An Unliganded Thyroid Hormone Receptor Causes Severe Neurological Dysfunction
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An unliganded thyroid hormone receptor causes severe neurological dysfunction Koshi Hashimoto*, Flavio H. Curty*, Patricia P. Borges*, Charlotte E. Lee†, E. Dale Abel†, Joel K. Elmquist†, Ronald N. Cohen*, and Fredric E. Wondisford*‡ *Section of Endocrinology and Metabolism, Department of Medicine, University of Chicago, Chicago, IL 60637; and †Division of Endocrinology and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02215 Edited by Michael G. Rosenfeld, University of California at San Diego, La Jolla, CA, and approved December 26, 2000 (received for review September 21, 2000) Congenital hypothyroidism and the thyroid hormone (T3) resis- in pGEM 7Z was linearized with SalI before electroporation into tance syndrome are associated with severe central nervous system embryonic stem cells derived from 129͞SvJ mice (Genome (CNS) dysfunction. Because thyroid hormones are thought to act Systems), and the cells were selected in media containing G418. principally by binding to their nuclear receptors (TRs), it is unex- One hundred ninety-two G418-resistant colonies were plained why TR knock-out animals are reported to have normal screened with the use of Southern blot analysis and a 1-kb CNS structure and function. To investigate this discrepancy further, BglII͞EcoRI radiolabeled probe from a region outside of the aT3 binding mutation was introduced into the mouse TR- locus by targeting vector in intron 4. Homologous recombination was homologous recombination. Because of this T3 binding defect, the detected in three clones, which were subsequently injected into mutant TR constitutively interacts with corepressor proteins and blastocysts of the C57͞BL6 strain. PCR with a Neo and an exon mimics the hypothyroid state, regardless of the circulating thyroid 7 oligonucleotide primer was performed on embryonic stem cell hormone concentrations. Severe abnormalities in cerebellar devel- DNA from all three clones. PCR products were subcloned, and opment and function and abnormal hippocampal gene expression DNA sequencing confirmed the ⌬337T mutation in the exon 6. and learning were found. These findings demonstrate the specific Male chimeric mice were mated with female C57͞BL6 mice as and deleterious action of unliganded TR in the brain and suggest described (11), and germline transmission was obtained from all the importance of corepressors bound to TR in the pathogenesis of three clones. All mice were propagated in the C57͞BL6 back- hypothyroidism. ground strain, and direct comparisons were made with littermate controls. All aspects of animal care were approved by the Institutional hyroid hormone (T3) deficiency in early life affects central Tnervous system (CNS) development and function, resulting Animal Care and Use Committee of the Beth Israel Deaconess in decreased intelligence and movement disorders in humans. In Medical Center (Boston, MA). Animals were maintained on a 12 h light͞12 h dark schedule (light on at 06:00 h) and fed rodent models of hypothyroidism, similar findings have been laboratory chow and water ad libitum. reported (1). Maternal hypothyroidism also has an impact on CNS development in utero, even when fetal thyroid function is RNA Analysis. Total RNA from tissues was isolated after homog- normal (2). Finally, in patients with the syndrome of resistance enization in guanidinium thiocyanate. For Northern analysis, to thyroid hormone (RTH), learning disorders and mental total RNA was fractionated in formaldehyde-containing agarose retardation have been reported (3). In this disorder, a mutant T 3 gels, transferred to nylon membrane (GeneScreen PLUS; NEN), receptor (TR-) expressed on one allele dominantly interferes and hybridized with a radiolabeled probe. For brain and cere- with the function of the other TR isoforms (TR-1, TR-2, and ␣ bellum RNA samples, myelin basic protein (12), brain-derived TR- 1) and causes tissue hypothyroidism (3, 4). neurotrophic factor (BDNF), and tyrosine protein kinase recep- To explore the mechanism of TR signaling in vivo,TR tor B (TrkB) cDNA probes were used. The latter two cDNA knock-out mouse models have been generated (5–9). Presum- probes were obtained by reverse transcription–PCR of total ably in these models of tissue hypothyroidism, nuclear signaling mouse brain. For the BDNF probe, a sense primer (nt 123–148) should be abolished because of the lack of TRs. Paradoxically, and an antisense primer (nt 654–629) were used (GenBank however, these animals have a relatively mild phenotype com- accession no. NM007540). For the TrkB probe, a sense primer pared with animals rendered hypothyroid by thyroid ablation (nt 121–146) and an antisense primer (Ϫnt 648–623) were used and, apart from deafness caused by an inner ear defect (10), are (GenBank accession no. M33385). cDNA probes were con- reported to have normal CNS structure and function. This firmed by DNA sequencing. discrepancy suggests that the unliganded TR may play a central RNase protection assays