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Stimulation of tubulin synthesis by thyroid hormone in the developing rat brain
Biochimica et Biophysica Acta, 763 (1983) 93-98 Elsevier
93
BBA 11194
S T I M U L A T I O N OF TUBULIN S Y N T H E S I S BY T H Y R O I D H O R M O N E IN T H E D E V E L O P I N G RAT BRAIN S. CHAUDHURY and P.K. SARKAR
Department of Cell Biology, lndian Institute of Chemical Biology, Jadavpur, Calcutta-700032 (India) (Received November 10th, 1982) (Revised manuscript received May 10th, 1983)
Key words: Tubulin synthesis; Thyroid hormone," Development," (Rat brain)
The effect of triiodothyronine on the biogenesis of tubulin has been studied in the developing rat brain. In organ cultures of brains from newborn rats, the hormone stimulates the incorporation of [t4C]leucine into tubulin by 60-80% within 2 h in the absence of any significant change in total protein synthesis. This stimulation is strictly age-dependent (only brains from late fetal or newborn rats are sensitive), dose-dependent (stimulation increases progressively and reaches a maximum level with physiological dose of the hormone) and displays tissue specificity. The temporal correspondence of the sensitivity of the rat brain to triiodothyronine with the period of normal rise in the level of tubulin and that of the maximal level of nuclear triiodothyronine receptors in the brain strongly suggests the involvement of the hormone in regulating the biogenesis of tubulin during the differentiation and maturation of neonatal rat brain. Thyroid hormones are known to be required for development and maturation of mammalian brains, early at the neonatal stage (see Ref. 1 for reviews). In rats, this period is limited to the first 10-12 days after birth [2] and is associated with rapid neuronal proliferation and outgrowth of dendrites and axons [3,4]. The molecular basis of thyroid hormone action in the developing brain is unknown. Since the assembly of neurotubules is required for the growth of axons and dendrites [5-7] and the neurotubule protein, tubulin, represents the major protein in the cellular processes of the maturing neurons [5,8], we suspected that the thyroid hormones might exert their effect in the developing brain by modulating the biogenesis or assembly of neurotubules. Supportive evidence for such speculation was available from the recent studies of Fellous et al. [9] who showed that hypothyroidism in neonatal rats retards the ability of brain tubulin to polymerize in vitro and that treatment with the thyroid hormones restores normal polymerization.
In the present communication we show that triiodothyronine (T3) elicits an age-dependent induction in the synthesis of tubulin in organ cultures of developing rat brain and that this effect is temporally coincident with the period of rapid rise in the level of tubulin and maturation of the brain.
Experimental procedures
Organ culture and labeling Brains from white albino rats or their offspring of indicated ages were dissected aseptically, freed from blood vessels and connective tissues, and cultured at 37°C in Tyrode's physiological solution containing 5000 u n i t s / m l 1% penicillin/5 /~g/ml streptomycin mixture in a New Brunswick Gyratory shaker (70 r p m / m i n ) as described previously [10,11]. A single brain was cultured in 5 ml media in each flask except for brains from fetuses, where two brains were used per flask. Whole brains were cultured and during the 2-3 h culture period, no loss of functional or histological integrity of the
94 tissue occurred. No differences were detectable between longitudinal sections of paraffin-embedded eosin-hematoxylin stained freshly dissected neonatal brains and those stained identically after 2 h culture. Protein synthesis during culture continued linearly at least up to 2 h (see Results) and more than 95% of the cells derived from the cultured tissue were viable as judged by Trypan blue exclusion test. Prior to addition of tissue, label and hormone, the media was equilibrated at 37°C and gassed with 95% air/5% CO 2. For experiments with actinomycin D, the tissues were preincubated for 15 min in the presence of the drug before adding hormone. Following the culture period, the tissues were washed thoroughly with ice-cold Tyrode's solution and either used immediately (colchicine binding assay for tubulin) or frozen until analysis (vinblastine assay for tubulin). For the colchicine assay of tubulin, brains were cultured with and without hormone or drugs in the absence of any radioactive label. Freshly harvested brains were homogenized in a Dounce-type homogenizer using 0.5-1 ml of buffer 1 (10 mM sodium phosphate/10 mM MgC12 (pH 7.0)) containing 0.1 mM GTP, and the homogenates were centrifuged at 30000 × g for 15 min and the supernatants used for assay of tubulin. Similar results were obtained when 100000 x g (1 h) supernatants were employed for the assay. Aliquots of the supernatants were incubated in a total volume of 0.25 ml for 2 h at 37°C with 1 • 10- s M non-radioactive colchicine and 0.2/~Ci [3H]colchicine (New England Nuclear; spec. act. 6 Ci/mmol). Addition of T~ to supernatams during incubation had no effect on the colchicine-binding activity. Bound [3H]colchicine was determined by the DEAE-impregnated filter paper (Whatman DE-81) assay of Weisenberg et al. [12]. When pure tubulin was incubated with [3H]colchicine, tool colchicine b o u n d / m o l tubulin is about 0.6. Saturatir~g levels of colchicine were used in our binding studies and to ensure that colchicine was not limiting, two concentrations of each sample (25-100 >1) containing 50-200 ~g of proteins were assayed. Samples were diluted with 1 ml of buffer 1 containing 1. 10 5 M colchicine and absorbed to the 2.4 cm diameter DEAE filters directly over 1-2 rain applying very gentle suction.
