Expression of neurotrophins and the trk family of neurotrophin receptors in normal and hypothyroid...

ELSEVIER Molecular Brain Research 27 (1994) 249-257

MOLECULAR BRAIN

RESEARCH

Research report

Expression of neurotrophins and the trk family of neurotrophin receptors in normal and hypothyroid rat brain

Manuel Alvarez-Dolado, Teresa Iglesias, Angeles Rodr~guez-Pefia, Juan Bernal, Alberto Mufioz *

Instituto de Investigaciones Biom~dicas (C. S.I. C,), Arturo Duperier 4, 28029 Madrid, Spain

Accepted 26 July 1994

Abstract

Thyroid hormone deficiency has dramatic effects on rat brain maturation. The expression of genes encoding neurotrophins and the trk family of neurotrophin receptors has been evaluated in several brain regions of normal and of neonatal or adult hypothyroid rats to analyze whether they are subject to thyroid hormone action. We found that hypothyroidism decreased trk mRNA levels in its major site of expression, the striatum, on postnatal days 5 (P5; 45%) and 15 (P15; 25%) and also in adults (35%). In contrast, no differences in trkB or trkC mRNAs levels were observed in any brain region at studied ages. According to previous reports, p75LNGFR mRNA was elevated in hypothyroid cerebellum as compared to age-matched controls on P5 and P15. We have also observed a distinct pattern for neurotrophin genes. The level of NGF mRNA was 20-50% lower in the cortex, hippocampus, and cerebellum of hypothyroid rats on neonatal hypothyroid rats on P15 and also after adult-onset hypothyroidism. Treatment of neonatally-induced hypothyroid rats with a single injection of triiodothyronine led to the recovery of hippocampal but not cortex NGF mRNA levels to that of control animals. On the contrary, no differences in the relatively high expression of the two mRNAs encoding BDNF were observed in any brain area. In contrast to a recent report, we did not find a reduction in brain NT-3 mRNA levels in hypothyroid animals. If any, the effect of thyroid deficiency in the hippocampus and cortex seems to be an early upregulation of NT-3 expression. In summary, our results support a region-specific developmental regulation by thyroid hormone of the expression of particular neurotrophic factors and their receptors in the rat brain. The observed alterations may contribute to the abnormal brain maturation caused by hypothyroidism.

Keywords: trk; p75LNGFR; Low-affinity nerve growth factor receptor; NT-3; Neurotrophin-3; BDNF; Brain-derived nerve factor; Hypothyroidism

1. Introduction

Thyroid hormone is an important regulator of mam- malian brain maturation. Lack of adequate levels of thyroid hormone during the fetal and neonatal periods leads to mental deficiency in humans and multiple brain abnormalities in experimental animals such as the rat [12,13,14,41,47]. These abnormalities include biochemical, cellular and behavioural alterations localized throughout the whole brain: reduction in mean size of neuronal cell bodies and decrease density and abnormal distribution of dendritic spines of the apical shaft of pyramidal cells in the forebrain, reduction in

* Corresponding author. Fax: (34) (1) 585 45 87.

0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 9 - 3 2 8 X ( 9 4 ) 0 0 1 7 1 - 5

cell number in the hippocampus and olfactory bulb, diminished arborization of cerebellar Purkinje cells, changes in cholinergic markers in the basal forebrain, retarded and diminished myelination in all regions. Despite the importance of the drastic effects caused by hypothyroidism the molecular basis of thyroid hormone action in the brain remains mostly unknown. The complex array of alterations described may be the result of the direct regulation by thyroid hormone of a relatively high number of genes or, alternatively, of the regulation of a few key genes in turn controlling secondary patterns of gene expression in different cell types or brain areas. Search for thyroid hormone-regulated genes based on differential or subtractive hybridization approaches has not been very successful and only a few genes regulated by thyroid hormone in rodents during

