-type response element, represented a functional T3 response element. Our results demonstrate that T3 is a factor that positively regulates C/EBPalpha gene expression in a direct fashion.