Identification of thyroid hormone response elements in vivo using mice expressing a tagged thyroid hormone receptor α1.

TRα1 (thyroid hormone receptor α1) is well recognized for its importance in brain development. However, due to the difficulties in predicting TREs (thyroid hormone response elements) in silico and the lack of suitable antibodies against TRα1 for ChIP (chromatin immunoprecipitation), only a few direct TRα1 target genes have been identified in the brain. Here we demonstrate that mice expressing a TRα1-GFP (green fluorescent protein) fusion protein from the endogenous TRα locus provide a valuable animal model to identify TRα1 target genes. To this end, we analysed DNA-TRα1 interactions in vivo using ChIP with an anti-GFP antibody. We validated our system using established TREs from neurogranin and hairless, and by verifying additional TREs from known TRα1 target genes in brain and heart. Moreover, our model system enabled the identification of novel TRα1 target genes such as RNF166 (ring finger protein 166). Our results demonstrate that transgenic mice expressing a tagged nuclear receptor constitute a feasible approach to study receptor-DNA interactions in vivo, circumventing the need for specific antibodies. Models like the TRα1-GFP mice may thus pave the way for genome-wide mapping of nuclear receptor-binding sites, and advance the identification of novel target genes in vivo.

Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions.

Thyroid hormone is well known for its profound direct effects on cardiovascular function and metabolism. Recent evidence, however, suggests that the hormone also regulates these systems indirectly through the central nervous system. While some of the molecular mechanisms underlying the hormone's central control of metabolism have been identified, its actions in the central cardiovascular control have remained enigmatic. Here, we describe a previously unknown population of parvalbuminergic neurons in the anterior hypothalamus that requires thyroid hormone receptor signaling for proper development. Specific stereotaxic ablation of these cells in the mouse resulted in hypertension and temperature-dependent tachycardia, indicating a role in the central autonomic control of blood pressure and heart rate. Moreover, the neurons exhibited intrinsic temperature sensitivity in patch-clamping experiments, providing a new connection between cardiovascular function and core temperature. Thus, the data identify what we believe to be a novel hypothalamic cell population potentially important for understanding hypertension and indicate developmental hypothyroidism as an epigenetic risk factor for cardiovascular disorders. Furthermore, the findings may be beneficial for treatment of the recently identified patients that have a mutation in thyroid hormone receptor α1.

Serum copper as a novel biomarker for resistance to thyroid hormone.

Thyroid hormone action is mediated by the thyroid hormone receptors TRα1 and TRß. Defects in TRß lead to RTH (resistance to thyroid hormone) ß, a syndrome characterized by high levels of thyroid hormone and non-suppressed TSH (thyroid-stimulating hormone). However, a correct diagnosis of RTHß patients is difficult as the clinical picture varies. A biochemical serum marker indicative of defects in TRß signalling is needed and could simplify the diagnosis of RTHß, in particular the differentiation to TSH-secreting pituitary adenomas, which present with clinically similar symptoms. In the present paper we show that serum copper levels are regulated by thyroid hormone, which stimulates the synthesis and the export of the hepatic copper-transport protein ceruloplasmin into the serum. This is accompanied by a concerted reduction in the mRNA levels of other copper-containing proteins such as metallothioneins 1 and 2 or superoxide dismutase 1. The induction of serum copper is abolished in genetically hyperthyroid mice lacking TRß and human RTHß patients, demonstrating an important role of TRß for this process. Together with a previously reported TRα1 specific regulation of serum selenium, we show that the ratio of serum copper and selenium, which is largely independent of thyroid hormone levels, volume changes or sample degradation, can constitute a valuable novel biomarker for RTHß. Moreover, it could also provide a suitable large-scale screening parameter to identify RTHα patients, which have not been identified to date.

Loss of thyroid hormone receptor ß is associated with increased progenitor proliferation and NeuroD positive cell number in the adult hippocampus.

