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Endocrinology Vol. 147, No. 10 4893-4903
Copyright © 2006 by The Endocrine Society

Increased Pro-Nerve Growth Factor and p75 Neurotrophin Receptor Levels in Developing Hypothyroid Rat Cerebral Cortex Are Associated with Enhanced Apoptosis

Ashok Kumar, Rohit Anthony Sinha, Meenakshi Tiwari, Lily Pal, Ashutosh Shrivastava, Rajesh Singh, Krishna Kumar, Sushil Kumar Gupta and Madan M. Godbole

Departments of Endocrinology (A.K., R.A.S., M.T., A.S., R.S., K.K., S.K.G., M.M.G.) and Pathology (L.P.), Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, India

Address all correspondence and requests for reprints to: Prof. Madan M. Godbole, Head, Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226 014, India. E-mail: madangodbole{at}yahoo.co.in.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormone insufficiency adversely affects cortical development; however, its effect on apoptosis modulation during cerebral cortex development is not understood. We investigated the effect of perinatal hypothyroidism on apoptosis and its mechanisms during rat cerebral cortex development. Primary hypothyroidism was induced by feeding methimazole (0.025% wt/vol) in the drinking water to pregnant and lactating rats and continued until the animals were killed (hypothyroid group). Cerebral cortices from pups were harvested at different postnatal ages (postnatal d 0, 8, 16, and 24 and adult), and apoptosis was quantitated by terminal deoxynucleotide transferase-mediated dUTP nick end labeling and cleaved caspase-3 immunoreactivity. Compared with the euthyroid, primary somatosensory cortex (S1) in the hypothyroid group exhibited enhanced apoptosis. In S1 of euthyroid rats, apoptotic cells were mostly found in cortical layers I–III and the proportion of apoptotic cells enhanced significantly in the hypothyroid group (P < 0.001). Most of the apoptotic cells were neurons, as assessed by double immunolabeling. A significantly increased activation of caspase-3 and -7, decreased levels of antiapoptotic proteins Bcl-2 and Bcl-xL, and increased levels of proapoptotic protein Bax was observed in the developing cerebral cortex of hypothyroid rats, compared with the euthyroid (P < 0.001). In addition, hypothyroidism significantly elevated the levels of 53-kDa pro-nerve growth factor (P < 0.001) and p75 neurotrophin receptor (P < 0.001) and decreased TrkA expression. Taken together, we provide evidence for the possible contribution of pro-nerve growth factor/p75 neurotrophin receptor pathway in hypothyroidism-enhanced apoptosis during rat cortical development. Thus, the present study may help in explaining the mechanism of the deleterious effect of thyroid hormone deficiency on cerebral cortex development in children.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE (TH) is essential for mammalian brain development. Neonatal hypothyroidism, caused by either iodine-deficiency disorder or congenital hypothyroidism, is the world’s leading cause of preventable mental retardation, the consequences of which depend on the specific timing of onset and duration of TH deficiency (1, 2). A delay in TH replacement or failure to treat the congenital hypothyroid neonate within the first few months after birth results in severe, irreversible intellectual deficits (3). Hypothyroidism in neonates is well documented in severe iodine-deficient areas (4, 5). Previous studies in rats suggested that perinatal hypothyroidism leads to poor development of cerebral cortex. Cells in the hypothyroid rat cortex are smaller and more closely aggregated than the euthyroid, due in part to an overall decrease in development of axonal and dendritic processes (6).

A critical role of TH in neuronal cell proliferation, migration, differentiation, and maturation has been well established (6). For example, in vitro studies have shown that T3 promotes survival and differentiation of septal (7), cerebellar (8), and sensory neurons (9). Although apoptosis is an important physiological process of brain development, how apoptosis is modulated under fetal and neonatal hypothyroidism is poorly understood. We and others have shown that neonatal hypothyroidism causes extensive apoptosis in the internal granular layer of the cerebellum (10, 11, 12, 13). Whether perinatal hypothyroidism in rats modulates apoptosis in the developing cerebral cortex has not been studied.

Apoptosis involves activation of a caspase cascade that directly causes disassembly of cellular structures (14, 15). Two different pathways of caspase activation leading to cell death have been identified, an extrinsic and an intrinsic pathway. The extrinsic pathway involves activation of death receptors, such as Fas by FasL and activation of caspase-8, which in turn activates caspase-3 (16). The intrinsic pathway involves mitochondrial release of cytochrome c, which forms the apoptosome complex by interacting with apoptosis protease-activating factor-1 and caspase-9 and, thereby, activates caspase-3 and -7 (17). Among the various proteins involved in apoptosis, the Bcl-2 family of proteins are the key regulators of apoptosis. Bcl-2 and Bcl-xL serve to protect cells from stimuli inducing cell death whereas Bax, Bcl-xS, Bad, and Bak promote apoptosis (18).

