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Effects of hypothyroidism on insulin-like growth factor-I expression during brain development in mice
Neuroscience Letters 293 (2000) 99±102
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Effects of hypothyroidism on insulin-like growth factor-I expression during brain development in mice Deborah A. Elder, Aysen F. Karayal, A. Joseph D'Ercole, Ali S. Calikoglu* Department of Pediatrics, Division of Endocrinology, CB 7220, 108 Burnett-Womack, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7220, USA Received 29 June 2000; received in revised form 23 August 2000; accepted 24 August 2000
Abstract Hypothyroidism has devastating consequences on brain development. While the mechanisms that mediate these effects are not known, several lines of evidence suggest that a reduction in insulin-like growth factor-I (IGF-I) expression and/or action has a role. To assess whether reduced IGF-I expression and/or actions mediates the brain pathology of congenital hypothyroidism, we induced hypothyroidism by treating pregnant mice and lactating dams with 0.1% propylthiouracil (PTU) in drinking water. Control and PTU-treated pups were sacri®ced on postnatal day (P) 7, 10 and 14, and IGF-I mRNA expression was assessed in the cerebral cortex and cerebellum by ribonuclease protection assay. To control for mRNA loading, the signal of IGF-I protected bands was normalized to those for cyclophillin. IGF-I mRNA expression in hypothyroid animals was decreased signi®cantly in cortex at P10 and P14 (42 and 60%, respectively). In the cerebellum, IGF-I mRNA expression was down-regulated at all ages studied, but the decrease was only statistically signi®cant at P7 (31% decreased). We conclude that hypothyroidism alters IGF-I expression in the developing brain. Furthermore, we speculate that IGF-I plays a role in mediating some thyroid hormone actions during brain development. q 2000 Published by Elsevier Science Ireland Ltd. Keywords: Thyroid hormone; Hypothyroidism; Insulin-like growth factor-I; Brain; Mice
Thyroid hormone (TH) has a signi®cant in¯uence on the structural, biochemical and functional development of the brain. In man, untreated congenital hypothyroidism leads to multiple morphologic and biochemical alterations in the brain and results in irreversible mental retardation. Studies in rodents show that reduced myelination, poor neuritic outgrowth and dendritic arborization are among the changes in the brain due to congenital hypothyroidism. The mechanisms of TH action on brain development, however, are incompletely de®ned. Furthermore, TH action may not be direct. TH is known to interact with growth factors, including, insulin-like growth factor-I (IGF-I), epidermal growth factor and neurotropins. It is possible, therefore, that these agents mediate TH actions during brain development. Several lines of experimental evidence in rodents suggest that a reduction in IGF-I expression and/or action may mediate the effects of TH on brain development. (1) Peak IGF-I expression in the rat brain occurs during late gestation through the weaning period, and this corresponds to the * Corresponding author. Tel.: 1919-966-4435 ext. 241; fax: 11919-966-2423. E-mail address: [email protected] (A.S. Calikoglu).
developmental period when TH action is critical for normal brain development [2,18,20]. (2) TH and IGF-I stimulate myelination [9,18,26]. Hypothyroid rats and mice exhibit decreased and delayed expression of myelin-associated protein mRNA, and ultimately a dramatic decrease in total brain myelin content [14,19]. Transgenic mice that overexpress IGF-I in the brain exhibit a 2.5-fold increase in total brain myelin, while there is decreased expression of myelinassociated genes when IGF binding protein-1 (IGFBP-1), an inhibitor of IGF action when present in molar excess, is ectopically expressed in the brains of transgenic mice [24]. (3) Both TH and IGF-I also appear to have similar effects of synaptogenesis and neuritic outgrowth [10,16]. Hypothyroid rats exhibit a dramatic decrease in Purkinje cell arborization and dendritic spines, while IGF-I has been shown to promote embryonic neuronal survival and neurite outgrowth in cultured cortical neurons [1]. (4) Hypothyroidism induced in neonatal rats is accompanied by lower circulating IGF-I levels [17]. Decreased serum IGF-I levels also have been observed in hypothyroid neonates and children [15]. To more directly assess the relationship between TH and
0304-3940/00/$ - see front matter q 2000 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 0) 01 50 8- 1
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D.A. Elder et al. / Neuroscience Letters 293 (2000) 99±102