Adult hippocampal neurogenesis is modulated by perturbations in thyroid hormone status; however the role of specific thyroid hormone receptors (TRs) in this process is not completely understood. We show here that loss of the TRß gene results in a significant increase in the proliferation of adult hippocampal progenitors, without any change in immature neuron number or in the neuronal and glial differentiation of progenitors. Using the mitotic marker 5'-bromo-2-deoxyuridine (BrdU) or the endogenous cell cycle marker, proliferating cell nuclear antigen (PCNA), we find a significant increase in the number of BrdU- and PCNA-immunopositive cells within the subgranular zone (SGZ) of the dentate gyrus subfield in TRß-/- mice. Further, we find that TRß-/- mice exhibit a significant increase in the numbers of NeuroD-positive cells within the SGZ, suggesting that the increased numbers of proliferating progenitors translate into enhanced numbers of neuroblasts. Interestingly, the number of BrdU-positive cells that persist 4 weeks post-BrdU injection is unaltered in TRß-/- mice, indicating that the enhanced proliferation does not result in increased hippocampal neurogenesis. This is also supported by the evidence of no change in the numbers of cells expressing markers of immature neurons such as doublecortin or polysialylated neural cell adhesion molecule. Furthermore, no change is observed in the neuronal or glial differentiation of BrdU-positive cells in the TRß-/- mice. Taken together, our results provide novel evidence for a role of TRß in modulating hippocampal progenitor cell division, and implicate this receptor in the effects of thyroid hormone on adult hippocampal neurogenesis.

The thyroid hormone receptor alpha1 protein is expressed in embryonic postmitotic neurons and persists in most adult neurons.

Thyroid hormone is essential for brain development where it acts mainly through the thyroid hormone receptor α1 (TRα1) isoform. However, the potential for the hormone to act in adult neurons has remained undefined due to difficulties in reliably determining the expression pattern of TR proteins in vivo. We therefore created a mouse strain that expresses TRα1 and green fluorescent protein as a chimeric protein from the Thra locus, allowing examination of TRα1 expression during fetal and postnatal development and in the adult. Furthermore, the use of antibodies against other markers enabled identification of TRα1 expression in subtypes of neurons and during specific stages of their maturation. TRα1 expression was first detected in postmitotic cells of the cortical plate in the embryonic telencephalon and preceded the expression of the mature neuronal protein NeuN. In the cerebellum, TRα1 expression was absent in proliferating cells of the external granular layer, but switched on as the cells migrated towards the internal granular layer. In addition, TRα1 was expressed transiently in developing Purkinje cells, but not in mature cells. Glial expression was found in tanycytes in the hypothalamus and in the cerebellum. In the adult brain, TRα1 expression was detected in essentially all neurons. Our data demonstrate that thyroid hormone, unexpectedly, has the capacity to play an important role in virtually all developing and adult neurons. Because the role of TRα1 in most neuronal cell types in vivo is largely unknown, our findings suggest that novel functions for thyroid hormone remain to be identified in the brain.

Unliganded thyroid hormone receptor alpha1 impairs adult hippocampal neurogenesis.

Thyroid hormone regulates adult hippocampal neurogenesis, a process involved in key functions, such as learning, memory, and mood regulation. We addressed the role of thyroid hormone receptor TRα1 in adult hippocampal neurogenesis, using mice harboring a TRα1 null allele (TRα1(-/-)), overexpressing TRα1 6-fold (TRα2(-/-)), and a mutant TRα1 (TRα1(+/m)) with a 10-fold lower affinity to the ligand. While hippocampal progenitor proliferation was unaltered, TRα1(-/-) mice exhibited a significant increase in doublecortin-positive immature neurons and increased survival of bromodeoxyuridine-positive (BrdU(+)) progenitors as compared to wild-type controls. In contrast, the TRα1(+/m) and the TRα2(-/-) mice, where the overexpressed TRα1 acts as an aporeceptor, showed a significant decline in surviving BrdU(+) progenitors. TRα1(-/-) and TRα2(-/-) mice showed opposing effects on neurogenic markers like polysialylated neural cell adhesion molecule and stathmin. The decreased progenitor survival in the TRα2(-/-) and TRα1(+/m) mice could be rescued by thyroid hormone treatment, as was the decline in neuronal differentiation seen in the TRα1(+/m) mice. These mice also exhibited a decrease in NeuroD(+) cell numbers in the dentate gyrus, suggesting an effect on early postmitotic progenitors. Our results provide the first evidence of a role for unliganded TRα1 in modulating the deleterious effects of hypothyroidism on adult hippocampal neurogenesis.