were performed (RPAIII kit; Am- role in mediating the phenotypic changes of hypothyroidism in bion, Austin, TX) with the use of [␣-32P]UTP-labeled antisense the CNS. riboprobes. The protected fragments were fractionated on a  Materials and Methods denaturing 6% polyacrylamide gel. For analysis of TR- expres- sion, a 500-bp genomic probe containing exon 6 and 5Ј and 3Ј   Generation of TR- Mutant Mice. The mouse TR genomic locus intronic DNA was used. For analysis of c-erbA-␣ expression, a was isolated as a 70-kb clone from a 129͞SvJ P1 genomic DNA library (Genome Systems, St. Louis). A mouse TR- Exon 6 fragment was obtained with the use of the PCR and intronic This paper was submitted directly (Track II) to the PNAS office. primers flanking exon 6. This fragment was subcloned, and the Abbreviations: T3, thyroid hormone; TR, thyroid hormone receptor; RTH, resistance to ⌬337T mutation was introduced by site-directed mutagenesis thyroid hormone; CNS, central nervous system; BDNF, brain-derived neurotrophic factor; (CLONTECH). An EcoRI͞BglII fragment, containing the exon wt, wild type; TSH, thyroid-stimulating hormone; H&E, hematoxylin͞eosin; Pcp-2, Purkinje cell protein-2; MBP, myelin basic protein; TrkB, tyrosine protein kinase receptor B. 6 mutation, was assembled and subcloned into pGEM 7Z ‡To whom reprint requests should be addressed. E-mail: fwondisf@medicine. (Promega). A neomycin resistance gene cassette (NEO), driven bsd.uchicago.edu. by the phosphoglycerate kinase promoter, was inserted into The publication costs of this article were defrayed in part by page charge payment. This intron 5 at an EcoRI site. Introduction of the NEO cassette article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. resulted in a new BglII site in intron 5. The targeting construct §1734 solely to indicate this fact. 3998–4003 ͉ PNAS ͉ March 27, 2001 ͉ vol. 98 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.051454698 Downloaded by guest on October 2, 2021 250-bp probe containing the A͞B coding region of mouse Tests of CNS Function. The beam-balance task was used to assess c-erbA-␣ was used. In RNase protection assays, an antisense the more complex components of vestibulomotor function and mouse cyclophilin riboprobe was included in the hybridization coordination as described for mice (17). Briefly, the mouse was reaction as a control. Laser densitometry (Molecular Dynamics) placed on a narrow plastic beam (0.7 cm), and its ability to was used to correct RNA levels with the use of this internal maintain equilibrium was scored as follows: 0, does not attempt standard. to balance; 1, hangs on the beam and falls off; 2, hangs on the beam without falling; 3, hugs the beam without falling; 4, grasps Gel Mobility Shift Assay. Gel mobility shift analysis of TR-1 the side of the beam and͞or has unsteady movements; and 5, interactions was conducted with the nuclear hormone receptor exhibits steady posture on the beam. Mice were pretrained twice, interaction domains of N-CoR (human amino acids correspond- 24 h before the measurement. ing to murine 2063–2300) and SMRT (human amino acids The rotarod task for rats (18) was modified for use in mice 2098–2507). Wild-type (wt) and mutant TR-1 proteins were (17). The rotarod device consists of a clear 36-mm-diameter derived from in vitro transcription͞translation reactions in rabbit motorized rod rotating at a speed of 18 rpm. Performance on the reticulocyte lysate (Promega). Interacting domains of the core- task was assessed by measuring the time until the mouse falls pressors were synthesized as glutathione S-transferase fusion usboff or grips the device without attempting to walk on the rod. protein and column purified (13). Twenty nanograms of the All mice were acclimated to the rotarod for 2 days before indicated corepressor was used in the indicated lanes. Interac- measurement. tions were tested on a radiolabeled DRϩ4 element as previously The Morris water maze test involves a platform hidden in a described (14). In some lanes, T3 was added at a concentration round pan of opaque water. The apparatus and testing procedure of 100 nM. for mice were previously described (5). Briefly, a pool was filled with water containing 10% (wt͞vol) skimmed milk. A hidden Serum Assays. Thyroid hormone levels (total and free T3 and T4) platform was placed 1 cm below the water surface so that it was were measured in triplicate by radioimmunoassays (ImmuChem- not visible to the mouse. Mice were pretrained twice on the coated tube kits; ICN). The thyroid-stimulating hormone (TSH) apparatus, 1 day before the test. level was determined by a specific mouse TSH RIA (15), with the use of radiolabeled rat TSH antigen (reference preparation, Results AFP98991) and a mouse TSH- antibody (hormone distribution Generation and Characterization of TR Knock-in Mice. To prove the program, National Institute of Diabetes and Digestive and importance of the unliganded TR in mammalian development Kidney Diseases, Bethesda, MD). Statistical comparisons used and function, we generated a knock-in model of tissue hypothy- the unpaired t test.