Role of Thyroid Hormone and Nerve Growth Factor in the Development of Choline Acetyltransferase and Other Cell-Specific Marker Enzymes in the Basal Forebrain of the Rat

The effects of treatment with L-thyroxine (subcutaneously 0.3 microgram/g body weight daily from birth, i.e., day 1) and 2.5S nerve growth factor (NGF; intraventricularly 2 micrograms on 1, 3, 5, 7, and 9 postnatal days), separately and together, were studied on the biochemical development of different cell types in the basal forebrain of 10-day-old rats. The development of cholinergic, gamma-aminobutyric acid-ergic (GABAergic), and glutamatergic neurons was monitored respectively in terms of choline acetyltransferase (ChAT), glutamate decarboxylase (GAD), and glutaminase activities, whereas glutamine synthetase (GS) and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase) activities were used to judge the maturation of astroglial and oligodendroglial cells. Treatment with either thyroid hormone or NGF from birth significantly increased the expression of ChAT activity in the basal forebrain of neonatal rats. When both agents were administered to the same animal, in agreement with our earlier in vitro findings, the stimulation in ChAT activity was much greater than the sum of the individual effects. In hypothyroid rats, significant effects of NGF at the low doses used were not detectable, although the increase of ChAT activity induced by thyroxine was potentiated by NGF in these animals. Under the present experimental conditions neither thyroxine nor NGF treatment had an appreciable effect on the activities of glutaminase, GS, and lactate dehydrogenase. However, the administration of thyroxine markedly increased CNPase activity in normal rats, whereas in hypothyroid rats the effect on both CNPase and GAD was also significant. Similar elevations in CNPase and GAD activities were not observed after NGF treatment, suggesting that the effect of NGF was specific to the cholinergic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of neonatal hypothyroidism on rat brain gene expression.

To define at the molecular biological level the effects of thyroid hormone on brain development we have examined cDNA clones of brain mRNAs and identified several whose expression is altered in hypothyroid animals during the neonatal period. Clones were identified with probes prepared by subtractive or differential hybridization, and those corresponding to mRNAs altered in hypothyroidism were further studied by Northern blot analysis. Using RNA prepared from whole brains, no effect of hypothyroidism was found on the expression of the astroglial gene coding for glial fibrillary acidic protein. Among genes of neuronal expression, no significant alterations were found in the steady state levels of mRNAs coding for neuron-specific enolase, microtubule-associated protein-2, Tau, or nerve growth factor. N-CAM mRNA increased slightly in hypothyroid brains. In contrast a 2- to 3-fold decrease was found in the mRNA coding for a novel neuronal gene, RC3. This is the first neuronal gene known to be significantly altered at the mRNA level by thyroid hormone deprivation. The abundance of the mRNAs for the major myelin proteins proteolipid protein, myelin basic protein, and myelin-associated glycoprotein, expressed by oligodendrocytes, were also decreased in hypothyroid brains. Developmental studies on RC3 and myelin-associated glycoprotein expression indicated that the corresponding mRNAs accumulate in the brain of normal rats during the first 15-20 days of neonatal life. A similar accumulation occurred in hypothyroid brains, but at much reduced levels. The results demonstrate that thyroid hormone controls the steady state levels of particular mRNAs during brain development.     

Morphological and morphometric studies of early myelination in the cervical dorsal funiculus of the newborn mouse spinal cord