The filters were then rinsed three times with 3 ml cold buffer 1 by mild suction, dried and counted using toluene containing 4 g/1 of omnifluor (New England Nuclear). For the vinblastine assay, frozen brains prelabeled with 2/,tCi/ml ['4C]leucine (spec. act. 280 m C i / m m o l , Bhaba Atomic Research Centre, Bombay) in the presence or absence of hormones, were homogenized with ice-cold buffer 2 (10 mM sodium phosphate, pH 7.0) in a Dounce-type homogenizer and centrifuged at 100000 x g for 1 h. The supernatants were filtered using Millipore type HA, 0.45 /,m filters and from the filtrates, tubulin was quantitated by combined vinblastine precipitation [10,13] and sodium dodecyl sulfate polyacrylamide gel electrophoresis [14]. 1 mg of supernatant protein was used for precipitation in duplicate and the precipitates were analysed by slab gel electrophoresis. The band comigrated in the gel with standard tubulin was sliced and the radioactivity counted using toluene/Triton X-100 (3:1, v / v ) containing 4 g / l of Omnifluor. The procedure has been shown to be highly efficient for the quantitation of tubulin from labeled supernatants and proven to be linear in the range of protein concentration 1 2 nag per assay [10]. Protein estimates were carried out according to Lowry et al. [ 15] and incorporation of [ 14C]leucine into total protein was determined by precipitation with 7.5% trichloroacetic acid. The precipitates were collected on Millipore filters (Type HA, 0,45 /,m), washed and the filters were counted as described before using a Packard scintillation counter. Results
In the rat, tubulin concentration increases 2--3fold between late fetal stage ( - 5 day) to early postnatal ( + 10 day) stage [16] and this period also corresponds to the period of thyroid hormone dependent maturation of the brain [3,4]. We therefore determined the effect of T 3 on the biogenesis of tubulin in organ cultures of brains of rats from late fetal and early postnatal stages. In this series of experiments, tissues were labeled with 2.0 ~ C i / ml of [~4C]leucine for 2 h in the presence or absence of 4.5 nM triiodothyronine (T3). Incorporation into total protein was determined by tri-
95 TABLE I m" 18o 'o
DOSE-RESPONSE P A T T E R N FOR T H E S T I M U L A T I O N O F T U B U L I N SYNTHESIS BY T R I I O D O T H Y R O N I N E (T3) IN T H E N E W B O R N R A T BRAIN
X
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Brains from newborn (0-24 h) rats were labeled with 2 I~Ci/ml of {~4C]leucine in media containing increasing concentrations (0 4.5 nM) of T 3 for 2 h. Radioactivity incorporated into total protein and tubulin were determined by precipitation with trichloroacetic acid and by vinblastine assay, respectively. Data represent mean ± S.D. of three experiments.
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Concentration of q~ in the culture medium (nM)
cpm per mg total protein ( x l O -3)
cpm in tubulin per mg total protein ( x l O 2)
0(Control) 0.045 0.45 4.5
132.2± 8 132.8±10 135.1±10 134.5± 8
28.4±2 31.3±2 48.6±4 51.5±4
12 Adult
Fig. 1. Age-related stimulation of tubulin synthesis by T 3 in the embryonic rat brain. Organ cultures of rat brains of indicated ages were labeled for 2 h with 2 # C i / m l [taClleucine in the presence (hatched bars) or absence (open bars) of 4.5 n M T 3. Incorporation into tubulin (lower panel) as determined by vinblastine assay and total protein (upper panel) are shown. Data for brains from - 5 to 3-day-old rats represents Mean ± S.D. from three experiments and those for other ages are averages from replicate experiments with the bar representing variation from the average.
chloroacetic acid precipitation and tubulin was quantitated by the combined vinblastine precipitation and SDS polyacrylamide gel electrophoresis as described in 'Methods'. The results showed an age-dependent stimulation in the synthesis of
tubulin by T3 (Fig. 1, lower panel) in the absence of any significant effect in total protein synthesis (Fig. 1, upper panel). In control cultures, both total protein and tubulin synthesis progressively declined with age. However, in the presence of the hormone, total protein synthesis remained within + 10% of the controls at all ages; tubulin synthesis was stimulated by 60-80% in the brains from newborn (0-24-hold) rats and 40-50% in brains from late fetal ( - 2 day) or early postnatal (+ 1-day-old) rats. The uptake of [14C]leucine in the brains from newborn rats remained essentially unaltered by T3 (250000-280000 c p m / m g protein in both control and hormone-treated tissues). No stimulation of tubulin synthesis was detectable in brains from an
T A B L E Ii T I M E KINETICS F O R T H E S T I M U L A T I O N OF T U B U L I N SYNTHESIS BY T R I I O D O T H Y R O N I N E (T3) IN T H E NEWB O R N RAT BRAIN Brains were labeled with 2 / L C i / m l [14C]leucine in the presence or absence (control) of 4.5 nM T 3 for the indicated periods of time. Total protein and tubulin were quantitated as described in legends to Table I. Data represent mean ± S.D. from three experiments. Length of culture time
cpm per mg total protein ( x 10-3)
cpm in tubulin per mg total protein ( × 10 2)
(min)
Control
T3-treated
Control
T3-treated
30 60 120 180
30.1±2 52.2±2 127.8±5 153.3±5
31.1±3 55.3±3 129.1±5 165.1±5
8.3±2 16.1±2 29.2±2 36.3±3
13.4±2 26.1±3 52.3±2 58.5±5
96 TABLE Ill EFFECT OF T 3 ON THE STIMULATION OF C O L C H I C I N E B I N D I N G ACTIVITY IN BRAINS FROM FETAL ( - 5 DAY), NEWBORN A N D 5-DAY-OLD RATS Brains were cultured for 2 h in the presence or absence of the indicated concentrations of T 3. Colchicine assay was performed as described in Experimental procedures. Except for the 5-day-old brains data represents mean _+S.D. from three experiments. Figures in parenthesis represent stimulation over control, n.d., not determined. Concentration of T 3 in the culture medium (nM)
