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Endocrinology, doi:10.1210/en.2006-1131
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Endocrinology Vol. 148, No. 4 1745-1753
Copyright © 2007 by The Endocrine Society

Alteration of Cerebellar Neurotropin Messenger Ribonucleic Acids and the Lack of Thyroid Hormone Receptor Augmentation by staggerer-Type Retinoic Acid Receptor-Related Orphan Receptor-{alpha} Mutation

Chun-Hong Qiu, Noriaki Shimokawa, Toshiharu Iwasaki, Ishwar S. Parhar and Noriyuki Koibuchi

Department of Integrative Physiology (C.-H.Q., N.S., T.I., N.K.), Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan; and School of Medicine and Health Science (I.S.P.), Monash University, Malaysia, 46150 Petaling Jaya, Malaysia

Address all correspondence and requests for reprints to: Noriyuki Koibuchi, M.D., Ph.D., Department of Integrative Physiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail: nkoibuch{at}med.gunma-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mutant mouse staggerer (sg) harbors a deletion within the gene encoding the retinoic acid receptor-related orphan receptor-{alpha} (ROR{alpha}). Homozygotes show aberrant cerebellar development. However, the mechanisms responsible for the cerebellar defect are still poorly understood. In the present study, the involvement of neurotropins (NTs), including nerve growth factor, brain-derived neurotropic factor, NT-3 and NT-4/5, and their receptors, which play a crucial role in brain development, on the cerebellar defects of sg mice was studied by semiquantitative RT-PCR and in situ hybridization histochemistry. An evident alteration of these mRNA levels was observed in both heterozygotes and homozygotes. Such difference was most evident in the internal granule cell layer. Because the changes in NT expression as well as morphological alterations in sg cerebellum are similar to those in hypothyroid animals, the effect of mutant ROR{alpha} (RORsg) on transcriptional regulation through the thyroid hormone (TH) response element or the ROR response element (RORE) was then studied. RORsg neither activated the transcription through RORE nor suppressed ROR{alpha}-induced transcription, indicating that it does not function as a dominant negative inhibitor. On the other hand, although wild-type ROR{alpha} augmented TH receptor (TR){alpha}1/ß1-mediated transcription through various TH response elements, RORsg was not effective in augmenting TR action. These results suggest that the cerebellar defect of the sg mouse is partly caused by the altered expression of NTs and the lack of augmentation of TR-mediated transcription by ROR{alpha} as well as the absence of ROR{alpha} action through RORE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RETINOIC ACID RECEPTOR (RAR)-related orphan receptor-{alpha} (ROR{alpha}) is a member of the nuclear receptor (NR) superfamily that requires specific ligands to activate the transcription of target genes (1). ROR{alpha} activates transcription as a monomer and/or homodimer on an ROR response element (RORE) composed of a half-site core motif PuGGTCA that is preceded by a 5' AT-rich sequence (2). Upon binding to RORE, it activates transcription in a ligand-dependent manner. A recent report showed that cholesterol and 7-dehydrocholesterol may be its endogenous ligands (3). Because these compounds are ubiquitously present in vivo, ROR{alpha} may behave as if it is a constitutively active NR.

ROR{alpha} is widely expressed in the central nervous system. Particularly, a high level of expression in the Purkinje cells of rodent cerebellum has been reported (4, 5). A genetic deletion corresponding to the ligand-binding domain (LBD) of ROR{alpha} results in aberrant cerebellar development in the staggerer (sg) mutant mouse (6, 7). The sg mouse shows severe cerebellar ataxia, which correlates with reduced numbers and poor dendritic arbors of Purkinje cells and degeneration of granule cells (8, 9). In addition, ROR{alpha}–/– mice also exhibit severe cerebellar ataxia that is also caused by massive cerebellar degeneration, particularly within Purkinje cells (10), indicating that ROR{alpha} is essential for normal cerebellar development. Because the target genes for ROR{alpha} have not yet been fully determined, the mechanism of cerebellar defect by the sg mutation is poorly understood. Interestingly, abnormal cerebellar development similar to those seen in sg and ROR{alpha}–/– mice is also observed in perinatal hypothyroid rodents (10, 11). We have previously reported that thyroid hormone (TH) accelerates the expression of ROR{alpha} in the developing cerebellum, and ROR{alpha} augments TH-receptor (TR)-mediated transcription (12, 13). Furthermore, TH treatment does not induce thymidine kinase activity in sg mice, which is normally induced in proliferating granule cells by interaction with Purkinje cells (14). These results suggest an involvement of cross-talk between ROR{alpha} and the TR signaling pathway in regulating cerebellar development. However, the candidate genes that are regulated through such cross-talk and play important roles in cerebellar development are not known.