Nerve growth factor (NGF) regulates neuronal survival and cell death via binding to two different receptors, viz. TrkA, a tyrosine kinase-receptor, and p75 neurotrophin receptor (NTR), a member of the tumor necrosis factor receptor family (19, 20). NGF is synthesized as a precursor, pro-NGF, which undergoes posttranslational processing to generate mature 13.5-kDa NGF-ß (21). The processing of pro-NGF also generates a number of high-molecular-weight intermediate forms whose biological roles are not well understood (21). Pro-NGF isoforms and not mature NGF-ß are the predominant forms in human and rat brains and several peripheral tissues (22). Recent studies have shown that pro-NGF is a specific ligand for p75NTR, which can induce apoptosis in superior cervical ganglion neurons, vascular smooth muscle cells (23), and oligodendrocytes (24). In addition, sortilin, a member of the Vps10p-domain family, receptor is required for pro-NGF-induced apoptosis (25). Neonatal as well as adult-onset hypothyroidism has been shown to modulate the levels of NGF in the rat cerebral cortex, hippocampus, and cerebellum (10, 26). Previous studies have reported conflicting results of NGF expression in various parts of the brain under a hypothyroid state: increased (10, 27), decreased (26), or no change (28). Despite the importance of NGF and its receptors in cell survival and cell death, their role in the survival of cortical neurons under TH deficiency is still not known.

This study for the first time demonstrates that perinatal hypothyroidism enhances apoptosis in developing cerebral cortex due to increased caspase-3 and -7 activation, up-regulation of a proapoptotic molecule (Bax) and down-regulation of antiapoptotic molecules (Bcl-2 and Bcl-xL). Furthermore, p75NTR and 53-kDa pro-NGF levels were elevated and TrkA mRNA expression reduced under the hypothyroid state, suggesting the involvement of a pro-NGF/p75NTR-mediated pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies
Phenylmethylsulfonyl fluoride, Hoechst 33258, proteinase K, protease inhibitor cocktail, and 2-mercapto-1-methylimidazole (MMI) were procured from Sigma Chemical Co. (St. Louis, MO). All other reagents were of molecular biology grade and of high purity. Rabbit polyclonal antibodies against caspase-7 (kindly gifted by Dr. Xiao-Ming Sun, University of Leicester, Leicester, UK), p75NTR (kindly gifted by Dr. M. V. Chao, Skirball Institute of Biomolecular Medicine, New York, NY), and sortilin (kindly gifted by Dr. C. M. Petersen, Aarhus University, Aarhus, Denmark) were used. Antibodies against Bcl-xL, Bax, Bcl-2, caspase-3, and NGF (Sc-548) and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against cleaved caspase-3 was purchased from Cell Signaling Technology (Beverly, MA).

Animals and treatments
Sprague Dawley rats were housed in a 12-h day and light cycle environment with ad libitum availability of chow diet and tap water. The pregnant rats were divided into two groups, the euthyroid and hypothyroid (n = 20 in each group). Hypothyroidism was induced in rats using a protocol described earlier (12). Briefly, MMI (0.025% wt/vol) was given to the pregnant rats in drinking water from gestational d 8 and continued until the animals were killed. Pups from both groups, the euthyroid and the hypothyroid, were killed at postnatal d 0 (P0) (n = 28), P8 (n = 23), P16 (n = 21), P24 (n = 15), and P90 (adult rat; n = 9). For replacement group (MMI+T4), 4-d-old pups kept on an MMI regimen were injected daily with T4 (1.5 µg/100 g body weight) ip and killed at P8 (n = 5). Total T4 (TT4) and total T3 (TT3) were measured in the serum of the rat pups by RIA using DPC kits (DPC, New York, NY). All animal procedures performed above were approved by the Institutional Guidelines for Animal Care and Research.

Detection of apoptosis
In situ detection of apoptosis was performed by terminal deoxynucleotide-transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and cleaved caspase-3 immunoreactivity (ir). In eu- and hypothyroid groups, five to seven pups from two to three litters were taken at each developmental stage (P0, P8, P16, and P24), with an exception at P90 (adult) where two to three animals were studied. Pups and adult rats were anesthetized and transcardially perfused with normal saline (0.9% NaCl wt/vol, 15–20 ml for pups and 50 ml for adults) followed by 4%-paraformldehyde in 0.1 M phosphate buffer (20–25 ml for pups and 100 ml for adults) and postfixed in paraformaldehyde at 4 C for 4 h. Tissues were then dehydrated in an alcohol series for paraffin embedding by standard protocol. Coronal sections (3 µm) were cut from the rat brain containing the primary somatosensory cortex (S1) from the pups and adult rats at different developmental stages and mounted on poly-L-lysine-coated slides. One series was processed for TUNEL, and parallel series were processed for cresyl violet staining and immunofluorescence studies. Sections from the eu- and hypothyroid rats were always processed in parallel.

For the TUNEL assay, a DNA fragmentation detection kit (BD Biosciences, Palo Alto, CA) was used. Coronal sections were deparaffinized, rehydrated, and treated with proteinase K (20 µg/ml) for 10 min. A labeling reaction was then performed with fluorescein-labeled dUTP in the presence of TdT at 37 C for 1 h. The sections were counterstained with Hoechst 33258 (0.5 µg/ml), a nuclear stain (blue fluorescence). Stained cells were then visualized under a fluorescence microscope (Nikon Instech Co. Ltd., Kawasaki, Kanagawa, Japan).