250 M. Ah,arez-Dolado et al./Molecular Brain Research 27 (1994) 249-257

the neonatal period have been characterized in the last years [42]. They include the cytoskeletal tau and tubulin genes [1,2,17], the neuronal rat cortex 3 (RC3) gene [42,25,26], which is specifically expressed in cortex, hippocampus, striatum and olfactory bulb; the prostaglandin D2 synthetase [19] in the cortex and cerebellum; PCP-2, calbindin and myo- inos i to l - l , 4 ,5 - t r iphos -

phate receptor [54], three cerebellar genes; NGFI-A [46]; and myelin genes [42,15,51]. The surprisingly low number of genes found to be altered by hypothyroidism contrasts with the abundance of thyroid hormone receptors in the brain [5,39] and the wide phenotypic alterations caused by thyroid hormone deficiency. This fact can be interpreted as supporting the notion of the existence of a reduced set of thyroid-regulated brain genes playing important regulatory actions during development, In line with this hypothesis, we decided to investigate the possible effects of thyroid hormone deficiency in vivo on the regional expression of genes encoding neurotrophins and their receptors during the critical perinatal period of rat brain development.

Neurotrophins are responsible for the survival and differentiation of defined neuronal populations during central nervous system (CNS) development [4,56,59]. Thus, neurotrophins are essential for the formation of the complex cellular networks which are the basis of brain structure and function. A few neurotrophins have been characterized and cloned including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4 /5 (NT-4/5) and ciliary neurotrophic factor (CNTF). The first step required for their biological activity is the binding to receptors localized in the membrane of target cells. Specific high affinity receptors exist for each neurotrophin [3]: the trk proto-oncogene product (gpl40 "k) for NGF, and the highly related trkB product (gp145 "k~) for BDNF and NT-4/5, and trkC product (gp145 t~kc) for NT-3. In addition, a low affinity receptor (p75LNGFR) for NGF, BDNF, NT-3 and NT-4/5 is expressed by different cell types [52]. CNTF has a distinct unrelated high affinity receptor (CNTF- R) [11]. Members of the trk family (trk, trkB, trkC) have distinct patterns of expression in the CNS [3,50]. The key roles played by the p75NGFR and trk gene family in the formation and function of the nervous system is clearly shown by the dramatic effects of their individual inactivation in mouse development (see [55] for a review).

The effect of the thyroid status on the expression of some neurotrophin genes has been studied by several groups but the information derived from these studies is still fragmentary. Following thyroxine injection, an increase in NGF and NT-3 expression has been found in the adult rat hippocampus [20] and of NGF also in the perinatal cerebellum [18]. Some of these effects may be posttranscriptional since thyroxine seems to

increase the brain NGF protein content [60,61] despite the fact that hypothyroidism causes only a minor reduction in NGF mRNA levels in the whole brain [42]. More recently, NT-3 has been reported to be induced by triiodothyronine in developing rat cerebellar granule cells both in cell culture and in vivo [32]. In the latter study, hypothyroidism leads to a 50% reduction in NT-3 mRNA in neonatal rats. Interactions between thyroid hormone and NGF have been previously described both in rat brain [45,9] and in cultured PC12 cells [43]. On the other hand, trkB gene is subject to regulation by the thyroid hormone receptor /c-erbA [44] and by retinoic acid [28] in neuroblastoma cell lines.

In order to help clarify this subject, we have analyzed the expression of all the currently cloned neurotrophin genes and the three members of the trk gene family of their receptors in five brain regions (cortex, hippocampus, striatum, diencephalum + mesence- phalum (dien. + mesen.), and cerebellum) from control and either neonatally- or adult-induced hypothyroid rats at different timepoints of development. Several findings are reported which are either novel, or in agreement with previous partial findings by other authors, whereas others do not confirm previously reported data.

2. Materials and methods

2.1. Induction of hypothyroidism

Wistar rats raised in our animal facilities were used. The mainte- nance and handling of the animals were as recommended by the NIH Guide-Lines on the Care and Use of Laboratory Animals. To induce neonatal hypothyroidism a combination of chemical and surgical thyroidectomy (Tx) was used. Methylmercaptoimidazol (MMI; 0.02%) was administered in the drinking water of the dams from the 9th day after conception and was continued until the animals were sacrified. In addition, to ensure cerebral hypothyroidism, the neonates were surgically thyroidectomized on postnatal day 5 (P5). On P5, hypothyroidism was caused by MMI only, whereas on P15 hypothyroidism was the combined result of MMI treatment plus surgical thyroidectomy performed ten days before. This protocol resulted in profound hypothyroidism which can be demonstrated by measuring thyroid hormone level in brain throughout the postnatal period [42,51] and by estimation of other features of early hypothyroidism, i.e., decreased growth (see Table 1) and delayed appear- ance of developmental landmarks such as eye opening. To study the effect of thyroid hormone treatment, P15 hypothyroid animals were intraperitoneally administered 100 p~g of 3,5,3'triiodothyronine (T3) and they were saerified four hours later according to Mano et al. [37]. To induce hypothyroidism in adult animals we either added 0.02% MMI to the drinking water from day P40 until their sacrifice on day P60 or did a combination of surgical thyroidectomy at day P40 followed by the same MMI treatment. The animals were killed by decapitation, the brains were quickly removed and put on a chilled surface, and individual regions were dissected out and frozen in liquid N 2.

