Remyelination protects neurons from DLK-mediated neurodegeneration.

Chronic demyelination is theorized to contribute to neurodegeneration and drive progressive disability in demyelinating diseases like multiple sclerosis. Here, we describe two genetic mouse models of inducible demyelination, one distinguished by effective remyelination, and the other by remyelination failure and persistent demyelination. By comparing these two models, we find that remyelination protects neurons from apoptosis, improves conduction, and promotes functional recovery. Chronic demyelination of neurons leads to activation of the mitogen-associated protein kinase (MAPK) stress pathway downstream of dual leucine zipper kinase (DLK), which ultimately induces the phosphorylation of c-Jun in the nucleus. Both pharmacological inhibition and CRISPR/Cas9-mediated disruption of DLK block c-Jun phosphorylation and the apoptosis of demyelinated neurons. These findings provide direct experimental evidence that remyelination is neuroprotective and identify DLK inhibition as a potential therapeutic strategy to protect chronically demyelinated neurons.

Thyroid hormone rewires cortical circuits to coordinate body-wide metabolism and exploratory drive.

Animals adapt to varying environmental conditions by modifying the function of their internal organs, including the brain. To be adaptive, alterations in behavior must be coordinated with the functional state of organs throughout the body. Here we find that thyroid hormone- a prominent regulator of metabolism in many peripheral organs- activates cell-type specific transcriptional programs in anterior regions of cortex of adult mice via direct activation of thyroid hormone receptors. These programs are enriched for axon-guidance genes in glutamatergic projection neurons, synaptic regulators across both astrocytes and neurons, and pro-myelination factors in oligodendrocytes, suggesting widespread remodeling of cortical circuits. Indeed, whole-cell electrophysiology recordings revealed that thyroid hormone induces local transcriptional programs that rewire cortical neural circuits via pre-synaptic mechanisms, resulting in increased excitatory drive with a concomitant sensitization of recruited inhibition. We find that thyroid hormone bidirectionally regulates innate exploratory behaviors and that the transcriptionally mediated circuit changes in anterior cortex causally promote exploratory decision-making. Thus, thyroid hormone acts directly on adult cerebral cortex to coordinate exploratory behaviors with whole-body metabolic state.

Promyelinating drugs promote functional recovery in an autism spectrum disorder mouse model of Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome is an autism spectrum disorder caused by autosomal dominant mutations in the human transcription factor 4 gene (TCF4). One pathobiological process caused by murine Tcf4 mutation is a cell autonomous reduction in oligodendrocytes and myelination. In this study, we show that the promyelinating compounds, clemastine, sobetirome and Sob-AM2 are effective at restoring myelination defects in a Pitt-Hopkins syndrome mouse model. In vitro, clemastine treatment reduced excess oligodendrocyte precursor cells and normalized oligodendrocyte density. In vivo, 2-week intraperitoneal administration of clemastine also normalized oligodendrocyte precursor cell and oligodendrocyte density in the cortex of Tcf4 mutant mice and appeared to increase the number of axons undergoing myelination, as EM imaging of the corpus callosum showed a significant increase in the proportion of uncompacted myelin and an overall reduction in the g-ratio. Importantly, this treatment paradigm resulted in functional rescue by improving electrophysiology and behaviour. To confirm behavioural rescue was achieved via enhancing myelination, we show that treatment with the thyroid hormone receptor agonist sobetirome or its brain penetrating prodrug Sob-AM2, was also effective at normalizing oligodendrocyte precursor cell and oligodendrocyte densities and behaviour in the Pitt-Hopkins syndrome mouse model. Together, these results provide preclinical evidence that promyelinating therapies may be beneficial in Pitt-Hopkins syndrome and potentially other neurodevelopmental disorders characterized by dysmyelination.

Maternal Administration of the CNS-Selective Sobetirome Prodrug Sob-AM2 Exerts Thyromimetic Effects in Murine MCT8-Deficient Fetuses.