For immunofluorescence, after deparaffinization and rehydration steps, antigen retrieval was performed in a microwave oven using 10 mM citrate buffer (pH 6.0). Sections were blocked with 10% normal sheep serum for 20 min. To localize the apoptotic cells, sections were incubated with a rabbit polyclonal cleaved caspase-3 antibody (1:250; Cell Signaling Technology) overnight at 4 C. After washing with PBS, the sections were incubated with Rhodamine Red-conjugated antirabbit IgG (red) secondary antibody for 1 h in the dark. To investigate whether the apoptotic cells are neurons, the sections were double immunostained with cleaved caspase-3 antibody and a mouse monoclonal neurofilament antibody (1:50; Sigma). Then the sections were incubated with Rhodamine Red-conjugated antirabbit IgG (red) and fluroescein-isothiocyanate-conjugated antimouse IgG (green) antibodies for 1 h in the dark. The sections were then counterstained with Hoechst 33258, mounted with antifade medium, and visualized under a fluorescence microscope. Sections not incubated with primary antibody were used as negative controls.

TUNEL-positive cells were counted in five randomly selected fields, spanning from layer I to white matter of S1. Five to seven sections per animal were analyzed at each developmental stage. At least 10,000 cells in each section were scored. The apoptotic index was expressed as the number of TUNEL-positive cells per 100 nuclei (Hoechst stained). Image-Pro Plus 5.1 software (Media Cybernetics Inc., Silver Spring, MD) was used for image capturing and cell counting.

Protein extraction and Western blotting
For Western blotting, neocortex from five to seven pups (in both groups, eu- and hypothyroid) from two different litters were harvested at each developmental stage, snap-frozen in liquid nitrogen, and stored at –80 C until further investigation. Cytosolic proteins were extracted from the neocortex of eu- and hypothyroid rats at different developmental stages, as described previously (12). In the early neonatal period (P0 and P8), neocortex from two to three pups was pooled to get a sufficient amount of sample. For preparation of tissue homogenates, the cerebral cortices were washed once with PBS and suspended in 10 vol of lysis buffer [10 mM Tris-Cl (pH 7.5), 50 mM sodium chloride, 1% Triton-X-100 and containing phenylmethylsulfonyl fluoride (1 mM) and protease inhibitor cocktail (a mixture of 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatinA, E-64, bestatin, leupeptin, and aprotinin)] and kept on ice for 10 min. Then the tissues were homogenized using a Teflon homogenizer and centrifuged at 12,000 x g for 15 min at 4 C, and the supernatant was collected. Protein concentration was determined in the above samples, the cytosolic and the tissue homogenate, using a protein assay kit (Bio-Rad, Hercules, CA). Cytosolic or tissue homogenate proteins (50–100 µg) were subjected to 12 or 15% SDS-PAGE and electrotransferred onto nitrocellulose membrane. The membranes were incubated with primary antibodies followed by incubation with horseradish-peroxidase-conjugated secondary antibodies. The signals were detected using an enhanced chemiluminescence detection system (Amersham Biosciences, Little Chalfont, UK). Relative expression of each gene was determined by densitometric analysis using LabWorks 4.0 software (UVP Ltd., Cambridge, UK).

RT-PCR
Total RNA was isolated from the neocortex of three to five pups (at each developmental stage from eu- and hypothyroid rats) from two different litters after single-step RNA isolation method using TRI reagent (MRC Inc., Cincinnati, OH). Total RNA (2 µg) was reverse transcribed to cDNA using oligo-(dT)16 primers with the Thermoscript-RT-PCR kit (Invitrogen) following the manufacturer’s instructions. The cDNA was then amplified using 35 cycles with the following conditions: for TrkA, denaturation at 94 C for 30 sec, annealing at 63 C for 15 sec, and extension at 72 C for 1 min; and for ß-actin, denaturation at 94 C for 15 sec, annealing at 48 C for 15 sec, and extension at 72 C for 30 sec. Primers used were 5'-TGGCTGCCTTCGCCTCAACCAG-3' (TrkA forward) 5'-GGTGGACACAGGTATCAC TG-3' (TrkA reverse), 5'-GCATGGGTCAGAAGGAT-3' (ß-actin forward), and 5'-CCAATGGTGATGACCTG-3' (ß-actin reverse). The PCR products were electrophoresed on 2% agarose gel and visualized by ethidium bromide staining. ß-Actin was used as an internal control. For negative control, reverse transcriptase was omitted from the reaction mixture.

Statistical analysis
Statistical analysis was performed by using SPSS software version 11 (SPSS, Inc., Chicago, IL). The data are presented as a mean ± SE. Significant differences between groups were compared by two-way ANOVA, factors being developmental stages and experimental groups (euthyroid vs. hypothyroid). One-way ANOVA was then used to identify developmental stages affected by the hypothyroidism, followed by Turkey’s or Duncan post hoc test. P value < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Assessment of hypothyroidism
TT4 and TT3 levels were significantly low in the hypothyroid compared with the euthyroid group (P < 0.001) (Table 1Go). The pups from euthyroid dams recorded normal growth and size gain during development; whereas those born from hypothyroid dams showed retarded size and weight gain and showed typical features of hypothyroidism such as delayed eye opening, hair growth, and staggered walk (data not shown). Cresyl-violet-stained sections showed marked differences in the cytoarchitecture of hypothyroid rat pups compared with the euthyroid pups. The borders between the layers were well defined in S1 of euthyroid rats, whereas in the hypothyroid group, the borders between the layers were blurred (Fig. 1AGo). At higher magnification, we found that pyramidal neurons in S1 of hypothyroid rats were smaller than those in the euthyroid group (data not shown).