IGF-I, we asked whether TH in¯uences the expression of IGF-I mRNA during brain development, and therefore, whether IGF-I might be a mediator of some TH actions on brain development. We induced hypothyroidism in mice from late in fetal life and determined whether TH de®ciency in¯uences early postnatal IGF-I expression in the cerebral cortex and cerebellum±brain regions known to be affected in congenital hypothyroidism. C57/B6 mice were housed under standard conditions of 12-h light and 12-h dark with ambient temperature adjusted to 258C. Congenital hypothyroidism was induced by treating pregnant females with 0.1% propylthiouracil (PTU; Sigma, St. Louis, MO) in their drinking water from day 12 after conception (as determined by the presence of post-coital vaginal plug) until the time of weaning. Pups were allowed to nurse ad libitum. All study protocols were reviewed and approved by the University of North Carolina at Chapel Hill Institutional Review Committee for Animal Studies. We have shown that this method is effective and markedly decreases thyroxine to undetectable levels during the ®rst 10 days of life, and to levels 3 standard deviations below the mean levels of control mice after postnatal day (P) 10 [7]. Age-matched control and PTU-treated pups were sacri®ced at P7, P10, and P14. In addition, adult mice were treated with 0.1% PTU drinking water for 6 weeks, sacri®ced, and compared to control adult mice. Brains were rapidly removed and weighed. Cerebral cortex (CTX) and cerebellum (CB) were dissected, frozen separately in liquid nitrogen and stored at 2808C until RNA was extracted. Total RNA was extracted using a commercial kit, (Trizol reagent, Life Technologies, Grand Island, NY). A mouse IGF-I riboprobe was generated with PCR using primers speci®c for mouse IGF-I exon 3 cDNA and labeled with biotin using Biotin-14-CTP (Ambion Corp., Austin, TX). This probe gives a protected fragment of 170 nt in ribonuclease protection assay (RPA). A mouse cyclophillin riboprobe (Ambion Corp., Austin, TX), giving a 102 nt protected fragment was labeled with biotin in a similar manner.
RPA was performed using a commercial kit (RPA II, Ambion Corp., Austin, TX). Brie¯y, total RNA (30 mg) was hybridized with speci®c biotin-labeled RNA antisense probes for IGF-I and cyclophillin at 458C for 16 h. After hybridization, samples were treated with RNases A and T1, and protected fragments separated in 8% acrylamide/8M urea gels prior to being transferred to positively charged membranes by electroblotting. After washing, the signal was detected according to manufactures recommendations (Ambion Inc., Austin, TX). The density of the signals was quanti®ed densitometrically using the Image Pro Analysis System (Image Pro, Media Cybernetic, Silver Spring, MD) and normalized to cyclophillin. IGF-I mRNA abundance was calculated as the ratio of the IGF-I band to that of cyclophillin in the same lane. Data are expressed as the mean ^ SEM. Comparisons were made by unpaired Student's t-test using Stat View-II program (Abacus Concepts, Berkeley, CA). Compared with controls at P7 (Fig. 1), IGF-I mRNA abundance in CTX of TH de®cient mice showed a modest increase (25%) that was not statistically signi®cant (0.542 ^ 0.101 arbitrary units in hypothyroid mice, compared to 0.432 ^ 0.06 in controls; mean ^ SEM; P NS). At P10 and P14 IGF-I mRNA in TH de®cient mice was decreased by 42 and 60%, respectively (P10: 0.580 ^ 0.051, compared to 0.997 ^ 0.137 in controls, P , 0:01; P14: 0.190 ^ 0.017, compared to 0.460 ^ 0.145 in controls, P , 0:05). In CB, IGF-I mRNA abundance in TH de®cient mice was signi®cantly decreased at P7 by 31%, and appeared to be lower at P10 and P14 (12 and 28%, respectively), although these decreases did not meet tests of statistical signi®cance (P7: 0.253 ^ 0.019, compared to 0.365 ^ 0.029 in controls, P 0:006; P10: 0.115 ^ 0.020, compared to 0.131 ^ 0.028 in controls, P NS; P14: 0.318 ^ 0.039, compared to 0.438 ^ 0.061 in controls P NS). In mice in whom hypothyroidism was induced during adult life, IGF-I mRNA abundance in CTX was similar to that in controls (1.82 ^ 0.18, compared to 1.82 ^ 0.11 in controls, P NS). In CB of adult hypothyroid mice, IGF-I mRNA abundance was lower than in controls, but did not meet
Fig. 1. IGF-I mRNA abundance in the cerebral cortex of control (C) and hypothyroid (H) mice at postnatal day (P) 7, 10 and 14. Photomicrographs of representative gels from ribonuclease protection assay are shown. Each lane was loaded with 30 mg total RNA. The upper arrow indicates the 170 nt IGF-I mRNA protected fragment and lower arrow indicates the 102 nt cyclophillin (CYC) protected fragment. Because experiments were performed with mRNA samples from the mice of the same age, the gels shown come from separate experiments.
