Nuclear triiodothyronine receptors in the developing rat brain

Molecular and Cellukzr Endocrinology,

o Elsevier/North-Holland

11 (1978) 31-41 Scientific Publishers, Ltd.

NUCLEAR TRIIODOTHYRONINE BRAIN

RECEPTORS IN THE DEVELOPING RAT

T. VALCANA ’ and P.S. TIMIRAS ’ 1 Department of Human and Animal Physiology, School of Natural Sciences and Mathematics, University of Patras, Patras, Greece, and 2 Department of Physiology-Anatomy, University of California, Berkeley, CaliJ 94720, U.S.A. Received 10 November 1977; accepted 31 January 1978

This study examines whether the high sensitivity of the developing brain to thyroid hormones and the purported decline in sensitivity in adulthood, are correlated with changes in the density and affinity characteristics of specific nuclear Ts receptors. The authors have found that the nuclei of cerebral hemispheres have a high density of Ts receptors at birth (212 f 28 X 10-l 7 mol/pg DNA) which declines to adult levels by the end pf the second postnatal week (115 f 7 X 10-l 7 mol/pg DNA), remaining at this level until 6 months of age. Even though no significant changes were detected in the equilibrium dissociation constant (Kd) during the early period of development, comparison of neonatal with the adult brain reveals a decrease in Kd (neonatal, 3.9 X lo-r0 M; adult, 2.3 X lo-lo M). In the developing animal, neonatal thyroidectomy increased the number of binding sites in the nucleus by 36-44%. It is concluded that the high number of nuclear Ts receptors in the first week of postnatal life is correlated with the high dependence of brain tissue on thyroid hormones and that the decline in brain sensitivity may be associated with the decline in nuclear T3 receptors. The high affinity and density of nuclear receptors in adult brain tissue relative to the developing brain and liver, respectively, point to a continued regulatory role of thyroid hormones in brain. Keywords:

brain Ts receptors; age-related changes; ‘ihyroidal status.

The brain is markedly dependent on thyroid hormones for its overall growth and its biochemical and morphological development (Gee1 and Timiras, 1970; Balais et al., 1971; Sokoloff and Kennedy, 1973). In contrast, the mature brain is not responsive to these hormones, at least in terms of the biochemical parameters known to be regulated by thyroid hormones in the developing tissue (Fazekas et al., 1951; Ismail-Beigi and Edehnan, 1971; Valcana, 1974). Although we know that many biochemical processes in the brain are altered by lack of thyroid hormones during development, we have not been able to single out a primary locus of thyroid hormone action in this tissue. Recently, specific thyroid hormone receptors have been identified in nuclei of tissues known to be thyroid-hormone-responsive and it has been hypothesized that the nucleus is the locus of thyroid hormone action. The 31

32

T. Valcana, P.S. Timiras

nuclear thyroid hormone receptors are acidic chromatin proteins (60 000-70 000 Mr) that bind triiodothyronine (Ta) with greater affinity than thyroxine (T4); however, the steps subsequent to binding that lead to the physiological expression of thyroid hormone action have not as yet been delineated (Surks et al., 1973; Oppenheimer et al., 1974a, b; Samuels et al., 1974; Thomopoulos et al., 1974). It has been demonstrated that nuclei isolated from the rat adult brain contain highaffinity T3 receptors with binding characteristics similar to those found in liver nuclei (Oppenheimer et al., 1974a, b; Valcana and Timiras, 1976a; Eberhardt et al., 1976, 1978). In contrast to brain nuclei, specific Ta- or T4-binding is not found in brain mitochondria or in synaptosomal and microsomal fractions (Eberhardt et al., 1976; Lazarus et al., 1976). Further, we have shown that T3 receptors are more abundant in nuclei isolated from the pituitary and the cerebral hemispheres than they are in liver, in the whole brain, or in brain regions such as the cerebellum, the mesodiencephalon and the hypothalamus (Eberhardt et al., 1976; Valcana and Timiras, 1976a,b). In the present study, we have sought to determine whether the extreme dependence of the brain on thyroid hormones during development, and its relative insensitivity to these hormones in adulthood, are associated with maturational changes in the number and/or affinity properties of nuclear Ta receptors. In addition, we have attempted to establish whether thyroid gland activity influences the ontogeny and/or the binding characteristics of brain receptors. Changes in the binding of Ts to receptors of nuclei isolated from the cerebral hemispheres were followed in vitro at various intervals from 1 day to 6 months of age. The hormonal influence was studied by comparing Ta-binding in nuclei from control and neonatally thyroidectomized rats.

