Pranab Kumar Sarkar

Hypothyroidism in the developing rat brain is associated with marked oxidative stress and aberrant intraneuronal accumulation of neurofilaments

The effects of hypothyroidism on parameters of oxidative stress and on intraneuronal distribution of neurofilaments have been investigated in the developing rat brain. Progressive hypothyroidism during the first 4 weeks of postnatal development led to an increase in superoxide dismutase and catalase activity, decline in the level of glutathione and mitochondrial cytochrome c oxidase activity and increase in the level of .OH radical along with enhanced protein carbonylation and lipid peroxidation. Immunocytochemical staining of cryostat sections of normal and hypothyroid cerebella from 25 day postnatal rats with anti neurofilament (NF) light chain (L) antibody showed aberrant accumulation of neurofilaments in the perikaryon of the hypothyroid Purkinje neurons in contrast to relatively uniform distribution in the controls. The morphological and biochemical alterations in the neurons of the developing hypothyroid brain are comparable to those seen in several neurodegenerative diseases.

Regulation of actin and tubulin gene expression by thyroid hormone during rat brain development

In the developing brain the active neurite outgrowth during the early phase of synaptogenesis is associated with a thyroid hormone dependent expression of tubulin and actin. In this study, the molecular mechanism of thyroid hormone (TH) action on actin and tubulin gene expression in the developing rat brain has been investigated by comparing the steady state levels of both mRNAs with their respective rates of transcription in cerebra from normal and hypothyroid animals. The developmental profile of actin as well as tubulin mRNAs in both normal and hypothyroid brains display a biphasic pattern, increasing progressively during the first week after birth and declining thereafter. However, hypothyroidism resulted in a significant reduction in the steady state levels of both mRNAs during the first postnatal week. During the second and third weeks, in contrast to their rapid decline in the normal controls, the corresponding decrease in the hypothyroid cerebra was retarded and prolonged resulting in their higher levels under TH-deficient condition. Kinetics of stimulation of actin and tubulin mRNAs in the 5-day hypothyroid cerebra following injection of the optimal dose of TH (200 micrograms T3/100 g body wt.) demonstrated elevation of both mRNAs within 1 h indicating a possible role of TH at the transcriptional level. In vitro transcription experiments by nuclear run off assay unambiguously confirmed that actin gene transcription is depressed in the hypothyroid cerebra compared to normal control. This reduced rate of transcription could be significantly induced in the hypothyroid cerebra by incubation of hypothyroid nuclei with T3 prior to transcription. In contrast, except for a reduced transcription in 5-day hypothyroid nuclei, no effect on tubulin gene transcription was evident at any other age. Moreover preincubation of hypothyroid nuclei from all three ages with T3 had no stimulatory effect on tubulin gene transcription. Analysis of age related changes in the rates of transcription of actin and tubulin genes with their corresponding steady state mRNA levels in normal and hypothyroid developing brain provides strong evidence that although additional modes of regulation may be operative, transcription represents an important level of control for thyroidal regulation of actin gene expression while tubulin gene expression is primarily regulated at post-transcriptional level.

Induction of brain tubulin by triidothyronine: dual effect of the hormone on the synthesis and turnover of the protein

The relative effects of triiodothyronine (T3) on the synthesis and turnover of tubulin in the developing rat and chick brain have been examined. Measurements of rates of turnover of radiolabeled tubulin in the hormone-sensitive tissues show that the half-life of tubulin turnover is 4-6 h in the absence of T3 and 10-12 h in the presence of the hormone. Analysis of short-term kinetic data on the stimulation of tubulin by T3 in the chick brain show that the rapid induction is due to a dual effect of the hormone on the metabolism of tubulin--an increase in the rate of synthesis as well as a decline in its rate of turnover.

Role of Thyroid Hormone in the Morphological Differentiation and Maturation of Astrocytes: Temporal Correlation with Synthesis and Organization of Actin

