Decreased hepatic thyroid hormone signaling in systemic and liver-specific but not brain-specific accelerated aging due to DNA repair deficiency in mice.

Background: Thyroid hormone signaling is essential for development, metabolism, and response to stress but declines during aging, the cause of which is unknown. DNA damage accumulating with time is a main cause of aging, driving many age-related diseases. Previous studies in normal and premature aging mice, due to defective DNA repair, indicated reduced hepatic thyroid hormone signaling accompanied by decreased type 1 deiodinase (DIO1) and increased DIO3 activities. We investigated whether aging-related changes in deiodinase activity are driven by systemic signals or represent cell- or organ-autonomous changes. Methods: We quantified liver and plasma thyroid hormone concentrations, deiodinase activities and expression of T3-responsive genes in mice with a global, liver-specific and for comparison brain-specific inactivation of Xpg, one of the endonucleases critically involved in multiple DNA repair pathways. Results: Both in global and liver-specific Xpg knockout mice, hepatic DIO1 activity was decreased. Interestingly, hepatic DIO3 activity was increased in global, but not in liver-specific Xpg mutants. Selective Xpg deficiency and premature aging in the brain did not affect liver or systemic thyroid signaling. Concomitant with DIO1 inhibition, Xpg -/- and Alb-Xpg mice displayed reduced thyroid hormone-related gene expression changes, correlating with markers of liver damage and cellular senescence. Conclusions: Our findings suggest that DIO1 activity during aging is predominantly modified in a tissue-autonomous manner driven by organ/cell-intrinsic accumulating DNA damage. The increase in hepatic DIO3 activity during aging largely depends on systemic signals, possibly reflecting the presence of circulating cells rather than activity in hepatocytes.

Unraveling the Role of Thyroid Hormones in Seasonal Neuroplasticity in European Starlings (Sturnus vulgaris).

Thyroid hormones clearly play a role in the seasonal regulation of reproduction, but any role they might play in song behavior and the associated seasonal neuroplasticity in songbirds remains to be elucidated. To pursue this question, we first established seasonal patterns in the expression of thyroid hormone regulating genes in male European starlings employing in situ hybridization methods. Thyroid hormone transporter LAT1 expression in the song nucleus HVC was elevated during the photosensitive phase, pointing toward an active role of thyroid hormones during this window of possible neuroplasticity. In contrast, DIO3 expression was high in HVC during the photostimulated phase, limiting the possible effect of thyroid hormones to maintain song stability during the breeding season. Next, we studied the effect of hypothyroidism on song behavior and neuroplasticity using in vivo MRI. Both under natural conditions as with methimazole treatment, circulating thyroid hormone levels decreased during the photosensitive period, which coincided with the onset of neuroplasticity. This inverse relationship between thyroid hormones and neuroplasticity was further demonstrated by the negative correlation between plasma T3 and the microstructural changes in several song control nuclei and cerebellum. Furthermore, maintaining hypothyroidism during the photostimulated period inhibited the increase in testosterone, confirming the role of thyroid hormones in activating the hypothalamic-pituitary-gonadal (HPG) axis. The lack of high testosterone levels influenced the song behavior of hypothyroid starlings, while the lack of high plasma T4 during photostimulation affected the myelination of several tracts. Potentially, a global reduction of circulating thyroid hormones during the photosensitive period is necessary to lift the brake on neuroplasticity imposed by the photorefractory period, whereas local fine-tuning of thyroid hormone concentrations through LAT1 could activate underlying neuroplasticity mechanisms. Whereas, an increase in circulating T4 during the photostimulated period potentially influences the myelination of several white matter tracts, which stabilizes the neuroplastic changes. Given the complexity of thyroid hormone effects, this study is a steppingstone to disentangle the influence of thyroid hormones on seasonal neuroplasticity.

Thyroid Hormone Transporter Deficiency in Mice Impacts Multiple Stages of GABAergic Interneuron Development.