The early myelination of the dorsal funiculus at the level of the 4th cervical spinal cord was ultrastructurally studied in the one-day-old mouse. It was found that the fibers were mainly unmyelinated. However, some early myelinated fibers were scattered among unmyelinated fibers. In the initial stage of the myelination, the axon was partially contracted by a piece of cytoplasmic process of the oligodendroglial cell. The two lips of the oligodendroglial process then extended and converged, enwrapping the axon completely and forming the first contact point. With the anchorage of that contact point, the two lips of the process became elongated and enfolded by each other, and produced the internal and external tongues of the future myelin sheath. More contact points were formed at a regular interval by the regional fusion of the two external surface layers of the opposed cytoplasmic membranes of adjacent tongue processes. With the advanced bidirectional spiralization of the two tongue processes, many contact points were found between the adjacent lamellae of the concentrically arranged oligodendroglial process; simultaneously, the cleft between the neighboring contact points disappeared and formed the initial sites of the intraperiod line. During the early myelination, one single axon ensheathed concentrically by two different oligodendroglial processes as well as several axons enwrapped by a continuous spiral myelin sheath of one oligodendroglial cell were frequently observed. The cross-sectional areas of unmyelinated axons varied from 0.01 to 0.2 micron 2, with a median of 0.07 micron 2; whereas, that of promyelinated axons ranged from 0.09 to 1.4 micron 2, with a median at 0.61 micron 2. These data support the suggestion that the axon calibre is a critical factor for the initiation of central myelination.     

Simultaneous inhibition of guinea pig brain 2′, 3′-cyclic nucleotide 3′-phosphohydrolase and myelin protein synthesis by 2′-adenosine monophosphate

Although the function of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) in myelin is unknown, the enzyme has been implicated in the metabolism of myelin proteins. Using 2′-AMP to inhibit CNPase, we examined the effect of reduced enzyme activity on the $\text{in vitro}$ incorporation of ¹⁴C-leucine into brain proteins. The results of this study revealed that (1) guinea pig brain homogenates incorporate leucine into protein from a sucrose medium in a linear fashion, (2) all brain fractions (cytosol, myelin, and microsomes) are labelled within 1 hr, (3) 2′-AMP inhibition of CNPase by 50% results in a similar inhibition of brain protein synthesis, and (4) the reduced protein synthesis is accompanied by a shift in label from myelin proteins to those found in the microsomes. These results are consistent with a role for CNPase in myelin protein synthesis.     

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.     

Myelin abnormalities in mice deficient in galactocerebroside and sulfatide

Myelin sheath formation depends on appropriate axo-glial interactions that are mediated by myelin-specific surface molecules. In this study, we have used quantitative morphological analysis to determine the roles of the prominent myelin lipids galactocerebroside (GalC) and sulfatide in both central and peripheral myelin formation, exploiting mutant mice incapable of synthesizing these lipids. Our results demonstrate a significant increase in uncompacted myelin sheaths, the frequency of multiple cytoplasmic loops, redundant myelin profiles, and Schmidt-Lanterman incisures in the CNS of these mutant mice. In contrast, PNS myelin appeared structurally normal in these animals; however, at post-natal day 10, greater than 10% of the axons withered and pulled away from their myelin sheaths. These results indicate that GalC and sulfatide are critical to the formation of CNS myelin. In contrast, PNS myelin formation is not dependent on these lipids; however, GalC and sulfatide appear to be instrumental in maintaining Schwann cell-axon contact during a specific developmental window.     

Thyroid hormone stimulates rat pro-natriodilatin mRNA levels in primary cardiocyte cultures

Pro-natriodilatin (PND) is the precursor for atrial natriuretic peptide (ANP), a hormone which plays an important role in cardiovascular homeostasis. Since the effects of thyroid hormone (T3) on the cardiovascular and renal systems appear to mimic those elicited by ANP, we studied the effect of T3 on PND gene expression using rat neonatal cardiocytes in primary cultures. Treatment of cardiocytes for 48 h with T3 (5 X 10(-9) M) results in a maximal increase in PND mRNA levels; this increase is two fold in atrial and four fold in ventricular cell cultures. These results taken together with a previous report showing decreased plasma ANP in hypothyroid and increased plasma ANP in hyperthyroid rats suggest that at least some of the cardiovascular and renal effects of T3 may be mediated by a T3-dependent increase in PND gene expression.     