D.A. Elder et al. / Neuroscience Letters 293 (2000) 99±102
Fig. 2. IGF-I mRNA abundance in the cerebral cortex and cerebellum quanti®cation by ribonuclease protection assay. Data are expressed as the percentage of control mice. The values represent mean ^ SEM from 4±6 animals. *P , 0:05; **P , 0:01, compared to controls.
tests of statistical signi®cance (1.74 ^ 0.32, compared to 2.76 ^ 0.49 in controls, P NS). In summary, IGF-I mRNA expression in hypothyroid mice was decreased signi®cantly in CTX at P10 and P14 (42 and 60%, respectively), while in the CB IGF-I mRNA expression appeared to be down regulated at all ages studied, but the decrease was statistically signi®cant only at P7 (31%) (Fig. 2). Our ®ndings demonstrate that congenital TH de®ciency alters IGF-I mRNA abundance in murine brain in a developmentally speci®c fashion. We found changes in IGF-I expression in the ®rst 2 weeks of postnatal life when the brain is vulnerable to environmental insult. The pattern of change in IGF-I expression induced by TH de®ciency also appeared to differ between the CTX and CB, with the decrease in IGF-I expression occurring earlier in the CB. TH status is known to in¯uence serum IGF-I levels [13]. Serum IGF-I levels are low in humans with hypothyroidism and normalized after TH replacement [22]. This decrease in serum IGF-I is most likely a re¯ection of reduced growth hormone (GH) secretion in TH de®ciency. Eighteen-day-old rats made hypothyroid by maternal methimazole treatment exhibit a 50% reduction of serum IGF-I that is corrected with either TH or GH replacement [17]. Similarly, serum levels of IGFBP-3, a GH dependent IGFBP that carries most of the IGF-I circulating in serum, are lower in children with congenital hypothyroidism, and replacement TH increased serum IGFBP-3 levels signi®cantly [4,15]. These observations suggest that TH modulates GH secretion which in turn in¯uences IGF-I levels. We believe, however, that the change in IGF-I mRNA in the brain is a direct consequence of TH de®ciency, rather than an indirect response to reduced GH secretion. Several lines of evidence support this conclusion. (1) Studies of fetal hypothalamus in vitro show that T3 stimulates the produc-
101
tion of IGF-I [3]. (2) In experiments utilizing hypopituitary mice, T4 administration alone increased serum IGF-I, as it did when used to treat hypophysectomized or thyroidectomized rats [8,12]. Both T3 and GH, however, appear to be necessary to normalize serum IGF-I levels in adult hypophysectomized rats [6,21]. (3) TH replacement increases hepatic IGF-I mRNA in hypothyroid adult rats, while GH administration to hypothyroid rats fails to correct the reduction in liver IGF-I mRNA [17,23]. The mechanism(s) for the developmental and region speci®c changes in IGF-I mRNA abundance is not clear. It seems possible that differences in the regional distribution of TH receptors in brain regions play a role [5,11]. Another possibility is that hypothyroidism alters the ontogeny of IGF-I expression in brain. IGF-I expression in CTX peaks in the ®rst week of life in mice and then gradually declines [25]. If TH de®ciency delays the peak in CTX IGF-I expression (from P4 to P7), the observed decline in hypothyroid mice at P7 would coincide with the reduced IGF-I expression in normal controls at this time, and likely result in no alteration between IGF-I mRNA abundance in hypothyroid and normal mice. We conclude that IGF-I expression in the developing brain is altered by TH de®ciency in an age-dependent fashion. Furthermore, changes in IGF-I expression induced by TH de®ciency differ between CTX and CB. Our ®ndings suggest that IGF-I may mediate some TH actions during brain development. It also seems likely that decreased IGF-I expression contributes to the brain abnormalities induced by hypothyroidism. This work was supported by NIH grants DK-02506 (to ASC) and HD-08299 (to AJD). We thank Dr Dionisios Chrysis for his help in preparation of the IGF-I probe. [1] Aizenman, Y. and de Vellis, J., Brain neurons develop in a serum and glial free environment: effects of transferrin, insulin, insulin-like growth factor-I and thyroid hormone on survival, growth and differentiation, Brain Res., 406 (1987) 32±42. [2] Bach, M.A., Shen-Orr, Z., Lowe Jr., W.L., Roberts