METHODS Animals and their treatment

Long-Evans rats of both sexes, kept on a standard rat chow diet, were used in this study. The ontogeny of nuclear thyroid hormone receptors in the cerebral hemispheres was studied in rats at critical stages of brain development (1,6,8 and 13 days), at the period of brain and endocrine maturation (1 and 2 months) and in adulthood (6 months). The effects of thyroid hormone deficiency were studied in neonatally thyroidectomized animals of 13-14 and 30-33 days of age. Neonatal hypothyroidism was accomplished by radiothyroidectomy at birth as described in our previous studies (Valcana and Timiras, 1969). Untreated Iittermates served as controls. Isolation of nuclei

The cerebral hemispheres including the entire neocortex, hippocampus, amyg-

33

Tg receptors in developing brain

Table 1 Triiodothyronine (TJ) binding to cerebral nuclei from developing rat brain Age 1 day 6 days 9 days 13 days 33 days 2-6 months

Protein : DNA

Ts-binding sites (IV) (10-l ’ mol/MgDNA)

(Kd)

212 f 28 a) (5) 190 r 20 (2) 97 f 6 (3) P < 0.01 115? 7 (6) P = 0.02 106 + 18 (5) P= 0.05 120+ 9 (10) P< 0.01

3.9 f 0.80 (5) 6.1 + 0.01 (2) 5.7 f 0.19 (3)

2.11 r 0.20 (6) 2.28 f 0.15 (3) 1.98 + 0.15 (3)

4.2 * 0.41 (6)

2.09 f 0.06 (6)

3.6 2 0.77 (5)

1.98 t 0.14 (5)

2.31 + 0.37 (10) P = 0.05

2.30 + 0.16 (9)

(10-l O mol/l)

a) Numbers represent the means f S.E. from the number of samples shown in parenthesis. Each nuclear sample was obtained from several animals (see Methods) and was assayed in duplicate. P value indicates significance of difference from lday-old values.

dala, septum and caudate, were dissected from the brain immediately after decapitation of the animal. At least 5 g of brain tissue (cerebral hemispheres) were used in each experiment, thus requiring the pooling of tissue from several animals (50 for l-day-old, 25 for 6- and g-day-old, 10 for 13- and 35-day-old and 7-10 for adults), depending on the age interval studied. The tissue was homogenized in 0.32 M sucrose and centrifuged at 1000 g for 10 min. The nuclear pellet was washed 3 times in 0.32 M sucrose and each time the debris and the supematant was discarded. The debris consisted mainly of unbroken neural cells, red cells and membraneous contaminants; it was easily distinguished from the pink-colored mass of nuclei,and was removed by suction. The nuclei were subsequently washed in Triton X-100 buffer containing 25 mM KHZP04, 1 mM MgClz, 20 mM /3-mercaptoethanol, 0.5% Triton X-100, pH 7.5, and finally washed once with Triton-free buffer of the above composition. The integrity and purity of nuclei were verified by phase-contrast and electron microscopy and by the determination of the protein : DNA ratio. The ratio of protein : DNA for all ages studied was approximately 2 (table 1). By all other criteria used the preparations were pure. Protein was measured by the method of Lowry et al. (195 l), DNA by the method of Burton (1956). T3 nuclear binding in vitro

The binding of T3 to nuclei isolated from the cerebral hemispheres was determined in vitro (within l-2 h after isolation) using the competitive binding assay system of Samuels and Tsai (1973) as modified by Eberhardt et al. (1978). In preliminary experiments we determined the dependence of T,-binding on incubation temperature and the magnitude of excess unlabeled T3 to be utilized for