The neurotropin (NT) family, including nerve growth factor (NGF), brain-derived neurotropic factor (BDNF), NT-3, and NT-4/5, profoundly affects the development of the central nervous system. Three different tyrosine kinase receptors have been identified, which are designated trkA, -B, and -C. These receptors primarily bind NGF, BDNF and NT-4/5, and NT-3, respectively (15). In the developing cerebellum, BDNF and NT-3 particularly serve important functions. BDNF promotes migration and neurite elongation of external granule cells, whereas NT-3 promotes neurite branching of mature granule cells (16). Both BDNF and NT-3 are able to promote Purkinje cell survival in culture. Furthermore, mice harboring a deletion of the BDNF gene show abnormal cerebellar development similar to that in sg and hypothyroid animals (17, 18). We and other investigators have previously reported that the expression of BDNF and NT-3 is depressed in hypothyroid rodent cerebellum (19, 20). Replacing BDNF or NT-3, in part, prevents hypothyroidism-induced abnormal cerebellar development (20, 21), indicating that these factors may play an essential role in TH-mediated cerebellar development. However, no report has shown the change in NT expression in developing sg mouse cerebellum.

In the present study, we studied the change in NT and their receptor expression using semiquantitative PCR and in situ hybridization histochemistry. The sg homozygotes used in the present study usually die around the third or fourth weeks after birth. In addition, there is a distinct critical period of TH-dependent cerebellar development, which is the first 2 wk of postnatal life in the rodent and is completed by postnatal d 21 (P21) (22). For this reason, mice were killed at P2, P7, P15, and P21 for analysis. Furthermore, we examined the effect of mutant ROR{alpha} expressed in sg mouse (RORsg) on transcription through various TH response elements (TREs) or RORE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The sg mice used in the present study were purchased from Jackson Laboratory (Bar Harbor, ME). Initially, their genetic background was B6C3Fe-a/a-Rorasg. Then, the genetic background was adjusted by crossing with C57BL/6J inbred mice purchased from CLEA Japan Inc. (Tokyo, Japan) for more than 10 generations. The animals were bred in the Animal Facility of Gunma University Graduate School of Medicine. The animal experimentation protocol in the present study was approved by the Animal Care and Experimentation Committee, Gunma University Showa Campus. Adult heterozygotes were mated for a suitable reproduction period. Pups were weighed and killed at P2, P7, P15, and P21 by decapitation under diethyl ether anesthesia. Their tails were cut to extract DNA from which to check the genotype by PCR according to the protocol described elsewhere (6). The cerebella were dissected out, weighed, and rapidly frozen on dry ice and stored at –80 C for RT-PCR. For in situ hybridization and immunohistochemistry, some brains were fixed in 4% paraformaldehyde in PBS (0.2 M, pH 7.40). C57BL/6J (CLEA Japan) mice were used as a wild-type control and treated the same as the sg mice.

Immunohistochemistry
The fixed brains were embedded in paraffin, and 8-µm-thick sections were cut and mounted on saline-coated glass slides. After deparaffinization and rehydration, the sections were incubated in 10 mM citric acid (pH 6.0) for antigen activation and in 0.1 M PBS containing 0.3% H2O2 for 30 min to prevent endogenous peroxidase activity. Then, sections were incubated with the mouse anti-calbindin-D28K antibody (clone 9848; Sigma-Aldrich, St. Louis, MO) overnight at 4 C (1:1000 dilution). Next, sections were incubated with biotinylated horse antimouse IgG (1:220 dilution; Vector Laboratories, Burlingame, CA) for 45 min. After rinsing in PBS, the sections were incubated with avidin-biotin complex (Vector) for 1 h and visualized by 3,3'-diaminobenzidine (0.5 mg/ml Tris-HCl containing 0.01% H2O2). Sections were then immersed into 0.5% cresyl violet for 1–3 min, dehydrated by an increasing graded series of ethanol, cleared in xylene, and placed under coverslips.