Jr., C.T. and LeRoith, D., insulin-like growth factor-I mRNA levels are developmentally regulated in speci®c regions of the rat brain, Brain Res. Mol. Brain Res., 10 (1991) 43±48. [3] Binoux, M., Faivre-Bauman, A., Lassarre, C., Barret, A. and Tixier-Vidal, A., Triiodothyronine stimulates the production of insulin-like growth factor (IGF) by fetal hypothalamus cells cultured in serum-free media, Dev. Brain Res., 21 (1985) 319±321. [4] Bona, G., Rapa, G., Boccardo, G., Silvestrol, L. and Chiorboli, E., IGF-I and IGFBP-3 in congenital and acquired hypothyroidism after long-term replacement treatment, Panminerva Med., 40 (1998) 103±106. [5] Bradley, D.J., Towel, H.C. and Young III, W.S., Spatial and temporal expression of a- and b-thyroid receptor mRNA, including the b2-subtype, in the developing mammalian nervous system, J. Neurosci., 12 (1992) 2288±2302. [6] Burstein, P.J., Draznin, B., Johnson, C.J. and Schalch, D.S., The effect of hypothyroidism on growth, serum growth hormone, the growth hormone-dependent somatomedin,
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D.A. Elder et al. / Neuroscience Letters 293 (2000) 99±102 insulin-like growth factor, and its carrier protein in rats, Endocrinology, 104 (1979) 1107±1111. Calikoglu, A.S., Gutierrez-Ospina, G. and D'Ercole, A.J., Congenital hypothyroidism delays the formation and retards the growth of the mouse primary somatic sensory cortex, Neurosci. Lett., 213 (1996) 132±136. Chernausek, S.D., Underwood, L.E. and Van Wyk, J.J., In¯uence of hypothyroidism on growth hormone binding by rat liver, Endocrinology, 111 (1982) 1534±1538. D'Ercole, A.J., Ye, P., Calikoglu, A.S. and Gutierrez-Ospina, G., The role of the insulin-like growth factors in the central nervous system, Mol. Neurobiol., 13 (1996) 227±255. Eayrs, J.T. and Taylor, S.H., The effect of thyroid de®ciency induced by methyl thiouracil on the maturation of the central nervous system, J. Anat., 850 (1951) 350±358. Forest, D., HallboÈoÈk, F., Persson, H. and VennstroÈm, B., Distinct functions for thyroid hormone receptors a and b in brain development indicated by differential expression of receptor genes, EMBO J., 10 (1991) 269±275. Gaspard, T., Wondergem, R., Hamamdzic, M. and Klitgaard, H.M., Serum somatomedin stimulation in thyroxine-treated hypophysectomized rats, Endocrinology, 102 (1978) 606± 611. Holder, A.T. and Wallis, M., Action of growth hormone, prolactin and thyroxine on serum somatomedin-like activity and growth in hypopituitary dwarf mice, J. Endocrinol., 74 (1977) 223±229. Ibarrola, N. and Rodriguez-Pena, A., Hypothyroidism coordinately and transiently affects myelin protein gene expression in most rat brain regions during postnatal development, Brain Res., 752 (1997) 285±293. Kandemir, N., Yordam, N. and Oguz, H., Age-related differences in serum insulin-like growth factor-I (IGF-I) and IGFbinding protein-3 levels in congenital hypothyroidism, J. Pediatr. Endocrinol. Metab., 10 (1997) 379±385. Legrand, J., Morphogenetic actions of thyroid hormone, Trends Neurosci., 2 (1979) 234±236. NaÈntoÈ-Salonen, K., Glasscock, G.F. and Rosenfeld, R.C., The effects of thyroid hormone on insulin-like growth factor (IGF) and IGF-binding proteins (IGFBPs) expression in the neonatal rat: prolonged high expression of IGFBP-2 in
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methimazole induced congenital hypothyroidism, Endocrinology, 129 (1991) 2563±2570. Porter®eld, S.P. and Hendrich, C.E., The role of thyroid hormones in prenatal and neonatal neurological development±current perspectives, Endocr. Rev., 14 (1993) 94±105. Rosman, N.P. and Malone, M.J., Brain myelination in experimental hypothyroidism, In G.D. Grave (Ed.), Thyroid Hormones and Brain Development, Raven Press, New York, 1977, pp. 169±198. Rotein, H., Differential expression of insulin-like growth factor-I genes in rat central nervous system, Proc. Natl. Acad. Sci. USA, 85 (1988) 265±269. Schalch, D.S., Heinrich, U.E., Draznin, B., Johnson, C.J. and Miller, L.L., Role of the liver in regulating somatomedin activity: hormonal effects on the synthesis and release of insulin-like growth factor and its carrier protein by the isolated perfused rat liver, Endocrinology, 104 (1979) 1143±1151. S®ell, J.P., Taylor, A.M., Zini, M., Maheshwari, H.G., Ross, R.J.M. and