Background: Monocarboxylate transporter 8 (MCT8) deficiency is a rare X-linked disease where patients exhibit peripheral hyperthyroidism and cerebral hypothyroidism, which results in severe neurological impairments. These brain defects arise from a lack of thyroid hormones (TH) during critical stages of human brain development. Treatment options for MCT8-deficient patients are limited and none have been able to prevent or ameliorate effectively the neurological impairments. This study explored the effects of the TH agonist sobetirome and its CNS-selective amide prodrug, Sob-AM2, in the treatment of pregnant dams carrying fetuses lacking Mct8 and deiodinase type 2 (Mct8/Dio2 KO), as a murine model for MCT8 deficiency. Methods: Pregnant dams carrying Mct8/Dio2 KO fetuses were treated with 1 mg of sobetirome/kg body weight/day, or 0.3 mg of Sob-AM2/kg body weight/day for 7 days, starting at embryonic day 12.5 (E12.5). As controls, pregnant dams carrying wild-type and pregnant dams carrying Mct8/Dio2 KO fetuses were treated with daily subcutaneous injections of vehicle. Dams TH levels were measured by enzyme-linked immunosorbent assay (ELISA). Samples were extracted at E18.5 and the effect of treatments on the expression of triiodothyronine (T3)-dependent genes was measured in the placenta, fetal liver, and fetal cerebral cortex by real-time polymerase chain reaction. Results: Maternal sobetirome treatment led to spontaneous abortions. Sob-AM2 treatment, however, was able to cross the placental as well as the brain barriers and exert thyromimetic effects in Mct8/Dio2 KO fetal tissues. Sob-AM2 treatment did not affect the expression of the T3-target genes analyzed in the placenta, but it mediated thyromimetic effects in the fetal liver by increasing the expression of Dio1 and Dio3 genes. Interestingly, Sob-AM2 treatment increased the expression of several T3-dependent genes in the brain such as Hr, Shh, Dio3, Kcnj10, Klf9, and Faah in Mct8/Dio2 KO fetuses. Conclusions: Maternal administration of Sob-AM2 can cross the placental barrier and access the fetal tissues, including the brain, in the absence of MCT8, to exert thyromimetic actions by modulating the expression of T3-dependent genes. Therefore, Sob-AM2 has the potential to address the cerebral hypothyroidism characteristic of MCT8 deficiency from fetal stages and to prevent neurodevelopmental alterations in the MCT8-deficient fetal brain.

TREM2 is thyroid hormone regulated making the TREM2 pathway druggable with ligands for thyroid hormone receptor.

Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease-associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. Here, we report that TREM2 is a thyroid hormone-regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone and synthetic thyroid hormone agonists (thyromimetics). Our findings report the endocrine regulation of TREM2 by thyroid hormone, and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small-molecule therapeutic agents.

TREM2 is thyroid hormone regulated making the TREM2 pathway druggable with ligands for thyroid hormone receptor.

Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. We report here that TREM2 is a thyroid hormone regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone. Both endogenous thyroid hormone and sobetirome, a synthetic thyroid hormone agonist drug, suppress pro-inflammatory cytokine production from myeloid cells including macrophages that have been treated with the SARS-CoV-2 spike protein which produces a strong, pro-inflammatory phenotype. Thyroid hormone agonism was also found to induce phagocytic behavior in microglia, a phenotype consistent with activation of the TREM2 pathway. The thyroid hormone antagonist NH-3 blocks the anti-inflammatory effects of thyroid hormone agonists and suppresses microglia phagocytosis. Finally, in a murine experimental autoimmune encephalomyelitis (EAE) multiple sclerosis model, treatment with Sob-AM2, a CNS-penetrating sobetirome prodrug, results in increased Trem2 expression in disease lesion resident myeloid cells which correlates with therapeutic benefit in the EAE clinical score and reduced damage to myelin. Our findings represent the first report of endocrine regulation of TREM2 and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small molecule therapeutic agents.

A CNS-Targeting Prodrug Strategy for Nuclear Receptor Modulators.

The blood-brain barrier is a major impediment for targeted central nervous system (CNS) therapeutics, especially with carboxylic acid-containing drugs. Nuclear receptor modulators, which often feature carboxylic acid motifs for target engagement, have emerged as a class of potentially powerful therapeutics for neurodegenerative CNS diseases. Herein is described a prodrug strategy that directs the biodistribution of parent drug nuclear receptor modulators into the CNS while masking them as functional receptor ligands in the periphery. This prodrug strategy targets a specific amidase, fatty acid amide hydrolase (FAAH), an enzyme with enriched expression in the CNS. Our results demonstrate that this prodrug strategy can be generalized to a variety of carboxylic acid-containing drug structures that satisfy the structural requirements of blood-brain barrier diffusion and FAAH substrate recognition.