Cortical interneuron neurogenesis is strictly regulated and depends on the presence of thyroid hormone (TH). In particular, inhibitory interneurons expressing the calcium binding protein Parvalbumin are highly sensitive toward developmental hypothyroidism. Reduced numbers of Parvalbumin-positive interneurons are observed in mice due to the combined absence of the TH transporters Mct8 and Oatp1c1. To unravel if cortical Parvalbumin-positive interneurons depend on cell-autonomous action of Mct8/Oatp1c1, we compared Mct8/Oatp1c1 double knockout (dko) mice to conditional knockouts with abolished TH transporter expression in progenitors of Parvalbumin-positive interneurons. These conditional knockouts exhibited a transient delay in the appearance of Parvalbumin-positive interneurons in the early postnatal somatosensory cortex while cell numbers remained permanently reduced in Mct8/Oatp1c1 dko mice. Using fluorescence in situ hybridization on E12.5 embryonic brains, we detected reduced expression of sonic hedgehog signaling components in Mct8/Oatp1c1 dko embryos only. Moreover, we revealed spatially distinct expression patterns of both TH transporters at brain barriers at E12.5 by immunofluorescence. At later developmental stages, we uncovered a sequential expression of first Oatp1c1 in individual interneurons and then Mct8 in Parvalbumin-positive subtypes. Together, our results point to multiple cell-autonomous and noncell-autonomous mechanisms that depend on proper TH transport during cortical interneuron development.

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination.

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development.

Uncovering a 'sensitive window' of multisensory and motor neuroplasticity in the cerebrum and cerebellum of male and female starlings.

Traditionally, research unraveling seasonal neuroplasticity in songbirds has focused on the male song control system and testosterone. We longitudinally monitored the song behavior and neuroplasticity in male and female starlings during multiple photoperiods using Diffusion Tensor and Fixel-Based techniques. These exploratory data-driven whole-brain methods resulted in a population-based tractogram confirming microstructural sexual dimorphisms in the song control system. Furthermore, male brains showed hemispheric asymmetries in the pallium, whereas females had higher interhemispheric connectivity, which could not be attributed to brain size differences. Only females with large brains sing but differ from males in their song behavior by showing involvement of the hippocampus. Both sexes experienced multisensory neuroplasticity in the song control, auditory and visual system, and cerebellum, mainly during the photosensitive period. This period with low gonadal hormone levels might represent a 'sensitive window' during which different sensory and motor systems in the cerebrum and cerebellum can be seasonally re-shaped in both sexes.

Deiodinases: How Nonmammalian Research Helped Shape Our Present View.

Iodothyronine deiodinases are enzymes capable of activating and inactivating thyroid hormones (THs) and have an important role in regulating TH action in tissues throughout the body. Three types of deiodinases (D1, D2, and D3) were originally defined based on their biochemical characteristics. Cloning of the first complementary DNAs in the 1990s (Dio1 in rat and dio2 and dio3 in frog) allowed to confirm the existence of 3 distinct enzymes. Over the years, increasing genomic information revealed that deiodinases are present in all chordates, vertebrates, and nonvertebrates and that they can even be found in some mollusks and annelids, pointing to an ancient origin. Research in nonmammalian models has substantially broadened our understanding of deiodinases. In relation to their structure, we discovered for instance that biochemical properties such as inhibition by 6-propyl-2-thiouracil, stimulation by dithiothreitol, and temperature optimum are subject to variation. Data from fish, amphibians, and birds were key in shifting our view on the relative importance of activating and inactivating deiodination pathways and in showing the impact of D2 and D3 not only in local but also whole body T3 availability. They also led to the discovery of new local functions such as the acute reciprocal changes in D2 and D3 in hypothalamic tanycytes upon photostimulation, involved in seasonal rhythmicity. With the present possibilities for rapid and precise gene silencing in any species of interest, comparative research will certainly further contribute to a better understanding of the importance of deiodinases for adequate TH action, also in humans.

Embryonic thermal manipulation has short and long-term effects on the development and the physiology of the Japanese quail.