Semiquantitative RT-PCR
Total RNA was extracted from P2, P7, P15, and P21 cerebella using RNeasy (QIAGEN, Hilden, Germany). Specific primers for BDNF, NGF, NT-3, NT-4/5, and trkA, -B, and -C are shown in Table 1Go. To define the linear range for PCR amplification, the optimal number of PCR cycles was decided based on the abundance of each target transcript in varying cycle numbers of different groups (Table 1Go). The RT-PCR was carried out as described in the instruction manual of the QIAGEN OneStep RT-PCR kit with some modifications. In one reaction, 50–100 ng template RNA, 0.6 µM sense and antisense primers, and 0.5 µl RT-PCR enzyme mix were added in a final volume of 25 µl. RT-PCR were done using the TAKARA PCR Thermal Cycler SP, TP-400 (Takara Holdings Inc., Kyoto, Japan). All PCR products were detected and analyzed by the Electrophoresis Documentation and Analysis System 290 (Kodak, Norwalk, CT). The PCR results for each sample were normalized by GAPDH mRNA level as an internal control. All experiments were repeated three times to confirm the consistency of results.


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  		TABLE 1. Primer sequences, annealing temperatures, and amplification cycles for RT-PCR

 
In situ hybridization histochemistry
cDNAs for mouse BDNF, NT-3, and trkB were prepared by RT-PCR and subcloned into pBluescript (Stratagene, La Jolla, CA). Linearized plasmids were then used for the generation of digoxigenin (DIG)-labeled riboprobes. In the transcription reaction, 1 µg linearized plasmid, 2 µl T3 or T7 RNA polymerase mix (Ambion Inc., Austin, TX), and 2 µl DIG RNA labeling mix (Roche, Basel, Switzerland) were added in a final volume of 20 µl, according to the instruction manual.

The 8-µm-thick paraffin sections were prepared under RNase-free conditions and used for pretreatment in 4% paraformaldehyde/PBS and digestion of proteinase K (10 µg/µl for 15 min at 37 C). The sections were then hybridized with the hybridization buffer (12) containing DIG-labeled riboprobes (50–100 ng/section) and 47% formamide overnight at 58–62 C. Sections were incubated with alkaline-phosphatase-conjugated anti-DIG Fab fragments (1:500 dilution; Roche) after being stringently washed in 2x SSC (1x SSC contains 0.15 M sodium chloride, 0.015 M sodium citrate) and 0.1x SSC and RNase (10 µg/ml) treatment. The hybridization signals were visualized by a nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Roche) mixture.

Transient transfection-based reporter assays
The cDNA for mouse ROR{alpha} was kindly provided by Dr. B. A. Hamilton, Whitehead Institute, Cambridge, MA. Expression vectors for mouse ROR{alpha} (amino acids 1–1577) and RORsg (amino acids 1–824) were constructed by inserting PCR-generated fragments into the BamHI-EcoRV site of the pcDNA3 expression vector (Invitrogen Life Technologies Inc., Carlsbad, CA). For PCR amplification, sense (5'-AAACATGGAGTCAGCTCCGGCA-3') for mouse ROR{alpha} and RORsg and antisense (5'-TTACCCATCGATTTGCATGGC-3') for ROR{alpha} and (5'-TTATAGTTCTGCCATGGACAC-3') with a stop codon for RORsg were used with a BamHI site on the 5' end and an EcoRV site on the 3' end. Expression vectors for human TRß1 and rat TR{alpha}1 have been described previously (13, 23). The luciferase (LUC) reporter constructs, the chick lysozyme TRE (F2), DR4, or palindrome-thymidine kinase minimum promoter (TK)-LUC and RORE-TK-LUC were described previously (13).