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  		TABLE 1. Circulating total T3 and total T4 in pups and adults in the experimental groups

 

Figure 1
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  		FIG. 1. Hypothyroidism enhances apoptosis in the developing cerebral cortex. A, Photomicrographs of cresyl-violet-stained coronal sections showing the cytoarchitecture of the primary somatosensory cortex in 8-d-old euthyroid (a) and hypothyroid (b) rats. Scale bar, 300 µm. Horizontal lines indicate borders between layers. B, Representative photomicrographs of coronal sections of primary somatosensory cortex showing TUNEL-positive cells in the euthyroid (a–e) and hypothyroid (f–j) rat pup at P0 (a and f), P8 (b and g), P16 (c and h), P24 (d and i), and adult (e and j) stages. Note the increase in TUNEL-positive cells in the hypothyroid group (f–j) compared with the euthyroid group (a–e). Scale bar, 20 µm (a–j). C, Proportion of TUNEL-positive cells in the primary somatosensory cortex in the euthyroid and hypothyroid rats at different developmental stages. TUNEL-positive cells were counted in five different areas spanning from layer I to white matter, and at least 10,000 cells in each section were scored. The apoptotic index was expressed as number of TUNEL-positive cells per 100 nuclei (Hoechst stained). Results are expressed as means ± SE of five to seven rats at each developmental stage. Significant differences compared with age-matched euthyroid pups are indicated: *, P < 0.05; **, P < 0.01; {psi}, P < 0.001.

 
Hypothyroidism enhances apoptosis in developing rat cerebral cortex
To investigate whether fetal and postnatal hypothyroidism modulates the extent of apoptosis, TUNEL assay was performed on paraffin-embedded tissue sections prepared from rat pups from the eu- and hypothyroid groups. Apoptotic cells were counted in a total of five randomly selected areas of S1 at each developmental stage. In the euthyroid group, the proportion of apoptotic cells was maximally observed at P0 stage (6.9 ± 0.3%), which progressively decreased with the age of rats (P8–P24) to reach adulthood levels at which stage it was minimum (Fig. 1Go, B and C). In the hypothyroid group, the overall proportion of apoptotic cells increased significantly compared with the euthyroid (P < 0.001). The increase in the proportion of apoptotic cells was observed throughout the developmental period (from birth to P24 stage) in the hypothyroid cerebral cortex, the highest increase being at P8 stage (Fig. 1Go, B and C; euthyroid 6.52 ± 0.353% vs. hypothyroid 8.84 ± 0.258%).

To investigate the radial distribution of the apoptotic cells in rat S1, cleaved caspase-3 immunofluorescence (red) was used as a marker of apoptosis (29). To identify the cortical layers, sections were counterstained with Hoechst 33258 (blue). In the merged images, pink indicates the localization of cleaved caspase-3 in the nuclei (Fig. 2AGo). In the euthyroid group at all the developmental stages, in S1, the majority of the cleaved caspase-3-ir cells were located in the superficial layers I–III (Fig. 2AGo, left). The maximal number of cleaved caspase-3-ir cells were observed at P0 stage (6.28 ± 0.93%) and then decreased progressively with the age of rats, being minimal in the adult rats (Fig. 2BGo). In the hypothyroid group, the proportion of cleaved caspase-3-ir cells were increased significantly in the superficial layers I–III of S1 throughout the developmental period (P0–P24) compared with the euthyroid group (P < 0.001; Fig. 2BGo). No significant difference was observed at the adult stage between the two groups.


Figure 2
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  		FIG. 2. A, Representative photomicrographs of coronal sections of primary somatosensory cortex showing the distribution of cleaved caspase-3-ir (apoptotic, red) cells in the euthyroid and hypothyroid rat pup at P0 (a–d), P8 (e–h), P16 (i–l), P24 (m–p), and adult (q–t) stages. Sections were counterstained with Hoechst (blue), and images of cleaved caspase-3-ir (red) and Hoechst (not shown) were merged (right). In the merged figures, pink indicates localization of cleaved caspase-3-ir in the nuclei. Note the increase in apoptotic cells (pink) at all the stages in the hypothyroid group (right) compared with the euthyroid group (left). Horizontal lines indicate the borders between layers. Scale bar, 50 µm (a–t). I, Layer I; II–III, layers II and III. B, Proportion of cleaved-caspase-3-ir cells in the primary somatosensory cortex in the euthyroid and hypothyroid rats at different developmental stages. Cleaved caspase-3-ir cells were counted in five different areas in layers I–III. At least 10,000 cells in each section were scored. Results are expressed as means ± SE of five to seven rats at each developmental stage. Significant differences compared with age-matched euthyroid pups are indicated: *, P < 0.05; **, P < 0.01; {psi}, P < 0.001.

 
Hypothyroidism induces apoptosis in neurons of developing cerebral cortex
To determine whether apoptosis occurs in the neurons of hypothyroid cortex, double immunofluorescence for cleaved caspase-3 and neurofilament was performed at P0 stage in S1 and counterstained with Hoechst 33258. Overlaid images of cleaved caspase-3 (red), neurofilament (green), and Hoechst (blue) revealed that most of the caspase-3-positive cells were neurons (shown by yellow arrows; Fig. 3Go, D and H). Caspase-3-ir was also observed in few nonneuronal cells (Fig. 3HGo).