Galactosphingolipids and axono-glial interaction in myelin of the central nervous system

The myelin of central and peripheral nervous system of UDP-galactose-ceramide galactosyltransferase deficient mice (cgt ^-/-) is completely depleted of its major lipid constituents, galactocerebrosides and sulfatides. The deficiency of these glycolipids affects the biophysical properties of the myelin sheath and causes the loss of the rapid saltatory conduction velocity of myelinated axons. With the onset of myelination, null mutant cgt ^-/- mice develop fatal neurological defects. CNS and PNS analysis of cgt ^-/- mice revealed (1) hypomyelination of axons of the spinal cord and optic nerves, but no apoptosis of oligodendrocytes, (2) redundant myelin in younger mice leading to vacuolated nerve fibers in cgt ^-/- mice, (3) the occurrence of multiple myelinated CNS axons, and (4) severely distorted lateral loops in CNS paranodes. The loss of saltatory conduction is not associated with a randomization of voltage-gated sodium channels in the axolemma of PNS fibers. We conclude that cerebrosides (GalC) and sulfatides (sGalC) play a major role in CNS axono-glial interaction. A close axono-glial contact is not a prerequisite for the spiraling and compaction process of myelin. Axonal sodium channels remain clustered at the nodes of Ranvier independent of the change in the physical properties of myelin membrane devoid of galactosphingolipids. Increased intracellular concentrations of free ceramides do not trigger apoptosis of oligodendrocytes.     

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in cat peripheral nerve

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) in desheathed, saline perfused cat sciatic nerve is reported. CNPase specific activity was enriched in the total particulate (P₂) fraction and was low in the soluble (S₂) fraction. “Light-myelin” floating above the 0.60 M sucrose phase had the highest CNPase activity, 2.5-fold over the crude homogenate (CH). By contrast, enzyme activity in “heavy myelin” floating above the 0.85 M sucrose interface was equal to that of the CH and accounted for only 12% of total activity. CNPase activity in the membranes floating above the 0.25 and 0.60 and 0.85 M sucrose phases comprised nearly 70% of the total CNPase activity. The “light myelin” fraction floating above the 0.60 M sucrose accounted for approx. 51% of the total CNPase activity. SDS-PAGE of membranes individually harvested from above the 0.25 and 0.60 and 0.85 M sucrose phases revealed the presence of myelin-specific proteins (P₀, P₁; and P₂). Electron microscope examination demonstrated the presence of myelin in each membrane fraction. The results of this study show that the majority of CNPase activity is associated with “light myelin” in cat peripheral nerve.     

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.     

Effect of thyroid hormone and sex status on epidermal growth factor concentrations in the submandibular gland of a congenitally hypothyroid mouse model

Studies were performed to explore the role of thyroid hormone and sex status on epidermal growth factor concentrations in the submandibular gland of a congenitally hypothyroid mouse model designated hyt/hyt. The animals were studied at 20, 30 and 40 days of postnatal age. The euthyroid animals were homozygous or heterozygous for the hypothyroid gene. The homozygous euthyroid animals displayed a pattern of submandibular gland epidermal growth factor concentrations similar to those previously described in other mouse species and showed the expected sex differences. The hypothyroid animals had measurable but very low submandibular gland epidermal growth factor concentrations without sexual dimorphism. Serum thyroxine concentrations in the heterozygotes were comparable to those in the homozygous euthyroid animals, yet the animals had a delayed increase in epidermal growth factor concentrations combined with a later expression of female-male differences. The timing of the sex differences in submandibular gland epidermal growth factor concentrations followed a pattern similar to that seen for the timing of the first litter in these three genetically distinct groups. This infers the timing of the onset of puberty and suggests a role of androgens in the changes seen in epidermal growth factor concentrations. We conclude that thyroid hormone and sex status in this mouse model influence the pattern and concentrations of submandibular gland epidermal growth factor concentrations.     