N- and O-Acetylated 3-Iodothyronamines Have No Metabolic or Thermogenic Effects in Male Mice.

INTRODUCTION: Injection of 3-iodothyronamine into experimental animals profoundly affects their metabolism and body temperature. As 3-iodothyronamine is rapidly acetylated in vivo after injection, it was hypothesized that the metabolites N- or O-acetyl-3-iodothyronamines could constitute the active hormones. METHODS: Adult male mice were injected once daily with one of the metabolites (5 mg/kg body weight intraperitoneally dissolved in 60% DMSO in PBS) or solvent. Metabolism was monitored by indirect calorimetry, body temperature by infrared thermography, and body composition by nuclear magnetic resonance analysis. Signaling activities in brown fat or liver were assessed by studying target gene transcription by qPCR including uncoupling protein 1 or deiodinase type 1 or 2, and Western blot. RESULTS: The markers of metabolism, body composition, or temperature tested were similar in the mice injected with solvent and those injected with one of the acetylated 3-iodothyronamines. CONCLUSIONS: In our experimental setup, N- and O-acetyl-3-iodothyronamine do not constitute compounds contributing to the metabolic or temperature effects described for 3-iodothyronamine. The acetylation of 3-iodothyronamine observed in vivo may thus rather serve degradation and elimination purposes.

Pharmacological Complementation Remedies an Inborn Error of Lipid Metabolism.

X-linked adrenoleukodystrophy (X-ALD) is a rare, genetic disease in which increased very long chain fatty acids (VLCFAs) in the central nervous system (CNS) cause demyelination and axonopathy, leading to neurological deficits. Sobetirome, a potent thyroid hormone agonist, has been shown to lower VLCFAs in the periphery and CNS. In this study, two pharmacological strategies for enhancing the effects of sobetirome were tested in Abcd1 KO mice, a murine model with the same inborn error of metabolism as X-ALD patients. First, a sobetirome prodrug (Sob-AM2) with increased CNS penetration lowered CNS VLCFAs more potently than sobetirome and was better tolerated with reduced peripheral exposure. Second, co-administration of thyroid hormone with sobetirome enhanced VLCFA lowering in the periphery but did not produce greater lowering in the CNS. These data support the conclusion that CNS VLCFA lowering in Abcd1 knockout mice is limited by a mechanistic threshold related to slow lipid turnover.

Myelin repair stimulated by CNS-selective thyroid hormone action.

Oligodendrocyte processes wrap axons to form neuroprotective myelin sheaths, and damage to myelin in disorders, such as multiple sclerosis (MS), leads to neurodegeneration and disability. There are currently no approved treatments for MS that stimulate myelin repair. During development, thyroid hormone (TH) promotes myelination through enhancing oligodendrocyte differentiation; however, TH itself is unsuitable as a remyelination therapy due to adverse systemic effects. This problem is overcome with selective TH agonists, sobetirome and a CNS-selective prodrug of sobetirome called Sob-AM2. We show here that TH and sobetirome stimulated remyelination in standard gliotoxin models of demyelination. We then utilized a genetic mouse model of demyelination and remyelination, in which we employed motor function tests, histology, and MRI to demonstrate that chronic treatment with sobetirome or Sob-AM2 leads to significant improvement in both clinical signs and remyelination. In contrast, chronic treatment with TH in this model inhibited the endogenous myelin repair and exacerbated disease. These results support the clinical investigation of selective CNS-penetrating TH agonists, but not TH, for myelin repair.

Evidence for hormonal control of heart regenerative capacity during endothermy acquisition.

Tissue regenerative potential displays striking divergence across phylogeny and ontogeny, but the underlying mechanisms remain enigmatic. Loss of mammalian cardiac regenerative potential correlates with cardiomyocyte cell-cycle arrest and polyploidization as well as the development of postnatal endothermy. We reveal that diploid cardiomyocyte abundance across 41 species conforms to Kleiber's law-the ¾-power law scaling of metabolism with bodyweight-and inversely correlates with standard metabolic rate, body temperature, and serum thyroxine level. Inactivation of thyroid hormone signaling reduces mouse cardiomyocyte polyploidization, delays cell-cycle exit, and retains cardiac regenerative potential in adults. Conversely, exogenous thyroid hormones inhibit zebrafish heart regeneration. Thus, our findings suggest that loss of heart regenerative capacity in adult mammals is triggered by increasing thyroid hormones and may be a trade-off for the acquisition of endothermy.