In vertebrates, the embryonic environment is known to affect the development and the health of individuals. In broiler chickens, the thermal-manipulation (TM) of eggs during the incubation period was shown to improve heat tolerance at slaughter age (35 days of age) in association with several modifications at the molecular, metabolic and physiological levels. However, little is known about the Japanese quail (Coturnix japonica), a closely related avian species widely used as a laboratory animal model and farmed for its meat and eggs. Here we developed and characterized a TM procedure (39.5°C and 65% relative humidity, 12 h/d, from days 0 to 13 of incubation) in quails by analyzing its short and long-term effects on zootechnical, physiological and metabolic parameters. Heat-tolerance was tested by a heat challenge (36°C for 7h) at 35 days of age. TM significantly reduced the hatching rate of the animals and increased mortality during the first four weeks of life. At hatching, TM animals were heavier than controls, but lighter at 25 days of age for both sexes. Thirty-five days after hatching, TM decreased the surface temperature of the shank in females, suggesting a modulation of the blood flow to maintain the internal temperature. TM also increased blood partial pressure and oxygen saturation percentage at 35 days of age in females, suggesting a long-term modulation of the respiration physiology. Quails physiologically responded to the heat challenge, with a modification of several hematologic and metabolic parameters, including an increase in plasma corticosterone concentration. Several physiological parameters such as beak surface temperature and blood sodium concentration revealed that TM birds responded differently to the heat challenge compared to controls. Altogether, this first comprehensive characterization of TM in Japanese quail showed durable effects that may affect the response of TM quails to heat.

Between- and Within-Individual Variation of Maternal Thyroid Hormone Deposition in Wild Great Tits (Parus major).

Maternal hormones are often considered a mediator of anticipatory maternal effects; namely, mothers adjust maternal hormone transfer to prepare the offspring for the anticipated environment. The flexibility for mothers to adjust hormone transfer is therefore a prerequisite for such anticipatory maternal effects. Nevertheless, previous studies have focused only on the average differences of maternal hormone transfer between groups and neglected the substantial individual variation, despite the fact that individual plasticity in maternal hormone transfer is actually the central assumption. In this study, we studied the between- and within-individual variation of maternal thyroid hormones (THs) in egg yolk of wild great tits (Parus major) and estimated the individual plasticity of maternal yolk THs across environmental temperature, clutch initiation dates, and egg laying order using linear mixed effects models. Interestingly, our models provide statistical evidence that the two main THs-the main biologically active hormone T3 and T4, which is mostly considered a prohormone-exhibited different variation patterns. Yolk T3 showed significant between-individual variation on the average levels, in line with its previously reported moderate heritability. Yolk T4, however, showed significant between-clutch variation in the pattern over the laying sequence, suggesting a great within-individual plasticity. Our findings suggest that the role and function of the hormone within the endocrine axis likely influences its flexibility to respond to environmental change. Whether the flexibility of T4 deposition brings a fitness advantage should be examined along with its potential effects on offspring, which remain to be further investigated.

Age-Dependent Changes in Glucose Homeostasis in Male Deiodinase Type 2 Knockout Zebrafish.

Thyroid hormones (THs) are crucial regulators of glucose metabolism and insulin sensitivity. Moreover, inactivating mutations in type 2 deiodinase (DIO2), the major TH-activating enzyme, have been associated with type 2 diabetes mellitus in both humans and mice. We studied the link between Dio2 deficiency and glucose homeostasis in fasted males of two different Dio2 knockout (KO) zebrafish lines. Young adult Dio2KO zebrafish (6 to 9 months) were hyperglycemic. Both insulin and glucagon expression were increased, whereas ß and α cell numbers in the main pancreatic islet were similar to those in wild-types. Insulin receptor expression in skeletal muscle was decreased at 6 months, accompanied by a strong downregulation of hexokinase and pyruvate kinase expression. Blood glucose levels in Dio2KO zebrafish, however, normalized around 1 year of age. Older mutants (18 to 24 months) were normoglycemic, and increased insulin and glucagon expression was accompanied by a prominent increase in pancreatic islet size and ß and α cell numbers. Older Dio2KO zebrafish also showed strongly decreased expression of glucagon receptors in the gastrointestinal system as well as decreased expression of glucose transporters GLUT2 and GLUT12, glucose-6-phosphatase, and glycogen synthase 2. This study shows that Dio2KO zebrafish suffer from transient hyperglycemia, which is counteracted with increasing age by a prominent hyperplasia of the endocrine pancreas together with decreases in hepatic glucagon sensitivity and intestinal glucose uptake. Further research on the mechanisms allowing compensation in older Dio2KO zebrafish may help to identify new therapeutic targets for (TH deficiency-related) hyperglycemia.

