Expression of thyroid hormone receptors mRNAs in rat cerebral hemisphere neuronal cultures

Developmental Brain Research, 69 (1992) 173-177

173

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00

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Expression of thyroid hormone receptors mRNAs in rat cerebral hemisphere neuronal cultures J. Puymirat, S. l ' H e r e a u l t and J.H. D u s s a u l t Department of Ontogenesis and Molecular Biology, CHUL Research Center, Sainte-Foy (Canada) (Accepted 9 June 1992)

Key words: Brain culture; Neuron; Brain development; c-erbA; Thyroid hormone receptor

We have studied the expression of the a and/3 thyroid receptors mRNAs (TR-mRNAs) in cerebral hemisphere neuronal cultures, initiated from 15-day-old rat embryos, by northern analysis. In the cultures grown in the absence of t.-triiodothyronine (L-T3), the a2 TR-mRNAs were the predominant form of TR-mRNAs and were approximately 8 to 20-fold higher than the levels of the al TR-mRNAs, depending on the age of the cultures. The levels of a2 TR-mRNAs significantly increased by 1.8 fold between day 8 and 15 and remained on a plateau value thereafter until day 22. Over the same time period, there were no significant changes on the levels of al TR-mRNAs. The ratio a l / a l + a2 TR-mRNAs decreased between day 8 and 15. The/31 TR-mRNAs increased by 8 fold between day 8 and day 22. On day 8, the 01 TR-mRNAs were 1.8 fold lower than the levels of the a l TR-mRNAs while they were 6 fold higher on day 22. L-T3 treatment of the cultures had no effect on the levels of the a l , a2 and/31 TR-mRNAs. The differential temporal expression of the a l and/31 TR-mRNAs suggests distinct functions for both types of T3 receptors in neuronal maturation.

INTRODUCTION The importance of thyroid hormones during brain development is well established. The most striking effects are observed during brain development where the absence of thyroid hormones produces multiple morphological and biochemical alterations (Legrand~5). However, little is known on the mechanisms by which these hormones produce their effects. It is generally accepted that thyroid hormones act through specific nuclear receptors (TR) 22. Displacement analyses have demonstrated the presence of L-triiodothyronine (L-T3) binding sites in the developing 5'2°'3L34and the adult rat brain 6,s.13,2z3°'36.L-T3binding sites have also been found in cultures of neurons and astrocytes ~6'23'25.Two thyroid receptor genes have been identified and classified in a and /3 subtypes, based on their localization on human chromosomes 17 and 3, respectively t9,29'33'sS. Alternate splicing of the a gene is responsible for the formation of a 5.0-kb mRNA which codes for a T3 binding protein (al) and two 2.6-kb mRNAs which code for two proteins (a2) which do not bind L-T3. Alternate splicing of the/3 gene generates two 6.0-kb

mRNAs which code for two proteins (/31 and/32), both of which bind T3. mRNAs coding for two L-T3 binding proteins (TRal and TRBI) and for the a2 variant have been demonstrated in the adult rat brain 2'1sA9'32'33. Furthermore, the a and/3 TR-mRNAs have a distinct pattern of expression in the developing rat brain 21'20's2. The al TR-mRNAs are the predominant form of TR-mRNAs between the 15th fetal day and postnatal day 7 whereas the ~1 TR-mRNAs become predominant thereafter 26'32. 1"o date, the expression of TRmRNAs in specific nerve cell populations remains unknown. We report here the expression of a and /3 TR-mRNAs in rat cerebral hemisphere neuronal cell cultures. Although it has been reported that L-T3 does not regulate TR-mRNAs in the adult rat brain 32, we examined this problem in vitro, since we cannot exclude that L-T3 regulates TR-mRNAs in specific nerve cell populations in the developing rat brain. MATERIALS AND METHODS Cerebral hemisphere cultures Neuronal cultures. Primary cultures of dissociated cerebral hemisphere cells from 15- to 16-day-old Sprague-Dawley rat fetuses were

Correspondence: J. Puymirat, Ontogenesis and Molecular Biology, RC-9300, CHU Laval, 2705 Bd Laurier, Ste-Foy, Qua., Canada G1V 4G2. Fax: (1) (418) 654-2748.