Figure 3
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  		FIG. 3. Photomicrographs of coronal sections of primary somatosensory cortex showing cleaved caspase-3 and neurofilament immunofluorescence labeling in the euthyroid (A–D) and hypothyroid (E–H) rat pup at P0. Overlaid images (D and H; white) of cleaved caspase-3 (A and E; red), neurofilament (B and F; green), and Hoechst (C and G; blue) immunolabeling showed that most of the cleaved caspase-3-positive cells are neurons (H; yellow arrows). Scale bar, 25 µm (A–H).

 
Hypothyroidism enhances the activation of caspase-3 and -7
Caspase-3 expression and activation were assessed by Western blotting using different antibodies specific for pro-caspase-3 (32 kDa) and its cleaved subunit (p18). In the euthyroid group, expression of pro-caspase-3 was maximally observed at P0 stage and gradually decreased with development to reach very low levels of expression observed during adulthood. A similar developmental pattern of pro-caspase-3 expression was also observed in hypothyroid rats, although its expression levels were approximately 20% less than the age-matched euthyroid pups at P0 and P8 stage (Fig. 4AGo, top). Whereas the euthyroid groups exhibited a constant (basal) level of cleaved subunit of caspase-3 throughout development, the levels of cleaved caspase-3 subunit increased significantly in the hypothyroid group (P < 0.001) and was more pronounced between P0 and P16 stages (Fig. 4AGo, middle, and Fig. 4BGo).


Figure 4
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  		FIG. 4. Hypothyroidism enhances the activation of caspase-3 and -7, shown by Western blot analysis for caspase-3 (A) and caspase-7 (B) in the euthyroid and hypothyroid rat cerebral cortex at postnatal period (P0, P8, P16, and P24) and adulthood (A). A, Cytosolic proteins (50 µg; top) and tissue homogenate (100 µg; middle) were run on 15% SDS-PAGE and transferred to nitrocellulose membranes. For detection of caspase-3 expression and its activation, membranes were incubated with two different antibodies, a rabbit polyclonal caspase-3 antibody (top), which recognizes mainly pro-caspase-3, and the second antibody specific for its cleaved subunit (middle). B, Cytosolic proteins (50 µg) were run on 15% SDS-PAGE and transferred to nitrocellulose membranes and probed with a rabbit caspase-7 antibody. ß-Actin (bottom) was used as a loading control (A and B). C and D, Densitometric analysis of relative levels of the cleaved subunits of caspase-3 and caspase-7. Abundance of cleaved caspase-3 (C) is expressed as a ratio relative to euthyroid adult, and abundance of cleaved caspase-7 (D) is expressed as a ratio relative to P0. Bars represent the mean of the respective individual ratios ± SE (n = 4–5 rats at each developmental stage). Significant differences compared with age-matched euthyroid pups are indicated: *, P < 0.05; **, P < 0.01; {psi}, P < 0.001. ND, Not detected.

 
Next, caspase-7 expression and activation were assessed by Western blotting using a rabbit polyclonal antibody that recognizes pro-caspase-7 (35 kDa) and its cleaved subunits (22 and 12 kDa). In the euthyroid group, caspase-7 activation was observed throughout development, being highest at P0 stage and gradually declining with age (Fig. 4CGo). In the hypothyroid group, activation of caspase-7 was more pronounced and levels of the cleaved subunits were significantly elevated compared with the euthyroid group (P < 0.001). Interestingly, in adult rat cerebral cortex, caspase-7 activation was detected only in the hypothyroid group but not in the euthyroid group (Fig. 4Go, C and D).

Hypothyroidism down-regulates Bcl-2 and Bcl-xL and up-regulates Bax in cerebral cortex
To study the role of Bcl-2 family proteins in hypothyroidism-enhanced apoptosis in cerebral cortex, the expression of Bcl-2, Bcl-xL, and Bax was assessed by Western blotting. In the euthyroid group, expression of Bcl-2 was maximal at P0 stage, decreased sharply at P8 stage, and was maintained at a low level until adulthood (Fig. 5AGo). In the hypothyroid group, Bcl-2 expression was significantly reduced at P0 stage when compared with the euthyroid group (P < 0.001), although its levels were comparable at later stages of development between these two groups (Fig. 5Go, A and B). High levels of Bcl-xL expression was observed in the developing as well as adult euthyroid rat cortex, and its expression was markedly less in the hypothyroid group (P < 0.001; Fig. 5Go, A and C). On the other hand, in the euthyroid group, the proapoptotic protein Bax exhibited a development-dependent decrease in expression that became undetectable in the adult cortex (Fig. 5AGo), whereas Bax expression was significantly enhanced in the cortex of the hypothyroid group during development (P0–P16 stages) (P < 0.001; Fig. 5Go, A and D).


Figure 5
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  		FIG. 5. Hypothyroidism down-regulates Bcl-2 and Bcl-xL and up-regulates Bax. A, Tissue homogenate (50 µg) from euthyroid and hypothyroid pups were run on 15% SDS-PAGE, transferred to nitrocellulose membranes, and probed with a Bcl-2, Bcl-xL, or Bax antibody. ß-Actin was used as a loading control (bottom). Densitometric analysis of Bcl-2 (B) expression is presented as a ratio relative to euthyroid adult cerebral cortex, and Bcl-xL (C) and Bax (D) are expressed as ratios relative to P0 stage. Bax expression was not detected in the adult stage and hence is not plotted. Bars represent the mean of the respective individual ratios ± SE (n = 4–5 rats at each developmental stage). Significant differences compared with age-matched euthyroid pups are indicated: **, P < 0.01; {psi}, P < 0.001.