Disruption of deiodinase type 2 in zebrafish disturbs male and female reproduction.

Thyroid hormones are crucial mediators of many aspects of vertebrate life, including reproduction. The key player is the biologically active 3,5,3'-triiodothyronine (T3), whose local bio-availability is strictly regulated by deiodinase enzymes. Deiodinase type 2 (Dio2) is present in many tissues and is the main enzyme for local T3 production. To unravel its role in different physiological processes, we generated a mutant zebrafish line, completely lacking Dio2 activity. Here we focus on the reproductive phenotype studied at the level of offspring production, gametogenesis, functioning of the hypothalamic-pituitary-gonadal axis and sex steroid production. Homozygous Dio2-deficient zebrafish were hypothyroid, displayed a delay in sexual maturity, and the duration of their reproductive period was substantially shortened. Fecundity and fertilization were also severely reduced. Gamete counts pointed to a delay in oogenesis at onset of sexual maturity and later on to an accumulation of oocytes in mutant ovaries due to inhibition of ovulation. Analysis of spermatogenesis showed a strongly decreased number of spermatogonia A at onset of sexual maturity. Investigation of the hypothalamic-pituitary-gonadal axis revealed that dysregulation was largely confined to the gonads with significant upregulation of igf3, and a strong decrease in sex steroid production concomitant with alterations in gene expression in steroidogenesis/steroid signaling pathways. Rescue of the phenotype by T3 supplementation starting at 4 weeks resulted in normalization of reproductive activity in both sexes. The combined results show that reproductive function in mutants is severely hampered in both sexes, thereby linking the loss of Dio2 activity and the resulting hypothyroidism to reproductive dysfunction.

The Role of Maternal Thyroid Hormones in Avian Embryonic Development.

During avian embryonic development, thyroid hormones (THs) coordinate the expression of a multitude of genes thereby ensuring that the correct sequence of cell proliferation, differentiation and maturation is followed in each tissue and organ. Although THs are needed from the start of development, the embryonic thyroid gland only matures around mid-incubation in precocial birds and around hatching in altricial species. Therefore, maternal THs deposited in the egg yolk play an essential role in embryonic development. They are taken up by the embryo throughout its development, from the first day till hatching, and expression of TH regulators such as distributor proteins, transporters, and deiodinases in the yolk sac membrane provide the tools for selective metabolism and transport starting from this level. TH receptors and regulators of local TH availability are expressed in avian embryos in a dynamic and tissue/cell-specific pattern from the first stages studied, as shown in detail in chicken. Maternal hyperthyroidism via TH supplementation as well as injection of THs into the egg yolk increase TH content in embryonic tissues while induction of maternal hypothyroidism by goitrogen treatment results in a decrease. Both increase and decrease of maternal TH availability were shown to alter gene expression in early chicken embryos. Knockdown of the specific TH transporter monocarboxylate transporter 8 at early stages in chicken cerebellum, optic tectum, or retina allowed to reduce local TH availability, interfering with gene expression and confirming that development of the central nervous system (CNS) is highly dependent on maternal THs. While some of the effects on cell proliferation, migration and differentiation seem to be transient, others result in persistent defects in CNS structure. In addition, a number of studies in both precocial and altricial birds showed that injection of THs into the yolk at the start of incubation influences a number of parameters in posthatch performance and fitness. In conclusion, the data presently available clearly indicate that maternal THs play an important role in avian embryonic development, but how exactly their influence on cellular and molecular processes in the embryo is linked to posthatch fitness needs to be further explored.

Knockdown of the thyroid hormone transporter MCT8 in chicken retinal precursor cells hampers early retinal development and results in a shift towards more UV/blue cones at the expense of green/red cones.