174 prepared as previously described ~. Alter mechanical dissociation in serum-stlaplemented medium, the cells were centrifugated and resuspended in chemically defined medium (CDM). An aliquot of the cell suspension containing 3 x 10(' cells was plated in 10 ml of CDM on 100 mm diameter tissue culture dishes which had been previously coated with gelatin (250/~g/ml), incubated overnight with polylysine (10p.g/ml), rinsed with phosphate-buffered saline (PBS) and preincubated with 10% fetal calf serum (FCS) stripped of thyroid hormones according to the procedure of Samuels et al. 2~. The composition of the CDM was that previously described for similar cultures TM. Cultures were maintained in a humidified atmosphere of 5% CO 2, 95% air at 37°C. The medium was renewed 3 days after seeding and every third day thereafter. On day 6, cytosine arabinosine (I × 10 -~ M) was added to the cultures in order to suppress the overgrowth of astrocytes. In experiments with thyroid hormones, L-T3 was added at the initiation of the culture and renewed at every change of the medium. Based on previous dose-response studies, L-T3 was used at a final concentration of 3x 10 -s M TM in these conditions, 80 to 85% of the cells were neurofilament positives attesting their neuronal phenotype. The remaining cells were GFAp positives attesting their astrocyte phenotype TM.

Northern hh)t hybridization Probes. Synthetic 48-base oligodeoxyribonucleotide probes specific to /tit TRal (sequence 1611-1658, 1721-17683"~), TRa2 (sequence 1637-1684t4), common to TRal and TRa2 (sequence 682-72914 ), and specific for TR#II (sequence 1412-1459, 1601-1648 II) were prepared on an applied Biosystems DNA synthetizer and purified on an 8 M urea/8 M polyacrylamide gel. Probes were 3' end labeled by using terminal deoxynucleotidyl transferase and [a-'~2P]dATP (NEN, 30(X) Ci/mmol) and purified through ncnsorb column (NEN). Prepamthm oJ" RNAs. Total RNA from 8-, 15-, and 22-day-old cullures grown in the absence or in the presence of I.-T3, were isolated by using guanidium isothiocyanate and poly(A) + RNA by oligoduoxyribothymidylate cellulose chromatogrui)hy as described 17. For each determination, five dishes were pooled, tlyhridization pnJcedl(r(,. 5 /~g of poly(A) ÷ from neuronal cultur,~ were subjected to ulectrophoresis in a I% agarose/fl)rmaldehyde (20 mM MOPS, 5 mM sodium acetate, I mm EDTA, pH 7.0, and 0,66 M formaldehyde), The RNAs wer~ transfered by vacuum onto a nylon membrane (Hybon) at 50 V fi)r 4 h, Th~n the RNAs weru fixed by UV irradiation G)llow~d by bathing at 80°C h)r 2 h. The blots w~rc prehybridizud at 37°C overnight in 50% duioniz~d t'ormamid~, 50 mM NaH,PO~, pH %4, I mM EDTA, 0,75 M NaCI, I xDenhardt's. tlybridizations were done overnight at 37°C with 2 x 10¢' cpm/ml of '~"P-labeled probes in the prehybridation solution, Blots were washed twice at room temperature in I xSSC, then once with I xSSC, I% SDS for 20 rain at 42°C and twice with ! x SSC, I% SDS for 20 rain at 50°C. The blots were autoradiographed on Kodak XAR-5 film and the intensity of the band were quantified by video densitometry. The blots were dehybridized in 5 mM Tris, pH 8.0, 0.2 mM EDTA, 0,05% sodium pyrophosphalc, 0,Ix Denhardt's, at 68°C for 3 h and rehybridized with alternate prob~, In order to standardize the procedure, the following protocol was used: each blot was made in duplicate. The first blot was hybridized with the /~ probe and the films were exposed at -70°(2. After dehybridization, the blots were rehybridizcd with the t~ probe in the same condition as above and the films were exposed at -70°C for the same time. The second blot was fi~t hybridized with the t~ probe G)llowed by the # probe, Variations in the degrees of labeling probes haw ~)een corrected by using hybridization of poly(A ~ ) from pituitary tumor cell lines (GHI), as external standard in each experiment. RNAs' req.avery was determined by hybridization of the membrane with cyclophilin probe and all values were normalized to the cyclophilin signal4. The poly(A ~ ) concentration was measured by absorbance at 260 nm. For DNA determination, four parallel dishes for each experiment were used to determine the amount of DNA, according to the procedure described by Burton et al). Results were expressed as a,'britrary densitometric units ( l e D ) / mg of DNA. Quantitative data were expressed as the mean + SEM.