Valcavi, R., Effects of hypothyroidism and hyperthyroidism on insulin-like growth factors and growth hormone on IGF binding proteins, J. Clin. Endocrinol. Metab., 76 (1993) 950±953. Wolf, M., Ingbar, S.H. and Moses, A.C., Thyroid hormone and growth hormone interact to regulate insulin-like growth factor-I messenger ribonucleic acid and circulating levels in the rat, Endocrinology, 125 (1989) 2905±2914. Ye, P., Carson, J. and D'Ercole, A.J., In vivo actions of insulin-like growth factor-I (IGF-I) on brain myelination: studies of IGF-I and IGF binding protein-1 (IGFBP-1) in transgenic mice, J. Neurosci., 15 (1995) 7344±7356. Ye, P., Umayahara, Y., Ritter, D., Bunting, T., Auman, H., Rotwein, P. and D'Ercole, A.J., Regulation of insulin-like growth factor-I gene (IGF-I) expression in the brain of transgenic mice expressing an IGF-I-luciferase fusion gene, Endocrinology, 138 (1997) 5466±5475. Ye, P., Xing, Y., Dai, Z. and D'Ercole, A.J., In vivo actions of insulin-like growth factors-I (IGF-I) on cerebellum development in transgenic mice: evidence that IGF-I increases proliferation of granule cell progenitors, Dev. Brain Res., 95 (1996) 44±54.
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Effects of hypothyroidism on insulin-like growth factor-I expression during brain development in mice Deborah A. Elder, Aysen F. Karayal, A. Joseph D'Ercole, Ali S. Calikoglu* Department of Pediatrics, Division of Endocrinology, CB 7220, 108 Burnett-Womack, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7220, USA Received 29 June 2000; received in revised form 23 August 2000; accepted 24 August 2000
Abstract Hypothyroidism has devastating consequences on brain development. While the mechanisms that mediate these effects are not known, several lines of evidence suggest that a reduction in insulin-like growth factor-I (IGF-I) expression and/or action has a role. To assess whether reduced IGF-I expression and/or actions mediates the brain pathology of congenital hypothyroidism, we induced hypothyroidism by treating pregnant mice and lactating dams with 0.1% propylthiouracil (PTU) in drinking water. Control and PTU-treated pups were sacri®ced on postnatal day (P) 7, 10 and 14, and IGF-I mRNA expression was assessed in the cerebral cortex and cerebellum by ribonuclease protection assay. To control for mRNA loading, the signal of IGF-I protected bands was normalized to those for cyclophillin. IGF-I mRNA expression in hypothyroid animals was decreased signi®cantly in cortex at P10 and P14 (42 and 60%, respectively). In the cerebellum, IGF-I mRNA expression was down-regulated at all ages studied, but the decrease was only statistically signi®cant at P7 (31% decreased). We conclude that hypothyroidism alters IGF-I expression in the developing brain. Furthermore, we speculate that IGF-I plays a role in mediating some thyroid hormone actions during brain development. q 2000 Published by Elsevier Science Ireland Ltd. Keywords: Thyroid hormone; Hypothyroidism; Insulin-like growth factor-I; Brain; Mice
Thyroid hormone (TH) has a signi®cant in¯uence on the structural, biochemical and functional development of the brain. In man, untreated congenital hypothyroidism leads to multiple morphologic and biochemical alterations in the brain and results in irreversible mental retardation. Studies in rodents show that reduced myelination, poor neuritic outgrowth and dendritic arborization are among the changes in the brain due to congenital hypothyroidism. The mechanisms of TH action on brain development, however, are incompletely de®ned. Furthermore, TH action may not be direct. TH is known to interact with growth factors, including, insulin-like growth factor-I (IGF-I), epidermal growth factor and neurotropins. It is possible, therefore, that these agents mediate TH actions during brain development. Several lines of experimental evidence in rodents suggest that a reduction in IGF-I expression and/or action may mediate the effects of TH on brain development. (1) Peak IGF-I expression in the rat brain occurs during late gestation through the weaning period, and this corresponds to the * Corresponding author. Tel.: 1919-966-4435 ext. 241; fax: 11919-966-2423. E-mail address: [email protected] (A.S. Calikoglu).