Thyroid hormones (THs) play a crucial role in coordinating brain development in vertebrates. They fine-tune processes like cell proliferation, migration, and differentiation mainly by regulating the transcriptional activity of many essential genes. Regulators of TH availability thereby define the cellular concentration of the bioactive 3,5,3'-triiodothyronine, which binds to nuclear TH receptors. One important regulator, the monocarboxylate transporter 8 (MCT8), facilitates cellular TH uptake and is known to be necessary for correct brain development, but data on its potential role during retinal development is lacking. The retinal cyto-architecture has been conserved throughout vertebrate evolution, and we used the chicken embryo to study the need for MCT8 during retinal development. Its external development allows easy manipulation, and MCT8 is abundantly expressed in the retina from early stages onwards. We induced MCT8 knockdown by electroporating a pRFP-MCT8-RNAi vector into the retinal precursor cells (RPCs) at embryonic day 4 (E4), and studied the consequences for early (E6) and late (E18) retinal development. The empty pRFP-RNAi vector was used as a control. RPC proliferation was reduced at E6. This resulted in cellular hypoplasia and a thinner retina at E18 where mainly photoreceptors and horizontal cells were lost, the two predominant cell types that are born around the stage of electroporation. At E6, differentiation into retinal ganglion cells and amacrine cells was delayed. However, since the proportion of a given cell type within the transfected cell population at E18 was similar in knockdown and controls, the partial loss of some cell types was most-likely due to reduced RPC proliferation and not impaired cell differentiation. Photoreceptors displayed delayed migration at first, but had successfully reached the outer nuclear layer at E18. However, they increasingly differentiated into short wavelength-sensitive cones at the expense of medium/long wavelength-sensitive cones, while the proportion of rods was unaltered. Improperly formed sublaminae in the inner plexiform layer additionally suggested defects in synaptogenesis. Altogether, our data echoes effects of hypothyroidism and the loss of some other regulators of TH availability in the developing zebrafish and rodent retina. Therefore, the expression of MCT8 in RPCs is crucial for adequate TH uptake during cell type-specific events in retinal development.

Insights from zebrafish deficiency models to understand the impact of local thyroid hormone regulator action on early development.

Thyroid hormones (THs) stimulate and coordinate a wide range of processes to ensure normal development, mainly by binding of the most active TH 3,5,3'-triiodothyronine (T3) to nuclear receptors resulting in changes in gene transcription. Local TH action is monitored at three distinct levels by different types of regulators: transmembrane transporters (TH influx and efflux), deiodinases (TH activation and inactivation) and nuclear receptors (TH signalling). Since TH regulators are strongly conserved among vertebrate species, the externally and rapidly developing zebrafish (Danio rerio) has become one of the favourite models to study their role in TH-dependent development. Most regulators are expressed in zebrafish from early stages in development in a dynamic and tissue-specific pattern. Transient or permanent disruption of a given regulator severely perturbs development of multiple organs. These zebrafish deficiency models help to explain why, next to overall hypo-/hyperthyroidism, inactivating mutations in the genes encoding TH regulators such as MCT8 and THRA/B have irreversible adverse effects on human development. Zebrafish are also increasingly used as a high-throughput model to assess the toxicity of various xenobiotics and their impact on development. While adverse effects on TH metabolism and gene expression have been shown, information on direct interaction with TH regulators is scarce, albeit essential to fully understand their mechanism of action. For the future, the combination of novel gene silencing tools, fluorescent reporter lines and (single-cell) transcriptomics holds promise for new zebrafish models to further elucidate the role of each TH regulator in vertebrate development.

A new method for measuring thyroid hormones using nano-LC-MS/MS.