 
Hypothyroidism increases the levels of glycosylated pro-NGF in developing cerebral cortex
Given the well established interaction between TH and NGF (26), we evaluated the expression of NGF in the developing cerebral cortex by Western blot analysis using a rabbit polyclonal antibody raised against the N terminal of NGF. In the euthyroid group, two isoforms of pro-NGF (approximately 53 and 32 kDa) were detected in both the developmental and adult stages (Fig. 6AGo). Low-molecular-weight pro-NGF isoform (32 kDa) was developmentally up-regulated in both eu- and hypothyroid groups. It was barely detectable at P0 and P8 stage in the euthyroid group and even at P16 stage in the hypothyroid group. The levels, however, rose dramatically at later stages (P24 and adulthood) in both the groups (Fig. 6AGo). The 53-kDa form of pro-NGF exhibited comparable levels of expression in the euthyroid group all throughout development leading up to the adult stage. However, the 53-kDa pro-NGF was several-fold more abundant in the cortex of the hypothyroid group during development but dramatically decreased at P24 stage. Reduction of 53-kDa pro-NGF continued until adult stage in the hypothyroid group, which exhibited nearly 9-fold lower levels compared with the euthyroid group (Fig. 6BGo). Mature NGF (14 kDa), however, was not detectable in either group.


Figure 6
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  		FIG. 6. Western blot analysis of pro-NGF. A, Cytosolic proteins (50 µg) were separated on 15% SDS-PAGE, transferred onto nitrocellulose membrane, and probed with a NGF antibody raised against the N terminal. The bottom panel shows ß-actin expression (loading control). Two isoforms of pro-NGF, 32 and 53 kDa, were detected. B, Relative density of pro-NGF (53 kDa) is expressed as a ratio relative to P0 stage. Each bar represents the mean of the respective individual ratios ± SE (n = 3 rats at each developmental stage). Significant differences compared with age-matched euthyroid pups are indicated: {psi}, P < 0.001.

 
Hypothyroidism increases the levels of p75NTR but decreases TrkA expression
The NGF binding to p75NTR induces cell death, whereas binding to TrkA receptor promotes cell survival (19). Thus, we evaluated the effect of TH deficiency on the expression of p75NTR and TrkA receptors. In the euthyroid group, from a readily detectable level at P0, p75NTR was hugely down-regulated in the developmental window of P8–P16 stages followed by an up-regulation at P24 that was comparable to adult stage (Fig. 7Go, A and B). In the hypothyroid group, p75NTR expression was significantly higher at P0 compared with the euthyroid group (relative density for euthyroid, 1.01 ± 0.07; for hypothyroid, 1.60 ± 0.1; P < 0.001) (Fig. 7Go, A and B). Interestingly, p75NTR levels remained at higher levels in the hypothyroid group between the P8 and P16 window, which is in stark contrast to a sharp down-regulation of p75NTR observed in the case of the euthyroid group (Fig. 7Go, A and B). p75NTR expression in the hypothyroid group then declined at P24 and adult stage compared with the euthyroid group. The expression pattern of p75NTR in the hypothyroid group was thus entirely different from that of the euthyroid group. The expression of sortilin, an essential co-receptor for pro-NGF (25), was found to be unchanged throughout development and adulthood in both eu- and hypothyroid groups (Fig. 7AGo, middle).


Figure 7
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  		FIG. 7. Effect of perinatal hypothyroidism on the expression of NGF receptors. A, Cytosolic proteins (50 µg) were separated on 12% SDS-PAGE, transferred onto nitrocellulose membrane, and probed with a p75NTR (top) or sortilin (middle) antibody. The bottom panel shows ß-actin expression (loading control). B, Relative expression of p75NTR levels is expressed as a ratio relative to P0. Bars represent the mean of the respective individual ratios ± SE (n = 4–5 rats at each developmental stage). Significant differences compared with age-matched euthyroid pups are indicated: *, P < 0.05; **, P < 0.01; {psi}, P < 0.001. C, Western blotting for p75NTR in the rat cerebral cortex of 8-d-old euthyroid (E), hypothyroid (H), and T4 replacement group (MMI+T4). D, RT-PCR for TrkA mRNA expression. ß-Actin mRNA was used as an internal control (n = 3).

 
To investigate whether the increased levels of p75NTR in hypothyroid pups is caused by either decreased levels of circulating TH or an independent effect of MMI, T4 was administered to 4-d-old hypothyroid pups. Restoration of euthyroid status in the MMI+T4 group was evident by the normalization of serum TT4 and TT3 levels at P8 stage (serum TT4 for euthyroid, 30.16 ± 2.4 nmol/liter; for hypothyroid, 12.57 ± 1.4 nmol/liter; for MMI+T4, 40.27 ± 5.3 nmol/liter; serum TT3 levels for euthyroid, 0.543 ± 0.064 nmol/liter; for hypothyroid, 0.257 ± 0.032 nmol/liter; and for MMI+T4, 0.527 ± 0.061 nmol/liter). Expression of p75NTR was then studied in the euthyroid, hypothyroid, and MMI+T4 groups at P8 stage. In comparison with the euthyroid group, expression of p75NTR was significantly increased in the hypothyroid group, which was attenuated by T4 supplementation (MMI+T4 group) (Fig. 7CGo).