Increased depressive behaviour in mice harboring the mutant thyroid hormone receptor alpha 1.

Clinical evidence indicates that hypothyroidism contributes to mood disorders. The present study tested if the mutant thyroid hormone receptor alpha 1 (TRalpha1) that causes a receptor-mediated hypothyroidism in the brain affects depressive and anxious behaviour in mice. Mice heterozygous for the TRalpha1 allele (TRalpha1+/m), yielding a receptor protein with a 10-fold reduced affinity to triiodothyronine (T3), and wildtype (wt) mice were subjected to several paradigms specifically testing depressive and anxious behaviour. Mutant and wt mice were either treated with T3 or vehicle. Untreated TRalpha1+/m animals displayed reduced locomotion, higher rates of helplessness in the shuttle box-, greater levels of anxiety in the startle response- and dark light box behavioural paradigms when compared to wt mice. Continuous T3-substitution therapy was effective in alleviating anxious and depressive behaviour without affecting locomotion in mutant mice. Notably, continuous T3-substitution reduced overall locomotion and increased helpless behaviour in wt mice when compared to untreated wt mice. The data suggest that receptor-mediated hypothyroidism caused by an unliganded thyroid hormone receptor alpha 1 leads to a depressive and anxious phenotype in mice, which is responsive to continuous T3-substitution and that an iatrogeneously induced hyperthyreoidism by continuous T3-administration leads to a hypolocomotive and depressive phenotype.

A mutant thyroid hormone receptor alpha1 alters hippocampal circuitry and reduces seizure susceptibility in mice.

Thyroid hormone deficiency during early developmental stages causes a multitude of functional and morphological deficits in the brain. In the present study we investigate the effects of a mutated thyroid hormone receptor TR alpha 1 and the resulting receptor-mediated hypothyroidism on the development of GABAergic neurotransmission and seizure susceptibility of neuronal networks. We show that mutant mice have a strong resistance to seizures induced by antagonizing the GABA(A) receptor complex. Likewise the hippocampal network of mutant mice shows a decreased likelihood to transform physiological into pathological rhythmic network activity such as seizure-like interictal waves. As we demonstrate the cellular basis for this behavior is formed by the excitatory nature of GABAergic neurotransmission in the mutant mice, possibly caused by altered Cl(-) homeostasis, and/or the altered patterning of calretinin-positive cells in the hippocampal hilus. This study is, to our knowledge, the first to show an effect of maternal and early postnatal hypothyroidism via TR alpha 1 on the development of GABAergic neurotransmission and susceptibility to epileptic seizures.

Interference of a mutant thyroid hormone receptor alpha1 with hepatic glucose metabolism.

Mice expressing the mutant thyroid hormone receptor TRalpha1R384C, which has a 10-fold reduced affinity to the ligand T(3), exhibit hypermetabolism due to an overactivation of the sympathetic nervous system. To define the consequences in the liver, we analyzed hepatic metabolism and the regulation of liver genes in the mutant mice. Our results showed that hepatic phosphoenolpyruvate-carboxykinase was up-regulated and pyruvate kinase mRNA down-regulated, contrary to what observed after T(3) treatment. In contrast, mice expressing a mutant TRalpha1L400R specifically in the liver did not show a dysregulation of these genes; however, when the TRalpha1L400R was expressed ubiquitously, the hepatic phenotype differed from TRalpha1R384C animals, suggesting that the localization of the mutation plays an important role for its consequences on glucose metabolism. Furthermore, we observed that glycogen stores were completely depleted in TRalpha1R384C animals, despite increased gluconeogenesis and decreased glycolysis. Exposure of the mutant mice to high maternal levels of thyroid hormone during fetal development leads to a normal liver phenotype in the adult. Our results show how genetic and maternal factors interact to determine the metabolic setpoint of the offspring and indicate an important role for maternal thyroid hormone in the susceptibility to metabolic disorders in adulthood.