[3H]Colchicine bound per mg protein (cpm x 10-2) Fetal ( - 5 day)
Newborn
5-day-old
0 0.045 0.45 4.5
27.4 + 1 n.d. n.d. 30.1 +_2 ( + 10%)
56.4 _+4 67.0 + 5 ( + 20%) 79.4 _+5 ( + 39%) 85.4 + 6 ( + 51%)
49.0, 59.6 n.d. n.d. 58.4, 62.6 ( + 10%)
earlier period of gestation ( - 5 day) or in brains from older, including adult, rats. In view of the maximum stimulation of tubulin synthesis by T 3 in brains from newborn rats, the dose dependency and time-course of the hormonal effect were determined in organ cultures of brains from newborn rats. The dose-response pattern of this phenomenon is shown in Table I. Maximal stimulation of tubulin synthesis by ~ was observed at 4.5 nM and with 0.45 nM T 3, 80-90% of this stimulation could be achieved. No further increase in rate of tubulin synthesis could be achieved with higher concentration (45 nM) of the hormone (data not shown). Total protein synthesis was not significantly altered by ~ in any of the doses examined. Table II shows the time-course data for the synthesis of tubulin and total protein in brains from newborn rats cultured in presence or absence of 4.5 nM T 3. Treatment with hormone progressively increased synthesis of tubulin between the first 2 h of culture. During this period, total protein synthesis remained essentially unaffected. A definitive increase in tubulin synthesis could be seen within 30 60 min of the exposure to hormone and by 120 min the response appeared to reach the maximal level (60-80% of control). No significant enhancement of response was noticed at the 180 min time-point, although incorporation into both tubulin and total protein increased to some extent. The lack of additional effect after 2 h could be clue to the fact that protein synthesis in the explanted
organ tends to decline slowly after 2 - 3 h in culture. In the experiments described above, tubulin was quantitated from 100000 × g supernatants of labeled brain by vinblastine precipitation and gel electrophoresis. Since vinblastine is known to precipitate small amounts of actin and few other minor proteins in addition to tubulin [17], we further examined the effect of ~ on the level of tubulin by using another independent quantitative assay based on the [3H]colchicine binding of tubulin. Using the colchicine assay, we redetermined the age-dependency, dose-response characteristics and the tissue specificity of the hormonal effect. The results (Table III) are similar to those obtained previously from the assay based on vinblastine precipitation. Of the three ages examined ( - 5 day, newborn and + 5 day) significant stimulation in the level of tubulin by T 3 was seen only in the case of the newborn brain, following the doseresponse pattern similar to that seen in Table I. Furthermore, in tissues such as kidney and lung from newborn rats, no stimulation of tubulin by T 3 was detectable by colchicine assay (data not shown). Since the effects of T 3 are generally mediated by interaction of the hormone with the genome [18], we further examined by colchicine assay the effect of inhibition of transcription by 20 ~tg/ml of actinomycin D (which inhibited 75% of the incorporation of [3H]uridine into RNA) on the stimulation of tubulin synthesis by T 3. Data in Table IV
97 TABLE IV EFFECT OF INHIBITION OF TRANSCRIPTION ON THE STIMULATION OF TUBULIN (COLCHICINE-BINDING ACTIVITY) BY TRIIODOTHYRONINE (T3) IN CULTURES OF BRAINS FROM NEWBORN RATS Brains from newborn rats were cultured for 120 min in the presence or absence of 4.5 nM T3 and 20 #g/ml actinomycin D as indicated. Colchicine assay was performed as described in Experimental procedures. Data represent mean +S.D. from three experiments except for case 2 where two experiments were done. Figures in parenthesis represent stimulation over control. Addition in the culture medium
[3H]Colchicine bound per mg protein (cpm x 10 2)
1 None (Control) 2 Control plus 20 ktg/ml actinomycin D 3 4.5nMT 3 4 4.5 nM T3 plus 20/~g/ml actinomycin D
56.4+_ 4 61.6, 55.5 (+ 4%) 85.4_+ 6(+51%) 61.3 _+ 3 ( + 10%)
show that the enhancement of tubulin by T 3 requires transcription of new R N A . In these experiments, T~ stimulated the synthesis of tubulin by about 50% over control and in the presence of the drug, the stimulation was only about 10%. Finally, to determine the effect of T 3 on proteins other than tubulin, we analysed 100000 × g supernatants of brains from newborn rats cultured for 2 h in the presence or absence of 4.5 nM T 3 by disc gel electrophoresis using 7.570 polyacrylamide gels according to Weber and Osborne [14]. Comparison of the distribution of radioactivity in the sliced gel revealed no significant difference in the profile except in the region of marker tubulin which was stimulated by about 50% in the T 3t r e a t e d sample. However, since this procedure does not resolve a- and /3-tubulin, further analysis by more sophisticated methods are necessary to determine whether a- or /3-tubulin is preferentially stimulated by T 3. Discussion
The main conclusion from the present investigation - the induction of tubulin synthesis by thyroid h o r m o n e s - is established and supported by the results of several different types of experi-