M. Alvarez-Dolado et al . / Molecular Brain Research 27 (1994) 249-257 251

2.2. RNA extraction and Northern analysis

Poly(A) ÷ RNA was obtained from pools of different brain regions from a variable number of animals depending on the weight of each region. Thus, five cortices were pooled from adult (P60) rats while as many as thirty-four striata from P5 rats were used for each RNA extraction. Purification of poly(A) + RNA was done using oligo d(T) cellulose chromatography as described by Vennstr6m and Bishop [58]. To prepare total RNA we used the guanidinium iso- thyocyanate-phenol-chloroform procedure [7]. RNAs were fraction- ated in formaldehyde agarose gels and blotted onto nylon (Nytran, Schleicher and Schuell) membranes following standard techniques [36]. As controls for the amount and integrity of RNA present on the filters, blots were stained in a 0.02% Methylene blue solution made in 0.3 M sodium acetate and rehybridized with a cyclophilin (Cy) cDNA probe. Cyclophilin mRNA is constant during postnatal development and is not influenced by thyroid hormone [42]. Sizes of respective mRNAs were calculated using RNA ladder as markers (BRL). Membranes were exposed to Hyperfilm T M MP films (Amersham). Autoradiograms were analyzed using a La Cie scanner connected to a Macintosh Ilci computer using Adobe Photoshop T M

2.0 and NIH Image programs.

2.3. Source and labelling of cDNA probes

The following probes were used: a 464 bp XbaI-SacI fragment corresponding to the extracellular domain of mouse trk cDNA in pDM97 [38] from Dr. L. Parada; a 483 bp EcoRI fragment containing the extracellular domain of the mouse trkB cDNA in pFRK16 [29] and a 600 bp AccI fragment in PGEM3Zf(+) corresponding to the extracellular domain of the mouse trkC cDNA [31], both from Dr. M. Barbacid; a 800 bp fragment of p75LNGFR cDNA in pNGFR.1 [49] from Dr. E. Shooter; the full-length rat NGF cDNA cloned in pXMRN and that of rat NT-4 in pBS-KS from Drs. E. Arenas/H. Persson; a 1127 bp EcoRI fragment containing the rat BDNF cDNA in pSK-rB (C1) [35] and a 800 bp XhoI fragment containing the rat NT-3 cDNA in pSP70 [34] from Dr. G.D. Yan- copoulos; and the 660 bp EcoRI-XbaI fragment containing the rat CNTF cDNA in pBS-SK [53] from Dr. H. Thoenen. The rat probes used for thyroid hormone-regulated brain genes were the full-length RC3 cDNA cloned in priG327 [62] and myelin associated glycoprotein (MAG) clone 1B236-18 [30], and for control of loading onto the filters we used the full-length cyclophilin (Cy) cDNA [10], all donated by Dr. J.G. Sutcliffe. All probes were labeled by the random priming method [16]. Hybridizations were carried out overnight at 65°C in 7% SDS, 500 mM sodium phosphate buffer, pH 7.2, and 1 mM EDTA according to Church and Gilbert [8]. Filters were washed twice for 30 rain each in 1% SDS and 40 mM sodium phosphate buffer, pH 7.2, at 65 °C.