developmental period when TH action is critical for normal brain development [2,18,20]. (2) TH and IGF-I stimulate myelination [9,18,26]. Hypothyroid rats and mice exhibit decreased and delayed expression of myelin-associated protein mRNA, and ultimately a dramatic decrease in total brain myelin content [14,19]. Transgenic mice that overexpress IGF-I in the brain exhibit a 2.5-fold increase in total brain myelin, while there is decreased expression of myelinassociated genes when IGF binding protein-1 (IGFBP-1), an inhibitor of IGF action when present in molar excess, is ectopically expressed in the brains of transgenic mice [24]. (3) Both TH and IGF-I also appear to have similar effects of synaptogenesis and neuritic outgrowth [10,16]. Hypothyroid rats exhibit a dramatic decrease in Purkinje cell arborization and dendritic spines, while IGF-I has been shown to promote embryonic neuronal survival and neurite outgrowth in cultured cortical neurons [1]. (4) Hypothyroidism induced in neonatal rats is accompanied by lower circulating IGF-I levels [17]. Decreased serum IGF-I levels also have been observed in hypothyroid neonates and children [15]. To more directly assess the relationship between TH and
0304-3940/00/$ - see front matter q 2000 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 0) 01 50 8- 1
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D.A. Elder et al. / Neuroscience Letters 293 (2000) 99±102
IGF-I, we asked whether TH in¯uences the expression of IGF-I mRNA during brain development, and therefore, whether IGF-I might be a mediator of some TH actions on brain development. We induced hypothyroidism in mice from late in fetal life and determined whether TH de®ciency in¯uences early postnatal IGF-I expression in the cerebral cortex and cerebellum±brain regions known to be affected in congenital hypothyroidism. C57/B6 mice were housed under standard conditions of 12-h light and 12-h dark with ambient temperature adjusted to 258C. Congenital hypothyroidism was induced by treating pregnant females with 0.1% propylthiouracil (PTU; Sigma, St. Louis, MO) in their drinking water from day 12 after conception (as determined by the presence of post-coital vaginal plug) until the time of weaning. Pups were allowed to nurse ad libitum. All study protocols were reviewed and approved by the University of North Carolina at Chapel Hill Institutional Review Committee for Animal Studies. We have shown that this method is effective and markedly decreases thyroxine to undetectable levels during the ®rst 10 days of life, and to levels 3 standard deviations below the mean levels of control mice after postnatal day (P) 10 [7]. Age-matched control and PTU-treated pups were sacri®ced at P7, P10, and P14. In addition, adult mice were treated with 0.1% PTU drinking water for 6 weeks, sacri®ced, and compared to control adult mice. Brains were rapidly removed and weighed. Cerebral cortex (CTX) and cerebellum (CB) were dissected, frozen separately in liquid nitrogen and stored at 2808C until RNA was extracted. Total RNA was extracted using a commercial kit, (Trizol reagent, Life Technologies, Grand Island, NY). A mouse IGF-I riboprobe was generated with PCR using primers speci®c for mouse IGF-I exon 3 cDNA and labeled with biotin using Biotin-14-CTP (Ambion Corp., Austin, TX). This probe gives a protected fragment of 170 nt in ribonuclease protection assay (RPA). A mouse cyclophillin riboprobe (Ambion Corp., Austin, TX), giving a 102 nt protected fragment was labeled with biotin in a similar manner.