34

T. Valcana, P.S. Timiras

determination of nonspecific binding. Based on the results of these experiments (described in detail below) and those from our previous studies (Eberhardt et al., 1978), all subsequent binding assays were performed as follows. Nuclei (20-100 /_tgDNA) were incubated in 1 ml of buffer of the above composition (minus Triton) and with various concentrations of L-[1251]triiodothyronine. L[1251]Ta (spec. act. 400-500 Ci/r.cg)was purchased from Abbott Laboratories and was 95% pure, as assessed by paper chromatography (Taurog and Chaikoff, 1957). Eight to ten concentration values in the range 50-1000 X lo-l2 M were examined. At all concentrations of radioactive Ts examined, identical incubations were carried out with 100 M excess of unlabeled T3 (100 M cold Ts excess was found as effective as 1000 M excess in displacing labeled Ta). Duplicate assays were performed at each concentration employed. The experiments were repeated several times in order to determine the significance of difference in the data obtained from various experimental groups. At the end of incubation, samples were centrifuged at 10 000 g for 10 min. The supernatant was sampled and counted, and the free hormone concentration was determined by utilizing the efficiency of the counting, the decay constant, and the specific activity of the radioactive hormone. This procedure is possible inasmuch as no metabolism of T3 takes place under similar incubation conditions (Samuels and Tsai, 1973) and the radioactivity in this fraction behaves as free Ta, as shown by Sephadex G-25 gel chromatography and acrylamide gel electrophoresis. The pellets were subsequently washed once in phosphate buffer containing 0.5% Triton X-100, centrifuged at 10 000 g for 10 min, and the supernatant was discarded. The pellets were then counted for determination of bound radioactivity which was expressed as moles bound per ng DNA. The specific binding was determined by taking the difference between the total bound radioactivity and the radioactivity that could not be competitively displaced by incubating the nuclei in loo-fold molar excess unlabeled Ta.

RESULTS In accordance with our previous findings (Eberhardt et al., 1978), these studies also indicate that at 37’C,equilibriumis reached by 10 min (fig. 1). We have utilized 37’C and 30 min incubation time in order to assure time for dissociation of any endogenously bound hormone (Surks and Oppenheimer, 1976) thus permitting proper comparison of control and hypothyroid conditions. The maximal binding capacity of nuclei (AJ) and the equilibrium dissociation constant (Kd) were similar whether incubation was carried out at 37’C or at 22’C (data not shown). Table 1 depicts changes in the apparent nuclear binding sites per pg DNA (N) and equilibrium dissociation constants (Kd) from birth to 6 months of age. At birth, N is high, decreasing to adult levels within the first two weeks of postnatal life with no further significant changes from 13 days to 6 months of age. The Kd does not change significantly early in development; however, it declines significantly in

35

T3 receptors in developing brain

201

I 0

I IO

I

I 20

1

I

I

I

II

40

30

Time (minuter)

Fig. 1. Timecourse of specific binding of [ 1251]Ta to nuclei isolated from the cerebral hemispheres of control (oo) and hypothyroid (o- - I- - - 0) 13-day-old rats. Nuclei (27 pg DNA, control; 29 fig DNA, hypothyroid) were incubated with 5 X IO-r0 M [1251]T3 in 1 ml phosphate buffer, at 37’C. Points represent mean + SD. of two determinations.

nuclei from animals older than one month of age (table 1). The binding of Ts to nuclei obtained from cerebral hemispheres of control and hypothyroid animals is illustrated in figs. l-5. Fig. 1 depicts the change in binding with incubation time. These data show that the binding by hypothyroid nuclei is higher than that of controls at all time intervals studied, even after equilibrium is reached. Fig. 2 shows the dependence of specific binding on T3 concentration in the incubation medium for nuclei isolated from control and hypothyroid 13-dayTable 2 Effect of neonatal thyroidectomy

Age (days) 13 30-33

on ‘Ts binding to cerebral nuclei

Experimental group

Ts-binding sites (N) (10-l ’ mol/pg DNA)

(Kd) ( lo-r0

Control Hypothyroid Control Hypothyroid

116 i 167 * 113i 154 *

4.2 3.7 2.8 2.2

8 a) 8 b) 9 19 b)

(4) (4) (3) (3)

mol/l)

r 0.48 * 0.52 + 0.17 * 0.11

a) Values are means f SE. from a number of samples shown in parenthesis. Each nuclear sample was obtained from several animals (see Methods) and was assayed in duplicate. b) Significantly different from control: P = 0.05 by Student’s t-test for paired data.