CV-1 cells were maintained in DMEM supplemented with 10% TH-free fetal bovine serum and penicillin/streptomycin at 37 C, 5% CO2. Cells were plated in 24-well culture plates 2 d before transfection. Transfection was carried out with expression plasmid (0.02 µg/well) and reporter plasmid (0.2 µg/well) using the calcium-phosphate precipitation method as described previously (23). Cytomegalovirus-ß-galactosidase plasmid (0.1 µg/well) was used as an internal control. Then, 16–24 h after transfection, the medium was changed and T3 was added to a final concentration of 10–7 M unless otherwise indicated. After 24 h, cells were harvested to measure luciferase and ß-galactosidase activities (24, 25). Total amount of transfected DNA for each well was balanced by adding pcDNA3 plasmids. The luciferase activity was normalized to ß-galactosidase activity and then calculated as relative luciferase activity. All transfection studies were repeated at least three times in triplicate.

Statistical analysis
Quantitative data were expressed as a mean ± SEM of the individuals in each experimental group. Statistical analysis was done using ANOVA. Post hoc comparison was performed using Duncan’s multiple range tests. P value < 0.05 or 0.01 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Delayed postnatal general and cerebellar growth in sg mouse
Although the small size of the sg mouse compared with the wild-type mouse has been reported from the first day of birth (8), there are no detailed data on the change in body weight or cerebellar weight during postnatal development. Thus, these were measured at P2, P7, P15, and P21. A significant difference between homozygote and wild-type mice was observed in body weight from P7 and in cerebellar weight from P15. The greatest reduction was observed at P21 (37% in body weight and 37% in cerebellar weight). No statistical significance in the body and cerebellar weights between heterozygote and wild-type mice was observed (Fig. 1Go).


Figure 1
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  		FIG. 1. Changes in body weight and cerebellar weight in wild-type (Wt), heterozygote (He), and homozygote (Ho) mice during postnatal development. Body weight and cerebellar weight for Wt ({square}), He (bullet), and Ho ({blacktriangleup}) mice were measured at P2, P7, P15, and P21, respectively. Data are expressed as mean ± SEM (n = 3). *, P < 0.05 compared with control groups (same age).

 
Aberrant cerebellar morphology in sg mouse
To examine the morphological changes in developing sg mouse cerebellum, immunohistochemistry using the antibody for calbindin-D28k, which is specifically expressed in Purkinje cells, was performed. The layer formation of the cerebellar cortex was examined using cresyl violet staining. Morphological alterations of the Purkinje and granule cell layers in sg mouse were detected starting from P7 (Fig. 2Go). The Purkinje cell dendrite was poorly developed in heterozygote and homozygote mice. The molecular layer in these mice was not distinguishable from other layers. At P15, ectopic Purkinje cells in the homozygote with small soma and poor dendritic arborization, and a thicker external granule cell layer (EGL) than that of the wild-type mouse were detected. On the other hand, no clear morphological difference was observed between heterozygote and wild-type cerebella. At P21, the layer formation was severely disrupted in the homozygote. The Purkinje cells failed to form an organized monolayer at the surface of the internal granule cell layer (IGL) and were mixed with granule cells in the IGL. Furthermore, the EGL was observed by P21 in the homozygote. On the other hand, no significant morphological difference between heterozygote and wild-type mice was observed at P21.


Figure 2
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  		FIG. 2. Morphological alterations in postnatal cerebellum. Sagittal sections of cerebellum (vermis) at P7, P15, and P21 were stained with mouse anti-calbindin-D28K (1:1000) and cresyl violet. He, Heterozygote; Ho, homozygote; Wt, wild-type. Bar, 50 µm.