Finally, TrkA mRNA expression was evaluated by RT-PCR. In the euthyroid group, TrkA mRNA expression was observed throughout development and adulthood, and its expression attained a peak at P16 stage (Fig. 7DGo). In the hypothyroid group, TrkA mRNA expression was significantly low during all stages of development compared with the euthyroid group. In contrast, TrkA mRNA levels in the hypothyroid group at adult stage were approximately 2.5-fold higher than the euthyroid group, suggesting an altered expression pattern of TrkA expression in the hypothyroid group (Fig. 7DGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The development of cerebral cortex is severely impaired by TH deficiency during the fetal and neonatal period, leading to altered migration, differentiation, and synaptogenesis. Apoptosis is a cardinal feature of brain development, although the role of TH deficiency on apoptosis modulation in cerebral cortex remains to be elucidated. Therefore, the present study was performed on chemical-induced hypothyroidism where pregnant rats, fetuses, and offspring were hypothyroid. This model reflects a situation of a severely hypothyroid mother bearing a child with congenital hypothyroidism left without treatment after birth or that of a myxoedematous cretin born in an area of severe iodine deficiency that is accompanied by other environmental factor that destroys the thyroid gland (30). The latter situation is still very common in rural areas of developing countries, where prompt and adequate TH replacement is not available (31).

The present study for the first time shows that perinatal hypothyroidism enhances apoptosis in the developing rat cerebral cortex. Hypothyroidism enhanced the apoptosis in the superficial layers (layers I–III) of primary somatosensory cortex, the apoptotic cells being predominantly neurons. Cerebral cortices in the hypothyroid group exhibited significantly increased activation of caspase-3 and -7, decreased levels of antiapoptotic proteins Bcl-2 and Bcl-xL, and increased levels of proapoptotic protein Bax. In addition, hypothyroidism significantly increased 53-kDa pro-NGF and p75NTR levels accompanied by decreased TrkA expression.

The increase in apoptosis during rat cerebral cortex development in the hypothyroid state is over the constitutive apoptosis that occurs in cerebral cortex development in euthyroid rats. Previous studies in hypothyroid rats during development have also shown an enhanced apoptosis in cerebellum (10, 11, 12, 13) and hippocampus (32, 33). There had been only in vitro reports in literature suggesting that TH promotes the survival of neurons (7, 8, 9) as well as nonneuronal cells such as microglia (34) and oligodendrocytes during development (35). The present study shows that even in euthyroid rats, during postnatal development, apoptosis mostly occurs in the superficial layers (layers I–III) of S1, and the proportion of apoptotic cells were minimal in the deeper layers (data not shown). A similar ontogenic pattern of naturally occurring apoptosis is also reported elsewhere (36). Perinatal hypothyroidism enhanced the proportion of this naturally occurring apoptosis in the layers I–III of S1. Furthermore, double immunofluorescence labeling with cleaved caspase-3 and neurofilament revealed that hypothyroidism enhances apoptosis predominantly in neuronal cells and to a lesser extent in nonneuronal cells. Thus, our results indicate an important role of TH in the modulation of apoptosis during brain development. During brain development, neurons that fail to find appropriate targets and the target-derived neurotrophins undergo apoptosis (14). Fetal and neonatal hypothyroidism in rats has been shown to alter neuronal migration during neo-corticogenesis (37, 38). Thus, aberrant neuronal migration under a hypothyroid state may result in the failure of neurons to reach their targets, eventually leading to apoptosis.

The activation of the effector caspases, caspase-3 and -7, was observed during normal cortical development, coinciding with the extent of apoptosis. In the hypothyroid group, the increase in the activation of caspase-3 and -7 suggested their involvement in the hypothyroidism-enhanced apoptosis during cortical development.

Bcl-2 and Bcl-xL are crucial for the maintenance of neuronal survival (14). Bcl-2 and Bcl-xL are localized on the outer membrane of mitochondria and inhibit the release of caspase activators such as cytochrome c from mitochondria to the cytosol. In mitochondria, Bax forms heterodimers with Bcl-2 or Bcl-xL and induces the release of cytochrome c; thus, the ratio of pro- to antiapoptotic molecules determines the cell fate to undergo apoptosis (18). A decrease in Bcl-2 and Bcl-xL levels and an increase in Bax in the hypothyroid rat cortex appear to shift the rheostat toward apoptosis. Moreover, the decrease in Bcl-xL was pronounced (4- to 5-fold throughout the developmental period) in the hypothyroid cortex compared with the decrease in Bcl-2 (approximately 2.5-fold at P0 stage only), suggesting that Bcl-xL might play a major role in the inappropriately enhanced apoptosis observed in this group. We observed an increase in Bax expression during development (P0–P16) in the hypothyroid group. However, at P24 stage, apoptosis was observed even in the absence of Bax. There are other proapoptotic Bcl-2 family members such as Bid, Bad, and Bak that promote apoptosis even in the absence of Bax (18). Therefore, the possibility of the involvement of Bid, Bad, and Bak cannot be ruled out in hypothyroidism-enhanced apoptosis during cortical development. The alteration in the levels of Bcl-2, Bcl-xL, and Bax by hypothyroidism observed in this study is similar to that reported earlier in neonatal cerebellum (12) and hippocampus (32, 33).