Morphological changes and the molecular mechanisms associated with the maturation of astrocytes were studied under normal and thyroid hormone‐deficient conditions using long‐term (30 days) primary cultures derived from the neonatal rat brain. Immunocytochemical staining of cells with a monoclonal antibody specific to glial fibrillary acidic protein demonstrated for the first time that, similar to their maturation in vivo, astrocytes maintained in normal serum‐containing medium can undergo complete maturation involving two distinct stages of morphological differentiation (from radial glia to flat polygonal cells with epithelioid morphology and then to mature process‐bearing cells with stellate morphology). Deficiency of thyroid hormone delays the first step and totally blocks the second stage of differentiation in the maturation process. Comparative staining of normal and thyroid hormone‐deficient astrocytes with filamentous actin‐specific fluorescein isothiocyanate‐phalloidin and quantitation of the various forms of intracellular actin using an improved DNase I assay demonstrated that maturation of astroglial cells is associated with characteristic alterations in the level of cytoskeletal and non‐cytoskeletal filamentous (F) actin. In particular, the maintenance of the epithelioid form of the hypothyroid astrocytes is associated with a progressive increase in the level of cytoskeletal F‐actin and a concomitant decline in the level of non‐cytoskeletal F‐actin. Quantitation of actin mRNA by Northern blot analysis and studies on the rate of actin synthesis at various stages of differentiation showed that the initial transformation into the epithelioid form is associated with an increase in the rate of synthesis of actin and the expression of its mRNA, while the final transformation into the mature process‐bearing form is correlated with a decline in these parameters. The results indicate that thyroid hormone plays an obligatory role in promoting the differentiation and maturation of astrocytes, and that during this process the hormone regulates the expression of actin and its intracellular organization in a way conducive to morphological differentiation.

Thyroid hormone stimulates γ-glutamyl transpeptidase in the developing rat cerebra and in astroglial cultures

Hypothyroidism in the developing rat brain is associated with enhanced oxidative stress, one of the earliest manifestations of which is a decline in the level of glutathione (GSH). To investigate the role of thyroid hormone (TH) on GSH homeostasis, the effect of TH on γ‐glutamyl transpeptidase (γGT), the key enzyme involved in the catalysis of GSH, was studied. Hypothyroidism declined the specific activity of cerebral γGT at all postnatal ages examined (postnatal day 1–20) with a maximum inhibition of 42% at postnatal day 10. Intraperitoneal injection of TH to 15‐day‐old rat pups increased the specific activity of γGT by 25‐30% within 4–6 hr. Treatment of primary cultures of astrocytes by TH also enhanced the specific activity of γGT by 30–40% within 4–6 hr. The induction of γGT by TH was blocked by actinomycin D or cycloheximide. γGT is an ectoenzyme that is normally involved in the catabolism of GSH released by astrocytes. In the presence of the γGT‐inhibitor, acivicin, GSH released in the culture medium of astrocytes increased linearly for at least 6 hr and TH had no effect on this accumulation pattern. In the absence of acivicin, GSH content of the medium from TH‐treated cells was significantly lower than that of untreated controls due to activation of γGT by TH and a faster processing of GSH. Because the products of γGT reaction are putative precursors for neuronal GSH, the activation of γGT by TH may be conducive to GSH synthesis in neurons and their protection from oxidative stress.

Regulation of cytoskeletal proteins by thyroid hormone during neuronal maturation and differentiation

Primary cultures of neurons from 16- to 17-day-old embryonic rat cerebra were maintained for 3 weeks in thyroid hormone deficient (THdef) and thyroid hormone supplemented (THsup) media to investigate how TH regulates the cytoskeletal (CSK) proteins during neuronal differentiation and maturation. Two distinct phases of regulation of triton-insoluble CSK-proteins by TH were discernible--an early phase (days 4-8 of culture) when TH-deficiency resulted in down-regulation of these proteins and a late phase (days 16-20) involving up-regulation of these proteins. In contrast, the triton-soluble non-CSK proteins always remained up-regulated by TH. The two main effects of TH-deficiency were retarded neurite outgrowth and altered neuronal morphology. Of all the CSK proteins, actin was found to be predominantly sensitive. Alterations in the level of CSK actin during neuronal maturation were found to be parallel to changes in steady-state level of actin mRNA as well as actin synthesis. However, these TH-induced changes (up-regulation of actin during the early phase and down-regulation during the late phase) did not lead to parallel changes in the level of soluble G-actin which was comparable at both days 8 and 16 in THdef and THsup cultures. Quantitation of different forms of intracellular actin revealed that G-actin level declined by about 50% between days 8 and 16. In the case of THsup neurons, this reduction in G-actin was accompanied by a parallel increase in the non-CSK F-actin, whereas TH-deficiency resulted in a corresponding increase in CSK F-actin during the terminal differentiation of neurons. Thus TH regulates the biogenesis of CSK-proteins with a predominant effect on actin and the transformation of G-actin into non-CSK F-actin appears to be the key step in neuronal maturation which is affected by hypothyroidism.