Thyroid hormone receptor function in maturing ovine cardiomyocytes.

KEY POINTS: Plasma thyroid hormone (tri-iodo-l-thyronine; T3 ) concentrations rise near the end of gestation and is known to inhibit proliferation and stimulate maturation of cardiomyocytes before birth. Thyroid hormone receptors are required for the action of thyroid hormone in fetal cardiomyocytes. Loss of thyroid hormone receptor (TR)α1 abolishes T3 signalling via extracellular signal-related kinase and Akt in fetal cardiomyocytes. The expression of TRα1 and TRß1 in ovine fetal myocardium increases with age, although TRα1 levels always remain higher than those of TRß1. Near term fetal cardiac myocytes are more sensitive than younger myocytes to thyroid receptor blockade by antagonist, NH3, and to the effects of TRα1/α2 short interfering RNA. Although T3 is known to abrogate ovine cardiomyocyte proliferation stimulated by insulin-like growth factor 1, this effect is mediated via the genomic action of thyroid hormone receptors, with little evidence for non-genomic mechanisms. ABSTRACT: We have previously shown that the late-term rise in tri-iodo-l-thyronine (T3 ) in fetal sheep leads to the inhibition of proliferation and promotion of maturation in cardiomyocytes. The present study was designed to determine whether these T3 -induced changes are mediated via thyroid hormone receptors (TRs) or by non-genomic mechanisms. Fetal cardiomyocytes were isolated from 102 ± 3 and 135 ± 1 days of gestational age (dGA) sheep (n = 7 per age; term ∼145 dGA). Cells were treated with T3 (1.5 nm), insulin-like growth factor (IGF)-1 (1 µg mL-1 ) or a combination in the presence of TR antagonist NH3 (100 nm) or following short interfering RNA (siRNA) knockdown of TRα1/α2. Proliferation was quantified by 5-bromo-2'-deoxyuridine (BrdU) uptake (10 µm). Western blots measured protein levels of extracellular signal-related kinase (ERK), Akt, TRα1/ß1 and p21. Age specific levels of TRα1/ß1 were measured in normal hearts from fetuses [95 dGA (n = 8), 135 dGA (n = 7)], neonates (n = 8) and adult ewes (n = 7). TRα1 protein levels were consistently >50% more than TRß1 at each gestational age (P < 0.05). T3 reduced IGF-1 stimulated proliferation by ∼50% in 100 dGA and by ∼75% in 135 dGA cardiomyocytes (P < 0.05). NH3 blocked the T3  + IGF-1 reduction of BrdU uptake without altering the phosphorylation of ERK or Akt at both ages. NH3 did not suppress T3 -induced p21 expression in 100 dGA cardiomyocytes in 135 dGA cardiomyocytes, NH3 alone reduced BrdU uptake (-28%, P < 0.05), as well as T3 -induced p21 (-75%, P < 0.05). In both ages, siRNA knockdown of TRα1/α2 blocked the T3  + IGF-1 reduction of BrdU uptake and dramatically reduced ERK and Akt signalling in 135 dGA cardiomyocytes. In conclusion, TRs are required for normal proliferation and T3 signalling in fetal ovine cardiomyocytes, with the sensitivity to TR blockade being age-dependent.

Structure-Activity Relationships of Central Nervous System Penetration by Fatty Acid Amide Hydrolase (FAAH)-Targeted Thyromimetic Prodrugs.