36

T. Valcana,

P.S.Timiras

80

0 0

20

60

40

00

I00

Fm T3 WI-‘1 M)

Fig. 2. The dependence of [ 1251]Ts specific binding on the concentration of free [1251]Ts in the incubation medium in nuclei from control (o) and hypothyroid (o- - - - - -0) 13day-old rats. Nuclei (27 pg DNA, control; 29 pg DNA, hypothyroid) were isolated and assayed as described in Methods. The ordinate represents specitkaIly bound hormone per ng DNA. The abscissa represents the concentration of free Ta in the medium determined at the end of the incubation as described in Methods. Points represent means of duplicate determinations S.D.

2 0’

m

0 0

20

40 Free Tg (lO_”

60

80

100

M)

Fig. 3. The dependence of [tz5 I]Ts specific nuclear binding on the concentration of free [12sI]Ts in the incubation medium. Nuclei (18 pg DNA, control; 22 c(g DNA, hypothyroid) were isolated from the cerebral hemispheres of 3Oday-old control (o------e ) and hypothyroid (o- - - - - -0) rats. Ordinate and abscissa are as described in fig. 2.

37

T3 receptors in developing brain 5

I

1

I

o\

I

I

\ \ \

9 \

4

\ 0 \ \

0 \ \

-

3

\

l

\ \

l l

\

0

\

0

\ -\

0 \

\ \ 0

l

0 \ 0

l

\

l

b \

\

l

0 0

40 Bound T3

120

80

(IO-l7 moles/

Irg DNA

Fig. 4. Scatchard analysis of [ I* 51]T3 specific binding to cerebral nuclei from control (*-) and hypothyroid (c- - -,- - -0) 13day-old rats. The ordinate represents the ratio of specifically bound hormone per ccg DNA to the concentration of free hormone in the incubation medium. The abscissa represents the specifically bound hormone per r.cgDNA. Points represent means of duplicate determinations. These data correspond to those depicted in fig. 2.

T. Valcana, P.S. Timiras

0

I

0

I

I

40

Bound T3 (lo-l7

I

I

I

80 moleslug

I

120 DNA

Fig. 5. Scatchard analysis of [ 125l]Ts specific binding to cerebral nuclei from control (o------o) and hypothyroid (o- - - - - -0) 30day-old rats. The ordinate and abscissa are as described in fig. 2. These data correspond to those depicted in fig. 3.

old rats, while fig. 3 depicts this relationship in control and hypothyroid 30-day-old rats. Analysis of these data by the method of Scatchard (1949) is presented in figs. 4 and 5, respectively. The equilibrium dissociation constants and the number of binding sites were estimated from the Scatchard plots of binding data from several nuclear preparations from control and hypothyroid animals. Table 2 provides a summary of data on the effects of neonatal thyroid hormone deficiency on nuclear receptors. These data indicate that while there is no significant change in Kd the density of receptors is significantly higher in nuclei isolated from hypothyroid animals, an effect observed as early as 13 days post-thyroidectomy. Similar findings have been reported in studies of liver (DeGroot et al., 1977; Valcana and Timiras, 1978).