Regulation of α- and β-tubulin mRNAs in rat brain during synaptogenesis

Abstract Developmental alterations in α- and β-tubulin mRNA in polysomes from brains of −3 days (fetal) to 30-day-old rats were quantitated by using well-characterized chicken α- and β-tubulin cDNA probes, pT 1 and pT 2 having 80–90% homology with rat sequences. Northern blot analysis revealed a single major (> 95%) 1.8 kb mRNA for both α- and β-tubulin. Quantitation by slot hybridization indicates a virtually coordinate expression of α- and β-tubulin mRNA with a maximal level around day 5 after birth, which represents the mid-phase of synaptogenesis.

Thyroidal stimulation of tubulin and actin in primary cultures of neuronal and glial cells of rat brain

The influence of triiodothyronine (T3) on the level of tubulin and other proteins in primary cultures of neuronal (N) and glial (G) cells from rat brain has been investigated. Quantitation of tubulin by [3H]colchicine binding assay revealed that when cells from 1 day rat brain were cultured for 18 hr with physiological doses (0.5–5 nM) of T3, the hormone elicited 35–40% increase in the soluble (30,000 g supernatant) tubulin content of G cells only. This stimulation was age‐dependent and occurred neonatally at a time corresponding to the onset of synaptogenesis. In mouse and chick brain also, [3H]colchicine binding assay showed a similar selective stimulation of the soluble tubulin content of G cells by T3 with virtually no effect on N cells. However, SDS‐polyacrylamide gel electrophoresis of the total proteins in the 30,000 g supernatants from N and C cells of rat brain, labeled for 18 hr with [14C]leucine in the presence of T3, revealed that T3 elicited 2–3‐fold enhancement of radiolabeled tubulin in the N cells which is relatively greater than the 1.5‐fold increase seen in the G cells. Analysis of the autoradiograms of these labeled proteins also revealed that in addition to tubulin, T3 stimulated the accumulation of radiolabeled actin by 1.5‐ and 2‐fold in N cells and G cells respectively. Similar electro‐phoretic analysis of the solubilized labeled proteins in the 30,000 g pellets from N and G cells indicated that the failure to detect the stimulation of tubulin in the 30,000 g supernatants from N cells by [3H]colchicine binding assay could be at least partly due to rapid translocation of the dimeric soluble tubulin into insoluble membrane fractions or due to presence of higher oligometric forms of tubulin which are insensitive to [3H]colchicine binding assay.

Protein kinase A linked phosphorylation mediates triiodothyronine induced actin gene expression in developing brain

In the developing rat cerebra, triiodothyronine (T3) stimulates actin mRNA by acting predominantly at the level of transcription whereas tubulin mRNA is enhanced primarily by post-transcriptional regulation. We report here that in primary cultures of rat cerebra, the T3-induced actin gene expression is mediated by phosphorylation events. Inhibition of protein kinase A (PKA), but not of protein kinase C (PKC) or tyrosine kinase, totally blocked the induction of actin mRNA by T3. Under identical conditions, induction of tubulin mRNA by T3 was virtually unaffected by all the inhibitors. Activators of PKA, but not of PKC, potentiated the T3-induced actin gene expression, both at mRNA and protein level, by about 2-fold. In the absence of T3, neither the inhibitor nor the activator of PKA had any significant effect on this induction. The involvement of PKA in mediating the induction of actin mRNA by T3 was confirmed by transfecting primary cultures of rat cerebra with an expression vector encoding the protein kinase A inhibitor which totally abolished the induction. T3 is shown to enhance the phosphorylation of the thyroid hormone receptor, TRalpha, by about 2-fold but the level of phosphorylation of TRbeta remained virtually unaffected.

Ontogeny of glutamine synthetase in rat brain

The developmental pattern of glutamine synthetase (GS) in rat brain has been studied with respect to that of the cells involved in the synthesis of the enzyme. GS activity is very low until day 13 after birth, rises sharply between days 13 and 15, and slowly thereafter. In contrast, more than half of the total number of protoplasmic astrocytes—the cells involved in synthesis of GS—are formed by day 12, following which the rate of proliferation declines considerably. GS is precociously inducible by cortisol in purified protoplasmic astrocytes, in organ cultures of 6–13 day rat brain and in the brains of cortisoladministered 12 day rats. These results and the temporal coincidence of the period of increase of GS with the onset of the function of adrenal cortex suggest that GS activity in the developing rat brain is under steroidal control.