Thyroid hormone (TH) action is of clinical interest in treating demyelinating diseases of the central nervous system (CNS). Two amide prodrugs of sobetirome, a potent thyroid hormone agonist, were previously shown to significantly improve CNS selective distribution of the parent drug through hydrolysis in the CNS by fatty acid amide hydrolase (FAAH). This concept is elaborated upon here with a series of 29 amide prodrugs targeting FAAH. We identify that conservative aliphatic modifications such as the N-methyl (4), N-ethyl (5), N-fluoroethyl (15), and N-cyclopropyl (18) substantially favor selective CNS distribution of the parent drug in mice. Additionally, lead compounds exhibit moderate to good rates of hydrolysis at FAAH in vitro suggesting both enzymatic and physicochemical properties are important parameters for optimization. Both 4 and 15 were orally bioavailable while retaining appreciable CNS parent drug delivery following an oral dose. The pharmacokinetic parameters of 4 over 24 h postdose (i.v. and p.o.) were determined.

Radiation-primed hepatocyte transplantation in murine monogeneic dyslipidemia normalizes cholesterol and prevents atherosclerosis.

BACKGROUND & AIMS: Inherited abnormalities in apolipoprotein E (ApoE) or low-density lipoprotein receptor (LDLR) function result in early onset cardiovascular disease and death. Currently, the only curative therapy available is liver transplantation. Hepatocyte transplantation is a potential alternative; however, physiological levels of hepatocyte engraftment and repopulation require transplanted cells to have a competitive proliferative advantage of over host hepatocytes. Herein, we aimed to test the efficacy and safety of a novel preparative regimen for hepatocyte transplantation. METHODS: Herein, we used an ApoE-deficient mouse model to test the efficacy of a new regimen for hepatocyte transplantation. We used image-guided external-beam hepatic irradiation targeting the median and right lobes of the liver to enhance cell transplant engraftment. This was combined with administration of the hepatic mitogen GC-1, a thyroid hormone receptor-ß agonist mimetic, which was used to promote repopulation. RESULTS: The non-invasive preparative regimen of hepatic irradiation and GC-1 was well-tolerated in ApoE-/- mice. This regimen led to robust liver repopulation by transplanted hepatocytes, which was associated with significant reductions in serum cholesterol levels after transplantation. Additionally, in mice receiving this regimen, ApoE was detected in the circulation 4 weeks after treatment and did not induce an immunological response. Importantly, the normalization of serum cholesterol prevented the formation of atherosclerotic plaques in this model. CONCLUSIONS: Significant hepatic repopulation and the cure of dyslipidemia in this model, using a novel and well-tolerated preparative regimen, demonstrate the clinical potential of applying this method to the treatment of inherited metabolic diseases of the liver. LAY SUMMARY: Hepatocyte transplantation is a promising alternative to liver transplantation for the treatment of liver diseases. However, it is inefficient, as restricted growth of transplanted cells in the liver limits its therapeutic benefits. Preparative treatments improve the efficiency of this procedure, but no clinically-feasible options are currently available. In this study we develop a novel well-tolerated preparative treatment to improve growth of cells in the liver and then demonstrate that this treatment completely cures an inherited lipid disorder in a mouse model.

Quantification of Thyromimetic Sobetirome Concentration in Biological Tissue Samples.

Thyroid hormone is a principal regulator of essential processes in vertebrate physiology and homeostasis. Synthetic derivatives of thyroid hormone, known as thyromimetics, display desirable therapeutic properties. Thoroughly understanding how thyromimetics distribute throughout the body is crucial for their development and this requires appropriate bioanalytical techniques to quantify drug levels in different tissues. Here, we describe a detailed protocol for the quantification of the thyromimetic sobetirome using liquid chromatography tandem-mass spectrometry (LC-MS/MS).

Sobetirome and its Amide Prodrug Sob-AM2 Exert Thyromimetic Actions in Mct8-Deficient Brain.