Locomotor deficiencies and aberrant development of subtype-specific GABAergic interneurons caused by an unliganded thyroid hormone receptor alpha1.

Thyroid hormone (TH) deficiency during development causes severe and permanent neuronal damage, but the primary insult at the tissue level has remained unsolved. We have defined locomotor deficiencies in mice caused by a mutant thyroid hormone receptor alpha1 (TR alpha1) with potent aporeceptor activity attributable to reduced affinity to TH. This allowed identification of distinct functions that required either maternal supply of TH during early embryonic development or sufficient innate levels of hormone during late fetal development. In both instances, continued exposure to high levels of TH after birth and throughout life was needed. The hormonal dependencies correlated with severely delayed appearance of parvalbumin-immunoreactive GABAergic interneurons and increased numbers of calretinin-immunoreactive cells in the neocortex. This resulted in reduced numbers of fast spiking interneurons and defects in cortical network activity. The identification of locomotor deficiencies caused by insufficient supply of TH during fetal/perinatal development and their correlation with subtype-specific interneurons suggest a previously unknown basis for the neuronal consequences of endemic cretinism and untreated congenital hypothyroidism, and specifies TR alpha1 as the receptor isoform mediating these effects.

Hypermetabolism in mice caused by the central action of an unliganded thyroid hormone receptor alpha1.

Thyroid hormone, via its nuclear receptors TRalpha and TRbeta, controls metabolism by acting locally in peripheral tissues and centrally by regulating sympathetic signaling. We have defined aporeceptor regulation of metabolism by using mice heterozygous for a mutant TRalpha1 with low affinity to T3. The animals were hypermetabolic, showing strongly reduced fat depots, hyperphagia and resistance to diet-induced obesity accompanied by induction of genes involved in glucose handling and fatty acid metabolism in liver and adipose tissues. Increased lipid mobilization and beta-oxidation occurred in adipose tissues, whereas blockade of sympathetic signaling to brown adipose tissue normalized the metabolic phenotype despite a continued perturbed hormone signaling in this cell type. The results define a novel and important role for the TRalpha1 aporeceptor in governing metabolic homeostasis. Furthermore, the data demonstrate that a nuclear hormone receptor affecting sympathetic signaling can override its autonomous effects in peripheral tissues.

Thyroid status during skeletal development determines adult bone structure and mineralization.

Childhood hypothyroidism delays ossification and bone mineralization, whereas adult thyrotoxicosis causes osteoporosis. To determine how effects of thyroid hormone (T3) during development manifest in adult bone, we characterized TRalpha1(+/m)beta(+/-) mice, which express a mutant T3 receptor (TR) alpha1 with dominant-negative properties due to reduced ligand-binding affinity. Remarkably, adult TRalpha1(+/m)beta(+/-) mice had osteosclerosis with increased bone mineralization even though juveniles had delayed ossification. This phenotype was partially normalized by transient T3 treatment of juveniles and fully reversed in compound TRalpha1(+/m)beta(-/-) mutant mice due to 10-fold elevated hormone levels that allow the mutant TRalpha1 to bind T3. By contrast, deletion of TRbeta in TRalpha1(+/+)beta(-/ -) mice, which causes a 3-fold increase of hormone levels, led to osteoporosis in adults but advanced ossification in juveniles. T3-target gene analysis revealed skeletal hypothyroidism in TRalpha1(m/+)beta(+/-) mice, thyrotoxicosis in TRalpha1(+/+)beta(-/-) mice, and euthyroidism in TRalpha1(+/)beta(-/-) double mutants. Thus, TRalpha1 regulates both skeletal development and adult bone maintenance, with euthyroid status during development being essential to establish normal adult bone structure and mineralization.

Anxiety, memory impairment, and locomotor dysfunction caused by a mutant thyroid hormone receptor alpha1 can be ameliorated by T3 treatment.