:% ",)~i

0

,~-

5.0-2.6-,i I

1

2

3

4

Fig. I. Northern blot hybridization of rat brain poly(A) + using specific oligonucleotide probes, six p,g of Poly(A) + were subjected to electrophoresis, transfcred to nylon membrane and hybridized with specific probes. Lane h T R a l and TRa2 common probe; Lane 2: TRal-specific probe; Lane 3: TRa2-specific probe; Lane 4: TR/3 I-specific probe.

Statistical significance was evaluated by Student's t-test. P < 0.05 was considered statistically significant.

RESULTS

iiy!Jridization specificity Poly(A) + from adult rat brains were used to determine the specificity of the hybridization. The al spe.

cific probe detected mRNAs of 5 and 6 kb whereas the a2 specific probe recognized rnRNAs of 2.6 and 6 kb (Fig. I). The probe common to TRal and TRa2 recognized the al and a2 rnRNAs. The /31 specific probe recognized a mRNA of 6 kb. The size of mRNAs recognized by each probe corresponds to the expected size of al, a2, and /31 mRNAs reported previously zl'J. The 6 kb mRNA recognized by the aland a2-specific probes may correspond to a RNA precursor as suggested by others zt').

Expression of TR mRNAs in neuronal cell cultures in neuronal cultures grown in the absence of L-T3, the probes hybridized with mRNAs of the same size as found in the adult rat brain (Fig. 2A). The t~2TRmRNAs were the predominant form of TR-mRNAs and were approximately 8- to 20-fold higher than the alTR-mRNAs, depending on the age of the cultures (Fig. 2B). The levels of a2 TR-mRNAs significantly (P < 0.001) increased by 1.8 fold between day 8 and day 15 and remained on a plateau value thereafter until day 22. The alTR-mRNAs slightly decreased with time in culture, although this decrease was not

175

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-2.6

cyclophilin

-

w ww. 8

T3

-

b

15 +

-

+

22 -

+

B 3000

Ip B 2000

ai

Ila2

1000

0 8

18

22

DAY8

Fig. 2. Expression of TR mRNAs in neuronal cultures. A u t o r a d i o gram of Northern blots of poly(A) + prepared from 8-, 15- and 22-dapold neuronal cultures grown in the. absence or in the presence of L-T3, hybridized with/31, a common, and cyclophilin probes (A). Films were exposed 15 days and 2 h for TR-mRNAs and cyclophilin, respectively, Northern blots were quantified as described in Materials and Methods (B), Results are expressed as arbitrary unit/mg of DNA and represented the mean :1:S,E,M, of $ different experiments,

significant. The ratio al/al + a 2 TR-mRNAs decreased with time in cultures (Table I). In contrast, the development of /~1 TR-mRNAs greatly differs from that of the a l and a2TR-mRNAs. After 8 days in vitro, the/31 TR-mRNAs were approximately 1.8 fold lower than the levels of the alTR-mRNAs (Table I). Between day 8 and day 22, the level of/31 TR-mRNAs increased by 8 fold (P < 0.001) and became the major form of TR-mRNAs between the 15th and 22th day of culture. On day 22, the /31 TR-mRNAs were 6 fold higher than that of the alTR-mRNAs (P < 0.001) (Table I). As shown in Table I, L-T3 treatment had no significant effect on the levels of al, a2 and/31 TR-mRNAs, whatever the age of the cultures. DISCUSSION In the present study, we report the expression of TR-mRNAs in enriched neuronal cultures. Northern