Table 1 Reduction in body weight by hypothyroidism

Age Body weight (g) (mean + S.D.) Reduction

(days) Control Hypothyroid (%)

P5 12.4+ 1.2 10.0_+ 1.1 19.3 P15 24.8_+ 3.8 16.5+ 1.6 33.4

Adult (P60) (MMI) 244.5± 3.5 169.0+ 9,8 30.8 (Tx+ MMI) 198.7_+ 14.7 124.3+ 13.7 37.5

Seventy-five (P5) and 45 (P15) neonatally-induced hypothyroid or control rats, and 10 adult-induced hypothyroid or control rats were weighed on indicated ages. S.D., standard deviation.

w e i g h t was s l ight ly h i g h e r in Tx + M M I an ima l s t h a n in

t h o s e on ly t r e a t e d wi th M M I . It is h o w e v e r k n o w n tha t

m a m m a l i a n b r a in is ve ry wel l p r o t e c t e d f r o m the

d e l e t e r e o u s e f fec t s o f h y p o t h y r o i d i s m and tha t d i f fe r -

e n t phys io log ica l m e c h a n i s m s exist to k e e p c o n s t a n t

t he c o n c e n t r a t i o n o f t hy ro id h o r m o n e s in t h e b r a i n

e v e n w h e n t h e res t o f t h e body is h y p o t h y r o i d [6]. T o

e n s u r e tha t o u r p r o t o c o l was e f f i c i en t to p r o d u c e ce r e -

b ra l h y p o t h y r o i d i s m in n e o n a t a l ra ts we s t u d i e d the

e x p r e s s i o n o f t he n e u r o n a l R C 3 and the gl ial M A G

g e n e s wh ich h a v e b e e n r e c e n t l y s h o w n to be d o w n - r e g -

u l a t e d in t he h y p o t h y r o i d rat b r a i n on P15 [25,51]. A s

s h o w n in Fig. 1, t h e c o n c e n t r a t i o n s o f b o t h m R N A a re

d i s m i n i s h e d in t he s t r i a t u m o f t he h y p o t h y r o i d ra ts

u s e d in this s tudy. S t r i a t u m was c h o s e n for this con t ro l

D A Y S

S T A T U S C H

- 1 . 5 k b

RC3 - 1 . 0 k b

3 . R e s u l t s

3.1. Verification o f brain hypothyroidism

T h e va l id i ty o f t h e p r o t o c o l u s e d to i n d u c e hypo-

t h y r o i d i s m has b e e n p r ev ious ly s h o w n by m e a s u r i n g

thy ro id h o r m o n e leve ls in n e o n a t a l r a t b r a i n s [42,51].

I n d u c t i o n o f h y p o t h y r o i d i s m was first c h e c k e d by c o m -

p a r i n g b o d y w e i g h t s o f c o n t r o l and t r e a t e d an ima l s . A s

e x p e c t e d , w h o l e b o d y w e i g h t o f h y p o t h y r o i d ra ts was

l o w e r t h a n tha t o f c o r r e s p o n d i n g a g e - m a t c h e d con t ro l

ra ts at all ages s t u d i e d ( T a b l e 1). T h e r e d u c t i o n in b o d y

MAG - 2 . 6 k b

Fig. 1. Down-regulation of thyroid hormone-dependent brain genes in hypothyroid animals. Northern blot analyses of the RC3 and MAG mRNA levels in the striatum of control (C) and hypothyroid (H) rats on P15. Ten /~g of poly(A) + RNA purified from 22 pooled samples in each case was subjected to Northern blot analysis as described under Materials and methods. Films were exposed for 1 day. Size of bands corresponding to the expected mRNAs is indicated.

252 M. Ah,arez-Dolado et aL / Molecular Brain Research 27 (1994) 249-257

experiment since it has been found to be a target area for the action of thyroid hormone in the brain [25,51].

3.2. Influence of hypothyroidism on the trk family of high affinity neurotrophin receptors

First, we studied the expression of the trk gene in the different brain areas of control and hypothyroid rats. The levels of the 3.4 kb trk mRNA were highest in the striatum, much lower in the dien.+ mesen., and undetectable in the remaining regions (cortex, hippocampus, cerebellum). A consistent decrease was observed in the striatum of neonatally-induced hypothyroid animals (45% at P5; 25% at P15). Adult-onset hypothyroidism induced by Tx + MMI also resulted in similar changes (35% decrease; Fig. 2A). Similar re- suits were obtained in two experiments using different pooled sets of striata. Subsequent hybridization of the filters with the MAG and Cy probes confirmed the hypothyroid status of the striata and the equal loading of the gel allowing the estimation of the t rk/Cy ratio (Fig. 2B). A slight decrease in trk mRNA concentration was also observed in the dien. + mesen.