RPA was performed using a commercial kit (RPA II, Ambion Corp., Austin, TX). Brie¯y, total RNA (30 mg) was hybridized with speci®c biotin-labeled RNA antisense probes for IGF-I and cyclophillin at 458C for 16 h. After hybridization, samples were treated with RNases A and T1, and protected fragments separated in 8% acrylamide/8M urea gels prior to being transferred to positively charged membranes by electroblotting. After washing, the signal was detected according to manufactures recommendations (Ambion Inc., Austin, TX). The density of the signals was quanti®ed densitometrically using the Image Pro Analysis System (Image Pro, Media Cybernetic, Silver Spring, MD) and normalized to cyclophillin. IGF-I mRNA abundance was calculated as the ratio of the IGF-I band to that of cyclophillin in the same lane. Data are expressed as the mean ^ SEM. Comparisons were made by unpaired Student's t-test using Stat View-II program (Abacus Concepts, Berkeley, CA). Compared with controls at P7 (Fig. 1), IGF-I mRNA abundance in CTX of TH de®cient mice showed a modest increase (25%) that was not statistically signi®cant (0.542 ^ 0.101 arbitrary units in hypothyroid mice, compared to 0.432 ^ 0.06 in controls; mean ^ SEM; P NS). At P10 and P14 IGF-I mRNA in TH de®cient mice was decreased by 42 and 60%, respectively (P10: 0.580 ^ 0.051, compared to 0.997 ^ 0.137 in controls, P , 0:01; P14: 0.190 ^ 0.017, compared to 0.460 ^ 0.145 in controls, P , 0:05). In CB, IGF-I mRNA abundance in TH de®cient mice was signi®cantly decreased at P7 by 31%, and appeared to be lower at P10 and P14 (12 and 28%, respectively), although these decreases did not meet tests of statistical signi®cance (P7: 0.253 ^ 0.019, compared to 0.365 ^ 0.029 in controls, P 0:006; P10: 0.115 ^ 0.020, compared to 0.131 ^ 0.028 in controls, P NS; P14: 0.318 ^ 0.039, compared to 0.438 ^ 0.061 in controls P NS). In mice in whom hypothyroidism was induced during adult life, IGF-I mRNA abundance in CTX was similar to that in controls (1.82 ^ 0.18, compared to 1.82 ^ 0.11 in controls, P NS). In CB of adult hypothyroid mice, IGF-I mRNA abundance was lower than in controls, but did not meet
Fig. 1. IGF-I mRNA abundance in the cerebral cortex of control (C) and hypothyroid (H) mice at postnatal day (P) 7, 10 and 14. Photomicrographs of representative gels from ribonuclease protection assay are shown. Each lane was loaded with 30 mg total RNA. The upper arrow indicates the 170 nt IGF-I mRNA protected fragment and lower arrow indicates the 102 nt cyclophillin (CYC) protected fragment. Because experiments were performed with mRNA samples from the mice of the same age, the gels shown come from separate experiments.
D.A. Elder et al. / Neuroscience Letters 293 (2000) 99±102
Fig. 2. IGF-I mRNA abundance in the cerebral cortex and cerebellum quanti®cation by ribonuclease protection assay. Data are expressed as the percentage of control mice. The values represent mean ^ SEM from 4±6 animals. *P , 0:05; **P , 0:01, compared to controls.