ments. First, the induction is strictly age-dependent, as observed for m a n y other h o r m o n a l effects in developing tissues [19]. Second, the extent of induction is dose-dependent and reaches a maximal level between 0.45-4.5 nM, which is in the range of physiological concentrations of the h o r m o n e in rat brain [20]. In separate experiments (data not shown) we have determined that the nuclear receptors in adult rat brain can be saturated by incubation with 2.5-25 n M T 3. Third, the stimulation is tissue-specific and tissues other than brain, such as lung and kidney derived from the rats of inducible age, are totally insensitive to T 3. In the present study, enhancement of tubulin by ~ is found both by vinblastine and colchicine assays. It should be clarified that for vinblastine assay 100 000 x g supernatants from brains labeled for 2 h with 14C-labeled amino acids are used and thus only the newly synthesized labeled tubulin is measured. On the other hand, for the colchicine assay, unlabeled supernatants are used and the total tubulin content is measured by binding to [3H]colchicine. Thus the extent of stimulation by in the vinblastine assay (60-80% of the newly synthesized tubulin) is less than that observed by colchicine assay (1.5-fold stimulation of the total tubulin). Further investigation on the mechanism of this stimulation and in particular, studies on the effect of T 3 on the synthesis and stability of tubulin in normal and hypothyroid rat brains, are expected to clarify this problem. These results are consistent with the observations of Fellous et al. [9] that thyroid h o r m o n e s are required very early after birth for normal microtubule assembly and neurite growth. Their experiments clearly established that microtubule assembly is retarded in the hypothyroid rat brain and suggested that the limiting factor is p r o b a b l y one of the microtubule associated protein ' t a u ' [21]. The present studies indicate that thyroid h o r m o n e might control the assembly/biogenesis of the neurotubule proteins by regulating both the level of tubulin and the microtubule-associated proteins. Gonzales and Geel [22] examined the effect of hypothyroidism on the concentration and biochemical properties of tubulin in 12-25-day-old developing rats and found no evidence of any defect in tubulin synthesis in brains from 12-25-
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This research was supported by grants from the Council of Scientific and Industrial Research, Government of India. References
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Age ( Days )
Fig. 2. Temporal relation between the stimulation of tubulin synthesis by T3 (present study), binding capacity of nuclear rl~ receptors [23] and the period of rise in tubulin [16] in the developing rat brain.
day-old hypothyroid rats. Francon et al. [9] also reported no difference in tubulin content per g whole brain for 15-day-old control and hypothyroid rats. In both of the above studies, the effect of thyroid hormone deficiency in the late fetal or newborn rats was not determined. It should be recalled that the most critical period of thyroid hormone action in rats is limited to the first 10 days after birth when the pyramidal neuronal differentiation occur [3,4] and this period also coincides with the period at which the tubulin concentration reaches the peak level [16]. Fig. 2. depicts an interesting temporal correlation between the stimulatory effect of T~ on tubulin synthesis, the level of nuclear receptors for T 3 and the developmental profile of tubulin in the rat brain. The binding capacity of the nuclear T 3 receptors in rat brain rises rapidly between the fetal stage (2 days before birth) and early postnatal stage (2 days after birth) and declines thereafter [23]. The coincidence of the period of maximal sensitivity of the brain to stimulation of tubulin synthesis by T3 with the period of rise in the level of ~ receptors and that of tubulin in the rat brain strongly indicate that the thyroidal induction of tubulin synthesis in the developing brain is a natural ontogenic phenomenon.
1 Grave, G.D. (1977) Thyroid Hormones and Brain Development, Raven Press, New York 2 Sokoloff, L. and Kennedy, C. (1973) in Biology of Brain Dysfunction (Graniet G.E., ed.), pp. 295-305, Plenum Press, New York 3 Bass, N.H., Pelton, E.W. and Young, E. (1977) in Thyroid Hormones and Brain Development (Grave, G.E, ed.), pp. 199-214, Raven Press, New York 4 Lauder, J.M. (1977) in Thyroid Hormones and Brain Development (Grave G.D., ed.), p. 235 254, Raven Press, New York 5 Daniels, M.P. (1972) J. Cell Biol. 53, 164 176 6 Yamada, K.M., Spooner, B.S. and Wessels, N.K. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 1206 1212 7 Seeds, N.S. and Maccioni, R.B. (1978) J. Cell Biol. 76, 547 555 8 Peters, A. and Vaughn, J.E. (1967) J. Cell Biol. 32, 113-119 9 Fellous, A., Lennon, A . M , Francon, J. and Nunez, J. (1979) Eur. J. Biochem. 101, 365-376 10 Chaudhury, S., Chaudhury, L. and Sarkar, P.K. (1982) Dev. Brain Res. 4, 241-243 11 Soh, B.M. and Sarkar, P.K. (1978) Dev. Biol. 64, 316-328 12 Weisenberg, R.C., Borisy, G.G. and Taylor, E.W. (1968) Biochemistry 7, 4466-4478 13 Feit, H., Dutton, G.R., Barondes, S.H. and Shelanski, M.L. (1971) J. Cell Biol. 51, 138-147 14 Weber, K. and Osborne, M. (1969) J. Biol. Chem. 244, 44O6 4412 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol Chem. 193, 265-275 16 Nunez, J., Fellous, A., Francon, J. and Lennon, A.M. (1975) in Microtubules and Microtubule lnhibitors (Berges, M. and De Brabander, M., eds.), pp. 269-278, North-Holland Publishing Co., Amsterdam 17 Wilson, L., Bryan, J., Ruby, A. and Mazia, D. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 807 812 18 Oppenheimer, J.H. (1979) Science 203, 971-979 19 Litwack, G. (1970) in Biochemical Actions of Hormones, Academic Press, New York 20 Schwartz, H.L. and Oppenheimer, J.H. (1978) Endocrinology 103, 267-273 21 Francon, J., Fellous, A., Lennon, A. and Nunez, J. (1977) Nature (Lond.) 266, 188 190 22 Gonzales, L W . and Geel, S.E. (1978) J. Neurochem. 30, 237-245 23 Schwartz, H.L. and Oppenheimer, J.H. (1978) Endocrinology 103, 943 948