Parallel filters containing RNAs from the same five brain regions and equally double-checked for MAG and Cy expression were used for the trkB and trkC genes. In both cases no differences were observed in the levels of any of the multiple mRNAs encoded. Fig. 2C shows the expression of the series of 7.5, 7.0, 4.8, 3.6, 2.4, 1.6 and 0.9 kb mRNAs corresponding to trkB [40] in the hippocampus (a region where all the expected transcripts were detected) of control and hypothyroid animals. The same result was observed in the other regions (not shown). In the case of trkC the same levels were detected for the subgenomic 5.5 and 5.0 mRNAs (not shown) in the cortex, cerebellum, hippocampus and dien. + mesen.. However, the large genomic 14 kb mRNA [57] was difficult to detect probably because of its large size.

3.3. p75LNGFR

We next analyzed the expression of the low affinity neurotrophin receptor p75LNGFR in the brain of normal and hypothyroid rats. According to reported data [17,33], the 3.7 kb p75LNGFR mRNA was found to be

A DAYS STATUS

trk 3.4 kb -

MAG 2.6 kb,

Cy l . l kb

PS _.PAL Ad C H C H C H

C

trkB

DAYS P5 ~ Ad

STATUS C H C H C H

7.5 kb. 7.0 kb. 4.8 kb.

3.6 kb.

2.4 k b

1.6 kb

0.9 kb

B '1 MAO 2.6kb

0 C " H C "H C H 1.1kb P5 PI5 Ad

A G E (days)

Fig. 2. Effect of neonatal and adult hypothyroidism on the expression of trk and trkB genes. Northern blot analyses of the trk, trkB, MAG and Cy m R N A levels in control (C) and hypothyroid (H) rats. A: poly(A) ÷ R N A (10/xg) purified from 34 (P5), 22 (P15) or 12 (adult) pooled striata at indicated days were subjected to Northern blot analysis. Filter was reprobed with MAG to check the hypothyroid status and with Cy for control of loading and normalization. Exposure times: 2 weeks for trk, 2 days for MAG and one for Cy. B: ratio between trk and Cy m R N A levels at the t imepoints studied. Mean values of two separate experiments are shown. C: poly(A) + R N A (10/zg for P5 and P15, 5 /xg for adult) purified from 20 (P5, P15) or 6 (adult) pooled hippocampi at indicated days were analyzed in Northern blots as before. Films were exposed for 1 day in all three cases. Size of bands corresponding to the expected m R N A s is indicated.

M. Alvarez-Dolado et al. / Molecular Brain Research 27 (1994) 249-257 253

developmentally down-regulated in the cerebellum with higher levels on P5 than on later ages. The message followed the same trend in hypothyroid rats but its concentration was higher than in normal animals at all ages. The largest differences were found on P15, with mRNA concentrations in hypothyroid cerebella that were about three times higher than those in normal animals (Fig. 3). An additional unchanged band corresponding to a 1.2 kb mRNA was detected on P5. In the other brain areas studied, only a weaker expression was found in the dien. + mesen, which was slightly increased by hypothyroidism. Expression of p75LNGFR mRNA was undetectable in adult (P60) animals.

3.4. Neurotrophins

The NGF mRNA encoding the physiological ligand of the trk protein was detected in all brain regions except the striatum. In the cortex, hippocampus, and cerebellum of neonatal hypothyroid animals, the 1.3 kb NGF mRNA levels were disminished on P15 as com-

p75LNGFR DAYS p.~ Pl$ Ad

STATUS C H C H C H

3.7 kb

pared to control animals (20-50%) (Fig. 4A,B). This reduction was also found in adult-onset hypothyroidism in the same areas. A different result was found for BDNF. The levels of BDNF 4.0 and 1.6 kb mRNAs are not affected by the hypothyroid status in any brain area. Fig. 4C shows the results obtained in the cortex and hippocampus of neonatally- and adult-induced hypothyroid animals and their respective controls.

To study whether thyroid hormone could restore the normal levels of NGF mRNA, 15 days-old hypothyroid rats were injected with 100 /xg triiodothyronine and sacrified four hours later. As seen in Fig. 5, this acute hormonal treatment restored the NGF mRNA levels in the hippocampus to that existing in control animals. In contrast,'this treatment failed to increase NGF mRNA levels in the cortex of the same animals.