This paper describes a novel mass spectrometry based analytical method for analyzing thyroid hormones (THs). Thyroid hormones play a critical role in the regulation of many biological processes such as growth, metabolism and development. Several analytical methods using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) have previously been developed to measure THs, especially in humans. For biomedical and toxicological research using small animal models, and in ecophysiological research using wild species where sample volume is limiting, sensitive methods are needed. In this study, we developed a nano-LC-MS/MS method enabling quantification of low concentrations of two key THs, thyroxine (T4) and 3,3',5-triiodothyronine (T3). The method was tested with egg yolk samples. We used a low flow rate (300 nl/min) to obtain maximal sensitivity of the method. The limit of quantitation was 10.6 amol for T4 and 17.9 amol for T3. The method shows good linearity (r > 0.99), repeatability and reproducibility (CVs <10%). We also reanalyzed yolk samples with radioimmunoassay for a comparison of the newly developed and previously used methods. Finally, we applied the methodology to measure hormones in egg yolk extracts in multiple avian species, and report interesting variation in maternal TH deposition. The newly developed nano-LC-MS/MS method is thus suitable for measuring THs in low concentrations and across species.

Thyroid Hormone Transporters MCT8 and OATP1C1 Control Skeletal Muscle Regeneration.

Thyroid hormone (TH) transporters are required for the transmembrane passage of TH in target cells. In humans, inactivating mutations in the TH transporter MCT8 cause the Allan-Herndon-Dudley syndrome, characterized by severe neuromuscular symptoms and an abnormal TH serum profile, which is fully replicated in Mct8 knockout mice and Mct8/Oatp1c1 double-knockout (M/O DKO) mice. Analysis of tissue TH content and expression of TH-regulated genes indicate a thyrotoxic state in Mct8-deficient skeletal muscles. Both TH transporters are upregulated in activated satellite cells (SCs). In M/O DKO mice, we observed a strongly reduced number of differentiated SCs, suggesting an impaired stem cell function. Moreover, M/O DKO mice and mice lacking both transporters exclusively in SCs showed impaired skeletal muscle regeneration. Our data provide solid evidence for a unique gate-keeper function of MCT8 and OATP1C1 in SC activation, underscoring the importance of a finely tuned TH signaling during myogenesis.

Forever young: Endocrinology of paedomorphosis in the Mexican axolotl (Ambystoma mexicanum).

The Mexican axolotl (Ambystoma mexicanum) is a salamander species that does not undergo metamorphosis, resulting in the retention of juvenile characteristics in the mature breeding stage (paedomorphosis). Here we review the endocrinological studies investigating the proximate cause of axolotl paedomorphosis with a focus on the hypothalamo-pituitary-thyroid (HPT) axis. It is well established that axolotl paedomorphosis is a consequence of low activity of the HPT axis. The pituitary hormone thyrotropin (TSH) is capable of inducing metamorphosis in the axolotl, which indicates that all processes and interactions in the HPT axis below the pituitary level are functional, but that TSH release is impaired. In metamorphosing species, TSH secretion is largely controlled by the hypothalamic neuropeptide corticotropin-releasing hormone (CRH), which seems to have lost its thyrotropic activity in the axolotl. However, preliminary experiments have not yet confirmed a role for faulty CRH signalling in axolotl paedomorphosis. Other hypothalamic factors and potential pituitary inhibitors need to be investigated to identify their roles in amphibian metamorphosis and axolotl paedomorphosis.

An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish.

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.

From zebrafish to human: A comparative approach to elucidate the role of the thyroid hormone transporter MCT8 during brain development.

Monocarboxylate transporter 8 (MCT8) facilitates transmembrane transport of thyroid hormones (THs) ensuring their action on gene expression during vertebrate neurodevelopment. A loss of MCT8 in humans results in severe psychomotor deficits associated with the Allan-Herndon-Dudley Syndrome (AHDS). However, where and when exactly a lack of MCT8 causes the neurological manifestations remains unclear because of the varying expression pattern of MCT8 between specific brain regions and cells. Here, we elaborate on the animal models that have been generated to elucidate the mechanisms underlying MCT8-deficient brain development. The absence of a clear neurological phenotype in Mct8 knockout mice made it clear that a single species would not suffice. The evolutionary conservation of TH action on neurodevelopment as well as the components regulating TH signalling however offers the opportunity to answer different aspects of MCT8 function in brain development using different vertebrate species. Moreover, the plethora of tools for genome editing available today facilitates gene silencing in these animals as well. Studies in the recently generated mct8-deficient zebrafish and Mct8/Oatp1c1 double knockout mice have put forward the current paradigm of impaired TH uptake at the level of the blood-brain barrier during peri- and postnatal development as being the main pathophysiological mechanism of AHDS. RNAi vector-based, cell-specific induction of MCT8 knockdown in the chicken embryo points to an additional function of MCT8 at the level of the neural progenitors during early brain development. Future studies including also additional in vivo models like Xenopus or in vitro approaches such as induced pluripotent stem cells will continue to help unravelling the exact role of MCT8 in developmental events. In the end, this multispecies approach will lead to a unifying thesis regarding the cellular and molecular mechanisms responsible for the neurological phenotype in AHDS patients.