 
The change in cerebellar NT mRNAs levels
To examine the involvement of NTs on the cerebellar development of the sg mouse, the expression of NT mRNAs was studied using the semiquantitative RT-PCR method. The BDNF and NT-3 transcripts were more abundant than those of NGF and NT-4/5. At P2, greater levels of NGF mRNA were detected in both heterozygote and homozygote mice than in the wild-type mouse. No significant difference among the three groups was detected at P15 and P21 (Fig. 3AGo). The BDNF mRNA level in the wild-type mouse started to increase from P15 and reached a peak at P21 (Fig. 3BGo). In heterozygotes, however, it remained stable at P15 and slightly increased at P21. No increase in BDNF mRNA level was observed in homozygotes. Compared with the wild-type mouse, lower levels in both heterozygotes (65%) and homozygotes (45%) were observed at P21. NT-3 levels reached a peak at P15, and lower levels in homozygotes were detected (Fig. 3CGo). NT-4/5 mRNA levels in homozygotes were significantly lower at P21 compared with those of the wild-type mouse, and no significant difference was observed between heterozygote and wild-type mice (Fig. 3DGo).


Figure 3
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  		FIG. 3. Expression of NT mRNAs during postnatal development of the cerebellum. The mRNA levels of NGF (A), BDNF (B), NT-3 (C), and NT-4/5 (D) in the cerebella of wild-type (Wt, {square}), heterozygote (He, bullet), and homozygote (Ho, {blacktriangleup}) mice were analyzed by semiquantitative RT-PCR. Values of NT mRNAs are expressed as relative OD levels, which are normalized with the corresponding density of GAPDH mRNA. The mRNA levels of each gene were summarized in three independent experiments in triplicate. Lanes in the lower part of the panels show the representative amplified band of each group. Data are expressed as mean ± SEM (n = 3). *, P < 0.05 compared with control groups.

 
The change in cerebellar NT receptor mRNAs levels
Next, the expression of trkA, -B, and -C mRNAs in developing cerebellum was studied (Fig. 4Go). The trkA mRNA levels are lower in wild-type cerebellum, and thus the number of amplification cycles was greater than that for other mRNAs (Table 1Go). It was relatively higher earlier in postnatal life and decreased with age. However, higher levels in the homozygote than in the wild-type mouse were detected and remained stable (Fig. 4AGo). The trkB mRNA level did not show significant changes in the wild-type mouse, whereas greater levels were observed in the homozygote at P15 and P21 (Fig. 4BGo). On the other hand, no significant difference in trkB mRNA level between the heterozygote and wild-type mice was detected. The trkC mRNA showed a peak level at P15 with no significant difference among the three groups throughout the study (Fig. 4CGo).


Figure 4
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  		FIG. 4. Expression of NT receptor mRNAs during the postnatal development of the cerebellum. The mRNA levels of trkA (A), trkB (B), and trkC (C) in the cerebella of wild-type (Wt, {square}), heterzygote (He, bullet), and homozygote (Ho, {blacktriangleup}) mice were analyzed by semiquantitative RT-PCR. Values of NT receptor mRNAs are expressed as relative OD levels, which are normalized with the corresponding density of GAPDH mRNA. The mRNA levels of each gene were summarized in three independent experiments in triplicate. Lanes in the lower part of the panels show the representative amplified band of each group. Data are expressed as mean ± SEM (n = 3). *, P < 0.05 compared with control groups.

 
Localization of BDNF, NT-3, and trkB mRNA in the postnatal cerebellum
Next, the regional expression of NTs and their receptor mRNAs in developing mouse cerebellum was studied. The RT-PCR study showed that the mRNA levels for all NTs and their receptors except for trkC were altered. However, levels for NGF, NT-4/5, and trkA mRNAs were too low to detect by in situ hybridization. On the other hand, BDNF, NT-3, and trkB mRNAs were abundantly expressed. Furthermore, BDNF and NT-3 play particularly important roles in cerebellar development (17, 18). Thus, we examined their localization by in situ hybridization histochemistry.