As for NT-3, highest levels of its 1.4 kb mRNA were found in the hippocampus and cerebellum, lower in the dien. + mesen, and cortex, and were undetectable in the striatum. After normalization with Cy mRNA levels, a slightly but consistently higher expression was found in the hippocampus, dien. + mesen., and cortex, but not cerebellum, of hypothyroid animals on P5 and P15 (Fig. 6). In contrast, these differences were not observed in adult-onset hypothyroidism. Two independent experiments were carried out using samples obtained from three to twenty-two individuals depending on the brain region.

Finally, in agreement with their low expression we were unable to detect the NT4/5 and CNTF mRNAs in our Northern blots.

1.2 kb 4. Discussion

I " .

0.5

Z

0"

ii !.iiii. _ C It

P5

:::::::-::::;:::::

ii!iiiiiiiiiiiii iii r iiii iiiii :iiiil

C H P15

AGE (days)

!

C H Ad

Fig. 3. Increase of p75LNGFR mRNA levels in the neonatal hypothyroid cerebellum. Northern blot analyses of the p75LNGFR mRNA in control (C) and hypothyroid (H) rats at different timepoints. Ten /zg of poly(A) + RNA purified from 22 (P5), 12 (P15) or 3 (adult) pooled cerebella were loaded. Filter was reprobed with Cy and the ratio between p75LNGFR and Cy mRNA levels was determined by densitometry. Exposure time was 1 day for both genes. Size of bands is indicated.

We have investigated the effect of neonatal and adult-onset hypothyroidism on the expression of all the currently known neurotrophin and neurotrophin receptor genes in different rat brain regions. By comparing to age-matched controls, several differences have been found particularly in the early postnatal period. Given the key roles played by these genes during the process of brain maturation, these differences may contribute to the alterations caused by the lack of thyroid hormone causes on rat brain development and function.

Our data show that decreased trk mRNA levels exist in the striatum of both neonatal and adult hypothyroid animals. This result is in agreement with the correlation between some of the clinical manifestations observed in neurological cretins and alterations in this cerebral area [12] and also with the reported sensitivity of the cholinergic cells of the basal ganglia to thyroid hormone [21]. These cells express maximum levels of gpl40 trk [24], which is the high affinity receptor for NGF. We also observed a decrease in NGF expression in the cortex, hippocampus and cerebellum in both

254 M. Ah,arez-Dolado et al. / Molecular Brain Research 27 (1994) 249-257

neonatal hypothyroidism on P15 and adult-onset hypothyroidism. Moreover, NGF mRNA levels in hypothyroid animals are restored to control values by an acute thyroid hormone treatment that is able to increase RXR/3 mRNA levels in hypothyroid rat brain [37]. The fact that cortex is not sensitive to this treatment raises the possibility of region-specific effects of or differences in sensitivity to thyroid hormone. Supporting this, RC3 mRNA levels in the striatum have been found to be more sensitive to hypothyroidism than in the cortex. Our data are in line with data from others [20] showing an upregulation by thyroid hormone of NGF mRNA levels in the hippocampus (and cortex) of adult rats. In both cases, the observed changes are small and of unclear statistical significance which is probably the reason why in a previous work [42] we did not observe changes in NGF mRNA in whole cerebra of hypothyroid rats. However, giving the key activity of the NGF and trk genes, a small variation in NGF, which has been reported to regulate trk expression

[24], combined with the observed changes in trk mRNA concentration may be biologically significant. Since thyroid hormone affects the differentiation of basal forebrain [21,27] and hippocampal pyramidal [22,23] neurons and thyroid hormone and NGF cooperate in the development of basal ganglia [45] and also of other brain areas such as the hippocampus, cerebellum and olfactory bulb [9], changes in the expression of trk and NGF genes may be of great importance for the formation and function of specific neuronal populations of these areas in the developing CNS.