93
BBA 11194
S T I M U L A T I O N OF TUBULIN S Y N T H E S I S BY T H Y R O I D H O R M O N E IN T H E D E V E L O P I N G RAT BRAIN S. CHAUDHURY and P.K. SARKAR
Department of Cell Biology, lndian Institute of Chemical Biology, Jadavpur, Calcutta-700032 (India) (Received November 10th, 1982) (Revised manuscript received May 10th, 1983)
Key words: Tubulin synthesis; Thyroid hormone," Development," (Rat brain)
The effect of triiodothyronine on the biogenesis of tubulin has been studied in the developing rat brain. In organ cultures of brains from newborn rats, the hormone stimulates the incorporation of [t4C]leucine into tubulin by 60-80% within 2 h in the absence of any significant change in total protein synthesis. This stimulation is strictly age-dependent (only brains from late fetal or newborn rats are sensitive), dose-dependent (stimulation increases progressively and reaches a maximum level with physiological dose of the hormone) and displays tissue specificity. The temporal correspondence of the sensitivity of the rat brain to triiodothyronine with the period of normal rise in the level of tubulin and that of the maximal level of nuclear triiodothyronine receptors in the brain strongly suggests the involvement of the hormone in regulating the biogenesis of tubulin during the differentiation and maturation of neonatal rat brain. Thyroid hormones are known to be required for development and maturation of mammalian brains, early at the neonatal stage (see Ref. 1 for reviews). In rats, this period is limited to the first 10-12 days after birth [2] and is associated with rapid neuronal proliferation and outgrowth of dendrites and axons [3,4]. The molecular basis of thyroid hormone action in the developing brain is unknown. Since the assembly of neurotubules is required for the growth of axons and dendrites [5-7] and the neurotubule protein, tubulin, represents the major protein in the cellular processes of the maturing neurons [5,8], we suspected that the thyroid hormones might exert their effect in the developing brain by modulating the biogenesis or assembly of neurotubules. Supportive evidence for such speculation was available from the recent studies of Fellous et al. [9] who showed that hypothyroidism in neonatal rats retards the ability of brain tubulin to polymerize in vitro and that treatment with the thyroid hormones restores normal polymerization.
In the present communication we show that triiodothyronine (T3) elicits an age-dependent induction in the synthesis of tubulin in organ cultures of developing rat brain and that this effect is temporally coincident with the period of rapid rise in the level of tubulin and maturation of the brain.
Experimental procedures
Organ culture and labeling Brains from white albino rats or their offspring of indicated ages were dissected aseptically, freed from blood vessels and connective tissues, and cultured at 37°C in Tyrode's physiological solution containing 5000 u n i t s / m l 1% penicillin/5 /~g/ml streptomycin mixture in a New Brunswick Gyratory shaker (70 r p m / m i n ) as described previously [10,11]. A single brain was cultured in 5 ml media in each flask except for brains from fetuses, where two brains were used per flask. Whole brains were cultured and during the 2-3 h culture period, no loss of functional or histological integrity of the
94 tissue occurred. No differences were detectable between longitudinal sections of paraffin-embedded eosin-hematoxylin stained freshly dissected neonatal brains and those stained identically after 2 h culture. Protein synthesis during culture continued linearly at least up to 2 h (see Results) and more than 95% of the cells derived from the cultured tissue were viable as judged by Trypan blue exclusion test. Prior to addition of tissue, label and hormone, the media was equilibrated at 37°C and gassed with 95% air/5% CO 2. For experiments with actinomycin D, the tissues were preincubated for 15 min in the presence of the drug before adding hormone. Following the culture period, the tissues were washed thoroughly with ice-cold Tyrode's solution and either used immediately (colchicine binding assay for tubulin) or frozen until analysis (vinblastine assay for tubulin). For the colchicine assay of tubulin, brains were cultured with and without hormone or drugs in the absence of any radioactive label. Freshly harvested brains were homogenized in a Dounce-type homogenizer using 0.5-1 ml of buffer 1 (10 mM sodium phosphate/10 mM MgC12 (pH 7.0)) containing 0.1 mM GTP, and the homogenates were centrifuged at 30000 × g for 15 min and the supernatants used for assay of tubulin. Similar results were obtained when 100000 x g (1 h) supernatants were employed for the assay. Aliquots of the supernatants were incubated in a total volume of 0.25 ml for 2 h at 37°C with 1 • 10- s M non-radioactive colchicine and 0.2/~Ci [3H]colchicine (New England Nuclear; spec. act. 6 Ci/mmol). Addition of T~ to supernatams during incubation had no effect on the colchicine-binding activity. Bound [3H]colchicine was determined by the DEAE-impregnated filter paper (Whatman DE-81) assay of Weisenberg et al. [12]. When pure tubulin was incubated with [3H]colchicine, tool colchicine b o u n d / m o l tubulin is about 0.6. Saturatir~g levels of colchicine were used in our binding studies and to ensure that colchicine was not limiting, two concentrations of each sample (25-100 >1) containing 50-200 ~g of proteins were assayed. Samples were diluted with 1 ml of buffer 1 containing 1. 10 5 M colchicine and absorbed to the 2.4 cm diameter DEAE filters directly over 1-2 rain applying very gentle suction.
