Cystic fibrosis as a bowel cancer syndrome and the potential role of CK2

Thyroid hormones are major regulators of postnatal brain development. Thyroid hormones act through nuclear receptors to modulate the expression of specific genes in the brain. We have used microarray analysis to identify novel responsive genes in 14-day-old hypothyroid rat brains, and discovered that synaptosomal-associated protein of 25 kDa (SNAP-25) was one of the thyroid hormone-responsive genes. SNAP-25 is a presynaptic plasma membrane protein and an integral component of the vesicle docking and fusion machinery mediating secretion of neurotransmitters and is required for neuritic outgrowth and synaptogenesis. Using microarray analysis we have shown that SNAP-25 was down-regulated in the hypothyroid rat brain compared with the age-matched controls. Real-time RT-PCR and western blotting analysis confirmed that SNAP-25 mRNA and protein levels decreased significantly in the developing hypothyroid rat brain. Our data suggest that in the developing rat brain, SNAP-25 expression is regulated by thyroid hormone, and thyroid hormone deficiency can cause decreased expression of SNAP-25 and this may on some level account for the impaired brain development seen in hypothyroidism.     

Regulation of Myelin Basic Protein (Arginine) Methyltransferase by Thyroid Hormone in Myelinogenic Cultures of Cells Dissociated from Embryonic Mouse Brain

Abstract: The ontogenetic expression of myelin basic protein (arginine) methyltransferase in myelinogenic cultures of cells dissociated from embryonic mouse brain is highly dependent on the presence of thyroid hormone. Restoration of myelin basic protein methyltransferase to normal activities occurred 16 h after the addition of 100 n M l -3,5,3'-triiodothyronine to hypothyroid medium. These data demonstrate that thyroid hormone can regulate a posttranslational event. On the other hand, histone (arginine) methyltransferase has a different temporal activity pattern, which is not coordinated with myelination, and is not influenced by the lack of thyroid hormone. These data, which suggest the existence of two methyltransferases, were substantiated by demonstrating that the total amount of methylation of added myelin basic protein and histone is the same whether they are incubated together or separately. The requirement of thyroid hormone for the expression of the myelin basic protein methyltransferase and not for histone methyltransferase suggests that thyroid hormone preferentially regulates myelin-associated events in these cultures.     

Differential thyroid hormone-regulated rat thyrotropin beta gene expression detected by blot hybridization

In the rat, there is a single TSH beta-subunit gene represented by three exons interrupted by two introns. This gene contains two promoters which determines the synthesis of two mRNAs with 5'-untranslated regions that differ by 43 base pairs. This study evaluates the steady state levels of these TSH beta mRNAs in various thyroidal states. Blot hybridization analyses of pituitary mRNA with synthetic probes designed to detect either one or both TSH beta mRNAs were performed. One probe corresponds to 24 bases in the 5'-untranslated region of mRNA1 and a second corresponds to 25 nucleotides in the coding region and detects both mRNA1 and mRNA2. These studies indicate the presence of TSH beta mRNA species of indistinguishable size consistent with the presence of two TSH beta mRNAs that contain slightly different 5'-untranslated regions. Comparison of pituitary RNA obtained from normal and hypothyroid rats reveals that the shorter mRNA (mRNA2) is increased approximately 6- to 8-fold with hypothyroidism while the abundance of the longer mRNA (mRNA1) is relatively unchanged. Treatment of either normal or hypothyroid animals with T3 decreases the abundance of mRNA2 while again mRNA1 is relatively unaffected. Thus, although both mRNAs are detected, only one mRNA is dramatically altered by thyroidal status. Therefore, the single rat TSH beta gene is transcribed into two mRNAs via the use of alternative promoters of which only one is markedly regulated by thyroid hormones.     

Alterations in mRNA Expression of Myelin Proteins in the Sciatic Nerves and Brains of Streptozotocin-induced Diabetic Rats

Diabetic neuropathy is the most common complication of diabetes. We examined the levels and the mRNA expression of myelin proteins in the sciatic nerves and the brains of streptozotocin-induced diabetic rats. The diabetic rats exhibited a decrease in body weight, elevation of the blood glucose level and a decrease in motor nerve conduction velocity at 2 weeks after streptozotocin injection. In the sciatic nerves of diabetic rats, the level of P0 protein and its mRNA expression were markedly reduced at 20 weeks after the injection. In the brains, the levels of proteolipid protein and myelin-associated glycoprotein and their mRNA expression were selectively decreased at 20 weeks after the injection. This affected expression of myelin proteins was found even when no histological abnormalities were detectable. Considering the functional significance of myelin proteins, this impairment of protein expression is possibly involved in the pathogenesis of diabetic neuropathy, including that in brain disorders.     