In contrast to these results, no alterations in the expression of the genes coding for the trkB receptor and its ligand BDNF were found. To our knowledge, this is the first study on the effect of hypothyroidism on these two widely expressed genes thought to play multiple and critical roles in diverse brain regions. Other authors [20] also failed to detect alterations in BDNF mRNA levels upon thyroid hormone treatment of adult rats. The lack of effect indicates that hypothyroidism

1 . 3 k b -

NGF DAYS - ] ~ ~ ,'~d

S T A T U S C 11 C" 11 (? n

C BDNF

DAYS p~ pl.~ 'td

S[ 'A'FUS (" I I ( II ( II

4.0 kb

A

C O R T E X

H I P P O C A M P U S

1.6 kb -

C O R T E X

4.0 kb -

,.3,b.[ ¸ii :il, '̧̧¸̧ ̧I CEREBELLUM

16 kb -

H I P P O C A M P U S

B

m

Z

2.5 ¸ C O R T E X H I P P O C A M P U S

c ~ H ¢ ' t t ' c tt ' " ' P5 PI5 Ad P5 Pl5 Ad

AGE (days)

C E R E B E L L l M

P5 PI5 Ad

Fig. 4. Effect of neonatal and adult hypothyroidism on the NGF and BDNF m R N A levels in different brain regions. Northern blot analyses of the NGF and BDNF m R N A s in the indicated brain regions of control (C) and hypothyroid (H) rats. A: for cortex, 10/xg of poly(A) + RNA (P5 and P15) or 20 /zg of total R N A (adult) purified from five pooled samples were loaded; for hippocampus, 10 (P5 and P15) or 5 (adul t ) /zg of poly(A) ÷ RNA purified from 20 (P5 and P15) or 6 (adult) pooled samples; for cerebellum, 10/zg of poly(A) + R N A purified from 22 (P5), 12 (P15) or 3 (adult) pooled samples were loaded. Filters were probed with NGF as described under Materials and methods. Exposure times: 2 weeks for NGF, 1 day for Cy. B: ratio between NGF and Cy m R N A levels in the different regions at the timepoints indicated. C: for cortex, 10 /zg of poly(A) ÷ RNA (P5 and P15) or 20/zg of total R N A (adult) purified from 5 pooled samples were loaded; for hippocampus, 10 (P5 and P15) or 5 (adul t ) /zg of poly(A) ÷ R N A purified from 20 (P5 and P15) or 6 (adult) pooled samples were loaded. Filters were probed with BDNF as described under Material and methods. Films were exposed for 2 weeks. Size of bands corresponding to the expected mRNAs is indicated.

M. Alvarez-Dolado et al. /Molecular Brain Research 27 (1994) 249-257 255

A HIPPOCAMPU~;

STATUS C H H+T3

NGF 1.3 kb

C H H+T3

Cy

B 1"

" • 0.5- t -

C H H+T3 C H H*T3

Fig. 5. Effect of a single thyroid hormone dose on NGF mRNA accumulation in the rat hippocampus and cortex. A: hypothyroid animals were injected with a single intraperitoneal injection of 100 /zg triiodothyronine on postnatal day 15. Animals were sacrified 4 h later. The hippocampi and cortices from control (C), hypothyroid (H), and T3-injected hypothyroid (H+T3) rats were collected and pooled in groups of 12 and 4 respectively for preparation of poly(A) ÷ RNA and Northern blotting (10 p.g poly(A) + RNA per lane). Probing and exposure times were as in legend for Fig. 4. B: ratio between NGF and Cy mRNA levels in hippocampus and cortex of control (open columns), hypothyroid (dotted columns) and T3-injected hypothyroid (dashed columns) rats.

does not cause a gene ra l inh ib i t ion of n e u r o t r o p h i n express ion. Ra the r , it a p p e a r s tha t some specif ic a l te r - a t ions in n e u r o t r o p h i n activi ty may occur in the hypo thyro id status.

Similarly, hypo thyro id i sm seems to have no majo r effects on the express ion of t rkC and NT-3. A s po in t e d out above, we however could not de t ec t the large genomic t rkC m R N A and no f i rm conclus ions can be drawn. W e obse rved tha t n e o n a t a l hypo thyro id i sm causes an increase in NT-3 m R N A concen t r a t i on in h i p p o c a m p u s and cortex. No changes were d e t e c t e d in the o t h e r reg ions s tudied. These da t a a re in d i sagree- m e n t wi th a r ecen t r e p o r t sugges t ing a posi t ive regula- t ion by thyro id h o r m o n e of the NT-3 gene bo th in vivo and in vi t ro [32]. W e have not found the r educ t ion in NT-3 m R N A levels desc r ibed in this r epo r t in the ce r ebe l l um of hypo thyro id rats. The reasons for this d i sc repancy may be r e l a t ed to the d i f fe ren t p ro toco ls used to i nduced hypo thyro id i sm (p ropy l th iourac i l in- s t ead of M M I , low-iodine die t ) and to the large doses of t r i i odo thy ron ine a d m i n i s t e r e d in the a fo remen- t i oned work. A n add i t iona l exp lana t ion may be the use by these au thor s of G A P - 4 3 as con t ro l gene in the i r hybr id iza t ions , s ince it has been r e p o r t e d tha t G A P - 4 3