Thyroid hormone regulation of flavocoenzyme biosynthesis

The means by which thyroid hormone regulates flavocoenzyme biosynthesis was studied in hyper-, eu-, and hypothyroid rats by determining the activities of flavocoenzyme-forming enzymes, viz., flavokinase and FAD synthetase, as well as those of flavocoenzyme-degrading enzymes, viz., FMN phosphatase and FAD pyrophosphatase. Flavokinase activity was increased in hyperthyroid animal and decreased in hypothyroid animals. Correspondence of flavokinase activity with the amount of a high-affinity flavin-binding protein quantitated immunologically in hypo-, eu-, and hyperthyroid rats indicated that the thyroid response is caused by an increased amount of enzyme; moreover, the concomitant decrease in a low-affinity flavin-binding protein suggests an inactive precursor form of flavokinase. FAD synthetase activity showed a similar but less pronounced trend than flavokinase. Activities of FMN phosphatase and FAD pyrophosphatase were not influenced by thyroid hormone. Overall results indicate that the mechanism of thyroid hormone regulation of flavocoenzyme level is in the steps of biosynthesis, especially at flavokinase, rather than in degradation steps.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

The activity of 2'',3''-cyclic nucleotide 3''-phosphohydrolase in the corpus callosum, subcortical white matter, and spinal cord in infants dying from sudden infant death syndrome

Abstract: The activity of 2',3'-cyclic nucleotide 3'-phos-phohydrolase (CNPase) has been determined in corpus callosum, subcortical white matter, and spinal cord of infants whose death was attributed to the sudden infant death syndrome (SIDS), and compared with enzyme activity in other cases in which the cause of death was not associated with respiratory distress. In nearly half the SIDS cases, CNPase activity and oligodendroglial cell numbers were reduced before the onset of myelination, but only in the corpus callosum. In other SIDS cases, enzyme activity and cell numbers were the same as in non-SIDS cases. If the expression of CNPase activity reflects glioblast differentiation to oligodendrocytes with myelinating potential, then this transformation is abnormal in certain SIDS cases, as also evidenced in cases of prolonged neonatal respiratory insufficiency and gives rise to a subsequent deficit of myelin in the corpus callosum.     

Hippocampal Up-Regulation of Apolipoprotein D in a Rat Model of Maternal Hypo- and Hyperthyroidism: Implication of Oxidative Stress

Thyroid disorders impair various functions of the hippocampus where thyroid hormone receptors are localized in the brain. Hyper and hypothyroidism are associated with large changes in brain oxidative stress. Apolipoprotein D (APOD) is a conserved glycoprotein that increased in response to oxidative stress in the brain and has been suggested function as an antioxidant in the brain. Thus, the goal of this work was to explore the effect of maternal hypo- and hyperthyroidism on the Apod expression in the pup’s brain regarding changes in oxidative stress. For induction hypo and hyperthyroidism in adult female rats, 100 ppm propylthiouracil (PTU) and 8 ppm levothyroxine administrated 1 month before copulation to the week 3 after delivery in drinking water. The hippocampal region of rat pups was isolated and used for immunohistochemistry and quantitative RT-PCR on postnatal day (PND)5, PND10 and PND20. Results revealed that APOD over-expressed in both hypo- and hyperthyroid groups on PND5, PND10, and PND20. There was a proportional increase between the Apod expression and oxidative stress in the hyperthyroid group but not the hypothyroid in different days. Regarding the wide functions of thyroid hormones, oxidative stress does not suggest to be the only mechanism that involves Apod gene expression in thyroid disturbances.

     

Proliferation of bone marrow pro-B cells is dependent on stimulation by the pituitary/thyroid axis

The frequency and absolute number of pro-B, pre-B, and B cells in the bone marrow of the hypothyroid strain of mice are significantly reduced compared with those of their normal littermates. To investigate why this is the case, various B cell developmental processes were examined in the thyroid hormone-deficient mice. These studies revealed that the frequency of pro-B cells in the S-G2/M phase of the cell cycle was significantly reduced in hypothyroid mice. That thyroid hormone deficiency was responsible for this proliferation defect was established by demonstrating that treatment of hypothyroid mice with thyroxine resulted in a specific increase in the frequency and total number of cycling pro-B cells. The latter effect was paralleled by increases in the frequency and number of bone marrow B lineage cells. Additional in vitro experiments revealed that at least some thyroid hormone effects were directly mediated on the bone marrow. Taken together, these data demonstrate that thyroid hormones are required for normal B cell production in the bone marrow through regulation of pro-B cell proliferation and establish a role for the pituitary/thyroid axis in B cell development.