The filters were then rinsed three times with 3 ml cold buffer 1 by mild suction, dried and counted using toluene containing 4 g/1 of omnifluor (New England Nuclear). For the vinblastine assay, frozen brains prelabeled with 2/,tCi/ml ['4C]leucine (spec. act. 280 m C i / m m o l , Bhaba Atomic Research Centre, Bombay) in the presence or absence of hormones, were homogenized with ice-cold buffer 2 (10 mM sodium phosphate, pH 7.0) in a Dounce-type homogenizer and centrifuged at 100000 x g for 1 h. The supernatants were filtered using Millipore type HA, 0.45 /,m filters and from the filtrates, tubulin was quantitated by combined vinblastine precipitation [10,13] and sodium dodecyl sulfate polyacrylamide gel electrophoresis [14]. 1 mg of supernatant protein was used for precipitation in duplicate and the precipitates were analysed by slab gel electrophoresis. The band comigrated in the gel with standard tubulin was sliced and the radioactivity counted using toluene/Triton X-100 (3:1, v / v ) containing 4 g / l of Omnifluor. The procedure has been shown to be highly efficient for the quantitation of tubulin from labeled supernatants and proven to be linear in the range of protein concentration 1 2 nag per assay [10]. Protein estimates were carried out according to Lowry et al. [ 15] and incorporation of [ 14C]leucine into total protein was determined by precipitation with 7.5% trichloroacetic acid. The precipitates were collected on Millipore filters (Type HA, 0,45 /,m), washed and the filters were counted as described before using a Packard scintillation counter. Results
In the rat, tubulin concentration increases 2--3fold between late fetal stage ( - 5 day) to early postnatal ( + 10 day) stage [16] and this period also corresponds to the period of thyroid hormone dependent maturation of the brain [3,4]. We therefore determined the effect of T 3 on the biogenesis of tubulin in organ cultures of brains of rats from late fetal and early postnatal stages. In this series of experiments, tissues were labeled with 2.0 ~ C i / ml of [~4C]leucine for 2 h in the presence or absence of 4.5 nM triiodothyronine (T3). Incorporation into total protein was determined by tri-
95 TABLE I m" 18o 'o
DOSE-RESPONSE P A T T E R N FOR T H E S T I M U L A T I O N O F T U B U L I N SYNTHESIS BY T R I I O D O T H Y R O N I N E (T3) IN T H E N E W B O R N R A T BRAIN
X
n- ~ 1 2 0 LU n
i-
o-
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Brains from newborn (0-24 h) rats were labeled with 2 I~Ci/ml of {~4C]leucine in media containing increasing concentrations (0 4.5 nM) of T 3 for 2 h. Radioactivity incorporated into total protein and tubulin were determined by precipitation with trichloroacetic acid and by vinblastine assay, respectively. Data represent mean ± S.D. of three experiments.
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cpm per mg total protein ( x l O -3)
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0(Control) 0.045 0.45 4.5
132.2± 8 132.8±10 135.1±10 134.5± 8
28.4±2 31.3±2 48.6±4 51.5±4
12 Adult
Fig. 1. Age-related stimulation of tubulin synthesis by T 3 in the embryonic rat brain. Organ cultures of rat brains of indicated ages were labeled for 2 h with 2 # C i / m l [taClleucine in the presence (hatched bars) or absence (open bars) of 4.5 n M T 3. Incorporation into tubulin (lower panel) as determined by vinblastine assay and total protein (upper panel) are shown. Data for brains from - 5 to 3-day-old rats represents Mean ± S.D. from three experiments and those for other ages are averages from replicate experiments with the bar representing variation from the average.
chloroacetic acid precipitation and tubulin was quantitated by the combined vinblastine precipitation and SDS polyacrylamide gel electrophoresis as described in 'Methods'. The results showed an age-dependent stimulation in the synthesis of
tubulin by T3 (Fig. 1, lower panel) in the absence of any significant effect in total protein synthesis (Fig. 1, upper panel). In control cultures, both total protein and tubulin synthesis progressively declined with age. However, in the presence of the hormone, total protein synthesis remained within + 10% of the controls at all ages; tubulin synthesis was stimulated by 60-80% in the brains from newborn (0-24-hold) rats and 40-50% in brains from late fetal ( - 2 day) or early postnatal (+ 1-day-old) rats. The uptake of [14C]leucine in the brains from newborn rats remained essentially unaltered by T3 (250000-280000 c p m / m g protein in both control and hormone-treated tissues). No stimulation of tubulin synthesis was detectable in brains from an
T A B L E Ii T I M E KINETICS F O R T H E S T I M U L A T I O N OF T U B U L I N SYNTHESIS BY T R I I O D O T H Y R O N I N E (T3) IN T H E NEWB O R N RAT BRAIN Brains were labeled with 2 / L C i / m l [14C]leucine in the presence or absence (control) of 4.5 nM T 3 for the indicated periods of time. Total protein and tubulin were quantitated as described in legends to Table I. Data represent mean ± S.D. from three experiments. Length of culture time
cpm per mg total protein ( x 10-3)
cpm in tubulin per mg total protein ( × 10 2)
(min)
Control
T3-treated
Control
T3-treated
30 60 120 180
30.1±2 52.2±2 127.8±5 153.3±5
31.1±3 55.3±3 129.1±5 165.1±5
8.3±2 16.1±2 29.2±2 36.3±3
13.4±2 26.1±3 52.3±2 58.5±5
96 TABLE Ill EFFECT OF T 3 ON THE STIMULATION OF C O L C H I C I N E B I N D I N G ACTIVITY IN BRAINS FROM FETAL ( - 5 DAY), NEWBORN A N D 5-DAY-OLD RATS Brains were cultured for 2 h in the presence or absence of the indicated concentrations of T 3. Colchicine assay was performed as described in Experimental procedures. Except for the 5-day-old brains data represents mean _+S.D. from three experiments. Figures in parenthesis represent stimulation over control, n.d., not determined. Concentration of T 3 in the culture medium (nM)