The affinity of transthyretin for T3 or T4 does not determine which form of the hormone accumulates in the choroid plexus.

Normal development of the brain is dependent on the required amounts of thyroid hormones (THs) reaching specific regions of the brain during each stage of ontogeny. Many proteins are involved with regulation of TH bioavailability in the brain: the TH distributor protein transthyretin (TTR), TH transmembrane transporters (e.g. MCT8, MCT10, LAT1, OATP1C1) and deiodinases (D1, D2 and D3) which either activate or inactivate THs. Previous studies revealed that in mammals, T4, but not T3, accumulated in the choroid plexus and then entered the cerebrospinal fluid. In all mammalian species studied so far, TTR binds T4 with higher affinity than T3, whereas TTR in non-mammalian vertebrates binds T3 with higher affinity than T4. We investigated if the form of TH preferentially bound by TTR influenced the form of the TH that accumulated in the choroid plexus and consequently other areas of the brain. We measured the mRNA levels corresponding to TTR, MCT8, MCT10, LAT1, OATP1C1, D1, D2 and D3 in the brains of chickens at 11days post-hatching. TTR, D3 and OATP1C1 expression were found to be highly concentrated in the choroid plexus. D1, MCT8 and MCT10 mRNA levels were slightly greater in the choroid plexus than in other areas of the brain while D2 mRNA levels were lower. LAT1 mRNA was evenly expressed throughout the brain. Therefore, the choroid plexus appears to be a structure which exhibits sophisticated control of TH levels within the brain. We also measured the uptake of intravenously injected 125I-T3 and 125I-T4 into brains of chickens of the same age. 125I-T4 but not 125I-T3 accumulated in the choroid plexus and optic lobes. Therefore, the form of TH preferentially bound by TTR does not determine the form of TH that accumulates in the choroid plexus and other areas of the brain. As for mammals, T3 present in the avian brain therefore seems mainly produced locally by conversion of T4 into T3 by D2.

The Thyroid Hormone Inactivating Type 3 Deiodinase Is Essential for Optimal Neutrophil Function: Observations From Three Species.

Neutrophils are essential effector cells of the innate immune system that have recently been recognized as thyroid hormone (TH) target cells. Cellular TH bioavailability is regulated by the deiodinase enzymes, which can activate or inactivate TH. We have previously shown that the TH inactivating enzyme type 3 deiodinase (D3) is present in neutrophils. Furthermore, D3 knockout (D3KO) mice show impaired bacterial killing upon infection. We hypothesized that D3 plays a role in neutrophil function during infection by actively regulating local TH availability. We measured TH concentrations in cerebrospinal fluid (CSF) from patients with bacterial meningitis and controls. Bacterial meningitis resulted in marked changes in CSF TH levels, characterized by a strong increase of thyroxine and reverse-triiodothyronine concentrations. This altered TH profile was consistent with elevated D3 activity in infiltrating neutrophils at the site of infection. D3 knockdown in zebrafish embryos with pneumococcal meningitis resulted in increased mortality and reduced neutrophil infiltration during infection. Finally, stimulated neutrophils from female D3KO mice exhibited impaired NADPH-oxidase activity, an important component of the neutrophil bacterial killing machinery. These consistent findings across experimental models strongly support a critical role for reduced intracellular TH concentrations in neutrophil function during infection, for which the TH inactivating enzyme D3 appears essential.