Identification of a mammalian homologue of the fungal Tom70 mitochondrial precursor protein import receptor as a thyroid hormone-regulated gene in specific brain regions

Thyroid hormone is an important regulator of mammalian brain maturation. By differential display PCR, we isolated a cDNA clone (S2) that is specifically up-regulated in the striatum of neonatal hypothyroid rats. S2 was identified as KIAA0719, the first human gene distantly homologous to the fungal Tom70, which encodes a member of the translocase mitochondrial outer membrane complex involved in the import of preproteins into the mitochondria. By northern and in situ hybridization studies, KIAA0719 was found to be up-regulated in the striatum, nucleus accumbens, and discrete cortical layers of 15-day-old hypothyroid rats. In contrast, lower expression was found in the olfactory tubercle, whereas no differences were detected in other brain regions. Significantly, treatment of hypothyroid animals with single injections of thyroxine restored the normal levels of KIAA0719 expression. Moreover, treatment of control animals with thyroxine led to a reduced expression, demonstrating a negative hormonal regulation in vivo. Thus, KIAA0719 gene expression is regulated by thyroid hormone in the neonatal rat brain in a region-specific fashion. Given the role of the homologous Tom70 gene, the alteration of KIAA0719 expression may contribute to the changes in mitochondrial morphology and physiology caused by hypothyroidism in the developing rat brain.     

Prenatal exposure to thyroid hormone is necessary for normal postnatal development of murine heart and lungs

Maternal hypothyroxinemia during early pregnancy poses an increased risk for poor neuropsychological development of the fetus. We tested the hypothesis that maternal hypothyroidism before the onset of fetal thyroid function also affects postnatal development of heart and lungs. This question was addressed in transgenic mice that express herpes simplex virus thymidine kinase in their thyroidal follicle cells. Treatment with ganciclovir rendered these mice severely hypothyroid because viral thymidine kinase converts ganciclovir into a cytotoxic nucleoside analog. Since ganciclovir crosses the placenta, it also destroyed the thyroid of transgenic embryos while leaving the thyroids of nontransgenic littermates unaffected. Hypothyroidism of both mother and fetus did not affect prenatal heart and lung development. However, the postnatal switch from beta- to alpha-myosin heavy chain (beta- and alpha-MHC, respectively) gene expression and the increase of SERCA-2a mRNA expression did not occur in the ventricular myocardium of either the transgenic (thyroid destroyed) or nontransgenic (intact thyroid) offspring of hypothyroid mothers. Similarly, postnatal animals of the latter two groups retained elevated surfactant protein (SP) A, B, and C mRNA levels in their alveolar epithelium. In hypothyroid pups from hypothyroid mothers, these changes were accompanied by decreased alveolar septation. Our study shows that these effects of maternal hypothyroidism become manifest after birth and are aggravated by the concomitant existence of neonatal hypothyroidism.     

Hypothyroidism induces expression of the peptide transporter PEPT2

The kidney is a target organ for thyroid hormone action and a variety of renal transport processes are altered in response to impaired thyroid functions. To investigate the effect of thyroid hormone on the expression of the renal proximal tubular high-affinity-type H(+)-peptide cotransporter (PEPT2) in rats, hypothyroidism was induced in animals by administration of methimazole (0.05%) via drinking water. After 7 weeks of treatment, hypothyroidism was confirmed by determining serum free T(3) and free T(4) concentrations. Northern blotting was used to examine the expression of PEPT2 mRNA in kidney tissues from hypothyroid rats compared to control rats. Hypothyroidism resulted in an increased level of total renal PEPT2 mRNA (121.1+/-3.3% vs. control 100+/-2.8%; p=0.008). The mRNA results were confirmed by immuno-blotting, which demonstrated significantly increased protein levels (162% vs. control 100%; p<0.01). Immunohistochemistry also revealed increased PEPT2 protein levels in the proximal tubules of treated compared to non-treated rats. In summary, PEPT2 is the first proximal tubule transporter protein that shows increased expression in states of hypothyreosis. As PEPT2 reabsorbs filtered di- and tripeptides and peptide-like drugs, the present findings may have important implications in nutritional amino acid homeostasis and for drug dynamics in states of altered thyroid function.     

Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain

The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA⁺RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T₃-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T₄ intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005)     

Myelin-Associated Oligodendrocytic Basic Protein mRNAs Reside at Different Subcellular Locations

The mRNAs for two myelin proteins, myelin basic protein (MBP) and myelin-associated oligodendrocytic basic protein (MOBP)-81A, are uniquely located at sites where myelin sheaths are assembled. Here, we use subcellular fractionation to show that four MOBP mRNAs, like MBP mRNA, are located at sites of myelin sheath assembly, and that three other MOBP mRNAs are located in oligodendrocyte soma. The MOBP-81 protein is found in myelin and in another subcellular fraction, whereas other myelin proteins, including MBP, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin-associated glycoprotein, are largely restricted to myelin. Different MBP mRNAs are generated by alternative splicing. All of them contain an RNA transport sequence (RTS) that directs them to sites in oligodendrocytes, where myelin sheaths are assembled. Consequently, all are enriched in myelin. After fractionation, four MOBP mRNAs, MOBP-71, MOBP-81A, MOBP-99, and MOBP-169 (identified in this study), are enriched in myelin. These mRNAs contain a common exon, exon 8b, which has a nucleotide sequence that is similar to MBP mRNA RTS. This sequence likely directs these mRNAs to sites of myelin sheath assembly. Three other MOBP mRNAs, MOBP-69, MOBP-81B, and MOBP-170, lack this exon. Their subcellular distribution indicates that they are largely retained in oligodendrocyte soma. We conclude that the distribution of MOBPs in oligodendrocytes is strongly influenced by alternative splicing of the corresponding mRNAs.     

Myelin genes are downregulated in canine fucosidosis

The processes regulating the complex neurodegenerative cascade of vacuolation, neuroinflammation, neuronal loss and myelin deficits in fucosidosis, a neurological lysosomal storage disorder, remain unclear. To elucidate these processes the gene expression profile of the cerebral cortex from untreated and intrathecal enzyme replacement therapy treated fucosidosis pups and age-matched unaffected controls were examined. Neuroinflammation and cell death processes were identified to have a major role in fucosidosis pathophysiology with 37% of differentially expressed (DE) genes involved in these processes. Critical, specific, early decreases in expression levels of key genes in myelin assembly were identified by gene expression profiling, including myelin-associated glycoprotein (MAG), myelin and lymphocyte protein (MAL), and oligodendrocyte myelin paranodal and inner loop protein (OPALIN). These gene expression changes may be indicative of early neuronal loss causing reduced electrical impulses required for oligodendrocyte maturation.     

Influence of thyroid hormone on the in vitro translational activity of specific mRNAs in the rat heart

The influence of thyroid hormone on the translational activity of specific cardiac mRNA was determined by in vitro translation of RNA isolated from the heart of normal, hypothyroid, and 3,3',5-triiodo-L-thyronine-injected hypothyroid rats. Proteins synthesized in vitro in the presence of [35S]methionine were separated by two-dimensional gel electrophoresis and quantitated by a novel scanning procedure using digital matrix photometry. A total of 421 translational products were detected by fluorography and changes in the predominance of 12 of these were influenced by the thyroid state of the animals. The relative predominance of 8 species was increased in euthyroid animals, whereas 4 translational products were increased in hypothyroid animals. The majority of these thyroid hormone-related alterations occurred in spot pairs of similar molecular weights, but slightly different isoelectric points. In contrast, the relative predominance of mRNAs coding for the major contractile proteins, light chain 1, light chain 2, tropomyosin, actin, and myosin heavy chain was not altered by the thyroid status of the animals. The relative levels of these abundant mRNA species remained unaltered in spite of a thyroid hormone-related increase in total RNA levels. In vivo effects of thyroid hormone on cardiac RNA levels are complex. In addition to a general increase in total RNA and mRNA levels, increases or attenuations in the predominance of a small number of specific mRNA species are observed when euthyroid and hypothyroid animals are compared.     

Ablation of Neuronal Ceramide Synthase 1 in Mice Decreases Ganglioside Levels and Expression of Myelin-associated Glycoprotein in Oligodendrocytes

Ceramide synthase 1 (CerS1) catalyzes the synthesis of C18 ceramide and is mainly expressed in the brain. Custom-made antibodies to a peptide from the C-terminal region of the mouse CerS1 protein yielded specific immunosignals in neurons but no other cell types of wild type brain, but the CerS1 protein was not detected in CerS1-deficient mouse brains. To elucidate the biological function of CerS1-derived sphingolipids in the brain, we generated CerS1-deficient mice by introducing a targeted mutation into the coding region of the cers1 gene. General deficiency of CerS1 in mice caused a foliation defect, progressive shrinkage, and neuronal apoptosis in the cerebellum. Mass spectrometric analyses revealed up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of myelin-associated glycoprotein was also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic myelin-associated glycoprotein by neuronal gangliosides is due to the C18 acyl membrane anchor of CerS1-derived precursor ceramides. A behavioral analysis of CerS1-deficient mice yielded functional deficits including impaired exploration of novel objects, locomotion, and motor coordination. Our results reveal an essential function of CerS1-derived ceramide in the regulation of cerebellar development and neurodevelopmentally regulated behavior.