[3H]Colchicine bound per mg protein (cpm x 10-2) Fetal ( - 5 day)
Newborn
5-day-old
0 0.045 0.45 4.5
27.4 + 1 n.d. n.d. 30.1 +_2 ( + 10%)
56.4 _+4 67.0 + 5 ( + 20%) 79.4 _+5 ( + 39%) 85.4 + 6 ( + 51%)
49.0, 59.6 n.d. n.d. 58.4, 62.6 ( + 10%)
earlier period of gestation ( - 5 day) or in brains from older, including adult, rats. In view of the maximum stimulation of tubulin synthesis by T 3 in brains from newborn rats, the dose dependency and time-course of the hormonal effect were determined in organ cultures of brains from newborn rats. The dose-response pattern of this phenomenon is shown in Table I. Maximal stimulation of tubulin synthesis by ~ was observed at 4.5 nM and with 0.45 nM T 3, 80-90% of this stimulation could be achieved. No further increase in rate of tubulin synthesis could be achieved with higher concentration (45 nM) of the hormone (data not shown). Total protein synthesis was not significantly altered by ~ in any of the doses examined. Table II shows the time-course data for the synthesis of tubulin and total protein in brains from newborn rats cultured in presence or absence of 4.5 nM T 3. Treatment with hormone progressively increased synthesis of tubulin between the first 2 h of culture. During this period, total protein synthesis remained essentially unaffected. A definitive increase in tubulin synthesis could be seen within 30 60 min of the exposure to hormone and by 120 min the response appeared to reach the maximal level (60-80% of control). No significant enhancement of response was noticed at the 180 min time-point, although incorporation into both tubulin and total protein increased to some extent. The lack of additional effect after 2 h could be clue to the fact that protein synthesis in the explanted
organ tends to decline slowly after 2 - 3 h in culture. In the experiments described above, tubulin was quantitated from 100000 × g supernatants of labeled brain by vinblastine precipitation and gel electrophoresis. Since vinblastine is known to precipitate small amounts of actin and few other minor proteins in addition to tubulin [17], we further examined the effect of ~ on the level of tubulin by using another independent quantitative assay based on the [3H]colchicine binding of tubulin. Using the colchicine assay, we redetermined the age-dependency, dose-response characteristics and the tissue specificity of the hormonal effect. The results (Table III) are similar to those obtained previously from the assay based on vinblastine precipitation. Of the three ages examined ( - 5 day, newborn and + 5 day) significant stimulation in the level of tubulin by T 3 was seen only in the case of the newborn brain, following the doseresponse pattern similar to that seen in Table I. Furthermore, in tissues such as kidney and lung from newborn rats, no stimulation of tubulin by T 3 was detectable by colchicine assay (data not shown). Since the effects of T 3 are generally mediated by interaction of the hormone with the genome [18], we further examined by colchicine assay the effect of inhibition of transcription by 20 ~tg/ml of actinomycin D (which inhibited 75% of the incorporation of [3H]uridine into RNA) on the stimulation of tubulin synthesis by T 3. Data in Table IV
97 TABLE IV EFFECT OF INHIBITION OF TRANSCRIPTION ON THE STIMULATION OF TUBULIN (COLCHICINE-BINDING ACTIVITY) BY TRIIODOTHYRONINE (T3) IN CULTURES OF BRAINS FROM NEWBORN RATS Brains from newborn rats were cultured for 120 min in the presence or absence of 4.5 nM T3 and 20 #g/ml actinomycin D as indicated. Colchicine assay was performed as described in Experimental procedures. Data represent mean +S.D. from three experiments except for case 2 where two experiments were done. Figures in parenthesis represent stimulation over control. Addition in the culture medium
[3H]Colchicine bound per mg protein (cpm x 10 2)
1 None (Control) 2 Control plus 20 ktg/ml actinomycin D 3 4.5nMT 3 4 4.5 nM T3 plus 20/~g/ml actinomycin D
56.4+_ 4 61.6, 55.5 (+ 4%) 85.4_+ 6(+51%) 61.3 _+ 3 ( + 10%)
show that the enhancement of tubulin by T 3 requires transcription of new R N A . In these experiments, T~ stimulated the synthesis of tubulin by about 50% over control and in the presence of the drug, the stimulation was only about 10%. Finally, to determine the effect of T 3 on proteins other than tubulin, we analysed 100000 × g supernatants of brains from newborn rats cultured for 2 h in the presence or absence of 4.5 nM T 3 by disc gel electrophoresis using 7.570 polyacrylamide gels according to Weber and Osborne [14]. Comparison of the distribution of radioactivity in the sliced gel revealed no significant difference in the profile except in the region of marker tubulin which was stimulated by about 50% in the T 3t r e a t e d sample. However, since this procedure does not resolve a- and /3-tubulin, further analysis by more sophisticated methods are necessary to determine whether a- or /3-tubulin is preferentially stimulated by T 3. Discussion
The main conclusion from the present investigation - the induction of tubulin synthesis by thyroid h o r m o n e s - is established and supported by the results of several different types of experi-