blot analysis is often considered as a non-suitable method for the comparison of different mRNAs. However, using the same oligonucleotides as probes for Northern blot hybridization, we have obtained a similar expression of the levels of each form of TR-mRNAs (Puymirat j.26) as those reported by Strait et al. 32, using solution hybridization, in the developing rat brain. This indicates that the methodology used in the present study is valid and that our results represent the relative levels of TR-mRNAs. The expression of TRmRNAs observed in neuronal cultures is not a function of the 15% astrocyte contaminant. (1) Cultures were grown in the presence of AraC, a drug which kills dividing cells. (2) In pure astrocyte cultures, we have shown that the levels of a l RNAs are 3-fold higher than that of/31-mRNAs whatever the age of the cultures and that the levels of/31 mRNAs did not change with time in cultures (Lebel et al., submitted). Furthermore, the/31 but not the a mRNAs are up-regulated by T3. These data indicate that the expression of TR-mRNAs reported in the present study represents the expression in neurons. This study reveals two points: (1) the levels of mRNAs ( a l +/31) encoding for active T3 receptors increased with time in culture. This agrees with the increase of the T3 binding capacity, observed in similar cultures 16. (2) The differential expression of the a and /3 gene during neuronal maturation. This pattern of expression is very similar with that observed in rive, in the developing cerebral hemispheres 2t'. The al TRmRNAs are the predominant form of TR-mRNAs after 8 days in cultures and before postnatal day 7, in the developing cerebral hemispheres. The /31 TRmRNAs become the predominant TR.mRNAs between the second and the third week in vitro and between postnatal day 7 and 15, in the developing cerebral hemispheres 26. This indicates that the expression of TR-mRNAs in the developing cerebral hemispheres reflects the expression in neurons and that neurons in cultures maintain their biological clock and differentiate on a time schedule similar to one observed in situ. The differential expression of the al and/31 TR-mRNAs in neuronal cultures suggests specific functions for both type of T3 receptors duriag neuronal differentiation. The al are the predominant mRNAs that code for T3 receptors during the first week of cultures, when neurons still divide, whereas the/31 TR~mRNAs become predominant when mitosis has ceased. This result implicates TRs with distinct functions in early neuronal maturation as well as in the last steps of neuronal maturation. It is therefore possible that the a l receptors could be implicated in the effects of thyroid hormones on the control of neuronal

176 cell proliferation and migration tS. This hypothesis is supported by: (1) in vivo data showing that the a l TR-mRNAs are the major T R - m R N A s in the developing cerebral hemispheres during neuronal cell proliferation and migration 2~'. (2) Recent data showing that the a I receptors are predominantly located in proliferative astrocytes (Lebel et al, submitted). (3) It is of note that the a receptors are also the predominant T3 receptors in nerve cell lines (neuroblastoma NB41A3). In contrast, the rapid rise of the fil T R - m R N A s between the second and the third week of cultures coincides with the maximal morphological and biochemical effects of L-T3 on different neuronal cell populations 1'7''~'").Thus, it is therefore possible that the filTRs could be involved in the effects of L-T3 on the last steps of neuronal differentiation, including neurite outgrowth, synaptogenesis, and the development of neurotransmitter activities. To discuss about the physiological role of both types u~' T3 receptors, it is necessary to take into account the presence of the inactive form of TR. Recent studies have shown by transfection experiments that the a2cDNA products can block the effect of L-T3 mediates by the a l and fil receptors ~2. These observations have prompted speculation that the high levels of a 2 in the brain accounts for the non-responsive nature of this tissue to T3. Here, we show that the a 2 mRNAs are the major forms of T3 receptors in neurons, although these cells have been shown to respond to T3. Indeed, we have previously shown that L-T3 increases the neurite length of neurofilament positive cells, demonstrating that this effect is a general effect on neuronal cells 7. These observations do not support the hypothesis of a blocking effect of the a 2 variant on the effects of L-T3 mediated by the a l and /31 receptors, However, there is no data on the cellular localization of the a l , a2, and/31 proteins. In particular, we do not know if the a and fl gene products could be co-expressed by specific neuronal cell subpopulations, Immunocytochew:':fl studies using specific antibodies raised against each form of T3 receptors are necessary, in order to determine the function of the a 2 variants. Acknowlt,dgemcms. Supported by CRM du Canada (no. 1187).

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