During neuronal development, neurotrophins are essential in promoting neuronal survival and differentiation. Pro- and mature neurotrophins exert opposite functions by differential interactions with Trks and p75 receptor (21, 39). In this study, the accumulation of 53-kDa pro-NGF in the neonatal rat cerebral cortex was observed under the hypothyroid state. A similar induction and secretion of pro-NGF has also been shown after spinal cord injury, where pro-NGF triggered apoptosis in cultured cells through a p75NTR-mediated pathway (40). Recently, it has been shown that 53-kDa pro-NGF is a glycosylated form of pro-NGF, which accumulates in the cerebral cortex of Alzheimer’s disease patients and has the ability to induce apoptosis through a p75NTR-mediated pathway (41). We show that pro-NGF and not the mature NGF is a major form in rat brain, thus corroborating earlier findings (22, 42). In this study, 53-kDa pro-NGF was positively correlated with apoptosis in rat cerebral cortex, whereas 32-kDa pro-NGF showed a negative correlation (data not shown). Hence, it is possible that both forms of pro-NGF may regulate apoptosis through different mechanisms. Additional studies are needed to further elucidate the functions of both the pro-NGF isoforms in apoptosis regulation.

Pro-NGF promotes the formation of a signaling complex comprising endogenous receptors sortilin and p75NTR, required for apoptosis. Sortilin binds pro-NGF with an affinity in the low nanomolar range, but the affinity increases approximately 20-fold in the cells that coexpress p75NTR (25). In the present study, sortilin was constitutively expressed in the cerebral cortex and was not affected by hypothyroidism. Perinatal hypothyroidism increased the levels of p75NTR several-fold in the early postnatal period (P0–P16). Our results of hypothyroidism-induced enhanced expression of p75NTR in the developing cerebral cortex are in agreement with earlier studies (10, 26). In accordance with previous studies (43, 44), TrkA mRNA expression was developmentally regulated in the rat cerebral cortex. However, TrkA mRNA expression was significantly low in the hypothyroid group; a similar decrease (20–45%) in TrkA expression was also reported in the striatum (26). In p75NTR-mediated cell death, the decision of cell survival vs. apoptosis depends critically on the ratio of p75NTR and TrkA (19). The p75NTR/TrkA ratio was increased in the hypothyroid rat cortex (data not shown), which could shift the balance toward cell death. Moreover, to ensure that the increase in p75NTR expression, observed in the hypothyroid group, is not due to an independent effect of MMI, the expression of p75NTR was studied after T4 supplementation in the hypothyroid pups. The marked attenuation in the enhanced p75NTR expression in MMI+T4 group compared with MMI-treated group suggested that low levels of TH and not MMI per se mediated the p75NTR increase.

p75NTR is structurally a member of the Fas/tumor necrosis factor receptor 1 receptor family, but unlike other members of this family, caspase-8 is not required for p75NTR-mediated cell death (45). In agreement with the above study, no alteration was observed in the levels of FasL and caspase-8 activation in the hypothyroid group (data not shown). Increased reactive oxygen species generation and oxidative stress was also observed in the developing hypothyroid rat cerebral cortex (Kumar, A., R. A. Sinha, A. Pathak, and M. M. Godbole, unpublished data). Oxidative stress is known to stimulate the expression of NGF and p75NTR in astrocytes and neuroblastoma cells (46, 47). Therefore, it seems plausible that hypothyroidism-induced reactive oxygen species generation may increase the levels of pro-NGF and thereby activate p75NTR pathway.

In conclusion, the present study demonstrates that perinatal hypothyroidism enhances apoptosis in the developing rat cerebral cortex through an increase in caspase activation and alteration in the levels of Bcl-2 family proteins. Moreover, hypothyroidism increased the levels of 53-kDa pro-NGF and p75NTR, accompanied by a decreased TrkA expression. Taken together, these results may explain the cellular mechanism of the deleterious effect of perinatal hypothyroidism on the development of cerebral cortex.


   Acknowledgments
 
We thank Dr. Naibedya Chattopadhyay, Central Drug Research Institute, Lucknow, for critical reading of the manuscript.


   Footnotes
 
This work was supported by the Council of Scientific and Industrial Research, New Delhi, Grant 37(1165)/03/EMR-II. A.K. is a University Grants Commission-Senior Research Fellow, and this work is a part of his Ph.D. thesis.

Present address for R.S.: Center for Apoptosis Research, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

Disclosure statement: All contributing authors have nothing to declare.

First Published Online June 22, 2006

Abbreviations: ir, Immunoreactive; MMI, 2-mercapto-1-methylimidazole; NGF, nerve growth factor; NTR, neurotrophin receptor; P0, postnatal d 0; S1, primary somatosensory cortex; TH, thyroid hormone; TT4, total T4; TT3, total T3; TUNEL, terminal deoxynucleotide-transferase (TdT)-mediated dUTP nick end labeling.

Received January 9, 2006.

Accepted for publication May 31, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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