BACKGROUND: Loss of function mutations in the thyroid hormone (TH)-specific cell membrane transporter, the monocarboxylate transporter 8 (MCT8), lead to profound psychomotor retardation and abnormal TH serum levels, with low thyroxine (T4) and high triiodothyronine (T3). Several studies point to impaired TH transport across brain barriers as a crucial pathophysiological mechanism resulting in cerebral hypothyroidism. Treatment options for MCT8-deficient patients are limited and are focused on overcoming the brain barriers. The aim of this study was to evaluate the ability of the TH analog sobetirome and its prodrug Sob-AM2 to access the brain and exert thyromimetic actions in the absence of Mct8. METHODS: Juvenile wild-type (Wt) mice and mice lacking Mct8 and deiodinase type 2 (Mct8/Dio2KO) were treated systemically with daily injections of vehicle, 1 mg of sobetirome/kg body weight/day, or 0.3 mg of Sob-AM2/kg body weight/day for seven days. Sobetirome content was measured using liquid chromatography-tandem mass spectrometry, and T4 and T3 levels by specific radioimmunoassays. The effect of sobetirome treatment in the expression of T3-dependent genes was measured in the heart, liver, and cerebral cortex by real-time polymerase chain reaction. RESULTS: Sob-AM2 treatment in Mct8/Dio2KO animals led to 1.8-fold more sobetirome content in the brain and 2.5-fold less in plasma in comparison to the treatment with the parent drug sobetirome. Both sobetirome and Sob-AM2 treatments in Mct8/Dio2KO mice greatly decreased plasma T4 and T3 levels. Dio1 and Ucp2 gene expression was altered in the liver of Mct8/Dio2KO mice and was not affected by the treatments. In the heart, Hcn2 but not Atp2a2 expression was increased after treatment with the analogs. Interestingly, both sobetirome and Sob-AM2 treatments increased the expression of several T3-dependent genes in the brain such as Hr, Abcd2, Mme, and Flywch2 in Mct8/Dio2KO mice. CONCLUSIONS: Sobetirome and its amide prodrug Sob-AM2 can access the brain in the absence of Mct8 and exert thyromimetic actions modulating the expression of T3-dependent genes. At the peripheral level, the administration of these TH analogs results in the depletion of circulating T4 and T3. Therefore, sobetirome and Sob-AM2 have the potential to address the cerebral hypothyroidism and the peripheral hyperthyroidism characteristic of MCT8 deficiency.

Hypothalamic-Pituitary-Thyroid Axis Perturbations in Male Mice by CNS-Penetrating Thyromimetics.

Thyromimetics represent a class of experimental drugs that can stimulate tissue-selective thyroid hormone action. As such, thyromimetics should have effects on the hypothalamic-pituitary-thyroid (HPT) axis, but details of this action and the subsequent effects on systemic thyroid hormone levels have not been reported to date. Here, we compare the HPT-axis effects of sobetirome, a well-studied thyromimetic, with Sob-AM2, a newly developed prodrug of sobetirome that targets sobetirome distribution to the central nervous system (CNS). Similar to endogenous thyroid hormone, administration of sobetirome and Sob-AM2 suppress HPT-axis gene transcript levels in a manner that correlates to their specific tissue distribution properties (periphery vs CNS, respectively). Dosing male C57BL/6 mice with sobetirome and Sob-AM2 at concentrations ≥10 µg/kg/d for 29 days induces a state similar to central hypothyroidism characterized by depleted circulating T4 and T3 and normal TSH levels. However, despite the systemic T4 and T3 depletion, the sobetirome- and Sob-AM2-treated mice do not show signs of hypothyroidism, which may result from the presence of the thyromimetic in the thyroid hormone-depleted background.

T3 Induces Both Markers of Maturation and Aging in Pancreatic ß-Cells.

Previously, we showed that thyroid hormone (TH) triiodothyronine (T3) enhanced ß-cell functional maturation through induction of Mafa High levels of T3 have been linked to decreased life span in mammals and low levels to lengthened life span, suggesting a relationship between TH and aging. Here, we show that T3 increased p16Ink4a (a ß-cell senescence marker and effector) mRNA in rodent and human ß-cells. The kinetics of Mafa and p16Ink4a induction suggested both genes as targets of TH via TH receptors (THRs) binding to specific response elements. Using specific agonists CO23 and GC1, we showed that p16Ink4a expression was controlled by THRA and Mafa by THRB. Using chromatin immunoprecipitation and a transient transfection yielding biotinylated THRB1 or THRA isoforms to achieve specificity, we determined that THRA isoform bound to p16Ink4a , whereas THRB1 bound to Mafa but not to p16Ink4a On a cellular level, T3 treatment accelerated cell senescence as shown by increased number of ß-cells with acidic ß-galactosidase activity. Our data show that T3 can simultaneously induce both maturation (Mafa) and aging (p16Ink4a ) effectors and that these dichotomous effects are mediated through different THR isoforms. These findings may be important for further improving stem cell differentiation protocols to produce functional ß-cells for replacement therapies in diabetes.

Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function.

Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-ß1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.