T3 receptors in developing brain

39

DISCUSSION Our interpretation of the observed changes in nuclear Ts receptors with development as well as with hypothyroidism is based on the fact that we are assaying total nuclear binding and not only free receptors and that the receptor-hormone complex formed in vivo does dissociate invitro and can indeed rebind the hormone. Although we have no direct data on whether the latter property is maintained, we do know, from our studies in liver (where we observe similar changes due to hypothyroidism), that the hormone bound to nuclei after injection of T3 in vivo does dissociate in vitro and that equilibrium is reached within lo-15 min under incubation conditions similar to those employed here (Valcana and Timiras, 1978). In addition (unpublished data), the in vitro binding to brain nuclei isolated from animals injected with 18 pg Ta per 100 g body weight, 2 h prior to sacrifice, was similar to that of controls. The high density of nuclear Ts receptors found in the cerebral hemispheres during the first week of brain development may be correlated with the critical dependence of the brain on thyroid hormones at this time. The decline in the number of brain receptors to adult levels by the second week may underlie the decline, with age, in the sensitivity of the brain to thyroid hormones. Developmental changes in nuclear thyroid hormone receptors have also been found in other tissues. For example, a high density of nuclear Ts receptors has been found in the tadpole tail at the peak of metamorphosis, followed by a decline in the adult (Yoshizato and Frieden, 1975). The decline in Kd with development observed in this study, has also been observed in nuclei isolated from heart tissue (Tsai and Chen, 1976). In contrast to brain, developing liver shows an increase in nuclear T3 receptor density with no significant change in Kd (De Groot et al., 1977; Valcana and Timiras, 1978). Although the decline in nuclear Ta receptor density may explain the decline in brain sensitivity to thyroid hormones, the fact that the affinity and the number of T3 receptors in the cerebral hemispheres remains relatively high in the adult brain relative to those of other thyroid-hormone-responsive tissues, such as the liver (Eberhardt et al., 1978; Valcana and Timiras, 1978) argues for a continued role of T3 in the mature brain. Indeed there are several lines of evidence, clinical, behavioral and experimental, that indicate that the adult brain tissue does depend on thyroid hormones for regulation of neural function (Timiras and Woodbury, 1956; Prange, 1974; Jacoby et al., 1975; Ito et al., 1977). On the other hand, the mere presence of receptors does not constitute proof of their biological role and relevance in the adult brain, and the properties of brain nuclear receptors and their role in the adult brain must be delineated further. The hormonal activity depends also on the number of occupied nuclear sites, which could be lower in the adult relative to the developing brain and/or liver, due to a decrease, with development, in the Ts transport to the nucleus and/or cytoplasm. The aspects of Ta transport to the nucleus are not as yet clear; however, the study of Timiras and Luckock (1974) indicates that there is a decrease in the uptake of T3 by brain tissue with development.

40

T. Valcana. P.S. Timiras

Neonatal hypothyroidism is associated with a marked increase in the binding capacity of brain nuclei (figs. l-5, table 2); this change, also found in the liver (Valcana and Timiras, 1978), can be demonstrated in the adult animal as well, but only if hypothyroidism is prolonged beyond 2 weeks (data not presented here). Previous reports in the literature, however, have indicated that liver nuclear receptors are not altered with thyroidal status in terms of their density or affinity for Ta (Dillman et al., 1974; Silva et al., 1975; Spindler et al., 1975). Our results indicate that only severe and/or prolonged deviations from normal thyroidal status induce changes in nuclear binding capacity. In the studies mentioned above, hypothyroidism was not prolonged (2 weeks) and therefore thyroid hormones may not have been sufficiently depleted from the circulation and/or tissue stores to affect receptor changes. Neonatal hypothyroidism represents a severe condition because thyroidal activity is interrupted at an early developmental age when thyroidal synthetic capacity and circulating hormone levels are low, the pituitary-thyroid axis has not yet developed (Beltz and Reineke, 1968; Cons et al., 1975; Dussault and Labrie, 1975), and the animal does not receive exogenous T3 through food sources. In this condition the effects are demonstrated as early as 13 days post-thyroidectomy (table 2); in the adult state, however, the effects are not evident until at least 3 weeks post-thyroidectomy (Valcana and Timiras, 1978). Our data are interpreted as indicative of a regulatory role of thyroid hormones on their nuclear receptors; that is, an increase may occur in the nuclear binding sites in hypothyroidism. That hormones influence the composition of chromatin in terms of their own receptors is known for estrogens in their target tissues (Teng and Hamilton, 1970; O’Malley and Means, 1974; Cohen and Hamilton, 1975; Chan and O’Malley, 1976) and has also been suggested for thyroid hormones (Samuels et al., 1976). Proof that thyroid hormones influence their own receptors would be of considerable importance to the clarification of the mechanisms involved in thyroidnuclear receptor interactions. These studies must also be expanded to include the effects of the hyperthyroid state, for a better understanding of the proposed regulatory role of T3 on its nuclear receptor.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the technical assistance of Ms. Carole Miller. We also thank Ms. Ming-Ming Ho for her help with electromicroscopy of the nuclear preparations. The newborn animals were generously supplied by Ms. Jacqueline Ehlert of the Physiology-Anatomy Colony, University of California, Berkeley, U.S.A.

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TCJreceptors in developing brain

41

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