The localization of BDNF, NT-3, and trkB mRNAs in the cerebellar cortex is shown in Fig. 5Go. Because a significant difference in the expression of NT-3 and trkB was observed at P15, and BDNF at P21 by RT-PCR, photomicrographs at these time points are shown. Hybridization signals for NT-3 and BDNF mRNA were mainly located in granule cells, including the EGL and IGL. No significant hybridization signals were found in Purkinje cells, which is consistent with previous findings (26, 27). In homozygotes, weaker signals for both NT-3 and BDNF mRNA in the IGL than those of the wild-type mouse were observed. However, a strong signal for BDNF was detected in the EGL. The trkB mRNA was found in most granule cells and some Purkinje cells (arrows). A strong signal was observed in the EGL, which could account for the relatively high level of its mRNA in homozygotes at P15, because the EGL volume is greater in this group.


Figure 5
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  		FIG. 5. Distribution of NT-3, BDNF, and trkB mRNA in the postnatal cerebellum. NT-3 (A–D, P15), BDNF (E–H, P21), and trkB (I–L, P15) mRNA expressions in the cerebella of wild-type (Wt), heterozygote (He), and homozygote (Ho) mice were analyzed by in situ hybridization. The control sections with the sense cRNA probes are shown. Bar, 50 µm.

 
Effect of RORsg on ROR{alpha} or TR-mediated transcription
The similarity of cerebellar morphology and NT expression profile between sg and hypothyroid mice suggests the involvement of cross-talk between ROR{alpha} and the TR signaling pathway. To examine the effect of RORsg on ROR{alpha}- or TR-mediated transcription, a transient transfection-based reporter assay was performed. Expression vectors used in the present study are shown in Fig. 6AGo. First, the transcriptional activity of ROR{alpha} and/or RORsg on a RORE was studied (Fig. 6BGo). As expected, RORsg did not induce significant transcription on the RORE, whereas ROR{alpha} significantly activated it. When RORsg was cotransfected with ROR{alpha} (ROR{alpha}:RORsg = 1:1, 1:3, or 1:5), RORsg did not suppress transcription induced by ROR{alpha} even with a 5-fold greater amount.


Figure 6
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  		FIG. 6. The effect of mutant ROR{alpha} (RORsg) on wild-type ROR{alpha} or TR-mediated transcription. A, The structure of RORsg used in the present study. B, Expression vectors encoding wild-type ROR{alpha} and/or RORsg at various ratios (ROR{alpha}:RORsg = 1:1, 1:3, or 1:5) were cotransfected with RORE-LUC into CV-1 cells. Total amounts of DNA for each well were balanced by adding pcDNA3. Data represent mean ± SEM of experiments performed in triplicate. *, Statistically significant (P < 0.01). C, Expression vectors encoding TRß1(C-1) or TR{alpha}1(C-2), wild-type ROR{alpha}, and/or RORsg were cotransfected with F2-TRE-LUC into CV-1 cells. Cells were grown in the absence or presence of 10–7 M T3. Total amounts of DNA for each well were balanced by adding pcDNA3. Data represent mean ± SEM. *, Statistically significant (P < 0.01).

 
Next, the effect of RORsg on TR{alpha}1- or TRß1-mediated transcription on F2 (Fig. 6CGo-1 and -2), DR4, and palindrome was studied. Although TR{alpha}1- or TRß1-mediated transcription was significantly augmented by ROR{alpha}, such effect by RORsg was not observed. Furthermore, RORsg did not repress the augmentation of TR-mediated transcription induced by ROR{alpha}. These results indicate that RORsg does not function as a dominant-negative inhibitor. In addition, the augmentation of TR-mediated transcription by ROR{alpha} was completely eliminated by RORsg mutation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study show the alteration of NTs and their receptor mRNA levels in developing sg mouse cerebellum. Because NTs play an essential role in cerebellar development, the cerebellar defect in the sg mouse may be at least in part induced by the altered expression of NTs and their receptors. The altered expression profile was similar to those of hypothyroid animals, indicating an involvement of cross-talk between ROR{alpha} and the TR signaling pathway. The unchanged TR-mediated transcription after cotransfection of RORsg indicates that it could not augment the expression of TR target genes, which may induce abnormal cerebellar development similar to that in hypothyroid animals.

