Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy ( Necturus maculosus ) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus . Strikingly, we found that Necturus TRΑ and TRΒ genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRΑ and TRΒ are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75694/1/j.1525-142X.2006.00099.x.pd

A Fine Regulation of the Hippocampal Thyroid Signalling Protects Hypothyroid Mice against Glial Cell Activation

Adult-onset hypothyroidism is associated with learning and cognitive dysfunctions, which may be related to alterations in synaptic plasticity. Local reduced levels of thyroid hormones (THs) may impair glia morphology and activity, and promote the increase of pro-inflammatory cytokine levels mainly in the hippocampus. Given that neuroinflammation induces memory impairments, hypothyroidism-related glia dysfunction may participate in brain disorders. Thus, we investigated the mechanisms linking hypothyroidism and neuroinflammation, from a protective perspective. We induced hypothyroidism in adult C57BL/6J and wild-derived WSB/EiJ male mice by a seven-week propylthiouracil (PTU) treatment. We previously showed that WSB/EiJ mice were resistant to high-fat diet (HFD)-induced obesity, showing no neuroinflammatory response through adaptive abilities, unlike C57BL/6J. As PTU and HFD treatments are known to induce comparable inflammatory responses, we hypothesized that WSB/EiJ mice might also be protected against hypothyroidism-induced neuroinflammation. We showed that hypothyroid WSB/EiJ mice depicted no hippocampal neuroinflammatory response and were able to maintain their hippocampal thyroid signalling despite low circulatisng TH levels. In contrast, C57BL/6J mice exhibited disturbed hippocampal TH signalling, accompanied by neuroinflammation and memory impairment. Our results reinforce the preponderance of the hippocampal TH regulatory system over TH circulating levels in the hippocampal glial reactivity

Liver X receptor regulation of thyrotropin-releasing hormone transcription in mouse hypothalamus is dependent on thyroid status.

Reversing the escalating rate of obesity requires increased knowledge of the molecular mechanisms controlling energy balance. Liver X receptors (LXRs) and thyroid hormone receptors (TRs) are key physiological regulators of energetic metabolism. Analysing interactions between these receptors in the periphery has led to a better understanding of the mechanisms involved in metabolic diseases. However, no data is available on such interactions in the brain. We tested the hypothesis that hypothalamic LXR/TR interactions could co-regulate signalling pathways involved in the central regulation of metabolism. Using in vivo gene transfer we show that LXR activation by its synthetic agonist GW3965 represses the transcriptional activity of two key metabolic genes, Thyrotropin-releasing hormone (Trh) and Melanocortin receptor type 4 (Mc4r) in the hypothalamus of euthyroid mice. Interestingly, this repression did not occur in hypothyroid mice but was restored in the case of Trh by thyroid hormone (TH) treatment, highlighting the role of the triiodothyronine (T3) and TRs in this dialogue. Using shLXR to knock-down LXRs in vivo in euthyroid newborn mice, not only abrogated Trh repression but actually increased Trh transcription, revealing a potential inhibitory effect of LXR on the Hypothalamic-Pituitary-Thyroid axis. In vivo chromatin immunoprecipitation (ChIP) revealed LXR to be present on the Trh promoter region in the presence of T3 and that Retinoid X Receptor (RXR), a heterodimerization partner for both TR and LXR, was never recruited simultaneously with LXR. Interactions between the TR and LXR pathways were confirmed by qPCR experiments. T3 treatment of newborn mice induced hypothalamic expression of certain key LXR target genes implicated in metabolism and inflammation. Taken together the results indicate that the crosstalk between LXR and TR signalling in the hypothalamus centres on metabolic and inflammatory pathways

Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus.

The beta thyroid hormone receptor (TRbeta), but not TRalpha1, plays a specific role in mediating T(3)-dependent repression of hypothalamic TRH transcription. To investigate the structural basis of isoform specificity, we compared the transcriptional regulation and DNA binding obtained with chimeric and N-terminally deleted TRs. Using in vivo transfection assays to follow hypothalamic TRH transcription in the mouse brain, we found that TRbeta1 and chimeras with the TRbeta1 N terminus did not affect either transcriptional activation or repression from the rat TRH promoter, whereas N-terminally deleted TRbeta1 impaired T(3)-dependent repression. TRalpha1 or chimeras with the TRalpha1 N terminus reduced T(3)-independent transcriptional activation and blocked T(3)-dependent repression of transcription. Full deletion of the TRalpha1 N terminus restored ligand-independent activation of transcription. No TR isoform specificity was seen after transcription from a positive thyroid hormone response element. Gel mobility assays showed that all TRs tested bound specifically to the main negative thyroid hormone response element in the TRH promoter (site 4). Addition of neither steroid receptor coactivator 1 nor nuclear extracts from the hypothalamic paraventricular nuclei revealed any TR isoform specificity in binding to site 4. Thus N-terminal sequences specify TR T(3)-dependent repression of TRH transcription but not DNA recognition, emphasizing as yet unknown neuron-specific contributions to protein-promoter interactions in vivo

GW3965 represses hypothalamic <i>Trh</i> and <i>Mc4r</i> transcriptional activity only in euthyroid newborn mice.