m R N A levels a re inc reased in hypothyro id i sm [17], a resul t also found by us.

In the case of the p 7 5 L N G F R gene, our da ta agree with those pub l i shed recent ly indica t ing an increase in its m R N A levels in the hypo thyro id b ra in [17]. As d iscussed by these authors , this inc rease may be the consequence r a the r than a cause of a r e t a r d e d cerebe l - lar m a t u r a t i o n ( r e t a r d e d d i s a p p e a r a n c e of the ex te rna l g ranu la r layer), the resul t of changes on o the r processes or, a l ternat ively , a c o m p e n s a t o r y reac t ion to a l t e r ed ce l lu la r funct ions. Since the exact funct ion of this low affinity r e c e p t o r for several ne u ro t roph in s is still unknown, no c lear conclus ions can be drawn about the s ignif icance of this observa t ion .

The set of da t a p r e s e n t e d here r ep re sen t the resul t of an extensive s tudy on the express ion in several pa r t i cu l a r b ra in regions of a ser ies of genes encod ing for ne u ro t roph in s and the i r r ecep to r s p laying impor- tan t roles in the deve lop ing CNS. O u r resul ts indica te the conven ience of ex tend ing this s tudy to the new m e m b e r s of this impor t an t class of genes to be discov- e r ed in the next future.

Acco rd ing to one of our work ing hypotheses , we have d e m o n s t r a t e d tha t the express ion of a subset of n e u r o t r o p h i n and n e u r o t r o p h i n r e c e p t o r genes is af- f ec ted by the lack of thyro id hormone . This could be the basis for some of the a l t e ra t ions obse rved in the

e ~

CORTEX CEREBELLUM

c H c H c H P 5 P l 5 Ad

H1PPOCAMPUS

f i l l f2_lt P5 PI5 Ad

C it ¢ It c t t p5 p15 Ad

DIEN. + MESEN.

• , °

C H C H C H P5 Pl5 Ad

AGE ( days )

Fig. 6. NT-3 mRNA levels in neonatally- and adult-induced hypothyroid animals. Ratio between NT-3 and Cy mRNA levels in different brain regions of control (C) and hypothyroid (H) rats as determined by densitometric scanning of Northern blots containing the same amounts of poly(A) + mRNAs or total RNA as described in legend for Fig. 4. Two separate experiments using RNAs from 5 cortices, or 22 (P5), 12 (P15) and 3 (adult) cerebella, or 20 (P5 and P15) and 6 (adult) hippocampi, or 5 dien.+ mesen, gave very similar results.

256 M. Alvarez-Dolado et al. / Molecular Brain Research 27 (1994) 249-257

hypothyroid brain. Thyroid hormone may regulate multiple aspects of brain maturation involving different cell populations and processes by controlling the expression of a reduced number of key genes. These genes could in turn regulate the expression or activity of other different genes in distinct cells during development. Although our data do not unambiguosly estab- lish this mechanism, they are compatible with such an hypothesis to explain the complex and still mostly unknown mechanism of action of thyroid hormone in the brain.

Acknowledgements

We thank all who appear in Materials and methods for generously supplying us with plasmids and probes. We also acknowledge Dr. Gabriella Morreale for her helpful discussions and critical reading of the manuscript. This work was supported by Grants from the Plan Nacional de I + D (SAF92/0396) and Funda- ci6n Ram6n Areces. In addition, we would like to thank Margarita Gonzfilez for her excellent technical assistance and Fernando Nufiez and Pablo Sefior for the care of animals. M.A.-D. was recipient of a predoc- toral fellowship from the Universidad Aut6noma de Madrid.

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