tests of statistical signi®cance (1.74 ^ 0.32, compared to 2.76 ^ 0.49 in controls, P NS). In summary, IGF-I mRNA expression in hypothyroid mice was decreased signi®cantly in CTX at P10 and P14 (42 and 60%, respectively), while in the CB IGF-I mRNA expression appeared to be down regulated at all ages studied, but the decrease was statistically signi®cant only at P7 (31%) (Fig. 2). Our ®ndings demonstrate that congenital TH de®ciency alters IGF-I mRNA abundance in murine brain in a developmentally speci®c fashion. We found changes in IGF-I expression in the ®rst 2 weeks of postnatal life when the brain is vulnerable to environmental insult. The pattern of change in IGF-I expression induced by TH de®ciency also appeared to differ between the CTX and CB, with the decrease in IGF-I expression occurring earlier in the CB. TH status is known to in¯uence serum IGF-I levels [13]. Serum IGF-I levels are low in humans with hypothyroidism and normalized after TH replacement [22]. This decrease in serum IGF-I is most likely a re¯ection of reduced growth hormone (GH) secretion in TH de®ciency. Eighteen-day-old rats made hypothyroid by maternal methimazole treatment exhibit a 50% reduction of serum IGF-I that is corrected with either TH or GH replacement [17]. Similarly, serum levels of IGFBP-3, a GH dependent IGFBP that carries most of the IGF-I circulating in serum, are lower in children with congenital hypothyroidism, and replacement TH increased serum IGFBP-3 levels signi®cantly [4,15]. These observations suggest that TH modulates GH secretion which in turn in¯uences IGF-I levels. We believe, however, that the change in IGF-I mRNA in the brain is a direct consequence of TH de®ciency, rather than an indirect response to reduced GH secretion. Several lines of evidence support this conclusion. (1) Studies of fetal hypothalamus in vitro show that T3 stimulates the produc-
101
tion of IGF-I [3]. (2) In experiments utilizing hypopituitary mice, T4 administration alone increased serum IGF-I, as it did when used to treat hypophysectomized or thyroidectomized rats [8,12]. Both T3 and GH, however, appear to be necessary to normalize serum IGF-I levels in adult hypophysectomized rats [6,21]. (3) TH replacement increases hepatic IGF-I mRNA in hypothyroid adult rats, while GH administration to hypothyroid rats fails to correct the reduction in liver IGF-I mRNA [17,23]. The mechanism(s) for the developmental and region speci®c changes in IGF-I mRNA abundance is not clear. It seems possible that differences in the regional distribution of TH receptors in brain regions play a role [5,11]. Another possibility is that hypothyroidism alters the ontogeny of IGF-I expression in brain. IGF-I expression in CTX peaks in the ®rst week of life in mice and then gradually declines [25]. If TH de®ciency delays the peak in CTX IGF-I expression (from P4 to P7), the observed decline in hypothyroid mice at P7 would coincide with the reduced IGF-I expression in normal controls at this time, and likely result in no alteration between IGF-I mRNA abundance in hypothyroid and normal mice. We conclude that IGF-I expression in the developing brain is altered by TH de®ciency in an age-dependent fashion. Furthermore, changes in IGF-I expression induced by TH de®ciency differ between CTX and CB. Our ®ndings suggest that IGF-I may mediate some TH actions during brain development. It also seems likely that decreased IGF-I expression contributes to the brain abnormalities induced by hypothyroidism. This work was supported by NIH grants DK-02506 (to ASC) and HD-08299 (to AJD). We thank Dr Dionisios Chrysis for his help in preparation of the IGF-I probe. [1] Aizenman, Y. and de Vellis, J., Brain neurons develop in a serum and glial free environment: effects of transferrin, insulin, insulin-like growth factor-I and thyroid hormone on survival, growth and differentiation, Brain Res., 406 (1987) 32±42. [2] Bach, M.A., Shen-Orr, Z., Lowe Jr., W.L., Roberts Jr., C.T. and LeRoith, D., insulin-like growth factor-I mRNA levels are developmentally regulated in speci®c regions of the rat brain, Brain Res. Mol. Brain Res., 10 (1991) 43±48. [3] Binoux, M., Faivre-Bauman, A., Lassarre, C., Barret, A. and Tixier-Vidal, A., Triiodothyronine stimulates the production of insulin-like growth factor (IGF) by fetal hypothalamus cells cultured in serum-free media, Dev. Brain Res., 21 (1985) 319±321. [4] Bona, G., Rapa, G., Boccardo, G., Silvestrol, L. and Chiorboli, E., IGF-I and IGFBP-3 in congenital and acquired hypothyroidism after long-term replacement treatment, Panminerva Med., 40 (1998) 103±106. [5] Bradley, D.J., Towel, H.C. and Young III, W.S., Spatial and temporal expression of a- and b-thyroid receptor mRNA, including the b2-subtype, in the developing mammalian nervous system, J. Neurosci., 12 (1992) 2288±2302. [6] Burstein, P.J., Draznin, B., Johnson, C.J. and Schalch, D.S., The effect of hypothyroidism on growth, serum growth hormone, the growth hormone-dependent somatomedin,
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