The transcriptional properties of unliganded thyroid hormone receptors are thought to cause the misdevelopment during hypothyroidism of several functions essential for adult life. To specifically determine the role of unliganded thyroid hormone receptor alpha1 (TRalpha1) in neuronal tissues, we introduced a mutation into the mouse TRalpha1 gene that lowers affinity to thyroid hormone (TH) 10-fold. The resulting heterozygous mice exhibit several distinct neurological abnormalities: extreme anxiety, reduced recognition memory, and locomotor dysfunction. The anxiety and memory deficiencies were relieved by treatment with high levels of TH in adulthood, an effect that correlated with a normalization of GABAergic inhibitory interneurons in the hippocampal CA1 region. In contrast, a post-natal TH treatment was necessary and sufficient for ameliorating the adult locomotor dysfunction. Here, the hormone treatment normalized the otherwise delayed cerebellar development. The data thus identify two novel and distinct functions of an unliganded TRalpha1 during development and adulthood, respectively.

Methodological development: structured outcome assessment and community risk monitoring (SORM).

This paper describes an effort to develop a clinical tool for the continuous monitoring of risk for violence in forensic mental health clients who have left their institutions and who are dwelling in the community on a conditional release basis. The model is called Structured Outcome Assessment and Community Risk Monitoring (SORM). The SORM consists of 30 dynamic factors and each factor in SORM is assessed in two ways: The current absence, presence or partial och intermittent presence of the factors, which is an actuarial (systematized and 'objective') assessment. Secondly, the risk effect, i.e. whether the presence/absence of factors currently increases, decreases or is perceived as unrelated to violence risk, is a clinical (or impressionistic) assessment. Thus, the factors considered via the SORM can be coded as risk factors or protective factors (or as factors unimportant to risk of violence) depending on circumstances that apply in the individual case. Further, the SORM has a built-in module for gathering idiographical information about risk-affecting contextual factors. The use of the SORM and its potential as a risk monitoring instrument is illustrated via preliminary data and case vignettes from an ongoing multicenter project. In this research project, patients leaving any of the 9 participating forensic hospitals in Sweden is assessed at release on a variety of static background factors, and the SORM is then administered every 30 days for 2 years.

Effective generation of transgenic mice by Bovine papillomavirus type 1 based self-replicating plasmid that is maintained as extrachromosomal genetic element in three generations of animals.

The objective of our study was to analyze the efficiency and the properties of the inheritance of the Bovine papillomavirus type 1 (BPV1) replicator-based plasmid used as vector system for generation of transgenic animals. Previously, we have characterized a series of self-replicating plasmid vectors containing all viral factors necessary and sufficient for stable extrachromosomal replication of the BPV1 genome in the tissue culture system. We also demonstrated that the designed replicating vector system has a considerable benefit in the transgene expression, if compared to the regular expression vector. The vector, which showed the highest stability and maintenance function in the tissue culture was chosen for generation of the transgenic mice by pronuclear injections of the circular supercoiled plasmid. This method resulted in successful production of transgenic animals. Transmission efficiency of the vectors into the F(1) generation of animals varied between 0 and 48%, whereas transmission into the F(2) generation was uniformly near 50%. The maintenance of the vector-plasmids in the F(2) generation of transgenic animals as extrachromosomal genetic element was demonstrated by rescue of the plasmid into the Escherichia coli.

Retardation of post-natal development caused by a negatively acting thyroid hormone receptor alpha1.

Most patients with the syndrome resistance to thyroid hormone (RTH) express a mutant thyroid hormone receptor beta (TRbeta) with transdominant negative transcriptional effects. Since no patient with a mutant TRalpha has been identified, we introduced a point mutation into the mouse thyroid hormone receptor (TRalpha1) locus originally found in the TRbeta gene, that reduces ligand binding 10-fold. Heterozygous 2- to 3-week- old mice exhibit a severe retardation of post-natal development and growth, but only a minor reduction in serum thyroxine levels. Homozygous mice died before 3 weeks of age. Adult heterozygotes overcome most of these defects except for cardiac function abnormalities, suggesting that other factors compensate for the receptor defect. However, the additional deletion of the TRbeta gene in this mouse strain caused a 10-fold increase in serum thyroxine, restored hormonal regulation of target genes for TRs, and rescued the growth retardation. The data demonstrate a novel array of effects mediated by a dominant negative TRalpha1, and may provide important clues for identification of a potentially unrecognized human disorder and its treatment.