<p><b>A</b> and <b>B</b>. Transcriptional repression of <i>Trh</i> (A) and <i>Mc4r</i> (B) promoters via LXR activation by subcutaneous injection of GW3965 (12.5 or 25 mg/Kg) in euthyroid newborn mice. <b>C</b> and <b>D</b>. Lack of effect of subcutaneous injection of GW3965 (12.5 or 25 mg/Kg) on the transcription from <i>Trh</i> (C) and <i>Mc4r</i> (D) promoters in hypothyroid newborn mice. <b>A–B/C–D</b>. <i>In vivo</i> gene reporter assays: one-day-old euthyroid (A and B) or hypothyroid (C and D) pups were subcutaneously injected by GW3965 at 12.5 or 25 mg/Kg of body weight, and co-transfected 24 h after in the hypothalamic region of the brain (Paraventricular nuclei, PVN) with 4 µl of a solution of PEI–complexed TRH-f.luc (0.2 µg/pup)/MC4R-r.luc (0.8 µg/pup). Firefly and Renilla luciferase activities were measured 24 h later. Representative experiments are shown. n = 10 per group. Non-parametric permutation test was used to assess statistical significance. *, p<0.05, **, p<0.01. <b>E</b> and <b>F</b>. Transcriptional repression of <i>Trh</i> (E) and <i>Mc4r</i> (F) promoters via LXR activation by ICV injection of GW3965 (10⁻⁶ or 10⁻⁷ M) in euthyroid newborn mice. <b>G</b> and <b>H</b>. Lack of effect of I.C.V injection of GW3965 (10⁻⁶ or 10⁻⁷ M) on the <i>Trh</i> (G) and <i>Mc4r</i> (H) transcriptions in hypothyroid newborn mice. <b>E–F/G–H.</b> One-day-old euthyroid (E and F) or hypothyroid (G and H) pups were co-transfected in the hypothalamic region of the brain (PVN) with a solution of PEI–complexed TRH-f.luc (0.2 µg/pup)/MC4R-r.luc (0.8 µg/pup) with GW3965 at 10⁻⁷ or 10⁻⁶ M in the transfection mix. Firefly and Renilla Luciferase activities were measured 24 h later. n = 10 per group, pools of three independent experiments are represented. Non-parametric permutation test was used to assess statistical significance. *, p<0.05, **, p<0.01; ***, p<0.001.</p

Overexpression of TRα1, but not TRα2, in the hypothalamus modulates circulating T₄ levels.

<p>pSG5-TRα1 (TRα1) or pSG5-TRα2 (TRα2) were transfected (100 ng/pup) into the hypothalamus of hypothyroid 2-day old mice. Serum was collected at the ages shown (3, 5 or 7 days old) and pooled (four individual samples per pool). At 5 days post-transfection (7 days old mice), T₄ circulating levels are significantly decreased when TRα1 is overexpressed and not modified by TRα2 overexpression. Means ± SEM of pooled samples are given, n≥4 for each point. **, p<0.01.</p

T₄ treatment of hypothyroid dams restores the GW3965-dependent repression of <i>Trh</i> promoter of newborn offspring.

<p><b>A.</b> One-day-old hypothyroid pups, from dams treated with T₄ at 12 µg/ml in drinking water 24 h before giving birth, were transfected in the hypothalamus with a solution of PEI-complexed TRH-f.luc (0.2 µg/pup)/MC4R-r.luc (0.8 µg/pup) with GW3965 at 10⁻⁶ M in the transfection mix. Firefly and Renilla luciferase activities were measured 24 h later. A representative experiment is shown. n = 10 per group. Non-parametric Mann-Whitney test was used to assess statistical significance. *, p<0.05. <b>B.</b> One-day-old hypothyroid pups, from dams treated with T₄ at 12 µg/ml in drinking water 48 h before giving birth, were co-transfected in the hypothalamus with a solution of PEI-complexed TRH-f.luc (0.2 µg/pup) MC4R-r.luc (0.8 µg/pup) with GW3965 at 10⁻⁶ M in the transfection mix. Firefly and Renilla luciferase activities were measured 24 h later. A representative experiment is shown. n = 10 per group. Non-parametric permutation test was used to assess statistical significance. **, p<0.01.</p

Subcutaneous T₃ treatment of hypothyroid newborn mice restores the GW3965-dependent repression of the <i>Trh</i> promoter.

<p>One-day-old hypothyroid pups were subcutaneously injected by T₃ (2.5 µg/g bw) and co-transfected 24 h after in the hypothalamic region of the brain (Paraventricular nuclei, PVN) with a solution of PEI-complexed TRH-f.luc (0.2 µg/pup)/MC4R-r.luc (0.8 µg/pup) with GW3965 at 10⁻⁶ M in the transfection mix. Firefly and Renilla luciferase activities were measured 24 h later. n = 10 pups per group. A representative experiment is shown. Non-parametric permutation test was used to assess statistical significance, **, p<0.01.</p