Morphological changes in the cerebellum of the sg mouse during postnatal development (Fig. 2Go) demonstrated that both Purkinje and granule cells were affected. The aberrant cerebellar development observed in the present study is consistent with previous studies using sg mice with a different genetic background (28, 29). In the present study, however, sg mice with a C57BL/6J background show a more severe phenotype. Their general growth was greatly retarded, and homozygote mice die around the weaning period, although heterozygote mice did not show any aberrant general growth. These results indicate that although the genetic background may affect their phenotype, abnormal cerebellar development seen in the present study is mainly caused by ROR{alpha} mutation. However, the reason the mice with this background die soon after weaning is not known. The more severe staggerer gait and hypotonia may have hampered them from taking a sufficient amount of solid food. In fact, when the mouse was not separated from the mother, it survived for about 1 wk longer than the weaned mice.

Whether ROR{alpha} directly regulates the expressions of NTs and their receptor genes is not known. ROR{alpha} is strongly expressed in Purkinje cells throughout postnatal development (5, 30). It is also weakly expressed at a certain developmental period in the EGL, IGL, and molecular layer (4, 5). On the other hand, as shown in the present study, BDNF and NT-3 are strongly expressed in the EGL and IGL. Interaction between granule and Purkinje cells is indispensable for proper cerebellar development. Through a synaptic connection between parallel fibers from granule cells and Purkinje cell dendrites, the development of both types of neurons is promoted (31). In sg mice, such connection is disrupted, because Purkinje cells are not able to develop normal dendritic spines, although parallel fibers are able to attach to the dendrite (32, 33). Thus, failure of synaptic formation may inhibit proper development of granule cells, which then alter the expression of NTs, particularly within the IGL. In addition, there is some evidence from mutant, irradiation, and virus studies that Purkinje cells also exert control over both the onset and the cessation of proliferation of the EGL (34). Thus, the prolonged existence of the EGL and relatively stronger BDNF and trkB expressions within this region at P15 and P21 observed in the present study may be also caused by Purkinje cell defect, although the exact cause for such abnormality is not known. Multiple possibilities such as impaired migration, enhanced cell proliferation, or diminished apoptosis could be considered. On the other hand, although ROR{alpha} expression is relatively weaker than that in Purkinje cells, cultured granule cells from sg mice behave differently from those of wild-type mice (14, 35). The ability to form clusters is weaker (35), and TH did not induce thymidine kinase activity (14). Thus, the possibility that ROR{alpha} may directly regulate NT expression in granule cells cannot be excluded. In any case, considering the roles of BDNF and NT-3 in cerebellar development, alteration of expression of these genes may play a critical role in generating cerebellar defects in the sg mouse. On the other hand, strong expression of BDNF and trkB in the remaining EGL at P15 and P21 in homozygotes may in part compensate for their decrease in the IGL, thereby reducing abnormal changes.

ROR{alpha} is a member of the NR superfamily that regulates a wide range of biological processes such as cell proliferation, differentiation, and homeostasis of many organs, including the central nervous system (36, 37, 38). A deletion within the LBD of the ROR{alpha} gene causes the expression of LBD-truncated mutant ROR{alpha} (RORsg) (6), which induces cerebellar defect and gene expression patterns similar to those of hypothyroid animals. TH action is exerted by binding to TR, which is also a member of the NR superfamily (36). The similarity of cerebellar defect and gene expression profiles indicates a possible involvement of interaction between TR and the ROR{alpha} signaling pathway. Although the specific gene that is regulated through such interaction has not yet been identified, several possible mechanisms that cause the sg phenotype can be considered. First, ROR{alpha} may regulate TH action by modulating TR gene expression. This possibility is not likely, because TR seems to be normally expressed in the sg mouse (6). To completely exclude this possibility, however, administration of TH into the sg mouse to examine whether abnormal cerebellar phenotype is rescued, is required. We have attempted such treatment. However, heterozygote mothers were so sensitive that slight handling of their pups resulted in cannibalism. Second, TR may regulate ROR{alpha} expression. Because TH regulates ROR{alpha} expression during the first two postnatal weeks, TH might exert its effect by regulating the expression of the ROR{alpha} gene, which then regulates the expression of its target genes or augments TR-mediated transcription (12, 39). However, TRE or RORE within the promoter region of NT genes has not yet been identified. In addition, as stated above, the possibility that ROR{alpha} or TR-ROR{alpha} interaction may indirectly regulate NT gene expression through the Purkinje-granule cell interaction cannot be excluded.