ments. First, the induction is strictly age-dependent, as observed for m a n y other h o r m o n a l effects in developing tissues [19]. Second, the extent of induction is dose-dependent and reaches a maximal level between 0.45-4.5 nM, which is in the range of physiological concentrations of the h o r m o n e in rat brain [20]. In separate experiments (data not shown) we have determined that the nuclear receptors in adult rat brain can be saturated by incubation with 2.5-25 n M T 3. Third, the stimulation is tissue-specific and tissues other than brain, such as lung and kidney derived from the rats of inducible age, are totally insensitive to T 3. In the present study, enhancement of tubulin by ~ is found both by vinblastine and colchicine assays. It should be clarified that for vinblastine assay 100 000 x g supernatants from brains labeled for 2 h with 14C-labeled amino acids are used and thus only the newly synthesized labeled tubulin is measured. On the other hand, for the colchicine assay, unlabeled supernatants are used and the total tubulin content is measured by binding to [3H]colchicine. Thus the extent of stimulation by in the vinblastine assay (60-80% of the newly synthesized tubulin) is less than that observed by colchicine assay (1.5-fold stimulation of the total tubulin). Further investigation on the mechanism of this stimulation and in particular, studies on the effect of T 3 on the synthesis and stability of tubulin in normal and hypothyroid rat brains, are expected to clarify this problem. These results are consistent with the observations of Fellous et al. [9] that thyroid h o r m o n e s are required very early after birth for normal microtubule assembly and neurite growth. Their experiments clearly established that microtubule assembly is retarded in the hypothyroid rat brain and suggested that the limiting factor is p r o b a b l y one of the microtubule associated protein ' t a u ' [21]. The present studies indicate that thyroid h o r m o n e might control the assembly/biogenesis of the neurotubule proteins by regulating both the level of tubulin and the microtubule-associated proteins. Gonzales and Geel [22] examined the effect of hypothyroidism on the concentration and biochemical properties of tubulin in 12-25-day-old developing rats and found no evidence of any defect in tubulin synthesis in brains from 12-25-
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This research was supported by grants from the Council of Scientific and Industrial Research, Government of India. References
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Fig. 2. Temporal relation between the stimulation of tubulin synthesis by T3 (present study), binding capacity of nuclear rl~ receptors [23] and the period of rise in tubulin [16] in the developing rat brain.
day-old hypothyroid rats. Francon et al. [9] also reported no difference in tubulin content per g whole brain for 15-day-old control and hypothyroid rats. In both of the above studies, the effect of thyroid hormone deficiency in the late fetal or newborn rats was not determined. It should be recalled that the most critical period of thyroid hormone action in rats is limited to the first 10 days after birth when the pyramidal neuronal differentiation occur [3,4] and this period also coincides with the period at which the tubulin concentration reaches the peak level [16]. Fig. 2. depicts an interesting temporal correlation between the stimulatory effect of T~ on tubulin synthesis, the level of nuclear receptors for T 3 and the developmental profile of tubulin in the rat brain. The binding capacity of the nuclear T 3 receptors in rat brain rises rapidly between the fetal stage (2 days before birth) and early postnatal stage (2 days after birth) and declines thereafter [23]. The coincidence of the period of maximal sensitivity of the brain to stimulation of tubulin synthesis by T3 with the period of rise in the level of ~ receptors and that of tubulin in the rat brain strongly indicate that the thyroidal induction of tubulin synthesis in the developing brain is a natural ontogenic phenomenon.
1 Grave, G.D. (1977) Thyroid Hormones and Brain Development, Raven Press, New York 2 Sokoloff, L. and Kennedy, C. (1973) in Biology of Brain Dysfunction (Graniet G.E., ed.), pp. 295-305, Plenum Press, New York 3 Bass, N.H., Pelton, E.W. and Young, E. (1977) in Thyroid Hormones and Brain Development (Grave, G.E, ed.), pp. 199-214, Raven Press, New York 4 Lauder, J.M. (1977) in Thyroid Hormones and Brain Development (Grave G.D., ed.), p. 235 254, Raven Press, New York 5 Daniels, M.P. (1972) J. Cell Biol. 53, 164 176 6 Yamada, K.M., Spooner, B.S. and Wessels, N.K. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 1206 1212 7 Seeds, N.S. and Maccioni, R.B. (1978) J. Cell Biol. 76, 547 555 8 Peters, A. and Vaughn, J.E. (1967) J. Cell Biol. 32, 113-119 9 Fellous, A., Lennon, A . M , Francon, J. and Nunez, J. (1979) Eur. J. Biochem. 101, 365-376 10 Chaudhury, S., Chaudhury, L. and Sarkar, P.K. (1982) Dev. Brain Res. 4, 241-243 11 Soh, B.M. and Sarkar, P.K. (1978) Dev. Biol. 64, 316-328 12 Weisenberg, R.C., Borisy, G.G. and Taylor, E.W. (1968) Biochemistry 7, 4466-4478 13 Feit, H., Dutton, G.R., Barondes, S.H. and Shelanski, M.L. (1971) J. Cell Biol. 51, 138-147 14 Weber, K. and Osborne, M. (1969) J. Biol. Chem. 244, 44O6 4412 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol Chem. 193, 265-275 16 Nunez, J., Fellous, A., Francon, J. and Lennon, A.M. (1975) in Microtubules and Microtubule lnhibitors (Berges, M. and De Brabander, M., eds.), pp. 269-278, North-Holland Publishing Co., Amsterdam 17 Wilson, L., Bryan, J., Ruby, A. and Mazia, D. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 807 812 18 Oppenheimer, J.H. (1979) Science 203, 971-979 19 Litwack, G. (1970) in Biochemical Actions of Hormones, Academic Press, New York 20 Schwartz, H.L. and Oppenheimer, J.H. (1978) Endocrinology 103, 267-273 21 Francon, J., Fellous, A., Lennon, A. and Nunez, J. (1977) Nature (Lond.) 266, 188 190 22 Gonzales, L W . and Geel, S.E. (1978) J. Neurochem. 30, 237-245 23 Schwartz, H.L. and Oppenheimer, J.H. (1978) Endocrinology 103, 943 948