Sukanya Chaudhury

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.

Differential effects of hypothyroidism on Na-K-ATPase mRNA α isoforms in the developing rat brain

In the developing rat cerebrum, the level of different isoforms of Na-K-ATPase mRNA increases significantly during the first three postnatal weeks, which represent the critical period of synaptogenesis and myelination—the two thyroid hormone-sensitive maturational events. To determine the possible functional relationship of these isoforms with maturational events in the developing brain and their mode of regulation by T3, we have examined the effect of hypothyroidism on the expression of the different α-isoforms (α1, α2, and α3) of Na-K-ATPase mRNA covering the first 3 wk of postnatal development. Quantitation of these mRNAs from cerebra of 1-, 5-, 10-, 15-, and 20-d-old normal and hypothyroid rats by Northern blot analysis indicate that α3 mRNA is not only predominantly expressed throughout this entire period of study but also represents the species which is most severely affected in the hypothyroid brain. The relative sensitivity for the expression of these mRNAs to T3 were α3>α1>α2. These results, together with the report of predominant expression of the α3 isoform in neuronal cells, suggest specific functional involvement of this isoform with the decisive maturational events in the rat brain. Kinetic studies on in vivo induction of Na-K-ATPase α-mRNAs by T3 in the 15-d-old hypothyroid rat shows clear stimulation of all the isoforms within 1 h of the administration of the optimal dose (200 µg T3/100 g body wt) suggesting a direct, possibly transcriptional effect of the hormone on the expression of these genes.

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.

Thyroidal regulation of different isoforms of NaKATPase in glial cells of developing rat brain.

The developmental profile of the different isoforms of NaKATPase have been investigated during the first three weeks of postnatal development using primary cultures of isolated glial cells derived from neonatal rat cerebra. Northern and Western blot analysis show that the expression of four isoforms (alpha1, alpha2, beta1 and beta2) in these cells increases progressively between 5 to 20 days of culture. Comparison of the mRNA levels of these isoforms in thyroid hormone deficient (TH def) and thyroid hormone supplemented (TH sup) cells cultured for 5-10 days, revealed for the first time that all four isoforms are sensitive to T3 in the glial cells. Furthermore immunocytochemical staining of these cells with isoform specific NaKATPase antibodies also showed that the localization of the different isoforms in the TH def cells were altered in comparison to that in the TH sup cells. These results establish glial cells as the target cells for the regulation of NaKATPase by TH in the developing brain.

Transcriptional and post-transcriptional regulation of Na+, K+-ATPase α isoforms by thyroid hormone in the developing rat brain

Transcriptional and post-transcriptional events involved in the induction of sodium potassium adenosine triphosphatase (Na+,K(+)-ATPase) by thyroid hormone (T3) were investigated. In vitro transcription of nuclei isolated from cerebra of 5- and 15-day-old normal and hypothyroid rats showed that transcription of all alpha mRNA isoforms (alpha 1, alpha 2 and alpha 3) of Na+,K(+)-ATPase are sensitive to T3. This is evidenced by a 50-70% reduction in the rates of transcription of alpha 1 and alpha 3 mRNA and 20-40% reduction of alpha 2 mRNA in nuclei from hypothyroid cerebra compared with those from normal controls. Preincubation of nuclei from hypothyroid cerebra with T3 prior to transcription also showed an increase in the rates of transcription of these mRNAs. At the post-transcriptional level, T3 enhanced the half life of alpha 3 mRNA by 1.5-fold with no discernible effect on alpha 1 and alpha 2 mRNA.

Tubulin and glial fibrillary acidic protein gene expression in developing fetal human brain at midgestation

Developmental alterations in the expression of glial fibrillary acidic protein (GFAP) and α-tubulin were examined at the level of mRNA and protein in human fetal brain between weeks 13–23 of gestation. Except for a transient increase at week 15, GFAP expression in the cytoskeletal (CSK) fraction was low until week 17, when it increased steadily to week 23, corresponding to the phase of glial proliferation. The developmental profile of α-tubulin in the CSK fraction displayed a biphasic pattern, with an initial rise between weeks 13–16 coinciding with the early phase of neuroblast multiplication, and a second rise between weeks 17–23 corresponding to the phase of glial proliferation. No significant difference in the spatial distribution of α-tubulin was found in different region of brain but GFAP expression varied with a higher level in cerebellum than that in cerebrum at late midgestation.

Modulation of tubulin synthesis by tri-iodothyronine in the embryonic chick brain

In organ cultures of embryonic chick brain, tri-iodothyronine is shown to elicit an age-dependent stimulation of the synthesis of tubulin. In cultures of brains from 8-day embryos where the response is maximal, a 70-100% increase in the rate of synthesis of tubulin is elicited by T3 within 2 h. The stimulatory response is dose-dependent, requires transcription, and is temporally coincident with the early phase of the normal ontogenic rise in level of tubulin and of intense neuronal differentiation in the embryonic chick brain. The overall results indicate the involvement of T3 in regulating the biogenesis of tubulin in the developing brain.

Age-related changes in tubulin biosynthesis in the embryonic chick brain

Abstract The rates of synthesis of tubulin and total protein have been measured in organ cultures of brains from 6–14-day embryonic chicks. Total protein synthesis declines progressively with age but tubulin synthesis peaks between days 6 and 7 and then declines. The turnover of tubulin in 7-day brain is significantly faster than that in the 12-day brain. Thus, alterations in the rates of synthesis as well as degradation of tubulin occur in the developing brain.

Thyroidal regulation of different isoforms of NaKATPase in the primary cultures of neurons derived from fetal rat brain

The developmental profile of the different isoforms of NaKATPase have been investigated using primary cultures of isolated neurons initiated from 17 day old fetal rat brain. Northern blot analysis showed that the expression of three alpha isoforms (alpha(1), alpha(2) and alpha(3)) and two beta isoforms (beta(1) and beta(2)) increased progressively and reached a peak between 12 to 16 days of culture. Comparison of the mRNA levels of these isoforms in the cells maintained in thyroid hormone deficient (TH def) and thyroid hormone supplemented (TH sup) media for 6-12 days, revealed for the first time that in the neurons three alpha and two beta isoforms of NaKATPase are sensitive to TH. Furthermore immunocytochemical staining of these cells with isoform specific NaKATPase antibodies showed that the uniform distribution of alpha(2), alpha(3) and beta(2) isoforms in the neuronal processes require the presence of TH. These results establish neurons as the target cells for the regulation of NaKATPase by TH in the developing brain.