In the present study, wild-type mouse ROR{alpha} augmented TR-mediated transcription, which is consistent with our previous study showing the augmentation of TR action by human ROR{alpha} (13). Such augmentation by RORsg was not observed. Furthermore, RORsg did not suppress wild-type ROR{alpha}-mediated transcription on an RORE and augmentation of TR action by wild-type ROR{alpha} on various TREs, indicating that RORsg does not act as a dominant-negative inhibitor. Because both ROR{alpha} and TR are coexpressed in the normal developing brain, TR-regulated gene expression may be augmented by ROR{alpha} within many cells in wild-type animals, whereas such augmentation cannot occur in the sg mouse. That the phenotype of the sg heterozygote mouse is very mild can be explained from the reporter assay result showing that RORsg does not act in a dominant negative manner. The more severe phenotype of the sg homozygote than that of the hypothyroid animal is probably due to the failure of RORE-mediated transcription in addition to the absence of augmentation of TR action by ROR{alpha}.

Although this and a previous study have shown the augmentation of TR action by ROR{alpha} (13), the molecular mechanisms of such augmentation have not yet been clarified. The potential interaction of TR and ROR{alpha} was examined by electromobility shift assay on several TREs (13). However, no apparent TR-ROR{alpha} heterodimer formation was observed, although ROR{alpha} showed a strong augmentation of TR action on such TREs. We also examined the interaction of ROR{alpha} with TR or other NRs, using the glutathione S-transferase (GST) pull-down assay (13). Although GST-TR-LBD interacted with ROR{alpha}, all other GST-fused NRs such as RARß, retinoid X receptor ß, and estrogen receptor {alpha} interacted with ROR{alpha}. Thus, such binding may be nonspecific. Binding of ROR{alpha} and RARß is, in particular, not likely, because RAR-induced transcription on the RAR response element was not augmented by ROR{alpha} and a RAR/ROR{alpha} heterodimer was not seen on a RAR response element by electromobility shift assay (40). Although attempts to clarify the mechanisms of ROR{alpha} action on TR-mediated transcription have not yet been successful, the results of the present study have shown the possible involvement of TR on ROR{alpha} mutation-induced cerebellar defect in the sg mouse. In other words, ROR{alpha} may be indispensable for the full function of TR action in the developing cerebellum.

Overall, our study showed the changes in gene expression profiles of NTs and their receptors due to ROR{alpha} mutation. Our study also showed that RORsg could neither activate gene expression on a RORE nor augment TR-mediated transcription. Taken together with the facts that NTs play critical roles in cerebellar development and that the cerebellar phenotype in sg is similar to that of hypothyroid animals, the present results indicate that NTs and TR may play key roles in inducing abnormal cerebellar phenotype in the sg mouse.


   Acknowledgments
 
We thank Dr. B. A. Hamilton for providing mouse ROR{alpha} cDNA. We are also grateful to Dr. Behenaz Yousefi, Dr. Satoshi Ogawa, Mr. Wataru Miyazaki, and Ms. Misae Ota for their technical assistance.


   Footnotes
 
This research was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) (to N.K. and T.I.) and Initiatives for Attractive Education in Graduate Schools from MEXT (to C.-H.Q.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 11, 2007

Abbreviations: BDNF, Brain-derived neurotropic factor; DIG, digoxigenin; EGL, external granule cell layer; GST, glutathione S-transferase; IGL, internal granule cell layer; LBD, ligand-binding domain; NGF, nerve growth factor; NR, nuclear receptor; NT, neurotropin; P21, postnatal d 21; RAR, retinoic acid receptor; ROR{alpha}, RAR-related orphan receptor-{alpha}; RORE, ROR response element; RORsg, mutant ROR{alpha}; sg, staggerer; TH, thyroid hormone; TR, TH receptor; TRE, TH response element.

Received August 17, 2006.

Accepted for publication January 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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